The Prostate 75:1187–1196 (2015)
Metformin Represses Androgen-Dependent and Androgen-Independent Prostate Cancers by Targeting Androgen Receptor Yan Wang,1 Gaolei Liu,1 Dali Tong,1 Henna Parmar,2 Donald Hasenmayer,2 Wenqiang Yuan,1 Dianzheng Zhang,2 and Jun Jiang1* 1
Department of Urology, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing, PR China 2 Department of Bio-Medical Sciences, Philadelphia College of osteopathic Medicine, Philadelphia, Pennsylvania
BACKGROUND. Metformin has been reported to inhibit the growth of different types of cancers, including prostate cancer. We were interested to understand if the effect of metformin on prostate cancer is AR-dependent and, if so, whether metformin could act synergistically with the other anti-AR agents to serve as a therapeutic regimen with high efficacy and low toxicity. METHODS. Cell viabilities and apoptosis were determined by MTT assay and annexin V-FITC staining, respectively, when the two human prostate cancer cell lines, the androgen-dependent LNCaP and the androgen-independent 22RV1 were treated with metformin alone or in combination with bicalutamide. Quantitative RT-PCR and western blotting assays were conducted to examine metformin effects on AR mRNA and protein levels, respectively. Chromatin immunoprecipitation (ChIP) assays were conducted to confirm the recruitment of AR to the ARE(s) located on the promoter region of the AR target gene PSA. RESULTS. Metformin treatment reduced cell viability and enhanced apoptosis for both cell lines and additive effects were observed when LNCaP cells were treated with combined metformin and bicalutamide. Metformin down-regulated full-length AR protein in LNCaP cells. Both full-length and the truncated AR (AR-v7) were down-regulated by metformin in CWR22Rv1 cells. In both LNCaP and CWR22Rv1 cells, metformin repressed AR signaling pathway not by affecting AR protein degradation/stability, but rather through down-regulating the levels of AR mRNAs. CONCLUSIONS. Metformin represses prostate cancer cell viability and enhances apoptosis by targeting the AR signaling pathway. Combinations of metformin and other anti-AR agents pose a potentially promising therapeutic approach for treatment of prostate cancers, especially the castrate-resistant prostate cancer, with high efficacy and low toxicity. Prostate 75:1187–1196, 2015. # 2015 Wiley Periodicals, Inc. KEY WORDS:
prostate cancer; androgen receptor; metformin; castration resistance
Grant sponsor: National Natural Science Foundation of China; Grant number: 81402120; Grant sponsor: Chongqing Science and Technology Commission; Grant number: 2011GZ0047. Conflict of interest: The authors declare that they have no conflict of interest.
Correspondence to: Jun Jiang, Department of Urology, Institute of Surgery Research, Daping Hospital, Third Military Medical University. No. 10 Changjiangzhilu, Yuzhong District, Chongqing 400042, PR China. E-mail address: [email protected] Received 19 February 2015; Accepted 17 March 2015 DOI 10.1002/pros.23000 Published online 20 April 2015 in Wiley Online Library (wileyonlinelibrary.com).
ß 2015 Wiley Periodicals, Inc.
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Prostate cancer is one of the biggest threats to men’s health in the western world and it accounts for the second largest number of male cancer-related deaths in the United States. Currently, androgen deprivation therapy (ADT) is the gold standard treatment for advanced prostate cancer. ADT temporarily reduces prostate cancer progression with concomitant decrease of serum prostate-specific antigen (PSA). Unfortunately, almost all patients treated with ADT will relapse into castration-resistant prostate cancer (CRPC), a disease status characterized by rising PSA levels and tumor cell recurrence, within a period of 6 months–3 years [1]. However, CRPC is not completely refractory to further hormonal manipulation as the androgen receptor (AR) continues to play an important role in CRPC progression [2,3]. It has been demonstrated that complex mechanisms including AR gene amplification/overexpression, intra-tumoral androgen synthesis, overexpression of AR coactivators, aberrant kinase pathway activation, AR mutation, and expression of constitutively active AR splice variants exist in prostate cancers [4]. Therefore, AR is an attractive therapeutic target for different prostate cancers including CRPC. Metformin is one of the most well-established drugs for the treatment of type II diabetes. In addition to its capacity to treat diabetes, data from epidemiological studies suggest that metformin also possesses remarkable antitumor properties and is capable of exerting these effects on a wide spectrum of cancers including; breast cancer, ovarian cancer, colon cancer, and prostate cancer. When compared to non-metformin users, metformin users were associated with 9% lower risk of prostate cancer; 18% reduction in the biochemical recurrence (BCR); and reduced cancer-specific mortality as well as overall survival [5]. Recent publications have shown that metformin can even reduce the serum PSA levels in CRPC patients resistant to both Abiraterone and Enzalutamide [6]. Furthermore, accumulative evidence suggests that the use of metformin alone or in combination with bicalutamide can inhibit cell proliferation and induce apoptosis in prostate cancer cells [7,8]. Although metformin is currently undergoing clinical trials aimed at the treatment of prostate cancer, the molecular mechanisms underlying these beneficial functions remain unclear. This gap in our understanding continues to prevent further application of metformin in prostate cancer therapy. Given the accumulating data regarding the anticancer effect of metformin and the important roles played by AR in CRPC, we set to dissect the effects The Prostate
of metformin on AR activity. In this study, we demonstrated that metformin reduces AR transcriptional activity by down-regulating both full-length AR and the truncated AR-V7 proteins in both the androgen-dependent and the androgen-independent prostate cancer cells. Mechanistically, metformin does not function by affecting AR protein stability/ degradation or nuclear translocation, but by downregulating total AR mRNA levels. Here, combinatorial treatment of low dose metformin in conjunction with bicalutamide supplementation is shown to additively repress the growth of prostate cancer cells. These findings not only reveal a novel molecular mechanism of metformin’s effects on prostate cancer, but also imply that co-treatment of low concentrations metformin in concert with other anti-AR agents is an attractive and effective approach for prostate cancer therapy with high efficiency and low toxicity. MATERIALS AND METHODS Cell Culture LNCaP and CWR22Rv1 human prostate cancer cells were purchased from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. Both cell lines were cultured in RPMI 1640 (Hyclone, Logan, UT) supplemented with 10% FBS (Invitrogen, Carlsbad, CA) at 37°C in incubators with humidified air and 5% carbon dioxide. For DHT induction, the cells were cultured in phenol red-free RPMI supplemented with 10% charcoal stripped FBS (Invitrogen) and 10 nM DHT (Sigma). For the analysis of the mRNA levels, LNCaP cells were seeded in 6-well plates 2105 cells/well, treated with 10 nM DHT and different concentrations (0 mM, 1 mM, 5 mM, 10 mM and 20 mM) of metformin (Sigma). For the measurement of the protein levels, both LNCaP and CWR22Rv1 cells were seeded at 4105 cells/well in 6-well plates, treated with different concentrations (0 mM, 1 mM, 5 mM, 10 mM and 20 mM) of metformin with or without 10 nM DHT, respectively. Quantitative Real-Time PCR Total RNA was isolated using RNA extraction kit (Bioteke Inc, Beijing, China). Reverse transcriptase PCR was performed using the 1st Stand cDNA Synthesis (OriGene, Rockville, MD). PCRs were conducted using the qSTAR SYBR Master Mix (OriGene). Primers for full-length AR were 5’-GACGACCAGATGGCTGTCATT-3’ (forward) and 5’-GGGCGAAGTAGAGCATCCT-3’ (reverse). Primers for AR-v7 were 5’-
Metformin, AR and Prostate Cancer CCATCTTGTCGTCTTCGGAAATGTTA-3’ (forward), and 5’-TTTGAATGAGGCAAGTCAGCCTTTCT-3’ (reverse). Primers for PSA were 5’-GTGTGTGGACCTCCATGTTATT-3’ (forward) and 5’-CCACTCACCTTTCCCCTCAAG-3’ (reverse). Primers for Nkx3.1 were 5’-CCCACACTCAGGTGATCGAG-3’ (forward) and 5’GAGCTGCTTTCGCTTAGTCTT-3’ (reverse). Primers for the internal control, GAPDH, were 5’-AAGGTGAAGGTCGGAGTCAAC-3’ (forward) and 5’-GGGGTCATTGATGGCAACAATA-3’ (reverse).
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Cell Viability Assay Cells were seeded at a 0.5104 density per well in 96-well plates and exposed to metformin (2 mM) and bicalutamide (5 mM) for 7 days, 20 ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) dye (5 mg/ml, Sigma) was added to each well then kept at 37°C for 4 hr. After the media was removed, the resulting formazan crystals were solubilized in 200 ml dimethyl sulfoxide (DMSO). The density of the solubilized formazan was read at 490 nm spectrophotometrically (Bio-Rad, Hercules, CA).
Western Blot Cells were lysed in a RIPA buffer containing a protease inhibitor cocktail tablet and phosphotase inhibitor cocktail (KeyGEN BioTECH, NanJing, China). Protein levels were determined by BCA assay (Thermo–Fisher Scientific). 15 mg of protein were separated by SDS–PAGE and transferred onto PVDF membrane (PALL, NY). After 1 hr of incubation in blocking solution (5% BSA in PBS), the membranes were incubated with the appropriate antibodies: AR (Epitomics, CA), Bcl-2 (Epitomics), Cyclin D1 (Fantibody, China) and b-actin (Cell Signaling, MA).
Protein Stability Assay For the protein stability assays, CWR22Rv1 cells were pre-treated with 20 mg/ml cycloheximide (CHX, Sigma) for 30 min to stop protein synthesis followed by the treatment of 10 mM metformin for different time periods. For proteasome pathway, CWR22Rv1 cells were treated with metformin (10 mM), proteasome inhibitor MG132 (2 mM), or metformin (10 mM) þ MG132 (2 mM) for either 24 hr or 48 hr. For caspase-3 pathway, 22RV1 cells were pretreated with caspase-3 inhibitor z-VAD (20 mM) for 2 hr, followed by metformin (10 mM) for 48 hr. The protein levels were estimated by western blots as described above.
Chromatin Immunoprecipitation (ChIP) Assay LNCaP cells were treated with 1% formaldehyde/ RPMI for 10 min at room temperature. Nuclear extracts were prepared and sonicated on ice to generate DNA fragments less than 500 bp. For immunoprecipitation, the AR antibody (Epitomics) was added to DNA–protein complexes and incubated overnight at 4°C with rotation, and chromatin–antibody complexes were incubated with protein A/G agarose beads for overnight at 4°C with rotation. The bound cross-linked DNA–protein complexes were reversed by Proteinase K (Invitrogen) digestion at 65°C for 2 hr and DNA was purified and used as a template for PCR. Primer sequences for amplification of the PSA enhancer region were 5’-GTGTGTGGACCTCCATGTTATT-3’ (forward) and 5’-CCACTCACCTTTCCCCTCAAG-3’ (reverse). Human RPL30 exon3 was amplified with primers #7014 purchased from Cell Signal Technology (Danvers, MA) and served as positive control. Statistical Analysis All the experiments were analyzed using GraphPad Prism 5.0. All data were presented as means standard deviation (SD). P < 0.05 was considered statistically significant. RESULTS
Apoptosis Assay
Metformin Inhibits Prostate Cancer Cell Growth and Induces Cell Apoptosis
CWR22Rv1 Cells were seeded at a density of 2105 cells/well in six-well plates, and treated with metformin (10 mM) for 48 hr. The cells were then washed with cold PBS for three times, resuspended in Annexin V binding buffer and incubated with Annexin V–FITC and PI (KeyGEN BioTECH) for 10 min at 37°C in the dark and analyzed by a FACScan (BD Biosciences, NJ).
In order to determine if metformin exerts any effects on prostate cancer viability, we treated both the hormone-dependent prostate cancer cell line, LNCaP, and the hormone-independent prostate cancer cell line, CWR22v1, with vehicle, 2 mM metformin, 5 mM bicalutamide, or a combinational treatment of metformin and bicalutamide for 7 days in the presence or absence of 10 nM DHT. The Prostate
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The respective cell viabilities were determined by MTT assays. As shown in Figure 1A, LNCaP cell viability was reduced to a similar degree after either treatment with 2 mM metformin or 5 mM bicalutamide when compared to the control. Additionally, a additive effect was observed when metformin and bicalutamide were administered simultaneously. In contrast, the viability of the CWR22Rv1 cells was not affected upon treatment with bicalutamide alone (Figure 1B) but was significantly reduced by metformin. Furthermore, the additive effect produced by metformin and bicalutamide on LNCaP viability was not observed in the CWR22v1 cells. These results indicate that metformin exerts its effect on the prostate cancer
cells though a different mechanism than that of the general anti-androgen agents. In order to determine if metformin represses prostate cancer cell viability by enhancing cancer cell apoptosis, we analyzed the cell cycle status of the CWR22Rv1 cells as well as the percentage of apoptotic cells by flow cytometry. As shown in Figure 1C, metformin treatment increased both early and late apoptotic populations with an overall fourfold increase in apoptosis compared to the control. These observations were further substantiated by the findings that treatment with metformin alone or in combination with bicalutamide down-regulated both Bcl-2 and cyclin D1, suggesting that metformin induced prostate cancer cell
Fig. 1. Metformin inhibits prostate cancer cell growth and induces apoptosis. (A) LNCaP cells were seeded into a 96-well plate, cultured with CS-FBS and treated with 10 nM DHT. Metformin (2 mM), bicalutamide (5 mM), and metforminþ bicalutamide were added for 1, 3, 5, or 7 days. (B) CWR22Rv1 cells were seeded into a 96-well plate, treated with metformin (2 mM), bicalutamide (5 mM), and metforminþ bicalutamide for 1, 3, 5, or 7 days. Cell viability was determined by MTT assay. (C) CWR22Rv1 cells were treated with metformin (10 mM) for 48 hr and Annexin V-FITC assays and PI staining were conducted. Early apoptotic cells (Q3) and late apoptotic cells (Q2). (D) CWR22Rv1 cells were treated metformin (10 mM), bicalutamide (10 mM), and metforminþ bicalutamide for 48 hr. The protein levels of Bcl-2 and Cyclin D1 were analyzed by western blot. *: P < 0.05; **: P < 0.01. The Prostate
Metformin, AR and Prostate Cancer G1/S arrest and apoptotic death. Bicalutamide treatment alone, however, did not affect Bcl-2 or cyclin D1 (Figure 1D). These data collectively demonstrated that metformin reduces the cell viability of both androgen-dependent and androgenindependent prostate cancer cells and promotes cell cycle arrest and induces apoptosis in androgenindependent prostate cancer cells. Metformin Inhibits AR Transcriptional Activity and Reduces AR/ARE Interaction AR plays important roles in both androgendependent and androgen-independent cancers. In order to determine if metformin exerts its effect on prostate cancer through AR, we first examined the mRNA levels of two well-established AR target genes, PSA, and Nkx3.1 in LNCaP cells. As shown in Figure 2A, DHT treatment induced the levels of both mRNAs more than two times comparing to the basal levels. When LNCaP were treated with DHT and increasing concentrations of metformin (0–20 mM) for 24 hr, the mRNA levels of PSA and NKX3.1 were reduced approximately 45–80% and 35–75%, respec-
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tively. Similarly, when the androgen-independent CWR22v1 cells were treated with the increasing metformin concentrations, both PSA and Nkc3.1 were observed to be down regulated (Figure 2B). These results imply that metformin not only counteracts DHT-induced AR activity, but also the basal level AR activity in a dose-dependent manner. To further elucidate the mechanism of metformin-induced AR inhibition, ChIP assays were performed to assess metformin’s effect on AR recruitment to the PSA promoter region. As shown in Figure 2C, AR association with the PSA promotor was significantly increased when cells were treated with DHT compared to the control, and this hormone-induced AR recruitment was significantly reduced when the cells were treated with metformin. Assays for the negative control using the nonspecific antibody IgG did not precipitate any detectable amount of DNA; and assays for the positive control showed equal amounts of AR were recruited to the Human RPL30 exon3 region across treatment groups. Collectively, these results demonstrated that metformin is capable of inhibiting AR transcriptional activity by reducing AR recruitment to the promoter of its target gene.
Fig. 2. Metformin inhibits AR transcriptional activity. (A) LNCaP cells were cultured with CS-FBS and treated with 10 nM DHT and different concentrations of metformin for 24 hr. (B) CWR22Rv1 cells were treated with different concentrations of metformin for 24 hr. Total RNA was isolated and used as a template for qRT-PCR to estimate the mRNA levels of PSA and NKX3.1. (C) LNCaP cells were cultured with CS-FBS and treated with 10 nM DHT (H) alone or in combination with 10 mM metformin (HM) overnight. Whole cell lysates were subjected to CHIP assays with antibody against AR. Purified DNA was used for PCR amplification of the enhancer region of the PSA promoter. *: P < 0.05; **: P < 0.01. The Prostate
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Metformin Down-Regulates AR Protein Levels Since metformin treatment inhibited both AR recruitment and AR transcriptional activity, we then decided to examine whether metformin affects overall AR expression. First, western blot assays were conducted in LNCaP cells to compare total AR protein levels before and after metformin treatment. As expected that hormone treatment enhanced overall AR protein levels. Metformin treatment, however, was able to counteract the hormone-mediated AR induction and down-regulated AR protein levels dose-dependently (Figure 3A, upper panel). Next, the cells were treated with 10 mM metformin for increasing duration. Western blot analyses showed that metformin down-regulated total AR protein levels in a time-dependent manner (Figure 3 A, bottom panel). Here, androgen-induced AR expression was significantly reduced after 24 hr of 10 mM metformin treatment. In addition to the full length protein, AR exists as a multitude of smaller splice variants. Recent
studies have suggested that these AR splice variants, especially ARV7, could be one of the most prominent factors influencing the development of CRPC. As CWR22Rv1 are known to express both AR and ARV7 [9], we decided to assess the effects of metformin on both the full-length AR and the truncated AR-V7. Lysates prepared from CWR22Rv1 cells treated with increasing concentrations of metformin for increasing time periods were analyzed by western blot assays with the antibody specifically against the N-terminus of AR. Similar to what was observed with full length AR in LNCaP cells, Figure 3B shows that metformin treatment down-regulated both full length and truncated AR (AR-v7) in a dose- andtime-dependent manner in the CWR22Rv1 cells. Metformin Does not Affect AR Protein Stability nor Nuclear Translocation but AR mRNA Levels In order to determine if metformin-mediated AR down-regulation was the result of decreased AR
Fig. 3. Metformin down-regulating AR protein levels in LNCaP and CW22RV1 cells. (A) LNCaP cells were cultured with CS-FBS and treated with 10 nM DHT and different concentrations of metformin for 24 hr, or with 10 nM DHT þ 5 mM metformin and for different time periods. (B) CWR22Rv1 cells were treated with different concentrations (0 mM, 5 mM, 10 mM and 20 mM) of metformin for 12 hr, 24 hr, 36 hr and 48 hr. Cell lysates were analyzed with western blot assays with specific antibodies against N-terminus of AR and b-actin. The Prostate
Metformin, AR and Prostate Cancer stability, we conducted a series of experiments in the absence of de novo protein synthesis. First, we monitored AR protein levels subsequent to treatment with cycloheximide (CHX), an inhibitor of protein biosynthesis in eukaryotes. Results from the western blot assays (Figure 4A) showed that when the CWR22Rv1 cells were treated with CHX (50 mM) for 12 and 24 hr, the intensities of both the full-length AR and the truncated AR-V7 bands decreased significantly and in a time-dependent manner. Additionally, treating the cells with 10 mM metformin did not further reduce the intensities of either band. To further substantiate these findings, we treated the CWR22vRv1 cells with metformin (10 mM) in the presence and absence of the proteasome inhibitor
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MG132 (2 mM). As shown in Figure 4B, the addition of MG132 did not affect metformin-mediated AR down-regulation. These data collectively demonstrate that metformin is down-regulating AR protein levels through a mechanism other than enhancing protein degradation. Next, we examined the possibility that metformin affects AR nuclear translocation. LNCaP cells were cultured in growth media supplemented with CS-FBS and treated with 10 nM DHT and increasing concentrations of metformin for 24 hr. Whole cell lysates and nuclear and cytoplasmic extracts were prepared and analyzed by western blot. As shown in Figure 4C, metformin treatment counteracted hormone-mediated AR induction, but did not inhibit AR nuclear translocation. Finally, AR tran-
Fig. 4. Metformin inhibits AR activity not by enhancing AR degradation or nuclear translocation, but repressing AR mRNA levels. (A) CWR22Rv1 cells were pre-treated with CHX (50 mg/ml) for 30 min before addition of metformin (10 mM) and continuously treated for 0 hr, 12 hr and 24 hr. (B) CWR22Rv1 cells were treated with MG132 (2 mM) with or without metformin (10 mM) for 24 hr and 48 hr. (C) LNCaP cells were cultured with CS-FBS and treated with 10 nM R1881 and different concentrations of metformin for 24 hr. The proteins of whole cell lysate, cytoplasm and nucleus extracts were analyzed by western blot. (D) LNCaP cells were cultured with CS-FBS and treated with 10 nM DHT and different concentrations of metformin for 24 hr. (E) CWR22Rv1 cells were treated with different concentrations of metformin for 24 hr. Total RNA was isolated and used for qRT-PCR to estimate the mRNA levels of AR in LNCaP cells and AR and AR-V7 in CWR22Rv1, respectively. *: P < 0.05; **: P < 0.01. The Prostate
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script levels were assessed in order to determine if metformin-mediated AR down-regulation was occurring at the mRNA level. The results from the RT-PCR assays revealed that AR mRNA levels were significantly down-regulated when the LNCaP cells were treated with metformin (Figure 4D), and the mRNA levels of both full-length AR as well as the truncated AR-V7 in CWR22Rv1 cells were also down regulated by metformin in a dose-dependent manner (Figure 4E). These data collectively demonstrated that metformin represses AR protein levels and transcriptional activities not by affecting AR protein degradation or AR nuclear translocation, but by downregulating the levels of mRNA encoding the fulllength AR in the androgen-dependent cancer cells and both full-length AR and the truncated AR-V7 in the hormone-independent prostate cancer cells. DISCUSSION Metformin is commonly prescribed for the treatment of type II diabetes because it lowers the levels of both glucose and insulin in the bloodstream. Numerous preclinical, epidemiological and clinical studies suggest that metformin usage is associated with low or no risk [10]. More importantly for patients with prostate cancer, taking metformin usually has better prognoses because it inhibits the growth of prostate cancer cells [8]. Given the fact that metformin affects a wide spectrum of pathways, it is very likely that metformin exerts its anticancer effects through targeting of multiple signaling pathways. Unfortunately, wider application of metformin as a preventive and therapeutic tool for the treatment of prostate cancer has so far been prevented by our limited knowledge of the specific molecular mechanisms underlying its beneficial properties. In this study, we demonstrate for the first time that (i) metformin significantly inhibits AR transcriptional activity and down-regulates AR target gene expression; and that (ii) a combination of low-dose metformin in conjunction with bicalutamide supplementation inhibits prostate cancer growth and induce cancer cell apoptosis. Given the additive benefits observed for the combinatory treatments over short-, mid- and long-term periods, combinations of low-dose metformin and other anti-androgenic reagents could be become a plausible regimen in prostate cancer therapy. In this study, we demonstrate that metformin inhibits prostate cancer cell growth and induces apoptosis by down-regulating AR protein levels and repressing the AR signaling pathway. Mechanistically, metformin exerts its effects on AR not by affecting AR degradation/stability, AR nuclear translocation or recruitment to AR target genes, but by down-regulating AR mRNA The Prostate
levels. However, it is not known at this stage if metformin is down-regulating AR mRNA through inhibiting AR transcription or enhancing AR mRNA degradation, or both. Further elucidation of the mechanism underlying metformin-mediated repression of AR mRNA levels is critical to better our understanding of metformin-mediated AR regulation. Nonetheless, metformin-mediated AR mRNA down-regulation as well as metformin-mediated AR pathway attenuation was observed in both the hormone-dependent and the hormone-independent prostate cancers, implying that metformin may exert its effects on both early and late stages of prostate cancers. Results from more recently published studies imply that AR variants lacking the ligand binding domain (LBD) may play unique roles in the development in CRPC [11]. Seven AR splice variants have been identified and named AR-V1 to AR-V7. Despite their structural and functional diversity, all of these AR splice variants are constitutively active. In other words, they activate AR signaling pathways in an androgen-independent manner. Among them, AR-V1 and AR-V7 draw the most attention due to being detected in CRPC at levels of 20-fold higher than that in the hormone naive prostate tumors [12]. These AR variants co-occupy the AREs in the promoter and/or enhancer regions of canonical androgenresponsive genes. Even more importantly, increased AR-Vs were observed in tumors that developed resistance to enzalutamide [13]. Additionally, AR-V7-positive patients had lower PSA response rates than AR-V7-negative patients and shorter PSA progression-free survival, clinical or radiographic progression-free survival, and overall survival, implying that suppressing AR-V7 could be a plausible strategy against CRPC development and progression. Furthermore, Daniel et al. found that metformin usage is also independently associated with a reduction in the rate of CRPC development in patients experiencing biochemical failure [14]. In the current study, we demonstrated that metformin treatment not only down-regulates full-length AR and the recruitment of AR to the ARE in LNCaP cells, but also lowered the truncated AR-V7 in the CRPC cell line CWR22RV1. These data suggest that metformin supplementation may be a valuable therapeutic strategy in both ADT-responsive prostate cancer as well as CRPC. Numerous studies suggest that metformin inhibits the rapamycin complex 1 (mTORC1) pathway, which plays an important role in metabolism, growth and proliferation of different cells including cancer cells [15]. Furthermore, it was suggested that metformin inhibits mTORC1 through inhibiting the insulin/ IGF receptor signaling pathway, increasing tuberous
Metformin, AR and Prostate Cancer sclerosis complex protein 2 (TSC2) and modifying cellular metabolism in cancer cells [16]. Additional evidence suggest that AMP-activated protein kinase (AMPK) and liver kinase B1 (LKB1) also play important role in metformin-inhibited mTORC1-mediated prostate cancer cell proliferation [17,18]. However, it has also been reported that the negative regulation of mTOR by metformin in prostate cancer cells is AMPK activation-independent, and rather DNA-damageinducible transcript 4 protein (DDIT4, REDD1)dependent [19]. Colquhoun et al. reported a direct anti-proliferative effect produced by metformin through the activation of phospho-AMPK with subsequent inhibition of downstream mTOR signaling in the AR-positive cells [8]. In addition, the signal transducer and activator of transcription (STAT)-3 plays a unique role in prostate cancer by alternative mechanisms than through AR interactions with its primary ligand, IL-6 cytokine, AKT/PI3K/PTEN, MAPK, EGFR, and Hsps have also been suggested [20]. Inhibition of AMP-activated protein kinase (AMPK) was also reported to activate PI3K-Akt-mTOR survival pathway and increase AR expression [21]. Since multiple lines of evidence suggest that metformin inhibition of the mTORC1 pathway in cancer cell proliferation is associated with inhibition of STAT3 and activation of AMPK [18], it will be intriguing to discover whether the metformin-mediated repression of AR mRNA is also mediated through the mTORC1STAT3/AMPK axis. CONCLUSION In our study, we demonstrated that metformin alone or in combination with bicalutamide inhibits the growth of both androgen-dependent and androgen-independent prostate cancer cells. In the androgen-independent prostate cancer cells, metformin perturbs the cell cycle (G1/S cell cycle arrest) and enhances apoptosis. These results collectively indicate that metformin has a great potential to be a valuable and efficient adjunct agent for both ADT as well as the treatment of CRPC. REFERENCES 1. Siegel R,Naishadham D, Jemal A. Cancer statistics. CA Cancer J Clin. 2013;63:11–30. 2. Andersen RJ, Mawji NR, Wang J, Wang G, Haile S, Myung JK, Watt K, Tam T, Yang YC, Ba~ nuelos CA, Williams DE, McEwan IJ, Wang Y, Sadar MD. Regression of castrate-recurrent prostate cancer by a small-molecule inhibitor of the amino-terminus domain of the androgen receptor. Cancer Cell. 2010;17:535–546. 3. Chen Y, Sawyers CL, Scher HI. Targeting the androgen receptor pathway in prostate cancer. Curr Opin Pharmacol. 2008;8: 440–448.
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4. Attard G, Cooper CS, de Bono JS. Steroid hormone receptors in prostate cancer: A hard habit to break? Cancer Cell. 2009;16:458–462. 5. Yu H, Yin L, Jiang X, Sun X, Wu J, Tian H, Gao X, He X. Effect of metformin on cancer risk and treatment outcome of prostate cancer: A meta-analysis of epidemiological observational studies. PLoS ONE 2014;9:e116327. DOI: 10.1371/journal. pone.0116327. eCollection [PubMed - in process]. 6. Rothermundt C, Hayoz S, Templeton AJ, Winterhalder R, Strebel RT, B€ artschi D, Pollak M, Lui L, Endt K, Schiess R, R€ uschoff JH, Cathomas R, Gillessen S. Metformin in chemotherapy-naive castration-resistant prostate cancer: A multicenter phase 2 trial (SAKK 08/09). Eur Urol 2014;66: 468–474. 7. Ben Sahra I, Laurent K, Loubat A, Giorgetti-Peraldi S, Colosetti P, Auberger P, Tanti JF, Le Marchand-Brustel Y, Bost F. The antidiabetic drug metformin exerts an antitumoral effect in vitro and in vivo through a decrease of cyclin D1 level. Oncogene 2008;27:3576–3586. 8. Colquhoun AJ, Venier NA, Vandersluis AD, Besla R, Sugar LM, Kiss A, Fleshner NE, Pollak M, Klotz LH, Venkateswaran V. Metformin enhances the antiproliferative and apoptotic effect of bicalutamide in prostate cancer. Prostate Cancer Prostatic Dis 2012;15:346–352. 9. Aloysius H, Hu L. Targeted Prodrug Approaches for Hormone Refractory Prostate Cancer. Med Res Rev 2014; Dec 22 DOI: 10.1002/med.21333. [Epub ahead of print]. 10. Hwang IC, Park SM, Shin D, Ahn HY, Rieken M, Shariat SF. Metformin Association with Lower Prostate Cancer Recurrence in Type 2 Diabetes: A Systematic Review and Meta-analysis. Asian Pac J Cancer Prev 2015;16:595–600. 11. Hu R, Dunn TA, Wei S, Isharwal S, Veltri RW, Humphreys E, Han M, Partin AW, Vessella RL, Isaacs WB, Bova GS, Luo J. Ligand-independent androgen receptor variants derived from splicing of cryptic exons signify hormone-refractory prostate cancer. Cancer Res 2009;69:16–22. 12. Cao B, Qi Y, Zhang G, Xu D, Zhan Y, Alvarez X, Guo Z, Fu X, Plymate SR, Sartor O, Zhang H, Dong Y. Androgen receptor splice variants activating the full-length receptor in mediating resistance to androgen-directed therapy. Oncotarget 2014;5: 1646–1656. 13. Antonarakis ES, Lu C, Wang H, Luber B, Nakazawa M, Roeser JC, Chen Y, Mohammad TA, Chen Y, Fedor HL, Lotan TL, Zheng Q, De Marzo AM, Isaacs JT, Isaacs WB, Nadal R, Paller CJ, Denmeade SR, Carducci MA, Eisenberger MA, Luo J. ARV7 and resistance to enzalutamide and abiraterone in prostate cancer. N Engl J Med 2014;371:1028–1038. 14. Spratt DE, Zhang C, Zumsteg ZS, Pei X, Zhang Z, Zelefsky MJ. Metformin and prostate cancer: Reduced development of castration-resistant disease and prostate cancer mortality. Eur Urol 2013;63:709–716. 15. Chiang GG, Abraham RT. Targeting the mTOR signaling network in cancer. Trends Mol Med. 2007;13:433–442. 16. Gong J, Robbins LA, Lugea A, Waldron RT, Jeon CY, Pandol SJ. Diabetes, pancreatic cancer, and metformin therapy. Front Physiol 2014; Nov. 07 5:426. DOI: 10.3389/fphys 17. Memmott RM, Mercado JR, Maier CR, Kawabata S, Fox SD, Dennis PA. Metformin prevents tobacco carcinogen-induced lung tumorigenesis. Cancer Prev Res (Phila) 2010;3: 1066–1076.
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The Prostate
Metformin Represses Androgen-Dependent and Androgen-Independent Prostate Cancers by Targeting Androgen Receptor Yan Wang,1 Gaolei Liu,1 Dali Tong,1 Henna Parmar,2 Donald Hasenmayer,2 Wenqiang Yuan,1 Dianzheng Zhang,2 and Jun Jiang1* 1
Department of Urology, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing, PR China 2 Department of Bio-Medical Sciences, Philadelphia College of osteopathic Medicine, Philadelphia, Pennsylvania
BACKGROUND. Metformin has been reported to inhibit the growth of different types of cancers, including prostate cancer. We were interested to understand if the effect of metformin on prostate cancer is AR-dependent and, if so, whether metformin could act synergistically with the other anti-AR agents to serve as a therapeutic regimen with high efficacy and low toxicity. METHODS. Cell viabilities and apoptosis were determined by MTT assay and annexin V-FITC staining, respectively, when the two human prostate cancer cell lines, the androgen-dependent LNCaP and the androgen-independent 22RV1 were treated with metformin alone or in combination with bicalutamide. Quantitative RT-PCR and western blotting assays were conducted to examine metformin effects on AR mRNA and protein levels, respectively. Chromatin immunoprecipitation (ChIP) assays were conducted to confirm the recruitment of AR to the ARE(s) located on the promoter region of the AR target gene PSA. RESULTS. Metformin treatment reduced cell viability and enhanced apoptosis for both cell lines and additive effects were observed when LNCaP cells were treated with combined metformin and bicalutamide. Metformin down-regulated full-length AR protein in LNCaP cells. Both full-length and the truncated AR (AR-v7) were down-regulated by metformin in CWR22Rv1 cells. In both LNCaP and CWR22Rv1 cells, metformin repressed AR signaling pathway not by affecting AR protein degradation/stability, but rather through down-regulating the levels of AR mRNAs. CONCLUSIONS. Metformin represses prostate cancer cell viability and enhances apoptosis by targeting the AR signaling pathway. Combinations of metformin and other anti-AR agents pose a potentially promising therapeutic approach for treatment of prostate cancers, especially the castrate-resistant prostate cancer, with high efficacy and low toxicity. Prostate 75:1187–1196, 2015. # 2015 Wiley Periodicals, Inc. KEY WORDS:
prostate cancer; androgen receptor; metformin; castration resistance
Grant sponsor: National Natural Science Foundation of China; Grant number: 81402120; Grant sponsor: Chongqing Science and Technology Commission; Grant number: 2011GZ0047. Conflict of interest: The authors declare that they have no conflict of interest.
Correspondence to: Jun Jiang, Department of Urology, Institute of Surgery Research, Daping Hospital, Third Military Medical University. No. 10 Changjiangzhilu, Yuzhong District, Chongqing 400042, PR China. E-mail address: [email protected] Received 19 February 2015; Accepted 17 March 2015 DOI 10.1002/pros.23000 Published online 20 April 2015 in Wiley Online Library (wileyonlinelibrary.com).
ß 2015 Wiley Periodicals, Inc.
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Prostate cancer is one of the biggest threats to men’s health in the western world and it accounts for the second largest number of male cancer-related deaths in the United States. Currently, androgen deprivation therapy (ADT) is the gold standard treatment for advanced prostate cancer. ADT temporarily reduces prostate cancer progression with concomitant decrease of serum prostate-specific antigen (PSA). Unfortunately, almost all patients treated with ADT will relapse into castration-resistant prostate cancer (CRPC), a disease status characterized by rising PSA levels and tumor cell recurrence, within a period of 6 months–3 years [1]. However, CRPC is not completely refractory to further hormonal manipulation as the androgen receptor (AR) continues to play an important role in CRPC progression [2,3]. It has been demonstrated that complex mechanisms including AR gene amplification/overexpression, intra-tumoral androgen synthesis, overexpression of AR coactivators, aberrant kinase pathway activation, AR mutation, and expression of constitutively active AR splice variants exist in prostate cancers [4]. Therefore, AR is an attractive therapeutic target for different prostate cancers including CRPC. Metformin is one of the most well-established drugs for the treatment of type II diabetes. In addition to its capacity to treat diabetes, data from epidemiological studies suggest that metformin also possesses remarkable antitumor properties and is capable of exerting these effects on a wide spectrum of cancers including; breast cancer, ovarian cancer, colon cancer, and prostate cancer. When compared to non-metformin users, metformin users were associated with 9% lower risk of prostate cancer; 18% reduction in the biochemical recurrence (BCR); and reduced cancer-specific mortality as well as overall survival [5]. Recent publications have shown that metformin can even reduce the serum PSA levels in CRPC patients resistant to both Abiraterone and Enzalutamide [6]. Furthermore, accumulative evidence suggests that the use of metformin alone or in combination with bicalutamide can inhibit cell proliferation and induce apoptosis in prostate cancer cells [7,8]. Although metformin is currently undergoing clinical trials aimed at the treatment of prostate cancer, the molecular mechanisms underlying these beneficial functions remain unclear. This gap in our understanding continues to prevent further application of metformin in prostate cancer therapy. Given the accumulating data regarding the anticancer effect of metformin and the important roles played by AR in CRPC, we set to dissect the effects The Prostate
of metformin on AR activity. In this study, we demonstrated that metformin reduces AR transcriptional activity by down-regulating both full-length AR and the truncated AR-V7 proteins in both the androgen-dependent and the androgen-independent prostate cancer cells. Mechanistically, metformin does not function by affecting AR protein stability/ degradation or nuclear translocation, but by downregulating total AR mRNA levels. Here, combinatorial treatment of low dose metformin in conjunction with bicalutamide supplementation is shown to additively repress the growth of prostate cancer cells. These findings not only reveal a novel molecular mechanism of metformin’s effects on prostate cancer, but also imply that co-treatment of low concentrations metformin in concert with other anti-AR agents is an attractive and effective approach for prostate cancer therapy with high efficiency and low toxicity. MATERIALS AND METHODS Cell Culture LNCaP and CWR22Rv1 human prostate cancer cells were purchased from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. Both cell lines were cultured in RPMI 1640 (Hyclone, Logan, UT) supplemented with 10% FBS (Invitrogen, Carlsbad, CA) at 37°C in incubators with humidified air and 5% carbon dioxide. For DHT induction, the cells were cultured in phenol red-free RPMI supplemented with 10% charcoal stripped FBS (Invitrogen) and 10 nM DHT (Sigma). For the analysis of the mRNA levels, LNCaP cells were seeded in 6-well plates 2105 cells/well, treated with 10 nM DHT and different concentrations (0 mM, 1 mM, 5 mM, 10 mM and 20 mM) of metformin (Sigma). For the measurement of the protein levels, both LNCaP and CWR22Rv1 cells were seeded at 4105 cells/well in 6-well plates, treated with different concentrations (0 mM, 1 mM, 5 mM, 10 mM and 20 mM) of metformin with or without 10 nM DHT, respectively. Quantitative Real-Time PCR Total RNA was isolated using RNA extraction kit (Bioteke Inc, Beijing, China). Reverse transcriptase PCR was performed using the 1st Stand cDNA Synthesis (OriGene, Rockville, MD). PCRs were conducted using the qSTAR SYBR Master Mix (OriGene). Primers for full-length AR were 5’-GACGACCAGATGGCTGTCATT-3’ (forward) and 5’-GGGCGAAGTAGAGCATCCT-3’ (reverse). Primers for AR-v7 were 5’-
Metformin, AR and Prostate Cancer CCATCTTGTCGTCTTCGGAAATGTTA-3’ (forward), and 5’-TTTGAATGAGGCAAGTCAGCCTTTCT-3’ (reverse). Primers for PSA were 5’-GTGTGTGGACCTCCATGTTATT-3’ (forward) and 5’-CCACTCACCTTTCCCCTCAAG-3’ (reverse). Primers for Nkx3.1 were 5’-CCCACACTCAGGTGATCGAG-3’ (forward) and 5’GAGCTGCTTTCGCTTAGTCTT-3’ (reverse). Primers for the internal control, GAPDH, were 5’-AAGGTGAAGGTCGGAGTCAAC-3’ (forward) and 5’-GGGGTCATTGATGGCAACAATA-3’ (reverse).
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Cell Viability Assay Cells were seeded at a 0.5104 density per well in 96-well plates and exposed to metformin (2 mM) and bicalutamide (5 mM) for 7 days, 20 ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) dye (5 mg/ml, Sigma) was added to each well then kept at 37°C for 4 hr. After the media was removed, the resulting formazan crystals were solubilized in 200 ml dimethyl sulfoxide (DMSO). The density of the solubilized formazan was read at 490 nm spectrophotometrically (Bio-Rad, Hercules, CA).
Western Blot Cells were lysed in a RIPA buffer containing a protease inhibitor cocktail tablet and phosphotase inhibitor cocktail (KeyGEN BioTECH, NanJing, China). Protein levels were determined by BCA assay (Thermo–Fisher Scientific). 15 mg of protein were separated by SDS–PAGE and transferred onto PVDF membrane (PALL, NY). After 1 hr of incubation in blocking solution (5% BSA in PBS), the membranes were incubated with the appropriate antibodies: AR (Epitomics, CA), Bcl-2 (Epitomics), Cyclin D1 (Fantibody, China) and b-actin (Cell Signaling, MA).
Protein Stability Assay For the protein stability assays, CWR22Rv1 cells were pre-treated with 20 mg/ml cycloheximide (CHX, Sigma) for 30 min to stop protein synthesis followed by the treatment of 10 mM metformin for different time periods. For proteasome pathway, CWR22Rv1 cells were treated with metformin (10 mM), proteasome inhibitor MG132 (2 mM), or metformin (10 mM) þ MG132 (2 mM) for either 24 hr or 48 hr. For caspase-3 pathway, 22RV1 cells were pretreated with caspase-3 inhibitor z-VAD (20 mM) for 2 hr, followed by metformin (10 mM) for 48 hr. The protein levels were estimated by western blots as described above.
Chromatin Immunoprecipitation (ChIP) Assay LNCaP cells were treated with 1% formaldehyde/ RPMI for 10 min at room temperature. Nuclear extracts were prepared and sonicated on ice to generate DNA fragments less than 500 bp. For immunoprecipitation, the AR antibody (Epitomics) was added to DNA–protein complexes and incubated overnight at 4°C with rotation, and chromatin–antibody complexes were incubated with protein A/G agarose beads for overnight at 4°C with rotation. The bound cross-linked DNA–protein complexes were reversed by Proteinase K (Invitrogen) digestion at 65°C for 2 hr and DNA was purified and used as a template for PCR. Primer sequences for amplification of the PSA enhancer region were 5’-GTGTGTGGACCTCCATGTTATT-3’ (forward) and 5’-CCACTCACCTTTCCCCTCAAG-3’ (reverse). Human RPL30 exon3 was amplified with primers #7014 purchased from Cell Signal Technology (Danvers, MA) and served as positive control. Statistical Analysis All the experiments were analyzed using GraphPad Prism 5.0. All data were presented as means standard deviation (SD). P < 0.05 was considered statistically significant. RESULTS
Apoptosis Assay
Metformin Inhibits Prostate Cancer Cell Growth and Induces Cell Apoptosis
CWR22Rv1 Cells were seeded at a density of 2105 cells/well in six-well plates, and treated with metformin (10 mM) for 48 hr. The cells were then washed with cold PBS for three times, resuspended in Annexin V binding buffer and incubated with Annexin V–FITC and PI (KeyGEN BioTECH) for 10 min at 37°C in the dark and analyzed by a FACScan (BD Biosciences, NJ).
In order to determine if metformin exerts any effects on prostate cancer viability, we treated both the hormone-dependent prostate cancer cell line, LNCaP, and the hormone-independent prostate cancer cell line, CWR22v1, with vehicle, 2 mM metformin, 5 mM bicalutamide, or a combinational treatment of metformin and bicalutamide for 7 days in the presence or absence of 10 nM DHT. The Prostate
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The respective cell viabilities were determined by MTT assays. As shown in Figure 1A, LNCaP cell viability was reduced to a similar degree after either treatment with 2 mM metformin or 5 mM bicalutamide when compared to the control. Additionally, a additive effect was observed when metformin and bicalutamide were administered simultaneously. In contrast, the viability of the CWR22Rv1 cells was not affected upon treatment with bicalutamide alone (Figure 1B) but was significantly reduced by metformin. Furthermore, the additive effect produced by metformin and bicalutamide on LNCaP viability was not observed in the CWR22v1 cells. These results indicate that metformin exerts its effect on the prostate cancer
cells though a different mechanism than that of the general anti-androgen agents. In order to determine if metformin represses prostate cancer cell viability by enhancing cancer cell apoptosis, we analyzed the cell cycle status of the CWR22Rv1 cells as well as the percentage of apoptotic cells by flow cytometry. As shown in Figure 1C, metformin treatment increased both early and late apoptotic populations with an overall fourfold increase in apoptosis compared to the control. These observations were further substantiated by the findings that treatment with metformin alone or in combination with bicalutamide down-regulated both Bcl-2 and cyclin D1, suggesting that metformin induced prostate cancer cell
Fig. 1. Metformin inhibits prostate cancer cell growth and induces apoptosis. (A) LNCaP cells were seeded into a 96-well plate, cultured with CS-FBS and treated with 10 nM DHT. Metformin (2 mM), bicalutamide (5 mM), and metforminþ bicalutamide were added for 1, 3, 5, or 7 days. (B) CWR22Rv1 cells were seeded into a 96-well plate, treated with metformin (2 mM), bicalutamide (5 mM), and metforminþ bicalutamide for 1, 3, 5, or 7 days. Cell viability was determined by MTT assay. (C) CWR22Rv1 cells were treated with metformin (10 mM) for 48 hr and Annexin V-FITC assays and PI staining were conducted. Early apoptotic cells (Q3) and late apoptotic cells (Q2). (D) CWR22Rv1 cells were treated metformin (10 mM), bicalutamide (10 mM), and metforminþ bicalutamide for 48 hr. The protein levels of Bcl-2 and Cyclin D1 were analyzed by western blot. *: P < 0.05; **: P < 0.01. The Prostate
Metformin, AR and Prostate Cancer G1/S arrest and apoptotic death. Bicalutamide treatment alone, however, did not affect Bcl-2 or cyclin D1 (Figure 1D). These data collectively demonstrated that metformin reduces the cell viability of both androgen-dependent and androgenindependent prostate cancer cells and promotes cell cycle arrest and induces apoptosis in androgenindependent prostate cancer cells. Metformin Inhibits AR Transcriptional Activity and Reduces AR/ARE Interaction AR plays important roles in both androgendependent and androgen-independent cancers. In order to determine if metformin exerts its effect on prostate cancer through AR, we first examined the mRNA levels of two well-established AR target genes, PSA, and Nkx3.1 in LNCaP cells. As shown in Figure 2A, DHT treatment induced the levels of both mRNAs more than two times comparing to the basal levels. When LNCaP were treated with DHT and increasing concentrations of metformin (0–20 mM) for 24 hr, the mRNA levels of PSA and NKX3.1 were reduced approximately 45–80% and 35–75%, respec-
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tively. Similarly, when the androgen-independent CWR22v1 cells were treated with the increasing metformin concentrations, both PSA and Nkc3.1 were observed to be down regulated (Figure 2B). These results imply that metformin not only counteracts DHT-induced AR activity, but also the basal level AR activity in a dose-dependent manner. To further elucidate the mechanism of metformin-induced AR inhibition, ChIP assays were performed to assess metformin’s effect on AR recruitment to the PSA promoter region. As shown in Figure 2C, AR association with the PSA promotor was significantly increased when cells were treated with DHT compared to the control, and this hormone-induced AR recruitment was significantly reduced when the cells were treated with metformin. Assays for the negative control using the nonspecific antibody IgG did not precipitate any detectable amount of DNA; and assays for the positive control showed equal amounts of AR were recruited to the Human RPL30 exon3 region across treatment groups. Collectively, these results demonstrated that metformin is capable of inhibiting AR transcriptional activity by reducing AR recruitment to the promoter of its target gene.
Fig. 2. Metformin inhibits AR transcriptional activity. (A) LNCaP cells were cultured with CS-FBS and treated with 10 nM DHT and different concentrations of metformin for 24 hr. (B) CWR22Rv1 cells were treated with different concentrations of metformin for 24 hr. Total RNA was isolated and used as a template for qRT-PCR to estimate the mRNA levels of PSA and NKX3.1. (C) LNCaP cells were cultured with CS-FBS and treated with 10 nM DHT (H) alone or in combination with 10 mM metformin (HM) overnight. Whole cell lysates were subjected to CHIP assays with antibody against AR. Purified DNA was used for PCR amplification of the enhancer region of the PSA promoter. *: P < 0.05; **: P < 0.01. The Prostate
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Metformin Down-Regulates AR Protein Levels Since metformin treatment inhibited both AR recruitment and AR transcriptional activity, we then decided to examine whether metformin affects overall AR expression. First, western blot assays were conducted in LNCaP cells to compare total AR protein levels before and after metformin treatment. As expected that hormone treatment enhanced overall AR protein levels. Metformin treatment, however, was able to counteract the hormone-mediated AR induction and down-regulated AR protein levels dose-dependently (Figure 3A, upper panel). Next, the cells were treated with 10 mM metformin for increasing duration. Western blot analyses showed that metformin down-regulated total AR protein levels in a time-dependent manner (Figure 3 A, bottom panel). Here, androgen-induced AR expression was significantly reduced after 24 hr of 10 mM metformin treatment. In addition to the full length protein, AR exists as a multitude of smaller splice variants. Recent
studies have suggested that these AR splice variants, especially ARV7, could be one of the most prominent factors influencing the development of CRPC. As CWR22Rv1 are known to express both AR and ARV7 [9], we decided to assess the effects of metformin on both the full-length AR and the truncated AR-V7. Lysates prepared from CWR22Rv1 cells treated with increasing concentrations of metformin for increasing time periods were analyzed by western blot assays with the antibody specifically against the N-terminus of AR. Similar to what was observed with full length AR in LNCaP cells, Figure 3B shows that metformin treatment down-regulated both full length and truncated AR (AR-v7) in a dose- andtime-dependent manner in the CWR22Rv1 cells. Metformin Does not Affect AR Protein Stability nor Nuclear Translocation but AR mRNA Levels In order to determine if metformin-mediated AR down-regulation was the result of decreased AR
Fig. 3. Metformin down-regulating AR protein levels in LNCaP and CW22RV1 cells. (A) LNCaP cells were cultured with CS-FBS and treated with 10 nM DHT and different concentrations of metformin for 24 hr, or with 10 nM DHT þ 5 mM metformin and for different time periods. (B) CWR22Rv1 cells were treated with different concentrations (0 mM, 5 mM, 10 mM and 20 mM) of metformin for 12 hr, 24 hr, 36 hr and 48 hr. Cell lysates were analyzed with western blot assays with specific antibodies against N-terminus of AR and b-actin. The Prostate
Metformin, AR and Prostate Cancer stability, we conducted a series of experiments in the absence of de novo protein synthesis. First, we monitored AR protein levels subsequent to treatment with cycloheximide (CHX), an inhibitor of protein biosynthesis in eukaryotes. Results from the western blot assays (Figure 4A) showed that when the CWR22Rv1 cells were treated with CHX (50 mM) for 12 and 24 hr, the intensities of both the full-length AR and the truncated AR-V7 bands decreased significantly and in a time-dependent manner. Additionally, treating the cells with 10 mM metformin did not further reduce the intensities of either band. To further substantiate these findings, we treated the CWR22vRv1 cells with metformin (10 mM) in the presence and absence of the proteasome inhibitor
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MG132 (2 mM). As shown in Figure 4B, the addition of MG132 did not affect metformin-mediated AR down-regulation. These data collectively demonstrate that metformin is down-regulating AR protein levels through a mechanism other than enhancing protein degradation. Next, we examined the possibility that metformin affects AR nuclear translocation. LNCaP cells were cultured in growth media supplemented with CS-FBS and treated with 10 nM DHT and increasing concentrations of metformin for 24 hr. Whole cell lysates and nuclear and cytoplasmic extracts were prepared and analyzed by western blot. As shown in Figure 4C, metformin treatment counteracted hormone-mediated AR induction, but did not inhibit AR nuclear translocation. Finally, AR tran-
Fig. 4. Metformin inhibits AR activity not by enhancing AR degradation or nuclear translocation, but repressing AR mRNA levels. (A) CWR22Rv1 cells were pre-treated with CHX (50 mg/ml) for 30 min before addition of metformin (10 mM) and continuously treated for 0 hr, 12 hr and 24 hr. (B) CWR22Rv1 cells were treated with MG132 (2 mM) with or without metformin (10 mM) for 24 hr and 48 hr. (C) LNCaP cells were cultured with CS-FBS and treated with 10 nM R1881 and different concentrations of metformin for 24 hr. The proteins of whole cell lysate, cytoplasm and nucleus extracts were analyzed by western blot. (D) LNCaP cells were cultured with CS-FBS and treated with 10 nM DHT and different concentrations of metformin for 24 hr. (E) CWR22Rv1 cells were treated with different concentrations of metformin for 24 hr. Total RNA was isolated and used for qRT-PCR to estimate the mRNA levels of AR in LNCaP cells and AR and AR-V7 in CWR22Rv1, respectively. *: P < 0.05; **: P < 0.01. The Prostate
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script levels were assessed in order to determine if metformin-mediated AR down-regulation was occurring at the mRNA level. The results from the RT-PCR assays revealed that AR mRNA levels were significantly down-regulated when the LNCaP cells were treated with metformin (Figure 4D), and the mRNA levels of both full-length AR as well as the truncated AR-V7 in CWR22Rv1 cells were also down regulated by metformin in a dose-dependent manner (Figure 4E). These data collectively demonstrated that metformin represses AR protein levels and transcriptional activities not by affecting AR protein degradation or AR nuclear translocation, but by downregulating the levels of mRNA encoding the fulllength AR in the androgen-dependent cancer cells and both full-length AR and the truncated AR-V7 in the hormone-independent prostate cancer cells. DISCUSSION Metformin is commonly prescribed for the treatment of type II diabetes because it lowers the levels of both glucose and insulin in the bloodstream. Numerous preclinical, epidemiological and clinical studies suggest that metformin usage is associated with low or no risk [10]. More importantly for patients with prostate cancer, taking metformin usually has better prognoses because it inhibits the growth of prostate cancer cells [8]. Given the fact that metformin affects a wide spectrum of pathways, it is very likely that metformin exerts its anticancer effects through targeting of multiple signaling pathways. Unfortunately, wider application of metformin as a preventive and therapeutic tool for the treatment of prostate cancer has so far been prevented by our limited knowledge of the specific molecular mechanisms underlying its beneficial properties. In this study, we demonstrate for the first time that (i) metformin significantly inhibits AR transcriptional activity and down-regulates AR target gene expression; and that (ii) a combination of low-dose metformin in conjunction with bicalutamide supplementation inhibits prostate cancer growth and induce cancer cell apoptosis. Given the additive benefits observed for the combinatory treatments over short-, mid- and long-term periods, combinations of low-dose metformin and other anti-androgenic reagents could be become a plausible regimen in prostate cancer therapy. In this study, we demonstrate that metformin inhibits prostate cancer cell growth and induces apoptosis by down-regulating AR protein levels and repressing the AR signaling pathway. Mechanistically, metformin exerts its effects on AR not by affecting AR degradation/stability, AR nuclear translocation or recruitment to AR target genes, but by down-regulating AR mRNA The Prostate
levels. However, it is not known at this stage if metformin is down-regulating AR mRNA through inhibiting AR transcription or enhancing AR mRNA degradation, or both. Further elucidation of the mechanism underlying metformin-mediated repression of AR mRNA levels is critical to better our understanding of metformin-mediated AR regulation. Nonetheless, metformin-mediated AR mRNA down-regulation as well as metformin-mediated AR pathway attenuation was observed in both the hormone-dependent and the hormone-independent prostate cancers, implying that metformin may exert its effects on both early and late stages of prostate cancers. Results from more recently published studies imply that AR variants lacking the ligand binding domain (LBD) may play unique roles in the development in CRPC [11]. Seven AR splice variants have been identified and named AR-V1 to AR-V7. Despite their structural and functional diversity, all of these AR splice variants are constitutively active. In other words, they activate AR signaling pathways in an androgen-independent manner. Among them, AR-V1 and AR-V7 draw the most attention due to being detected in CRPC at levels of 20-fold higher than that in the hormone naive prostate tumors [12]. These AR variants co-occupy the AREs in the promoter and/or enhancer regions of canonical androgenresponsive genes. Even more importantly, increased AR-Vs were observed in tumors that developed resistance to enzalutamide [13]. Additionally, AR-V7-positive patients had lower PSA response rates than AR-V7-negative patients and shorter PSA progression-free survival, clinical or radiographic progression-free survival, and overall survival, implying that suppressing AR-V7 could be a plausible strategy against CRPC development and progression. Furthermore, Daniel et al. found that metformin usage is also independently associated with a reduction in the rate of CRPC development in patients experiencing biochemical failure [14]. In the current study, we demonstrated that metformin treatment not only down-regulates full-length AR and the recruitment of AR to the ARE in LNCaP cells, but also lowered the truncated AR-V7 in the CRPC cell line CWR22RV1. These data suggest that metformin supplementation may be a valuable therapeutic strategy in both ADT-responsive prostate cancer as well as CRPC. Numerous studies suggest that metformin inhibits the rapamycin complex 1 (mTORC1) pathway, which plays an important role in metabolism, growth and proliferation of different cells including cancer cells [15]. Furthermore, it was suggested that metformin inhibits mTORC1 through inhibiting the insulin/ IGF receptor signaling pathway, increasing tuberous
Metformin, AR and Prostate Cancer sclerosis complex protein 2 (TSC2) and modifying cellular metabolism in cancer cells [16]. Additional evidence suggest that AMP-activated protein kinase (AMPK) and liver kinase B1 (LKB1) also play important role in metformin-inhibited mTORC1-mediated prostate cancer cell proliferation [17,18]. However, it has also been reported that the negative regulation of mTOR by metformin in prostate cancer cells is AMPK activation-independent, and rather DNA-damageinducible transcript 4 protein (DDIT4, REDD1)dependent [19]. Colquhoun et al. reported a direct anti-proliferative effect produced by metformin through the activation of phospho-AMPK with subsequent inhibition of downstream mTOR signaling in the AR-positive cells [8]. In addition, the signal transducer and activator of transcription (STAT)-3 plays a unique role in prostate cancer by alternative mechanisms than through AR interactions with its primary ligand, IL-6 cytokine, AKT/PI3K/PTEN, MAPK, EGFR, and Hsps have also been suggested [20]. Inhibition of AMP-activated protein kinase (AMPK) was also reported to activate PI3K-Akt-mTOR survival pathway and increase AR expression [21]. Since multiple lines of evidence suggest that metformin inhibition of the mTORC1 pathway in cancer cell proliferation is associated with inhibition of STAT3 and activation of AMPK [18], it will be intriguing to discover whether the metformin-mediated repression of AR mRNA is also mediated through the mTORC1STAT3/AMPK axis. CONCLUSION In our study, we demonstrated that metformin alone or in combination with bicalutamide inhibits the growth of both androgen-dependent and androgen-independent prostate cancer cells. In the androgen-independent prostate cancer cells, metformin perturbs the cell cycle (G1/S cell cycle arrest) and enhances apoptosis. These results collectively indicate that metformin has a great potential to be a valuable and efficient adjunct agent for both ADT as well as the treatment of CRPC. REFERENCES 1. Siegel R,Naishadham D, Jemal A. Cancer statistics. CA Cancer J Clin. 2013;63:11–30. 2. Andersen RJ, Mawji NR, Wang J, Wang G, Haile S, Myung JK, Watt K, Tam T, Yang YC, Ba~ nuelos CA, Williams DE, McEwan IJ, Wang Y, Sadar MD. Regression of castrate-recurrent prostate cancer by a small-molecule inhibitor of the amino-terminus domain of the androgen receptor. Cancer Cell. 2010;17:535–546. 3. Chen Y, Sawyers CL, Scher HI. Targeting the androgen receptor pathway in prostate cancer. Curr Opin Pharmacol. 2008;8: 440–448.
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4. Attard G, Cooper CS, de Bono JS. Steroid hormone receptors in prostate cancer: A hard habit to break? Cancer Cell. 2009;16:458–462. 5. Yu H, Yin L, Jiang X, Sun X, Wu J, Tian H, Gao X, He X. Effect of metformin on cancer risk and treatment outcome of prostate cancer: A meta-analysis of epidemiological observational studies. PLoS ONE 2014;9:e116327. DOI: 10.1371/journal. pone.0116327. eCollection [PubMed - in process]. 6. Rothermundt C, Hayoz S, Templeton AJ, Winterhalder R, Strebel RT, B€ artschi D, Pollak M, Lui L, Endt K, Schiess R, R€ uschoff JH, Cathomas R, Gillessen S. Metformin in chemotherapy-naive castration-resistant prostate cancer: A multicenter phase 2 trial (SAKK 08/09). Eur Urol 2014;66: 468–474. 7. Ben Sahra I, Laurent K, Loubat A, Giorgetti-Peraldi S, Colosetti P, Auberger P, Tanti JF, Le Marchand-Brustel Y, Bost F. The antidiabetic drug metformin exerts an antitumoral effect in vitro and in vivo through a decrease of cyclin D1 level. Oncogene 2008;27:3576–3586. 8. Colquhoun AJ, Venier NA, Vandersluis AD, Besla R, Sugar LM, Kiss A, Fleshner NE, Pollak M, Klotz LH, Venkateswaran V. Metformin enhances the antiproliferative and apoptotic effect of bicalutamide in prostate cancer. Prostate Cancer Prostatic Dis 2012;15:346–352. 9. Aloysius H, Hu L. Targeted Prodrug Approaches for Hormone Refractory Prostate Cancer. Med Res Rev 2014; Dec 22 DOI: 10.1002/med.21333. [Epub ahead of print]. 10. Hwang IC, Park SM, Shin D, Ahn HY, Rieken M, Shariat SF. Metformin Association with Lower Prostate Cancer Recurrence in Type 2 Diabetes: A Systematic Review and Meta-analysis. Asian Pac J Cancer Prev 2015;16:595–600. 11. Hu R, Dunn TA, Wei S, Isharwal S, Veltri RW, Humphreys E, Han M, Partin AW, Vessella RL, Isaacs WB, Bova GS, Luo J. Ligand-independent androgen receptor variants derived from splicing of cryptic exons signify hormone-refractory prostate cancer. Cancer Res 2009;69:16–22. 12. Cao B, Qi Y, Zhang G, Xu D, Zhan Y, Alvarez X, Guo Z, Fu X, Plymate SR, Sartor O, Zhang H, Dong Y. Androgen receptor splice variants activating the full-length receptor in mediating resistance to androgen-directed therapy. Oncotarget 2014;5: 1646–1656. 13. Antonarakis ES, Lu C, Wang H, Luber B, Nakazawa M, Roeser JC, Chen Y, Mohammad TA, Chen Y, Fedor HL, Lotan TL, Zheng Q, De Marzo AM, Isaacs JT, Isaacs WB, Nadal R, Paller CJ, Denmeade SR, Carducci MA, Eisenberger MA, Luo J. ARV7 and resistance to enzalutamide and abiraterone in prostate cancer. N Engl J Med 2014;371:1028–1038. 14. Spratt DE, Zhang C, Zumsteg ZS, Pei X, Zhang Z, Zelefsky MJ. Metformin and prostate cancer: Reduced development of castration-resistant disease and prostate cancer mortality. Eur Urol 2013;63:709–716. 15. Chiang GG, Abraham RT. Targeting the mTOR signaling network in cancer. Trends Mol Med. 2007;13:433–442. 16. Gong J, Robbins LA, Lugea A, Waldron RT, Jeon CY, Pandol SJ. Diabetes, pancreatic cancer, and metformin therapy. Front Physiol 2014; Nov. 07 5:426. DOI: 10.3389/fphys 17. Memmott RM, Mercado JR, Maier CR, Kawabata S, Fox SD, Dennis PA. Metformin prevents tobacco carcinogen-induced lung tumorigenesis. Cancer Prev Res (Phila) 2010;3: 1066–1076.
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