Inhibiting Multiple Deubiquitinases to Reduce Androgen Receptor Expression in Prostate Cancer Cells

Prostate cancer (PCa), a leading cause of cancer-related death in men, becomes resistant to androgen deprivation therapy by inducing androgen receptor (AR) activity, which is known as castration-resistant PCa (CRPC). Enzalutamide is an approved drug that inhibits AR activity and increases overall survival. However, resistance to enzalutamide develops rapidly often by increasing AR activity, suggesting that new therapies are required for CRPC. We investigated whether betulinic acid (BA), a small molecule from plants that inhibits multiple deubiquitinases (DUBs), reduces AR, and selectively kills PCa cells, can provide an adjuvant strategy for CRPC. Our data indicated that BA reduced AR protein stability and mRNA expression, making it an attractive agent for CRPC. BA decreased AR mRNA possibly by inhibiting a histone 2A DUB thereby increasing ubiquitinated histone 2A, a transcriptional repressor. We identified multiple and specific DUBs inhibited by BA either in PCa cells or using recombinant DUBs. Similar results were obtained using another multi-DUB inhibitor WP1130, suggesting that these DUB inhibitors can decrease AR expression and increase PCa-specific death. Our results also suggest that combining multi-DUB inhibitors BA or WP1130 with enzalutamide may provide a novel strategy for CRPC by further decreasing AR expression and increasing apoptotic cell death.</p

Abstract 334: Mcl-1 protects prostate cancer cells from chemotherapy-induced DNA damage

Abstract Mcl-1 (myeloid cell leukemia sequence 1 [Bcl2-related]) is predominantly localized in the cytoplasm and protects cells from apoptosis by blocking mitochondrial outer membrane permeabilization. There is evidence that nuclear localization of Mcl-1 has pleiotropic effects independent of apoptosis, including functional roles in DNA damage response. Mcl-1 is highly expressed in castration-resistant prostate cancer (CRPC), resulting in resistance to apoptosis and association with poor prognosis. The role of nuclear Mcl-1 in mediating sensitivity to chemotherapy-induced DNA damage and apoptotic cell death in prostate cancer (PCa) is not known. The novel antimitotic agent ENMD-1198 is a more stable and potent derivative of 2-methoxyestradiol (2ME2). We confirmed that 1198 was a more potent inhibitor of androgen-dependent LNCaP and castration-resistant DU145 and PC3 PCa cell viability compared to 2ME2 and induced apoptotic cell death in docetaxel-resistant PC3 cells that overexpress the multi-drug resistance protein (MDR). The ability of 1198 to increase the proteasomal-mediated degradation of Mcl-1 correlated with enhanced apoptotic cell death and shRNA knockdown of Mcl-1 further sensitized LNCaP and PC3 cells to 1198-mediated cell death. We then showed that the combination of 1198 with betulinic acid (BA, a plant-derived small molecule that can increase apoptosis specifically in cancer but not in normal cells) reduced nuclear Mcl-1, increased a pro-death Mcl-1 isoform, increased gamma histone 2AX (marker of DNA damage), and enhanced apoptotic cell death in human PC3 CRPC cells. In the TRAMP mouse model of PCa, the 1198 (75 mg/kg) + BA (10 mg/kg) combination significantly reduced PCa weights compared to 1198, BA alone, and vehicle controls. However, the reduced PCa weights between the 1198 + BA combination and BA alone was more associated with decreased Mcl-1 and increased gamma-H2AX (DNA damage) immunostaining compared to cleaved caspase-3 (apoptosis), CD31(angiogenesis), or Ki67 (proliferation). Furthermore, our results showed that blocking the DNA damage response signaling pathway with an ATM kinase (KU55933) or DNA-PK (NU7441) inhibitor lowered apoptotic cell death induced by the 1198 + BA combination in PC3 cells. Overall, our results demonstrate that nuclear Mcl-1 has an important role in protecting PCa cells from DNA damage and that agents such as 1198 and BA that reduce total/nuclear Mcl-1 and increase pro-death Mcl-1 isoforms will sensitize PCa cells to antimitotic DNA damaging agents. Citation Format: Carlos Perez-Stable, Teresita Reiner, Alicia de las Pozas, Ricardo Parrondo. Mcl-1 protects prostate cancer cells from chemotherapy-induced DNA damage. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 334. doi:10.1158/1538-7445.AM2014-33

Abstract 2571: ABT-737, a small molecule Bcl-2/Bcl-xL antagonist, increases docetaxel-mediated apoptosis in human prostate cancer cells

Abstract Castration-resistant prostate cancer (CRPC) express high levels of the anti-apoptotic proteins Bcl-2 and Bcl-xL, resulting in drug resistance and association with poor prognosis. Docetaxel (Doc), an antimitotic drug that is the first-line treatment strategy for CRPC, is known to provide a small survival benefit but patients eventually become resistant to Doc with little hope for survival. It is apparent that Doc chemotherapy alone is not enough to counteract the high levels of Bcl-2/Bcl-xL present in CRPC. ABT-737 is a small molecule that binds to Bcl-2/Bcl-xL with high affinity and disrupts their interaction with pro-apoptotic Bax/Bak, thus enhancing pro-apoptotic signals when combined with other drugs. We investigated whether ABT-737 can increase Doc-mediated apoptosis in CRPC cells. Our results in androgen-dependent LNCaP and CRPC PC3 cell lines indicate that ABT-737 (1 μM) increases Doc (1 nM)-mediated cell death and caspase-dependent apoptosis, as determined by trypan blue exclusion, annexin-FITC/propidium iodide flow cytometry, and Western blot analysis. Our data suggests that Doc's anti-cancer effect results from prolonged cyclin B1/Cdk1 activation and inactivation of Bcl-2/Bcl-xL by phosphorylation, resulting in increased apoptosis. We show a correlation between high phosphorylation (P) of Bcl-2(Ser70)/Bcl-xL(Ser62) and increased apoptosis by Doc and Doc + ABT-737 treatments. Furthermore, the Cdk1 inhibitor purvalanol A blocks Doc + ABT-737-mediated increases in P-Bcl-2/P-Bcl-xL and subsequent apoptotic cell death. Lentivirus pLKO.1 shRNA stable knockdown of Bax but not Bak lowers cell death LNCaP and PC3 cells after treatment with Doc + ABT-737. In contrast to LNCaP and PC3, the CRPC DU145 cell line is null for Bax, expresses high levels of anti-apoptotic Mcl-1, and is more resistant to ABT-737. Retrovirus pBABE stable expression of Bax in DU145 cells restores sensitivity to Doc + ABT-737 compared to the empty vector control cells. We conclude that ABT-737 can sensitize CRPC cells to Doc treatment and may provide an important combination chemotherapy strategy for improving overall survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2571. doi:10.1158/1538-7445.AM2011-257

ABT-737, a small molecule Bcl-2/Bcl-xL antagonist, increases antimitotic-mediated apoptosis in human prostate cancer cells

Castration-resistant prostate cancer (CRPC) expresses high levels of the anti-apoptotic proteins Bcl-2, Bcl-xL and Mcl-1, resulting in resistance to apoptosis and association with poor prognosis. Docetaxel, an antimitotic drug that is the first-line treatment strategy for CRPC, is known to provide a small survival benefit. However, docetaxel chemotherapy alone is not enough to counteract the high levels of Bcl-2/Bcl-xL/Mcl-1 present in CRPC. ABT-737 is a small molecule that binds to Bcl-2/Bcl-xL (but not Mcl-1) with high affinity and disrupts their interaction with pro-apoptotic Bax/Bak, thus enhancing apoptosis. Our results indicate that ABT-737 can sensitize androgen-dependent LNCaP and CRPC PC3 cells to docetaxel- and to the novel antimitotic ENMD-1198-mediated caspase-dependent apoptosis. CRPC DU145 cells, however, are more resistant to ABT-737 because they are Bax null and not because they express the highest levels of anti-apoptotic Mcl-1 (associated with ABT-737 resistance). Knockdown of Bax or Bak in LNCaP indicates that ABT-737-induced antimitotic enhancement of apoptosis is more dependent on the levels of Bax than Bak. Furthermore, we find that the ability of docetaxel to increase cyclin B1/Cdk1-mediated phosphorylation of Bcl-2/Bcl-xL and decrease Mcl-1 is required for ABT-737 to enhance apoptosis in PC3 cells, as determined by addition of Cdk1 inhibitor purvalanol A and expression of shRNA specific for cyclin B1. Overall, our data suggests that the high levels of anti-apoptotic proteins in Bax-expressing CRPC cells can be overcome by targeting Bcl-2/Bcl-xL with ABT-737 and Mcl-1 with antimitotics

Progression of prostate cancer from a subset of p63-positive basal epithelial cells in FG/Tag transgenic mice

Transgenic mice that allow targeting of SV40 T antigen (Tag) to the prostate provide a unique model to identify cancer-initiating cells and follow their progression from a normal cell phenotype into prostate cancer cells. We have developed the FG/Tag transgenic mouse model of prostate cancer using the human fetal globin (FG) promoter linked to Tag. Immunohistochemistry results show that before the development of prostate intraepithelial neoplasia (PIN), a subset of p63(+) basal epithelial cells expresses Tag. As in the case of human prostate cancer, there is a loss of p63(+) basal cells with neoplastic progression, and a long period of time is required for PIN lesions to develop into palpable prostate tumors. Other immunohistochemistry results show cellular heterogeneity in FG/Tag PIN lesions and primary tumors with neuroendocrine differentiation. Cell lines derived from primary prostate tumors showed characteristics of a neuroendocrine-epithelial intermediate cell type. The FG promoter has high transcriptional activity in intermediate (DU 145, PC-3) and p63(+) basal epithelial (LHSR-AR) prostate cancer cells. Therefore, the unexpected development of prostate cancer in the FG/Tag mice may be due to the presence of DNA elements in the FG promoter that can target Tag to specific basal or intermediate cells. We conclude that FG/Tag mouse is a unique model of prostate cancer because the initiating cells are a subset of p63(+) basal (possibly stem cells), which may be the true cells of origin for carcinogenesis in aggressive human prostate cancer

Low dose combinations of 2-methoxyestradiol and docetaxel block prostate cancer cells in mitosis and increase apoptosis

Clinical trials have shown that chemotherapy with docetaxel (Doc) combined with prednisone can improve survival of patients with androgen-independent prostate cancer (AI-PC). It is likely that the combination of Doc with other novel agents would also improve the survival of AI-PC patients. We investigated whether the combination of Doc and 2-methoxyestradiol (2ME2), an endogenous metabolite of estradiol promising for cancer therapy, can increase apoptotic cell death in prostate cancer cells. Low concentration 2ME2 (0.5−1 μM) + Doc (0.05−0.1 nM) combinations inhibit cell growth, increase G2/M cell cycle arrest, and increase apoptosis more effectively than the single concentrations in a variety of human AI-PC cells. Effects on apoptosis were associated with an increase in p53 protein and a decrease in cyclin A-dependent kinase activity. We then investigated whether the combination of 2ME2 + Doc can increase apoptotic cell death and inhibit the growth of prostate tumors in the FG/Tag transgenic mouse model of AI-PC. Doses of 2ME2 and Doc that increase mitotic cell cycle arrest result in an increase in apoptosis and lower primary prostate tumor weights in FG/Tag mice. High dose 2ME2 + Doc combinations did not increase G2/M cell cycle arrest or apoptosis in AI-PC cell lines and in the FG/Tag mice more than the single drugs. Overall, our data indicate that low dose 2ME2 + Doc combinations may provide a treatment strategy that can improve therapeutic efficacy against AI-PC while reducing toxicity often seen in patients treated with Doc

Abstract 4679: Betulinic acid inhibits deubiquitinases to increase the degradation of pro-survival proteins and enhance prostate cancer-specific apoptosis

Abstract Inhibition of the ubiquitin proteasome (UPS) pathway is a valid anti-cancer target but the alternative strategy to increase activity in order to degrade pro-survival proteins is much less developed. Betulinic acid (BA) is a naturally occurring small molecule with multiple mechanisms for increasing apoptosis in cancer cells, making it an attractive anti-prostate cancer agent. There is evidence that BA can activate the UPS pathway to degrade multiple pro-survival proteins such as androgen receptor (AR), but the mechanism is not clear. Our western blot data in prostate cancer (PC) cells indicates that 10 μM BA decreases the levels of multiple pro-survival proteins including AR, cyclins, and cyclin-dependent kinases. Inhibition of the UPS pathway with 1 μM MG132 blocks the BA-mediated decrease of AR and AKT proteins and lowers cell death, suggesting a dependence on the UPS pathway. In TRAMP transgenic mice, BA treatment (10 mg/kg) inhibits tumor growth, increases apoptosis, decreases angiogenesis and proliferation, and lowers AR and cyclin D1 protein levels (immunohistochemistry) without toxic side effects to normal tissues. However, BA does not increase UPS activity (Proteasome-Glo Chymotrypsin-like Cell-Based Assay; Promega) in LNCaP PC cells suggesting an alternative mechanism for increasing protein degradation. BA-mediated inhibition of deubiquitinase (DUB) activity, as determined by DUB-Glo Protease Assay (Promega) and DUB labeling with HA-ubiquitin vinyl sulfone, results in the accumulation of poly-ubiquitinated (Ub) proteins that are recognized by the UPS pathway and degraded. The cyclin D1 T286A mutant that cannot be degraded by the UPS pathway is resistant to BA-mediated degradation. In non-cancer BJ fibroblast, however, BA does not inhibit DUB activity nor increases total poly-Ub proteins and this is associated with no effect on cell death. Our data suggests that BA-mediated inhibition of DUBs and induction of apoptotic cell death specifically in PC but not in non-cancer cells will provide an effective non-toxic and clinically selective agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4679. doi:1538-7445.AM2012-467

Increased expression of cyclin B1 sensitizes prostate cancer cells to apoptosis induced by chemotherapy

Chemotherapeutic drugs ideally should take advantage of the differences between transformed and normal cells and induce apoptosis only in cancer cells. One such difference may be the overexpression of cyclin B1 protein in cancer cells, which is required for the proper progression through mitosis. Previously, we showed that treatment of human prostate cancer cells with 2-methoxyestradiol (2-ME) or docetaxel results in an accumulation of cyclin B1 protein and an increase in cyclin B1 kinase activity, followed by induction of apoptotic cell death. Inhibition of cyclin B1 kinase lowers apoptosis induced by 2-ME and docetaxel. In this study, we established a positive correlation between cyclin B1 protein and apoptosis induced by chemotherapy in prostate cancer cells. There is minimal cyclin B1 and induction of apoptosis by chemotherapy in nontransformed cells. LNCaP and PC-3 prostate cancer cells stably overexpressing cyclin B1 are more sensitive to apoptosis induced by chemotherapy. LNCaP cells expressing cyclin B1 small interfering RNA to lower cyclin B1 protein or dominant negative cyclin-dependent kinase 1 to inhibit cyclin B1 kinase show a decrease in apoptosis. Increased sensitivity to apoptosis by overexpression of cyclin B1 may be due to lower Bcl-2, higher p53, and decreased neuroendocrine differentiation. We suggest that a cancer-specific mechanism whereby 2-ME and docetaxel may exert anti-prostate cancer activity is the deregulated activation of cyclin B1 kinase, leading to the induction of apoptotic cell death. Our results also suggest that higher levels of cyclin B1 in prostate cancer cells may be a good prognostic marker for chemotherapy

Abstract B14: Human Mut T homolog 1 (MTH1) as a novel facilitator of KRAS-driven lung cancer

Abstract Non-small cell lung carcinoma (NSCLC), a chemoresistant form of lung cancer, is characterized by oncogenic KRAS mutations that confer multiple tumor-promoting traits. Several of these traits are mediated by oncogene-induced reactive oxygen species (ROS). However, oncogenic ROS also produce DNA damage, a major effector of tumor suppressor mechanisms. We previously reported overexpression of the mammalian 8-oxo-dGTP triphosphatase, human MutT Homolog 1 (MTH1), inhibits RAS oncogene-induced senescence (OIS) and any associated DNA damage without substantially affecting ROS levels. Here we report that MTH1 expression is elevated in NSCLC tumors relative to adjacent normal tissue, particularly in tumors with high KRAS expression. We show that MTH1 overexpression facilitates transformation of lung epithelial cells by oncogenic KRAS. We further report that MTH1 suppression in NSCLC cell lines reduces proliferation and tumorigenicity either via DNA strand break-induced elevation of the p53/p21 pathway or by inhibition of Akt activation in p53-nonfunctional tumor cells. Collectively our results indicate MTH1 as a novel potential chemotherapeutic target in NSCLC with activating KRAS mutations. Citation Format: Asmita Patel, Katherine Halvorsen, Wayne Balkan, Alexander Cohen, Teresita Reiner, David Robbins, Dao Nguyen, Priyamvada Rai. Human Mut T homolog 1 (MTH1) as a novel facilitator of KRAS-driven lung cancer. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B14. doi: 10.1158/1557-3125.RASONC14-B14</jats:p