Regulates Prostate Cancer Research Articles

A role for paracrine interleukin-6 signaling in the tumor microenvironment in prostate tumor growth.

Interleukin-6 (IL-6) is a mediator of inflammation that can facilitate prostate cancer progression. We previously demonstrated that IL-6 is present in the prostate tumor microenvironment and is restricted almost exclusively to the stromal compartment. The present study examined the influence of paracrine IL-6 signaling on prostate tumor growth using allograft models of mouse prostate cancer (TRAMP-C2), colon cancer (MC38), and melanoma (B16) cell lines in wildtype (WT) and IL-6 knockout (IL-6-/- ) mice. Cells were implanted into WT or IL-6-/- mice and tumor sizes were measured at a 3 to 4 day interval. Serum, tumors, and other organs were collected for IL-6 analysis by ELISA and RNA in situ hybridization (RISH). There was a significant reduction in TRAMP-C2 and B16 tumor size grown in IL-6-/- mice versus WT mice (P = 0.0006 and P = 0.02, respectively). This trend was not observed for the MC38 cell line. RISH analysis of TRAMP-C2 tumors grown in WT mice showed that cells present in the tumor microenvironment were the primary source of IL-6 mRNA, not the TRAMP-C2 cells. Serum IL-6 ELISA analyses showed an increase in the circulating levels of IL-6 in WT mice bearing TRAMP-C2 tumors. Similar phospho-STAT3 expression and tumor vascularization were observed in TRAMP-C2 tumors grown in WT and IL-6-/- mice. Our results are consistent with previous studies in prostate cancer patients demonstrating that paracrine IL-6 production in the tumor microenvironment may influence tumor growth. Additionally, these data provide evidence that elevated systemic IL-6 levels may be involved in tumor growth regulation in prostate cancer, and are not simply caused by or indicative of tumor burden.

Expression of Androgen Receptor Variant 7 (AR-V7) in Circulated Tumor Cells and Correlation with Drug Resistance of Prostate Cancer Cells.

BackgroundProstate cancer is a common type of malignant tumor invading the male reproductive-urinary system, which has increasing incidence worldwide. Androgen receptor variant 7 (AR-V7) participates in regulating prostate cancer cell proliferation and gene expression. This study aimed to investigate the expression of AR-V7 in circulated tumor cells (CTCs) in patients with prostate cancer and to assess its correlation with drug sensitivity against enzalutamide or abiraterone.Material/MethodsBlood samples of prostate cancer patients were collected for separating CTCs, in which mRNA expression level of full-length AR and AR-V7 was measured to analyze their correlation with enzalutamide or abiraterone resistance. Progression-free survival (PFS) of patients with different AR-V7 expression levels was compared. AR-V7 was overexpressed in transfected prostate cancer cells, and its effects on proliferation were analyzed by clonal formation assay.ResultsqRT-PCR showed AR-V7 overexpression in a total of 13 patients; 76.92% of these patients developed drug resistance, the distal metastasis of which was significantly higher than that in the group with AR-V7 downregulation, with lower PFS (p<0.01). In cultured prostate cancer cells, AR-V7 upregulation resulted in a significantly higher clonal formation rate than in the control group with enzalutamide-containing medium (p<0.05).ConclusionsIn prostate cancer cells, AR-V7 expression is correlated with drug resistance, as AR-V7 upregulation leads to enhanced proliferation potency of cancer cells, indicating unfavorable prognosis of patients.

Neoadjuvant sipuleucel-T induces both Th1 activation and immune regulation in localized prostate cancer

ABSTRACT Sipuleucel-T is the only FDA-approved immunotherapy for metastatic castration-resistant prostate cancer. The mechanism by which this treatment improves survival is not fully understood. We have previously shown that this treatment can induce the recruitment of CD4 and CD8 T cells to the tumor microenvironment. In this study, we examined the functional state of these T cells through gene expression profiling. We found that the magnitude of T cell signatures correlated with the frequency of T cells as measured by immunohistochemistry. Sipuleucel-T treatment was associated with increased expression of Th1-associated genes, but not Th2-, Th17 – or Treg-associated genes. Post-treatment tumor tissues with high CD8+T cell infiltration was associated with high levels of CXCL10 expression. On in situ hybridization, CXCL10+ cells colocalized with CD8+T cells in post-treatment prostatectomy tumor tissue. Neoadjuvant sipuleucel-T was also associated with upregulation of immune inhibitory checkpoints, including CTLA4 and TIGIT, and downregulation of the immune activation marker, dipeptidylpeptidase, DPP4. Treatment-associated declines in serum PSA were correlated with induction of Th1 response. In contrast, rises in serum PSA while on treatment were associated with the induction of multiple immune checkpoints, including CTLA4, CEACAM6 and TIGIT. This could represent adaptive immune resistance mechanisms induced by treatment. Taken together, neoadjuvant sipuleucel-T can induce both a Th1 response and negative immune regulation in the prostate cancer microenvironment.

Long noncoding RNA LOXL1-AS1 regulates prostate cancer cell proliferation and cell cycle progression through miR-541-3p and CCND1

Prostate cancer is one of the most frequent malignancies affecting men. Long non-coding RNAs (lncRNAs) are involved in the pathogenesis of prostate cancer. LncRNA LOXL1-AS1 participates in the pathogenesis of the exfoliation syndrome. However, the role of LOXL1-AS1 in cancer remains largely unknown. Here, we found that LOXL1-AS1 down-regulation inhibited prostate cancer cell proliferation and cell cycle progression. RNA sequencing analysis revealed that it regulates the expression of cell cycle-related genes. LOXL1-AS1 is predominantly distributed in the cytoplasm, where it interacts with miR-541-3p. In addition, miR-541-3p targets the cell cycle regulator CCND1 in prostate cancer cells. LOXL1-AS1 down-regulation inhibits the expression of CCND1 and cell cycle progression, whereas these effects are abolished upon miR-541-3p suppression. In summary, our study revealed that LOXL1-AS1 regulates prostate cancer cell proliferation and cell cycle progression through miR-541-3p and CCND1. Modulation of their levels may be used to treat prostate cancer.

TRIP13 is a predictor for poor prognosis and regulates cell proliferation, migration and invasion in prostate cancer

Thyroid hormone receptor interactor 13 (TRIP13) has been reported to be overexpressed in serval types of human cancers, and regulate tumor cell proliferation, migration and invasion. However, the role of TRIP13 in prostate cancer was still unclear. In our study, the correlation between TRIP13 expression and clinical parameters including prognosis was evaluated in 160 prostate cancer patients. Moreover, the MTT assay, cell migration and invasion assays were performed to assess the effect of TRIP13 on prostate cancer cell biological behaviour. In our results, the expression status of TRIP13 was observed to be elevated in prostate cancer tissue samples through analyzing microarray (GSE55945). Furthermore, mRNA and protein TRIP13 expression were confirmed to be overexpressed in prostate cancer tissue samples and cell lines. High-expression of TRIP13 was correlated with present lymph node involvement, distant metastasis, high Gleason score, levels of serum PSA and poor prognosis in prostate cancer patients. The gain-of-function and loss-of-function studies suggested that TRIP13 functioned as oncogene to regulate prostate cancer cell proliferation, migration, invasion through controlling YWHAZ and epithelial-mesenchymal transition (EMT)-associated genes. In conclusion, TRIP13 is correlated with clinical progression and poor prognosis, and serves as oncogene in prostate cancer.

KLF4, a miR-32-5p targeted gene, promotes cisplatin-induced apoptosis by upregulating BIK expression in prostate cancer

BackgroundChemotherapeutic insensitivity remains a big challenge in prostate cancer treatment. Recently, increasing evidence has indicated that KLF4 plays a key role in prostate cancer. However, the potential biological role of KLF4 in Chemotherapeutic insensitivity of prostate cancer is still unknown.MethodsThe role of KLF4 in cisplatin-induced apoptosis was detected by western blotting and a cell counting kit (CCK8). The potential molecular mechanism of KLF4 in regulating prostate cancer chemosensitivity was investigated by RNA sequencing analysis, q-RT-PCR, western blotting and chromatin immunoprecipitation (ChIP). The expression level of KLF4 mediated by miR-32-5p was confirmed by bioinformatic analysis and luciferase assays.ResultsHere, we found that KLF4 was induced by cisplatin in prostate cancer cells and that the increase in KLF4 promoted cell apoptosis. Further mechanistic studies revealed that KLF4 directly bound to the promoter of BIK, facilitating its transcription. Additionally, we also found that the gene encoding KLF4 was a direct target of miR-32-5p. The downregulation of miR-32-5p in response to cisplatin treatment promoted KLF4 expression, which resulted in a increase in the chemosensitivity of prostate cancer.ConclusionThus, our data revealed that KLF4 is an essential regulator in cisplatin-induced apoptosis, and the miR-32-5p-KLF4-BIK signalling axis plays an important role in prostate cancer chemosensitivity.

MiRNA-205 Nanoformulation Sensitizes Prostate Cancer Cells to Chemotherapy.

The therapeutic application of microRNA(s) in the field of cancer has generated significant attention in research. Previous studies have shown that miR-205 negatively regulates prostate cancer cell proliferation, metastasis, and drug resistance. However, the delivery of miR-205 is an unmet clinical need. Thus, the development of a viable nanoparticle platform to deliver miR-205 is highly sought. A novel magnetic nanoparticle (MNP)-based nanoplatform composed of an iron oxide core with poly(ethyleneimine)-poly(ethylene glycol) layer(s) was developed. An optimized nanoplatform composition was confirmed by examining the binding profiles of MNPs with miR-205 using agarose gel and fluorescence methods. The novel formulation was applied to prostate cancer cells for evaluating cellular uptake, miR-205 delivery, and anticancer, antimetastasis, and chemosensitization potentials against docetaxel treatment. The improved uptake and efficacy of formulations were studied with confocal imaging, flow cytometry, proliferation, clonogenicity, Western blot, q-RT-PCR, and chemosensitization assays. Our findings demonstrated that the miR-205 nanoplatform induces significant apoptosis and enhancing chemotherapeutic effects in prostate cancer cells. Overall, these study results provide a strong proof-of-concept for a novel nonviral-based nanoparticle protocol for effective microRNA delivery to prostate cancer cells.

Abstract A079: MicroRNA-139 regulates prostate cancer aggressiveness by targeting IGF1R

Abstract Background: Dysregulated microRNA (miRNA) expression has been implicated in prostate cancer progression. We previously identified a panel of five miRNAs associated with biochemical recurrence and metastasis following prostatectomy based on NGS-based whole miRNome discovery and qPCR-based validation analysis. In this analysis, we examine the effect of miR-139-5p, one of the downregulated miRNAs identified in the panel, in greater detail. Methods: Using a cohort of 585 patients treated with radical prostatectomy, we examined the prognostic significance of miR-139 (dichotomized around the median) using the Kaplan Meier method and Cox proportional hazard models. We validated these results using The Cancer Genome Atlas (TCGA) data. We created cell lines that overexpressed miR-139 for functional assays. Finally, we examined pathways through which miR-139 may function using prediction algorithms and confirmed targets by Western blotting and reporter assays. Results: MiR-139 downregulation was significantly associated with a variety of accepted prognostic factors in prostate cancer, including Gleason score, pathologic stage, margin positivity, and lymph node status. MiR-139 was associated with prognosis: the cumulative incidence of biochemical recurrence and metastasis was significantly lower among patients with high miR-139 expression (p=0.0004 and 0.038, respectively). After adjusting for known prognostic factors, patients with high miR-139 expression had significantly lower risk of recurrence (HR 0.77, 95% 0.58-1.04). Validation in the TCGA dataset showed a significant association between dichotomized miR-139 expression and biochemical recurrence (OR 0.52, 95% CI 0.33-0.82). Overexpression of miR-139 in PC3 and DU145 prostate cancer cells led to a significant reduction in cell proliferation and migration compared to control cells. IGF1R was identified as a potential target of miR-139 based on previous work in colorectal and non-small cell lung cancers. Reduced luciferase reporter activity was observed upon co-transfection of the 3′ UTR of IGF1Rβ with miR-139 mimic compared to co-transfection with control mimic. Furthermore, Western blotting of PC3 cells overexpressing miR-139 revealed reduced IGF1Rβ protein expression, as well as reduced expression of its downstream pathway proteins pAKT and pERK. Cell cycle analysis indicated a significantly increased number of cells arrested in G2/M phase in PC3 cells overexpressing miR-139. This was accompanied by an increase in β-galactosidase stained senescent cells and p21 protein expression. Conclusions: miR-139 is associated with improved prognosis in patients with localized prostate cancer. This appears to be mediated through an IGF1R pathway leading to increased p21 expression, resulting in prostate cancer cell senescence from G2 arrest. Citation Format: Yutaka Amemiya, Christopher J. Wallis, Tania Benatar, Elizabeth Kobylecky, Linda Sugar, Christopher Sherman, Robert Nam, Arun K. Seth. MicroRNA-139 regulates prostate cancer aggressiveness by targeting IGF1R [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 A079.

Toll-Like Receptor 4 (TLR4)/Cyclooxygenase-2 (COX-2) Regulates Prostate Cancer Cell Proliferation, Migration, and Invasion by NF-κB Activation.

BackgroundToll-like receptor 4 (TLR4)-mediated signaling has been implicated in invasion, metastasis, and survival of various cancers. Activation of TLR4 can promote cyclooxygenase-2 (COX-2) and nuclear factor-κB (NF-κB). However, little is known about the effects of TLR4/COX-2 in prostate cancer (PCa).Material/MethodsIn our study, TLR4 and COX-2 expressions were detected by quantitative real-time reverse transcription PCR (qRT-PCR) in PCa tissues (n=34). Cell proliferation was measured by Cell Counting Kit-8 (CCK-8) and carboxyfluorescein succinimidyl ester (CFSE) assays. The migration and invasion abilities were detected by wound healing and Transwell assays. qRT-PCR and western blot assays were performed to detect TLR4, COX-2, matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitor of matrix metalloproteinases (TIMP)-1, epithelial-cadherin (E-cadherin), vimentin, NF-κB (p65), and p-p65 expressions.ResultsThe results revealed that TLR4 and COX-2 were upregulated in PCa tissues; Silencing of TLR4 or COX-2 inhibited PCa cell proliferation, migration, and invasion, and TLR4 siRNAs combined with COX-2 siRNAs synergistically suppressed PCa cell proliferation, migration, and invasion. Silencing of TLR4 or COX-2 also downregulated MMP-2, MMP-9, and E-cadherin expressions, and upregulated TIMP-1 and vimentin expressions. In addition, silencing of TLR4 or COX-2 inhibited p65 phosphorylation and had a synergistic effect.ConclusionsWe demonstrated that TLR4/COX-2 inhibits PCa cell proliferation, migration, and invasion by regulating NF-κB.

LncRNA TINCR is associated with clinical progression and serves as tumor suppressive role in prostate cancer.

IntroductionTerminal differentiation-induced non-coding RNA (TINCR) has been suggested to have aberrant expression in multiple human cancers, and functions as tumor suppressor or promoter in various types of human tumors depending on the specific cancer types. The expression status and biological function of TINCR in prostate cancer is still unknown.Materials and methodsIn our study, we detected TINCR expression in prostate cancer tissue samples and cell lines, and analyzed the association between TINCR expression and clinical parameters in 160 prostate cancer patients. Moreover, we conducted gain-of-function and loss-of-function studies in prostate cancer cell to explore the biological function and molecular mechanism of TINCR.ResultsIn our results, low-expression TINCR was observed in prostate cancer, and correlated with advanced clinical T stage, lymph node involvement, distant metastasis, high Gleason score and poor prognosis in prostate cancer patients. Moreover, levels of TINCR expression were negatively associated with TRIP13 mRNA and protein expressions in prostate cancer tissues, and negatively regulated the TRIP13 mRNA and protein expressions in prostate cancer cell lines. TINCR inhibits prostate cancer cell proliferation, migration and invasion via suppressing TRIP13 expression.ConclusionTINCR plays a tumor suppressive role in regulating prostate cancer cell proliferation, migration and invasion through modulating TRIP13 expression.

Loss of miR-516a-3p mediates upregulation of ABCC5 in prostate cancer and drives its progression.

To gain a comprehensive understanding of whether ABCC5 can regulate prostate cancer (PCa) progression, we performed microarray data analyses and identified that ABCC5 was drastically increased in primary PCa relative to normal samples, metastatic PCa relative to primary PCa, and castration-resistant PCa relative to hormone naïve PCa, respectively. Multivariate Cox regression analysis suggested that ABCC5 overexpression in PCa was an independent prognostic factor for both poor biochemical recurrence-free and overall survival. We demonstrated that ABCC5 knockdown significantly inhibits PCa cell proliferation, migration, and invasion in vitro and suppresses tumor growth and metastasis in vivo. We also demonstrated that miR-516a-3p was significantly downregulated in PCa. We finally demonstrated that ABCC5 was a direct target of miR-516a-3p. miR-516a-3p overexpression can phenotypically copy ABCC5 knockdown-induced phenotypes, whereas forced expression of ABCC5 can drastically reverse the inhibitory effects of miR-516a-3p. miR-516a-3p may modulate the sensitivity of cancer cells to adriamycin and docetaxel by targeting ABCC5 with important implications in the design of new therapeutic agents. Taken together, our results indicated that loss of miR-516a-3p expression and thus uncontrolled ABCC5 upregulation might drive PCa progression and influence chemosensitivity.

Abstract 2130: Novel ADAM9 variant induces prostate cancer metastasis

Abstract Prostate cancer often progresses from an androgen to castration-refractory prostate cancer and metastasizes to the bone and lymph nodes. However, there is no biomarker to predict the initiation of cancer metastasis. Therefore, it is an unmet need to understand molecules which induce metastasis to replace the prediction by PSA. Previous studies by us demonstrate the elevation of ADAM9 in PCa malignant progression. However, the role of ADAM9 in the tumor microenvironment is still not fully understood. Using laser capture microdissection of prostate tumor and stromal cells, indicating the increased of ADAM9 and reactive oxygen species in tumor-associated stromal cells. These inductions correlated with the increased expression of SOD-2 in tumor-associated stromal cells. Both electron microscope analysis and conditioned medium of stromal cells demonstrated the increased of soluble ADAM9 level in the medium. ELISA analysis of patient serum confirmed the increased of ADAM9 serum levels in prostate cancer patients. PCR and sequencing analysis confirmed a novel soluble ADAM9 (sADAM9v2) with deletion of membrane domain that induces cell migration through the activation of FAK and AKT phosphorylation. Our studies demonstrated the release of sADAM9 in tumor microenvironment correlated with reactive oxygen stress and regulate prostate cancer migration and metastasis. In conclusion, our studies showed that sADAM9 release by cancer-associated stromal cells could be a potential biomarker for prostate cancer progression. Citation Format: Chia-Ling Hsieh, Shian-Ying Sung. Novel ADAM9 variant induces prostate cancer metastasis [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 2130.

Abstract 4292: Negative regulation of oncogenes by a novel tumor suppressor microRNA in prostate cancer

Abstract Background: MicroRNAs are small noncoding RNAs that regulate the expression of &amp;gt;60% of all human genes, either inhibiting target mRNA translation or inducing its degradation. MicroRNAs act as tumor suppressors or oncogenes in various cancers. The main objective of this study was to investigate the role of microRNA-588 (miR-588) in prostate cancer (PC). Methods: The methods employed in this study include quantitative-real time PCR; western blot; fluorescence-activated cell sorting assays for cell cycle distribution and apoptosis; assays for cell viability, migration and invasiveness of prostate cancer cells. Luciferase reporter assays and in-vivo study in nude mice was also performed. Results: The expression of miR-588 was significantly suppressed or silenced in PC tissue samples and cell lines when compared with normal tissues and a non-malignant cell line. Similar results were observed by analyzing the publicly available TCGA data sets for prostate adenocarcinoma. Functionally ectopic expression of miR-588 induced G0/G1 cell cycle arrest and apoptosis and suppressed cell proliferation. miR-588 exerted these functional effects by directly targeting the oncogenic Cyclin A2 that is involved in cancer cell cycle and proliferation. In silico algorithm showed a complimentary binding sequence in the 3'UTR of Cyclin A2 for miR-588. Over-expression of miR-588 significantly suppressed the luciferase activity of reporter plasmid containing the wild type 3'UTR sequences of cyclin A2 complementary to miR-588, which was abolished by mutations in these 3'UTR regions. miR-588 overexpression also significantly reduced the expression of cyclin A2 at both mRNA and protein levels. A significant decrease in the expression of various cell cycle pathway genes such as CycE1, MCM2, MCM4, CDC7 and CDT1 was also observed. These genes are involved in promoting cell cycle and proliferation and are overexpressed in prostate cancer. In vivo intracardiac implantation of PC3-MLuc-C6 prostate cancer cells constitutively expressing miR-588 showed a significant inhibition of the metastatic dissemination of these cells compared to the control miR expressing cells. Conclusion: This is the first study demonstrating that miR-588 is significantly silenced and acts as a tumor suppressor in prostate cancer. Reconstitution of silenced miR-588 may contribute to novel therapeutic approaches for regulating prostate cancer. Citation Format: Nadeem Bhat, Altaf A. Dar, Sharanjot Saini, Varahram Shahryari, Priyanka Kulkarni, Pritha Dasgupta, Soichiro Yamamura, Yuichiro Tanaka, Taku Kato, Yutaka Hashimoto, Marisa Shiina, Z. Laura Tabatabai, Guoren Deng, Rajvir Dahiya, Shahana Majid. Negative regulation of oncogenes by a novel tumor suppressor microRNA in prostate cancer [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 4292.

Abstract 3747: Translating the functional interactions of checkpoint kinase 2 and the androgen receptor into more effective therapies for the treatment of prostate cancer

Abstract Prostate cancer remains the most diagnosed cancer among men in the United States behind skin cancer, and advanced prostate cancer is the third leading cause of cancer-related deaths, with a 5-year survival rate of 26%. Radiation is the standard of care for the treatment of prostate cancer at the early and late stages. Checkpoint kinase 2 (CHK2) is a serine/threonine protein kinase whose main function is regulating the DNA damage response (DDR) induced by ionizing radiation. The androgen receptor (AR) is a major driver of prostate cancer, even at the castration-resistant stage of the disease. The development of the second-generation anti-androgen enzalutamide, which is a selective AR antagonist, highlights the enduring importance of the AR. We have previously demonstrated that CHK2 is a critical negative regulator of prostate cancer cell growth, androgen sensitivity, and AR transcriptional activity. We have now uncovered novel molecular interactions between CHK2 and AR that provide mechanistic insight into our observation that CHK2 regulates prostate cancer growth. The AR directly interacts with CHK2, and that interaction increases with radiation. We found that the interaction of CHK2 and AR occurs at sites of DNA damage. The binding of CHK2 with AR can be disrupted with CHK2 kinase inhibitors suggesting that the kinase activity of CHK2 is required. This was verified using kinase-impaired CHK2 variants, including the K373E variant associated with 4.2% of prostate cancer. Furthermore, the radiation-induced increase in CHK2-AR interactions requires AR phosphorylation on both serine 81 and serine 308. Interestingly, CHK2-depletion in LNCaP cells increases ionizing radiation induced AR expression and DNA damage. Together, these data provide the rationale for targeting the CHK2-AR signaling axis to improve the effectiveness of prostate cancer therapies. The combination of CHK2 or CDK1 inhibitors with androgen deprivation therapy (ADT) and radiation shows an additive effect on the repression of tumor cell growth. Nearly every patient with disseminated prostate cancer will relapse following ADT and develop incurable castration-resistant prostate cancer. We have uncovered the molecular details of a signaling axis involving CHK2 and AR that, when perturbed in combination with ADT and/or ionizing radiation, effectively inhibits prostate cancer cell growth. This may enable resensitization of castration-resistant prostate cancer to the currently approved treatment options. Citation Format: Huy Q. Ta, Natalia Dworak, Rosalie Sleppy, Jeffery A. Allende, Daniel Gioeli. Translating the functional interactions of checkpoint kinase 2 and the androgen receptor into more effective therapies for the treatment of prostate cancer [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 3747.

Abstract 1084: TRAF4-mediated NGF receptor TrkA ubiquitination regulates prostate cancer metastasis

Abstract Receptor tyrosine kinases (RTKs) are important drivers of cancers. In addition to genomic alterations, aberrant activation of wild type RTKs play an important role in driving cancer progression. However, the underlying mechanisms of how RTKs drive prostate cancer remains incompletely characterized. Here we show that non-proteolytic ubiquitination of RTK regulates its kinase activity and contributes to RTK-mediated prostate cancer metastasis. TRAF4, a RING domain E3 ubiquitin ligase, is highly expressed in metastatic prostate cancer. We demonstrated here that it is a key player in regulating RTK mediated prostate cancer metastasis. We further identified TrkA, a neurotrophin receptor tyrosine kinase, as a TRAF4-targeted ubiquitination substrate that promotes cancer cell invasion and genetic ablation of TrkA abolished TRAF4-dependent cell invasion. TRAF4 promoted atypical K27 and K29-linked ubiquitination at the TrkA kinase domain and increased its kinase activity. Mutation of TRAF4-targeted ubiquitination sites abolished TrkA tyrosine auto-phosphorylation and its interaction with downstream effector proteins. TRAF4 knockdown also suppressed nerve growth factor-stimulated TrkA downstream p38 MAPK activation and subsequent invasion-associated gene expression. Furthermore, elevated TRAF4 levels significantly correlated with increased NGF-stimulated invasion-associated gene expression in prostate cancer patients, indicating this signaling axis is significantly activated during oncogenesis. Our results revealed a post-translational modification mechanism contributing to aberrant non-mutated receptor tyrosine kinase activation in cancer cells. Citation Format: Ramesh Singh, Dileep Karri, Jiangyong Shao, Subhamoy Dasgupta, Shixia Huang, Dean P. Edwards, Bert W. O'Malley, Ping Yi. TRAF4-mediated NGF receptor TrkA ubiquitination regulates prostate cancer metastasis [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 1084.

Fatty Acids and Calcium Regulation in Prostate Cancer.

Prostate cancer is a widespread malignancy characterized by a comparative ease of primary diagnosis and difficulty in choosing the individualized course of treatment. Management of prostate cancer would benefit from a clearer understanding of the molecular mechanisms behind the transition to the lethal, late-stage forms of the disease, which could potentially yield new biomarkers for differential prognosis and treatment prioritization in addition to possible new therapeutic targets. Epidemiological research has uncovered a significant correlation of prostate cancer incidence and progression with the intake (and often co-intake) of fatty acids and calcium. Additionally, there is evidence of the impact of these nutrients on intracellular signaling, including the mechanisms mediated by the calcium ion as a second messenger. The present review surveys the recent literature on the molecular mechanisms associated with the critical steps in the prostate cancer progression, with special attention paid to the regulation of these processes by fatty acids and calcium homeostasis. Testable hypotheses are put forward that integrate some of the recent results in a more unified picture of these phenomena at the interface of cell signaling and metabolism.

TRAF4-mediated ubiquitination of NGF receptor TrkA regulates prostate cancer metastasis.

Receptor tyrosine kinases (RTKs) are important drivers of cancers. In addition to genomic alterations, aberrant activation of WT RTKs plays an important role in driving cancer progression. However, the mechanisms underlying how RTKs drive prostate cancer remain incompletely characterized. Here we show that non-proteolytic ubiquitination of RTK regulates its kinase activity and contributes to RTK-mediated prostate cancer metastasis. TRAF4, an E3 ubiquitin ligase, is highly expressed in metastatic prostate cancer. We demonstrated here that it is a key player in regulating RTK-mediated prostate cancer metastasis. We further identified TrkA, a neurotrophin RTK, as a TRAF4-targeted ubiquitination substrate that promotes cancer cell invasion and found that inhibition of TrkA activity abolished TRAF4-dependent cell invasion. TRAF4 promoted K27- and K29-linked ubiquitination at the TrkA kinase domain and increased its kinase activity. Mutation of TRAF4-targeted ubiquitination sites abolished TrkA tyrosine autophosphorylation and its interaction with downstream proteins. TRAF4 knockdown also suppressed nerve growth factor (NGF) stimulated TrkA downstream p38 MAPK activation and invasion-associated gene expression. Furthermore, elevated TRAF4 levels significantly correlated with increased NGF-stimulated invasion-associated gene expression in prostate cancer patients, indicating that this signaling axis is significantly activated during oncogenesis. Our results revealed a posttranslational modification mechanism contributing to aberrant non-mutated RTK activation in cancer cells.

Regulation of prostate cancer by hormone-responsive G protein-coupled receptors.

Regulation of prostate cancer by androgen and androgen receptor (AR), and blockade of AR signaling by AR antagonists and steroidogenic enzyme inhibitors have been extensively studied. G protein-coupled receptors (GPCRs) are a family of membrane receptors that regulate almost all physiological processes. Nearly 40% of FDA-approved drugs in the market target GPCRs. A variety of GPCRs that mediate reproductive function have been demonstrated to be involved in the regulation of prostate cancer. These GPCRs include gonadotropin-releasing hormone receptor, luteinizing hormone receptor, follicle-stimulating hormone receptor, relaxin receptor, ghrelin receptor, and kisspeptin receptor. We highlight here GPCR regulation of prostate cancer by these GPCRs. Further therapeutic approaches targeting these GPCRs for the treatment of prostate cancer are summarized.

MiR-203 over-expression promotes prostate cancer cell apoptosis and reduces ADM resistance.

Extra-cellular signal regulated kinase (ERK)/mitogen activated protein kinase (MAPK) signaling pathway is widely involved in cell proliferation, apoptosis, and drug resistance. MAPK kinase 1 (MEK1) is the upstream protein kinase of ERK that can activate ERK/MAPK signaling pathway. microRNA 203 (MiR-203) down-regulation is found to be associated with prostate cancer pathogenesis. Bioinformatics analysis showed the complementary targeted relationship between miR-203 and the 3'-UTR of MEK1 mRNA. This study explored the role of miR-203 in regulating prostate cancer cell proliferation, apoptosis, and ADM resistance through affecting MEK1 expression. Dual luciferase assay confirmed the targeted relationship between miR-203 and MEK1. MiR-203, MEK1, p-ERK1/2, and B cell lymphoma 2 (Bcl-2) expressions were compared in normal prostate epithelial cells PrEC, prostate cancer cells PC-3M, and drug resistance cells PC-3M/ADM. PC-3M, PC-3M/ADM cell apoptosis and proliferation were detected by using flow cytometry under ADM treatment at IC50 concentration of PC-3M cells. PC-3M cells were cultured in vitro and divided into four groups, including microRNA-normal control (miR-NC), miR-203 mimic, small interfere NC (si-NC), and si-MEK1. MiR-203 targeted and inhibited MEK1 expression. MiR-203 levels and cell apoptosis were significantly lower, while MEK1, p-ERK1/2, Bcl-2, and cell proliferation were significantly higher in PC-3M/ADM cells compared to the PC-3M cells. MiR-203 mimic and/or si-MEK1 transfection significantly reduced MEK1, p-ERK1/2, and Bcl-2 levels, attenuated cell proliferation, induced cell apoptosis, and decreased drug resistance. MiR-203 elevation suppressed prostate cancer PC-3M cell proliferation, promoted apoptosis, and weakened ADM resistance through targeted inhibiting MEK1 expression to alleviate ERK/MAPK signaling pathway and Bcl-2 expression.

URG11 Regulates Prostate Cancer Cell Proliferation, Migration, and Invasion.

Upregulated gene 11 (URG11), a new gene upregulated by hepatitis B virus X protein, is involved in the development and progression of several tumors, including liver, stomach, lung, and colon cancers. However, the role of URG11 in prostate cancer remains yet to be elucidated. By determined expression in human prostate cancer tissues, URG11 was found significantly upregulated and positively correlated with the severity of prostate cancer, compared with that in benign prostatic hyperplasia tissues. Further, the mRNA and protein levels of URG11 were significantly upregulated in human prostate cancer cell lines (DU145, PC3, and LNCaP), compared with human prostate epithelial cell line (RWPE-1). Moreover, by the application of siRNA against URG11, the proliferation, migration, and invasion of prostate cancer cells were markedly inhibited. Genetic knockdown of URG11 also induced cell cycle arrest at G1/S phase, induced apoptosis, and decreased the expression level of β-catenin in prostate cancer cells. Overexpression of URG11 promoted the expression of β-catenin, the growth, the migration, and invasion ability of prostate cancer cells. Taken together, this study reveals that URG11 is critical for the proliferation, migration, and invasion in prostate cancer cells, providing the evidence of URG11 to be a novel potential therapeutic target of prostate cancer.