Castration-resistant Growth Research Articles

PiRNA-4447944 promotes castration-resistant growth and metastasis of prostate cancer by inhibiting NEFH expression through forming the piRNA-4447944-PIWIL2-NEFH complex.

Castration-resistant prostate cancer (CRPC) is the leading cause of prostate cancer (PCa)-related death in males, which occurs after the failure of androgen deprivation therapy (ADT). PIWI-interacting RNAs (piRNAs) are crucial regulators in many human cancers, but their expression patterns and roles in CRPC remain unknown. In this study, we performed small RNA sequencing to explore CRPC-associated piRNAs using 10 benign prostate tissues, and 9 paired hormone-sensitive PCa (HSPCa) and CRPC tissues from the same patients. PiRNA-4447944 (piR-4447944) was discovered to be highly expressed in CRPC group compared with HSPCa and benign groups. Functional analyses revealed that piR-4447944 overexpression endowed PCa cells with castration resistance ability in vitro and in vivo, whereas knockdown of piR-4447944 using anti-sense RNA suppressed the proliferation, migration and invasion of CRPC cells. Additionally, enforced piR-4447944 expression promoted in vitro migration and invasion of PCa cells, and reduced cell apoptosis. Mechanistically, piR-4447944 bound to PIWIL2 to form a piR-4447944/PIWIL2 complex and inhibited tumor suppressor NEFH through direct interaction at the post-transcriptional level. Collectively, our study indicates that piR-4447944 is essential for prostate tumor-propagating cells and mediates androgen-independent growth of PCa, which extends current understanding of piRNAs in cancer biology and provides a potential approach for CRPC treatment.

MP20-03 microRNA-15b-5p/ MUSCARINIC RECEPTORS/ YAP SIGNALING CONTRIBUTES TO CASTRATION-RESISTANT GROWTH OF PROSTATE CANCER

You have accessJournal of UrologyCME1 Apr 2023MP20-03 microRNA-15b-5p/ MUSCARINIC RECEPTORS/ YAP SIGNALING CONTRIBUTES TO CASTRATION-RESISTANT GROWTH OF PROSTATE CANCER Yusuke Goto, Shunichi Asai, Shinichi Sakamoto, Tomokazu Sazuka, Yusuke Imamura, Naohiko Seki, and Tomohiko Ichikawa Yusuke GotoYusuke Goto More articles by this author , Shunichi AsaiShunichi Asai More articles by this author , Shinichi SakamotoShinichi Sakamoto More articles by this author , Tomokazu SazukaTomokazu Sazuka More articles by this author , Yusuke ImamuraYusuke Imamura More articles by this author , Naohiko SekiNaohiko Seki More articles by this author , and Tomohiko IchikawaTomohiko Ichikawa More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003245.03AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Prostate cancer (PCa) innervation contributes to the progression of PCa. However, the precise impact of innervation on PCa cells is still poorly understood. We have recently shown that muscarinic receptors promote castration-resistant growth of PCa through FAK-YAP signaling axis. However, mechanisms of upregulation of muscarinic receptors leading to castration-resistant growth of PCa cells were unclear. METHODS: Small RNA-sequencing and bioinformatics screening were used to identify candidate microRNAs to regulate muscarinic receptors. Luciferase reporter assay was performed to see direct regulation of muscarinic receptors by microRNAs. Western blot and qRT-PCR verified the effect of the microRNA on downstream YAP signaling. Cell proliferation and sphere assay demonstrated the effect of microRNA on the muscarinic-receptor-driven castration-resistant growth of PCa. RESULTS: Small RNA-seq showed that 255 microRNAs were significantly downregulated under castrated condition in LNCaP cells. Using bioinformatic approach, microRNA-15b-5p was found to be one of the candidate microRNAs that directly regulate m3. microRNA-15b-5p was confirmed to downregulate with castrate condition, in contrast to the upregulation of muscarinic receptor 3 (m3) with castration in vitro. Indeed, microRNA-15b-5p was downregulated in castration-resistant PCa (CRPC) compared with hormone-sensitive PCa (HSPC) specimens. Moreover, microRNA-15b-5p directly binds to m3, and microRNA-15b-5p inhibited YAP activation which was induced by m3 stimulation. Furthermore, microRNA-15b-5p abolished castration-resistant growth of PCa cells which was induced by m3 stimulation, in 2D cell proliferation assay and 3D cell sphere assay. CONCLUSIONS: microRNA-15b-5p/m3/YAP signaling axis is a novel pathway which promotes castration-resistant growth of PCa. Our findings provide novel mechanisms of muscarinic-signal-driven CRPC progression. Source of Funding: JSPS KAKENHI Grant Number: 21K16753 © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e276 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Yusuke Goto More articles by this author Shunichi Asai More articles by this author Shinichi Sakamoto More articles by this author Tomokazu Sazuka More articles by this author Yusuke Imamura More articles by this author Naohiko Seki More articles by this author Tomohiko Ichikawa More articles by this author Expand All Advertisement PDF downloadLoading ...

MiR-15b-5p inhibits castration-resistant growth of prostate cancer cells by targeting the muscarinic cholinergic receptor CHRM3.

Cholinergic receptor muscarinic 3 (CHRM3)-mediated focal adhesion kinase/YES-associated protein (YAP) signalling is essential for the growth of castration-resistant prostate cancer (CRPC) cells. Here, we evaluated the molecular mechanisms through which CHRM3 overexpression facilitates castration-resistant growth. Small RNA sequencing combined with in silico analyses revealed that CHRM3 was a putative target of miR-15b-5p. Notably, androgen deprivation suppressed miR-15b-5p expression and increased CHRM3 expression. Moreover, miR-15b-5p bound directly to CHRM3 and inhibited YAP activation induced by CHRM3 stimulation. Furthermore, miR-15b-5p abolished the growth of CRPC cells induced by CHRM3 stimulation. We conclude that the miR-15b-5p/CHRM3/YAP signalling axis promotes the castration-resistant growth of prostate cancer.

Comprehensive genomics in androgen receptor-dependent castration-resistant prostate cancer identifies an adaptation pathway mediated by opioid receptor kappa 1

Castration resistance is a lethal form of treatment failure of prostate cancer (PCa) and is associated with ligand-independent activation of the androgen receptor (AR). It is only partially understood how the AR mediates survival and castration-resistant growth of PCa upon androgen deprivation. We investigated integrative genomics using a patient-derived xenograft model recapitulating acquired, AR-dependent castration-resistant PCa (CRPC). Sequencing of chromatin immunoprecipitation using an anti-AR antibody (AR-ChIP seq) revealed distinct profiles of AR binding site (ARBS) in androgen-dependent and castration-resistant xenograft tumors compared with those previously reported based on human PCa cells or tumor tissues. An integrative genetic analysis identified several AR-target genes associated with CRPC progression including OPRK1, which harbors ARBS and was upregulated upon androgen deprivation. Loss of function of OPRK1 retarded the acquisition of castration resistance and inhibited castration-resistant growth of PCa both in vitro and in vivo. Immunohistochemical analysis showed that expression of OPRK1, a G protein-coupled receptor, was upregulated in human prostate cancer tissues after preoperative androgen derivation or CRPC progression. These data suggest that OPRK1 is involved in post-castration survival and cellular adaptation process toward castration-resistant progression of PCa, accelerating the clinical implementation of ORPK1-targeting therapy in the management of this lethal disease.

GLI3 Is Stabilized by SPOP Mutations and Promotes Castration Resistance via Functional Cooperation with Androgen Receptor in Prostate Cancer.

Although the Sonic hedgehog (SHH) signaling pathway has been implicated in promoting malignant phenotypes of prostate cancer, details on how it is activated and exerts its oncogenic role during prostate cancer development and progression is less clear. Here, we show that GLI3, a key SHH pathway effector, is transcriptionally upregulated during androgen deprivation and posttranslationally stabilized in prostate cancer cells by mutation of speckle-type POZ protein (SPOP). GLI3 is a substrate of SPOP-mediated proteasomal degradation in prostate cancer cells and prostate cancer driver mutations in SPOP abrogate GLI3 degradation. Functionally, GLI3 is necessary and sufficient for the growth and migration of androgen receptor (AR)-positive prostate cancer cells, particularly under androgen-depleted conditions. Importantly, we demonstrate that GLI3 physically interacts and functionally cooperates with AR to enrich an AR-dependent gene expression program leading to castration-resistant growth of xenografted prostate tumors. Finally, we identify an AR/GLI3 coregulated gene signature that is highly correlated with castration-resistant metastatic prostate cancer and predictive of disease recurrence. Together, these findings reveal that hyperactivated GLI3 promotes castration-resistant growth of prostate cancer and provide a rationale for therapeutic targeting of GLI3 in patients with castration-resistant prostate cancer (CRPC). IMPLICATIONS: We describe two clinically relevant mechanisms leading to hyperactivated GLI3 signaling and enhanced AR/GLI3 cross-talk, suggesting that GLI3-specific inhibitors might prove effective to block prostate cancer development or delay CRPC.

Phenotypic characterization of two novel cell line models of castration-resistant prostate cancer.

Resistance to androgen deprivation therapies is a major driver of mortality in advanced prostate cancer. Therefore, there is a need to develop new preclinical models that allow the investigation of resistance mechanisms and the assessment of drugs for the treatment of castration-resistant prostate cancer. We generated two novel cell line models (LAPC4-CR and VCaP-CR) which were derived by passaging LAPC4 and VCaP cells in vivo and in vitro under castrate conditions. We performed detailed transcriptomic (RNA-seq) and proteomic analyses (SWATH-MS) to delineate expression differences between castration-sensitive and castration-resistant cell lines. Furthermore, we characterized the in vivo and in vitro growth characteristics of these novel cell line models. The two cell line derivatives LAPC4-CR and VCaP-CR showed castration-resistant growth in vitro and in vivo which was only minimally inhibited by AR antagonists, enzalutamide, and bicalutamide. High-dose androgen treatment resulted in significant growth arrest of VCaP-CR but not in LAPC4-CR cells. Both cell lines maintained AR expression, but exhibited distinct expression changes on the mRNA and protein level. Integrated analyses including data from LNCaP and the previously described castration-resistant LNCaP-abl cells revealed an expression signature of castration resistance. The two novel cell line models LAPC4-CR and VCaP-CR and their comprehensive characterization on the RNA and protein level represent important resources to study the molecular mechanisms of castration resistance.

Abstract 2294: NEK1 phosphorylation of YAP promotes its stabilization and transcriptional output

Abstract Background: Despite recent development in prostate cancer (PCa) therapeutics, castration-resistant prostate cancer still accounts for high morbidity and mortality in patients, partly because of incomplete knowledge of the major players that contribute to the androgen independent growth of prostate tumor. NIMA related kinase 1 (NEK1), a dual specificity kinase, mainly functions in maintaining genomic stability by regulating DNA damage response (DDR) and G2/M transition of the cells. We previously reported that tousled-like kinase 1 (TLK1) mediated phosphorylation of NEK1 on T141 can lead to rapid androgen independent progression of PCa cells upon anti-androgen treatment by activating DDR. However, activating DDR alone may not be sufficient to trigger androgen insensitive growth of PCa cells. We hypothesize that TLK1>NEK1 axis may also regulate hippo pathway oncoprotein YAP as a previous study reported that NEK1 can phosphorylate YAP homolog TAZ and upregulate YAP/TAZ transcriptional targets. Yes-associated protein (YAP) is a transcriptional co-activator and a major effector of hippo kinases that binds to transcription factor TEAD and promotes many oncogenic activities in the cells. Methods and Results: To determine if NEK1 can regulate Hippo pathway, our western blotting (WB) analysis revealed higher cleaved YAP (cl-YAP) and lower total YAP level in the NEK1-T141A hypoactive variant overexpressing LNCaP than wt-NEK1 overexpressing cells treated with bicalutamide (anti-androgen) and/or THD (a specific inhibitor of TLK1) which suggest that NEK1 may play a role in stabilizing YAP. TLK1 inhibition also resulted in the reduced expression of some YAP target genes such as CTGF, CDH2, Twist1, and P53AIP1. Co-IP assay revealed that NEK1 can interact with YAP regardless of its kinase activity. We generated a CRISPR-Cas9 mediated NEK1 knockout NT1 (a mouse PCa cell line) cells which showed reduced level of YAP in NEK1 KO clones as well as downregulation of YAP target genes such as CTGF, Zeb1, Twist1, and Cyr61. TLK1 knockdown and inhibition also resulted in reduced pNEK1 (T141) and total YAP level which suggested TLK1 mediated NEK1 activation is necessary for YAP stabilization. An IVK assay using [γ-32P] ATP demonstrated that catalytically active purified NEK1 can phosphorylate purified YAP1. MS analysis determined eight NEK1 mediated phospho-acceptor sites of YAP1: T83, T361, S366, S388, Y407, and T493. Y407 and T493 are the first ever reported phosphorylation sites on YAP1 especially by NEK1 which lies in the transactivation and PDZ binding domain. Our bioinformatics analysis revealed mRNA downregulation of YAP1, while upregulation of YAP protein level was observed in high-grade PCa, which is consistent with our model of post-transcriptional protein stabilization. Conclusion: Our data support that TLK1>NEK1 signaling promotes YAP stabilization and may contribute to castration resistant growth of PCa during AR blockade. Citation Format: Md Imtiaz Khalil, Ishita Ghosh, Vibha Singh, Jing Chen, Haining Zhu, Arrigo De Benedetti. NEK1 phosphorylation of YAP promotes its stabilization and transcriptional output [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2294.

MAPK4 promotes prostate cancer by concerted activation of androgen receptor and AKT.

Prostate cancer (PCa) is the second leading cause of cancer death in American men. Androgen receptor (AR) signaling is essential for PCa cell growth/survival and remains a key therapeutic target for lethal castration-resistant PCa (CRPC). GATA2 is a pioneer transcription factor crucial for inducing AR expression/activation. We recently reported that MAPK4, an atypical MAPK, promotes tumor progression via noncanonical activation of AKT. Here, we demonstrated that MAPK4 activated AR by enhancing GATA2 transcriptional expression and stabilizing GATA2 protein through repression of GATA2 ubiquitination/degradation. MAPK4 expression correlated with AR activation in human CRPC. Concerted activation of both GATA2/AR and AKT by MAPK4 promoted PCa cell proliferation, anchorage-independent growth, xenograft growth, and castration resistance. Conversely, knockdown of MAPK4 decreased activation of both AR and AKT and inhibited PCa cell and xenograft growth, including castration-resistant growth. Both GATA2/AR and AKT activation were necessary for MAPK4 tumor-promoting activity. Interestingly, combined overexpression of GATA2 plus a constitutively activated AKT was sufficient to drive PCa growth and castration resistance, shedding light on an alternative, MAPK4-independent tumor-promoting pathway in human PCa. We concluded that MAPK4 promotes PCa growth and castration resistance by cooperating parallel pathways of activating GATA2/AR and AKT and that MAPK4 is a novel therapeutic target in PCa, especially CRPC.

MYBL2 disrupts the Hippo-YAP pathway and confers castration resistance and metastatic potential in prostate cancer.

Rationale: Resistance to androgen-deprivation therapy (ADT) associated with metastatic progression remains a challenging clinical task in prostate cancer (PCa) treatment. Current targeted therapies for castration-resistant prostate cancer (CRPC) are not durable. The exact molecular mechanisms mediating resistance to castration therapy that lead to CRPC progression remain obscure.Methods: The expression of MYB proto-oncogene like 2 (MYBL2) was evaluated in PCa samples. The effect of MYBL2 on the response to ADT was determined by in vitro and in vivo experiments. The survival of patients with PCa was analyzed using clinical specimens (n = 132) and data from The Cancer Genome Atlas (n = 450). The mechanistic model of MYBL2 in regulating gene expression was further detected by subcellular fractionation, western blotting, quantitative real-time PCR, chromatin immunoprecipitation, and luciferase reporter assays.Results: MYBL2 expression was significantly upregulated in CRPC tissues and cell lines. Overexpression of MYBL2 could facilitate castration-resistant growth and metastatic capacity in androgen-dependent PCa cells by promoting YAP1 transcriptional activity via modulating the activity of the Rho GTPases RhoA and LATS1 kinase. Importantly, targeting MYBL2, or treatment with either the YAP/TAZ inhibitor Verteporfin or the RhoA inhibitor Simvastatin, reversed the resistance to ADT and blocked bone metastasis in CRPC cells. Finally, high MYBL2 levels were positively associated with TNM stage, total PSA level, and Gleason score and predicted a higher risk of metastatic relapse and poor prognosis in patients with PCa.Conclusions: Our results reveal a novel molecular mechanism conferring resistance to ADT and provide a strong rationale for potential therapeutic strategies against CRPC.

RGS2 is prognostic for development of castration resistance and cancer-specific survival in castration-resistant prostate cancer.

Regulator of G-protein signaling 2 (RGS2) is a multifaceted protein with a prognostic value in hormone-naïve prostate cancer (PC). It has previously been associated with the development of castration resistance. However, RGS2 expression in clinical specimens of castration-resistant prostate cancer (CRPC) and its clinical relevance has not been explored. In the present study, RGS2 was assessed in CRPC and in relation to the development of castration resistance. In the present study, RGS2 expression was evaluated with immunohistochemistry in patient materials of hormone-naïve and castration-resistant primary tumors, also in matched specimens before and after 3 months of androgen deprivation therapy (ADT). Cox regression and Kaplan-Meier curves were used to evaluate the clinical significance of RGS2 expression. RGS2 expression in association to castration-resistant growth was assessed experimentally in an orthotopic xenograft mouse model of CRPC. In vitro, hormone depletion of LNCaP and enzalutamide treatment of LNCaP, 22Rv1, and VCaP was performed to evaluate the association between RGS2 and the androgen receptor (AR). Stable RGS2 knockdown was used to evaluate the impact of RGS2 in association to PC cell growth under hormone-reduced conditions. Gene and protein expression were evaluated with quantitative polymerase chain reaction and Western blot analysis, respectively. RGS2 expression is increased in CRPC and enriched under ADT. Furthermore, a high RGS2 level is prognostic for poor cancer-specific survival for CRPC patients and significantly reduced failure-free survival (FFS) after an initiated ADT. Additionally, the prognostic value of RGS2 outperforms prostate-specific antigen (PSA) in terms of FFS. The present study furthermore suggests that RGS2 expression is reflective of AR activity. Moreover, low RGS2-expressing cells display hampered growth under hormone-reduced conditions, in line with the poor prognosis associated with high RGS2 expression. High levels of RGS2 are associated with aggressive forms of castration-resistant PC. The results demonstrate that a high level of RGS2 is associated with poor prognosis in association with castration-resistant PC growth. RGS2 alone, or in association with PSA, has the potential to identify patients that require additional treatment at an early stage during ADT.

Muscarinic receptors promote castration-resistant growth of prostate cancer through a FAK-YAP signaling axis.

Prostate cancer innervation contributes to the progression of prostate cancer (PCa). However, the precise impact of innervation on PCa cells is still poorly understood. By focusing on muscarinic receptors, which are activated by the nerve-derived neurotransmitter acetylcholine, we show that muscarinic receptors 1 and 3 (m1 and m3) are highly expressed in PCa clinical specimens compared to all other cancer types, and that amplification or gain of their corresponding encoding genes (CHRM1 and CHRM3, respectively) represent a worse prognostic factor for PCa progression free survival. Moreover, m1 and m3 gene gain or amplification are frequent in castration-resistant PCa (CRPC) compared with hormone-sensitive PCa (HSPC) specimens. This was reflected in HSPC-derived cells, which show aberrantly high expression of m1 and m3 under androgen deprivation mimicking castration and androgen receptor inhibition. We also show that pharmacological activation of m1 and m3 signaling is sufficient to induce the castration-resistant growth of PCa cells. Mechanistically, we found that m1 and m3 stimulation induces YAP activation through FAK, whose encoding gene, PTK2 is frequently amplified in CRPC cases. Pharmacological inhibition of FAK and knockdown of YAP abolished m1 and m3-induced castration-resistant growth of PCa cells. Our findings provide novel therapeutic opportunities for muscarinic-signal-driven CRPC progression by targeting the FAK-YAP signaling axis.

Nuclear receptor ERRα contributes to castration-resistant growth of prostate cancer via its regulation of intratumoral androgen biosynthesis.

Enhanced intratumoral androgen biosynthesis and persistent androgen receptor (AR) signaling are key factors responsible for the relapse growth of castration-resistant prostate cancer (CRPC). Residual intraprostatic androgens can be produced by de novo synthesis of androgens from cholesterol or conversion from adrenal androgens by steroidogenic enzymes expressed in prostate cancer cells via different steroidogenic pathways. However, the dysregulation of androgen biosynthetic enzymes in CRPC still remains poorly understood. This study aims to elucidate the role of the nuclear receptor, estrogen-related receptor alpha (ERRα, ESRRA), in the promotion of androgen biosynthesis in CRPC growth.Methods: ERRα expression in CRPC patients was analyzed using Gene Expression Omnibus (GEO) datasets and validated in established CRPC xenograft model. The roles of ERRα in the promotion of castration-resistant growth were elucidated by overexpression and knockdown studies and the intratumoral androgen levels were measured by UPLC-MS/MS. The effect of suppression of ERRα activity in the potentiation of sensitivity to androgen-deprivation was determined using an ERRα inverse agonist.Results: ERRα exhibited an increased expression in metastatic CRPC and CRPC xenograft model, could act to promote castration-resistant growth via direct transactivation of two key androgen synthesis enzymes CYP11A1 and AKR1C3, and hence enhance intraprostatic production of dihydrotestosterone (DHT) and activation of AR signaling in prostate cancer cells. Notably, inhibition of ERRα activity by an inverse agonist XCT790 could reduce the DHT production and suppress AR signaling in prostate cancer cells.Conclusion: Our study reveals a new role of ERRα in the intratumoral androgen biosynthesis in CRPC via its transcriptional control of steroidogenic enzymes, and also provides a novel insight that targeting ERRα could be a potential androgen-deprivation strategy for the management of CRPC.

Cotargeting the Cell-Intrinsic and Microenvironment Pathways of Prostate Cancer by PI3Kα/β/δ Inhibitor BAY1082439.

Targeting the PI3K pathway is a promising strategy for treating prostate cancers with PTEN-loss. However, current anti-PI3K therapies fail to show long lasting in vivo effects. We find that not only the PI3Kα- and PI3kβ-isoforms, but also PI3Kδ, are associated with the epithelial-mesenchymal transition (EMT), a critical process distinguishing indolent from aggressive prostate cancer. This suggests that cotargeting PI3Kα/β/δ could preempt the rebound activation of the parallel pathways induced by α- or β-isoform-selective inhibitor and prevent EMT. Indeed, BAY1082439, a new selective PI3Kα/β/δ inhibitor, is highly effective in vivo in inhibiting Pten-null prostate cancer growth and preventing EMT in the mutant Pten/Kras metastatic model. The anti-PI3Kδ property of BAY1082439 further blocks B-cell infiltration and lymphotoxin release, which are tumor microenvironment factors that promote castration-resistant growth. Together, our data suggest a new approach for the treatment of prostate cancer by targeting both tumor cells and tumor microenvironment with PI3Kα/β/δ inhibitor. Mol Cancer Ther; 17(10); 2091-9. ©2018 AACR.

Tumor Cell Autonomous RON Receptor Expression Promotes Prostate Cancer Growth Under Conditions of Androgen Deprivation

Current treatment strategies provide minimal results for patients with castration-resistant prostate cancer (CRPC). Attempts to target the androgen receptor have shown promise, but resistance ultimately develops, often due to androgen receptor reactivation. Understanding mechanisms of resistance, including androgen receptor reactivation, is crucial for development of more efficacious CRPC therapies. Here, we report that the RON receptor tyrosine kinase is highly expressed in the majority of human hormone-refractory prostate cancers. Further, we show that exogenous expression of RON in human and murine prostate cancer cells circumvents sensitivity to androgen deprivation and promotes prostate cancer cell growth in both in vivo and in vitro settings. Conversely, RON loss induces sensitivity of CRPC cells to androgen deprivation. Mechanistically, we demonstrate that RON overexpression leads to activation of multiple oncogenic transcription factors (namely, β-catenin and NF-κB), which are sufficient to drive androgen receptor nuclear localization and activation of AR responsive genes under conditions of androgen deprivation and support castration-resistant growth. In total, this study demonstrates the functional significance of RON during prostate cancer progression and provides a strong rationale for targeting RON signaling in prostate cancer as a means to limit resistance to androgen deprivation therapy.

Abstract B012: Histone demethylase JMJD1A promotes the generation of androgen receptor variant 7 (AR-V7) in prostate cancer cells

Abstract Generation of androgen receptor (AR) splicing variant, AR-V7, has been shown to contribute to the resistance of prostate cancer to androgen deprivation therapy and development of castration-resistant prostate cancer (CRPC). We and others have reported that histone demethylase JMJD1A promotes the transcriptional activity of AR by functioning as an AR coactivator. Here, we identified a new role for JMJD1A in the generation of AR-V7 in prostate cancer cells through interaction with HNRFPF. Knockdown of JMJD1A or HNRNPF reduced the mRNA and protein level of AR-V7 but had little effect on the full-length AR (AR-FL). Mechanistically, we found that JMJD1A and HNRNPF cooperatively bound to a polyG motif adjacent to the 5′ splicing site of Exon3b of AR gene, where they promoted the assembly of splicesome. Re-expression of AR-V7 in the JMJD1A-knockdown cells partly rescued the growth defect of prostate cancer cells in vitro and in the mouse xenograft model. The staining of AR-V7 paralleled that of JMJD1A or HNRNFP in a human prostate cancer tissue microarray. Together, our data reveal that JMJD1A enhances the castration-resistant growth of prostate cancer cells through coactivation of AR-FL and generation of AR-V7. Citation Format: Lingling Fan, Fengbo Zhang, Ladan Fazli, Martin Gleave, Xuesen Dong, Jianfei Qi. Histone demethylase JMJD1A promotes the generation of androgen receptor variant 7 (AR-V7) in prostate cancer cells [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B012.

BRCA1 Interacting Protein COBRA1 Facilitates Adaptation to Castrate-Resistant Growth Conditions.

COBRA1 (co-factor of BRCA1) is one of the four subunits of the negative elongation factor originally identified as a BRCA1-interacting protein. Here, we provide first-time evidence for the oncogenic role of COBRA1 in prostate pathogenesis. COBRA1 is aberrantly expressed in prostate tumors. It positively influences androgen receptor (AR) target gene expression and promoter activity. Depletion of COBRA1 leads to decreased cell viability, proliferation, and anchorage-independent growth in prostate cancer cell lines. Conversely, overexpression of COBRA1 significantly increases cell viability, proliferation, and anchorage-independent growth over the higher basal levels. Remarkably, AR-positive androgen dependent (LNCaP) cells overexpressing COBRA1 survive under androgen-deprivation conditions. Remarkably, treatment of prostate cancer cells with well-studied antitumorigenic agent, 2-methoxyestradiol (2-ME2), caused significant DNA methylation changes in 3255 genes including COBRA1. Furthermore, treatment of prostate cancer cells with 2-ME2 downregulates COBRA1 and inhibition of prostate tumors in TRAMP (transgenic adenocarcinomas of mouse prostate) animals with 2-ME2 was also associated with decreased COBRA1 levels. These observations implicate a novel role for COBRA1 in progression to CRPC and suggest that COBRA1 downregulation has therapeutic potential.

Abstract 1162: Castration-resistant prostate cancer bone metastasis model to assess new therapeutics

Abstract The role of androgens in the regulation of growth of primary prostate cancer is well established but less recognized in the context of advanced bone metastatic disease, which is the cause of high morbidity in patients. At advanced stage, prostate cancer cells typically lose their dependence on endogenous androgens leading to metastatic castration resistant prostate cancer (mCRPC). The aim of the study was to establish a novel mCRPC model resembling clinical aspects of the bone metastatic disease, which could be used for the evaluation of efficacy of new therapies. To obtain a model for castration resistant growth, 5-6 weeks old NOD.Scid male mice were divided into three study groups. Mice in two study groups were castrated either before inoculation of the cancer cells or at four weeks after inoculation when the tumors were already growing. The mice in one study group were left intact. All mice received an intratibial injection of 2x106 VCaP human prostate cancer cells originally derived from vertebral metastatic site. Tumor growth was followed biweekly for 16 weeks by PSA measurements and X-ray imaging of tumor induced bone changes (lesions). At endpoint, androgen dependent organs were weighed and tumor-bearing tibias were subjected to histological evaluation. At endpoint, a tumor take of 60%, 40% and 50% was observed in the intact mice and in the mice castrated one week before and four weeks after VCaP cell inoculation, respectively. Tumor take was assessed by PSA, X-ray and histology at endpoint. The PSA levels were higher in the intact mice and in the mice castrated at four weeks compared to the mice castrated before cancer cell inoculation. The PSA levels became detectable between 6-8 weeks after cancer cell inoculation in the intact mice and in the mice castrated at four weeks. In the mice castrated before cancer cell inoculation the PSA levels started to increase very late at 14-16 weeks. VCaP cells induced mainly new bone formation, an osteoblastic bone reaction typical for prostate cancer patients. In castrated mice, fewer bone lesions were observed compared to intact mice. The bone lesion areas quantified from X-ray images were larger in the intact mice compared to the mice castrated before cancer cell inoculation. As expected, the weight of androgen dependent organs was lower in the castrated mice compared to the intact mice. In conclusion, a model mimicking important clinical aspects of castration resistant prostate cancer bone metastases was established. Tumor take and growth rates indicated that early phases of tumor development into the bone are androgen dependent whereas tumor growth at later stage relies on the bone microenvironment. The results highlight the significance of the tumor microenvironment in establishing clinically relevant preclinical models for drug development. Citation Format: Tiina E. Kähkönen, Mari I. Suominen, Jussi M. Halleen, Jenni Bernoulli, Pascale Lejeune. Castration-resistant prostate cancer bone metastasis model to assess new therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1162.

Identification of Pik3ca Mutation as a Genetic Driver of Prostate Cancer That Cooperates with Pten Loss to Accelerate Progression and Castration-Resistant Growth.

Genetic alterations that potentiate PI3K signaling are frequent in prostate cancer, yet how different genetic drivers of the PI3K cascade contribute to prostate cancer is unclear. Here, we report PIK3CA mutation/amplification correlates with poor survival of patients with prostate cancer. To interrogate the requirement of different PI3K genetic drivers in prostate cancer, we employed a genetic approach to mutate Pik3ca in mouse prostate epithelium. We show Pik3caH1047R mutation causes p110α-dependent invasive prostate carcinoma in vivo Furthermore, we report that PIK3CA mutation and PTEN loss coexist in patients with prostate cancer and can cooperate in vivo to accelerate disease progression via AKT-mTORC1/2 hyperactivation. Contrasting single mutants that slowly acquire castration-resistant prostate cancer (CRPC), concomitant Pik3ca mutation and Pten loss caused de novo CRPC. Thus, Pik3ca mutation and Pten deletion are not functionally redundant. Our findings indicate that PIK3CA mutation is an attractive prognostic indicator for prostate cancer that may cooperate with PTEN loss to facilitate CRPC in patients.Significance: We show PIK3CA mutation correlates with poor prostate cancer prognosis and causes prostate cancer in mice. Moreover, PIK3CA mutation and PTEN loss coexist in prostate cancer and can cooperate in vivo to accelerate tumorigenesis and facilitate CRPC. Delineating this synergistic relationship may present new therapeutic/prognostic approaches to overcome castration/PI3K-AKT-mTORC1/2 inhibitor resistance. Cancer Discov; 8(6); 764-79. ©2018 AACR.See related commentary by Triscott and Rubin, p. 682This article is highlighted in the In This Issue feature, p. 663.

Nuclear Receptor LRH-1 Functions to Promote Castration-Resistant Growth of Prostate Cancer via Its Promotion of Intratumoral Androgen Biosynthesis.

Targeting of steroidogenic enzymes (e.g., abiraterone acetate targeting CYP17A1) has been developed as a novel therapeutic strategy against metastatic castration-resistant prostate cancer (CRPC). However, resistance to steroidal inhibitors inevitably develops in patients, the mechanisms of which remain largely unknown. Liver receptor homolog-1 (LRH-1, NR5A2) is a nuclear receptor, originally characterized as an important regulator of some liver-specific metabolic genes. Here, we report that LRH-1, which exhibited an increased expression pattern in high-grade prostate cancer and CRPC xenograft models, functions to promote de novo androgen biosynthesis via its direct transactivation of several key steroidogenic enzyme genes, elevating intratumoral androgen levels and reactivating AR signaling in CRPC xenografts as well as abiraterone-treated CRPC tumors. Pharmacologic inhibition of LRH-1 activity attenuated LRH-1-mediated androgen deprivation and anti-androgen resistance of prostate cancer cells. Our findings not only demonstrate the significant role of LRH-1 in the promotion of intratumoral androgen biosynthesis in CRPC via its direct transcriptional control of steroidogenesis, but also suggest targeting LRH-1 could be a potential therapeutic strategy for CRPC management.Significance: These findings not only demonstrate the significant role of the nuclear receptor LRH-1 in the promotion of intratumoral androgen biosynthesis in CRPC via its direct transcriptional control of steroidogenesis, but also suggest targeting LRH-1 could be a potential therapeutic strategy for CRPC management. Cancer Res; 78(9); 2205-18. ©2018 AACR.

MP64-11 CHEMOPREVENTIVE AND CHEMOTHERAPEUTIC POTENTIALS OF LUTEOLIN VIA REGULATION OF OXIDATIVE STRESS IN CASTRATION-RESISTANT PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology II1 Apr 2018MP64-11 CHEMOPREVENTIVE AND CHEMOTHERAPEUTIC POTENTIALS OF LUTEOLIN VIA REGULATION OF OXIDATIVE STRESS IN CASTRATION-RESISTANT PROSTATE CANCER Taku Naiki, Aya Naiki-Ito, Toshiki Etani, Keitaro Iida, Ryosuke Ando, Takashi Nagai, Noriyasu Kawai, Satoru Takahashi, and Takahiro Yasui Taku NaikiTaku Naiki More articles by this author , Aya Naiki-ItoAya Naiki-Ito More articles by this author , Toshiki EtaniToshiki Etani More articles by this author , Keitaro IidaKeitaro Iida More articles by this author , Ryosuke AndoRyosuke Ando More articles by this author , Takashi NagaiTakashi Nagai More articles by this author , Noriyasu KawaiNoriyasu Kawai More articles by this author , Satoru TakahashiSatoru Takahashi More articles by this author , and Takahiro YasuiTakahiro Yasui More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.2056AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Identifying effective treatments for castration-resistant prostate cancer (CRPC) is important to prevent its persistent progression following androgen deprivation therapy. In a previous study, we showed that changes in the expression of genes concerned with oxidative stress regulation induced carcinogenesis and castration-resistant growth in prostate cancer. Herein, we investigated the chemotherapeutic potential of luteolin, an anti-oxidative flavonoid, on prostate cancer and CRPC. METHODS Male transgenic rats for adenocarcinoma of prostate (TRAP) were divided into three groups (n = 12 in each group) and fed the following for 8 weeks: basal diet, diet containing 20 ppm luteolin, and diet containing 100 ppm luteolin. Prostate tissues were collected from each animal for histopathological, gene, and protein expression analysis.PCai cells established from the TRAP prostate tumor and 22Rv1 cells were subcutaneously implanted in castrated and non-castrated nude mice, and in vivo tumor growth was analyzed. Subsequently, the effect of luteolin on cell proliferation and apoptosis in PCai1 and 22Rv1 cells was analyzed. RESULTS Luteolin decreased the incidence of adenocarcinoma and suppressed its progression from PIN to adenocarcinoma in TRAP. An increased apoptosis index with activation of caspase-3 and 7 was observed with high grade-PIN in the luteolin-fed TRAP. Production of reactive oxygen species and mRNA expression of the oxidative stress defense genes Gpx2 and Ho-1 were reduced by luteolin. Furthermore, luteolin inhibited tumor growth in the xenograft model via apoptosis induction and Gpx2 down-regulation. Luteolin also suppressed PCai1 and 22Rv1 cell proliferation in a dose-dependent manner in vitro by caspase-3 and 7-dependent apoptosis. CONCLUSIONS Luteolin decreased cell proliferation in a dose-dependent manner, and induced apoptosis with caspase activation in both, PCai1 and 22Rv1 cells. Dietary luteolin also suppressed tumor growth by increasing apoptosis and inhibiting angiogenesis in the PCai1 xenograft model. These results suggest that luteolin is a potential therapeutic agent for CRPC. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e854 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Taku Naiki More articles by this author Aya Naiki-Ito More articles by this author Toshiki Etani More articles by this author Keitaro Iida More articles by this author Ryosuke Ando More articles by this author Takashi Nagai More articles by this author Noriyasu Kawai More articles by this author Satoru Takahashi More articles by this author Takahiro Yasui More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...