Castration-resistant Prostate Cancer Tissues Research Articles

MUC1 expression is associated with ST3GAL2 and negatively correlated with the androgen receptor in castration-resistant prostate cancer

Stage-specific embryonic antigen-4 (SSEA-4) is a developmentally regulated antigen, while expression level of SSEA-4 and / or its synthase ST3GAL2 is associated with prognosis in various malignancies. We have reported a prominent increase of SSEA-4 in castration-resistant prostate cancer (CRPC) and its negative correlation with the androgen receptor (AR). Meanwhile, loss of AR has increased to approximately 30% with the growing use of androgen receptor signaling inhibitor for metastatic CRPC (mCRPC). However, monitoring the progression status of AR-negative prostate cancer is a challenge because it does not produce prostate-specific antigen. Based on the negative relationship of expression between AR and SSEA-4, we hypothesized that a soluble molecule synchronized with SSEA-4 in expression could be a serum marker candidate for AR-negative prostate cancer. Thus, we investigated the molecular background of SSEA-4 expression by ST3GAL2-knockout in DU145 cells. Here we show that MUC1 is identified as a molecule associated with ST3GAL2 and expressed in AR-negative prostate cancer. A negative correlation of expression between AR and MUC1 was observed in prostate cancer cell lines and CRPC tissues. The average rate of MUC1 expression was nearly 60% in AR-negative prostate cancer cells in CRPC tissues. Level of serum CA15–3 (MUC1) was the highest in mCRPC among various stages and its higher level was associated with faster progression of mCRPC. Our results demonstrate that MUC1 is identified as a ST3GAL2-associated molecule and expressed in AR-negative CRPC cells. Furthermore, level of serum CA15–3 may reflect the progression status of mCRPC.

NFYA-mediated promotion of castration-resistant prostate cancer progression through EGR4 regulation

This research investigates the intricate involvement of nuclear Transcription Factor Y Subunit Alpha (NFYA) in the advancement of castration-resistant prostate cancer (CRPC) and its consequential impact on early Growth Response 4 (EGR4) expression. NFYA demonstrates a significant elevation in CRPC tissues and cell lines, displaying robust upregulation in metastatic prostate cancer (mPCa) samples, closely associated with the Gleason score. Immunohistochemistry validates heightened nuclear staining of NFYA in CRPC patients, highlighting its crucial role in the progression of advanced prostate cancer. Silencing NFYA through siRNA in androgen-independent cell lines markedly impedes cell growth and migration, emphasizing NFYA’s pivotal role in promoting CRPC behavior. RNA-seq analysis identifies EGR4 as a downstream target of NFYA, with both genes consistently upregulated in CRPC. Validating this finding, heightened expression of EGR4 is observed in CRPC samples. In vivo studies utilizing a mouse model demonstrate that NFYA silencing substantially inhibits LNCaP-AI/22RV1shNFYA xenograft tumor growth, accompanied by reduced expression of EGR4 and Ki67. This comprehensive study reveals the multifaceted role of NFYA in CRPC progression, elucidates its downstream impact on EGR4, and underscores the therapeutic potential of targeting NFYA to inhibit CRPC growth in vivo. These findings contribute valuable insights into potential therapeutic strategies for managing CRPC.

Silencing SPP1 in M2 macrophages inhibits the progression of castration-resistant prostate cancer via the MMP9/TGFβ1 axis.

M2 macrophages can promote the progression of castration-resistant prostate cancer (CRPC), but the specific mechanism is still unclear. Therefore, we are preliminarily exploring the molecular mechanism by which M2 macrophages regulate the progression of CRPC. The genes positively correlated with CRPC and with the most significant differences in the GEO32269 dataset were obtained. Database and immunofluorescence experiments were used to validate the localization of secreted phosphoprotein 1 (SPP1) in localized prostate cancer (PCa), hormone-sensitive prostate cancer (HSPC), and CRPC tumor tissues. The function of SPP1 in M2 macrophages was verified through cell scratch, Transwell, and an orthotopic PCa model. PCa database and Western blot were used to verify the relationship between SPP1 and matrix metallopeptidase 9 (MMP9), as well as the ability of MMP9 in M2 macrophages to promote epithelial-mesenchymal transition (EMT) in PCa cells. The primary localization of SPP1 in prostate and CRPC tissues is in macrophages. Silencing SPP1 expression in M2 macrophages promotes their polarization towards the M1 phenotype and significantly inhibits the malignant progression of PCa in vitro and in vivo. SPP1 promotes the expression of MMP9 through the PI3K/AKT signaling pathway in M2 macrophages. Furthermore, MMP9 enhances the EMT and migratory capabilities of PC3 cells by activating the TGFβ signaling pathway. We have found that the high expression of SPP1 in M2 macrophages promotes the progression of CRPC through cell-cell interactions. These findings can contribute to the development of novel therapeutic approaches for combating this deadly disease.

Abstract 1725: Role of PRMT5 in neuroendocrine prostate cancer development

Abstract Background: Neuroendocrine prostate cancer (NEPC) is a lethal subtype of prostate cancer responsible for an estimated 20-30% of castration resistant prostate cancer (CRPC) deaths. While NEPC can arise de novo, the majority of these cases emerge as treatment-induced NEPC (tNEPC). Our data establish PRMT5:MEP50 as a key epigenetic regulator in NEPC and provide a potential predictive biomarker for tNEPC. Methods: Here we generated an in vitro cell culture model and mouse model to mimic the clinical conditions in androgen signaling inhibitor (ASI) or androgen deprivation treatment (ADT) We developed an in vitro cell culture and in vivo model to mimic the clinical conditions in enzalutamide-resistant prostate cancer. Regulation and role of protein arginine methyltransferase 5 (PRMT5) and its cofactor methylosome protein 50 (MEP50) were determined by treating cells and mice tumors with doxycycline inducible-shRNAs and catalytic inhibitors followed by analysis of cell viability, neurite growth, and effects on neuroendocrine related gene transcription. Pro-NEPC development effects were analyzed by immunofluorescence, immunohistochemical analysis, and western blot. PRMT5:MEP50-dependent methyltransferase activity was further validated by observing histone deposition levels via western blot and ChIP-qPCR. For clinical correlation, PRMT5 and MEP50 expression levels were comprehensively analyzed in both CRPC and NEPC patient prostate tissue samples. Results: Our clinical and computational analyses have identified increased expression of PRMT5:MEP50 in NEPC. Overexpression of PRMT5 and MEP50 in prostate cancer cells is sufficient to induce neuroendocrine differentiation (NED) in prostate cancer cells. Gene depletion of PRMT5 and MEP50 prevented the NE phenotype induced by androgen receptor inhibition and resensitized the prostate cancer cells to ASI/ADT. PRMT5 and MEP50 facilitate chromatin modulation through the dimethylation of H4R3, which is highly correlated with the NED phenotype. Moreover, overexpression of PRMT5 and MEP50 in mouse prostate induces NEPC development, and gene downregulation can prevent NED in a xenograft mouse model. Elevated PRMT5 and MEP50 correlated with increased recurrence in prostate cancer patients receiving ADT and their recurrence probability after the treatment. Conclusion: Our data establish PRMT5:MEP50 as a key epigenetic regulator in NEPC and provide a potential predictive biomarker for tNEPC. Citation Format: Hye Seung Nam, Xuehong Deng, Chang-Deng Hu. Role of PRMT5 in neuroendocrine prostate cancer development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1725.

Abstract 2266: Detection of androgen receptor splice variants from clinical sequencing

Abstract Background: Androgen receptor splice variant-7 (AR-v7) has been widely studied as a biomarker of resistance to AR-targeted therapy in castration-resistant prostate cancer (CRPC). Various methods have been used to detect AR-v7 in CRPC specimens, including next generation sequencing (NGS) technology. However, NGS technologies applied in clinical sequencing have focused on using blood or fresh-frozen CRPC tissue specimens. There is not yet an AR-v7 detection system tailored to formalin-fixed paraffin-embedded (FFPE) tissue specimens commonly used for clinical sequencing in hospitals. Methods: In this respect, we propose a novel approach to identify AR-v7 in targeted RNA sequencing (RNA-seq) data derived from FFPE specimens. This approach can identify, in addition to AR-v7, all constitutively active AR splice variants (AR-Vs) that are co-expressed with AR-v7. In short, our two-step approach first gathers soft-clipped or divided reads that are adjacent to the splicing sites of AR-Vs and then yields the number of splitting reads that support the existence of AR-Vs. Next, the algorithm selects the paired-end reads whose one side and the other side are mapped to the preceding and following exons, respectively. The final number of spanning reads are calculated following to the removal of low-quality reads. Results: We validated the proposed approach using two large-scale, independent RNA-seq datasets of prostate cancer (PC) samples: 111 samples from Seoul National University Hospital (SNUH) and 558 samples from The Cancer Genome Atlas (TCGA) dataset. The majority of both datasets were localized PC tumors, especially in the SNUH dataset, where localized PC accounted for 91% of the total. Overall, our approach successfully identified samples with putative AR-Vs, including AR-v7, AR-v3, and AR-v9. Statistical analyses of 111 SNUH PC patients suggested potential detection threshold in clinical sequencing settings (at least 200 split reads). In the SNUH dataset, the AR-v7 positive group, which included 5 patients (5%) with values above the threshold, shared the following characteristics: higher AR-v7/full length AR expression levels, AR amplification and co-expression with AR-v3/AR-v9. AR-v7 positivity was experimentally validated by Sanger sequencing. The similar pattern was also found in the analysis of TCGA dataset. Conclusions: These results demonstrate that the detection system can successfully identify AR-v7 positivity not only for metastatic PC but also for patients with localized PC through clinical sequencing. We expect that the further in-depth analyses including larger samples and clinical outcomes can discover clinical applicability of AR-v7. Citation Format: Suhyun Hwangbo, Sungyoung Lee, Sheehyun Kim, Hongseok Yun. Detection of androgen receptor splice variants from clinical sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2266.

YAP1 Regulates the YAP1/AR/PSA Axis through Autophagy in Castration-Resistant Prostate Cancer and Mediates T-Cell Immune and Inflammatory Cytokine Infiltration.

The emergence of castration-resistant prostate cancer (CRPC) following androgen deprivation therapy (ADT) is associated with increased malignancy and limited treatment options. This study aims to investigate potential connections between immune cell infiltration and inflammatory cytokines with the YAP1/AR/PSA axis by exploring their interactions with autophagy. Our research reveals heightened levels of Yes-associated protein 1 (YAP1) expression in CRPC tissues compared with tissues from androgen-dependent prostate cancer (ADPC) and benign prostate hyperplasia (BPH). Additionally, a correlation was observed between YAP1 and PSA expressions in CRPC tissues, suggesting that YAP1 may exert a regulatory influence on PSA expression within CRPC. Enhanced YAP1 expression in C4-2 cells resulted in the upregulation of androgen receptor (AR) nuclear translocation and intracellular prostate-specific antigen (PSA) levels. Conversely, the suppression of YAP1 led to a decrease in PSA expression, suggesting that YAP1 may positively regulate the PSA in castration-resistant prostate cancer (CRPC) by facilitating AR nuclear import. The modulation of the autophagy activity exerts a significant impact on the expression levels of YAP1, the AR, and the PSA. Moreover, recent advancements in immunity and inflammation studies present promising avenues for potential therapies targeting prostate cancer (PC).

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.

YAP1 inhibits RSL3-induced castration-resistant prostate cancer cell ferroptosis by driving glutamine uptake and metabolism to GSH

High levels of YAP1 and ferroptosis activation in castration-resistant prostate cancer (CRPC) can inhibit CRPC progression and improve its sensitivity toward chemotherapeutics drugs. However, whether YAP1 regulates ferroptosis in CRPC cells and the underlying mechanisms are unknown. The protein levels of YAP1, SLC1A5, and GLS1 in benign prostatic hyperplasia (BPH), prostate cancer (PCa) that did not progress to CRPC, and CRPC tissue samples were evaluated using western blotting. In PC-3 and DU-145 cells, YAP1 overexpression vector, small-interfering RNA, specific inhibitor verteporfin, ferroptosis-inducer RSL3, SLC1A5-inhibitor V-9302, and GLS1-inhibitor CB-839 were used. Immunofluorescence, flow cytometry, dual-luciferase reporter gene, and related kits were used to investigate the effect of YAP1 on the ferroptosis activity in CRPC cells and its underlying mechanisms. YAP1 promoted extracellular glutamine uptake and subsequent production of glutamate and glutathione (GSH), and increases the GPX4 activity. For the activation of ferroptosis by RSL3, YAP1 decreased the levels of reactive oxygen species, malondialdehyde, and lipid peroxidation, and the proportion of dead cells. Mechanistically, YAP1 promoted the expression of SCL1A5 and GLS1 and further increased the GSH levels and GPX4 activity. Thus, inhibiting SLC1A5 or GLS1 activity could alleviate the antagonistic effect of YAP1 on the ferroptosis of RSL3-induced CRPC cells. In CRPC, the YAP1 level is high, which enters the nucleus and promotes the expressions of SLC1A5 and GLS1, thereby promoting cellular glutamine uptake and metabolism to generate glutamate and further synthesizing GSH, increasing GPX4 activity, improving cellular antioxidant capacity, and inhibiting cell death.

Knocking down SOX2 overcomes the resistance of prostate cancer to castration via notch signaling.

Castration-resistant prostate cancer (CRPC) is a terminal type of advanced cancer resistant to androgen deprivation therapy (ADT). Due to the poor therapeutic response of CRPC, novel treatment strategies are urgently required. This study aimed to clarify the regulatory roles of the SOX2/Notch axis in CRPC. For the evaluation of the SOX2, Notch, and Hey1 expression in the prostate cancer (PCa) and CRPC tissues, we conducted immunohistochemistry (IHC) analyses. RT-PCR, Western blotting, and immunofluorescence were performed to evaluate SOX2 and Notch expression in enzalutamide-resistant LNCaP cells (Enza-R). CCK-8, Transwell, Wound healing, and Western blotting assays were used to assess the viability, invasion, migration, cell cycle, and drug-resistant in Enza-R cells. Compared to the PCa tissues, CRPC tissues exhibited significantly elevated SOX2, Notch1, and Hey1 expression. SOX2-positive patients were more likely to develop bone metastases than SOX2-negative ones. Significant activation of the signaling associated with SOX2 and Notch was detected in Enza-R cells. The suppression of SOX2 clearly inactivated the Notch signaling and inhibited malignant behaviors, including proliferation, invasion, migration, and drug resistance in Enza-R cells. Theγsecretase inhibitor, GSI-IX, abrogated the enzalutamide resistance by inhibiting Notch signaling in vitro in vitro. Also, GSI-IX alone had a significant anti-tumor effect in Enza-R cells. We demonstrated that SOX2/Notch signaling was responsible for Enzalutamide resistance in CRPC. Targeting SOX2/Notch signaling might represent a new choice for the treatment and therapy of CRPC.

Anillin actin-binding protein expression correlates with poor prognosis for prostate cancer patients

ObjectiveOctamer transcription factor 1 (OCT1), a transcription factor that interacts with androgen receptor, is involved in prostate cancer (PCa) progression. The OCT1 target gene, Anillin actin-binding protein (ANLN), is highly expressed in castration-resistant PCa tissue; however, it remains unclear whether ANLN expression in hormone-sensitive PCa tissue could be used as a predictive biomarker for poor prognosis of patients. We aimed to investigate ANLN expression in PCa tissue obtained via radical prostatectomy and its correlation with clinical parameters. MethodsImmunohistochemical staining for ANLN was performed on 86 PCa specimens, followed by evaluation using immunoreactivity (IR) scores. Prognosis was analyzed by the log-rank test using the Kaplan–Meier method to generate a cancer-specific survival curve. The correlations between ANLN IR and clinical parameters as well as OCT1 IR were analyzed using the Chi-squared test. ResultsThe median IR score was 0 for ANLN. Accordingly, given the low median IR score, an IR score of ≥3 was defined as positive. There were 17 (19.8%) ANLN-positive cases, and these cases had a significantly poorer prognosis. Multivariate analysis revealed that the Gleason score, pathological tumor and lymph node stages, and positive ANLN expression were significant predictors of poor prognosis. Notably, patients with both positive ANLN and high OCT1 expression had a significantly decreased overall survival (p=0.001). ConclusionANLN, which is a OCT1 target gene especially in castration-resistant PCa, is expressed in a small number of hormone-sensitive PCa cases. Both positive ANLN expression and high OCT1 expression are significantly correlated with poor prognosis for PCa patients.

Abstract 3771: PiR-hsa-4447944 promotes the progression of prostate cancer through NEFH and induces androgen-independent growth

Abstract Introduction and Objective: Non-coding RNAs (ncRNAs) represent a diverse family of regulatory transcripts that drive tumorigenesis of prostate cancer (PCa) and various other types of cancers by their hyperactivity or diminished function. PIWI-interacting RNAs (piRNAs) are a class of small ncRNAs mechanistically similar to but much less studied than microRNAs. As the most diversified small ncRNAs, piRNAs are widely involved in the pathogenesis of different types of cancers. Our study aimed to investigate the role of piRNAs in the development of castration-resistant prostate cancer (CRPC). Methods: Small RNA sequencing was used to explore the CRPC associated piRNAs on a basis of 10 benign prostate tissues, 10 paired hormone-sensitive PCa tissues and CRPC tissues from the same patients. The top 12 upregulated piRNAs were validated through qRT-PCR in a bigger cohort. piR-hsa-4447944 was selected and investigated its biological function by gain of function in vitro. To study its loss of function role, an androgen-independent PCa cell (LNCaP-AI) was established. For mechanistic investigation, high-throughput RNA sequencing was conducted to identify dysregulated genes in PCa cells with control or piR-hsa-4447944 overexpression. Then, potential downstream targets were further predicted by miRanda program and validated by qRT-PCR, western-blot and dual-luciferase reporter assay. Results: piR-hsa-4447944 demonstrated a relatively higher expression level in both CRPC tissues and CRPC cell lines when compared with PCa tissues and cells. Functionally, overexpression of piR-hsa-4447944 could confer resistance to androgen deprivation and anti-androgen in PCa cells in vitro, whereas anti-sense RNA against the piR-hsa-4447944 could potentiate the sensitivity to androgen deprivation in LNCaP-AI cell. In addition, piR-hsa-4447944 showed the ability in promoting migration and invasion of PCa cells. Mechanistically, mRNA sequencing results identified a panel of downstream target-genes. Among them, neurofilament heavy chain (NEFH) was predicted and validated to have a direct binding relationship with piR-hsa-4447944, and was successfully verified to be negatively regulated by piR-hsa-4447944 in PCa cells. Conclusions: Our study showed, for the first time, that piR-hsa-4447944 can contribute to androgen insensitivity in CRPC. This sheds a new insight into the regulation of CRPC by a small ncRNA piRNA, which may form the basis of a novel target in the treatment of CRPC. Citation Format: Qiang Peng, Jingkai Sun, Peter Ka-Fung Chiu, Tingting Xie, Jeremy Yuen-Chun Teoh, Chi-Fai Ng. PiR-hsa-4447944 promotes the progression of prostate cancer through NEFH and induces androgen-independent growth. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3771.

Abstract 6555: Integrated bioinformatics analysis identified ASNS and DDIT3 as the therapeutic target in castration resistance prostate cancer

Abstract Background: Many studies have demonstrated the mechanisms of progression to castration-resistant prostate cancer (CRPC) and novel strategies for the treatment of CRPC. Despite these advances, the molecular mechanism underlying the progression to CRPC remain unclear, and no effective treatments for CRPC are currently available. Here, we characterized the pathways and key genes involved in the progression to CRPC to gain insight into the mechanisms and potential therapeutic targets of CRPC. Methods: Bicalutamide-resistant prostate cancer cells derived from LNCaP was generated and named Bical R. RNA sequencing was used to identify differentially expressed genes (DEGs) between LNCaP and Bical R. DAVID and Clue GO were used to perform functional enrichment analysis, and the Cytohubba plug-in Cytoscape was used to identify hub genes associated with progression to CRPC. To validate the expression of hub genes, we performed qRT-PCR and western blot analyses. Further validation was performed using GEO microarray (GSE 32269 and GSE28403). To elucidate the clinical usefulness of these genes, we assessed the disease-free survival of patients with prostate cancer from The Cancer Genome Atlas (TCGA) database. Results: In total, 631 DEGs (302 upregulated genes and 329 downregulated genes) were identified, and Kyoto Encyclopedia of Genes and Genomes pathways were analyzed based on DAVID and Clue GO. We found that the PI3K-Akt and MAPK signaling pathways are of great significance for investigating the mechanism of CRPC progression. Hub genes from protein-protein interaction network of upregulated DEGs (AGT, ASNS, ATF3, ATF4, DDIT3, EFNA5, and VEGFA) were identified. Among the hub genes, ASNS and DDIT3 were markedly up-regulated in CRPC cell lines and CRPC tissues, respectively. Additionally, patients with high expression of ASNS and DDIT3 showed worse disease-free survival in TCGA database. Conclusion: Our study revealed that the PI3K-Akt and MAPK signaling pathways may be important in the mechanism of CRPC progression, and ASNS and DDIT3 may be associated with progression to CRPC. These results may contribute to the development of potential therapeutic targets and mechanisms underlying the progression to CRPC to improve clinical efficacy for CRPC treatment. Acknowledgements: This work was supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government(MSIT) (No. 2022R1A2C2003513). Citation Format: Ae Ryang Jung, Sun Shin, U-Syn Ha, Mee Young Kim, Sung-Hoo Hong, Ji Youl Lee, Sae Woong Kim, Yeun-Jun Chung, Yong Hyun Park. Integrated bioinformatics analysis identified ASNS and DDIT3 as the therapeutic target in castration resistance prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6555.

MP20-12 PiR-hsa-24683 IS ESSENTIAL FOR PROSTATE TUMOR-PROPAGATING CELLS AND PROMOTE THE DEVELOPMENT OF CASTRATION RESISTANCE

You have accessJournal of UrologyCME1 Apr 2023MP20-12 PiR-hsa-24683 IS ESSENTIAL FOR PROSTATE TUMOR-PROPAGATING CELLS AND PROMOTE THE DEVELOPMENT OF CASTRATION RESISTANCE Qiang Peng, Peter Ka-Fung Chiu, Tingting Xie, Jeremy Yuen-Chun Teoh, and Chi-Fai Ng Qiang PengQiang Peng More articles by this author , Peter Ka-Fung ChiuPeter Ka-Fung Chiu More articles by this author , Tingting XieTingting Xie More articles by this author , Jeremy Yuen-Chun TeohJeremy Yuen-Chun Teoh More articles by this author , and Chi-Fai NgChi-Fai Ng More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003245.12AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: PIWI-interacting RNAs (piRNAs) are a class of small non-coding RNAs (sncRNAs) mechanistically similar to but much less studied than microRNAs. piRNAs represent the most diversified sncRNAs that are widely involved in the pathogenesis of different types of cancers including prostate cancer (PCa). Our study aimed to investigate the role of piRNAs in the development of castration-resistant prostate cancer (CRPC). METHODS: Small RNA sequencing was used to explore the CRPC associated piRNAs on a basis of 10 benign prostate tissues, 10 paired hormone-sensitive PCa tissues and CRPC tissues from the same patients. The top 8 upregulated piRNAs were validated through qRT-PCR in a bigger cohort. piR-hsa-24683 was selected and investigated its biological function by gain of function in vitro. To study its loss of function role, an androgen-independent PCa cell (LNCaP-AI) was established. For mechanistic investigation, the mRNA sequencing analysis of differentially expressed genes (DEGs) was conducted in PCa cells transfected with control/piR-hsa-24683 mimics. Then, potential downstream targets were further predicted by miRanda program and validated by qRT-PCR. RESULTS: piR-hsa-24683 demonstrated a relatively higher expression level in both CRPC tissues and CRPC cell lines when compared with PCa tissues and cells. Functionally, overexpression of piR-hsa-24683 could confer resistance to androgen deprivation and anti-androgen in PCa cells in vitro, whereas anti-sense RNA against the piR-hsa-24683 could potentiate the sensitivity to androgen deprivation in LNCaP-AI cell. In addition, piR-hsa-24683 showed the ability in promoting sphere formation, migration and invasion of PCa cells. Mechanistically, mRNA sequencing results identified a panel of downstream target-genes. Among them, serine/threonine kinase 3 (STK3) was predicted to have a direct binding relationship with piR-hsa-24683, and was successfully verified to be negatively regulated by piR-hsa-24683. CONCLUSIONS: Our study showed, for the first time, that piR-hsa-24683 can contribute to androgen insensitivity in CRPC. This sheds a new insight into the regulation of CRPC by a sncRNA piRNA, which may form the basis of a novel target in the treatment of CRPC. Source of Funding: UGC Research Matching Fund 8601404 © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e279 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Qiang Peng More articles by this author Peter Ka-Fung Chiu More articles by this author Tingting Xie More articles by this author Jeremy Yuen-Chun Teoh More articles by this author Chi-Fai Ng More articles by this author Expand All Advertisement PDF downloadLoading ...

A first-in-class HBO1 inhibitor WM-3835 inhibits castration-resistant prostate cancer cell growth in vitro and in vivo

The prognosis and overall survival of castration-resistant prostate cancer (CRPC) patients are poor. The search for novel and efficient anti-CRPC agents is therefore extremely important. WM-3835 is a cell-permeable, potent and first-in-class HBO1 (KAT7 or MYST2) inhibitor. Here in primary human prostate cancer cells-derived from CRPC patients, WM-3835 potently inhibited cell viability, proliferation, cell cycle progression and in vitro cell migration. The HBO1 inhibitor provoked apoptosis in the prostate cancer cells. It failed to induce significant cytotoxicity and apoptosis in primary human prostate epithelial cells. shRNA-induced silencing of HBO1 resulted in robust anti-prostate cancer cell activity as well, and adding WM-3835 failed to induce further cytotoxicity in the primary prostate cancer cells. Conversely, ectopic overexpression of HBO1 further augmented primary prostate cancer cell proliferation and migration. WM-3835 inhibited H3-H4 acetylation and downregulated several pro-cancerous genes (CCR2, MYLK, VEGFR2, and OCIAD2) in primary CRPC cells. Importantly, HBO1 mRNA and protein levels are significantly elevated in CRPC tissues and cells. In vivo, daily intraperitoneal injection of WM-3835 potently inhibited pPC-1 xenograft growth in nude mice, and no apparent toxicities detected. Moreover, intratumoral injection of HBO1 shRNA adeno-associated virus (AAV) suppressed the growth of primary prostate cancer xenografts in nude mice. H3-H4 histone acetylation and HBO1-dependent genes (CCR2, MYLK, VEGFR2, and OCIAD2) were remarkably decreased in WM-3835-treated or HBO1-silenced xenograft tissues. Together, targeting HBO1 by WM-3835 robustly inhibits CRPC cell growth.

Immunoproteasome inhibition prevents progression of castration-resistant prostate cancer.

Castration-resistant prostate cancer (CRPC) is refractory to hormone treatment. This study aims to explore the effect and underlying mechanisms of immunoproteasome inhibition,a novel immunotherapy, on the progression of CRPC. The immunoproteasome subunit LMP7 was silenced by using gene knockout or inhibited by theepoxyketone inhibitor ONX 0914 in a mouse CRPC tumour graft model and in interferon-γ-pretreated human CRPC cell lines in vitro. CRPC tissues reveal a significant "tumour-elicited" Th17-type inflammatory response which induces immunoproteasome subunit expression. LMP7 deficiency in host mice or in CRPC tumour grafts had no effect on the "tumour-elicited" Th17-type inflammatory response and tumour progression. However, the selective LMP7 inhibitor ONX 0914 strongly suppressed the "tumour-elicited" Th17-type inflammatory response and CRPC tumour progression. Treatment of wild-type mice receiving LMP7-deficient CRPC tumour grafts with ONX 0914 further suggested that immunoproteasome inhibition prevents CRPC progression through suppressing IL-17-induced angiogenesis and epithelial-mesenchymal transition via inactivation of COX-2/VEGF-A signalling and β-catenin/Snail signalling. Treatment of LMP7-deficient mice receiving wild-type CRPC tumour grafts with ONX 0914 and inhibition of LMP7 in PC3 and 22Rv.1 cells with ONX 0914 showed that immunoproteasome inhibition also prevents CRPC progression through inducing CRPC cell apoptosis via activation of the unfolded protein response. We define a critical role of the immunoproteasome in CRPC and propose immunoproteasome inhibition as a promising therapeutic approach to suppress CRPC progression.

H19 in Serum Extracellular Vesicles Reflects Resistance to AR Axis-targeted Therapy Among CRPC Patients.

We aimed to evaluate the changes of androgen receptor (AR) signaling-related long non-coding RNAs (lncRNAs) in serum extracellular vesicles (EVs) from prostate cancer (PC) patients, in order to identify novel biomarkers for AR axis-targeted therapy (ARAT)-resistance among castration-resistant PC (CRPC) patients. EVs were isolated from 2 patients before and after acquiring ARAT-resistance. RNA profiling of EVs was performed by RNA-sequencing. The expression levels of selected lncRNAs in EVs were analyzed by digital droplet PCR (ddPCR) in 58 localized and 14 metastatic PC patients at diagnosis, 7 ARAT-naïve and 6 ARAT-resistant CRPC patients. LncRNA H19 expression in PC tissue was examined using published data. In order to analyze the role of H19, the prognosis was analyzed in PC patients and proteomic analysis was performed in 22Rv1 PC cells. RNA-sequencing revealed that AR-regulated RNAs were most enriched in EVs after acquiring ARAT-resistance. Among them, up-regulation of AR signaling-related lncRNAs (PCAT1, H19, HOXA-11AS, ZEB1-AS1, ARLNC1, PART1, CTBP1-AS and PCA3) was confirmed by ddPCR. H19 contained in EVs (EV-H19) was significantly increased among ARAT-resistant patients compared to ARAT-naïve CRPC or metastatic PC patients. In PC tissue, H19 was negatively correlated with AR protein and AR-activity score and up-regulated in neuroendocrine CRPC tissue with low AR expression. Furthermore, EV-H19 expression was significantly associated with worse outcome to androgen-deprivation therapy. Proteomic analysis demonstrated that H19 knockdown enhanced PC-related protein expression. EV-H19 may negatively correlate with AR-signaling activity and could be a marker to diagnose ARAT-resistance among CRPC patients.

Ciliary Neurotrophic Factor Modulates Multiple Downstream Signaling Pathways in Prostate Cancer Inhibiting Cell Invasiveness.

Prostate cancer (PCa) remains the most common diagnosed tumor and is the second-leading cause of cancer-related death in men. If the cancer is organ-confined it can be treated by various ablative therapies such as RP (radical prostatectomy), RT (radiation therapy), brachytherapy, cryosurgery or HIFU (High-Intensity Focused Ultrasound). However, advanced or metastatic PCa treatment requires systemic therapy involving androgen deprivation, but such patients typically progress to refractory disease designated as castration-resistant prostate cancer (CRPC). Interleukin-6 (IL-6) has been established as a driver of prostate carcinogenesis and tumor progression while less is known about the role of ciliary neurotrophic factor (CNTF), a member of the IL-6 cytokine family in prostate cancer. Moreover, MAPK/ERK, AKT/PI3K and Jak/STAT pathways that regulate proliferative, invasive and glucose-uptake processes in cancer progression are triggered by CNTF. We investigate CNTF and its receptor CNTFRα expressions in human androgen-responsive and castration-resistant prostate cancer (CRPC) by immunohistochemistry. Moreover, we investigated the role of CNTF in proliferative, invasive processes as well as glucose uptake using two cell models mimicking the PCa (LNCaP cell line) and CRPC (22Rv1 cell line). Our results showed that CNTF and CNTFRa were expressed in PCa and CRPC tissues and that CNTF has a pivotal role in prostate cancer environment remodeling and as a negative modulator of invasion processes of CRPC cell models.

The ERα-NRF2 signalling axis promotes bicalutamide resistance in prostate cancer

BackgroundBicalutamide is a nonsteroidal antiandrogen widely used as a first-line clinical treatment for advanced prostate cancer (PCa). Although patients initially show effective responses to bicalutamide treatment, resistance to bicalutamide frequently occurs and leads to the development of castration-resistant PCa (CRPC). This research investigated the roles of the oestrogen receptor α (ERα)-nuclear factor E2-related factor 2 (NRF2) signalling pathway in bicalutamide resistance in PCa cells.MethodsWe performed bioinformatic analysis and immunohistochemical staining on normal and cancerous prostate tissue to evaluate ERα and NRF2 expression and their correlation. Gene expression and localization in PCa cell lines were further investigated using real-time reverse transcription PCR/Western blotting and immunofluorescence staining. We treated PCa cells with the ER inhibitor tamoxifen and performed luciferase reporter assays and chromatin immunoprecipitation (ChIP) assays to understand ERα-dependent NRF2 expression. Overexpression and knockdown of ERα and NRF2 were used to explore the potential role of the ERα-NRF2 signalling axis in bicalutamide resistance in PCa cells.ResultsWe found that the expression of ERα and NRF2 was positively correlated and was higher in human CRPC tissues than in primary PCa tissues. Treatment with oestrogen or bicalutamide increased the expression of ERα and NRF2 as well as NRF2 target genes in PCa cell lines. These effects were blocked by pretreatment with tamoxifen. ChIP assays demonstrated that ERα directly binds to the oestrogen response element (ERE) in the NRF2 promoter. This binding led to increased transcriptional activity of NRF2 in a luciferase reporter assay. Activation of the ERα-NRF2 signalling axis increased the expression of bicalutamide resistance-related genes. Inhibition of this signalling axis by knockdown of ERα or NRF2 downregulated the expression of bicalutamide resistance-related genes and inhibited the proliferation and migration of PCa cells.ConclusionsWe demonstrated the transcriptional interaction between ERα and NRF2 in CRPC tissues and cell lines by showing the direct binding of ERα to the ERE in the NRF2 promoter under oestrogen treatment. Activation of the ERα-NRF2 signalling axis contributes to bicalutamide resistance in PCa cells, suggesting that the ERα-NRF2 signalling axis is a potential therapeutic target for CRPC.CxbrQyr1MPyN3X1_gKxrKxVideo

Glucocorticoid receptor and androgen receptor-targeting therapy in patients with castration-resistant prostate cancer.

ObjectiveThe glucocorticoid receptor (GR) promotes resistance to androgen receptor (AR)-targeting therapies in castration-resistant prostate cancer (CRPC) by bypassing AR blockade. However, the clinical relevance of evaluating GR expression in patients with CRPC has not been determined. The present study investigated the association of relative GR expression in CRPC tissue samples with treatment response to AR-targeting therapy.MethodsLevels of GR, AR-FL, and AR-V7 mRNAs were measured in prostate cancer tissue from prospectively enrolled CRPC patients who were starting treatment. Patients were divided into groups with high and low AR-V7/AR-FL ratios and with high and low GR/AR-FL ratios. The primary endpoint was prostate-specific antigen (PSA) response rate to treatment.ResultsEvaluation of 38 patients treated with AR-targeting therapies showed that the PSA response rate was significantly higher in patients with low than high AR-V7/AR-FL ratios (77.8% vs. 25.0%, p=0.003) and in patients with low than high GR/AR-FL ratios (81.3% vs. 27.3%, p=0.003). Patients with low GR/AR-FL ratios had higher rates of PSA progression-free survival (46.0% vs. 22.4%, p=0.006), radiologic progression-free survival (28.9% vs. 10.0%, p=0.02), and overall survival (75.2% vs. 48.0%, p=0.037) than patients with high GR/AR-FL ratios. The association of GR/AR-FL ratio with PSA response to AR-targeting therapy remained significant in multivariable models. Evaluation of the 14 patients who received taxane chemotherapy showed that PSA response rates did not differ significantly in those with low and high AR-V7/AR-FL and GR/AR-FL ratios, although no definitive conclusions can be drawn due to the small number of patients.ConclusionRelative GR expression is associated with sensitivity to AR-targeting therapy and survival in patients with CRPC. Large-scale prospective validation and liquid biopsy-based studies are warranted.

Cathepsin K regulates the tumor growth and metastasis by IL-17/CTSK/EMT axis and mediates M2 macrophage polarization in castration-resistant prostate cancer

A common stage of advanced prostate cancer is castration-resistant prostate cancer (CRPC), greater understanding of which is required in order to address and solve the clinically difficult challenge. Cathepsin K (CTSK) is a cysteine protease that usually has a strong activity of degrading extracellular matrix and is related to osteoclast-mediated bone destruction. However, the mechanism of CTSK-regulation in CRPC is still unclear to us. The current study aimed to analyze the expression of differentially expressed genes (DEGs) in patient samples (from localized PC and CRPC). Interestingly, we found that CTSK to be significantly up-regulated in CRPC. Through further signal pathway enrichment analysis, we found that the IL-17 signaling pathway to be highly correlated with CTSK. The oncogenic functions of CTSK and IL-17 in CRPC were proven by a series of in vivo and in vitro experiments. Possible downstream molecules of CTSK were investigated, which could serve as control elements to regulate the expression of EMT, thereby facilitating the metastasis and excessive proliferation of PC cells. Expression of CTSK was related to high concentration of M2 tumor-associated macrophages (TAMs) M2 in CRPC. A CTSK-mediated feedback circuit between TAMs and CRPC tissues was indicated in the process of transfer, proving the possibility of CTSK could be use as an available therapeutic target for CRPC.