Paul Rivas

Dietary Resveratrol prevents development of high-grade prostatic intraepithelial neoplastic lesions: Involvement of SIRT1/S6K axis

SIRT1 (mammalian ortholog of the yeast silent information regulator 2) is a NAD-dependent histone deacetylase belonging to the multigene family of sirtuins. Anecdotal and epidemiologic observations provide evidence for beneficial effects of the calorie restriction mimetic resveratrol (RES), a SIRT1 activator in preventing cardiovascular diseases and cancer. Although SIRT1 possesses both tumorigenic and antitumorigenic potential, the molecular mechanisms underlying SIRT1-mediated tumor progression or inhibition are poorly understood. In this study, we investigated the role of SIRT1 in multiple human prostate cancer cell lines and prostate-specific PTEN knockout mouse model using resveratrol. Androgen-independent prostate cancer cell lines (C42B, PC3, and DU145) express higher levels of SIRT1 than androgen-responsive (LNCaP) and nontumorigenic prostate cells (RWPE-1). Resveratrol enhanced this expression without any significant effect on SIRT1 enzymatic activity. Inhibition of SIRT1 expression using shRNA enhanced cell proliferation and inhibited autophagy by repressing phosphorylation of S6K and 4E-BP1. These biologic correlates were reversed in the presence of resveratrol. Analysis of prostates from dietary intervention with resveratrol showed a significant reduction in prostate weight and reduction in the incidence of high-grade prostatic intraepithelial neoplastic (HGPIN) lesions by approximately 54% with no significant change in body weight. Consistent with the in vitro findings, resveratrol intervention in the PTEN knockout mouse model was associated with reduction in the prostatic levels of mTOR complex 1 (mTORC1) activity and increased expression of SIRT1. These data suggest that SIRT1/S6K-mediated inhibition of autophagy drives prostate tumorigenesis. Therefore, modulation of SIRT1/S6K signaling represents an effective strategy for prostate cancer prevention. Cancer Prev Res; 6(1); 27–39. ©2012 AACR.

Combined Targeting of STAT3/NF-κB/COX-2/EP4 for Effective Management of Pancreatic Cancer

Purpose: Near equal rates of incidence and mortality emphasize the need for novel targeted approaches for better management of patients with pancreatic cancer. Inflammatory molecules NF-κB and STAT3 are overexpressed in pancreatic tumors. Inhibition of one protein allows cancer cells to survive using the other. The goal of this study is to determine whether targeting STAT3/NF-κB crosstalk with a natural product Nexrutine can inhibit inflammatory signaling in pancreatic cancer. Experimental Design: HPNE, HPNE-Ras, BxPC3, Capan-2, MIA PaCa-2, and AsPC-1 cells were tested for growth, apoptosis, cyclooxygenase-2 (COX-2), NF-κB, and STAT3 level in response to Nexrutine treatment. Transient expression, gel shift, chromatin immunoprecipitation assay was used to examine transcriptional regulation of COX-2. STAT3 knockdown was used to decipher STAT3/NF-κB crosstalk. Histopathologic and immunoblotting evaluation was performed on BK5–COX-2 transgenic mice treated with Nexrutine. In vivo expression of prostaglandin receptor E-prostanoid 4 (EP4) was analyzed in a retrospective cohort of pancreatic tumors using a tissue microarray. Results: Nexrutine treatment inhibited growth of pancreatic cancer cells through induction of apoptosis. Reduced levels and activity of STAT3, NF-κB, and their crosstalk led to transcriptional suppression of COX-2 and subsequent decreased levels of prostaglandin E2 (PGE2) and PGF2. STAT3 knockdown studies suggest STAT3 as negative regulator of NF-κB activation. Nexrutine intervention reduced the levels of NF-κB, STAT3, and fibrosis in vivo. Expression of prostaglandin receptor EP4 that is known to play a role in fibrosis was significantly elevated in human pancreatic tumors. Conclusions: Dual inhibition of STAT3–NF-κB by Nexrutine may overcome problems associated with inhibition of either pathway. Clin Cancer Res; 20(5); 1259–73. ©2014 AACR.

Suppression of ribosomal protein RPS6KB1 by Nexrutine increases sensitivity of prostate tumors to radiation

Radiation therapy (XRT) is a standard treatment for prostate cancer (PCa). Although dose escalation increases local control, toxicity hampers further escalation. Broader improvement will be possible by the addition of adjuvant therapies, which can synergize with radiation and thus improve efficacy. We have identified a natural compound (Nexrutine, Nx) that inhibits the survival and growth of PCa cells in combination with radiation. Combination studies demonstrated strong interaction between Nx and radiation both in vitro in multiple PCa cell lines and in the Transgenic adenocarcinoma of mouse prostate (TRAMP) model. Nx potentiated growth inhibitory effects of IR by down regulating ribosomal protein S6K (RPS6KB1), CyclinD1, Chk1 and HIF-1 α and prolonging G2/M checkpoint block. RPS6KB1 is upregulated in prostate cancers and its expression is correlated with tumor grade. Knockdown of RPS6KB1 in PCa cells increased their sensitivity toward radiation-induced survival inhibition. Overall, we provide scientific evidence (i) in support of Nx as an adjuvant in PCa patients receiving XRT (ii) suggesting that RPS6KB1 is an important player in Nx-mediated combinatorial benefits and emphasizes that RPS6KB1 is a novel target for PCa treatment. These data underscore the need to test the agent in additional preclinical models to validate these observations.

MP87-15 INHIBITION OF RPS6KB1 AS A POTENTIAL ADJUVANT FOR PROSTATE CANCER RADIATION THERAPY

INTRODUCTION AND OBJECTIVES: While new prostate cancer (CaP) treatments (Abiraterone and Enzalutamide) have improved survival in castration resistant prostate cancer (CRPC), their benefits are short-lived and drug resistance develops likely due to numerous adaptive mutations. Accumulating evidence has established the androgen regulated TMPRSS2-ERG fusion as a common oncogenic driver that contributes to the early development and progression of over half of CaP. Therefore, ERG oncoprotein and ERG dependent pathways are promising targets for CaP therapy in early stages when cancer is most responsive to treatment. We previously identified a small molecule inhibitor, ERGi-USU, which selectively inhibits ERG protein and cell growth in ERG positive tumor cell lines and mouse xenograft models. In an effort to further develop ERGi-USU with enhanced efficacy we performed detailed structure-activity relationship (SAR) evaluation of ERGi-USU core structure and developed new derivatives. METHODS: Based on SAR of the core structure of ERGi-USU, 48 new derivatives were designed and synthesized by substitutions with alkyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl, heteroaryl or hydroxyl groups. The new ERGi-USU derivatives were evaluated for inhibition of cell growth and ERG protein levels in the TMPRSS2-ERG fusion harboring CaP cell line, VCaP. Four of these compounds have been selected for evaluation of ERG selectivity by defining IC50 in ERG positive malignant cells (VCaP, KG1, MOLT-4 and COLO320), ERG negative CaP cell line (LNCaP) or ERG positive normal primary endothelium-derived cells (HUVEC). RESULTS: Like parental compound, four new ERGi-USU derivatives exhibited inhibition of cell growth and ERG protein levels in ERG positive VCaP, KG1, MOLT-4 and COLO320 cell lines, with no or minimal effects on LNCaP and HUVEC cells. One of the new derivatives (ERGi-USU#6) showed increased efficacy for cell growth inhibition (IC501⁄40.074mM) compared to the parental ERGi-USU (IC501⁄40.200mM). Other three new compounds showed similar IC50 as the ERGi-USU. CONCLUSIONS: Comprehensive evaluation of ERGi-USU derivatives along with parental compound has continued to underscore selective inhibition of ERG positive tumor cells by these small molecules.

Abstract 1797: Downregulation of ribosomal protein S6 overcomes radioresistance in prostate cancer

Radiation Therapy (RT) is a definitive treatment for early-localized prostate cancer (PCA), but is associated with side effects which impair quality of life in addition to development of radioresistance. PI3K/Akt/mTOR signaling is one of the contributors to therapeutic resistance, including radioresistance. Ribosomal protein S6 (rpS6), a downstream effector of PI3K/Akt/mTOR signaling, mediates radioresistance by increasing protein synthesis, cell survival and epithelial mesenchymal transition. Also, increased activation of rpS6 is correlated with poor survival in PCA. Therefore, downregulation of rpS6 could decrease RT induced toxic side effects by sensitizing tumor cells. Based on published evidence demonstrating tumor growth inhibitory activities, we tested if Nexrutine® (Nx), an inexpensive OTC herbal supplement from Phellodendron amurense bark extract, could potentiate RT by inhibiting rpS6 activation. Using clonogenic assays, low dose RT in combination with Nx was found to have similar inhibition of surviving fraction compared to high dose RT in androgen independent PC-3 cells. Isobologram analysis of these data depicted strong synergism. In addition, increased activation of Akt/NFKB signaling molecules was observed in PC-3 cells exposed to radiation. The observed radiation-induced increase in these signaling molecules was either abolished (p-rpS6, p-NFKB and p-p70S6K) or decreased (p-Akt) in cells pretreated with Nx (8 hours) prior to RT. Additionally, Nx pretreatment prolonged the G2/M arrest caused by RT in PC-3 cells. Strikingly, knockdown of rpS6 in PC-3 cells reversed the observed effects of Nx, indicating the importance of rpS6 in mediating these changes. Furthermore, administration of Nx in combination with RT inhibited prostate tumor progression in transgenic adenocarcinoma of the mouse prostate (TRAMP) mice with no prominent toxicity. Notably, immunohistochemical analysis revealed decreased levels of p-mTOR, p70S6K, NFKB, Ki67 and Cyclin D1 in the prostate in combination group of animals compared to monotherapy. Taken together, our data suggest that Nx sensitizes PCA cells by down regulating rpS6 and delays progression to lethal disease. Remarkably, in a recent phase 0/1 study patients (81%) receiving Nx showed decreased PSA levels with no signs of grade 3 toxicity. Thus, Nx shows immense potential for use as an adjuvant in combination with conventional therapy for effective clinical management of PCA. Supported by NCCAM (R01 AT-007448) and VA-MERIT Award (I01 BX 000766; APK) Citation Format: Suleman S. Hussain, Paul Rivas, Roble G. Bedolla, Nikos Papanikolaou, Robert L. Reddick, Brad H. Pollock, Daniel C. Chan, Rita Ghosh, Addanki P. Kumar. Downregulation of ribosomal protein S6 overcomes radioresistance in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1797. doi:10.1158/1538-7445.AM2015-1797

Abstract 5392: Metabolic profiling of castrate-resistant prostate cancer reveals novel role for bile acids in driving castration resistance.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The 5-year survival for metastatic castrate-resistant prostate cancer patients is less than 30% despite significant progress in the understanding of prostate cancer biology and development of novel therapeutic agents. A major contributing factor for the observed low survival rate of patients with castrate resistant disease is the lack of knowledge regarding metabolic alterations and their underlying contributions during development of castrate-resistant phenotype. Studies conducted in our laboratory and others identified a potential role for 2-methoxyestradiol (2-ME2) to prevent prostate cancer development and progression through inhibition of the anti-apoptotic protein FLIP. The goal of the current study was to identify biochemical changes in response to castration and treatment with 2-ME2 in serum from transgenic adenocarcinoma of mouse prostate (TRAMP) mice using mass spectrometry based global profiling. We identified a total of 54 biochemicals of which 16 increased and 38 decreased in castrated animals compared to sham-castration. Treatment of sham-castrated animals with low and high doses of 2-ME2 altered 91 and 145 biochemicals respectively. On the other hand treatment of castrated animals modulated 89 and 106 biochemicals. Cumulative analysis of these data also identified alteration of 60 biochemicals associated with castration effect, 149 with treatment and 70 interactions between castration and treatment effects. Castration affected metabolites involved in variety of metabolic pathways including lipid, oxidative stress, energetics and bile acid. Given the data showing enhanced expression of FLIP in castrate-resistant prostate tumors and upregulation of bile acids in patients undergoing androgen deprivation therapy, we examined the activation of FLIP in prostate cancer cells in response to deoxycholic acid (DCA). Our data suggests that transcriptional activity of FLIP was higher in PC-3 cells treated with DCA. To the best of our knowledge, this is the first report demonstrating global metabolomic profiling of serum in response to castration and provide a framework for therapeutic targeting of bile acid metabolism. Supported by NIH CA 135451 (APK). Citation Format: Divya Chakravarthy, Paul Rivas, Brian Keppler, Jianhua Ruan, Rita Ghosh, Addanki Pratap Kumar. Metabolic profiling of castrate-resistant prostate cancer reveals novel role for bile acids in driving castration resistance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5392. doi:10.1158/1538-7445.AM2013-5392 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 3673: AMPK-SIRT1 axis: a potential therapeutic target for prostate cancer management.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Recently we have shown that resveratrol (RES) intervention prevents development of high-grade prostatic intraepithelial neoplastic (HGPIN) lesions in prostate-specific PTEN knockout mouse model targeting SIRT1/mTORC1 axis. It is known that insufficient nutrient supply combined with high proliferation keeps solid tumors including prostate under hypoxic and metabolic stress. Tumor cells adapt to survive under such conditions through activation of AMP-activated kinase (AMPK). AMPK has been reported to be activated in prostate tumors. Therefore targeting AMPK and associated signaling pathways will be a promising approach for prostate cancer management. Accordingly we investigated the role of AMPK in RES-induced growth inhibitory effects using multiple human prostate cancer cell lines and preclinical animal model. These data show that RES treatment (50 μM, 24 h) results in activation of SIRT1, significant inhibition of AMPK phosphorylation and cell survival in human prostate cancer cells (RWPE-1, LNCaP, C42B and DU145). The observed molecular changes were associated with induction of apoptosis and autophagy following treatment with RES. Further dietary administration of RES (0.1 and 2%) to 4-5 week old prostate-specific PTEN knockout mice for 11 and 14 weeks showed prevention of HGPIN development. Interestingly intervention for 7 weeks showed prevention of HGPIN development at lower dose but not at high dose. On the other hand, 28-week intervention had no significant effect on the development of HGPIN lesions. Immunohistochemical evaluation showed modulation of AMPK, pS6K and SIRT1 in the prostate. Overall these data provide novel insights into RES-induced prevention of HGPIN development via AMPK/SIRT1/mTORC1 axis. Supported in part by NIH (CA 137518 and 135451 APK). Citation Format: Guiming Li, Paul Rivas, Roble Bedolla, Robert L. Reddick, Rita Ghosh, Addanki P. Kumar. AMPK-SIRT1 axis: a potential therapeutic target for prostate cancer management. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3673. doi:10.1158/1538-7445.AM2013-3673

Abstract 2585: Resveratrol induces autophagy in prostate cancer cells and intervention suppresses the progression of PIN in animals

Resveratrol (3,4′-trihydroxystilbene), a natural product is present in significant concentrations in red wine, peanuts, walnuts etc. Emerging evidence indicates that resveratrol exerts antitumorigenic activity in various tumor models including prostate. Although multiple biological effects on proliferation, apoptosis and activation of SIRT1 have been demonstrated to be associated with resveratrol-induced activities, the precise mechanism associated with resveratrol-induced SIRT1 activation and cancer cell growth inhibition remains unclear. In addition, whether resveratrol intervention suppresses the development and progression of PIN (Prostatic Intraepithelial Neoplasia) is unknown. In this study, we examined the role of SIRT1 and the underlying mechanism involved in resveratrol-mediated biological effects. Our results show that although resveratrol significantly inhibited proliferation of multiple prostate cell lines including RWPE-1, C42B, PC3 and DU145 at higher concentrations (> 50 µM); however, at lower concentrations ( −/− mice, we found that dietary administration of resveratrol reduced the incidence of high-grade PIN lesions significantly. These findings implicated an important role for mTORC1/SIRT1 signaling axis in mediating resveratrol-induced upregulation of autophagy and inhibition of apoptosis. [Supported by NIH CA137578 (APK)] 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 2585. doi:1538-7445.AM2012-2585

Abstract 851: Interactions among transcription factors as a potential mechanism for inhibiting castrate-resistant prostate cancer (CRPCa) in transgenic adenocarcinoma of mouse prostate (TRAMP) through regulation of FLIP

Androgens are critical regulators of prostate differentiation and function as well as prostate cancer growth and survival. Prostate tumors initially regress in response to androgen-ablation therapy. However, most cancers eventually relapse with an androgen-depletion-independent phenotype that is often more aggressive than the original androgen-dependent tumor. Studies from our laboratory demonstrated that intervention with 2-ME 2 [2-methoxyestradiol] prevents early stage prostate cancer development and causes regression of established prostate tumors in the transgenic adenocarcinoma of mouse prostate model. Although 2-ME 2 was found to be safe, well tolerated, reduce or stabilize PSA levels when given to hormone refractory prostate cancer patients who had failed other treatments including hormone therapy, it was found to be less bioavailable. Accordingly second generation 2-ME 2 was developed using nanocrystal colloidal dispersion technology and has increased its bioavailability by 5-10 fold (EntreMed, Inc, Rockville, MD). In the current study we tested the ability of this newly formulated 2-ME 2 on the development and progression of castrate-resistant prostate cancer in TRAMP mice. 10-12 week old TRAMP mice were castrated and fed water containing different amounts of 2-ME 2 for 6 weeks. Efficacy was evaluated by magnetic resonance imaging, determining the prostate seminal vesicle complex volume and histological analysis of prostate tumor or tissue. Our results also show that 2-ME 2 inhibited development of CRPCa in these mice. Further molecular analysis using cell lines demonstrated 2-ME 2 treatment significantly inhibited DHT-stimulated cell proliferation in androgen-responsive LNCaP cells (p 2 (p 2 treatment reduced such binding to the FLIP promoter as evidenced by ChIP. 2-ME 2 treatment also modulated the interactions among these transcription factors as evidenced by Re-ChIP assays. Interestingly transcription factor CREB but not Sp1 seems to play a predominant role in androgen regulation of FLIP expression. Our findings suggest that 2-ME 2 affects androgen induced survival of prostate cancer cells through regulation of FLIP involving potential interactions between multiple transcription factors. Supported by ACS RSG-04-169 and NIH CA 135451 (APK). 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 851. doi:10.1158/1538-7445.AM2011-851