Sensitizes Prostate Cancer Cells Research Articles

Neuropilin-2 axis in regulating secretory phenotype of neuroendocrine-like prostate cancer cells and its implication in therapy resistance.

SUMMARYNeuroendocrine (NE)-like tumors secrete various signaling molecules to establish paracrine communication within the tumor milieu and to create a therapy-resistant environment. It is important to identify molecular mediators that regulate this secretory phenotype in NE-like cancer. The current study highlights the importance of a cell surface molecule, Neuropilin-2 (NRP2), for the secretory function of NE-like prostate cancer (PCa). Our analysis on different patient cohorts suggests that NRP2 is high in NE-like PCa. We have developed cell line models to investigate NRP2’s role in NE-like PCa. Our bioinformatics, mass spectrometry, cytokine array, and other supporting experiments reveal that NRP2 regulates robust secretory phenotype in NE-like PCa and controls the secretion of factors promoting cancer cell survival. Depletion of NRP2 reduces the secretion of these factors and makes resistant cancer cells sensitive to chemotherapy in vitro and in vivo. Therefore, targeting NRP2 can revert cellular secretion and sensitize PCa cells toward therapy.

Molecular Characterization and Clinical Relevance of N6-Methyladenosine Regulators in Metastatic Prostate Cancer.

Prostate cancer is a leading malignancy in the male population globally. N6-methylation of adenosine (m6A) is the most prevalent mRNA modification and plays an essential role in various biological processes in vivo. However, the potential roles of m6A in metastatic prostate cancer are largely unknown. In this study, we evaluated and identified two m6A modification patterns based on 21 m6A regulators in four public metastatic prostate cancer datasets. Different modification patterns correlated with distinct molecular characteristics. According to m6A-associated genes, we constructed a prognostic model, called m6Ascore, to predict the outcomes of patients with metastatic prostate cancer. We found that high m6A score level was related to dismal prognosis and characterized by higher cell cycle, DNA repair and mismatch repair pathway score. In vitro experiments confirmed that upregulation of METTL14, an m6A writer, enhanced the invasion, metastasis, and sensitivity of prostate cancer cells to poly (ADP-ribose) polymerase inhibitor. Conversely, down-regulation of potential target genes of m6A had the opposite effect. Finally, we validated that a higher m6A score was associated with a worse prognosis and a higher Gleason score in The Cancer Genome Atlas Program (TCGA) cohort. This work illustrated the nonnegligible role of m6A modification in multiple biological processes of metastatic prostate cancer. Evaluating the m6A risk scores of individual tumours will guide more effective judgement of prognosis as well as treatments for metastatic prostate cancer in clinical practice.

Abstract 2996: Pre-activation of autophagy impacts PARP inhibitor sensitivity of prostate cancer cells

Abstract Introduction: Advanced prostate cancer (APC) is one of the most common causes of cancer-related mortality in males largely related to the development of resistance to hormonal and chemotherapeutic based regimens. Recently, the PARP inhibitor (PARPi) olaparib was shown to improve progression-free and overall survival in patients with APC although resistance remains a barrier to cure. Therapeutic resistance in cancers can be mediated by autophagy, a cellular recycling process. Here we addressed the role of autophagy in the development of olaparib resistance in APC. Methods: Autophagy levels and olaparib sensitivity were evaluated in prostate cancer (PC) cell lines (LNCaP, C4-2B and PC-3) by western blots and clonogenic assays, respectively. We introduced a double-tagged (mCherry-GFP) LC3 protein in cells to monitor autophagic flux by confocal microscopy and evaluated the temporal importance of autophagy activation by rapamycin on olaparib sensitivity. We measured cell proliferation (Incucyte), cell cycle (flow cytometry) and DNA repair (microscopy). Finally, we confirmed the role of autophagy in olaparib sensitivity using CRISPR/Cas9 Atg16L1 knockout cell lines. Results: We found a correlation between the basal level of autophagy and olaparib sensitivity in PC cell lines. PC-3 cells were olaparib resistant and had a higher basal level of autophagy compared to LNCaP and C4-2B cells, which were PARPi sensitive. When autophagy was pre-activated before olaparib treatment, PC cell lines became more resistant by increasing homologous recombination activity. This autophagy-mediated resistance was partially regulated by a balance between SQSTM1 and FLNA protein levels in the nucleus. Inhibition of autophagy or post-activated autophagy prevented development of PARPi resistance. Conclusion: The autophagic flux is associated with PARPi resistance in PC cell lines by regulating DNA repair. Inhibiting autophagy may lead to new strategies to increase PARPi efficacy. Understanding the role of autophagy in PC progression may lead to strategies that prevent the development of PARPi resistance. Citation Format: Maxime Cahuzac, Benjamin Péant, Hubert Fleury, Anne-Marie Mes-Masson, Fred Saad. Pre-activation of autophagy impacts PARP inhibitor sensitivity of prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2996.

Abstract 5540: Tumor phenotypic plasticity and resistance to immune-mediated cytotoxicity in models of castration resistant prostate cancer

Abstract Androgen receptor (AR)-targeted therapies are routinely used to treat prostate cancer, including the use of next-generation hormonal therapies such as abiraterone or enzalutamide. Although these therapies are initially effective, a significant proportion of patients exhibit resistance to treatment, therefore developing castration-resistant prostate cancer (CRPC). While immune checkpoint blockade therapies have led to remarkable clinical responses in patients with several tumor types, CRPC remains resistant to immunotherapy. The characterization of tumor resistance mechanisms has led to the identification of various tumor variants that could emerge along the progression of prostate cancer, including tumors undergoing phenotypic plasticity. Our laboratory has previously shown that phenotypic plasticity is a driver of resistance to immunotherapy. Here we investigated whether changes in tumor phenotype could affect the response of CRPC to immune-based therapies, and ways these mechanisms can be mitigated. Models of resistance to enzalutamide (LNCAP-EnzaR) or abiraterone (LNCAP-AbiR) were derived from the androgen sensitive LNCAP prostate cancer cell line. Resistant and parental LNCAP cells were comparatively evaluated for phenotypic features via RT-PCR, ELISA, western blot, immunofluorescence, and RNAseq analysis. Changes in the susceptibility to NK-cell mediated cytotoxicity were evaluated with NK cells isolated from peripheral blood from healthy donors. LNCAP-EnzaR and LNCAP parental cells were also grown in vivo in NSG MHCI/II-deficient mice, and tumors were characterized for phenotypic markers and potential therapeutic targets. Our results demonstrated that acquisition of resistance to both enzalutamide and abiraterone was associated with a significant increase in mesenchymal tumor features, including increased levels of vimentin and fibronectin, and the loss of epithelial E-cadherin. The susceptibility of LNCAP-Enza-R and LNCAP-AbiR cells to NK-cell mediated cytotoxicity and antibody-dependent cell cytotoxicity (ADCC) were significantly reduced (up to 90%), compared with parental cells. RNAseq analysis identified several upregulated gene and gene pathways, including estrogen receptor 1 (ESR1), which was particularly overexpressed in LNCAP-EnzaR cells. In a xenograft model of LNCAP-EnzaR cells, we corroborated the maintenance of tumor phenotypic plasticity and the expression of actionable targets. Our data indicates that acquisition of resistance to AR inhibition is associated with significant reduction of susceptibility to immune attack, and the acquisition of tumor phenotypic plasticity features. Future studies plan to investigate approaches that revert tumor plasticity, including blockade of increased markers such as ESR1, TGF-beta or IL-8, for potential improvement of tumor susceptibility to immune attack in CRPC. Citation Format: Madeline Dahut, Kristen Fousek, Lucas Horn, Haiyan Qin, Jeffrey Schlom, Claudia Palena. Tumor phenotypic plasticity and resistance to immune-mediated cytotoxicity in models of castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5540.

Abstract 884: Cooperative actions of a novel ERβ ligand, OSU-ERb-12, and Enzalutamide in advanced prostate cancer

Abstract Background: Historically, estrogens were used therapeutically in prostate cancer (PCa) but discontinued due to serious side effects. Given estrogen receptor alpha (ERα) and beta (ERβ) exert opposing actions, it is unclear how estrogen actions control PCa growth. The OSU Drug Development Institute developed a novel, highly selective, ERβ ligand (OSU-ERB-12 (ERB12)) which has excellent in vivo PK/PD properties. Methods: A combination robotic high-throughput drug viability screen was undertaken in androgen deprivation therapy (ADT)-sensitive and resistant PCa cell models that combined multiple concentrations of ERB12 with 30 compounds including Enzalutamide (ENZA), and epigenomic regulators; potent combinations were examined by clonogenic assays. The ERB12 and ENZA combination was examined in 22RV1 by RNA-Seq and ATAC-Seq and CRISPR-editing undertaken to delete ERα or ERb. Result: All cell lines (LNCaP, 22RV1, PC-3, and DU145) were sensitive to high doses of ERB12 alone (1-10μM) whereas each cell line displayed unique responses to ERB12 drug interactions. 22RV1 cells responded with the greatest additive effects to combinations with ENZA and HDAC or DNMT inhibitors. Clonogenic assays also demonstrated significant suppression of colony formation by ERB12 combined with all three classes of drug. Also, 22RV1 cells with CRISPR-deleted ERα grew slower whereas faster with ERβ deletion. ATAC-Seq in 22RV1 cells revealed that the combination of ENZA (1μM) with ERB12 (100nM), but not 17b-estradiol (E2) + ENZA), led to unique gain of nucleosome free (NF) regions enriched for nuclear receptor motifs (e.g. AR, RARa, PPARa) and loss of CTCF motifs. These regions also significantly overlapped with ChromHMM-defined Active and Poised Enhancers. Parallel RNA-Seq also identified a unique ERB12/ENZA-dependent transcriptome significantly associated with repression of androgen and MYC signaling. MYC repression was confirmed at the protein level. Integration of ATAC-Seq and RNA-Seq data revealed antagonism between ERB12 and E2 treatments notably with ERB12/ENZA leading to gain of NF regions and increased expression of coregulators (e.g. RUNX1 and NCOA3) that were repressed by E2/ENZA. Finally, transcript-aware analyses identified unique and common ERB12/ENZA driven splice variation including coregulators NCOR2 and PRAME. Conclusion: Our data suggest that there is a cooperative action between ERB12 and ENZA in limiting cell proliferation and altering nucleosome positioning and leading to a unique transcriptome. ERB12, is an orally-available and well-tolerated drug that appears to antagonize the AR driven signaling pathways that lead to lethal prostate cancer. We propose that ERB12 could allow patients to take a lower, and more well-tolerated dose of ADT drugs, which at high dose can be very harsh on patients. Citation Format: Jaimie S. Gray. Cooperative actions of a novel ERβ ligand, OSU-ERb-12, and Enzalutamide in advanced prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 884.

Abstract 5657: Anti-hypertensive drugs increase TRAIL sensitivity-induced surface DR5 via autophagy inhibition pathway in prostate cancer cells

Abstract Angiotensin II type 1 receptor blockers(ARB) are used as antihypertensive agents, The cohort study found that among people with prostate cancer, thus taking Angiotensin II type 1 receptor blockers had reduced risk ratio. This study investigated that can ARB treatment enhance the TRAIL-mediated prostate cancer cell apoptosis, and the detailed molecular mechanisms. The results showed that ARB treatment increased the TRAIL-mediated prostate cancer cell apoptosis. In the mechanism study, ARB treatment increased DR5 protein expression level, surface DR5 protein amount, and DR5 protein stability. Furthermore, the Regulation of DR5 expression was mediated by autophagy flux inhibition followed by ARB treatment. Taken together, the results demonstrated that ARB treatment sensitized prostate cancer cells to TRAIL-induced apoptosis via autophagy inhibition and DR5 protein stabilization, and also suggest that the prostate cancer cells of ARB taken-patient may be sensitized to TRAIL-mediated apoptosis, and may support the mechanism of cohort study which showed reduction of prostate cancer risk ratio in patient taking angiotensin II type 1 receptor blockers. Citation Format: sang-youel park, Jong-Hun Kim, Jeong-Min Hong, Jae-Won Seol. Anti-hypertensive drugs increase TRAIL sensitivity-induced surface DR5 via autophagy inhibition pathway in prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5657.

A phase Ia/Ib study of talazoparib in combination with tazemetostat in metastatic castration-resistant prostate cancer (mCRPC).

TPS5098 Background: Enhancer of zeste homolog 2 (EZH2) is frequently overexpressed in metastatic castration-resistant prostate cancer (mCRPC), and is linked to lineage plasticity and therapy resistance. In pre-clinical studies, EZH2 directly regulates DNA damage repair (DDR) gene expression, and pharmacologic inhibition of EZH2 sensitizes prostate cancer cells to genotoxic stress as induced by poly-ADP ribose polymerase (PARP) inhibition. The PARP inhibitor talazoparib and EZH2 inhibitor tazemetostat are currently under study in mCRPC, and we are conducting a Phase 1 clinical trial of the combination. Methods: Phase 1a of the study will define the recommended phase 2 dose (RP2D) and Phase 1b will better assess safety and preliminary clinical activity of the combination at the RP2D. Eligible patients must have progressive disease after at least one secondary hormonal therapy and taxane-based chemotherapy (or felt not to be more appropriate for taxane), have disease evaluable for response (PSA ≥ 2 ng/ml or measurable disease by RECIST 1.1) and have a metastatic lesion amenable to biopsy adequate for next generation sequencing. In Phase 1a (n = 9-18), the starting doses are talazoparib 0.75 mg daily and tazemetostat 600 mg BID, with dose escalation/de-escalation of both agents by up to 2 dose levels [DLs] based on a 3+3 design. The RP2D is the maximum tolerated dose (MTD) or DL +2 (talazoparib 1 mg daily + tazemetostat 800 mg BID) if the MTD is not reached. After 6 patients are treated at the RP2D, phase 1b will enroll an additional 20 patients to an expansion cohort. The primary endpoint of safety and tolerability is based on incidence of dose-limiting toxicities [DLTs] and incidence and grade of adverse events [AEs] by CTCAE version 5.0. For the secondary endpoint of overall response rate (ORR; defined as PSA reduction by ≥ 50% OR radiographic response by RECIST 1.1), with a sample size of 26 (6 patients from dose escalation and 20 from expansion), we deem talazoparib+tazemetostat effective if ORR is ≥ 5/26 (19%). The probability of concluding that the treatment strategy effective is 0.11 if its true response rate is 10% and at least 0.93 if the true response rate exceeds 30%. Mandatory pre-treatment and on-treatment (8-week) biopsies will undergo targeted genetic sequencing, transcriptomic profiling, ChIP (Chromatin ImmunoPrecipitation)-seq, and immunohistochemistry (IHC) for DDR and differentiation markers; blood specimens will undergo circulating cell-free DNA and circulating tumor cell profiling – these studies will nominate possible predictive biomarkers for therapeutic response and serve as pharmacodynamic markers of combined PARP and EZH2 inhibition. The goal of this study is to expand treatment options in mCRPC through a novel approach to exploit EZH2 as a therapeutic target through co-targeting the DDR response. Enrollment began in July 2021. Clinical trial information: NCT04846478.

Radiosensitization of PC3 Prostate Cancer Cells by 5-Thiocyanato-2'-deoxyuridine.

Simple SummaryRadiation therapy is one of the main treatments for cancer. However, the success of treatment by radiation therapy is largely dependent on tumor radiosensitivity. To improve therapeutic outcomes, radiation therapy should be combined with the use of a radiosensitizer which enables irradiation at lower doses with higher efficacies. 5-Thiocyanato-2′-deoxyuridine has been reported as a potential radiosensitizer of DNA damage based on advanced radiation chemical studies. In this paper, for the first time, we demonstrate the radiosensitizing properties of this modified nucleoside at the cellular level. The tested analogue increases the sensitivity of prostate cancer cells to ionizing radiation which is, at least partially, related to an increase in the number of DNA double-strand breaks and cell cycle regulation.Purpose: The radiosensitizing properties of uracil analogs modified in the C5 position are very interesting in the context of their effectiveness and safety in radiation therapy. Recently, radiation chemical studies have confirmed that 5-thiocyanato-2′-deoxyuridine (SCNdU) undergoes dissociation induced by an excess electron attachment and established this nucleoside as a potential radiosensitizer. In this paper, we verify the sensitizing properties of SCNdU at the cellular level and prove that it can effectively enhance ionizing radiation-induced cellular death. Methods and Materials: Prostate cancer cells were treated with SCNdU and irradiated with X rays. The cytotoxicity of SCNdU was determined by MTT test. Cell proliferation was assessed using a clonogenic assay. Cell cycle analyses, DNA damage, and cell death analyses were performed by flow cytometry. Results: SCNdU treatment significantly suppressed the proliferation and increased the radiosensitivity of prostate cancer cells. The radiosensitizing effect expressed by the dose enhancement factor is equal to 1.69. Simultaneous exposure of cells to SCNdU and radiation causes an increase in the fraction of the most radiosensitive G2/M phase, enhancement of the histone H2A.X phosphorylation level, and apoptosis induction. Finally, SCNdU turned out to be marginally cytotoxic in the absence of ionizing radiation. Conclusions: Our findings indicate that SCNdU treatment enhances the radiosensitivity of prostate cancer cells in a manner associated with the cell cycle regulation, double strand formation, and a slight induction of apoptosis.

TFAP2C-Mediated lncRNA PCAT1 Inhibits Ferroptosis in Docetaxel-Resistant Prostate Cancer Through c-Myc/miR-25-3p/SLC7A11 Signaling.

Recent evidence has shown that the induction of ferroptosis is a new therapeutic strategy for advanced prostate cancer (PCa) when used as a monotherapy or in combination with second-generation antiandrogens. However, whether ferroptosis inducers are effective against docetaxel-resistant PCa remains unclear. In addition, the biological role and intrinsic regulatory mechanisms of long noncoding RNAs (lncRNAs) in ferroptosis and chemoresistance are not well understood. In this study, we established two acquired docetaxel-resistant PCa cell lines and found that docetaxel-resistant PCa cells developed tolerance toward ferroptosis. In addition, dysregulated lncRNAs in drug-resistant and -sensitive PCa cells were identified by RNA sequencing, and we identified that prostate cancer-associated transcript 1 (PCAT1) was highly expressed in the docetaxel-resistant PCa cell lines and clinical samples. Overexpression of PCAT1 inhibited ferroptosis and increased docetaxel resistance, which could be attenuated by PCAT1 knockdown. Furthermore, we revealed that PCAT1 inhibited ferroptosis by activating solute carrier family 7-member 11 (SLC7A11) expression via reducing iron accumulation and subsequent oxidative damage. Mechanistically, we demonstrated that PCAT1 interacted with c-Myc and increased its protein stability using nucleotides 1093-1367 of PCAT1 and 151-202 amino acids of c-Myc protein, thereby transcriptionally promoting SLC7A11 expression. In addition, PCAT1 facilitated SLC7A11 expression by competing for microRNA-25-3p. Finally, transcription factor AP-2 gamma (TFAP2C) activated PCAT1 expression at the transcriptional level to reduce ferroptosis susceptibility and enhance chemoresistance. Collectively, our findings demonstrated that TFAP2C-induced PCAT1 promotes chemoresistance by blocking ferroptotic cell death through c-Myc/miR-25-3p/SLC7A11 signaling.

A non-coding RNA balancing act: miR-346-induced DNA damage is limited by the long non-coding RNA NORAD in prostate cancer

BackgroundmiR-346 was identified as an activator of Androgen Receptor (AR) signalling that associates with DNA damage response (DDR)-linked transcripts in prostate cancer (PC). We sought to delineate the impact of miR-346 on DNA damage, and its potential as a therapeutic agent.MethodsRNA-IP, RNA-seq, RNA-ISH, DNA fibre assays, in vivo xenograft studies and bioinformatics approaches were used alongside a novel method for amplification-free, single nucleotide-resolution genome-wide mapping of DNA breaks (INDUCE-seq).ResultsmiR-346 induces rapid and extensive DNA damage in PC cells - the first report of microRNA-induced DNA damage. Mechanistically, this is achieved through transcriptional hyperactivation, R-loop formation and replication stress, leading to checkpoint activation and cell cycle arrest. miR-346 also interacts with genome-protective lncRNA NORAD to disrupt its interaction with PUM2, leading to PUM2 stabilisation and its increased turnover of DNA damage response (DDR) transcripts. Confirming clinical relevance, NORAD expression and activity strongly correlate with poor PC clinical outcomes and increased DDR in biopsy RNA-seq studies. In contrast, miR-346 is associated with improved PC survival.INDUCE-seq reveals that miR-346-induced DSBs occur preferentially at binding sites of the most highly-transcriptionally active transcription factors in PC cells, including c-Myc, FOXA1, HOXB13, NKX3.1, and importantly, AR, resulting in target transcript downregulation. Further, RNA-seq reveals widespread miR-346 and shNORAD dysregulation of DNA damage, replication and cell cycle processes.NORAD drives target-directed miR decay (TDMD) of miR-346 as a novel genome protection mechanism: NORAD silencing increases mature miR-346 levels by several thousand-fold, and WT but not TDMD-mutant NORAD rescues miR-346-induced DNA damage. Importantly, miR-346 sensitises PC cells to DNA-damaging drugs including PARP inhibitor and chemotherapy, and induces tumour regression as a monotherapy in vivo, indicating that targeting miR-346:NORAD balance is a valid therapeutic strategy.ConclusionsA balancing act between miR-346 and NORAD regulates DNA damage and repair in PC. miR-346 may be particularly effective as a therapeutic in the context of decreased NORAD observed in advanced PC, and in transcriptionally-hyperactive cancer cells.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

EIF4A3-Induced circARHGAP29 Promotes Aerobic Glycolysis in Docetaxel-Resistant Prostate Cancer through IGF2BP2/c-Myc/LDHA Signaling

Upregulation of a novel circRNA, circARHGAP29, promotes docetaxel resistance and glycolytic metabolism, suggesting it could be a prognostic biomarker and therapeutic target in chemoresistant prostate cancer.

Targeting Protein Arginine Methyltransferase 5 Suppresses Radiation-induced Neuroendocrine Differentiation and Sensitizes Prostate Cancer Cells to Radiation.

Prostate cancer remains the second leading cause of cancer death among American men. Radiotherapy is a potentially curative treatment for localized prostate cancer, and failure to control localized disease contributes to the majority of prostate cancer deaths. Neuroendocrine differentiation (NED) in prostate cancer, a process by which prostate adenocarcinoma cells transdifferentiate into neuroendocrine-like (NE-like) cells, is an emerging mechanism of resistance to cancer therapies and contributes to disease progression. NED also occurs in response to treatment to promote the development of treatment-induced neuroendocrine prostate cancer (NEPC), a highly aggressive and terminal stage disease. We previously demonstrated that by mimicking clinical radiotherapy protocol, fractionated ionizing radiation (FIR) induces prostate cancer cells to undergo NED in vitro and in vivo. Here, we performed transcriptomic analysis and confirmed that FIR-induced NE-like cells share some features of clinical NEPC, suggesting that FIR-induced NED represents a clinically relevant model. Furthermore, we demonstrated that protein arginine methyltransferase 5 (PRMT5), a master epigenetic regulator of the DNA damage response and a putative oncogene in prostate cancer, along with its cofactors pICln and MEP50, mediate FIR-induced NED. Knockdown of PRMT5, pICln, or MEP50 during FIR-induced NED and sensitized prostate cancer cells to radiation. Significantly, PRMT5 knockdown in prostate cancer xenograft tumors in mice during FIR prevented NED, enhanced tumor killing, significantly reduced and delayed tumor recurrence, and prolonged overall survival. Collectively, our results demonstrate that PRMT5 promotes FIR-induced NED and suggests that targeting PRMT5 may be a novel and effective radiosensitization approach for prostate cancer radiotherapy.

A phase Ia/Ib study of talazoparib in combination with tazemetostat in metastatic castration-resistant prostate cancer (mCRPC).

TPS195 Background: Enhancer of zeste homolog 2 (EZH2) is frequently overexpressed in metastatic castration-resistant prostate cancer (mCRPC), and is linked to lineage plasticity and therapy resistance. In pre-clinical studies, EZH2 directly regulates DNA damage repair (DDR) gene expression, and pharmacologic inhibition of EZH2 sensitizes prostate cancer cells to genotoxic stress as induced by poly-ADP ribose polymerase (PARP) inhibition. The PARP inhibitor talazoparib and EZH2 inhibitor tazemetostat are currently under study in mCRPC, and we are conducting a phase 1 clinical trial of the combination. Methods: Phase 1a of the study will define the recommended phase 2 dose (RP2D) and phase 1b will better assess safety and preliminary clinical activity of the combination at the RP2D. Eligible patients must have progressive disease after at least one secondary hormonal therapy and taxane-based chemotherapy (or felt not to be more appropriate for taxane), have disease evaluable for response (PSA ≥ 2 ng/ml or measurable disease by RECIST 1.1) and have a metastatic lesion amenable to biopsy adequate for next generation sequencing. In phase 1a (n=9-18), the starting doses are talazoparib 0.75 mg daily and tazemetostat 600 mg BID, with dose escalation/de-escalation of both agents by up to 2 dose levels [DLs] based on a 3+3 design. The RP2D is the maximum tolerated dose (MTD) or DL +2 (talazoparib 1 mg daily + tazemetostat 800 mg BID) if the MTD is not reached. After 6 patients are treated at the RP2D, phase 1b will enroll an additional 20 patients to an expansion cohort. The primary endpoint of safety and tolerability is based on incidence of dose-limiting toxicities [DLTs] and incidence and grade of adverse events [AEs] by CTCAE version 5.0. For the secondary endpoint of overall response rate (ORR; defined as PSA reduction by ≥ 50% OR radiographic response by RECIST 1.1), with a sample size of 26 (6 patients from dose escalation and 20 from expansion), we deem talazoparib+tazemetostat effective if ORR is ≥ 5/26 (19%). The probability of concluding that the treatment strategy effective is 0.11 if its true response rate is 10% and at least 0.93 if the true response rate exceeds 30%. Mandatory pre-treatment and on-treatment (8-week) biopsies will undergo targeted genetic sequencing, transcriptomic profiling, ChIP (Chromatin ImmunoPrecipitation)-seq, and immunohistochemistry (IHC) for DDR and differentiation markers; blood specimens will undergo circulating cell-free DNA and circulating tumor cell profiling – these studies will nominate possible predictive biomarkers for therapeutic response and serve as pharmacodynamic markers of combined PARP and EZH2 inhibition. The goal of this study is to expand treatment options in mCRPC through a novel approach to exploit EZH2 as a therapeutic target through co-targeting the DDR response. Enrollment began in July 2021. Clinical trial information: NCT04846478.

Targeting a splicing-mediated drug resistance mechanism in prostate cancer by inhibiting transcriptional regulation by PKC\u03b21

The androgen receptor (AR) is a central driver of aggressive prostate cancer. After initial treatment with androgen receptor signaling inhibitors (ARSi), reactivation of AR signaling leads to resistance. Alternative splicing of AR mRNA yields the AR-V7 splice variant, which is currently an undruggable mechanism of ARSi resistance: AR-V7 lacks a ligand binding domain, where hormones and anti-androgen antagonists act, but still activates AR signaling. We reveal PKCβ as a druggable regulator of transcription and splicing at the AR genomic locus. We identify a clinical PKCβ inhibitor in combination with an FDA-approved anti-androgen as an approach for repressing AR genomic locus expression, including expression of AR-V7, while antagonizing full-length AR. PKCβ inhibition reduces total AR gene expression, thus reducing AR-V7 protein levels and sensitizing prostate cancer cells to current anti-androgen therapies. We demonstrate that this combination may be a viable therapeutic strategy for AR-V7-positive prostate cancer.

In Vitro characterization of the endocrine disrupting effects of per- and poly-fluoroalkyl substances (PFASs) on the human androgen receptor

Per- and poly-fluoroalkyl substances (PFASs) are used extensively in a broad range of industrial applications and consumer products. While a few legacy PFASs have been voluntarily phased out, over 5000 PFASs have been produced as replacements for their predecessors. The potential endocrine disrupting hazards of most emerging PFASs have not been comprehensively investigated. In silico molecular docking to the human androgen receptor (hAR) combined with machine learning techniques were previously applied to 5206 PFASs and predicted 23 PFASs bind the hAR. Herein, the in silico results were validated in vitro for the five candidate AR ligands that were commercially available. Three manufactured PFASs namely (9-(nonafluorobutyl)− 2,3,6,7-tetrahydro-1 H,5 H,11 H-pyrano[2,3-f]pyrido[3,2,1-ij]quinolin-11-one (NON), 2-(heptafluoropropyl)− 3-phenylquinoxaline (HEP), and 2,2,3,3,4,4,5,5,5-nonafluoro-N-(4-nitrophenyl)pentanamide (NNN) elicited significant antiandrogenic effects at relatively low concentrations. We further investigated the mechanism of AR inhibition and found that all three PFASs inhibited AR transactivation induced by testosterone through a competitive binding mechanism. We then examined the antiandrogenic effects of these PFASs on AR expression and its responsive genes. Consistently, these PFASs significantly decreased the expression of PSA and FKBP5 and increased the expression of AR, similar to the effects elicited by a known competitive AR inhibitor, hydroxyflutamide. This suggests they are competitive antagonists of AR activity and western blot analysis revealed these PFASs decreased intracellular AR protein in androgen sensitive human prostate cancer cells. Hence, the findings presented here corroborate our published in silico approach and indicate these emerging PFASs may adversely affect the human endocrine system.

Caffeic acid phenethyl ester induces radiosensitization via inhibition of DNA damage repair in androgen-independent prostate cancer cells.

In the present study, we evaluated the radiomodulatory potential of caffeic acid phenethyl ester (CAPE), an active component of traditional herbal medicine propolis. CAPE has been identified as a potent anticancer agent in multiple cancer types and is reported to have the dual role of radioprotection and radiosensitization. However, the radiomodulatory potential of CAPE in prostate cancer (PCa), which eventually becomes radioresistant is not known. Therefore, we studied the effect of co-treatment of CAPE and gamma radiation on androgen-independent DU145 and PC3 cells. The combination treatment sensitized PCa cells to radiation in a dose-dependent manner. The radiosensitizing effect of CAPE was observed in both cell lines. CAPE enhanced the level of ionizing radiation (IR)-induced gamma H2AX foci and cell death by apoptosis. The combination treatment also decreased the migration potential of PCa cells. This was confirmed by increased expression of E-cadherin and decrease in vimentin expression. CAPE sensitized PCa cells to radiation in vitro and induced apoptosis, augmented phosphorylation of Akt/mTOR, and hampered cell migration. At the mechanistic level, co-treatment of CAPE and IR inhibited cell growth by decreasing RAD50 and RAD51 proteins involved in DNA repair. This resulted in enhanced DNA damage and cell death. CAPE might represent a promising new adjuvant for the treatment of hormone-refractory radioresistant PCa.

MiR-143 mediates abiraterone acetate resistance by regulating the JNK/Bcl-2 signaling pathway in prostate cancer.

Background: miR-143 is known to be downregulated in various cancer cells and tumors and generally plays a tumor-suppressor role. miR-143. However, the role of miR-143 in the mediation of the sensitivity of prostate cancer cells to abiraterone acetate remains unrevealed. Methods: The expression levels of miRNAs were determined by miRNA microarray and quantitative real-time PCR (qRT-PCR). The protein levels were assessed by Western blot assay. Cell viability and apoptosis were respectively measured by Cell Counting Kit-8 (CCK-8) assay and flow cytometry. Results: We identified that miR-143 was significantly downregulated in PC3-AbiR cells compared to PC3 cells. Overexpression of miR-143 promoted PC-AbiR sensitivity to abiraterone acetate in vitro and in vivo. We also revealed that miR-143 upregulation inhibited p-JNK (c-Jun N-terminal kinases) and increased p-Bcl2 (B-cell lymphoma 2), contributing to abiraterone acetate-induced apoptosis in PC3-AbiR cells. Finally, we showed that the combination of miR-143 and abiraterone acetate exerted the most profound tumor inhibition effect and prolonged the mice survival rate in PC3-AbiR tumor-bearing mice. Conclusion: Upregulation of miR-143 may serve as a new strategy to enhance the therapeutical effect of abiraterone acetate on prostate cancer patients who are resistant to abiraterone acetate.

STAT3 as a target for sensitizing prostate cancer cells to irradiation.

Radioresistance of prostate cancer (PCa) is a major factor leading to local failure of radiotherapy. STAT3 is an oncogenic protein that was recently found to be activated in PCa tumors. This study aimed to investigate the radiosensitization effect of targeting STAT3 in PCa tumors. Here, the radiosensitization effect of STAT3 blockade was investigated by clonogenic assay, flow cytometry and western blot analysis in human PCa cells in vitro and in vivo. We demonstrated that STAT3 blockade with a STAT3 inhibitor or siRNA increased the radiosensitivity of PCa cells and that radiation together with STAT3 blockade induced more apoptosis and double-strand breaks (DSBs) than radiation alone in LNCaP cells. In addition, radiation induced STAT3 activation and survivin expression in PCa cells, which was inhibited by STAT3 blockade. Transfection with survivin cDNA attenuated the radiosensitization effect of STAT3 blockade. These effects were further confirmed by in vivo studies, which showed that the STAT3 inhibitor enhanced the treatment efficacy of radiation on LNCaP xenografts with decreased STAT3 activation and survivin expression.These findings suggest that STAT3 blockade radiosensitizes PCa cells through regulation of survivin. Thus, our study has revealed STAT3 as a potential sensitizer for irradiation in PCa cells. Its clinical application as an adjuvant in radiotherapy of PCa should be explored in the future.

Insights of RKIP-Derived Suppression of Prostate Cancer.

Simple SummaryDespite an intensive research effort in the past few decades, prostate cancer (PC) remains a top cause of cancer death in men, particularly in the developed world. The major cause of fatality is the progression of local prostate cancer to metastasis disease. Treatment of patients with metastatic prostate cancer (mPC) is generally ineffective. Based on the discovery of mPC relying on androgen for growth, many patients with mPC show an initial response to the standard of care: androgen deprivation therapy (ADT). However, lethal castration resistant prostate cancers (CRPCs) commonly develop. It is widely accepted that intervention of metastatic progression of PC is a critical point of intervention to reduce PC death. Accumulative evidence reveals a role of RKIP in suppression of PC progression towards mPC. We will review current evidence and discuss the potential utilization of RKIP in preventing mPC progression.Prostate cancer (PC) is a major cause of cancer death in men. The disease has a great disparity in prognosis. Although low grade PCs with Gleason scores ≤ 6 are indolent, high-risk PCs are likely to relapse and metastasize. The standard of care for metastatic PC (mPC) remains androgen deprivation therapy (ADT). Resistance commonly occurs in the form of castration resistant PC (CRPC). Despite decades of research efforts, CRPC remains lethal. Understanding of mechanisms underpinning metastatic progression represents the overarching challenge in PC research. This progression is regulated by complex mechanisms, including those regulating PC cell proliferation, epithelial–mesenchymal transition (EMT), and androgen receptor (AR) signaling. Among this PC metastatic network lies an intriguing suppressor of PC metastasis: the Raf kinase inhibitory protein (RKIP). Clinically, the RKIP protein is downregulated in PC, and showed further reduction in mPC. In xenograft mouse models for PC, RKIP inhibits metastasis. In vitro, RKIP reduces PC cell invasion and sensitizes PC cells to therapeutic treatments. Mechanistically, RKIP suppresses Raf-MEK-ERK activation and EMT, and modulates extracellular matrix. In return, Snail, NFκB, and the polycomb protein EZH2 contribute to inhibition of RKIP expression. In this review, we will thoroughly analyze RKIP’s tumor suppression actions in PC.