Multi-modality imaging to assess metabolic response to dichloroacetate treatment in tumor models

Abstract

Reverting glycolytic metabolism is an attractive strategy for cancer therapy as upregulated glycolysis is a hallmark in various cancers. Dichloroacetate (DCA), long used to treat lactic acidosis in various pathologies, has emerged as a promising anti-cancer drug. By inhibiting the pyruvate dehydrogenase kinase, DCA reactivates the mitochondrial function and decreases the glycolytic flux in tumor cells resulting in cell cycle arrest and apoptosis. We recently documented that DCA was able to induce a metabolic switch preferentially in glycolytic cancer cells, leading to a more oxidative phenotype and decreasing proliferation, while oxidative cells remained less sensitive to DCA treatment. To evaluate the relevance of this observation in vivo, the aim of the present study was to characterize the effect of DCA in glycolytic MDA-MB-231 tumors and in oxidative SiHa tumors using advanced pharmacodynamic metabolic biomarkers. Oxygen consumption, studied by 17O magnetic resonance spectroscopy, glucose uptake, evaluated by 18F-FDG PET and pyruvate transformation into lactate, measured using hyperpolarized 13C-magnetic resonance spectroscopy, were monitored before and 24 hours after DCA treatment in tumor bearing mice. In both tumor models, no clear metabolic shift was observed. Surprisingly, all these imaging parameters concur to the conclusion that both glycolytic tumors and oxidative tumors presented a similar response to DCA. These results highlight a major discordance in metabolic cancer cell bioenergetics between in vitro and in vivo setups, indicating critical role of the local microenvironment in tumor metabolic behaviors.

Keywords: tumor metabolism, DCA, 17O MRS, hyperpolarized 13C-MRI, 18F-FDG PET

INTRODUCTION

Warburg metabolism (enhanced glycolysis in the presence of oxygen) is a common feature of several malignant tumors and is associated with cancer aggressiveness, invasiveness and poor prognosis []. Because of this high glycolytic rate in various cancers, targeting glucose metabolism is presented as an attractive anticancer approach endowed with a high specificity and limited undesirable side effects []. Indeed, conventional treatments rely on the rapid proliferation process present in cancer cells but also in healthy cells. Treatments targeting glycolytic metabolism should instead specifically alter metabolic adaptations that support the Warburg malignant phenotype, adaptations that are not shared by normal cells. To support drug development and assessment in clinical trials, there is a critical need for dedicated criteria evaluating tumor response to these emerging therapies. Moreover, for new cytostatic agents targeting tumor metabolism, the use of conventional anatomical imaging techniques is not optimal for treatment response assessment [] and only functional and molecular imaging techniques may offer the possibility of an early assessment of the tumor response [].

Recently, we have investigated the effects of dichloroacetate (DCA) in tumor cell lines presenting different metabolic profiles []. DCA is a promising molecule that promotes glucose oxidation over glycolysis by inhibiting the mitochondrial pyruvate dehydrogenase kinase (PDK) and has successfully reached clinical trials []. We found that 5 mM DCA was more effective in glycolytic-phenotype cancer cells, where reduction in cell proliferation was mediated by a reactivation of mitochondrial function and a decrease in glycolytic and pentose phosphate pathway fluxes. Our data suggested that DCA may benefit to patients with highly glycolytic tumors. Therefore, the objective of the present study was to assess the effect of DCA in these prototypical tumor models in vivo, namely the glycolytic MDA-MB-231 human breast cancer model and the oxidative SiHa human cervical cancer model. For this purpose, we used a multi-modality molecular imaging approach using several pharmacodynamic metabolic biomarkers. Oxygen consumption, studied by 17O magnetic resonance spectroscopy (17O MRS), glucose uptake, evaluated by 18F-fluorodeoxyglucose positron emission tomography (18F-FDG PET) and pyruvate transformation into lactate, measured during hyperpolarized 13C-magnetic resonance imaging (hyperpolarized 13C-MRI), were monitored before and after DCA treatment in tumor bearing mice. Surprisingly, in vivo models did not recapitulate the previously observed in vitro behavior.

RESULTS

To assess the impact of DCA treatment on the metabolism of the models in vivo, oxygen consumption (Figure ​(Figure1),1), glucose uptake (Figure ​(Figure2)2) and lactate flux (Figure ​(Figure3)3) were measured in MDA-MB-231 and SiHa tumors before and 24 hours after DCA treatment.

 

Figure 1

Effect of dichloroacetate on tumor oxygen consumption in vivo

Tumor H217O signal from representative MDA-MB-231 tumors A. and SiHa tumors B. acquired before, during and after a 2 min inhalation period of the 17O2gas. H217O signal is expressed as relative to the mean baseline signal before 17O2delivery. 17O2 metabolismis not modified by DCA treatment. C. Comparison of the rate of H217O signal after 17O2 delivery in tumors pre and post-treatment. Data are expressed as means ± SEM. Paired tests were two-sided.

 

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Figure 2

Effect of dichloroacetate on tumor glucose uptake in vivo

Representative 18F-FDG PET images showing MDA-MB-231 A-B. and SiHa C-D.tumor-bearing mouse imaged before and 24 hours after DCA treatment. Tumors are indicated by thin arrows. 18F-FDG uptake is expressed in %ID/g. Images were normalized. DCA does not alter 18F-FDG uptake in MDA-MB-231 and SiHa tumors. E. Comparison of 18F-FDG uptake before and after treatment. Data are expressed as means ± SEM. Paired tests were two-sided.

 

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Figure 3

Effect of dichloroacetate on tumor lactate production in vivo

Tumor lactate and pyruvate peak intensities after i.v. injection of hyperpolarized 1-13C pyruvate from representative MDA-MB-231 tumors A-B. and SiHa tumors C-D. Lactate production, measured by the Lac/Pyr ratio, in MDA-MB-231 and SiHa tumors before and after treatment E. Data are expressed as means ± SEM. Paired tests were two-sided.

In Figure ​Figure1,1, tumor H217O spectra are presented for representative MDA-MB-231 (Figure ​(Figure1A)1A) and SiHa tumors (Figure ​(Figure1B)1B) during 17O MRS experiments. The results were highly reproducible under the same conditions tested. The evolution of H217O signal, demonstrating the 17O2 metabolism in tumors, was similar before and after DCA treatment in MDA-MB-231 tumors (Figure ​(Figure1A)1A) and in SiHa tumors (Figure ​(Figure1B).1B). In both tumors models, we found that DCA treatment did not majorly impact oxygen consumption, as assessed by the rate of increase in H217O signal (Figure ​(Figure1C).1C). MDA-MB-231 tumors exhibited a slope of 1.02 10-3 ± 0.16 10-3 s-1and 0.91 10-3 ± 0.09 10-3 s-1 before and after treatment respectively (n=5, P=0.5366). For SiHa tumors, the slopes were 0.85 10-3 ± 0.14 10-3 s-1 under baseline condition and 0.79 10-3 ± 0.11 10-3 s-1 post-treatment (n =5, P=0.2892).

18F-FDG uptake (%ID/g) measured in both tumor models under baseline and post-treatment conditions are presented in Figure ​Figure2.2. In both tumor models, we found that DCA treatment did not lead to a significant change in the uptake of 18F-FDG (Figure 2A-2D), assessing a limited impact of DCA on glucose uptake (Figure ​(Figure2E).2E). %ID/g measured on PET images (mean ± SEM) were 1.88 ± 0.12 under baseline condition and 1.78 ± 0.11 after treatment for MDA-MB-231 tumors (n=7, P=0.2120) and 1.79 ± 0.21 under baseline condition and 1.89 ± 0.19 after treatment for SiHa tumors (n=7, P=0.0813).

The influence of DCA treatment on pyruvate transformation into lactate was measured after hyperpolarized 1-13C pyruvate injection during hyperpolarized 13C-MRS studies (Figure ​(Figure3).3). Representative pyruvate and lactate peak intensities over time of MDA-MB-231 tumors and SiHa tumors captured before and 24 hours after DCA treatment are shown in Figure 3A-3D. Lactate production was reduced after DCA treatment only in SiHa tumors (Figure ​(Figure3E).3E). Lactate/pyruvate ratio (Lac/Pyr) shifted from 0.55 ± 0.05 to 0.48 ± 0.04 in MDA-MB-231 tumors (n=7, P=0.3105) and from 0.82 ± 0.05 to 0.63 ± 0.07 in SiHa tumors (n=7, P=0.0348).

We also evaluated the magnitude of response to the treatment by measuring the variation within the above biomarkers between baseline and post-treatment conditions (Figure ​(Figure4).4). No differences in oxygen consumption and lactate flux measurements were observed between MDA-MB-231 and SiHa tumors during the treatment (P>0.05) (Figure 4A, 4C). Only a small but significant difference in behavior was observed for 18F-FDG uptake measurements (P=0.0240) (Figure ​(Figure4B).4B). MDA-MB-231 tumors decreased their 18F-FDG uptake after treatment (n=7, -5.4 ± 3.5 %) compared to SiHa tumors (n=7, +6.7 ± 3.1 %).

 

Figure 4

DCA does not significantly influence the metabolism of glycolytic tumors compared to oxidative tumors, as assessed by 17O2 metabolism A., 18F-FDG uptake B. and pyruvate transformation into lactate C. measurements in vivo

The magnitude of response to dichloroacetate (variation) is identical in both models, only a small difference in behavior is observed for 18F-FDG uptake. Data are expressed as means ± SEM. Unpaired tests were two-sided.

DISCUSSION

In this study, the impact of DCA on tumors presenting different metabolic profiles was evaluated using molecular imaging in vivo. Recent findings identified that DCA preferentially impairs glycolytic cells compared to oxidative cells. The purpose of the present study was to establish the relevance of these findings in vivo using the same prototypical tumor models as in our in vitro study [], namely the MDA-MB-231 human breast cancer model reported as glycolytic [] and the SiHa human cervical cancer model documented as oxidative []. The dose and administration scheme were selected based on previous reports attesting the efficacy of DCA in tumors [].

In vitro, we previously identified clear effects of DCA treatment on oxygen consumption, glucose consumption and lactate uptake in glycolytic MDA-MB-231 human breast cancer cells. On the other hand, the metabolic activity of oxidative SiHa human cervical cancer cells was not altered by DCA treatment. Using a multi-modality imaging project, we were not able to recapitulate these findings in vivo. Pre-treatment, MDA-MB-231 and SiHa tumors exhibit the same metabolic profile. As MDA-MB-231 and SiHa tumors were previously described as hypoxic under baseline condition [], we highlighted here that both tumor models exhibit a glycolytic phenotype under anaerobic condition. Post-treatment, glycolytic MDA-MB-231 tumors do not appear more impacted than oxidative SiHa tumors (Figure 1-4). Also, some marginal metabolic changes were identified such as a significant decreased lactate production in SiHa tumors (Figure ​(Figure3E)3E) or a decreased 18F-FDG uptake in MDA-MB-231 tumors (Figure ​(Figure4B).4B). Together, those findings did not highlight a clear metabolic shift in MDA-MB-231 tumors or in SiHa tumors treated with DCA during 24 hours (Supplementary Figure S1). This inability to observe any treatment response in vivo could not be attributed to any differences in growth rate between the tumor models under study (Supplementary Figure S2). Also, the measurement repeatability was formerly established using the same tumor models []. Our study demonstrated that the tumor metabolic response to DCA was dramatically different between in vitro and in vivo conditions.

Because of its good tolerability and safety, DCA has been universally exploited to lower lactate levels in acquired or congenital forms of lactic acidosis []. In 2007, Bonnet and colleagues investigated the effects of DCA in cancer and discovered that DCA was promoting apoptosis in vitro and decreasing tumor growth in vivo []. Since then, this orally available and cheap molecule has been further investigated in vitro, in vivo and successfully reached clinical trials. The first data available from the clinical trials indicate that DCA appears to be efficient in adults in solid and brain tumors []. However, no firm conclusions stand out in advanced non-small cell lung cancer []. In another recent study of Feuerecker and co-workers, promotion of tumor growth was even observed after DCA treatment in neuroblastoma tumors []. These studies indicate that response to DCA treatment may drastically vary among tumor types.

The redirection of glucose metabolism from glycolysis to oxidation leading to the inhibition of proliferation and the induction of caspase-mediated apoptosis was initially proposed as the generic mechanism of action of DCA. In a recent phase I study in patients with advanced solid tumors, decreased 18F-FDG uptake was observed after DCA therapy, supporting the use of 18F-FDG uptake as a potential biomarker of response to DCA []. Also, hyperpolarized 13C-pyruvate MRI has already been used in several preclinical studies to monitor DCA effect in solid tumors [], but also in cardiac [] and brain studies []. In the present study, 18F-FDG uptake was unchanged after DCA treatment (Figure ​(Figure2E).2E). This suggests that DCA treatment does not impair glucose uptake and phosphorylation but could potentially impact downstream transformation of glucose. However, no effects on bicarbonate production were detected that could demonstrate changes in energy metabolism from glycolysis to oxidative phosphorylation (Supplementary Figure S3). Recent findings suggested that DCA may also act by other mechanisms. While a possible disruption of the balance between fatty acid β-oxidation and glucose oxidation has already been suggested as an additional mechanism involved in the overall effects of DCA in vivo (as reviewed by []), PDK inhibitors may potentially induce other compensatory mechanisms that could limit the impact of such drugs on global tumor metabolism. The anti-cancer effects of DCA appear to rely on multiple mechanisms depending on the drug concentration, drug administration scheme [] and cell type []. Also, a change in PDK isoform expression between in vitro and in vivo model could also greatly influence the effect of DCA on tumor metabolism in vivo. Indeed, oncogene regulation and tumor microenvironment, like extracellular acidosis, can affect PDK isoform expression [], possibly leading to the expression of a PDK isoform less sensitive to DCA effects. Our findings are consistent with a recent study highlighting that tumor microenvironment could be as important as the (epi) genetic profile to shape the tumor phenotype []. Further investigations using relevant isogenic cell clones with ability to form tumors in vivo should be considered to determine the effects of DCA on energy metabolism in vivo.

In conclusion, our multi-modality imaging study identified major discordances between in vitro and in vivo metabolic responses to DCA treatment, in cancer models presenting distinct metabolic profiles. Results suggest preferring implanted tumors and spontaneous cancer models to study DCA treatment within the milieu of the tumor microenvironment. Overall, further investigations are required to elucidate the impact of different tumor microenvironments on metabolic effects of DCA and its impact for clinical use.

MATERIALS AND METHODS

Cell culture

MDA-MB-231 (human breast cancer) and SiHa (human cervix squamous cell carcinoma) cell lines (American Type Culture Collection [ATCC]), were routinely cultured in Dulbecco’s modified Eagle’s medium containing 4.5g/l glucose supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin.

Animal housing

Animal studies were undertaken in accordance with Belgian and the Université catholique de Louvain ethical committee regulations (agreements number UCL/2010/MD/001 and UCL/2014/MD/026). Hyperpolarized 13C-MRI experiments were carried out in compliance with the Guide for the Care and Use of Laboratory Animal Resources (National Research Council, 1996) and approved by the National Cancer Institute (NCI) Animal Care and Use Committee.

Tumor implantation and animal experiments

A total of 107 MDA-MB-231 cells or 107 SiHa cells, amplified in vitro, were collected by trypsinization, washed three times with Hanks balanced salt solution and resuspended in 200 μL of a 1:1 mixture of Matrigel (BD Biosciences) and Hanks balanced salt solution. For 17O MRS and PET scan experiments, the tumor cells were inoculated subcutaneously into the hind thigh of nude NMRI female mice (Janvier Le Genest-Saint-Isle, France). For hyperpolarized 13C-MRI experiments, the tumor cells were inoculated subcutaneously into the hind thigh of athymic nude female mice (Frederick Cancer Research Center, Animal Production, Frederick, MD, USA). The experiments were performed when tumors reached 7 mm (at this tumor size, necrosis was less than 5% as characterized by Hematoxylin Eosin staining).

To assess the effects of DCA on tumor metabolism using biomarkers, all animals have undergone imaging before and after treatment, with one day between each measurement. Dichloroacetate sodium (Sigma-Aldrich) was administered intraperitoneally (200 mg/kg) after baseline measurements to the mouse. Another dose was given 24 hours after the first dose injection. Post-treatment measurements were initiated 1 hour after treatment administration. This administration scheme is consistent with previous studies attesting the effects of DCA in tumors using hyperpolarized 13C-MRI []. The imaging protocol is summarized in Figure ​Figure55.

 

Figure 5

Experimental protocol

Mice were anesthetized by isoflurane inhalation (Forene, Abbot, England) mixed with air in a continuous flow (2 L/min). Animals were warmed (approximately 35°C) throughout the anesthesia period.

17O MRS experiments

For oxygen consumption experiments, 17O MRS was performed on an 11.7 T (Bruker, Biospec) controlled by Paravision 6.0 (Bruker, Ettlingen, Germany). Experiments were carried out using a 1H/17O Bruker surface coil system positioned over the tumor mass. Anatomical images were firstly acquired using a T2-weighted axial turbo RARE sequence (TR = 2500ms; TE = 30ms; rare factor = 8; NA = 2; FOV = 25x25mm2; resolution: 98 x 98 μm2; 1 mm slice thickness). 17O MRS measurements were carried out using a nonlocalized, single-pulse sequence (TR = 16.5 ms; NA = 600; repetition: 120; Tacq = 20 min; Acq BW = 5000 Hz; FA = 20°). For 17O MRS sequence, the 90° reference pulse was optimized previously on natural abundance H217O samples.

To measure tumor oxygen consumption during the 17O2 delivery, a total of 120 17O-spectra were collected in about 20 min, before, during and after a 2 min inhalation period of the 17O2 mixture. The integrals of the H217O peaks over time were measured using a home-made program written in Matlab (The MathWorks Inc., Natick, MA, USA). H217O signal was then expressed as relative to the mean baseline signal before 17O2 delivery. The mean signal of the final steady state (sfinal) during the post-inhalation period was calculated between 1100 and 1200 s. We considered that the steady state was reached when the signal stood between sfinal ± 5 % of signal variation. The slope during the linear incorporation phase was measured between 600 sec and the time point when steady state was reached.

PET/CT imaging

Whole-body PET imaging was performed on a dedicated small-animal PET scanner (Mosaic, Philips Medical Systems, Cleveland, USA) with a spatial resolution of 2.5 mm (FWHM). The PET scans were followed by whole-body acquisitions using a helical CT scanner (NanoSPECT/CT Small Animal Imager, Bioscan Inc., DC, USA). For each breathing condition, anesthetized mice were injected 120 μl intraperitoneally with 11.1-14.8 MBq of 18F-FDG (Betaplus Pharma, Brussels, Belgium). A 10 min transmission scan was first obtained in a single mode using a 370 MBq 137Cs source for attenuation correction. A 10 min static PET acquisition was then performed after a 60 min resting period. After the correction with attenuation factors obtained from the transmission scan, images were reconstructed using a fully 3D iterative algorithm (3D-RAMLA) in a 128 x 128 x 120 matrix, with a voxel size of 1 mm3. After PET acquisition, anesthetized animals were transferred on the same bed from the PET scanner to the CT scanner (x-ray tube voltage: 55 kVp; number of projections: 180; exposure time 1000 ms) for anatomical reference. The CT projections were reconstructed with a voxel size of 0.221 x 0.221 x 0.221 mm3. Regions of Interest (ROIs) were delineated on PET images using PMOD software (PMOD™, version 3.403, PMOD technologies Ltd, Zurich, Switzerland). 2D ROIs were established on consecutive transversal slices using a 50% isocontour tool (ROI including the pixel values larger than 50% of the maximum pixel) that semi-automatically defined a 3D Volume of Interest (VOI) around the tissue of interest. To avoid overestimation of the uptake within the VOI, PET/CT fused images where used to discriminate hot pixels coming from the neighboring tissues like urinary bladder. Using the mean uptake within this VOI, the global tracer uptake was assessed in tumors and expressed as percentage of injected dose per gram of tissue (%ID/g).

Hyperpolarized 13C-MRI studies

1-13C pyruvic acid (30 μL), containing 15 mM OXO63 and 2.5 mM gadolinium chelate ProHance (Bracco Diagnostics, Milano, Italy), was hyperpolarized at 3.35T and 1.4K using the Hypersense DNP polarizer (Oxford Instruments, Abingdon, UK) according to the manufacturer’s instructions. After 60-90 min, the hyperpolarized sample was rapidly dissolved in 4.5 mL of a superheated alkaline buffer that consisted of 50 mM Tris(hydroxymethyl)aminomethane, 75 mM NaOH, and 100 mg/L ethylenediaminetetraacetic acid. The hyperpolarized 1-13C pyruvate solution (96 mM) was intravenously injected through a catheter placed in the tail vein of the mouse (12 μL/g body weight). Hyperpolarized 13C MRI studies were performed on a 3T scanner (MR Solutions, Guildford, UK) using a home-built 13C solenoid leg coil. After the rapid injection of hyperpolarized 1-13C pyruvate, spectra were acquired every second for 240 seconds using a single pulse sequence. Data were analyzed in a model free approach using the lactate/pyruvate ratio, calculated from the areas under the curves of the 1-13C lactate peak and the 1-13C pyruvate peak [].

Statistical analysis

Analysis was performed using the GraphPad Prism 7 software. Results are expressed as means value of parameter ± SEM. All statistical tests were two-sided. Paired t-test was used to compare mean changes between groups (baseline vs. post-treatment) for each tumor model, and unpaired t-test was used to compare mean changes between the two tumor models. Results with P < 0.05 (*), <0.01 (**), or <0.001 (***) were considered to be statistically significant.

Crosstalk among proteome, acetylome and succinylome in colon cancer HCT116 cell treated with sodium dichloroacetate

Crosstalk among proteome, acetylome and succinylome in colon cancer HCT116 cell treated with sodium dichloroacetate

Abstract

Protein lysine acetylation and succinylation play important regulatory roles in cells, both of which or each other has a close relationship. Dichloroacetate (DCA), a well-known pyruvate dehydrogenase kinase (PDK) inhibitor, has the potential to be used as anti-cancer drugs for several tumors including colorectal cancer. However, little is known about the potential mechanism of DCA-based cancer therapy by protein posttranslational modifications (PTM) including global proteome, acetylome and succinylome. Here the combinations with stable isotope labeling (SILAC), antibody affinity enrichment and high resolution LC-MS/MS analysis were performed in human colon cancer HCT116 cells. The quantifiable proteome was annotated using bioinformatics. In total, 4,518 proteins, 1,436 acetylation sites, and 671 succinylation sites were quantified, respectively to DCA treatment. Among the quantified acetylated sites, 158 were with increased level (quantification ratio >1.5) and 145 with decreased level (quantification ratio <0.67). Meanwhile, 179 up-regulated and 114 down-regulated succinylated sites were identified. The bioinformatics analyses initially showed acetylation and succinylation were involved in a wide range of cellular functions upon DCA-based anti-cancer effects. Notably, protein-protein interaction network analyses demonstrated widespread interactions modulated by protein acetylation and succinylation. Taken together, this study may shed a light on understanding the mechanism of DCA-based cancer treatment.

Introduction

Protein posttranslational modifications (PTMs), which can alter structural, conformational and physicochemical properties of proteins, are key cellular events involved in many biological processes1,2. Among all the amino acids, lysine is a frequent target to be modified, which can be subjected to a variety of PTMs3. With the advances in high-resolution mass spectrometry (MS) and antibody-based affinity enrichment of lysine (Lys) residues, a lot of novel PTMs have been identified, such as ubiquitylation, butyrylation, succinylation, and glutarylation.

The acetylation of Lys residues in proteins has a role in transferring acetyl moiety from acetyl-CoA to its amino groups. Early studies of Lys acetylation mainly focused on histones and other transcription factors in the nucleus4,5,6. However, they have been found recently to occur in almost every compartment of a cell, such as the cytoplasm and mitochondria, and play a major role in metabolic regulation, including glycolysis, tricarboxylic acid (TCA) cycle, fatty acid metabolism and so on7,8. Succinylation refers to the transfer of succinyl group from the succinyl donor succinyl-CoA to the ε-amino group of specific lysine residue of the target protein, which is also considered to occur frequently like acetylation in several cellular events9. Most of the identified succinylated proteins are enzymes involved in kinds of metabolic pathways, which are important for the regulation of central metabolism such as fatty acid metabolism, amino acid degradation. It was reported that acetylation and succinylation were closely linked to central carbon metabolism10. Brian et al.11 revealed that majority of succinylation sites in bacteria, yeast, and mouse liver were also acetylated at the same position11. Yuta Mizuno et al.12 found that lysine acetylation and succinylation target most enzymes in central carbon metabolic pathways that are directly linked to glutamate production, and the extent of modification changed in response to glutamate overproduction12.

Dichloroacetate (DCA), an inhibitor of pyruvate dehydrogenase kinase (PDK), can reverse the Warburg effect and induce apoptosis in tumor cell by increasing the flux of pyruvate into the mitochondria and promoting glucose oxidation13. The increasing evidence in preclinical in vitro and in vivo indicates that DCA may be a promising selective anti-cancer agent. However, the potential mechanism of DCA-based cancer treatment is still unclear.

Given the widespread regulatory role of PTMs and the close relationship between acetylation and succinylation, we are interested to explore the regulative mechanism of DCA treatment by PTM. Here, an integrated system of SILAC labeling, HPLC fractionation and affinity enrichment followed by high resolution LC-MS/MS analysis were performed for the quantitative comparison of the global proteome, acetylome and succinylome in HCT116 cells with or without DCA treatment. Intensive bioinformatics analyses were then used to annotate those quantifiable modified targets in response to DCA treatment. Our studies indicate that the approach described above is powerful for identification of modified proteins and peptides. Therefore, the results provided a novel insight into DCA treatment on colon cancer cells.

Results

Analysis of lysine acetylation, and succinylation sites in HCT116 cells stimulated by DCA

To identify the profiles of the global proteome and lysine acetylome, succinylome, we carried out a 6-step workflow: (1) stable isotope labeling of HCT116 cells with or without DCA stimulation by SILAC; (2) protein extraction and trypsin digestion to yield peptides; (3) HPLC fractionation; (4) affinity enrichment of lysine acetylated and succinylated peptides; (5) LC-MS/MS analysis was used to identify the enriched peptides; (6) database search and bioinformatic analysis (Fig. 1).

Figure 1: Assaying DCA-based cancer treatment by PTM.

(A) The analytical strategy and method for DCA-responsive quantitative profiling of global proteome, acetylome and succinylome in HCT116 cell line33,34 (The Edraw Max V7.3 software is used to create the map in (A), https://www.edrawsoft.com/term-conditions.php). (B) Western blotting with anti-acetyl lysine antibody (up) and anti-succinyl lysine antibody (down).

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In this work, comparing cells with or without DCA treatment, 5,448 proteins identified and 4,518 proteins were quantified in aspects of global proteome. Among these, 244 proteins were increased and 269 proteins were decreased. In addition, we identified 1,484 lysine acetylation (Kac) sites in 860 proteins, among of which 1,436 Kac sites in 841 proteins were quantified. We also identified 680 lysine succinylation (Ksu) sites in 295 proteins, among of which 671 Ksu sites in 291 proteins were quantified. The identified acetylated and succinylated peptides showed different abundance depending on their lengths (Fig. 2B,D), match 860 acetylated and 295 succinylated proteins, respectively (Fig. 2A,C). Apparently, most of them were modified at sole site, consistent with the previous findings about post-translational modification14,15,16.

Figure 2: The distribution of acetylated proteins and succinylated proteins.

(A) Distribution of acetylated proteins based on their number of modified sites. (B) Distribution of acetylated peptides based on their length. (C) Distribution of succinylated proteins based on their number of modified sites. (D) Distribution of succinylated peptides based on their length.

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Figure 3 shows the MS/MS spectra of two representative acetylated and succinylated proteins, histone acetyltransferase p300 and 60 kDa heat shock protein, respectively. Histone acetyltransferase p300 is encoded by gene EP300, and functions as histone acetyltransferase, which can regulate transcription via chromatin remodeling by acetylating four core histones. 60 kDa heat shock protein encoded by gene HSPD1 is involved in mitochondrial protein import and macromolecular assembly. Meanwhile, it plays an important role in protein folding, refolding, and proper assembly of unfolded polypeptides generated under the stress conditions. The two proteins were obviously modified, implying their crucial function in response to DCA treatment.

Figure 3

Representative MS/MS spectra of Histone acetyltransferase p300 acetylation (A) and 60 kDa heat shock protein succinylation (B).

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DCA-responsive global proteome

DCA has been reported as anti-cancer drug. It is therefore intrigue to assay the DCA-responsive proteome in HCT116 cells. Here, 5,448 proteins were identified and 4,518 proteins were quantified in the cells. Among them, 244 proteins were DCA-increased and 269 proteins were DCA-decreased (Supplementary Information Table S1) when we set quantification ratio of >1.5 as up-regulated threshold and <0.67 as down-regulated threshold. To elucidate the possible roles of these proteins, we performed four types of enrichment-based clustering analyses: Gene Ontology (GO) functional classification, KEGG pathway analysis, protein domain enrichment-based analysis, and protein complex analysis. The quantified proteomic dataset were divided into four quantiles according to L/H ratio to generate four quantiles: Q1 (0~25%), Q2 (25~50%), Q3 (50~75%), and Q4 (75~100%). Enrichment analyses were performed separately based on the quantiles.

We analyzed the quantifiable proteome dataset for three enrichment GO categories: biological process, molecular function, and cellular compartment (Figure S1A~C). For the molecular function analysis (Figure S1A), we found that proteins with increased L/H ratios were enriched in carboxylic ester hydrolase activity, and structural constituent of cytoskeleton. Proteins with helicase activity, pyrophosphatase activity, and hydrolase activity were enriched in Q1 and Q2. The cellular compartment analysis was presented in Figure S1B, indicating that many proteins located in keratin filament, extracellular space, and cytosol were enriched in quantiles Q3 and Q4 with high L/H ratios, which may suggest that might indicate DCA treatment have obvious influence on cytoarchitecture located in these places. The decreased proteins were mainly focused on chromosome and spindle, condensed chromosome, suggesting DCA may participate in cell cycle and mitosis.

In the biological process category (Figure S1C), proteins in Q3 and Q4 quantiles were enriched in epidermis development, cell differentiation, and other developmental processes. In contrast, proteins in Q1 and Q2 quantiles were enriched in cell cycle process, cell division, mitosis and organelle fission. These findings were consistent with results of the cellular compartment analysis. Taken together, DCA-responsive protein modifications have impacts on various processes such as replication, transcription, cell proliferation and so on. Importantly, the influence of DCA on cell developmental process and differentiation suggest that the potential role of DCA may act as an anti-cancer agent.

The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the quantified proteins was also performed (Figure S1D). DCA-decreased proteins were mainly focused on cell cycle, DNA replication, steroid biosynthesis, pyrimidine metabolism, and p53 signaling pathway. While DCA-increased proteins were mainly involved in galactose metabolism, amino sugar and nucleotide sugar metabolism, as well as glycerolipid metabolism. It has been proved that DCA is the inhibitor of pyruvate dehydrogenase kinase (PDK)17. Besides, DCA has the ability to activate AMPK signaling pathway, which has a close relationship with glycolysis or gluconeogenesis18. The findings indicate that DCA has potential regulatory roles of participating in other kinds of glycometabolism.

Finally, the protein domain analysis indicated that the protein function domains involved in MCM N-terminal, mini-chromosome maintenance, and DNA-dependent ATPase were enriched in Q1 and Q2 quantiles, while protein domains participated in EGF-like conserved site, MCM N-terminal domain were enriched in Q3 and Q4 quantiles (Figure S1E).

DCA-responsive acetylome

Acetylation is a dynamic and reversible PTMs, which can transfer acetyl moiety from acetyl-CoA or acetyl phosphate to lysine residues at ε-amino groups in proteins18,19. Lysine acetylation was found in histones20, and in non-histones21, which has diverse functions like regulation of gene transcription, cell cycle, apoptosis, metabolic flux and so on15,22. It has been reported that DCA may have a relationship with acetylation23, however the direct evidence needs to be further investigated.

Here, the acetylation level of proteins in response to DCA stimulation was investigated by the combination of SILAC labeling, lysine acetylation antibody enrichment and LC-MS/MS analysis. Notably, 1,484 lysine acetylation sites in 860 protein groups were assayed, among which 1,436 sites in 841 proteins were quantified (Table S2). When setting quantification ratio of >1.5 as up-regulated threshold and <0.67 as down-regulated threshold, 158 lysine acetylation sites in 116 proteins were quantified as up-regulated targets and 145 lysine acetylation sites in 112 proteins were quantified as down-regulated targets. To our knowledge, this is the first profiling of lysine acetylation dataset in HCT116 cells under DCA treatment.

The enrichment-based clusting analyses were then performed to compare the functions of corresponding DCA-responsive proteins. All these quantified acetylated proteins were divided into four quantiles (Q1~4) as described above, including Gene Ontology, KEGG pathway, protein domain and protein motif (Fig. 4A~EFigure S2A,C).

Figure 4: Clustering analysis of the quantified acetylome based on the functional enrichment.

(A) Molecular function, (B) cellular compartment, (C) biological process, (D) KEGG pathway, and (E) protein domain. (F) Protein-protein interaction network of acetylated proteins clustered in ribosome. (G) Protein-protein interaction network of acetylated proteins clustered in cell cycle process.

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In GO functional classification (Fig. 4A–C), the analysis of molecular function (Fig. 4A) showed that increased proteins involved in structural constituent of ribosome, structural molecule activity, ATPase activity were changed significantly, while the decreased proteins participated in nucleic acid binding, heterocyclic compound binding, and other bindings were severely affected. The results were consistent with the notion that DCA can lead to the inhibition of cell proliferation and cause cell death24. In cellular compartment (Fig. 4B), the increased proteins were mostly enriched in cytosolic ribosome, ribosome. In contrast, decreased proteins were mainly enriched in nucleolus, chromatin, and DNA bending complex. In the biological process category (Fig. 4C), nuclear-transcribed mRNA catabolic process, aromatic compound catabolic process, mRNA catabolic process were highly enriched, while the processes related to chromatin organization, regulation of skeletal muscle tissue development were significantly enriched. These results were consistent with the former analysis of molecular function, suggesting DCA may play a role in RNA and protein synthesis and therefore have influence on the growth of tumors.

To identify cellular pathways affected by DCA treatment, we then performed a pathway clustering analysis using KEGG (Fig. 4D). The results showed that ribosome, oxidative phosphorylation, carbon metabolism were the most prominent pathways enriched in quantiles with high L/H ratios, suggesting DCA functions as a regulatory factor of protein biosynthesis and energy metabolism. It is notable that these results were consistent with the notion DCA can alter the ‘Warburg Effect’ into normal oxidative phosphorylation and thus inhibit tumor growth. But in quantiles with low L/H ratios, protein expression in pathways of viral carcinogenesis, systemic lupus erythematosus, RNA transport were severely decreased in response to DCA stimulation.

As domain structure is one of the most important functional features of protein, we next analyzed the domain features of the proteins changed after DCA adding (Fig. 4E). We found that domains in proteins involved in Histone-fold, Histone core, Histone H2B were remarkably enriched upon DCA treatment in down-regulated quantiles, suggesting DCA have great influence on transcription. But Zinc finger, translation protein SH3-like domain, ribosomal protein L2 domain 2 were mainly enriched in up-regulated quantiles.

To determine if there were specific amino acids adjacent to acetylated lysines, we compared the sequences flanking acetylated sites by heat-map. A strong bias of amino acid sequence, namely, Tyrosine (Y), Phenylalanine (F), Histidine (H), Tryptophan (W), was found in our data. In addition, isoleucine (I) and arginine (R) were overrepresented in the two and four to five positions behind Kac sites (Figure S2A,C), suggesting aromatic groups were common to be modified by acetyltransferases. These results were consistent with former report using mycobacterium tuberculosis as test subjects15 and indicated lysine acetylating may be conserved and widespread.

Protein-protein interaction network of acetylome proteins was established by using Cytoscape software (Figure S4A and Fig. 4F~G). Two kinds of important pathways (ribosome and cell cycle process) were listed in Fig. 4F~G, indicating that proteins participated in cell cycle process have mostly been down-regulated except MCM3, which had been acetylated at multiple sites. The acetylation of this protein can inhibit the initiation of DNA replication and cell cycle progression. Meanwhile, MKI67, a nuclear protein that is associated with and may be necessary for cellular proliferation, was acetylated at multiple sites and their levels were down-regulated remarkably25. These findings can partly explain how DCA cause cell cycle arrest and inhibit cell proliferation.

The correlation of DCA-responsive global proteome and acetylome

Upon the dataset of DCA-responsive proteome and acetylome, we performed the overlapping analysis between them. According to the quantitative results, 664 proteins were quantified both in proteome and acetylome, including a number of 1484 Kac sites. The quantitative ratios of proteome and acetylome were compared (Table S4) and the scatter diagram was shown in Figure S3A. The pearson’s correlation coefficient and the Spearman’s rank correlation coefficient were 0.235 and 0.236, respectively. The results demonstrate the global proteome and acetylome have little correlation with each other.

DCA-responsive succinylome

The succinylome in HCT116 cells was identified by combining the SILAC, immuneaffinity enrichment by a high-specificity antibody (PTM Biolabs), and high-resolution mass spectrometry. We identified 680 lysine succinylation sites in 295 protein groups, among which 671 sites in 291 proteins were quantified. Among them, 179 lysine succinylation sites in 108 proteins were increased and 114 lysine succinylation sites in 71 proteins were decreased (Table S3). Then all the quantified succinylated proteins were divided into four quantiles (Q1~4) according to L/H ratios as described above. The clustering analyses included GO analysis, KEGG pathway analysis, protein domain analysis and motif analysis (Fig. 5A~E and Figure S2B,D).

Figure 5: Clustering analysis of the quantified succinylome based on the functional enrichment.

(A) Molecular function, (B) cellular compartment, (C) biological process, (D) KEGG pathway, and (E) protein domain. (F) Protein-protein interaction network of acetylated proteins clustered in ribosome. (G) Protein-protein interaction network of acetylated proteins clustered in oxidative phosphorylation.

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For the molecular function analysis (Fig. 5A), we first found that increased proteins were highly enriched in oxidoreductase activity, hydrogen ion transmembrane transporter activity, substrate-specific transmembrane transporter activity, but for decreased proteins, they were mainly enriched in unfolded protein binding, protein binding and calcium ion binding. It was reported that plasma membrane oxidoreductase activity had a relationship with mitochondrial function and oxidative stress26, and excessive reactive oxygen species (ROS) could ultimately lead to ROS-mediated genomic instability and cancer27 as well as induce apoptosis. We propose that the enhanced oxidoreductase activity induced by DCA may be associated with its anti-cancer effect.

In the cellular compartment category (Fig. 5B), proteins located in the mitochondrial parts such as matrix, membrane, inner membrane were highly enriched in Q3 and Q4 quantiles, indicating that DCA-induced succinylation may have important roles in mitochondrial. However, proteins in Q1 and Q2 quantiles were mainly focused on pigment granule, melanosome and cytosol. The biological process of succinylation was displayed in Fig. 5C, which indicates that the up-regulated proteins were mainly enriched in small molecule metabolic process, oxidation-reduction process, cellular respiration. The decreased proteins were mainly concentrated on protein folding, cellular localization, and cellular component organization.

The KEGG pathway analysis for the succinylate proteins showed a number of important pathways (Fig. 5D). For proteins in Q1 and Q2 quantiles, the ribosome, viral carcinogenesis, TCA cycle, and glycolysis/gluconeogenesis pathways were highly enriched. In contrast, pathways of oxidative phosphorylation, Huntington’s disease, Alzheimer’s disease, and metabolism were highly enriched in Q3 and Q4 quantiles. These results implied succinylation has a close relationship with diseases and some metabolic pathways like oxidative phosphorylation, indicating succinylation may play an important role in DCA’s effects of turning ‘Warburg effect’ into normal oxidative phosphorylation. In addition, the pathways were highly enriched in Q3 and Q4 quantiles of acetylated proteins, implying the close relationship between these two PTMs. This was associated with the previous report, suggesting that acetylation and succinylation are closely overlapped16.

Figure 5E shows that protein domains involved in Histone-fold, Histone core, and Histone H2B were highly enriched in proteins with decreased Ksu sites, while several function domains (i.e., Zinc finger, LIM-type, Translation protein SH3-like, and Ribosomal protein L2) were highly enriched in proteins with increased Ksu sites.

Protein-protein interaction network of succinylated proteins was established by using Cytoscape software, and the global network of Ksu proteins was displayed in Figure S4B. The network indicated that succinylated proteins actively participated in ribosome and oxidative phosphorylation (Fig. 5F~G). Almost all succinylated proteins involved in ribosome were decreased and proteins involved in oxidative phosphorylation were increased. These findings imply that DCA-responsive succinylation play a significant role in protein synthesis and cellular energy metabolism.

To test whether conserved lysine succinylation motifs exist among lysine- acetylated proteins, we carried out an analysis of global succinylated proteins (Figure S2B,D). Three preferred sequence patterns were found as V*Ksu, I*Ksu, and R******Ksu. To our knowledge, V*Ksu and I*Ksu have already been observed to be existed in human, rat and E. coli, but neither of them were reported to be among the most common ones in the previous reports11. In addition, EK and Ksu*****K, two of the most common sequence patterns, were overrepresented in this study, suggesting the preferred sites for succinylation have some similarities even in different species.

The correlation of DCA-responsive global proteome and succinylome.

We next assayed the features of DCA-responsive global proteome and succinylome. Totally, 295 quantified proteins were found in both of them, including 636 Ksu sites. The quantitative ratios of proteome and succinylome under DCA treatment were compared (Table S5), and the scatter diagram was shown in Figure S3B. To be accurate, the pearson’s correlation coefficient and the Spearman’s rank correlation coefficient were 0.255 and 0.292, respectively. The results demonstrate there is litte correlation between the global proteome and succinylome.

The correlation of DCA-responsive acetylome and succinylome.

It was reported that each PLM can crosstalk with at least one other PLM and the co-occurrences of different PLMs at the same site were abundant28. Here, the DCA-responsive acetylome and succinylome in HCT116 cells were assayed to investigate their relationships. 91 proteins were both acetylated and succinylated, but they were not modified at the same site exactly. Among them, there were 151 lysine sites were modified with both acetylation and succinylation (Fig. 6A,B). The correlation between the L/H ratios of acetylome and succinylome (Table S6), as well as the scatter diagram were shown in Fig. 6C. The Spearman’s correlation coefficient and Pearson’s was 0.347 and 0.478, respectively. This indicated that DCA-responsive patterns of acetylated and succinylated sites were positively related. Notably, some important proteins participated in glycolysis, the main pathway through which DCA regulates tumor cell metabolism, were acetylated and succinylated simultaneously, such as pyruvate kinase muscle (PKM), phosphoglycerate kinase 1 (PGK1), lactate dehydrogenase B (LDHB), and enolase 1 (ENO1). This implies that the two modifications are involved in DCA’s effect on HCT116 cells.

Figure 6: Correlation of global proteome, acetylome and succinylome.

(A) Overlap between acetylated proteins and succinylated proteins. (B) Overlap between acetylated sites and succinylated sites. (C) Correlation of acetylome and succinylome. (D) Protein-protein interaction network between acetylated proteins and succinylated proteins.

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For assaying the relationship of acetylome and succinylome, the protein-protein interaction network was established. The global overview of network among Kac and Ksu proteins was performed by using Cytoscape (Fig. 6D). The complicated network demonstrates that the acetylated and succinylated proteins have a closed crosstalk, indicating that some important proteins underwent both acetylation and succinylation, such as dihydrolipoamide dehydrogenase (DLD), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and cytochrome C somatic (CYCS).

Discussion

DCA has been reported to be a promising anti-cancer drug29, but its regulatory mechanisms in tumor cells are still unknown. In this study, a SILAC-based quantitative proteomic approach was used to investigate the anti-cancer effect of DCA on the proteome, acetylome and succinylome in HCT116 colon cancer cells.

The alternation of the whole proteome in HCT116 cells showed that proteins participated in cell cycle, cell division and proliferation apparently decreased, which linked gene expression levels with the DCA-induced anti-cancer effects. In addition, The KEGG pathway analysis of the quantified proteins showed that the increased proteins were highly enriched in galactose metabolism, amino sugar and nucleotide sugar metabolism, consistent with DCA’s role of regulating glucose metabolism.

The quantitative acetylome analysis revealed 1436 DCA-responsive Kac sites in HCT116 cells, which would be the most comprehensive Kac profiling in HCT116 cells. It indicated that the decreased proteins mainly located in nucleolus, chromatin, and DNA bending complex, participating in nucleic acid binding, which implys that DCA can inhibit DNA replication and regulate protein biosynthesis by lysine acetylation. In addition, we observed that some proteins encoded by important myconcogene was significantly down-regulated.

In addition, among 680 lysine succinylation sites in 295 proteins, the DCA-increased proteins were mostly localized in mitochondrial and enriched in oxidoreductase activity, hydrogen ion transmembrane transporter activity, and substrate-specific transmembrane transporter activity. It has been reported that mitochondrial membrane potential and ROS production are dependent on the flux of electrons down the electron transport chain (ETC). Decreased entry of pyruvate would eventually result in decreased flux of electrons in the ETC and therefore reduced ROS production, contributing to the close of the redox-sensitive mitochondrial transition pore (MTP) and mitochondrial hyperpolarization which are considered to be closely related with tumor genesis30. These findings suggest that DCA may result in lysine succinylation and then increase the delivery of pyruvate into the mitochondria, which may help mitochondria-based glucose oxidation and mitochondrial depolarizing, thus returning the membrane potential towards the levels of normal cells, and exert its anti-cancer effect.

Considering the growing evidence suggests that lysine acetylation and succinylation may have a close relationship, we focused on several pathways undergoing the two modifications. Generally, the overlap between acetylome and succinylome were mainly related to oxidative phosphorylation, metabolic pathways, citrate cycle (TCA cycle), and carbon metabolism. Here, we discussed the TCA cycle in detail for its close relationship with DCA’s anti-cancer effect. It is well known that DCA can target PDK, which can connect glycolysis with TCA cycle by promoting catalyzing pyruvate to acetyl-CoA. We checked the modification of PDH (regulated by PDK) and eight enzymes participated in TCA cycle, including citric synthase, aconitate hydratase, isocitrate dehydrogenase, ketoglurate dehydrogenase, succinyl-CoA synthase, succinate dehydrogenase, fumarate hydrogenase, malate dehydrogenase in our dataset. Some of them were acetylated or succinylated (Fig. 7). However, PDK itself was not acetylated or succinylated in our study. Isocitrate dehydrogenase, which catalyzes the conversion of isocitrate to α-keto-glutarate, was involved both in acetylation and succinylation. There were a total of eight Ksu sites and 5 Kac sites on this enzyme. According to the previous report, mutagenesis of the succinylated lysine residues of this enzyme has a crucial role of the activity of the enzyme31. It is reasonable to suppose that the modifications described above will induce important changes of TCA cycle, especially when this step is irreversible and rate-limiting. But we didn’t find another two enzymes in citric synthase and ketoglurate dehydrogenase were acetylated or succinylated. In total, among the nine enzymes, seven were succinylated and five were acetylated in HCT116 cells after DCA treatment, respectively. These results suggest that the enzyme activity in metabolism has been positively regulated by acetylome and succinylome, thus mediating the drug effect.

Figure 7: Acetylated and succinylated enzymes involved in TCA cycle.

The enzymes identified as lysine-acetylated are marked with an asterisk. The enzymes identified as lysine-succinylated are underlined.

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Recent studies have reported that histone acetylation could induce the change of global proteome by epigenetics while non-histone acetylation of transcription factors could regulate proteome level through transcriptiome32,33. Our classification was not clear enough to show the direct relationship among global proteome, acetylome and succinylome. Obviously, to further confirm the view, more explicit classification and bioinformatic analysis should be done.

Conclusions

Here we present a large-scale quantitative analysis of DCA-responsive global proteome, acetylome and succinylome in HCT116 cells by using high sensitivity mass spectrometry and bioinformatic analysis. In total, we identified DCA-responsive 860 Kac proteins and 295 Ksu proteins in cells. Our work provides a database that can be used to examine what effect DCA on cancer cells, drug resistance. Notably, the correlation of global proteome, acetylome and succinylome may expand our understanding of DCA’s anti-cancer effect. Although the underlying mechanism of acetylome and succinylome remains to be further explored, the current study shed a light on several biological processes like TCA cycle, glycolysis, and other functions.

Methods and Materials

HCT116 culture and SILAC labeling

Cells were grown to 80% confluence in high glucose Dulbecco’s modified Eagle’s medium (with glutamine and sodium pyruvate) (Pierce, MA, USA) containing 10% fetal bovine serum (Gibco, CA, USA) at 37 °C with 95% air and 5% CO2. Then cells were labeled with ‘light isotopic lysine’ (12C-Lysine) or ‘heavy isotopic lysine’ (13C-Lysine) using a SILAC Protein Quantitation Kit (Pierce, MA, USA) according to manufacturer’s instructions. After cells were expanded in SILAC media to our desired cell number (~5 × 108), the ‘light’ labeled cells were then treated with 20 mM DCA (Sigma, MO, USA) and cultured for another 24 hours in SILAC media before harvesting.

Protein extraction, Trypsin Digestion and HPLC fractionation

The harvested labeled cells were lysed with 2 × NETN buffer (200 mM NaCl, 100 mM Tris-Cl, 2 mM EDTA, 1.0% NP-40, pH 7.2) supplemented with 0.5% Triton X-100 on ice for 30 min. The unbroken cells or debris were removed after centrifuge at 20,000 g for 10 min at 4 °C. After concentration measurement, same amounts of proteins labeled with ‘heavy’ or ‘light’ were mixed and the crude proteins were precipitated. After washing twice with ice-cold acetone, the air-dried precipitate was dissolved in 100 mM NH4HCO3 (pH 8.0) and then digested with trypsin (Promega, WI, USA) at an enzyme-to substrate ratio (1:50) at 37 °C for 16 hours. After that, DTT was added to final concentration 5 mM and then incubated at 50 °C for 30 min, then IAA was added with final concentration 15 mM followed by incubation at room temperature in total darkness for 30 min. The alkylation reaction was terminated by 30 mM cysteine at room temperature for another 30 min. Again Trypsin was added with ratio of trypsin to protein at 1:100 for digestion at 37 °C for 4 hours to ensure the complete digestion. The sample was then fractionated into fractions by high pH reverse-phase HPLC by using Agilent 300Extend C18 column (5 μm particles, 4.6 mm ID, 250 mm length). In brief, peptides obtained were separated into 80 fractions with a gradient of 2% to 60% acetonitrile in 10 mM ammonium bicarbonate (pH 10) for over 80 min first, then they were combined into 18 fractions and dried.

Affinity Enrichment of Lysine Acetylated and Succinylated Peptides

Tryptic peptides dissolved in NETN buffer were incubated with pre-washed antibody beads (PTM Biolabs) (4 times with NETN buffer and 2 times with ddH2O) at 4 °C overnight with gentle shaking. And then, the bound peptides were eluted from the beads with 0.1% TFA, and the eluted fractions were combined and vacuum-dried. The peptides obtained were cleaned with C18 ZipTips (Millipore, MA, USA) according to the manufacturer’s instructions, followed by LC-MS/MS analysis.

LC-MS/MS Analysis

Three parallel analyses for each fraction were performed. Peptides were dissolved in 0.1% FA (Sigma Fluka, MO, USA), directly loaded onto a reversed-phase pre-column (Acclaim PepMap 100, Thermo, MA, USA). Peptides were separated by using a reversed-phase analytical column (Acclaim PepMap RSLC). The gradient was comprised of a span starting from 6% to 23% solvent B (0.1% FA in 98% ACN) for 24 min, 23% to 35% for 8 min, 80% in 4 min, and at last holding at 80% for another 4 min. All of the above procedures were at a constant flow rate of 280 nL/min on EASY-nLC 1000 UPLC system, and the resulting peptides were analyzed by Q ExactiveTM Plus hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher, MA, USA).

The peptides were subjected to NSI source, followed by tandem mass spectrometry (MS/MS) in Q ExactiveTMPlus (Thermo, MA, USA) coupled online to the UPLC. We detected the intact peptides in the Orbitrap at a resolution of 70,000. Peptides were selected using NCE setting as 30; ion fragments were detected at a resolution of 17,500. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans was applied for the top 20 precursor ions above a threshold ion count of 1.5 E4 in the MS survey scan with 30.0 s dynamic exclusion. The electrospray voltage applied was 2.0 kV. Automatic gain control (AGC) was used to prevent overfilling of the ion trap; 5 E4 ions were accumulated for generation of MS/MS spectra. For MS scans, the m/z scan range was 350 to 1800.

Data processing

The data obtained was processed using MaxQuant with integrated Andromeda search engine (v.1.4.1.2). Tandem mass spectra were searched against SwissProt_human database concatenated with reverse decoy database. Mass error was set to 10 ppm for precursor ions and 0.02 Da for fragment ions. Carbamidomethylation on Cys was specified as fixed modification and oxidation on Met, acetylation or succinylation on Lys and protein N-terminal were specified as variable modifications. Minimum peptide length was set at 7. False discovery rate (FDR) thresholds for protein, peptides and modification sites were specified at 1%. All the other parameters in MaxQuant were the default values. The Kac and Ksu site localization probabilities were set as >0.75.

Bioinformatics Methods

Gene Ontology (GO) annotation proteome was derived from the UniProt-GOA database (http://www.ebi.ac.uk/GOA/). For proteins not annotated by UniProt-GOA database (http://www.ebi.ac.uk/GOA/), the InterProScan soft was used to annotated protein’s GO functional based on protein sequence alignment method. Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg/) was used to annotate protein pathway. Online service tools KAAS was used to annotate protein’s KEGG description. KEGG mapper was used to map the annotation results. Functional Annotation Tool of DAVID Bioinformatics Resources 6.7 to identify enriched GO, KEGG pathway and protein domain against the background of Homo sapiens. A two-tailed Fisher’s exact test was employed to test the enrichment of the protein-containing IPI entries against all IPI proteins. Correction for multiple hypothesis testing was carried out using standard false discovery rate control methods. The GO with a corrected p-value < 0.05 is considered significant. The protein complex database CORUM was used for protein complex analysis. Corrected p-value < 0.05 was considered significant when performing the bioinformatics analysis.

Long-term stabilization of stage 4 colon cancer using sodium dichloroacetate therapy

Long-term stabilization of stage 4 colon cancer using sodium dichloroacetate therapy
Akbar Khan, Douglas Andrews, Anneke C Blackburn
Akbar Khan, Douglas Andrews, Medicor Cancer Centres Inc., Toronto, ON M2N 6N4, Canada
Anneke C Blackburn, The John Curtin School of Medical Research, The Australian National University, Canberra, ACT 2601, Australia
Author contributions: Khan A treated the patient and wrote most of the case report; Andrews D treated the patient, designed the natural therapy protocols, and co-wrote the case report; Blackburn AC performed in vitro and in vivo work demonstrating DCA’s effects as a cytostatic agent, and wrote the parts of the case report dealing with the in vitro and in vivo DCA research.
Institutional review board statement: Not applicable.
Informed consent statement: The patient described in this manuscript has given consent to publish her case anonymously.
Conflict-of-interest statement: One of the authors (Khan) administers dichloroacetate therapy for cancer patients through Medicor Cancer Centres at a cost, and without profit. The clinic is owned by a family member of this author. The other authors have nothing to disclose.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Akbar Khan, MD, Medical Director, Medicor Cancer Centres Inc., 4576 Yonge St., Suite 301, Toronto, ON M2N 6N4, Canada. akhan@medicorcancer.com
Telephone: +1-416-2270037 Fax: +1-416-2271915
Received: April 30, 2016
Peer-review started: May 3, 2016
First decision: June 17, 2016
Revised: July 23, 2016
Accepted: August 6, 2016
Article in press: August 8, 2016
Published online: October 16, 2016
Abstract

Oral dichloroacetate sodium (DCA) has been investigated as a novel metabolic therapy for various cancers since 2007, based on data from Bonnet et al that DCA can trigger apoptosis of human lung, breast and brain cancer cells. Response to therapy in human studies is measured by standard RECIST definitions, which define “response” by the degree of tumour reduction, or tumour disappearance on imaging. However, Blackburn et al have demonstrated that DCA can also act as a cytostatic agent in vitro and in vivo, without causing apoptosis (programmed cell death). A case is presented in which oral DCA therapy resulted in tumour stabilization of stage 4 colon cancer in a 57 years old female for a period of nearly 4 years, with no serious toxicity. Since the natural history of stage 4 colon cancer consists of steady progression leading to disability and death, this case highlights a novel use of DCA as a cytostatic agent with a potential to maintain long-term stability of advanced-stage cancer.

Key Words: DichloroacetateCancerColonColorectalCytostaticStabilizationGrowth inhibitionIntravenous

Core tip: Oral dichloroacetate sodium (DCA) has been investigated as a novel metabolic therapy for various cancers. Response to therapy in human studies is measured by standard RECIST definitions, which define “response” by the degree of tumour reduction, or tumour disappearance on imaging. However, DCA can also act as a cytostatic agent, without causing apoptosis (programmed cell death). A case is presented in which oral DCA therapy resulted in tumour stabilization of stage 4 colon cancer in a 57 years old female for a period of nearly 4 years, with no serious toxicity.


Citation: Khan A, Andrews D, Blackburn AC. Long-term stabilization of stage 4 colon cancer using sodium dichloroacetate therapy. World J Clin Cases 2016; 4(10): 336-343


INTRODUCTION

The drug sodium dichloroacetate (DCA) has been investigated as a novel metabolic therapy for various cancers since 2007 when Bonnet et al[1] published a combined in vitro/in vivo rat study demonstrating the efficacy of DCA in treating human lung, breast and brain cancers by inhibition of mitochondrial pyruvate dehydrogenase kinase. Stacpoole et al[24] had previously published multiple studies involving DCA for the treatment of congenital lactic acidosis, which is composed of a collection of inherited mitochondrial diseases[5]. These studies established the safety profile of oral DCA in humans. DCA was discovered to be a safe drug with no cardiac, pulmonary, renal or bone marrow toxicity[4]. The most serious common side effect consists of peripheral neuropathy, which is reversible[6]. Delirium has been reported, and is reversible after discontinuation of DCA[7]. Asymptomatic and reversible liver enzyme elevation has been reported in a small percentage of patients[3]. The prior work in congenital lactic acidosis has enabled the rapid progression of DCA into the cancer clinic. Four reports have now been published of cancer clinical trials using DCA, indicating a growing recognition of the potential usefulness of DCA[811]. However, these trials treating late stage patients, have only been able to report on treatment for relatively short time periods.

In the initial 2007 paper by Bonnet et al[1], it was reported that DCA reduced mitochondrial membrane potential resulting in selective apoptosis in cancer cells. The mechanism identified was inhibition of aerobic glycolysis (the Warburg effect) and activation of mitochondrial potassium ion channels[1]. Further investigation of DCA confirmed anti-cancer activity in several cancer types including colon[12], prostate[13], ovarian[14], neuroblastoma[15], lung carcinoid[16], cervical[17], endometrial[18], cholangiocarcinoma[19], sarcoma[20] and T-cell lymphoma[21]. Other antineoplastic actions of DCA have also been suggested. These include angiogenesis blockade[22], changes in expression of HIF1-α[23], alteration of pH regulators V-ATPase and MCT1, and other cell survival regulators such as PUMA, GLUT1, Bcl2 and p53[24]. However, in the quest for cytotoxic activity, many in vitro reports use concentrations of DCA that are unlikely to be achieved clinically[25]. Some studies have used restricted concentrations and found DCA to be cytostatic rather than cytotoxic, but able to enhance apoptosis with other agents[2628]. In the report of successful in vivo DCA treatment of breast cancer, Sun et al[26] found DCA to be cytostatic, inhibiting proliferation without increasing apoptosis. DCA was able to significantly reduce metastatic burden in the lungs of rats in a highly metastatic in vivo model of breast cancer. This suggests a new role for DCA as a cancer stabilizing agent, similar to an anti-angiogenic therapy. However, to the authors’ knowledge, no human data has yet been published supporting the use of DCA for long-term maintenance of stable disease.

As a result of Bonnet’s groundbreaking DCA publication, in early 2007, Khan commenced the clinical use of DCA to treat cancer patients with a poor prognosis or who failed to respond to approved cancer therapies. A protocol of natural medications was developed to address the dose-limiting neurologic toxicity, in collaboration with a naturopathic physician (Andrews). The oral DCA regimen that was developed included three natural medications acetyl L-carnitine[2931], R-alpha lipoic acid[3234] and benfotiamine[3537], for the primary purpose of neuropathy prevention. Observational data collected from more than 300 cancer patients with advanced disease revealed measurable benefits from DCA therapy in 60%-70% of cases. The neuropathy risk with inclusion of natural neuroprotective agents was roughly 20% with 20-25 mg/kg per day dosing on a 2 wk on/1 wk off cycle. Reversible liver enzyme elevation was noted in approximately 2% in this patient group (clinic observational data published online at http://www.medicorcancer.com ).

A patient case illustrating the cytostatic effects of oral DCA treatment maintained over several years is presented. This patient had a poor prognosis (median survival of 9-12 mo for stage 4 colorectal cancer using aggressive conventional palliative chemotherapy)[38]. The patient was treated by Khan in cooperation with the naturopathic physician Andrews, who developed a protocol comprised of natural neuroprotective agents.

CASE REPORT

A 57 years old female attended the author’s clinic (Khan) in March 2012 seeking therapy for metastatic colorectal cancer. The patient was originally diagnosed with rectal cancer in mid-2010 when she consulted her doctor for new constipation and low back pain. Colonoscopy was attempted, but the colonoscope could not be advanced due to the presence of a partially-obstructing rectal tumour. Biopsy confirmed moderately differentiated colorectal adenocarcinoma. Computerized tomography (CT) scan at the time demonstrated stage 4 disease with multiple liver metastases up to 3 cm in diameter, possible tiny lung metastases and an annular rectal carcinoma that was not easily measured (margins of the cancer were difficult to distinguish from the surrounding tissues on CT scan). The patient underwent a loop ileostomy procedure to bypass the obstruction, and the rectal tumour was not excised. Surgery was followed by chemotherapy consisting of 5-fluorouracil, irinotecan, leucovorin and bevacizumab (FOLFIRI + bevacizumab). Initially the patient responded to chemotherapy with a reduction in liver metastases, a reduction of the primary rectal lesion, and a reduction of blood carcinoembryonic antigen (CEA) marker from 260.9 ng/mL before chemo to 3.5 ng/mL just prior to DCA therapy initiation. The response to chemo then began to plateau. By the time the patient presented to the author’s clinic, chemotherapy was causing minimal disease reduction, and was essentially just maintaining stability.

The patient was previously healthy, and had a 20 year smoking history. She consumed alcohol on occasion. There was a positive family history of colon cancer and gastric cancer. Medications included ongoing chemotherapy as described, hydrogen peroxide enemas, oral vitamin C, occasional oral vitamin D, time-release hydromorphone 32 mg twice a day, and short-acting hydromorphone 2-4 mg orally as needed for “breakthrough” pain. There were no allergies. Functional enquiry revealed some mild mouth sores related to the ongoing chemotherapy, mild diarrhea (expected with an ileostomy) and mild intermittent rectal bleeding. There was aching/burning lower back and sacral pain up to 6 out of 10 intensity, and mild right shoulder-tip pain exacerbated by chemotherapy (felt to be referred pain related to liver metastases).

Since the chemotherapy was still effective, and the patient was not experiencing any serious side effects, the initial approach was to support the patient’s existing therapy, not replace it. An integrative plan was made in cooperation with a naturopathic physician (Andrews). The plan consisted of addition of high dose oral vitamin D at 10000 international units per day, a change of oral vitamin C to vitamin C 50 g intravenous (i.v.) weekly, and addition of dichloroacetate sodium (DCA) 3000 mg i.v. (49 mg/kg) weekly (manufacturer: Tokyo Chemical Industry, United States). To reduce the risk of DCA side effects, 3 natural supplements were prescribed: Alpha lipoic acid (racemic) 500 mg i.v. with each DCA dose, oral R-alpha lipoic acid 150 mg 3 times a day, oral acetyl L-carnitine 500 mg 3 times a day, and oral benfotiamine 80 mg twice a day. Infusions were planned around chemotherapy infusions (separated by at least 2 d from chemotherapy) to avoid any potential interference or drug interactions. Lipoic acid was not given on chemotherapy days, or within 1 d before or after chemotherapy, since it is a powerful antioxidant and has the potential to reduce chemotherapy efficacy. Integrative therapy began in March 2012. No side effects were noted, so DCA was increased to 4000 mg i.v. (66 mg/kg) weekly. The only side effect noted at the higher DCA dose was mild post-infusion sedation.

Oral metformin was added to help sensitize the cancer to the chemotherapy, starting at 500 mg orally once a day with titration up to 500 mg 3 times a day[39]. Pregabalin was added to help control the neuropathic sacral pain (started 50 mg daily, titrated up to 50 mg 3 times a day). Chemotherapy side effects included nausea and vomiting (prior to initiation of metformin), and metformin was skipped on days when the patient felt unwell to prevent potential toxicity, should the patient become dehydrated.

Routine baseline blood tests were obtained including complete cell counts, standard metabolic panel, liver enzymes and bilirubin (Table 1). A baseline CT scan was available, which had been performed 2 mo prior to initiation of integrative therapy with DCA.

Table 1 Blood panel prior to dichloroacetate sodium therapy.
Blood test Value Units Normal range
Hemoglobin 131 g/L 115-155
White cell count 6.5 × 109/L 4.0-11.0
Platelets 202 × 109/L 145-400
Glucose 5.9 mmol/L 2.6-7.0
Urea 6.5 mmol/L 2.5-8.1
Creatinine 64 μmol/L 50-100
Calcium 2.38 mmol/L 2.20-2.65
Albumin 43 g/L 35-52
Bilirubin 15 μmol/L < 23
Sodium 140 mmol/L 136-146
Potassium 4.2 mmol/L 3.7-5.4
Chloride 102 mmol/L 95-108
Alkaline phosphatase 1861 U/L 35-122
LDH 167 U/L 110-215
GGT 3641 U/L < 36
AST 331 U/L < 31
ALT 31 U/L < 36

After 4 mo of integrative therapy as described, a new CT scan was performed (Figure 1), which was reported as “stable and unchanged”, but no measurements were given. An incidental finding of a gallstone was noted (also stable from prior scan). The patient became frustrated that no improvement was noted, and no detailed measurements were indicated in the CT report. An attempt was made to obtain a positron emission tomography scan to clarify live vs necrotic tumours, but government funding could not be obtained and the patient declined to pay privately for the scan.

Figure 1
Figure 1 Abdominal computerized tomography scan after 4 mo of integrative therapy with dichloroacetate sodium, 5-fluorouracil and natural medicines. Three slices with various measurable liver metastases shown. A: 23 mm × 33 mm liver metastasis; B: 15 mm diameter liver metastasis; C: 11.2 mm × 25 mm liver metastasis.

After some discussion, the patient elected to continue therapy, and obtain future CT scans at a different hospital. By September 2012, increasing chemotherapy side effects including fatigue, nausea and vomiting were noted. A new CT scan revealed that all the liver lesions were “either smaller or no longer identified”. The greatest tumour reduction was only 2 mm however (2.5 cm marker lesion in liver segment 4a reduced to 2.3 cm). There were no new lesions identified.

After review of the CT scan, the patient decided to stop all chemotherapy, as well as bevacizumab and metformin. DCA i.v. was continued, and the dose was increased to 4500 mg i.v. weekly. Nausea and vomiting resolved. Pain remained under control. A new CT scan was obtained after 3 mo, which demonstrated residual rectal tumour with stricture and proximal fecal loading (unchanged), and “liver metastases, not significantly changed”. The patient reported mild numbness of the fingers and toes. There was a further increase in asymptomatic liver enzyme elevations (Table 2). Both of these were diagnosed as DCA side effects. During therapy up to this point, CEA had shown mild fluctuations, but was considered stable overall (Figure 2).

Table 2 Blood panel during dichloroacetate sodium therapy, January 2013.
Blood test Value Units Normal range
Hemoglobin 134 g/L 115-155
White cell count 5.1 × 109/L 4.0-11.0
Platelets 1421 × 109/L 145-400
Glucose 5.5 mmol/L 2.6-7.0
Urea 4.1 mmol/L 2.5-8.1
Creatinine 57 μmol/L 50-100
Calcium 2.24 mmol/L 2.20-2.65
Albumin 39 g/L 35-52
Bilirubin 11 μmol/L < 23
Sodium 140 mmol/L 136-146
Potassium 4.2 mmol/L 3.7-5.4
Chloride 106 mmol/L 95-108
Alkaline phosphatase 2671 U/L 35-122
LDH 183 U/L 110-215
GGT 8371 U/L < 36
AST 1041 U/L < 31
ALT 100 U/L < 36
Figure 2
Figure 2 Graph of carcinoembryonic antigen through the course of therapy. CEA: Carcinoembryonic antigen.

DCA therapy was interrupted for 3 mo to allow resolution of DCA side effects. During this time, only natural therapies were given (prescribed by Andrews). Acetyl L-carnitine, benfotiamine and alpha lipoic acid were continued to accelerate recovery of DCA neuropathy. Oral curcumin[40] and honokiol (magnolia tree extract) were added in an attempt to maintain cancer control[41]. During the period when DCA was stopped, CEA increased from 4.1 to 5.1 ng/mL (Figure 2). Mild DCA neuropathy had resolved and liver enzymes began to improve.

By March 2013, due to concern over the cost of infusion therapy, it was decided to begin oral DCA therapy. A new baseline CT scan demonstrated a 1 mm increase in a liver segment 7 marker lesion, and a 1 mm increase in a marker aortocaval lymph node, but was reported as “stable appearance of the colon” and “stable liver metastases”.

Oral DCA was initiated at a dose of 500 mg (8.2 mg/kg) twice a day, and neuroprotective supplements consisting of oral acetyl L-carnitine, benfotiamine and R-alpha lipoic acid were continued. Supplements were given continuously, and DCA was given on a 2 wk on/1 wk off cycle.

In December 2013, the pain medication was transitioned from hydromorphone to methadone 10 mg 3 times a day, for simplicity, improved pain control and cost savings.

The patient continued on this regimen with regular CT scans every 3 to 6 mo. The patient became less compliant with regular blood testing due to a busy work schedule. She remained highly functional (ECOG level 1) with mild chronic DCA neuropathy that was controlled and did not affect her daily function. An attempt was made to increase DCA to 500 mg 3 times a day, but this resulted in significant asymptomatic liver enzyme elevation and increase in neuropathy. As a result, a DCA dose of 500 mg twice a day was resumed after a brief therapy interruption.

Ongoing CT scans continued to reveal stable disease (Figure 3), with no new lesions appearing. Overall CEA was not significantly changed from the initiation of DCA therapy (CEA of 3.5 at the start of DCA therapy, to CEA of 3.7 after nearly 4 years of therapy). General blood panel was also favourable at the 3 year mark (Table 3) and after the 4 year mark (Table 4).

Table 3 Blood panel during dichloroacetate sodium therapy, May 2015.
Blood test Value Units Normal range
Hemoglobin 134 g/L 115-155
White cell count 7.7 × 109/L 4.0-11.0
Platelets 173 × 109/L 145-400
Glucose 5.3 mmol/L 2.6-7.0
Urea 5.1 mmol/L 2.5-8.1
Creatinine 70 µmol/L 50-100
Calcium 2.37 mmol/L 2.20-2.65
Albumin g/L 35-52
Bilirubin 8 µmol/L < 23
Sodium 144 mmol/L 136-146
Potassium 4.1 mmol/L 3.7-5.4
Chloride 104 mmol/L 95-108
Alkaline phosphatase U/L 35-122
LDH 174 U/L 110-215
GGT 1561 U/L < 36
AST 30 U/L < 31
ALT 25 U/L < 36
Figure 3
Figure 3 Abdominal computerized tomography scan after 3 additional months of integrative therapy (dichloroacetate sodium + 5-fluorouracil + natural medicines), followed by nearly 4 years of dichloroacetate sodium without any concurrent conventional cancer therapies.Scans demonstrate absence of cancer re-growth and absence of new liver metastases. Same slices as Figure 1 are shown. A: 11.3 mm × 27.5 mm liver metastasis; B: No metastases visible; C: No metastases visible.
Table 4 Blood panel during dichloroacetate sodium therapy, April 2016.
Blood test Value Units Normal range
Hemoglobin 133 g/L 115-155
White cell count 5.2 × 109/L 4.0-11.0
Platelets 155 × 109/L 145-400
Glucose mmol/L 2.6-7.0
Urea 4.9 mmol/L 2.5-8.1
Creatinine μmol/L 50-100
Calcium 2.39 mmol/L 2.20-2.65
Albumin 42 g/L 35-52
Bilirubin 9 μmol/L < 23
Sodium 142 mmol/L 136-146
Potassium 4 mmol/L 3.7-5.4
Chloride 102 mmol/L 95-108
Alkaline phosphatase 101 U/L 35-122
LDH 156 U/L 110-215
GGT 1491 U/L < 36
AST 30 U/L < 31
ALT 28 U/L < 36

In summary, after receiving conventional chemotherapy for approximately 18 mo, the patient received intravenous DCA therapy with concurrent chemotherapy for approximately 6 mo, followed by intravenous and oral DCA therapy with no concurrent conventional cancer therapy for nearly 4 years. During the treatment with oral DCA, the patient experienced stable disease by CT scans, and stable disease by CEA tumour marker measurement. She also was clinically stable with no escalation of methadone dose, maintenance of ECOG level 1 function, stable mild DCA neuropathy, and she was able to run her own business successfully.

DISCUSSION

This case of DCA therapy in a patient with advanced stage 4 colon cancer demonstrates long-term stable disease according to clinical, biochemical and radiologic criteria. The duration of stability while on DCA without other active chemotherapy is currently 46 mo (nearly 4 years), with a survival time since the initial diagnosis of stage 4 colorectal cancer of 6 years. Based on the National Cancer Institute’s SEER cancer statistics review 1975-2011, the 5-year relative survival rate for females diagnosed with stage IV colon/rectal cancer was 14.4% (http://seer.cancer.gov/csr/1975_2013/ ). While it cannot be definitively concluded that DCA has been efficacious, survival for this length of time in the absence of ongoing chemotherapy would be of relatively low probability. Cytostatic rather than cytotoxic effects of DCA on colorectal and other cancer cells have been reported and support this clinical finding[23,27,4244]. To date the patient remains clinically well and she remains on DCA therapy.

In addition to the maintenance of stable disease, this case demonstrates the tolerability of oral DCA in a cancer patient for much longer time periods than those currently reported from the published clinical trials in cancer patients. Chu et al[11] reported on 24 patients treated for a median time of 2 mo at either 6.25 or 12.5 mg/kg BID, on continuous oral DCA without neuroprotective supplements. They concluded that the recommended phase 2 dose was 6.25 mg/kg BID (12.5 mg/kg per day), with careful monitoring of neuropathy being needed. Dunbar et al[9] recommended 5 mg/kg BID as a starting dose for most patients, with their trial administering 4, 8 or 12.5 mg/kg BID continuously (median time on DCA 34 d), also without neuroprotective supplements. The patient in this report took 500 mg BID, equivalent to 8.2 mg/kg BID, 2 wk on/1 wk off, but could not tolerate this dose three times a day (total of 25 mg/kg per day). Dunbar et al[9] suggest that genotyping for polymorphisms in GSTZ1, the DCA metabolizing enzyme in the liver which is inactivated with ongoing DCA usage[45], should be considered in determining the starting dose for patients. However further work is necessary to gather convincing numbers of genotypes and dose-tolerance data. A clinical trial of DCA in multiple myeloma patients is currently underway to contribute to this pool of data (Australia New Zealand Clinical Trials Register #ACTRN12615000226505, http://www.anzctr.org.au ). Further studies are required to determine the optimal dosage regime for maximum tolerable acute or chronic treatment with DCA, and indeed what dose is required for efficacy.

The case presented indicates DCA holds great promise as a cancer therapy. The patient achieved a significant benefit from her therapy, with mild side effects and no hematological, cardiac, pulmonary or renal toxicity. Some hepatic toxicity was observed (Table 2), which was easily managed by DCA therapy interruption followed by dose adjustment. Mild reversible peripheral neurotoxicity was reported. Natural therapies that were combined with DCA (acetyl L-carnitine, alpha lipoic acid and benfotiamine) assisted the patient with reduction of side effects, but are not known to function as cancer therapies.

As of this writing, no active clinical trials exist investigating the human use of DCA as a cytostatic agent. Due to the fact that DCA is off patent, raising adequate funds to support large scale human trials is a serious challenge. It is hoped that this case exemplifying the benefits of oral DCA will stimulate further clinical investigation.

Based on our clinical experience, combined with existing publications, off-label DCA therapy is an option for patients with limited available conventional treatments, once they understand and accept the risks and benefits of therapy. This case report shows that even in advanced stage disease, DCA has the potential to prolong life without impacting a patient’s quality of life, as compared to chemotherapy with its frequent debilitating side effects or compromise in physiological function. Given its reasonable cost and modest toxicity, DCA deserves further investigation.

ACKNOWLEDGMENTS

The authors wish to thank Dr. Humaira Khan for her assistance, and also the patient for her support and consent to publish her case.

COMMENTS
Case characteristics

The 57 years old female patient presented with constipation and lower back pain.

Clinical diagnosis

The patient was diagnosed with a partially-obstructing rectal cancer.

Laboratory diagnosis

Elevated carcinoembryonic antigen tumour marker.

Imaging diagnosis

Rectal mass seen on colonoscopy.

Pathological diagnosis

Moderately differentiated colorectal adenocarcinoma.

Treatment

Loop ileostomy followed by chemotherapy consisting of 5-fluorouracil, irinotecan, leucovorin and bevacizumab, then addition of dichloroacetate sodium (DCA), then DCA without chemo for nearly 4 years.

Related reports

Computerized tomography scan reports demonstrate reduction of cancer with combined chemotherapy + DCA, then stable disease for nearly 4 years with DCA and no chemo.

Term explanation

DCA: Dichloroacetate sodium; RECIST: Response evaluation criteria for solid tumours; ECOG: Eastern cooperative oncology group; SEER: Surveillance, epidemiology and end results.

Experiences and lessons

DCA is not only a pro-apoptotic drug, but may also act as a cytostatic agent, and can thus achieve long-term stabilization of advanced cancer without serious side effects, as illustrated by this rectal cancer case.

Peer-review

DCA, the sodium salt of dichloroacetate, is a cheap chemical compound that has shown some clear potential as an alternative cancer treatment, which has been used in a number of trials with people suffering from brain cancer, or glioblastoma. This is a well-written case report in which oral DCA therapy resulted in tumor stabilization of stage 4 colon cancer in a 57 years old female for a period of over 3 years, with no serious toxicity. This report covers what it promises to. The authors do a solid job of explaining the basics of DCA therapy and its role in different tumor types. Along with the addition of mechanisms of action against cancer cells and therapeutic potential of DCA, the authors provide a good resource for readers who are more unfamiliar with DCA therapy but also provide detail.

Footnotes

Manuscript source: Invited manuscript

Specialty Type: Medicine, research and experimental

Country of Origin: Australia

Peer-Review Report Classification

Grade A (Excellent): 0

Grade B (Very good): B,B

Grade C (Good): 0

Grade D (Fair): D

Grade E (Poor): 0

P- Reviewer: Lakatos PL, Song J, Zhu YL S- Editor: Ji FF L- Editor: A E- Editor: Wu HL

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Method of Treating Cancer using Dichloroacetate United States Patent Application 20090118370

Method of Treating Cancer using Dichloroacetate

United States Patent Application 20090118370

Kind Code: A1

Abstract: “The invention relates to the use of dichloroacetate and chemical equivalents thereof for the treatment of cancer by inducing apoptosis or reversing apoptosis-resistance in a cell Preferably, the dosage is 10-100 mg/kg Preferably, sodium dichloroacetate is used. The dichloroacetate may optionally be given in combination with a pro-apoptotic agent and/or a chemotherapeutic agent Preferably, the cancers treated are non-small cell lung cancer, glioblastoma and breast carcinoma.”

 

 

Inventors: Michelakis, Evangelos (Edmonton, CA)

Archer, Stephen (La Grange, IL, US)

 

 

Application Number: 11/911299

 

 

Publication Date: 05/07/2009

 

 

Filing Date: 04/11/2006

 

 

Export Citation: Click for automatic bibliography generation

 

 

Primary Class: 514/557

 

 

Other Classes: 600/300

 

 

International Classes: A61K31/19; A61B5/00; A61P35/00; A61K31/185; A61B5/00; A61P35/00

 

Description:

 

This patent application claims priority from U.S. Provisional Patent Application No. 60/669,884 filed Apr. 11, 2005, the content of which is hereby incorporated by reference herein.

 

FIELD OF THE INVENTION

 

The invention relates to the use of dichloroacetate and obvious chemical equivalents thereof in the treatment of cancer. Related uses and diagnostic and screening methods are also included in one aspect of the present invention.

 

BACKGROUND OF THE INVENTION

 

Most cancers are characterized by a resistance to apoptosis that makes them prone to proliferation and resistant to most cancer therapies. Most of the available cancer treatments aim to induce apoptosis but are highly toxic. There are two main categories of apoptosis: the receptor-mediated and the mitochondria-dependent apoptosis. Mitochondria-dependent apoptosis is not very well studied and only recently have the mitochondria been viewed as anything more than an organelle that produces energy. As such there is a need for a cancer therapy that can overcome apoptosis resistance in cancer cells.

 

SUMMARY OF THE INVENTION

 

A cell can become resistant to apoptosis in a variety of ways one of which is altering its metabolism and having hyperpolarized mitochondria. Since apoptosis is initiated by depolarization of mitochondria, the more hyperpolarized a mitochondrion is, the further it is from the depolarization threshold and the more resistant it is to the initiation of apoptosis.

 

In one embodiment the present inventors have surprisingly found that one can modulate mitochondrial function to treat cancer. In one embodiment, the present invention provides a method for inducing apoptosis in cancer. In another embodiment, the inventors provide a method for inducting apoptosis in cancer but normal cells. In another embodiment, the invention provides a method of reversing apoptosis resistance in cancer cells, such as cancer cells with hyperpolarized mitochondria. In one embodiment, the method comprises administering to cancer cells, in one embodiment cells having or suspected of having hyperpolarized mitochondria, an effective amount of dichloroacetate or salts thereof or obvious chemical equivalents thereof.

 

In one embodiment, the dichloroacetate or obvious chemical equivalent thereof is administered in combination with another pro-apoptotic agent and/or chemotherapeutic agent, and/or other cancer therapy.

 

In one embodiment, the invention provides a method for inducing apoptosis and/or reversing apoptosis resistance in a cancer cell, comprising administering to the cell an effective amount of dichloroacetate or obvious chemical equivalent thereof. In another embodiment, the invention provides a method for inhibiting proliferation of cancer cells, comprising administering to the cells an effective amount of dichloroacetate or obvious chemical equivalent thereof. In another embodiment, the invention provides a method of decreasing survivin in a cancer cell, comprising administering to the cell an effective amount of dichloroacetate or obvious chemical equivalent thereof. In another embodiment, the invention provides a method of increasing Kv1.5 protein in a cancer cell comprising administering to the cell an effective amount of dichloroacetate or obvious chemical equivalent thereof. In another embodiment, the invention provides a method of increasing AIF in a cancer cell comprising administering to the cell an effective amount of dichloroacetate or obvious chemical equivalent thereof. In another embodiment, the invention provides a method of increasing H 2 O 2 in a cancer cell comprising administering to the cell an effective amount of dichloroacetate or obvious chemical equivalent thereof. In another embodiment, the methods of the invention cancer cells, but not normal or non-cancerous cells are affected by the treatment with dichloroacetate or obvious chemical equivalent thereof.

 

In one embodiment, the present invention provides a method for treating a cancer. In another embodiment, the invention provides a method of treating a cancer associated with hyperpolarized mitochondria. In another embodiment the invention provides a method of treating cancer by restoring mitochondrial membrane potential (ΔΨm) (essentially depolarizing the hyperpolarized cancer cell mitochondria). This molecular metabolic therapy is accomplished by administering to a patient in need thereof a therapeutically effective amount of dichloroacetate or obvious chemical equivalent thereof. In another embodiment, the invention provides a use of dichloroacetate or obvious chemical equivalent thereof in the treatment of cancer.

 

In one embodiment, the dichloroacetate is a salt of dichloroacetic acid. In another embodiment, the dichloroacetic acid is a sodium salt of dichloroacetic acid.

 

In one embodiment, the cancer to be treated using the DCA or obvious chemical equivalent thereof is selected from the group consisting of: non-small cell lung cancer, glioblastoma and breast carcinoma.

 

In another embodiment, the dichloroacetate, or obvious chemical equivalent thereof, is administered in the form of a pharmaceutical composition comprising dichloroacetate or obvious chemical equivalent thereof and a pharmaceutically acceptable carrier. In yet another embodiment the invention provides a use of dichloroacetic acid or dichloroacetate or obvious chemical equivalent thereof in the preparation of a medicament or pharmaceutical composition for the treatment of cancer, such as a cancer associated with hyperpolarized mitochondria. In yet another embodiment, the dichloroacetate, or obvious chemical equivalent thereof, is administered orally.

 

In yet another embodiment, the dichloroacetate is administered in a water-based formulation. In one embodiment the water-based formulation of DCA comprises 0.0075 g of DCA/l to 7.5 g of DCA/l). In another embodiment the dichloroacetate or obvious chemical equivalent thereof is administered at a total daily dose of ˜25-50 mg/kg bid of dichloroacetate. In another embodiment the dose is 10-100 mg/kg given twice a day is administered to the patient. In one embodiment the dose is 25-50 mg bid.

 

In another embodiment, the invention constitutes a method for determining whether a cancer is associated with hyperpolarized mitochondria, which would predict its therapeutic response to dichloroacetate or obvious chemical equivalents thereof or similar compounds. In one embodiment such method comprises administering an effective amount of dichloroacetate, or chemical equivalent thereof to a cancer tissue sample from a patient and measuring its apoptosis sensitivity and mitochondrial membrane potential using confocal microscopy or flow cytometry. This diagnostic test would determine whether the individual patient could benefit from dichloroacetate or other therapies that cause apoptosis through similar mechanism.

 

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

British Journal of Cancer DCA Mini Review

British Journal of Cancer (2008) 99, 989–994. doi:10.1038/sj.bjc.6604554 www.bjcancer.com

Published online 2 September 2008

Minireview

Dichloroacetate (DCA) as a potential metabolic-targeting therapy for cancer

Link to BJC article: http://www.nature.com/bjc/journal/v99/n7/full/6604554a.html

E D Michelakis1, L Webster1 and J R Mackey2

 

1Department of Medicine, University of Alberta, Edmonton, Canada

2Department of Oncology, University of Alberta, Edmonton, Canada

 

Correspondence: Dr ED Michelakis, Department of Medicine, University of Alberta Hospital, 8440-112 Street, Edmonton, AB, Canada T6G 2B7; E-mail: evangelos.michelakis@capitalhealth.ca

 

Received 18 December 2007; Revised 28 April 2008; Accepted 4 July 2008; Published online 2 September 2008.

 

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Abstract

A paradigm shift is needed in cancer therapeutics

The metabolism of cancer cells

Glycolysis offers an early adaptation to the hypoxic microenvironment in carcinogenesis

Glycolysis is associated with resistance to apoptosis

Mitochondria and apoptosis

DCA reverses the mitochondrial remodeling, unlocking the cancer cells from a state of apoptosis resistance: preclinical work

DCA: mechanism of action and clinical experience

DCA: clinical testing in cancer?

Note to proof

References

Figures and Tables

 

The unique metabolism of most solid tumours (aerobic glycolysis, i.e., Warburg effect) is not only the basis of diagnosing cancer with metabolic imaging but might also be associated with the resistance to apoptosis that characterises cancer. The glycolytic phenotype in cancer appears to be the common denominator of diverse molecular abnormalities in cancer and may be associated with a (potentially reversible) suppression of mitochondrial function. The generic drug dichloroacetate is an orally available small molecule that, by inhibiting the pyruvate dehydrogenase kinase, increases the flux of pyruvate into the mitochondria, promoting glucose oxidation over glycolysis. This reverses the suppressed mitochondrial apoptosis in cancer and results in suppression of tumour growth in vitro and in vivo. Here, we review the scientific and clinical rationale supporting the rapid translation of this promising metabolic modulator in early-phase cancer clinical trials.

 

 

Keywords:

 

mitochondria, metabolism, apoptosis, potassium channels, positron emission tomography, glycolysis

 

 

Top of page

A paradigm shift is needed in cancer therapeutics

A paradigm shift is needed in cancer therapeutics

The metabolism of cancer cells

Glycolysis offers an early adaptation to the hypoxic microenvironment in carcinogenesis

Glycolysis is associated with resistance to apoptosis

Mitochondria and apoptosis

DCA reverses the mitochondrial remodeling, unlocking the cancer cells from a state of apoptosis resistance: preclinical work

DCA: mechanism of action and clinical experience

DCA: clinical testing in cancer?

Note to proof

References

Figures and Tables

 

Although some battles have been won since the declaration of the ‘war on cancer’ in 1971 in the United States, the war is ongoing. Despite enormous investments from industry and the public, oncology has an impressively poor success rate in the clinical development of effective investigational drugs; less than a third of that in cardiovascular or infectious diseases (Kamb et al, 2007). Drug development in oncology has typically focused on targets essential for the survival of all dividing cells, leading to narrow therapeutic windows. Non-essential targets offer more selectivity but little efficacy. It is extremely rare to find an essential target that is unique to cancer cells; the dependence of CML cells on Ableson kinase is only induced by a chromosomal translocation in the malignant clone, making the efficacy and selectivity of imatinib for CML an exception in cancer therapy (Kamb et al, 2007). The most important reason for the poor performance of cancer drugs is the remarkable heterogeneity and adaptability of cancer cells. The molecular characteristics of histologically identical cancers are often dissimilar and molecular heterogeneity frequently exists within a single tumour. The view that ‘there are many different types of cancers’ is increasingly shared by scientists and clinical oncologists. This has important implications, including the realisation that specific drugs have to be developed and tested for molecularly defined tumours and effects in one might not necessarily be relevant to another cancer.

 

 

The biggest challenge remains the selective induction of cell death (mainly apoptosis) in cancer but not normal cells. Pragmatically, an ideal anticancer therapy would be easily administered (possibly an orally available small molecule) and affordable. Most new anticancer drugs are prohibitively expensive not only for millions of patients from developing countries, but also for many patients without strong medical insurance in developed countries.

 

One way that the problem of heterogeneity of ‘proximal’ molecular pathways in cancer can be addressed is by targeting more ‘distal’ pathways that integrate several proximal signals, as long as the common distal pathways remain essential and specific to cancer cells. The unique metabolism of most solid tumours integrates many proximal pathways and results in a remodeling of mitochondria (where the regulation of energy production and apoptosis converge), to produce a glycolytic phenotype and a strong resistance to apoptosis. There is now growing evidence that the mitochondria might be primary targets in cancer therapeutics instead of simple bystanders during cancer development. This cancer-specific metabolic remodeling can be reversed by dichloroacetate (DCA), a mitochondria-targeting small molecule, that penetrates most tissues after oral administration (Bonnet et al, 2007; Pan and Mak, 2007). The molecular and direct metabolic response to DCA can also be followed by measuring glucose uptake in tumours by positron emission tomography (PET) imaging, non-invasively and prospectively. Such metabolic strategies might be able to shift the paradigm of experimental therapeutics in oncology.

 

The preclinical work on DCA (showing effectiveness in a variety of tumours and relatively low toxicity) (Bonnet et al, 2007), its structure (a very small molecule), the low price (it is a generic drug) and the fact that DCA has already been used in humans for more than 30 years, provide a strong rationale for rapid clinical translation. Here, we expand the scientific rationale and discuss several practical points that will be important in the clinical evaluation of DCA as anticancer therapy.

 

Top of page

The metabolism of cancer cells

A paradigm shift is needed in cancer therapeutics

The metabolism of cancer cells

Glycolysis offers an early adaptation to the hypoxic microenvironment in carcinogenesis

Glycolysis is associated with resistance to apoptosis

Mitochondria and apoptosis

DCA reverses the mitochondrial remodeling, unlocking the cancer cells from a state of apoptosis resistance: preclinical work

DCA: mechanism of action and clinical experience

DCA: clinical testing in cancer?

Note to proof

References

Figures and Tables

 

Most cancers are characterised by aerobic glycolysis (GLY), that is, they use glycolysis for energy production, despite the fact that oxygen is present. In 1929, Warburg first observed this (i.e., the Warburg effect) and suggested it resulted from mitochondrial dysfunction, preventing the mitochondria-based glucose oxidation (GO) (Warburg, 1930). Because GO is far more efficient in generating ATP compared with GLY (producing 36 vs 2 ATP per glucose molecule), cancer cells upregulate glucose receptors and significantly increase glucose uptake in an attempt to ‘catch up’. Positron emission tomography imaging has now confirmed that most solid tumours have significantly increased glucose uptake and metabolism, compared with non-cancerous tissues (Figure 1). This bio-energetic difference between cancer and normal cells, might offer a very selective therapeutic target, as GLY is not typically seen in normal tissues apart from skeletal muscle during strenuous exercise. However, this area of experimental oncology has remained controversial; the glycolytic profile has traditionally been viewed as a result of cancer progression, not a cause and therefore the interest in targeting tumour metabolism has been low. Furthermore, at first glance, the glycolytic profile of cancer is difficult to understand, using an evolutionary model of carcinogenesis. First, why would these highly proliferating and energy-demanding cells rely on GLY rather than the much more efficient GO? Second, GLY results in significant lactic acidosis, which might cause significant toxicity to the surrounding tissues and the cancer cells themselves. Recent advances have caused a rekindling of the metabolic hypothesis of cancer suggesting that these facts are not as conflicting as they appear at first (Gatenby and Gillies, 2004):

 

 

Figure 1.

 

Brain MRI showing a large glioblastoma tumour with areas of necrosis within the tumour and significant brain oedema. On the right, a corresponding FDG-Glucose PET from the same patient shows much higher glucose uptake within the tumour, compared with the surrounding brain tissue.

Full figure and legend (49K)

 

 

 

Top of page

Glycolysis offers an early adaptation to the hypoxic microenvironment in carcinogenesis

A paradigm shift is needed in cancer therapeutics

The metabolism of cancer cells

Glycolysis offers an early adaptation to the hypoxic microenvironment in carcinogenesis

Glycolysis is associated with resistance to apoptosis

Mitochondria and apoptosis

DCA reverses the mitochondrial remodeling, unlocking the cancer cells from a state of apoptosis resistance: preclinical work

DCA: mechanism of action and clinical experience

DCA: clinical testing in cancer?

Note to proof

References

Figures and Tables

 

Gatenby and Gillies (2004) recently proposed that as early carcinogenesis often occurs in a hypoxic microenvironment, the transformed cells have to rely on anaerobic GLY for energy production. Hypoxia-inducible factor (HIF) is activated in hypoxic conditions and it has been shown to induce the expression of several glucose transporters and most of the enzymes required for GLY (Semenza et al, 1994). For example, HIF induces the expression of pyruvate dehydrogenase kinase (PDK) (Kim et al, 2006), a gate-keeping enzyme that regulates the flux of carbohydrates (pyruvate) into the mitochondria. In the presence of activated PDK, pyruvate dehydrogenase (PDH) is inhibited, limiting the entry of pyruvate into the mitochondria, where GO can take place. In other words, activated PDK promotes completion of GLY in the cytoplasm with metabolism of pyruvate into lactate; inhibited PDK ensures an efficient coupling between GLY and GO.

 

Initially, tumours compensate by increasing glucose uptake into the cells. Furthermore, Gatenby and Gillies (2004) list a number of mechanisms through which lactic acidosis facilitates tumour growth: breakdown of extra-cellular matrix allowing expansion, increased cell mobility/metastatic potential and (along with HIF) activation of angiogenesis. Although tumours eventually become vascularised and are not significantly hypoxic anymore (although some tumours remain hypoxic at the core because the quality of the neo-vessel formation is poor) the aerobic glycolytic profile persists. This suggests that the (initially adaptive) metabolic remodeling confers a survival advantage to cancer cells. Indeed, recent evidence suggests that transformation to a glycolytic phenotype offers resistance to apoptosis (Plas and Thompson, 2002) (Kim and Dang, 2005, 2006).

 

Top of page

Glycolysis is associated with resistance to apoptosis

A paradigm shift is needed in cancer therapeutics

The metabolism of cancer cells

Glycolysis offers an early adaptation to the hypoxic microenvironment in carcinogenesis

Glycolysis is associated with resistance to apoptosis

Mitochondria and apoptosis

DCA reverses the mitochondrial remodeling, unlocking the cancer cells from a state of apoptosis resistance: preclinical work

DCA: mechanism of action and clinical experience

DCA: clinical testing in cancer?

Note to proof

References

Figures and Tables

 

Several of the enzymes involved in glycolysis are also important regulators of apoptosis and gene transcription, suggesting that links between metabolic sensors, cell death and gene transcription are established directly through the enzymes that control metabolism (Kim and Dang, 2005). For example, hexokinase activation leads to a significant suppression of apoptosis; activated hexokinase translocates from the cytoplasm to the mitochondrial membranes where it interacts with and suppresses several key components of mitochondria-dependent apoptosis (Pastorino et al, 2005). It is therefore not surprising that hexokinase is upregulated and activated in many cancers (Kim and Dang, 2006). How does this occur? The promoter of hexokinase contains both p53 and HIF response elements and both mutated p53 and activated HIF increase hexokinase expression (Mathupala et al, 1997). In addition, the oncogenic protein Akt is upregulated in many cancers and induces a glycolytic metabolic profile through a number of mechanisms (Elstrom et al, 2004). Akt increases both the expression and activity of hexokinase (Gottlob et al, 2001; Elstrom et al, 2004). The gene that normally antagonises Akt, PTEN, is mutated (loss of function mutation) in a large number of cancers. Very recent data revealed even more links between p53 and metabolism: p53 regulates the expression of a critical enzyme of GLY through the production of TIGAR and is also directly regulating the expression of a subunit of cytochrome c oxidase, an important element of complex IV of the electron transport chain in mitochondria (reviewed in (Pan and Mak, 2007)). In other words, the most common molecular abnormality in cancer, that is, the loss of p53 function, induces metabolic and mitochondrial changes, compatible with the glycolytic phenotype. Likewise, the c-myc transcription factor increases the expression of many enzymes of GLY and can induce this same metabolic phenotype (Kim and Dang, 2005, 2006).

 

To conclude, an evolutionary theory of carcinogenesis identifies metabolism and GLY as a critical and early adaptive mechanism of cancer cells against hypoxia, that persist because it offers resistance to apoptosis in cancer cells (Gatenby and Gillies, 2004). The genetic theory on carcinogenesis, also identifies GLY and metabolism as an end result of activation of many diverse oncogenes, including c-myc, Akt/PTEN and p53 (Pan and Mak, 2007). Therefore, it is possible that this metabolic phenotype is centrally involved in the pathogenesis of cancer and is not simply a ‘by-product’ of carcinogenesis. Although it is not clear whether this metabolic phenotype directly induces malignancy, it certainly ‘facilitates’ carcinogenesis (Kim and Dang, 2006). In addition, this metabolic signature is the common denominator of multiple and diverse pathways; which means that if it is therapeutically targeted it might offer selectivity for malignant cells of diverse cellular and molecular origins.

 

Top of page

Mitochondria and apoptosis

A paradigm shift is needed in cancer therapeutics

The metabolism of cancer cells

Glycolysis offers an early adaptation to the hypoxic microenvironment in carcinogenesis

Glycolysis is associated with resistance to apoptosis

Mitochondria and apoptosis

DCA reverses the mitochondrial remodeling, unlocking the cancer cells from a state of apoptosis resistance: preclinical work

DCA: mechanism of action and clinical experience

DCA: clinical testing in cancer?

Note to proof

References

Figures and Tables

 

Shifting metabolism away from mitochondria (GO) and towards the cytoplasm (GLY), might suppress apoptosis, a form of cell death that is dependent on mitochondrial energy production (Figure 2). Pro-apoptotic mediators, like cytochrome c and apoptosis-inducing factor, are protected inside the mitochondria. When the voltage- and redox-sensitive mitochondrial transition pore (MTP) opens, they are released in the cytoplasm and induce apoptosis, although it is possible that this can occur without MTP opening (Halestrap, 2005). Mitochondrial depolarisation and increased ROS are associated with opening of the MTP (Zamzami and Kroemer, 2001). Mitochondrial membrane potential and ROS production are dependent on the flux of electrons down the electron transport chain (ETC), which in turn are dependent on the production of electron donors (NADH, FADH2) from the Krebs’ cycle. Suppressing the entry of pyruvate into the mitochondria and thus the production of acetyl-CoA, will suppress both Krebs’ cycle and the ETC and thus MTP opening and apoptosis.

 

 

Figure 2.

 

A glycolytic environment is associated with an antiapoptotic and pro-proliferative state, characterizing most solid tumours. Increase entry of pyruvate into the mitochondria by either DCA or inhibition of LDH, promotes glucose oxidation, increased apoptosis and decreased proliferation and tumour growth (see text for discussion).

Full figure and legend (148K)

 

 

 

Mitochondria can also affect downstream mechanisms involved in proliferation and apoptosis. For example, mitochondria uptake can directly regulate intracellular Ca++, the increase of which is associated with increased proliferation and activation of many transcription factors. Also, the mitochondria-produced superoxide can be dismutated to H2O2 through the manganese superoxide dismutase and diffuse freely, activating plasma membrane K+ channels, thereby regulating the influx of Ca++ and the activity of caspases. K+ channels are transmembrane proteins allowing the passage of K+ ions through the plasma membrane. Closing of K+ channels or decreasing their expression results in an increase in [K+]i which, in turn, increases the tonic inhibition that cytosolic K+ exerts on caspases (Remillard and Yuan, 2004). The voltage-gated family of K+ channels (Kv) is redox-sensitive and therefore can be regulated by the mitochondria. For example, mitochondria-derived H2O2 can activate certain Kv channels, like Kv1.5 (Bonnet et al, 2007).

 

Top of page

DCA reverses the mitochondrial remodeling, unlocking the cancer cells from a state of apoptosis resistance: preclinical work

A paradigm shift is needed in cancer therapeutics

The metabolism of cancer cells

Glycolysis offers an early adaptation to the hypoxic microenvironment in carcinogenesis

Glycolysis is associated with resistance to apoptosis

Mitochondria and apoptosis

DCA reverses the mitochondrial remodeling, unlocking the cancer cells from a state of apoptosis resistance: preclinical work

DCA: mechanism of action and clinical experience

DCA: clinical testing in cancer?

Note to proof

References

Figures and Tables

 

We recently showed that several cancer cell lines (non-small cell lung cancer, breast cancer and glioblastoma) had hyperpolarised mitochondria, compared with non-cancer cell lines (Bonnet et al, 2007), a finding that was first described by Dr Chen at the Dana Farber Institute in the 1980s (Chen, 1988). This was associated with suppressed levels of mitochondria-derived ROS and decreased activity and expression of Kv channels. The Ca++-sensitive transcription factor NFAT was also active (i.e., nuclear) in the cancer cells. NFAT is a transcription factor that has been shown to increase the levels of the antiapoptotic bcl-2 and decrease the levels of the Kv channel Kv1.5. All of these features are compatible with an antiapoptotic state and could be secondary to a suppressed mitochondrial activity: decrease entry of pyruvate would eventually result in decrease flux of electrons in the ETC and therefore decreased ROS production, closing of the existing redox-sensitive Kv channels and increased intracellular Ca++. The decreased ROS could also contribute to closure of the redox-sensitive MTP and mitochondrial hyperpolarisation. The decreased entry of pyruvate into the mitochondria (and therefore the decreased GO) would result in compensatory GLY. Increased hexokinase levels would contribute to the hyperpolarisation of the mitochondria; increased hexokinase in a glycolytic environment is known to be translocated to the mitochondrial membrane, inhibiting the voltage-dependent anion channel (a component of the MTP), resulting in hyperpolarisation and suppression of apoptosis (Pastorino et al, 2005) (Figure 2).

 

Dichloroacetate activated the pyruvate dehydrogenase, which resulted in increased delivery of pyruvate into the mitochondria. As predicted, DCA increased GO and depolarised the mitochondria, returning the membrane potential towards the levels of the non-cancer cells, without affecting the mitochondria of non-cancerous cells (Figure 2). Remarkably, all the above features of the cancer cells were ‘normalised’ following the increase in GO and the mitochondrial depolarisation: ROS increased, NFAT was inactivated and function/expression of Kv channels was increased. Most importantly, apoptosis was induced in the cancer cells with both cytochrome c and apoptosis-inducing factor efflux from the mitochondria. This resulted in a decrease in tumour growth both in vitro and in vivo in xenotransplant models (Bonnet et al, 2007) (Figure 3). In addition to the induction of apoptosis by DCA in non-small cell lung cancer, breast cancer and glioblastoma cell lines reported in our original publication (Bonnet et al, 2007), very recently DCA was shown to induce apoptosis in endometrial (Wong et al, 2008) and prostate (Cao et al, 2008) cancer cells by largely the same mechanism, independently confirming our results. Furthemore, as predicted, activating mitochondria by DCA increases O2 consumption in the tumour and dramatically enhances the effectiveness of hypoxia-specific chemotherapies in animal models (Cairns et al, 2007).

 

 

Figure 3.

 

Dichloroacetate depolarises mitochondria and suppresses tumour growth in vivo. On the left, non-small cell lung cancer cells are loaded with TMRM before and after treatment with DCA (the higher the red fluorescence the higher the mitochondrial membrane potential; nuclei in blue). The same cells were injected in the flank of nude rats. On the right these rats are imaged with a rodent PET-CT (GammaMedica). Simultaneous CT and FDG-Glucose PET imaging shows that DCA therapy decreases both the size and the glucose uptake in the tumour.

Full figure and legend (149K)

 

 

 

It is important here to clarify that simply inhibiting GLY, will not promote pyruvate entry into the mitochondria, that is, it will not re-activate mitochondria. It will also be toxic to several non-cancerous tissues that depend on GLY for energy production. Inhibiting GLY (which has previously been tested as a potential treatment for cancer) results in ATP depletion and necrosis, not apoptosis, because apoptosis is an energy-consuming process, requiring active mitochondria (Xu et al, 2005). The ‘trick’ is to enhance the GLY to GO coupling, not just inhibit GLY. One of the ways that this can happen is by activating PDH, or inhibiting LDH, bringing pyruvate into the mitochondria and enhancing GO (Figure 2). This hypothesis is also supported by the recently published work that inhibition of LDH (by siRNA), which promotes the transfer of pyruvate into the mitochondria (in that sense mimicking DCA), also promotes cancer apoptosis and decreases tumour growth in vitro and in mice xenotransplants (Fantin et al, 2006).

 

Top of page

DCA: mechanism of action and clinical experience

A paradigm shift is needed in cancer therapeutics

The metabolism of cancer cells

Glycolysis offers an early adaptation to the hypoxic microenvironment in carcinogenesis

Glycolysis is associated with resistance to apoptosis

Mitochondria and apoptosis

DCA reverses the mitochondrial remodeling, unlocking the cancer cells from a state of apoptosis resistance: preclinical work

DCA: mechanism of action and clinical experience

DCA: clinical testing in cancer?

Note to proof

References

Figures and Tables

 

Dichloroacetate is a small molecule of 150 Da (see structure in Figure 3) explaining in part the high bioavailability of this drug and the fact that it can penetrate into the traditional chemotherapy sanctuary sites, including the brain. In vitro, DCA activates PDH by inhibition of PDK at concentration of 10–250 μM or 0.15–37.5 μg ml−1 in a dose-dependent fashion (Stacpoole, 1989). To date, four different isoforms of PDK have been identified that have variable expression and sensitivity to the inhibition by DCA (Sugden and Holness, 2003). The isozyme constitutively expressed in most tissues and with the highest sensitivity to DCA is PDKII; in our published preclinical work we showed that PDK2 inhibition with siRNA completely mimicked DCA effects (Bonnet et al, 2007).

 

Oral DCA can achieve 100% bioavailability. Many studies using IV and oral DCA aimed to identify the optimal dose for DCA. The end point measured was the decrease in lactate levels in both the blood and the cerebrospinal fluid. A decrease in lactate levels is the immediate result of the inhibition of PDK (and thus activation of PDH) by DCA. Several studies treated patients with DCA and directly measured PDH activity in muscle biopsies. Dichloroacetate administered at 35–50 mg kg−1 decreases lactate levels by more than 60% and directly activates PDH by 3–6 fold (Howlett et al, 1999; Parolin et al, 2000).

 

Although the pharmacokinetics of DCA in healthy volunteers follow a simple one-compartment model, they are more complex in severely abnormal states like severe lactic acidosis or cirrhosis. Dichloroacetate inhibits its own metabolism by an unknown mechanism, and the clearance of DCA decreases after multiple doses (Stacpoole et al, 2003). Although the initial half-life with the first dose is less than one hour, this half-life increases to several hours with subsequent doses. However, there is a plateau of this effect and DCA serum levels do not continue to rise with chronic use. This is also true for DCA metabolites (which do not have any biologic effect, at least on PDH). For example, the serum DCA levels after 5 years of continued treatment with oral DCA at 25 mg kg−1 are only slightly increased compared with the levels after the first several doses (and remain in the range of approximately 100 μg ml−1) (Mori et al, 2004). The effects on lactate levels are sustained and persist after the DCA levels decrease, because the inhibition of PDK is not immediately reversible; DCA ‘locks’ PDK in a sustained inactive state.

 

A large number of children and adults have been exposed to DCA over the past 40 years, including healthy volunteers and subjects with diverse disease states. Since its first description in 1969 (Stacpoole, 1969), DCA has been studied to alleviate the symptoms or the haemodynamic consequences of the lactic acidosis complicating severe malaria, sepsis, congestive heart failure, burns, cirrhosis, liver transplantation and congenital mitochondrial diseases. Single-arm and randomised trials of DCA used doses ranging from 12.5 to 100 mg kg−1 day−1 orally or intravenously (reviewed in (Stacpoole et al, 2003)). Although DCA was universally effective in lowering lactate levels, it did not alter the course of the primary disease (for example sepsis).

 

More than 40 nonrandomised trials of DCA in small cohorts of patients have been reported, but the first two randomised control trials of chronic oral therapy with DCA in congenital mitochondrial diseases were reported in 2006. In the first, a blinded placebo-controlled study was performed with oral DCA administered at 25 mg kg−1 day−1 in 30 patients with MELAS syndrome (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes) (Kaufmann et al, 2006). Most patients enrolled in the DCA arm developed symptomatic peripheral neuropathy, compared with 4 out of 15 in the placebo arm, leading to the termination of the study. Seventeen out of 19 patients had at least partial resolution of peripheral neurological symptoms by 9 months after discontinuation of DCA. This neurotoxicity resembled the pattern of length-dependent, axonal, sensorimotor polyneuropathy without demyelination. No other toxicities were reported. It is important to note that peripheral neuropathy often complicates MELAS because of primary or secondary effects on peripheral nerves; for example these patients also have diabetes and diabetes-related peripheral neuropathy.

 

In contrast, another randomised placebo-controlled double-blinded study failed to show any significant toxicity of DCA, including peripheral neuropathy. In this study only one of 21 children with congenital lactic acidosis treated with DCA orally at 25 mg kg−1 day−1 for 6 months demonstrated mild peripheral neuropathy. Serial nerve conduction studies failed to demonstrate any difference in incidence of neuropathy in the 2 arms (placebo vs DCA). Sleepiness and lethargy, muscular rigidity of the upper extremity and hand tremor were reported in one patient in each group (Stacpoole et al, 2006).

 

The higher incidence of peripheral neuropathy in adult MELAS patients may represent an intrinsic predisposition to this complication in MELAS or its associated conditions, that is, diabetes mellitus; this toxicity might also be age-dependent. In summary, peripheral neuropathy is a potential side effect of DCA that appears to be largely reversible. As peripheral neuropathy is a frequent complication of taxane, platinum and vinca-alkaloid chemotherapies, the risk for DCA-associated peripheral neuropathy may depend on whether cancer patients have prior or concurrent neurotoxic therapy.

 

Top of page

DCA: clinical testing in cancer?

A paradigm shift is needed in cancer therapeutics

The metabolism of cancer cells

Glycolysis offers an early adaptation to the hypoxic microenvironment in carcinogenesis

Glycolysis is associated with resistance to apoptosis

Mitochondria and apoptosis

DCA reverses the mitochondrial remodeling, unlocking the cancer cells from a state of apoptosis resistance: preclinical work

DCA: mechanism of action and clinical experience

DCA: clinical testing in cancer?

Note to proof

References

Figures and Tables

 

There is substantial evidence in preclinical in vitro and in vivo models that DCA might be beneficial in human cancer (Bonnet et al, 2007; Cairns et al, 2007; Cao et al, 2008; Wong et al, 2008). The concept is strengthened by the fact that LDH inhibition in mice with human cancer xenotransplants, also induced apoptosis and inhibited growth, improving survival (Fantin et al, 2006). There is also 40 years of human experience with mechanistic studies of DCA in human tissues after oral use, pharmacokinetic and toxicity data from randomised studies for 6 months, and 5-year use case reports. This supports an easy translation to early-phase clinical trials.

 

Dichloroacetate could be tested in a variety of cancer types. The realisation that (i) a diverse group of signalling pathways and oncogenes result in resistance to apoptosis and a glycolytic phenotype, (ii) the majority of carcinomas have hyperpolarised/remodeled mitochondria, and (iii) most solid tumours have increased glucose uptake on PET imaging, suggest that DCA might be effective in a large number of diverse tumours. However, direct preclinical evidence of anticancer effects of DCA has been published only with non-small cell lung cancer, glioblastoma and breast, endometrial and prostate cancer. In addition, the lack of mitochondrial hyperpolarisation in certain types of cancer, including oat cell lung cancer, lymphomas, neuroblastomas and sarcomas (Chen, 1988), suggest that DCA might not be effective in such cases. Cancers with limited or no meaningful therapeutic options like recurrent glioblastoma or advanced lung cancer should be on top of the list of cancers to be studied.

 

No patient with cancer has received DCA within a clinical trial. It is unknown whether previously studied dose ranges will achieve cytotoxic intra-tumoral concentrations of DCA. In addition, the overall nutritional and metabolic profile of patients with advanced cancer differs from those in the published DCA studies. Furthermore, pre-exposure to neurotoxic chemotherapy may predispose to DCA neurotoxicity. Carefully performed phase I dose escalation and phase II trials with serial tissue biopsies are required to define the maximally tolerated, and biologically active dose. Clinical trials with DCA will need to carefully monitor neurotoxicity and establish clear dose-reduction strategies to manage toxicities. Furthermore, the pharmacokinetics in the cancer population will need to be defined.

 

The preclinical experience with DCA monotherapy warrants clinical trials with DCA as a single agent or in direct comparison with other agents. However, as it ‘unlocks’ cancer cells from a state of apoptosis resistance, DCA might be an attractive ‘apoptosis-sensitizer’ agent. In that sense, DCA could both precede and be given concurrently with chemotherapy or radiation therapy, in an attempt to increase their effectiveness, decrease the required doses and limit the toxicity of standard therapies (Cairns et al, 2007).

 

The ability to approach metabolism as an integrator of many diverse signalling pathways, prompts consideration of the imaging and diagnostic studies that might track metabolic modulation. As discussed above, important questions that need to be answered in clinical trials using DCA include: (i) can PET be used as a predictor of clinical response or as a means of documenting non-invasively a reversal of the glycolytic phenotype in response to DCA? (ii) can mitochondrial membrane potential or the acute effects on DCA in fresh tumour biopsies, predict clinical response to DCA and facilitate patient selection?

 

Funding for such trials would be a challenge for the academic community as DCA is a generic drug and early industry support might be limited. Fundraising from philanthropies might be possible to support early phase I–II or small phase III trials. However, if these trials suggest a favourable efficacy and toxicity, the public will be further motivated to directly fund these efforts and national cancer organisations like the NCI, might be inspired to directly contribute to the design and structure of larger trials. It is important to note that even if DCA does not prove to be the ‘dawn of a new era’ (Pan and Mak, 2007), initiation and completion of clinical trials with a generic compound will be a task of tremendous symbolic and practical significance. At this point the ‘dogma’ that trials of systemic anticancer therapy cannot happen without industry support, suppresses the potential of many promising drugs that might not be financially attractive for pharmaceutical manufacturers. In that sense, the clinical evaluation of DCA, in addition to its scientific rationale, will be by itself another paradigm shift.

 

Top of page

Note to proof

A paradigm shift is needed in cancer therapeutics

The metabolism of cancer cells

Glycolysis offers an early adaptation to the hypoxic microenvironment in carcinogenesis

Glycolysis is associated with resistance to apoptosis

Mitochondria and apoptosis

DCA reverses the mitochondrial remodeling, unlocking the cancer cells from a state of apoptosis resistance: preclinical work

DCA: mechanism of action and clinical experience

DCA: clinical testing in cancer?

Note to proof

References

Figures and Tables

 

Since the acceptance of this review two important papers have confirmed the novel anticancer effects of DCA in prostate and endometrial cancers: Wong JY et al, Dichloroacetate induces apoptosis in endometrial cancer cells. Gynecol Oncol June 2008; 109(3): 394–402 and Gao et al, Dichloroacetate (DCA) sensitizes both wild-type and over expressing Bcl-2 prostate cancer cells in vitro to radiation. Prostate 1 August 2008; 68(11): 1223–1231.

 

Top of page

References

A paradigm shift is needed in cancer therapeutics

The metabolism of cancer cells

Glycolysis offers an early adaptation to the hypoxic microenvironment in carcinogenesis

Glycolysis is associated with resistance to apoptosis

Mitochondria and apoptosis

DCA reverses the mitochondrial remodeling, unlocking the cancer cells from a state of apoptosis resistance: preclinical work

DCA: mechanism of action and clinical experience

DCA: clinical testing in cancer?

Note to proof

References

Figures and Tables

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Cao W, Yacoub S, Shiverick KT, Namiki K, Sakai Y, Porvasnik S, Urbanek C, Rosser CJ (2008) Dichloroacetate (DCA) sensitizes both wild-type and over expressing Bcl-2 prostate cancer cells in vitro to radiation. Prostate 68: 1223–1231 | Article | PubMed | ChemPort |

Chen LB (1988) Mitochondrial membrane potential in living cells. Annu Rev Cell Biol 4: 155–181 | Article | PubMed | ISI | ChemPort |

Elstrom RL, Bauer DE, Buzzai M, Karnauskas R, Harris MH, Plas DR, Zhuang H, Cinalli RM, Alavi A, Rudin CM, Thompson CB (2004) Akt stimulates aerobic glycolysis in cancer cells. Cancer Res 64: 3892–3899 | Article | PubMed | ISI | ChemPort |

Fantin VR, St-Pierre J, Leder P (2006) Attenuation of LDH-A expression uncovers a link between glycolysis, mitochondrial physiology, and tumor maintenance. Cancer Cell 9: 425–434 | Article | PubMed | ChemPort |

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Gottlob K, Majewski N, Kennedy S, Kandel E, Robey RB, Hay N (2001) Inhibition of early apoptotic events by Akt/PKB is dependent on the first committed step of glycolysis and mitochondrial hexokinase. Genes Dev 15: 1406–1418 | Article | PubMed | ISI | ChemPort |

Halestrap A (2005) Biochemistry: a pore way to die. Nature 434: 578–579 | Article | PubMed | ChemPort |

Howlett RA, Heigenhauser GJ, Hultman E, Hollidge-Horvat MG, Spriet LL (1999) Effects of dichloroacetate infusion on human skeletal muscle metabolism at the onset of exercise. Am J Physiol 277: E18–E25 | PubMed | ChemPort |

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Screening for Prostate Cancer: U.S. Preventive Services Task Force Recommendation Statement

Screening for Prostate Cancer: U.S. Preventive Services Task Force Recommendation Statement

  1. 1.  Virginia A. Moyer, MD, MPH,
  2. 2.  on behalf of the U.S. Preventive Services Task Force*

+ Author Affiliations

1.   From theU.S.Preventive Services Task Force,Rockville,Maryland.

 

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Abstract

Description: Update of the 2008 U.S. Preventive Services Task Force (USPSTF) recommendation statement on screening for prostate cancer.

Methods: The USPSTF reviewed new evidence on the benefits and harms of prostate-specific antigen (PSA)–based screening for prostate cancer, as well as the benefits and harms of treatment of localized prostate cancer.

Recommendation: The USPSTF recommends against PSA-based screening for prostate cancer (grade D recommendation).

This recommendation applies to men in the generalU.S.population, regardless of age. This recommendation does not include the use of the PSA test for surveillance after diagnosis or treatment of prostate cancer; the use of the PSA test for this indication is outside the scope of the USPSTF.

The U.S. Preventive Services Task Force (USPSTF) makes recommendations about the effectiveness of specific clinical preventive services for patients without related signs or symptoms.

It bases its recommendations on the evidence of both the benefits and harms of the service, and an assessment of the balance. The USPSTF does not consider the costs of providing a service in this assessment.

The USPSTF recognizes that clinical decisions involve more considerations than evidence alone. Clinicians should understand the evidence but individualize decision making to the specific patient or situation. Similarly, the USPSTF notes that policy and coverage decisions involve considerations in addition to the evidence of clinical benefits and harms.

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Summary of Recommendation and Evidence

The USPSTF recommends against prostate-specific antigen (PSA)–based screening for prostate cancer (grade D recommendation).

See the Clinical Considerations section for a discussion about implementation of this recommendation.

See Figure 1 for a summary of the recommendation and suggestions for clinical practice. Table 1 describes the USPSTF grades, and Table 2 describes the USPSTF classification of levels of certainty about net benefit.

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Figure 1. Screening for Prostate Cancer: Clinical Summary ofU.S.Preventive Services Task Force Recommendation.

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Table 1. What the USPSTF Grades Mean and Suggestions for Practice

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Table 2. USPSTF Levels of Certainty Regarding Net Benefit

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Rationale

Importance

Prostate cancer is the most commonly diagnosed non–skin cancer in men in the United States, with a lifetime risk for diagnosis currently estimated at 15.9%. Most cases of prostate cancer have a good prognosis even without treatment, but some cases are aggressive; the lifetime risk for dying of prostate cancer is 2.8%. Prostate cancer is rare before age 50 years, and very few men die of prostate cancer before age 60 years. Seventy percent of deaths due to prostate cancer occur after age 75 years (1).

Detection

Contemporary recommendations for prostate cancer screening all incorporate the measurement of serum PSA levels; other methods of detection, such as digital rectal examination or ultrasonography, may be included. There is convincing evidence that PSA-based screening programs result in the detection of many cases of asymptomatic prostate cancer. There is also convincing evidence that a substantial percentage of men who have asymptomatic cancer detected by PSA screening have a tumor that either will not progress or will progress so slowly that it would have remained asymptomatic for the man’s lifetime. The terms “overdiagnosis” or “pseudo-disease” are used to describe both situations. The rate of overdiagnosis of prostate cancer increases as the number of men subjected to biopsy increases. The number of cancer cases that could be detected in a screened population is large; a single study in which men eligible for PSA screening had biopsy regardless of PSA level detected cancer in nearly 25% of men (2). The rate of overdiagnosis also depends on life expectancy at the time of diagnosis. A cancer diagnosis in men with shorter life expectancies because of chronic diseases or age is much more likely to be overdiagnosis. The precise magnitude of overdiagnosis associated with any screening and treatment program is difficult to determine, but estimates from the 2 largest trials suggest overdiagnosis rates of 17% to 50% for prostate cancer screening (3).

Benefits of Detection and Early Treatment

The primary goal of prostate cancer screening is to reduce deaths due to prostate cancer and, thus, increase length of life. An additional important outcome would be a reduction in the development of symptomatic metastatic disease. Reduction in prostate cancer mortality was the primary outcome used in available randomized, controlled trials of prostate cancer screening. Although 1 screening trial reported on the presence of metastatic disease at the time of prostate cancer diagnosis, no study reported on the effect of screening on the development of subsequent metastatic disease, making it difficult to assess the effect of lead-time bias on the reported rates.

Men with screen-detected cancer can potentially fall into 1 of 3 categories: those whose cancer will result in death despite early diagnosis and treatment, those who will have good outcomes in the absence of screening, and those for whom early diagnosis and treatment improves survival. Only randomized trials of screening allow an accurate estimate of the number of men who fall into the latter category. There is convincing evidence that the number of men who avoid dying of prostate cancer because of screening after 10 to 14 years is, at best, very small. Two major trials of PSA screening were considered by the USPSTF: the U.S. PLCO (Prostate, Lung, Colorectal, and Ovarian) Cancer Screening Trial and the ERSPC (European Randomized Study of Screening for Prostate Cancer). TheU.S.trial did not demonstrate any prostate cancer mortality reduction. The European trial found a reduction in prostate cancer deaths of approximately 1 death per 1000 men screened in a subgroup of men aged 55 to 69 years. This result was heavily influenced by the results of 2 countries; 5 of the 7 countries reporting results did not find a statistically significant reduction. All-cause mortality in the European trial was nearly identical in the screened and nonscreened groups.

There is adequate evidence that the benefit of PSA screening and early treatment ranges from 0 to 1 prostate cancer deaths avoided per 1000 men screened.

Harms of Detection and Early Treatment

Harms Related to Screening and Diagnostic Procedures

Convincing evidence demonstrates that the PSA test often produces false-positive results (approximately 80% of positive PSA test results are false-positive when cutoffs between 2.5 and 4.0 μg/L are used) (4). There is adequate evidence that false-positive PSA test results are associated with negative psychological effects, including persistent worry about prostate cancer. Men who have a false-positive test result are more likely to have additional testing, including 1 or more biopsies, in the following year than those who have a negative test result (5). Over 10 years, approximately 15% to 20% of men will have a PSA test result that triggers a biopsy, depending on the PSA threshold and testing interval used (4). New evidence from a randomized trial of treatment of screen-detected cancer indicates that roughly one third of men who have prostate biopsy experience pain, fever, bleeding, infection, transient urinary difficulties, or other issues requiring clinician follow-up that the men consider a “moderate or major problem”; approximately 1% require hospitalization (6).

The USPSTF considered the magnitude of these harms associated with screening and diagnostic procedures to be at least small.

Harms Related to Treatment of Screen-Detected Cancer

Adequate evidence shows that nearly 90% of men with PSA-detected prostate cancer in the United Stateshave early treatment with surgery, radiation, or androgen deprivation therapy (7, 8). Adequate evidence shows that up to 5 in 1000 men will die within 1 month of prostate cancer surgery and between 10 and 70 men will have serious complications but survive. Radiotherapy and surgery result in long-term adverse effects, including urinary incontinence and erectile dysfunction in at least 200 to 300 of 1000 men treated with these therapies. Radiotherapy is also associated with bowel dysfunction (9, 10).

Some clinicians have used androgen deprivation therapy as primary therapy for early-stage prostate cancer, particularly in older men, although this is not a U.S. Food and Drug Administration (FDA)–approved indication and it has not been shown to improve survival in localized prostate cancer. Adequate evidence shows that androgen deprivation therapy for localized prostate cancer is associated with erectile dysfunction (in approximately 400 of 1000 men treated), as well as gynecomastia and hot flashes (9, 10).

There is convincing evidence that PSA-based screening leads to substantial overdiagnosis of prostate tumors. The amount of overdiagnosis of prostate cancer is of important concern because a man with cancer that would remain asymptomatic for the remainder of his life cannot benefit from screening or treatment. There is a high propensity for physicians and patients to elect to treat most cases of screen-detected cancer, given our current inability to distinguish tumors that will remain indolent from those destined to be lethal (7, 11). Thus, many men are being subjected to the harms of treatment of prostate cancer that will never become symptomatic. Even for men whose screen-detected cancer would otherwise have been later identified without screening, most experience the same outcome and are, therefore, subjected to the harms of treatment for a much longer period of time (12, 13). There is convincing evidence that PSA-based screening for prostate cancer results in considerable overtreatment and its associated harms.

The USPSTF considered the magnitude of these treatment-associated harms to be at least moderate.

USPSTF Assessment

Although the precise, long-term effect of PSA screening on prostate cancer–specific mortality remains uncertain, existing studies adequately demonstrate that the reduction in prostate cancer mortality after 10 to 14 years is, at most, very small, even for men in what seems to be the optimal age range of 55 to 69 years. There is no apparent reduction in all-cause mortality. In contrast, the harms associated with the diagnosis and treatment of screen-detected cancer are common, occur early, often persist, and include a small but real risk for premature death. Many more men in a screened population will experience the harms of screening and treatment of screen-detected disease than will experience the benefit. The inevitability of overdiagnosis and overtreatment of prostate cancer as a result of screening means that many men will experience the adverse effects of diagnosis and treatment of a disease that would have remained asymptomatic throughout their lives. Assessing the balance of benefits and harms requires weighing a moderate to high probability of early and persistent harm from treatment against the very low probability of preventing a death from prostate cancer in the long term.

The USPSTF concludes that there is moderate certainty that the benefits of PSA-based screening for prostate cancer do not outweigh the harms.

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Clinical Considerations

Implementation

Although the USPSTF discourages the use of screening tests for which the benefits do not outweigh the harms in the target population, it recognizes the common use of PSA screening in practice today and understands that some men will continue to request screening and some physicians will continue to offer it. The decision to initiate or continue PSA screening should reflect an explicit understanding of the possible benefits and harms and respect the patients’ preferences. Physicians should not offer or order PSA screening unless they are prepared to engage in shared decision making that enables an informed choice by the patients. Similarly, patients requesting PSA screening should be provided with the opportunity to make informed choices to be screened that reflect their values about specific benefits and harms. Community- and employer-based screening should be discontinued. Table 3 presents reasonable estimates of the likely outcomes of screening, given the current approach to screening and treatment of screen-detected prostate cancer in theUnited States.

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Table 3. PSA-Based Screening for Prostate Cancer*

The treatment of some cases of clinically localized prostate cancer can change the natural history of the disease and may reduce morbidity and mortality in a small percentage of men, although the prognosis for clinically localized cancer is generally good regardless of the method of detection, even in the absence of treatment. The primary goal of PSA-based screening is to find men for whom treatment would reduce morbidity and mortality. Studies demonstrate that the number of men who experience this benefit is, at most, very small, and PSA-based screening as currently implemented in the United Statesproduces more harms than benefits in the screened population. It is not known whether an alternative approach to screening and management of screen-detected disease could achieve the same or greater benefits while reducing the harms. Focusing screening on men at increased risk for prostate cancer mortality may improve the balance of benefits and harms, but existing studies do not allow conclusions about a greater absolute or relative benefit from screening in these populations. Lengthening the interval between screening tests may reduce harms without affecting cancer mortality; the only screening trial that demonstrated a prostate cancer–specific mortality benefit generally used a 2- to 4-year screening interval (15). Other potential ways to reduce diagnostic- and treatment-related harms include increasing the PSA threshold used to trigger the decision for biopsy or need for treatment (12, 16), or reducing the number of men having active treatment at the time of diagnosis through watchful waiting or active surveillance (11). Periodic digital rectal examinations could also be an alternative strategy worthy of further study. In the only randomized trial demonstrating a mortality reduction from radical prostatectomy for clinically localized cancer, a high percentage of men had palpable cancer (17). All of these approaches require additional research to better elucidate their merits and pitfalls and more clearly define an approach to the diagnosis and management of prostate cancer that optimizes the benefits while minimizing the harms.

Patient Population Under Consideration

This recommendation applies to men in the general U.S.population. Older age is the strongest risk factor for the development of prostate cancer. However, neither screening nor treatment trials show benefit in men older than 70 years. Across age ranges, black men and men with a family history of prostate cancer have an increased risk of developing and dying of prostate cancer. Black men are approximately twice as likely to die of prostate cancer than other men in the United States(1), and the reason for this disparity is unknown. Black men represented a small minority of participants in the randomized clinical trials of screening (4% of enrolled men in the PLCO trial were non-Hispanic black; although the ERSPC and other trials did not report the specific racial demographic characteristics of participants, they likely were predominately white). Thus, no firm conclusions can be made about the balance of benefits and harms of PSA-based screening in this population. However, it is problematic to selectively recommend PSA-based screening for black men in the absence of data that support a more favorable balance of risks and benefits. A higher incidence of cancer will result in more diagnoses and treatments, but the increase may not be accompanied by a larger absolute reduction in mortality. Preliminary results from PIVOT (Prostate Cancer Intervention Versus Observation Trial), in which 30% of enrollees were black, have become available since the publication of the USPSTF’s commissioned evidence reviews. Investigators found no difference in outcomes due to treatment of prostate cancer in black men compared with white men (12).

Exposure to Agent Orange (a defoliant used in the Vietnam War) is considered to be a risk factor for prostate cancer, although few data exist on the outcomes or effect of PSA testing and treatment in these persons. Prostate cancer inVietnamveterans who were exposed to Agent Orange is considered a service-connected condition by the Veterans Health Administration.

The USPSTF did not evaluate the use of the PSA test as part of a diagnostic strategy in men with symptoms potentially suggestive of prostate cancer. However, the presence of urinary symptoms was not an inclusion or exclusion criterion in screening or treatment trials, and approximately one quarter of men in screening trials had bothersome lower urinary tract symptoms (nocturia, urgency, frequency, and poor stream). The presence of benign prostatic hyperplasia is not an established risk factor for prostate cancer, and the risk for prostate cancer among men with elevated PSA levels is lower in men with urinary symptoms than in men without symptoms (18).

This recommendation also does not include the use of the PSA test for surveillance after diagnosis or treatment of prostate cancer and does not consider PSA-based testing in men with known BRCA gene mutations who may be at increased risk for prostate cancer.

Screening Tests

Prostate-specific antigen–based screening in men aged 50 to 74 years has been evaluated in 5 unique randomized, controlled trials of single or interval PSA testing with various PSA cutoffs and screening intervals, along with other screening methods, such as digital rectal examination or transrectal ultrasonography (4, 19–22). Screening tests or programs that do not incorporate PSA testing, including digital rectal examination alone, have not been adequately evaluated in controlled studies.

The PLCO trial found a nonstatistically significant increase in prostate cancer mortality in the annual screening group at 11.5 and 13 years, with results consistently favoring the usual care group (19, 23).

A prespecified subgroup analysis of men aged 55 to 69 years in the ERSPC trial demonstrated a prostate cancer mortality rate ratio (RR) of 0.80 (95% CI, 0.65 to 0.98) in screened men after a median follow-up of 9 years, with similar findings at 11 years (RR, 0.79 [CI, 0.68 to 0.91]) (4, 15). Of the 7 centers included in the ERSPC analysis, only 2 countries (Sweden and the Netherlands) reported statistically significant reductions in prostate cancer mortality after 11 years (5 did not), and these results seem to drive the overall benefit found in this trial (Figure 2) (15). No study reported any factors, including patient age, adherence to site or study protocol, length of follow-up, PSA thresholds, or intervals between tests, that could clearly explain why mortality reductions were larger inSweden or theNetherlands than in other European countries or theUnited States (PLCO trial). Combining the results through meta-analysis may be inappropriate due to clinical and methodological differences across trials.

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Figure 2. Relative risk of prostate cancer death for men screened with PSA versus control participants, by country.

ERSPC = European Randomized Study of Screening for Prostate Cancer; PLCO =U.S.Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial.

No study found a difference in overall or all-cause mortality. This probably reflects the high rates of competing mortality in this age group, because these men are more likely to die of prostate cancer, as well as the limited power of prostate cancer screening trials to detect differences in all-cause mortality, should they exist. Even in the “core” age group of 55 to 69 years in the ERSPC trial, only 462 of 17 256 deaths were due to prostate cancer. The all-cause mortality RR was 1.00 (CI, 0.98 to 1.02) in all men randomly assigned to screening versus no screening. Results were similar in men aged 55 to 69 years (15). The absence of any trend toward a reduction in all-cause mortality is particularly important in the context of the difficulty of attributing death to a specific cause in this age group.

Treatment

Primary management strategies for PSA-detected prostate cancer include watchful waiting (observation and physical examination with palliative treatment of symptoms), active surveillance (periodic monitoring with PSA tests, physical examinations, and repeated prostate biopsy) with conversion to potentially curative treatment at the sign of disease progression or worsening prognosis, and surgery or radiation therapy (24). There is no consensus about the optimal treatment of localized disease. From 1986 through 2005, PSA-based screening likely resulted in approximately 1 million additional U.S. men being treated with surgery, radiation therapy, or both compared with the time before the test was introduced (7).

At the time of the USPSTF’s commissioned evidence review, only 1 recent randomized, controlled trial of surgical treatment versus observation for clinically localized prostate cancer was available (13). In the Scandinavian Prostate Cancer Group Study 4 trial, surgical management of localized, primarily clinically detected prostate cancer was associated with an approximate 6% absolute reduction in prostate cancer and all-cause mortality at 12 to 15 years of follow-up; benefit seemed to be limited to men younger than 65 years (13). Subsequently, preliminary results were reported from another randomized trial that compared external beam radiotherapy (EBRT) with watchful waiting in 214 men with localized prostate cancer detected before initiation of PSA screening. At 20 years, survival did not differ between men randomly assigned to watchful waiting or EBRT (31% vs. 35%; P = 0.26). Prostate cancer mortality at 15 years was high in each group but did not differ between groups (23% vs. 19%; P = 0.51). External beam radiotherapy did reduce distant progression and recurrence-free survival (25). In men with localized prostate cancer detected in the early PSA screening era, preliminary findings from PIVOT show that, after 12 years, intention to treat with radical prostatectomy did not reduce disease-specific or all-cause mortality compared with observation; absolute differences were less than 3% and not statistically different (12). An ongoing trial in the United Kingdom (ProtecT [Prostate Testing for Cancer and Treatment]) comparing radical prostatectomy with EBRT or active surveillance has enrolled nearly 2000 men with PSA-detected prostate cancer. Results are expected in 2015 (26).

Up to 0.5% of men will die within 30 days of having radical prostatectomy, and 3% to 7% will have serious surgical complications. Compared with men who choose watchful waiting, an additional 20% to 30% or more of men treated with radical prostatectomy will experience erectile dysfunction, urinary incontinence, or both after 1 to 10 years. Radiation therapy is also associated with increases in erectile, bowel, and bladder dysfunction (9, 10).

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Other Considerations

Research Needs and Gaps

Because the balance of benefits and harms of prostate cancer screening is heavily influenced by overdiagnosis and overtreatment, research is needed to identify ways to reduce the occurrence of these events, including evaluating the effect of altering PSA thresholds for an abnormal test or biopsy result on false-positive rates and the detection of indolent disease.

Similarly, research is urgently needed to identify new screening methods that can distinguish nonprogressive or slowly progressive disease from disease that is likely to affect the quality or length of life, because this would reduce the number of men who require biopsy and subsequent treatment of disease that has a favorable prognosis without intervention. Additional research is also needed to evaluate the benefits and harms of modifications of the use of existing prostate cancer screening tools. Research is needed to assess the effect of using higher PSA thresholds to trigger a diagnostic prostate biopsy, extending intervals between testing, and the role of periodic digital rectal examinations by trained clinicians. Although not well-studied, these strategies may reduce overdiagnosis and overtreatment, and evidence suggests that they may be associated with decreased mortality. Research is also needed to compare the long-term benefits and harms of immediate treatment versus observation with delayed intervention or active surveillance in men with screen-detected prostate cancer. Two randomized, controlled trials, PIVOT (27) and the ProtecT trial (28), are studying this issue. Preliminary results from PIVOT potentially support increasing the PSA threshold for recommending a biopsy or curative treatments in men subsequently diagnosed with prostate cancer.

Additional research is needed to determine whether the balance of benefits and harms of prostate cancer screening differs in men at higher risk of developing or dying of prostate cancer, including black men and those with a family history of the disease.

Accurately ascertaining cause of death in older persons can be problematic; as such, basing clinical recommendations on disease-specific mortality in the absence of an effect on all-cause mortality may not completely capture the health effect and goals of a screening and treatment program. Additional research is required to better assess and improve the reliability of prostate cancer mortality as a valid outcome measure in clinical trials, as well as the best application of the concomitant use of all-cause mortality.

Two large randomized, controlled trials of 5α-reductase inhibitors (finasteride and dutasteride) have shown that these drugs reduce the risk for prostate cancer in men receiving regular PSA tests. However, the observed reduction resulted from a decreased incidence of low-grade prostate cancer alone (Gleason score ≤6).The FDA has not approved finasteride and dutasteride for the prevention of prostate cancer, concluding that the drugs do not possess a favorable risk–benefit profile for this indication. The FDA cited associated adverse effects, including loss of libido and erectile dysfunction, but most important, it noted that there was an absolute increase in the incidence of high-grade prostate cancer in men randomly assigned to finasteride or dutasteride compared with control participants in both trials (29). Additional research would be useful to better understand whether these drugs are associated with the development of high-grade prostatic lesions, determine the effect of 5α-reductase inhibitors (or other potential preventive agents) on prostate cancer mortality, and identify the population of men that may benefit most from prostate cancer prevention (with these or other chemoprevention strategies).

Research is needed to better understand patient and provider knowledge and values about the known risks and benefits of prostate cancer screening and treatment, as well as to develop and implement effective informed decision-making materials that accurately convey the best evidence and can be instituted in primary care settings across varied patient groups (for example, by race, age, or family history).

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Response to Public Comments

A draft version of this recommendation statement was posted for public comment on the USPSTF Web site from 11 October to 13 December 2011. Commenters expressed concern that a grade D recommendation from the USPSTF would preclude the opportunity for discussion between men and their personal health care providers, interfere with the clinician–patient relationship, and prevent men from being able to make their own decisions about whether to be screened for prostate cancer. Some commenters asked that the USPSTF change its recommendation to a grade C to allow men to continue to make informed decisions about screening. Recommendations from the USPSTF are chosen on the basis of the risk–benefit ratio of the intervention: A grade D recommendation means that the USPSTF has concluded that there is at least moderate certainty that the harms of doing the intervention equal or outweigh the benefits in the target population, whereas a grade C recommendation means that the USPSTF has concluded that there is at least moderate certainty that the overall net benefit of the service is small. The USPSTF could not assign a grade C recommendation for PSA screening because it did not conclude that the benefits outweigh the harms. In the Implementation section, the USPSTF has clarified that a D recommendation does not preclude discussions between clinicians and patients to promote informed decision making that supports personal values and preferences.

Some commenters requested that the USPSTF provide more information about the consequences of avoiding PSA screening. A summary of the benefits and harms of screening can be found in Table 3. In summary, the USPSTF concluded that choosing not to have PSA testing will result in a patient living a similar length of life, with little to no difference in prostate cancer–specific mortality, while avoiding harms associated with PSA testing and subsequent diagnostic procedures and treatments.

Commenters were concerned that the USPSTF did not adequately consider a separate recommendation for black men. Additional information about this population can be found in the Patient Population Under Consideration section.

Many commenters mistakenly believed that the USPSTF either relied solely on the PLCO trial or published meta-analyses or did its own meta-analysis to reach its conclusions about the efficacy of PSA-based screening. Although the commissioned systematic evidence review summarized the findings of 2 previously published meta-analyses, because they met the minimum inclusion requirements for the report, neither the authors of that review nor the USPSTF did a new meta-analysis. The USPSTF is aware of the heterogeneity in the available randomized trials of prostate cancer screening and the limitations of meta-analysis in this situation. Both the ERSPC and PLCO trials were heavily weighted by the USPSTF in its considerations, because they had the largest populations and were of the highest quality, although both had important—but different—methodological limitations. The screening intervals, PSA thresholds, use of digital rectal examinations, enrollee characteristics, and follow-up diagnostic and treatment strategies used in the PLCO trial are most applicable to currentU.S.settings and practice patterns.

Commenters asked the USPSTF to consider evidence from the National Cancer Institute’s Surveillance, Epidemiology, and End Results (SEER) database, showing that prostate cancer mortality decreased by 40% in the United States between 1992 and 2007 (30). Many suggested that the decline must be attributable to the effect of screening, because PSA-based screening was introduced in theUnited States in the early 1990s and became widespread by the mid-1990s to late 1990s. The challenge of ecologic data is that it is impossible to reliably separate out the relative effects of any changes in screening, diagnosis, or treatment practices (or fundamental changes in the underlying risk of developing or dying of the disease in the population due to a multiplicity of other causes) that may have been occurring simultaneously over a given time period. All of these, including screening, may have played some role in the decline seen in mortality; however, only a randomized trial can determine with confidence the magnitude of effect that can be attributable to a given intervention. According to the SEER database, in the 1970s and 1980s, before the introduction of widespread PSA screening, prostate cancer mortality rates started at 29.9 cases per 100 000 men and showed a slow but constant increase over time. The reason for this increase is unknown. Mortality from prostate cancer peaked between 1991 and 1993—roughly the same time when PSA tests became a common clinical practice—at 39.3 cases per 100 000 men, and began to decline by approximately 1 to 2 cases per 100 000 men per year after this point (2007 rate, 24.0 cases per 100 000 men). Information from randomized trials suggests that any potential mortality benefit from screening will not occur for 7 to 10 years. As such, it would be very unlikely that any decline in mortality rates from 1990 to 2000 would be related to screening.

Some commenters believed that the USPSTF should have considered a reduction in morbidity due to prostate cancer as an outcome, not just mortality. The rate of metastatic disease should roughly parallel the rate of deaths; if a large difference in metastatic disease was present between the intervention and control groups of the ERSPC and PLCO trials at 11 and 13 years of follow-up, a larger effect on the reduction in mortality would have been expected. Although the USPSTF agrees that a demonstrated effect of PSA-based screening on long-term quality of life or functional status would be an important outcome to consider, insufficient data are available from screening trials to draw such a conclusion. The ERSPC trial provides information about the incidence of metastatic disease only at the time of diagnosis, rather than longitudinal follow-up for the development of such disease in screened versus unscreened populations. Data on quality of life are available from randomized treatment trials of early-stage prostate cancer and suggest that treatment with observation or watchful waiting provides similar long-term quality of life as early intervention, with marked reduction in treatment-related adverse effects (31, 32).

Many commenters asked the USPSTF to review a publication reporting that the efficacy of PSA-based screening in the PLCO trial was affected by comorbidity status (33); they believed that this provided evidence that PSA-based screening could be recommended for very healthy men. In the article, Crawford and colleagues (33) reported that the hazard ratio for death in men without comorbid conditions in the annual screening versus the usual care group was 0.56 (CI, 0.33 to 0.95). However, the PLCO investigators later reported, as part of their extended follow-up of the trial, that this finding was sensitive to the definition of comorbidity used (23). Crawford and colleagues chose an expanded definition of comorbidity that included both “standard” Charlson comorbidity index conditions and hypertension (even if it was well-controlled), diverticulosis, gallbladder disease, and obesity. When the analysis was repeated by using only validated measures of comorbidity (that is, Charlson comorbidity index conditions only), an interaction was no longer seen. Several researchers (including PLCO investigators) have questioned the biological plausibility of this finding by Crawford and colleagues, noting, among other reasons, that the positive interaction seems to be largely driven by the inclusion of hypertension and obesity, conditions that seem to convey minimal excess treatment risks or differences in treatment options. These researchers also note that although Crawford and colleagues initially argue that comorbid conditions lessen the effectiveness of treatment (thus, causing screening to be ineffective in less healthy men), participants in the usual care group with a greater degree of comorbidity actually had a statistically significant lower risk of dying of prostate cancer than healthier men (23, 34). Preliminary results from PIVOT also found that the effect of radical prostatectomy compared with observation did not vary by comorbidity or health status (12).

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Discussion

Burden of Disease

An estimated 240 890 U.S.men received a prostate cancer diagnosis in 2011, and an estimated 33 720 men died of the disease (35). The average age of diagnosis was 67 years and the median age of those who died of prostate cancer from 2003 through 2007 was 80 years; 71% of deaths occurred in men older than 75 years (1). Black men have a substantially higher prostate cancer incidence rate than white men (232 vs. 146 cases per 100 000 men) and more than twice the prostate cancer mortality rate (56 vs. 24 deaths per 100 000 men, respectively) (35).

Prostate cancer is a clinically heterogeneous disease. Autopsy studies have shown that approximately one third of men aged 40 to 60 years have histologically evident prostate cancer (36); the proportion increases to as high as three fourths in men older than 85 years (37). Most cases represent microscopic, well-differentiated lesions that are unlikely to be of clinical importance. Increased frequency of PSA testing, a lower threshold for biopsy, and an increase in the number of core biopsies obtained all increase the detection of lesions that are unlikely to be of clinical significance.

Scope of Review

The previous evidence update, done for the USPSTF in 2008, found insufficient evidence that screening for prostate cancer improved health outcomes, including prostate cancer–specific and all-cause mortality, for men younger than 75 years. In men aged 75 years or older, the USPSTF found adequate evidence that the incremental benefits of treatment of screen-detected prostate cancer are small to none and that the harms of screening and treatment outweigh any potential benefits (38). After the publication of initial mortality results from 2 large randomized, controlled trials of prostate cancer screening, the USPSTF determined that a targeted update of the direct evidence on the benefits of PSA-based screening for prostate cancer should be done (39). In addition, the USPSTF requested a separate systematic review of the benefits and harms of treatment of localized prostate cancer (10). Since the release of the USPSTF’s draft recommendation statement on prostate cancer screening and its supporting systematic evidence reviews, updated results from the ERSPC and PLCO trials and data on harms related to prostate biopsy from the ProtecT trial have become available; these publications were used to inform this final recommendation statement.

Accuracy of Screening

The conventional PSA cutoff of 4.0 μg/L detects many cases of prostate cancer; however, some cases will be missed. Using a lower cutoff detects more cases of cancer, but at the cost of labeling more men as potentially having cancer. For example, decreasing the PSA cutoff to 2.5 μg/L would more than double the number of U.S. men aged 40 to 69 years with abnormal results (16), and most of these would be false-positive results. It also increases the likelihood of detection of indolent tumors with no clinical importance. Conversely, increasing the PSA cutoff to greater than 10.0 μg/L would reduce the number of men aged 50 to 69 years with abnormal results from approximately 1.2 million to roughly 352 000 (16). There is no PSA cutoff at which a man can be guaranteed to be free from prostate cancer (40).

There are inherent problems with the use of needle biopsy results as a reference standard to assess the accuracy of prostate cancer screening tests. Biopsy detection rates vary according to the number of biopsies done during a single procedure; the more biopsies done, the more cancer cases detected. More cancer cases detected with a “saturation” biopsy procedure (≥20 core biopsies) tend to increase the apparent specificity of an elevated PSA level; however, many of the additional cancer cases detected this way are unlikely to be clinically important. Thus, the accuracy of the PSA test for detecting clinically important prostate cancer cases cannot be determined with precision.

Variations of PSA screening, including the use of age-adjusted PSA cutoffs, free PSA, and PSA density, velocity, slope, and doubling time, have been proposed to improve detection of clinically important prostate cancer cases. However, no evidence has demonstrated that any of these testing strategies improve health outcomes, and some may even generate harms. One study found that using PSA velocity in the absence of other indications could lead to 1 in 7 men having a biopsy with no increase in predictive accuracy (41).

Effectiveness of Early Detection and Treatment

Two poor-quality (high risk of bias) randomized, controlled trials initiated in the 1980s in Sweden each demonstrated a nonstatistically significant trend toward increased prostate cancer mortality in groups invited to screening (21, 22). A third poor-quality (high risk of bias) trial from Canada showed similar results when an intention-to-screen analysis was used (20). The screening protocols for these trials varied; all included 1 or more PSA tests with cutoffs ranging from 3.0 to 10.0 μg/L; in addition, digital rectal examination and transrectal ultrasonography were variably used.

The more recently published PLCO and ERSPC trials were the principal trials considered by the USPSTF. The fair-quality prostate component of the PLCO trial randomly assigned 76 685 men aged 55 to 74 years to annual PSA screening for 6 years (and concomitant digital rectal examination for 4 years) or to usual care. It used a PSA cutoff of 4.0 μg/L. Diagnostic follow-up for positive screening test results and treatment choices were made by the participant and his personal physician; 90% of men with prostate cancer diagnoses received active treatment (surgery, radiation, hormonal therapy, or some combination). After 7 years (complete follow-up), a nonstatistically significant trend toward increased prostate cancer mortality was seen in the screened group (RR, 1.14 [CI, 0.75 to 1.70]) compared with men in the control group (19). Similar findings were seen after 13 years (RR, 1.09 [CI, 0.87 to 1.36]) (23). The primary criticism of this study relates to the high contamination rate; approximately 50% of men in the control group received at least 1 PSA test during the study, although the investigators increased both the number of screening intervals and the duration of follow-up to attempt to compensate for the contamination effects. In addition, approximately 40% of participants had received a PSA test in the 3 years before enrollment, although subgroup analyses stratified by history of PSA testing before study entry did not reveal differential effects on prostate cancer mortality rates (19). Contamination may attenuate differences in the 2 groups but would not explain both an increased prostate cancer incidence and mortality rate in men assigned to screening.

The fair-quality ERSPC trial randomly assigned 182 160 men aged 50 to 74 years from 7 European countries to PSA testing every 2 to 7 years or to usual care. Prostate-specific antigen cutoffs ranged from 2.5 to 4.0 μg/L, depending on study center (1 center used a cutoff of 10.0 μg/L for several years). Subsequent diagnostic procedures and treatment also varied by center. Sixty six percent of men who received a prostate cancer diagnosis chose immediate treatment (surgery, radiation therapy, hormonal therapy, or some combination). Among all men who were randomly assigned, there was a borderline reduction in prostate cancer mortality in the screened group after a median follow-up of 9 years (RR, 0.85 [CI, 0.73 to 1.00]) (4). Similar results were reported after 11 years of follow-up and were statistically significant (RR, 0.83 [CI, 0.72 to 0.94]) (15). After a median follow-up of 9 years in a prespecified subgroup analysis limited to men aged 55 to 69 years, a statistically significant reduction in prostate cancer deaths was seen in the screened group (RR, 0.80 [CI, 0.65 to 0.98]) (4). After 11 years of follow-up, a similar reduction was seen (RR, 0.79 [CI, 0.45 to 0.85]); the authors estimated that 1055 men needed to be invited to screening and 37 cases of prostate cancer needed to be detected to avoid 1 death from prostate cancer (15). Of the 7 individual centers included in the mortality analysis, 2 (Sweden and the Netherlands) demonstrated statistically significant reductions in prostate cancer deaths with PSA screening. The magnitude of effect was considerably greater in these 2 centers than in other countries (Figure 2). Primary criticisms of this study relate to inconsistencies in age requirements, screening intervals, PSA thresholds, and enrollment procedures used among the study centers, as well as the exclusion of data from 2 study centers in the analysis. There is also concern that differential treatments between the study and control groups may have had an effect on outcomes. Of note, men in the screened group were more likely than men in the control group to have been treated in a university setting, and a control participant with high-risk prostate cancer was more likely than a screened participant to receive radiotherapy, expectant management, or hormonal therapy instead of radical prostatectomy (42). Furthermore, ascertainment of cause of death in men with a diagnosis of prostate cancer included men whose prostate cancer was detected at autopsy. How this cause-of-death adjudication process may affect estimates is unknown, but previous research has demonstrated difficulties in accurately ascertaining cause of death and that small errors could have an important effect on results (43, 44).

After publication of the initial ERSPC mortality results, a single center from within that trial (Göteburg, Sweden) reported data separately. Outcomes for 60% of this center’s participants were reported as part of the full ERSPC publication, and the subsequent country-specific results within the ERSPC trial reflect the separately reported results from Sweden (which included some men not included in the overall ERSPC trial) (45).

Few randomized, controlled trials have compared treatments for localized prostate cancer with watchful waiting. A randomized, controlled trial of 695 men with localized prostate cancer (Scandinavian Prostate Cancer Group Study 4) reported an absolute reduction in the risk for distant metastases (11.7% [CI, 4.8% to 18.6%]) in patients assigned to radical prostatectomy versus watchful waiting after 15 years of follow-up. An absolute reduction in prostate cancer mortality (6.1% [CI, 0.2% to 12.0%]) and a trend toward a reduction in all-cause mortality (6.6% [CI, −1.3% to 14.5%]) were also seen over this period. Subgroup analysis suggests that the benefits of prostatectomy were limited to men aged 65 years or younger. The applicability of these findings to cancer detected by PSA-based screening is limited, because only 5% of participants were diagnosed with prostate cancer through some form of screening, 88% had palpable tumors, and more than 40% presented with symptoms (13, 17). An earlier, poor-quality study found no mortality reduction from radical prostatectomy versus watchful waiting after 23 years of follow-up (46). Another randomized trial of 214 men with localized prostate cancer detected before initiation of PSA screening that compared EBRT versus watchful waiting presented preliminary mortality results after completion of the evidence review. At 20 years, the observed survival did not differ between men randomly assigned to watchful waiting and EBRT (31% vs. 35%; P = 0.26). Prostate cancer mortality at 15 years was high in each group but did not differ between groups (23% vs. 19%; P = 0.51). External beam radiotherapy did reduce distant progression and recurrence-free survival (25).

Preliminary results from PIVOT have also become available since the evidence review was completed. PIVOT, conducted in the United States, included men with prostate cancer detected after the initiation of widespread PSA testing and, thus, included a much higher percentage of men with screen-detected prostate cancer. The trial randomly assigned 731 men aged 75 years or younger (mean age, 67 years) with a PSA level less than 50 μg/L (mean, 10 μg/L) and clinically localized prostate cancer to radical prostatectomy versus watchful waiting. One third of participants were black. On the basis of PSA level, Gleason score, and tumor stage, approximately 43% had low-risk tumors, 36% had intermediate-risk tumors, and 21% had high-risk tumors. After a median follow-up of 10 years, prostate cancer–specific or all-cause mortality did not statistically significantly differ between men treated with surgery versus observation (absolute risk reduction, 2.7% [CI, −1.3% to 6.2%] and 2.9% [CI, −4.1% to 10.3%], respectively). Subgroup analysis found that the effect of radical prostatectomy compared with observation for both overall and prostate cancer–specific mortality did not vary by patient characteristics (including age, race, health status, Charlson comorbidity index score, or Gleason score), but there was variation by PSA level and possibly tumor risk category. In men in the radical prostatectomy group with a PSA level greater than 10 μg/L at diagnosis, there was an absolute risk reduction of 7.2% (CI, 0.0% to 14.8%) and 13.2% (CI, 0.9% to 24.9%) for prostate cancer–specific and all-cause mortality, respectively, compared with men in the watchful waiting group. However, prostate cancer–specific or all-cause mortality was not reduced among men in the radical prostatectomy group with PSA levels of 10 μg/L or less or those with low-risk tumors, and potential (nonstatistically significant) increased mortality was suggested when compared with the watchful waiting group (12).

Harms of Screening and Treatment

False-positive PSA test results are common and vary depending on the PSA cutoff and frequency of screening. After 4 PSA tests, men in the screening group of the PLCO trial had a 12.9% cumulative risk of receiving at least 1 false-positive result (defined as a PSA level greater than 4.0 μg/L and no prostate cancer diagnosis after 3 years) and a 5.5% risk of having at least 1 biopsy due to a false-positive result (47). Men with false-positive PSA test results are more likely than control participants to worry specifically about prostate cancer, have a higher perceived risk for prostate cancer, and report problems with sexual function for up to 1 year after testing (48). In 1 study of men with false-positive PSA test results, 26% reported that they had experienced moderate to severe pain during the biopsy; men with false-positive results were also more likely to have repeated PSA testing and additional biopsies during the 12 months after the initial negative biopsy (49). False-negative results also occur, and there is no PSA level that effectively rules out prostate cancer. This has, in part, led to recommendations for doing prostate biopsy at lower PSA thresholds than previously used in randomized screening trials (for example, <2.5 μg/L).

Harms of prostate biopsy reported by the Rotterdamcenter of the ERSPC trial include persistent hematospermia (50.4%), hematuria (22.6%), fever (3.5%), urinary retention (0.4%), and hospitalization for signs of prostatitis or urosepsis (0.5%) (50). The ProtecT study, an ongoing randomized, controlled trial evaluating the effectiveness and acceptability of treatments for men with PSA-detected, localized prostate cancer, found that 32% of men experienced pain; fever; blood in the urine, semen, or stool; infection; transient urinary difficulties; or other issues requiring clinician follow-up after prostate biopsy that they considered a “moderate or major problem.” At 7 days after biopsy, 20% of men reported that they would consider a future biopsy a “moderate or major problem” and 1.4% of men were hospitalized for complications (6). Similar findings were reported at 30 days after biopsy in a U.S. study of older, predominately white male Medicare beneficiaries (51).

The high likelihood of false-positive results from the PSA test, coupled with its inability to distinguish indolent from aggressive tumors, means that a substantial number of men undergo biopsy and are overdiagnosed with and overtreated for prostate cancer. The number of men who have biopsies is directly related to the number of men having PSA testing, the threshold PSA level used to trigger a biopsy, and the interval between PSA tests. Estimates derived from the ERSPC and PLCO trials suggest overdiagnosis rates of 17% to 50% of prostate cancer cases detected by the PSA test (3, 52, 53). Overdiagnosis is of particular concern because, although these men cannot benefit from any associated treatment, they are still subject to the harms of a given therapy. Evidence indicates that nearly 90% of U.S. men diagnosed with clinically localized prostate cancer through PSA testing have early treatment (primarily radical prostatectomy and radiation therapy) (7, 8).

Radical prostatectomy is associated with a 20% increased absolute risk for urinary incontinence and a 30% increased absolute risk for erectile dysfunction compared with watchful waiting (that is, increased 20% above a median rate of 6% and 30% above a median rate of 45%, respectively) after 1 to 10 years (9, 10). Perioperative deaths or cardiovascular events occur in approximately 0.5% or 0.6% to 3% of patients, respectively (9, 10). Comparative data on outcomes using different surgical techniques are limited; 1 population-based observational cohort study using the SEER database and Medicare-linked data found that minimally invasive or robotic radical prostatectomy for prostate cancer was associated with higher risks for genitourinary complications, incontinence, and erectile dysfunction than open radical prostatectomy (54).

Radiation therapy is associated with a 17% absolute increase in risk for erectile dysfunction (that is, increased 17% above a median rate of 50%) and an increased risk for bowel dysfunction (for example, fecal urgency or incontinence) compared with watchful waiting after 1 to 10 years; the effect on bowel function is most pronounced in the first few months after treatment (9, 10).

Localized prostate cancer is not an FDA-approved indication for androgen deprivation therapy, and clinical outcomes for older men receiving this treatment for localized disease are worse than for those who are conservatively managed (55). Androgen deprivation therapy is associated with an increased risk for impotence compared with watchful waiting (absolute risk difference, 43%), as well as systemic effects, such as hot flashes and gynecomastia (9, 10). In advanced prostate cancer, androgen deprivation therapy may generate other serious harms, including diabetes, myocardial infarction, or coronary heart disease; however, these effects have not been well-studied in men treated for localized prostate cancer. A recent meta-analysis of 8 randomized, controlled trials in men with nonmetastatic high-risk prostate cancer found that androgen deprivation therapy was not associated with increased cardiac mortality (56).

Estimate of Magnitude of Net Benefit

All but 1 randomized trial has failed to demonstrate a reduction in prostate cancer deaths with the use of the PSA test, and several—including the PLCO trial—have suggested an increased risk in screened men, potentially due to harms associated with overdiagnosis and overtreatment. In a prespecified subgroup of men aged 55 to 69 years in the ERSPC trial, a small (0.09%) absolute reduction in prostate cancer deaths was seen after a median follow-up of 11 years. The time until any potential cancer-specific mortality benefit (should it exist) for PSA-based screening emerges is long (at least 9 to 10 years), and most men with prostate cancer die of causes other than prostate cancer (57). No prostate cancer screening study or randomized trial of treatment of screen-detected cancer has demonstrated a reduction in all-cause mortality through 14 years of follow-up.

The harms of PSA-based screening for prostate cancer include a high rate of false-positive results and accompanying negative psychological effects, high rate of complications associated with diagnostic biopsy, and—most important—a risk for overdiagnosis coupled with overtreatment. Depending on the method used, treatments for prostate cancer carry the risk for death, cardiovascular events, urinary incontinence, erectile dysfunction, and bowel dysfunction. Many of these harms are common and persistent. Given the high propensity for physicians and patients to elect to treat screen-detected cancer, limiting estimates of the harms of PSA testing to the harms of the blood test alone, without considering other diagnostic and treatment harms, does not reflect current clinical practice in theUnited States.

The mortality benefits of PSA-based prostate cancer screening through 11 years are, at best, small and potentially none, and the harms are moderate to substantial. Therefore, the USPSTF concludes with moderate certainty that the benefits of PSA-based screening for prostate cancer, as currently used and studied in randomized, controlled trials, do not outweigh the harms.

How Does Evidence Fit With Biological Understanding?

Prostate-specific antigen–based screening and subsequent treatment, as currently practiced in the United States, presupposes that most asymptomatic prostate cancer cases will ultimately become clinically important and lead to poor health outcomes and that early treatment effectively reduces prostate cancer–specific and overall mortality. However, long-term, population-based cohort studies and randomized treatment trials of conservatively managed men with localized prostate cancer do not support this hypothesis. A review of the Connecticut Tumor Registry, which was initiated before the PSA screening era, examined the long-term probability of prostate cancer death among men (median age at diagnosis, 69 years) whose tumors were mostly incidentally identified at the time of transurethral resection or open surgery for benign prostatic hyperplasia. Men received observation alone or early or delayed androgen deprivation therapy. After 15 years of follow-up, the prostate cancer mortality rate was 18 deaths per 1000 person-years. For men with well-differentiated prostate cancer, it was 6 deaths per 1000 person-years; far more of these men had died of causes other than prostate cancer (75% vs. 7%) (58). An analysis of the SEER database after the widespread introduction of PSA-based screening examined the risk for death in men with localized prostate cancer who did not have initial attempted curative therapy. The 10-year prostate cancer mortality rate for well- or moderately differentiated tumors among men aged 66 to 69 years at diagnosis was 0% to 7%, depending on tumor stage, versus 0% to 22% for other causes. The relative proportion of deaths attributable to other causes compared with prostate cancer increased substantially with age at prostate cancer diagnosis (59). In the only randomized, controlled trial comparing early intervention versus watchful waiting that included men primarily detected by PSA testing, prostate cancer mortality at 12 years or more was infrequent (7%) and did not differ between men randomly assigned to surgery versus observation (12).

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Update of Previous USPSTF Recommendation

This recommendation replaces the 2008 recommendation (38). Whereas the USPSTF previously recommended against PSA-based screening for prostate cancer in men aged 75 years or older and concluded that the evidence was insufficient to make a recommendation for younger men, the USPSTF now recommends against PSA-based screening for prostate cancer in all age groups.

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Recommendations of Others

The American Urological Association recommends that PSA screening, in conjunction with a digital rectal examination, should be offered to asymptomatic men aged 40 years or older who wish to be screened, if estimated life expectancy is greater than 10 years (60). It is currently updating this guideline (61). The American Cancer Society emphasizes informed decision making for prostate cancer screening: Men at average risk should receive information beginning at age 50 years, and black men or men with a family history of prostate cancer should receive information at age 45 years (62). The American College of Preventive Medicine recommends that clinicians discuss the potential benefits and harms of PSA screening with men aged 50 years or older, consider their patients’ preferences, and individualize screening decisions (63). TheAmericanAcademy of Family Physicians is in the process of updating its guideline, and theAmericanCollege of Physicians is currently developing a guidance statement on this topic.

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Appendix 1: U.S. Preventive Services Task Force

Members of the U.S. Preventive Services Task Force at the time this recommendation was finalized† are Virginia A. Moyer, MD, MPH, Chair (Baylor College of Medicine, Houston, Texas); Michael L. LeFevre, MD, MSPH, Co-Vice Chair (University of Missouri School of Medicine, Columbia, Missouri); Albert L. Siu, MD, MSPH, Co-Vice Chair (Mount Sinai School of Medicine, New York, New York, and James J. Peters Veterans Affairs Medical Center, Bronx, New York); Linda Ciofu Baumann, PhD, RN (University of Wisconsin, Madison, Wisconsin); Kirsten Bibbins-Domingo, PhD, MD (University of California, San Francisco, San Francisco, California); Susan J. Curry, PhD (University of Iowa College of Public Health, Iowa City, Iowa); Mark Ebell, MD, MS (University of Georgia, Athens, Georgia); Glenn Flores, MD (University of Texas Southwestern, Dallas, Texas); Adelita Gonzales Cantu, RN, PhD (University of Texas Health Science Center, San Antonio, Texas); David C. Grossman, MD, MPH (Group Health Cooperative, Seattle, Washington); Jessica Herzstein, MD, MPH (Air Products, Allentown, Pennsylvania); Joy Melnikow, MD, MPH (University of California, Davis, Sacramento, California); Wanda K. Nicholson, MD, MPH, MBA (University of North Carolina School of Medicine, Chapel Hill, North Carolina); Douglas K. Owens, MD, MS (Stanford University, Stanford, California); Carolina Reyes, MD, MPH (Virginia Hospital Center, Arlington, Virginia); and Timothy J. Wilt, MD, MPH (University of Minnesota and Minneapolis Veterans Affairs Medical Center, Minneapolis, Minnesota). Former USPSTF members who contributed to the development of this recommendation include Ned Calonge, MD, MPH, andRosanne Leipzig,MD, PhD.

† For a list of current Task Force members, visit www.uspreventiveservicestaskforce.org/members.htm.

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Appendix 2: Assumptions and References Informing Table 3

Estimates of the number of prostate cancer deaths in screened and unscreened men are taken from the 11- and 13-year follow-up studies of the PLCO (23) and ERSPC (15) trials. False-positive rates for PSA tests are derived from the PLCO trial and the Finnish center of the ERSPC trial (47, 64). Information related to the harms of biopsy is derived from the work of Rosario and colleagues (6). The incidence of prostate cancer in a screened population is derived from the incidence seen in the screened group of the PLCO trial (23). Treatment rates for localized prostate cancer in the U.S. population are derived from the SEER program and the Cancer of the Prostate Strategic Urologic Research Endeavor registry (9, 10). Expected complication rates from prostatectomy and radiation therapy are derived from pooled estimates calculated in the evidence review done for the USPSTF (10).

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Article and Author Information

Disclaimer: Recommendations made by the USPSTF are independent of theU.S. government. They should not be construed as an official position of the Agency for Healthcare Research and Quality or the U.S. Department of Health and Human Services.

Financial Support: The USPSTF is an independent, voluntary body. The U.S. Congress mandates that the Agency for Healthcare Research and Quality support the operations of the USPSTF.

Potential Conflicts of Interest: Dr. Moyer: Support for travel to meetings for the study or other purposes: Agency for Healthcare Research and Quality; Consultancy: American Academy of Pediatrics. Disclosure forms from USPSTF members can be viewed at www.acponline.org/authors/icmje/ConflictOfInterestForms.do?msNum=M12-1086.

Requests for Single Reprints: Reprints are available from the USPSTF Web site (www.uspreventiveservicestaskforce.org).

* For a list of the members of the USPSTF, see Appendix 1.

This article was published at www.annals.org on 22 May 2012.

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