CRPC Cells Research Articles

Evaluating Metformin as a Potential Chemosensitizing Agent when Combined with Docetaxel Chemotherapy in Castration-resistant Prostate Cancer Cells.

Docetaxel, the first-line chemotherapy for metastatic castration-resistant prostate cancer (mCRPC), provides certain survival benefits, but is associated with significant toxicity. A novel therapeutic approach for mCRPC is combining docetaxel with a chemosensitizing agent. We hypothesized that metformin, a potential chemosensitizer, would improve docetaxel efficacy in CRPC cells. MTS assays were used to determine the effect of metformin-docetaxel treatment on PC3 and DU145 cell viability. Wound-healing and ATP concentration assays were used to evaluate cell migration and intracellular ATP levels following metformin-docetaxel treatment. Western blotting was used for mechanistic evaluation. Metformin-docetaxel treatment significantly reduced PC3 cell viability. Metformin-docetaxel treatment did not significantly affect cell migration or intracellular ATP levels. Western blotting revealed metformin-docetaxel treatment did not significantly change AMPK or P-AMPK expression patterns. Metformin may be an effective chemosensitizer for certain types of CRPC cells, but further investigation is needed.

Targeting AR Variant-Coactivator Interactions to Exploit Prostate Cancer Vulnerabilities.

Castration-resistant prostate cancer (CRPC) progresses rapidly and is incurable. Constitutively active androgen receptor splice variants (AR-Vs) represent a well-established mechanism of therapeutic resistance and disease progression. These variants lack the AR ligand-binding domain and, as such, are not inhibited by androgen deprivation therapy (ADT), which is the standard systemic approach for advanced prostate cancer. Signaling by AR-Vs, including the clinically relevant AR-V7, is augmented by Vav3, an established AR coactivator in CRPC. Using mutational and biochemical studies, we demonstrated that the Vav3 Diffuse B-cell lymphoma homology (DH) domain interacted with the N-terminal region of AR-V7 (and full length AR). Expression of the Vav3 DH domain disrupted Vav3 interaction with and enhancement of AR-V7 activity. The Vav3 DH domain also disrupted AR-V7 interaction with other AR coactivators: Src1 and Vav2, which are overexpressed in PC. This Vav3 domain was used in proof-of-concept studies to evaluate the effects of disrupting the interaction between AR-V7 and its coactivators on CRPC cells. This disruption decreased CRPC cell proliferation and anchorage-independent growth, caused increased apoptosis, decreased migration, and resulted in the acquisition of morphological changes associated with a less aggressive phenotype. While disrupting the interaction between FL-AR and its coactivators decreased N-C terminal interaction, disrupting the interaction of AR-V7 with its coactivators decreased AR-V7 nuclear levels.Implications: This study demonstrates the potential therapeutic utility of inhibiting constitutively active AR-V signaling by disrupting coactivator binding. Such an approach is significant, as AR-Vs are emerging as important drivers of CRPC that are particularly recalcitrant to current therapies. Mol Cancer Res; 15(11); 1469-80. ©2017 AACR.

Abstract 1180: Targeting the HSP40/HSP70 chaperone axis as a novel strategy to treat castration-resistant prostate cancer

Abstract Castration-resistant prostate cancer (CRPC) is frequently characterized by elevated expression of nuclear receptors able to at least partially maintain the androgen receptor (AR) transcriptional program. Elevated expression of a number of constitutively active AR splice variants lacking the ligand binding domain (LBD) (e.g., ARv7, which is ligand-independent and correlates with poor prognosis, reduced survival, and resistance to existing LBD-targeted standard of care therapy) is a frequent occurrence in CRPC. Thus, alternative approaches to disrupt AR signaling in CRPC are of great clinical importance, and a single strategy able to target AR and ARv7 remains a critical unmet need. As a steroid hormone nuclear receptor, the AR exists in an interactive and dynamic cycle with the molecular chaperones (heat shock proteins, HSPs) HSP40/HSP70/HSP90 for proper folding and remodeling of the AR LBD to bind ligand. Notably, HSP90 inhibitors promote AR degradation and display efficacy in prostate cancer xenograft models. Although it has been shown that ARv7 functions independently of HSP90, additional chaperone requirements of LBD-deficient ARv7 are not known. Thus, we tested the hypothesis that both AR and ARv7 are dependent on HSP40/HSP70 and that targeting these chaperones with specific inhibitors (C86 and JG98, respectively) will lead to AR/ARv7 destabilization and loss of transcriptional activity in models of CRPC. To determine if AR proteins associate with HSP40/HSP70, 22Rv1 CRPC cells (expressing endogenous AR and ARv7) were first transfected with FLAG-HSP40 or FLAG-HSP70. Immunoprecipitation with FLAG beads revealed AR and ARv7 associated with both chaperones, indicating potential functional dependence of these nuclear receptors on HSP40/HSP70. To further characterize these interactions, 22Rv1 lysate was probed with biotinylated-C86 and subjected to IP with streptavidin beads. C86 bound a significant fraction of HSP40 complexed with HSP70, AR, and ARv7. Excess unlabeled C86 or JG98 effectively competed away binding of HSP40/HSP70 to biotinylated-C86 with concomitant loss of associated AR and ARv7. Treatment of 22Rv1 cells with C86 or JG98 led to a time and dose-dependent decrease in AR and ARv7 protein, concomitant with a significant loss of viability. We also observed that HSP40/HSP70 inhibition markedly reduced AR and ARv7 transcriptional activity, as indicated by decreased AR (KLK3, TMPRSS2) and ARv7 (UBE2C) target gene expression. Finally, treatment of mice bearing 22Rv1 xenografts with JG231 (an analog of JG98 with enhanced PK properties) led to significantly smaller tumors relative to vehicle treated mice. Together, these data confirm the continued dependence of AR and ARv7 on HSP40/HSP70 molecular chaperones and they demonstrate the feasibility of targeting the HSP40/HSP70 axis to abrogate sustained AR-mediated signaling in CRPC. Citation Format: Michael A. Moses, Yeong Sang Kim, Genesis Rivera-Marquez, Matthew J. Watson, Sunmin Lee, Andrea Kravats, Sue Wickner, Jason Gestwicki, Jane Trepel, Len Neckers. Targeting the HSP40/HSP70 chaperone axis as a novel strategy to treat castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1180. doi:10.1158/1538-7445.AM2017-1180

Understanding docetaxel and cabazitaxel modes of action in prostate cancer.

e23149 Background: Despite the approval of several new agents, taxanes remains the main treatment with survival benefits for castration-refractory metastatic prostate cancer/mCRPC. Still their mechanisms of action and therapeutic efficacy remain incompletely characterized. In the present study, we proposed to compare docetaxel/Doc and cabazitaxel/Cab, delivered as monotherapy or in combination with the anti-angiogenic and anti-tumoral Pigment Epithelium-Derived Factor/PEDF, on CRPC cells in vitro and in vivo. Methods: CRPCcells were assessed for cell growth, cell cycle, and apoptosis under taxane/control treatment by cytotoxicity, PI incorporation and TUNEL assays. CL1 cells that express PEDF/control were used in vivo. Tumor cells were injected s.c. into CB17-SCID mice. After two weeks, mice were randomized into groups: 1) Placebo; 2) Doc 5mg/kg i.p. d4; and 3) Cab 5-1-0.5-0.1mg/kg i.p. d4, d1-7, d2, daily. To assess the anti-tumor effect of the combination, tumor cell migration and phagocytosis were measured by Boyden chamber and confocal microscopy analyses of CL1-RAW264.7 macrophages co-cultures. Results: Cab was more cytotoxic than Doc in all the cell lines tested.This effect was concomitant to increased cell death, but was not due to autophagy or necrosis. Inversely, we showed that apoptosis was superior in Cab-treated cells than in Doc treatment. In vivo, while 0.5 and 0.1 mg/kg Cab did not improve PEDF efficacy on tumor growth, 1mg/kg was very toxic. In contrast, PEDF combined with 5mg/kg Cab lead to stabilization of the disease and was also found to be drastically more efficient than PEDF/Doc in delaying the disease. In vitro, PEDF/Cab inhibited tumor cell migration at a significant superior level compared to PEDF/Doc. Finally, tumor cells phagocytosis was induced in PEDF/Cab suggesting that the combination may target macrophages within the tumor microenvironment. Conclusions: Our data demonstrated the greater anti-tumor efficacy of Cab compared to Doc. They also insist on the fact that PEDF/Cab could be used as a novel combined therapy for CRPC, and emphasize on the importance in evaluating the cytotoxicity of the novel combination tested and investigating the role of the tumor microenvironment in the anti-tumor effect.

MP83-13 ZOLEDRONIC ACID SENSITIZES CASTRATION-RESISTANT PROSTATE CANCER CELLS TO RADIOTHERAPY AND CHEMOTHERAPY BY DOWNREGULATING STAT1

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology II1 Apr 2017MP83-13 ZOLEDRONIC ACID SENSITIZES CASTRATION-RESISTANT PROSTATE CANCER CELLS TO RADIOTHERAPY AND CHEMOTHERAPY BY DOWNREGULATING STAT1 Takayuki Nakayama, Toshiki Kijima, Soichiro Yoshida, Fumitaka Koga, Kazunori Kihara, and Yasuhisa Fujii Takayuki NakayamaTakayuki Nakayama More articles by this author , Toshiki KijimaToshiki Kijima More articles by this author , Soichiro YoshidaSoichiro Yoshida More articles by this author , Fumitaka KogaFumitaka Koga More articles by this author , Kazunori KiharaKazunori Kihara More articles by this author , and Yasuhisa FujiiYasuhisa Fujii More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.2581AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Management of castration-resistant prostate cancer (CPRC) remains challenging due to the inevitable emergence of resistance to treatments including radiotherapy (RT) and chemotherapy (CT). We previously reported that zoledronic acid (ZOL) clinically potentiates the antitumor effects of RT in patients with renal cell carcinoma (Kijima et al, BJU Int 2009) and that this radiosensitization could occur through the osteoclast-independent inhibition of signal transducer and activator of transcription 1 (STAT1) (Kijima et al, PLoS One 2013). As the association between STAT1 overexpression and treatment resistance has been reported in several cancers, we investigated whether STAT1 is associated with resistance to RT and CT in CRPC cells and whether ZOL could overcome this resistance by downregulating STAT1. METHODS Baseline expression of STAT1 was compared between androgen-dependent LNCaP cells and androgen-independent LNCaP (LNCaP-CR), PC3, and DU145 cells. The effect of ZOL on STAT1 expression was evaluated by Western blot and real-time PCR. The sensitizing effects of ZOL on RT and CT (docetaxel) were examined by clonogenic assay and MTS assay with combination index analysis. To confirm the importance of STAT1 on radio- and chemo-sensitization by ZOL, both siRNA knockdown and forced expression by cDNA transfection were performed. RESULTS STAT1 levels were higher in androgen-independent cell lines (PC3, DU145) than in LNCaP cells (Figure A). STAT1 was gradually upregulated in LNCaP as these cells acquired androgen independency through continuous androgen ablation (Figure B). ZOL decreased STAT1 at the protein level (Figure C) through proteasome-mediated degradation and sensitized PC3 and DU145 to both RT and CT. Functional siRNA knockdown of STAT1 resulted in the sensitization of DU145 to RT and CT. Forced expression of STAT1 in LNCaP cells rendered them resistant to those therapies. CONCLUSIONS ZOL sensitizes CRPC cells to RT and CT by downregulating STAT1. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e1111 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Takayuki Nakayama More articles by this author Toshiki Kijima More articles by this author Soichiro Yoshida More articles by this author Fumitaka Koga More articles by this author Kazunori Kihara More articles by this author Yasuhisa Fujii More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

MP28-14 DECREASED EXPRESSION OF MALE SPECIFIC HISTONE DEMETHYLASE “KDM5D” IS PROGNOSTIC FOR DEVELOPMENT OF CASTRATION-RESISTANT PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Markers I1 Apr 2017MP28-14 DECREASED EXPRESSION OF MALE SPECIFIC HISTONE DEMETHYLASE “KDM5D” IS PROGNOSTIC FOR DEVELOPMENT OF CASTRATION-RESISTANT PROSTATE CANCER Kazumasa Komura, Seong Ho Jeong, Haruhito Azuma, Gwo-Shu Lee, Christopher Sweeney, and Philip Kantoff Kazumasa KomuraKazumasa Komura More articles by this author , Seong Ho JeongSeong Ho Jeong More articles by this author , Haruhito AzumaHaruhito Azuma More articles by this author , Gwo-Shu LeeGwo-Shu Lee More articles by this author , Christopher SweeneyChristopher Sweeney More articles by this author , and Philip KantoffPhilip Kantoff More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.827AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES We previously showed that decreased expression of the Lysine-Specific Demethylase ′KDM5D′ encoded on the Y chromosome was associated with docetaxel resistance (Komura et al, 2016, PNAS). We hypothesize that loss of KDM5D may significantly affect the epigenetic landscape in prostate cancer cells and facilitate the development of CRPC. METHODS To elucidate biological function of KDM5D, we performed RNAseq analysis in hormone sensitive LNCaP cells (KDM5D positive) and corresponding LNCaP-104R2 CRPC cells (KDM5D negative). Individual genes which were found as potential targets of KDM5D were further explored in a publically available clinical database. RESULTS We found 143 overlapping genes, which are upregulated by knockdown of KDM5D in LNCaP and down regulated by overexpression of KDM5D in 104R2 and 28 genes, which were down regulated by KDM5D knockdown in LNCaP and up-regulated by KDM5D overexpression in 104R2. Gene ontology (GO) analyses with FDR<0.05 from the 143 genes identified mitotic and cell cycle related genes as most commonly upregulated by loss of KDM5D (Figure 1). To validate the results, we explored the Taylor′s prostate cancer cohort with cBioportal. Of 8643 genes negatively correlated with KDM5D expression level (Pearson Correlation Coefficient < -0.3), 69 genes were identified in both our data and the Taylor′s cohort. Upregulation of these genes in CRPC was further confirmed in 2 publicly available datasets (PAD).2 (Figure 2). Finally in a PAD from a Mayo Clinic′s cohort (Illumina DASL Cancer Panel microarray) which included 8 genes out of the 69 genes, noted shorter cancer-specific mortality in pts with higher expression of those genes was demonstrated in all 8 genes. CONCLUSIONS Loss of KDM5D is associated with upregulation of mitotic and cell-cycle related genes which may lead to development of CRPC and serve as prognostic factor for its development. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e343-e344 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Kazumasa Komura More articles by this author Seong Ho Jeong More articles by this author Haruhito Azuma More articles by this author Gwo-Shu Lee More articles by this author Christopher Sweeney More articles by this author Philip Kantoff More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Abstract B22: Increased phosphorylation of eIF4E induces resistance to treatment with mTOR inhibitors together with AR antagonists in advanced prostate cancer

Abstract The anti-androgen bicalutamide is widely used in the treatment of advanced prostate cancer (CaP) in many countries, but its effect on castration resistant CaP (CRPC) is limited. We previously showed that resistance to bicalutamide results from increased activation of the mechanistic target of rapamycin (mTOR); indicating a role for mTOR inhibitors in CaP treatment. Interestingly, a clinical trial testing combinations of the mTOR inhibitor RAD001 with bicalutamide were initially effective (PSA decline&amp;gt;50%) in 62.5% CRPC patients, but many initial responders later developed resistance. Here we investigate causes for their difference in response. Evaluation of various CRPC cell lines identified resistant vs sensitive in vitro models, and revealed that increased eIF4E phosphorylation at S209 is associated with resistance to the combination. To recognize potential causes of resistance, we demonstrated using a human tumor xenograft mouse model, that bicalutamide use is associated with an increase in eIF4E(S209) phosphorylation. Investigations revealed that AR suppressed eIF4E phosphorylation, while the use of anti-androgens relieved this suppression, thereby triggering its increase. Additional investigation on the consequences of increased eIF4E phosphorylation in human prostatectomy samples revealed that increased eIF4E phosphorylation strongly correlated with the cell proliferation marker Ki67, indicating a role for eIF4E in tumor growth. Inhibition of eIF4E phosphorylation by siRNA suppression or treatment with CGP57380 (an inhibitor of MAPK interacting serine-threonine kinases Mnk1/2, the eIF4E upstream kinase,) sensitized CRPC cells to RAD001+bicalutamide, while eIF4E overexpression induced resistance. Our results demonstrate that bicalutamide treatment induced an increase in eIF4E phosphorylation by inhibition of AR which otherwise suppressed this phosphorylation. Increased eIF4E phosphorylation then induced resistance to the combination of anti-androgens with mTOR inhibitors by increasing proliferation. These results also indicate that resistance to RAD001+bicalutamide can be prevented by the concurrent use of a Mnk1/2 inhibitor. Citation Format: Leandro S. D'Abronzo, Michael Crapuchettes, Ryan Beggs, Ruth Vinall, Clifford Tepper, Salma Siddiqui, Maria Mudryj, Frank Melgoza, Blythe Durbin-Johnson, Ralph deVere White, Paramita Ghosh. Increased phosphorylation of eIF4E induces resistance to treatment with mTOR inhibitors together with AR antagonists in advanced prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr B22.

Modes of action of low-dose cabazitaxel in combination with the antiangiogenic and antitumoral pigment epithelium-derived factor in prostate cancer.

275 Background: Despite the approval of several new agents, taxanes remain the main alternative treatment for castration-refractory metastatic prostate cancer (mCRPC). In a previous work, we have demonstrated that cabazitaxel is more efficient in a low-dose setting than docetaxel in inhibiting the proliferation and inducing apoptosis of CRPC cells in vitro, and delaying tumor growth in vivo. In the present study, we have investigated the efficacy of low-dose cabazitaxel when combined with the anti-angiogenic and anti-tumoral Pigment Epithelium-Derived Factor (PEDF). Methods: CRPC CL1 cells (LNCaP-derivative) that express PEDF or control plasmid were used. Tumor cells were injected s.c. into C.B.17-SCID mice. After two weeks, mice were randomized into groups: 1) Placebo; 2) Docetaxel: 5mg/kg i.p. d4; and 3) Cabazitazel 5-1-0.5-0.1mg/kg i.p. d4, d1-7, d2, and daily. To investigate the molecular mechanisms involved in the anti-tumor effect of the combined treatment, tumor cell migration was measured using the inverted Boyden chamber assay. Tumor cell phagocytosis was also assessed in CL1-RAW264.7 macrophages co-cultures. Results: We showed that while 0.5 and 0.1 mg/kg cabazitaxel did not improve PEDF efficacy on tumor growth, 1mg/kg was extremely toxic, killing 1/2 animals within the first week of treatment. In contrast, we demonstrated that PEDF combined with 5mg/kg cabazitaxel lead to stabilization of the disease. PEDF/cabazitaxel was also found to be drastically more efficient than PEDF/docetaxel in delaying the disease. In vitro, PEDF/cabazitaxel inhibited tumor cell migration at a superior level compared to PEDF/docetaxel. Finally, PEDF/cabazitaxel induced more phagocytosis of the tumor cells by macrophages than PEDF/docetaxel suggesting that the combination may target macrophages within the tumor microenvironment. Conclusions: Our data demonstrated that PEDF with low-dose cabazitaxel could be used as a novel therapeutic combination to treat CRPC. Our results also emphasize on the importance in closely evaluating the cytotoxicity of the combination tested and investigating the role of the tumor microenvironment in the anti-tumor effect.

Polymorphisms of the androgen transporting gene SLCO2B1 and response to abiraterone acetate in mCRPC patients.

174 Background: Abiraterone Acetate (AA), an effective treatment for metastatic castration resistant prostate cancer (mCRPC), is highly variable in its effectiveness and it is well known that form of primary and acquired resistance to this agent, exist. This resistance may partly depend on the increased capacity of CRPC cells to synthesize testosterone and dihydrotestosterone from weak adrenal androgens (i.e. DHEA, DHEA-S) or de novo from cholesterol, in response to a chronic exposition to a low-testosterone environment. The organic anion-transporting polypeptides (OATPs) encoded by SLCO genes, mediate the cellular uptake of many compounds, including adrenal androgens. We hypothesized that germ-line variants of the androgen transporter gene SLCO2B1 (solute carrier organic anion transporter family member 2B1), altering the ability of the prostate cancer cell to stock adrenal precursors, may influence the response to AA of mCRPC patients. Methods: Three single nucleotide polymorphisms (SNPs), exonic SNP rs12422149 and intronic SNPs rs1789693 and rs1077858, were genotyped in a cohort of 21 consecutive patients with mCRPC who were treated with AA at the Sant’Andrea Hospital of Rome, Italy. The SNPs were detected in blood samples using pyrosequencing technique. The median TTBP (Time to Biochemical Progression), PFS (Progression Free Survival) and OS (Overall Survival) was estimated using the Kaplan-Meier method, with 95% CI. To test the association between SNPs and TTBP, PFS and OS the log-rank test was used. Results: The intronic polymorphism of SLCO2B1 rs1077858A &gt; G was associated with TTBP (p = 0.03) and PFS (p = 0.04) of mCRPC patients on AA therapy. Patients carrying the SLCO2B1 rs1077858 risk genotype (GG) exhibited a TTBP that was 8 months shorter than that of patients with the major allele of SNP rs1077858 (AA or GA) (TTBP: A/A+G/A = 12 (95% CI: 12-NA) vs G/G = 4 (95% CI: NA) months). Patients carrying genotype GG of SNP rs1077858 also showed an 11 months shorter PFS on AA therapy than patients with AA or GA genotype (PFS: GG = 5.1 (95% CI:NA) vs AA+GA = 16.7 (95%CI:14.9-NA) months). Conclusions: Genetic variants of SLCO2B1 may function as pharmacogenomic determinants of resistance to AA in mCRPC.

Effect of enzalutamide on sensitivity in prostate cancer cells to radiation by inhibition of DNA double strand break repair.

208 Background: Prostate cancer is the second leading cause of cancer-related deaths amongst men in North America. Data suggests that, following radiation therapy (XRT), androgen receptor (AR) enhances DNA damage repair and contributes to resistance of prostate cancer (PCa) cells to XRT. At present AR-pathway inhibition is the mainstay treatment of metastatic castration resistance prostate cancer (mCRPC). Enzalutamide (ENZA), a potent AR inhibitor is one of the approved drugs in this setting. The purpose of this study was to assess the potential radiosensitization of ENZA and its mechanism of action in hormone resistant PCa cells. Methods: The effect of ENZA alone or in combination with XRT was assessed on hormone-sensitive, (HS: LNCaP, PC3-T877A) and insensitive PCa cells (HI: PC3, PC3-AR V7, C4-2) using viability and clonogenic assays, cell cycle arrest and DNA damage analysis. Results: MTT assay demonstrates that ENZA significantly inhibits the proliferation of HS PCa cells in a dose dependent manner whereas CRPC required ENZA in combination with ADT (androgen deprivation therapy). Additionally, clonogenic assay proves that concurrent administration of ENZA or ADT+ENZA and XRT led to a supra-additive antitumor effect with the dose enhancement factor of 1.76±0.008 in LNCaP, 1.65±0.01 in PC3-T877A and 1.35±0.003 in C4-2 respectively at surviving fraction of 0.1. This effect was not observed in PC3 and PC3-AR V7 cells pre-treated with ENZA (in all cases DEF = 1 at SF = 0.1). Additionally, the level of γH2AX increased in HS cells and CRPC cells treated with ENZA/ADT+ENZA and XRT when compared to XRT alone. The enhanced H2AX activity remained unchanged up to 24 hours after combination treatment. Furthermore, there is an initial inhibition of DNA-PKcs in HS and CRPC cells treated with ENZA/ADT+ENZA administered before XRT. Conclusions: Our data suggest that the higher efficacy of ENZA/ENZA+ADT and XRT could be partially due to inhibition of DNA damage repair. Our results demonstrated a significant enhancement of XRT efficacy and confirms the rational for the ongoing combination clinical trials with XRT.

Silencing CAPN2 Expression Inhibited Castration-Resistant Prostate Cancer Cells Proliferation and Invasion via AKT/mTOR Signal Pathway.

The mRNA expression of CAPN2 was upregulated in CRPC cells (DU145 and PC3) than that in non-CRPC cells. Silencing CAPN2 expression could inhibit DU145 and PC3 cells proliferation by cell cycle arrest at G1 phase. Knockdown of CPAN2 level suppressed the migration and invasion capacity of CRPC cells by reducing matrix metalloproteinase-2 (MMP-2) and MMP-9 activation, as well as repressing the phosphorylation protein expression of AKT and mTOR. In addition, we found that the expression of CAPN2 was elevated in Pca tissues than that in normal control tissues. Therefore, we showed the important roles of CAPN2 in the development and progression in CRPC cells, suggesting a new therapeutic intervention for treating castration-resistant prostate cancer patients.

Triptolide Inhibits the AR Signaling Pathway to Suppress the Proliferation of Enzalutamide Resistant Prostate Cancer Cells.

Enzalutamide is a second-generation androgen receptor (AR) antagonist for the treatment of metastatic castration-resistant prostate cancer (mCRPC). Unfortunately, AR dysfunction means that resistance to enzalutamide will eventually develop. Thus, novel agents are urgently needed to treat this devastating disease. Triptolide (TPL), a key active compound extracted from the Chinese herb Thunder God Vine (Tripterygium wilfordii Hook F.), possesses anti-cancer activity in human prostate cancer cells. However, the effects of TPL against CRPC cells and the underlying mechanism of any such effect are unknown. In this study, we found that TPL at low dose inhibits the transactivation activity of both full-length and truncated AR without changing their protein levels. Interestingly, TPL inhibits phosphorylation of AR and its CRPC-associated variant AR-V7 at Ser515 through XPB/CDK7. As a result, TPL suppresses the binding of AR to promoter regions in AR target genes along with reduced TFIIH and RNA Pol II recruitment. Moreover, TPL at low dose reduces the viability of prostate cancer cells expressing AR or AR-Vs. Low-dose TPL also shows a synergistic effect with enzalutamide to inhibit CRPC cell survival in vitro, and enhances the anti-cancer effect of enzalutamide on CRPC xenografts with minimal side effects. Taken together, our data demonstrate that TPL targets the transactivation activity of both full-length and truncated ARs. Our results also suggest that TPL is a potential drug for CRPC, and can be used in combination with enzalutamide to treat CRPC.

TGF-β signal rewiring sustains epithelial-mesenchymal transition of circulating tumor cells in prostate cancer xenograft hosts.

Activation of TGF-β signaling is known to promote epithelial-mesenchymal transition (EMT) for the development of metastatic castration-resistant prostate cancer (mCRPC). To determine whether targeting TGF-β signaling alone is sufficient to mitigate mCRPC, we used the CRISPR/Cas9 genome-editing approach to generate a dominant-negative mutation of the cognate receptor TGFBRII that attenuated TGF-β signaling in mCRPC cells. As a result, the delicate balance of oncogenic homeostasis is perturbed, profoundly uncoupling proliferative and metastatic potential of TGFBRII-edited tumor xenografts. This signaling disturbance triggered feedback rewiring by enhancing ERK signaling known to promote EMT-driven metastasis. Circulating tumor cells displaying upregulated EMT genes had elevated biophysical deformity and an increase in interactions with chaperone macrophages for facilitating metastatic extravasation. Treatment with an ERK inhibitor resulted in decreased aggressive features of CRPC cells in vitro. Therefore, combined targeting of TGF-β and its backup partner ERK represents an attractive strategy for treating mCRPC patients.

Abstract 280: Synergistic in vitro activity of MOR209/ES414 in combination with enzalutamide

Abstract Metastatic castration-resistant prostate cancer (mCRPC) presents a daunting therapeutic challenge for patients who eventually progress after androgen ablation therapy. Although newer AR antagonists show a survival benefit, these patients ultimately relapse. Thus, it is critical to identify new therapies that will work in combination with AR antagonists or in AR-antagonist resistant settings. MOR209/ES414 is a bispecific ADAPTIR™ (modular protein technology) molecule currently under investigation in a phase 1 clinical trial in mCRPC, including patients that have progressed on enzalutamide. In pre-clinical studies, MOR209/ES414 has previously been shown to redirect T cell cytotoxicity against cells expressing Prostate Specific Membrane Antigen (PSMA). Here, we aimed to determine whether enzalutamide affects PSMA expression on CRPC cells in vitro, and could alter the effectiveness of MOR209/ES414 at inducing T-cell mediated tumor cell death. To address these questions, we utilized a CRPC cell line, 22Rv1, which has low expression of PSMA and is resistant to enzalutamide. Treatment of 22Rv1 cells with 10 μM enzalutamide for 3 weeks resulted in increased PSMA cell surface expression as measured by flow cytometry. 22Rv1 cells exposed to enzalutamide were also more sensitive to MOR209/ES414-mediated T-cell lysis, with a 25% increase in cytotoxicity over a 4 hour chromium release assay compared to 22Rv1 cells that were not exposed to enzalutamide. A concomitant decrease in EC50 for MOR209/ES414-mediated lysis from 0.8 pM to 0.5 pM was also observed in 22Rv1 cells exposed to enzalutamide. MOR209/ES414 and enzalutamide were next tested in combination using a PSMA-positive, enzalutamide-sensitive cell line, LNCaP cultured with primary T cells over several days. Suboptimal doses of MOR209/ES414 and enzalutamide were added to the cultures, and surviving LnCaP cells were quantitated 4 days later. Enzalutamide did not interfere with the ability of MOR209/ES414 to target T cells to LNCaP cells, and Combination Index analysis showed the activity of MOR209/ES414 and enzalutamide to be synergistic in vitro. These studies provide a rationale for further examination of combining MOR209/ES414 with enzalutamide, even in enzalutamide-resistant settings. Citation Format: Toddy Sewell, Michelle Blake, Jane A. Gross, Jan Endell, Johannes Weirather, John W. Blankenship. Synergistic in vitro activity of MOR209/ES414 in combination with enzalutamide. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 280.

Abstract 288: Low-dose cabazitaxel inhibits the growth of prostate cancer cells and enhances the anti-tumor properties of PEDF with greater efficacy than docetaxel

Abstract Despite recently approved novel agents, taxane-based chemotherapy remains the major therapeutic strategy for metastatic castration-resistant prostate cancer (mCRPC). Still mCRPC continues to be incurable. Furthermore, patients often experience severe side effects, prompting a re-evaluation of conventional regimen. Past studies have demonstrated promise for Low-Dose Metronomic (LDM) chemotherapy; schedule defined as the frequent administration of low doses of chemotherapeutic drugs with no prolonged drug-free breaks. Yet relative activities of LDM taxanes and combinations with known anti-neoplasic agents have to be investigated. Using CRPC cell lines (PC3, Du145 and the LNCaP-derivative CL1), we have shown that cabazitaxel-treated cells have a significantly lower EC50 compared to docetaxel, with Du145 cells presenting the greatest differences. In cell cycle analysis, both low-dose taxanes increased the sub-G1 cells population. However, the sub-G1 increase was significantly greater in cabazitaxel- than in docetaxel-treated Du145 cells, but not in PC3 and CL1. Accordingly, plasma membrane Annexin V elevation occurred in Du145 cells at lower doses of cabazitaxel than docetaxel validating a higher efficacy due to increased apoptosis. As other possible cell death mechanisms, autophagy and necrosis were investigated. Beclin1 expression levels remain unchanged in all three cell lines. In contrast, necrosis was stimulated in all the taxanes-treated cell lines in a dose dependent manner. In vivo, LDM cabazitaxel was significantly more efficient in delaying tumor growth than docetaxel. This effect was markedly increased when cabazitaxel was combined with the angio-inhibitor and anti-tumor Pigment Epithelium-Derived Factor (PEDF). Other results showed that PEDF and LDM chemotherapy combination induces more phagocytosis of CRPC cells when compared to single treatments. In conclusion, our data demonstrate a higher efficacy of cabazitaxel on CRPC both in vitro and in vivo, and suggest that LDM taxane chemotherapy/PEDF combination could be used as a novel therapeutic strategy for CRPC. Citation Format: Courtney L. Jarvis, Thomas Nelius, Dalia Martinez-Marin, Srirupa Cheerla, Stéphanie Filleur. Low-dose cabazitaxel inhibits the growth of prostate cancer cells and enhances the anti-tumor properties of PEDF with greater efficacy than docetaxel. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 288.

Abstract 2898: Elucidating the role of BHLHE40/DEC1/SHARP2/STRA13 in prostate cancer

Abstract Background: Basic-helix-loop-helix proteins (BHLH) are transcription factors that play important roles in critical developmental processes in many organisms by repressing the transcription of various target genes. Previous studies showed that overexpression of the BHLHE40 (also known as DEC1, STRA13 or SHARP2) transcriptional repressor results in growth suppression, cell cycle arrest, and cellular senescence indicating that BHLHE40 may have important tumor suppressor functions. Studies showed that the expression of BHLHE40 is indeed downregulated in some tumors, intriguingly however, it is also overexpressed in many such as lung cancer, breast cancer. Our overall goal is to elucidate the function of BHLHE40 in prostate cancer (CaP) and gain an understanding of how it is regulated. Methods: Initial studies were based on qPCR analysis to compare the expression levels of BHLHE40 in different cell lines ranging from androgen-dependent LNCaP cells to the castration resistant C4, C4-2, C4-2b, R1, Rv1, PC3 and PC3 cells that stably express the androgen receptor (AR) (PC3-AR). Further analysis were done by treating LNCaP and C4-2 cells with 1 nM dihydrotestosterone (DHT) in media containing fetal bovine serum (FBS), or charcoal stripped serum (CSS). Western blot analysis was done to check the level of BHLHE40 protein in these cell lines. Knock-down of AR, or the use of AR inhibitors Enzalutamide and/or Bicalutamide were used to further assess the regulation of BHLHE40 by the AR. Results: qPCR comparison of BHLHE40 transcript levels in LNCaP versus C4, C4-2, R1, Rv1, PC3 cells showed that while LNCaP cells have high transcript levels of BHLHE40, the CRPC cells all had very low transcript levels of this transcription factor. When comparing LNCaP to PC3 and PC3-AR cells, it was interesting to note that PC3-AR cells showed significantly higher transcript levels of BHLHE40 than PC3 implying that BHLHE40 may be regulated by the AR pathway. Treatment with 1 nM DHT on these cells showed that BHLHE40 increases over time following DHT treatment. In contrast, inhibition of AR by Enzalutamide (2 μM) showed reduced BHLHE40 levels, although Bicalutamide (10 μM) did not significantly altered the expression level of BHLHE40 in LNCaP cells. Conclusions: Our results indicate that BHLHE40 is regulated by the AR pathway, although whether this is direct or indirect is yet to be determined. This observation is significant, since it indicates that the AR may be utilizing this transcription factor to suppress the transcription of other genes. Future studies will attempt to determine if BHLHE40 may have an androgen-responsive-element (ARE) in its promoter and via ChIP assay to identify possible binding sites for AR in conjunction with luciferase assay to assess androgen-receptor mediated regulation. Citation Format: Zsofia Kiss, Paramita Ghosh. Elucidating the role of BHLHE40/DEC1/SHARP2/STRA13 in prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2898.

Abstract 1806: TRIM24 is an oncogenic transcriptional activator in prostate cancer

Abstract Androgen receptor (AR) signaling is a key driver of prostate cancer (PC). While androgen-deprivation therapy is transiently effective in advanced disease, tumors often progress to a lethal castration-resistant state (CRPC). We show that recurrent PC-driver mutations in SPOP stabilize the TRIM24 protein, which promotes proliferation under low androgen conditions. TRIM24 augments AR signaling, and AR and TRIM24 co-activated genes are significantly up-regulated in CRPC. Expression of TRIM24 protein increases from primary PC to CRPC, and both TRIM24 protein levels and the AR/TRIM24 gene signature predict disease-recurrence. Analyses in CRPC cells reveal that the TRIM24 bromodomain and the AR-interacting motif are essential to support proliferation. These data provide a rationale for therapeutic TRIM24 targeting in SPOP-mutant and CRPC patients. Citation Format: Anna C. Groner, Laura Cato, Jonas de Tribolet-Hardy, Tiziano Bernasocchi, Qing Zhong, Christian Fankhauser, Christine Fritz, Cédric Poyet, Ulrich Wagner, Levi A. Garraway, Peter J. Wild, Jean-Philippe Theurillat, Myles Brown. TRIM24 is an oncogenic transcriptional activator in prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1806.

TRIM24 Is an Oncogenic Transcriptional Activator in Prostate Cancer

Androgen receptor (AR) signaling is a key driver of prostate cancer (PC). While androgen-deprivation therapyis transiently effective in advanced disease, tumors often progress to a lethal castration-resistant state (CRPC). We show that recurrent PC-driver mutations in speckle-type POZ protein (SPOP) stabilize the TRIM24 protein, which promotes proliferation under low androgen conditions. TRIM24 augments ARsignaling, and AR and TRIM24 co-activated genes are significantly upregulated in CRPC. Expression of TRIM24 protein increases from primary PC to CRPC, and both TRIM24 protein levels and the AR/TRIM24 gene signature predict disease recurrence. Analyses in CRPC cells reveal that the TRIM24 bromodomain and the AR-interacting motif are essential to support proliferation. These data provide a rationale for therapeutic TRIM24 targeting in SPOP mutant and CRPC patients.

Alterations of androgen receptor-regulated enhancer RNAs (eRNAs) contribute to enzalutamide resistance in castration-resistant prostate cancer.

Enzalutamide is a second-generation anti-androgen for treatment of castration-resistant prostate cancer (CPRC). It prolongs survival of CRPC patients, but its overall survival benefit is relatively modest (4.8 months) and by 24 months most patients progress on enzalutamide. To date, however, the molecular mechanisms underlying enzalutamide resistance remain elusive. Herein, we report enzalutamide treatment-induced alterations of androgen receptor (AR)-regulated enhancer RNAs (AR-eRNAs) and their roles in enzalutamide-resistant growth and survival of CRPC cells. AR chromatin immunoprecipitation and high throughput sequencing (ChIP-seq) and RNA-seq analyses revealed that 188 and 227 AR-eRNAs were differentially expressed in enzalutamide-resistant LNCaP and C4-2 cells, respectively. The AR-eRNAs upregulated in C4-2 cells and downregulated in LNCaP cells were selected through meta-analysis. Expression of AR-eRNAs and related mRNAs in the loci of FTO, LUZP2, MARC1 and NCAM2 were further verified by real-time RT-PCR. Silencing of LUZP2 inhibited, but silencing of MARC1 increased the growth of enzalutamide-resistant C4-2 cells. Intriguingly, meta-analysis showed that expression of LUZP2 mRNA increased in primary tumors compared to normal prostate tissues, but decreased again in metastatic CRPC. Our findings suggest that eRNA alteration profiling is a viable new approach to identify functional gene loci that may not only contribute to enzalutamide-resistant growth of CRPC, but also serve as new targets for CRPC therapy.

MP66-11 LIM-SH3 DOMAIN PROTEIN 1 CORRELATES WITH CELL INVASION AND STEMNESS IN PROSTATE CANCER AND EARLY METASTASIS AFTER RADICAL PROSTATECTOMY

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology II1 Apr 2016MP66-11 LIM-SH3 DOMAIN PROTEIN 1 CORRELATES WITH CELL INVASION AND STEMNESS IN PROSTATE CANCER AND EARLY METASTASIS AFTER RADICAL PROSTATECTOMY Takashi Dejima, Ario Takeuchi, Kevin Tam, Masatoshi Eto, Martin Gleave, and Christopher Ong Takashi DejimaTakashi Dejima More articles by this author , Ario TakeuchiArio Takeuchi More articles by this author , Kevin TamKevin Tam More articles by this author , Masatoshi EtoMasatoshi Eto More articles by this author , Martin GleaveMartin Gleave More articles by this author , and Christopher OngChristopher Ong More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1285AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Prostate cancer is the second most frequently diagnosed cancer and the leading cause of cancer death in developed countries. LIM-SH3 domain protein 1 (LASP1) has been shown to promote progression and invasion in several malignancies. LASP1 has shown to be highly expressed in prostate cancer patients who are at high risk for developing metastases; however, the role of LASP1 in tumorigenesis is unclear. In this study, we investigated the mechanism of LASP1 for invasion and stemness in prostate cancer. METHODS We used the Affimetrix Exon Array system to determine LASP1 gene expression in tissue samples of prostate cancer patients. The LASP1 expression in several prostate cancer cell lines was assessed by Western blot analysis and quantitative reverse transcription-PCR. To assess LASP1 function, we generated stable LASP1 knockdown and overexpression cell lines. For our cell growth assay, we used anchorage-independent growth conditions in 24 well plates coated with poly-Hema and monitored growth by WST-8 assay. To determine invasiveness, we monitored movement of cell through Matrigel coated invasion chambers. To measure tumorigenicity, in vivo tumor formation was assessed in mice xenografted with LNCaP cells overexpressing LASP1 RESULTS Compared to low LASP1 expression, high LASP1 expression in prostate cancer is prone to early metastasis after radical prostatectomy (P=0.0423). High LASP1 correlated with prostate cancer-specific mortality after radical prostatectomy (P<0.001). Knockdown of LASP1 in VehA (CRPC cells) and PC3 cells inhibited cell growth. Furthermore, knockdown of LASP1 in PC3 cells significantly decreased cell invasion and was accompanied by a decrease in snail and slug expression. Overexpression of LASP1 in LNCaP cells promoted cell growth. Nanog, a stemness-related marker, was increased by knockdown of LASP1 and decreased by overexpression of LASP1. No tumors in 10 mice inoculated with LNCaP mock cells grew in size, whereas all tumors in 10 mice inoculated with LASP1-overexpressing LNCaP cells grew in size. CONCLUSIONS These results suggest that LASP1 induces tumorigenesis by promoting cell growth, invasion and stemness in prostate cancer. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e876-e877 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Takashi Dejima More articles by this author Ario Takeuchi More articles by this author Kevin Tam More articles by this author Masatoshi Eto More articles by this author Martin Gleave More articles by this author Christopher Ong More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...