Hormone-sensitive Prostate Cancer Cells Research Articles

Altered expression of vesicular trafficking machinery in prostate cancer affects lysosomal dynamics and provides insight into the underlying biology and disease progression.

This study focuses on the role of lysosomal trafficking in prostate cancer, given the essential role of lysosomes in cellular homoeostasis. Lysosomal motility was evaluated using confocal laser scanning microscopy of LAMP-1-transfected prostate cells and spot-tracking analysis. Expression of lysosomal trafficking machinery was evaluated in patient cohort databases and through immunohistochemistry on tumour samples. The roles of vesicular trafficking machinery were evaluated through over-expression and siRNA. The effects of R1881 treatment on lysosome vesicular trafficking was evaluated by RNA sequencing, protein quantification and fixed- and live-cell microscopy. Altered regulation of lysosomal trafficking genes/proteins was observed in prostate cancer tissue, with significant correlations for co-expression of vesicular trafficking machinery in Gleason patterns. The expression of trafficking machinery was associated with poorer patient outcomes. R1881 treatment induced changes in lysosomal distribution, number, and expression of lysosomal vesicular trafficking machinery in hormone-sensitive prostate cancer cells. Manipulation of genes involved in lysosomal trafficking events induced changes in lysosome positioning and cell phenotype, as well as differential effects on cell migration, in non-malignant and prostate cancer cells. These findings provide novel insights into the altered regulation and functional impact of lysosomal vesicular trafficking in prostate cancer pathogenesis.

Androgen receptor modulatory miR-1271-5p can promote hormone sensitive prostate cancer cell growth.

In most patients with advanced prostate cancer treated with hormonal therapy, androgen independence eventually emerges, leading to death. Androgen receptor signalling remains an important prostate cancer driver, even in the advanced disease stage. MicroRNAs (miRs), non-coding RNAs that regulate gene expression by inhibiting translation and/or promoting degradation of target mRNAs, can act as tumour suppressors or "oncomiRs" and modulate tumour growth. Because of their stability in tissues and in circulation, and their specificity, microRNAs have emerged as potential biomarkers, as well as therapeutic targets in cancer. We identified miR-1271-5p as an androgen receptor modulatory microRNA and we show it can promote hormone sensitive prostate cancer cell growth. Inhibition or overexpression of miR-1271-5p levels affects prostate cancer cell growth, apoptosis and expression of both androgen receptor target genes and other genes that are likely direct targets, dependent on androgen receptor status, and tumour stage. We conclude that miR-1271-5p has the potential to drive progression of hormone-dependent disease and that the use of specific inhibitors of miR-1271-5p may have therapeutic potential in prostate cancer.

Androgen receptor transcriptional activity is required for heregulin-1β–mediated nuclear localization of the HER3/ErbB3 receptor tyrosine kinase

Prostate cancer is initially regulated by the androgen receptor (AR), a ligand-activated, transcription factor, and is in a hormone-dependent state (hormone-sensitive prostate cancer (HSPC)), but eventually becomes androgen-refractory (castration-resistant prostate cancer (CRPC)) because of mechanisms that bypass the AR, including by activation of ErbB3, a member of the epidermal growth factor receptor family. ErbB3 is synthesized in the cytoplasm and transported to the plasma membrane for ligand binding and dimerization, where it regulates downstream signaling, but nuclear forms are reported. Here, we demonstrate in prostatectomy samples that ErbB3 nuclear localization is observed in malignant, but not benign prostate, and that cytoplasmic (but not nuclear) ErbB3 correlated positively with AR expression but negatively with AR transcriptional activity. In support of the latter, androgen depletion upregulated cytoplasmic, but not nuclear ErbB3, while invivo studies showed that castration suppressed ErbB3 nuclear localization in HSPC, but not CRPC tumors. Invitro treatment with the ErbB3 ligand heregulin-1β (HRG) induced ErbB3 nuclear localization, which was androgen-regulated in HSPC but not in CRPC. In turn, HRG upregulated AR transcriptional activity in CRPC but not in HSPC cells. Positive correlation between ErbB3 and AR expression was demonstrated in AR-null PC-3cells where stable transfection of AR restored HRG-induced ErbB3 nuclear transport, while AR knockdown in LNCaP reduced cytoplasmic ErbB3. Mutations of ErbB3's kinase domain did not affect its localization but was responsible for cell viability in CRPC cells. Taken together, we conclude that AR expression regulated ErbB3 expression, its transcriptional activity suppressed ErbB3 nuclear translocation, and HRG binding to ErbB3 promoted it.

Abstract 2625: KRAS activity-mediated mechanism of castration-resistant prostate cancer progression

Abstract Purpose: Prostate cancer is highly androgen-dependent, and androgen receptor (AR) signaling is suppressed by treatment. However, there are cases in which disease progresses rapidly at the end of treatment even after AR suppression, suggesting that the disease progresses by mechanisms different from AR. The aim of this study is to investigate the KRAS signaling pathway as one of the mechanisms. Methods: We used the hormone-sensitive prostate cancer (HSPC) cell line LNCaP and the castration-resistant prostate cancer (CRPC) cell line DU145. We performed KRAS knockdown by si-RNA and activation of KRAS signaling by EGF stimulation. To examine KRAS signaling, PCR and Western blotting were performed. The changes in proliferation and migration due to inhibition and activation of KRAS signaling were confirmed. Results: RT-PCR showed a fusion gene of ubiquitin-conjugating enzyme UBE2L3 and KRAS (UBE2L3-KRAS) in DU145. Western blotting showed increased phosphorylation of ERK and p38 downstream of KRAS in DU145. KRAS knockdown inhibited proliferation and migration in DU145 but not in LNCaP. In EGF-stimulated activation of KRAS signaling, Western blotting showed activation of downstream KRAS signaling in LNCaP, but no change in proliferation and migration in fetal bovine serum (FBS)-added medium. In contrast, EGF stimulation of DU145 further activated downstream KRAS signaling and promoted proliferation and migration. The AR-dependent LNCaP showed almost no effect of KRAS signaling activation on cancer progression, whereas DU145 showed that KRAS signaling is involved in cancer progression. Conclusion: While HSPC is highly AR-dependent and does not show any effect of KRAS on cancer progression, KRAS may be involved in cancer progression in some CRPCs. Citation Format: Taiki Kamijima, Kouji Izumi, Yoshifumi Kadono, Atsushi Mizokami. KRAS activity-mediated mechanism of castration-resistant prostate cancer progression [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 2625.

Androgen Deprivation Freezes Hormone-Sensitive Prostate Cancer Cells in a Reversible, Genetically Unstable Quasi-Apoptotic State, Bursting into Full Apoptosis upon Poly(ADP-ribose) Polymerase Inhibition.

Androgen deprivation therapy (ADT) is a powerful treatment for metastatic hormone-sensitive prostate cancer (mHSPC) patients, but eventually and inevitably, cancer relapses, progressing to the fatal castration-resistant (CR)PC stage. Progression implies the emergence of cells proliferating in the absence of androgen through still elusive mechanisms. We show here for the first time that ADT induces LNCaP mHSPC cells to collectively enter a metastable quasi-apoptotic state (QUAPS) consisting of partial mitochondrial permeabilization, limited BAX and caspase activation, and moderate induction of caspase-dependent dsDNA breaks; despite this, cells maintain full viability. QUAPS is destabilized by poly(ADP)-polymerase inhibition (PARPi), breaking off toward overt intrinsic apoptosis and culture extinction. Instead, QUAPS is rapidly and efficiently reverted upon androgen restoration, with mitochondria rapidly recovering integrity and cells collectively resuming normal proliferation. Notably, replication restarts before DNA repair is completed, and implies an increased micronuclei frequency, indicating that ADT promotes genetic instability. The recovered cells re-acquire insensitivity to PARPi (as untreated LNCaP), pointing to specific, context-dependent vulnerability of mHSPC cells to PARPi during ADT. Summarizing, QUAPS is an unstable, pro-mutagenic state developing as a pro-survival pathway stabilized by PARP, and constitutes a novel viewpoint explaining how ADT-treated mHSPC may progress to CRPC, indicating possible preventive countermeasures.

The Androgen Regulated lncRNA NAALADL2-AS2 Promotes Tumor Cell Survival in Prostate Cancer.

Castration resistance is the leading cause of death in men with prostate cancer. Recent studies indicate long noncoding RNAs (lncRNAs) to be important drivers of therapy resistance. The aim of this study was to identify differentially expressed lncRNAs in castration-resistant prostate cancer (CRPC) and to functionally characterize them in vitro. Tumor-derived RNA-sequencing data were used to quantify and compare the expression of 11,469 lncRNAs in benign, primary prostate cancer, and CRPC samples. CRPC-associated lncRNAs were selected for semi-quantitative PCR validation on 68 surgical tumor specimens. In vitro functional studies were performed by antisense-oligonucleotide-mediated lncRNA knockdown in hormone-sensitive prostate cancer (HSPC) and CRPC cell line models. Subsequently, cell proliferation, apoptosis, cell cycle, transcriptome and pathway analyses were performed using the appropriate assays. Transcriptome analysis of a prostate cancer tumor specimens unveiled NAALADL2-AS2 as a novel CRPC-upregulated lncRNA. The expression of NAALADL2-AS2 was found to be particularly high in HSPC in vitro models and to increase under androgen deprived conditions. NAALADL2-AS2 knockdown decreased cell viability and increased caspase activity and apoptotic cells. Cellular fractionization and RNA fluorescent in situ hybridization identified NAALADL2-AS2 as a nuclear transcript. Transcriptome and pathway analyses revealed that NAALADL2-AS2 modulates the expression of genes involved with cell cycle control and glycogen metabolism. We hypothesize that the nuclear lncRNA, NAALADL2-AS2, functions as a pro-survival signal in prostate cancer cells under pressure of targeted hormone therapy.

Increased MYBL2 expression in aggressive hormone-sensitive prostate cancer.

Loss of the histone demethylase KDM5D (lysine-specific demethylase 5D) leads to in vitro resistance of prostate cancer cells to androgen deprivation therapy (ADT) with and without docetaxel. We aimed to define downstream drivers of the KDM5D effect. Using chromatin immunoprecipitation sequencing (ChIP-seq) of the LNCaP cell line (androgen-sensitive human prostate adenocarcinoma) with and without silenced KDM5D, MYBL2-binding sites were analyzed. Associations between MYBL2 mRNA expression and clinical outcomes were assessed in cohorts of men with localized and metastatic hormone-sensitive prostate cancer. In vitro assays with silencing and overexpression of MYBL2 and KDM5D in androgen receptor (AR)-positive hormone-sensitive prostate cancer cell lines, LNCaP and LAPC4, were used to assess their influence on cellular proliferation, apoptosis, and cell cycle distribution, as well as sensitivity to androgen deprivation, docetaxel, and cabazitaxel. We found that silencing KDM5D increased histone H3 lysine K4 (H3K4) trimethylation and increased MYBL2 expression. KDM5D and MYBL2 were negatively correlated with some but not all clinical samples. Higher MYBL2 expression was associated with a higher rate of relapse in localized disease and poorer overall survival in men with metastatic disease in the CHAARTED trial. Lower MYBL2 levels enhanced LNCaP and LAPC4 sensitivity to androgen deprivation and taxanes. In vitro, modifications of KDM5D and MYBL2 altered cell cycle distribution and apoptosis in a cell line-specific manner. These results show that the transcription factor MYBL2 impacts in vitro hormone-sensitive prostate cancer sensitivity to androgen deprivation and taxanes, and lower levels are associated with better clinical outcomes in men with hormone-sensitive prostate cancer.

Lipin Expression Correlates to Differences in Phospholipid Profiles in Prostate Cancer Progression

PURPOSE The association between clinical outcomes of prostate cancer progression and lipid remodeling is not well understood. Although it is known that increases in select circulating lipids correlate to decreased patient survival, the mechanisms mediating alterations in these lipids are not fully explained. We addressed this gap-in-knowledge using in vitro models of non-cancerous, hormone-sensitive, and castration-resistant prostate cancer (CRPC) cell lines combined with quantitative HPLC-ESI-Orbitrap-MS lipidomic analysis. HYPOTHESIS We hypothesized that the expression of lipin (phosphatidic acid phosphatase), an enzyme mediating the conversion of phosphatidic acid to diacylglycerol, correlates to changes in lipidomic profiles in prostate cancer cells. We further hypothesized that changes in this enzyme influences the overall survivability of prostate cancer patients. METHODS Lipids were isolated using the Bligh-Dyer extraction from non-cancerous, hormone-sensitive, and CRPC cell lines. Lipids present in both cells and media were initially analyzed using an untargeted shotgun approach (ESI-MS), followed by a targeted-based quantitative HPLC-ESI-Orbitrap-MS approach. Post data acquisition for both shotgun and HPLC-ESI/MS/MS lipidomics were based on multiple methods including isotope, carbon number (to internal standards) and ionization efficiency-based corrections. Online resources and software's utilized included MetaboAnalyst, LIPIDMATCH, LIPID MAPS, XCMS, and MZmine. Gene alteration frequency and overall survival data were generated using cBioPortal for Cancer Genomics and GEPIA 2. Lipin expression in cells was determined using immunoblot analysis. RESULTS Our data showed significant changes in the abundance of lipid species between the various cell lines and a correlation between select lipid species and the progression of prostate cancer. Prominent among these were the levels of phosphatidic acid (PA) and diacylglycerol (DAG), a process controlled by lipin. Most notably, PA levels in cells tended to inversely correlate with DAG levels. These levels correlated to the decreased expression on lipin, specifically lipin-1, suggesting that decreased lipin-1 expression correlates to more aggressive prostate cancer. This hypothesis was supported by analysis of alteration in lipin genes expressed in numerous cohorts of prostate cancer patients, demonstrating that lipin mutations indicated a decrease in the overall survival of prostate cancer patients. CONCLUSIONS Distinct phospholipid classes were observed to have increased levels in prostate cancer cells, as compared to non-cancer cells. Preliminary data shows a correlation between the abundance of PA and DAG with lipin expression in cells representing more aggressive prostate cancers, which is supported by data from patient populations, suggesting that altered lipin expression correlates to decreased survival.

Huaier Extract Inhibits Prostate Cancer Growth via Targeting AR/AR-V7 Pathway.

The androgen receptor (AR) plays a pivotal role in prostatic carcinogenesis, and it also affects the transition from hormone sensitive prostate cancer (HSPC) to castration-resistant prostate cancer (CRPC). Particularly, the persistent activation of the androgen receptor and the appearance of androgen receptor splicing variant 7 (AR-V7), could partly explain the failure of androgen deprivation therapy (ADT). In the present study, we reported that huaier extract, derived from officinal fungi, has potent antiproliferative effects in both HSPC and CRPC cells. Mechanistically, huaier extract downregulated both full length AR (AR-FL) and AR-V7 mRNA levels via targeting the SET and MYND domain-containing protein 3 (SMYD3) signaling pathway. Huaier extract also enhanced proteasome-mediated protein degradation of AR-FL and AR-V7 by downregulating proteasome-associated deubiquitinase ubiquitin-specific protease 14 (USP14). Furthermore, huaier extract inhibited AR-FL/AR-V7 transcriptional activity and their nuclear translocation. More importantly, our data demonstrated that huaier extract could re-sensitize enzalutamide-resistant prostate cancer cells to enzalutamide treatment in vitro and in vivo models. Our work revealed that huaier extract could be effective for treatment of prostate cancer either as monotherapy or in combination with enzalutamide.

Significance of targeting the antiapoptotic pathway in castration-sensitive prostate cancer.

250 Background: Prostate cancer (PC) is a major health problem for men in the U.S. and is the second most common cause of cancer-related deaths in males. Although most PCs are initially sensitive to androgen-deprivation therapy (ADT), the duration of response is variable, and eventually, the cancer becomes resistant to ADT and progresses to metastatic castration-resistant prostate cancer (mCRPC). For mCRPC patients, many initially respond to second-line ARIs (eg. enzalutamide and abiraterone) or docetaxel-based chemotherapy however durable responses are rare. Therefore, it is vital to investigate additional therapeutic strategies to delay or prevent the transition of castration-sensitive prostate cancer (CSPC) to mCRPC. Methods: We treated castration-sensitive human PC cells with various anti-androgen inhibitors to investigate the direct association between Bcl2 expression and AR-pathway. We used a lentiviral-based over-expression method to develop BCL2 over-expressed experimental PC cell line systems and subjected them to various in -vitro and in vivo studies. We studied the combinational effect of Bcl2 and AR inhibitor on the in vitro growth of hormone-sensitive human PC cells and in vivo mice model. Results: We observed that treatment with androgen inhibits but ARIs (eg enzalutamide, apalutamide) restore Bcl2 expression in human CSPC cell lines indicating there is possible direct negative-regulation of the Bcl2 by the AR-signaling pathway. BCL2 over-expressed LNCaP cells show deregulation of the AR pathway, induces PSMA expression, and exhibit relative resistance to enzalutamide indicating that over-expression of BCL2 induces castration resistance in hormone-sensitive PC cells. Our cell growth inhibition assay showed an overall strong additive effect on growth inhibition with enzalutamide and the pharmacological Bcl2 inhibitor (venetoclax) combination on LNCaP cells and 22Rv1 cells. We also observed a negative association between BCL2 and AR pathway in clinical PC cohorts (Localized and mCRPC). In the isograft mice model, we showed the combination of enzalutamide and venetoclax significantly reduces subcutaneous prostate tumor growth and increases overall survival (~2 weeks) compare to control groups of mice. Moreover, using Isogenic cell lines (control and BCL2 over-expressed LNCaP) we showed higher uptake of [68Ga]-PSMA-11 in BCL2 over-expressed prostate tumors compared to control tumors in immunodeficient mice indicating that BCL2 over-expressed PC can monitor non-invasively by PSMA-PET imaging. Conclusions: Our current study develops a rationale for combining ADT with Bcl2-inhibitors for CSPC. We believe this combinatorial therapeutic approach will show great potential for future clinical trials of high-risk hormone-sensitive PC patients and may block the ADT-induced shift of CSPC to mCRPC.

V-ATPase Inhibition Decreases Mutant Androgen Receptor Activity in Castrate-resistant Prostate Cancer.

Prostate cancer is critically dependent on androgen receptor (AR) signaling. Despite initial responsiveness to androgen deprivation, most patients with advanced prostate cancer subsequently progress to a clinically aggressive castrate-resistant prostate cancer (CRPC) phenotype, typically associated with expression of splice-variant or mutant AR forms. Although current evidence suggests that the vacuolar-ATPase (V-ATPase), a multiprotein complex that catalyzes proton transport across intracellular and plasma membranes, influences wild-type AR function, the effect of V-ATPase inhibition on variant AR function is unknown.Inhibition of V-ATPase reduced AR function in wild-type and mutant AR luciferase reporter models. In hormone-sensitive prostate cancer cell lines (LNCaP, DuCaP) and mutant AR CRPC cell lines (22Rv1, LNCaP-F877L/T878A), V-ATPase inhibition using bafilomycin-A1 and concanamycin-A reduced AR expression, and expression of AR target genes, at mRNA and protein levels. Furthermore, combining chemical V-ATPase inhibition with the AR antagonist enzalutamide resulted in a greater reduction in AR downstream target expression than enzalutamide alone in LNCaP cells. To investigate the role of individual subunit isoforms, siRNA and CRISPR-Cas9 were used to target the V1C1 subunit in 22Rv1 cells. Whereas transfection with ATP6V1C1-targeted siRNA significantly reduced AR protein levels and function, CRISPR-Cas9-mediated V1C1 knockout showed no substantial change in AR expression, but a compensatory increase in protein levels of the alternate V1C2 isoform.Overall, these results indicate that V-ATPase dysregulation is directly linked to both hormone-responsive prostate cancer and CRPC via impact on AR function. In particular, V-ATPase inhibition can reduce AR signaling regardless of mutant AR expression.

Changes in phospholipid metabolism in exosomes of hormone-sensitive and hormone-resistant prostate cancer cells.

Background: To explore the changes in lipids in exosomes of hormone-sensitive and hormone-resistant prostate cancer cells and develop an inexpensive and rapid technique for screening lipid-based biomarkers of prostate cancer.Methods: Exosomes were extracted from LnCap, PC3 and DU-145 cells, and their lipid composition was analyzed quantitatively using high-throughput mass spectrometry. Exosomes released by LnCap prostate cancer cells were also purified using a modified procedure based on polyethylene glycol (PEG) precipitation.Results: Exosomes extracted from LnCap cells contained higher proportions of phosphatidyl choline, phosphatidyl ethanolamine and phosphatidyl inositol lipids than whole LnCap cells. Lysophosphatidylcholine, a harmful intermediate product of phosphatidylcholine metabolism in vivo, was not found in LnCap cells but in exosomes. Phospholipids were different in exosomes from LnCap, PC3 and DU-145 prostate cancer cells. The main lipid pathways involved, i.e., glycerophospholipid metabolism, autophagy, and ferroptosis pathways, were also different in these cells. Exosomes isolated by this modified PEG precipitation technique were similar in purity to those obtained using a commercial kit.Conclusions: This study demonstrates that phosphatidylcholine and its harmful product lysophosphatidylcholine may play important roles in hormone-sensitive prostate cancer. Phospholipid exosome metabolism was changed in hormone-sensitive and hormone-resistant prostate cancer cells. The LPC, lipid pathway of autophagy and ferroptosis may act as therapeutic targets. The possibility of purifying prostate cancer cell exosomes using modified PEG precipitation is suitable for cancer screening.

IL-4 Counteracts the Cytotoxic Effects of Peripheral Blood Mononuclear Cells on Hormone-sensitive Prostate Cancer Cells.

Proinflammatory cytokines play an essential role in the development and progression of prostate cancer (PCa). Especially interleukine (IL-)6 is involved in the development of aggressive PCa. Peripheral blood mononuclear cells (PBMC) have been reported to interact with cancer cells and subsequently lead to increased production of pro-inflammatory cytokines. However, the role of anti-nflammatory cytokines, such as IL-4 is still largely unexplored in prostate cancer. In the present study, we investigated the effects of IL-4 on PBMC co-cultured with PCa cells. PBMC were co-culured with the PCa cell lines LNCaP and LNCaP-IL6+. To avoid cell-cell contact, cancer and immune cells were separated using cell culture inserts with a 0.4 μm pore size membrane. Cell growth was assessed using the [3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyl tetrazolium bromide] (MTT) assay. Cytokine levels were measured using a BD™Cytometric Bead Array. Cell viability of LNCaP cells decreased massively when cells were co-cultured with PBMC. Pre-incubation with IL-4 could partly rescue the observed effect of cell viability of LNCaP cells co-cultured with PBMC. In contrast, cell viability of the LNCaP-IL6+ cell line was not affected when co-cultured with PBMC. IL-4 counteracts the cytotoxic effects of PBMC on hormone-sensitive LNCaP cells and is involved in the immune escape and development of aggressive phenotypes of PCa.

Downregulation of lysine-specific demethylase 1 enhances the sensitivity of hormone-sensitive prostate cancer cells to androgen deprivation therapy.

Lysine-specific demethylase 1 (LSD1) plays an important role in androgen receptor (AR) signaling, and LSD1 levels are associated with prostate cancer (PCa) progression. The present study investigated the association between the downregulation of LSD1 and the proliferation and invasiveness of PCa cells, as well as the effect of LSD1 on the androgen deprivation therapy (ADT)-induced apoptosis of PCa cells. The effect of the inhibition of LSD1 combined with ADT on PCa cell apoptosis was characterized. Furthermore, the mechanisms underlying LSD1-mediated apoptosis induced by ADT in PCa cells were investigated. Downregulation of LSD1 impaired the proliferation and invasiveness of PCa cells. Moreover, downregulation of LSD1 enhanced the apoptosis of PCa cells induced by bicalutamide in vitro. Downregulation of LSD1 decreased PSA expression, increased caspase 3 and Bax expression, decreased Bcl-2 expression and consequently enhanced castration-induced PCa cell apoptosis in vivo. These findings indicated that downregulation of LSD1 could effectively enhance the efficacy of ADT for hormone- sensitive PCa, demonstrating that this could be a promising adjunctive therapy with ADT for this disease.

Identification of a Radiosensitivity Molecular Signature Induced by Enzalutamide in Hormone-sensitive and Hormone-resistant Prostate Cancer Cells

Prostate cancer (PCa) is the most common cancer amongst men. A novel androgen receptor (AR) antagonist, enzalutamide (ENZA) has recently been demonstrated to enhance the effect of radiation (XRT) by impairing the DNA damage repair process. This study aimed to identify a radiosensitive gene signature induced by ENZA in the PCa cells and to elucidate the biological pathways which influence this radiosensitivity. We treated LNCaP (AR-positive, hormone-sensitive PCa cells) and C4-2 (AR-positive, hormone-resistant PCa cells) cells with ENZA alone and in combination with androgen deprivation therapy (ADT) and XRT. Using one-way ANOVA on the gene expression profiling, we observed significantly differentially expressed (DE) genes in inflammation-and metabolism-related genes in hormone-sensitive and hormone-resistant PCa cell lines respectively. Survival analysis in both the TCGA PRAD and GSE25136 datasets suggested an association between the expression of these genes and time to recurrence. These results indicated that ENZA alone or in combination with ADT enhanced the effect of XRT through immune and inflammation-related pathways in LNCaP cells and metabolic-related pathways in C4-2 cells. Kaplan–Meier analysis and Cox proportional hazard models showed that low expression of all the candidate genes except for PTPRN2 were associated with tumor progression and recurrence in a PCa cohort.

Abstract 858: Enzalutamide versus abiraterone as a radiosensitizer in hormone-sensitive prostate cancer cells

Abstract Prostate cancer treatment is based on the estimated risk of recurrence in the U.S. Combined androgen deprivation therapy (ADT) with radiation therapy (XRT) is the standard of care for high-risk localized PCa. However, a large percentage of tumors are resistance to ADT due to continued AR signaling. Abiraterone (ABI), an androgen synthesis inhibitor, and Enzalutamide (ENZA), a potent AR antagonist, are new treatment options for metastatic castration resistance prostate cancer (mCRPC) patients. The aim of this study is to compare the efficacy of ENZA or ABI as a radiosensitizer in XRT therapy on PCa cells. Methods: The effect of ENZA or ABI alone or in combination with XRT was assessed on hormone-sensitive (LNCaP, PC3-AR-T877A) and insensitive (PC3, PC3-AR V7) PCa cells using cell viability (MTT) and also clonogenic assays in different scheduling regimens: A- drug 24 h before XRT, B-drug 2h before XRT, or C- XRT followed by 24h later drug. Results: We first determined the effect of ENZA or ABI on MTT assays in androgen-dependent (AD) and androgen-independent (AI) PCa cell lines. The results of MTT assay showed that ENZA inhibited the growth of the four different cell lines, LNCaP, PC3-T877A, PC3 and PC3 AR-V7 with IC50 values of 20, 22, 50, and 45μmol/L, respectively, after 24 hours of treatment. The same effect was observed on ABI treated cell lines. Radiosensitivity was not significantly increased in AD and AI PCa cell lines by ABI (DEF=1.00, in all cases) while there was a supra-additive dose enhancement factor (DEF= 1.75±0.08) for hormone-sensitive cells treated with ENZA (Table 1). Conclusion: Our data indicates that ENZA acts as a much stronger radiosensitizer compared to ABI through different probable mechanisms of radiosensitivity. Table 1. Dose Enhancement Factors calculations by scheduling ProtocolABCENZAABIENZAABIENZAABIADLNCaP1.35±0.021.05±0.011.75±0.081.001.30±0.051.00PC3-AR T877A1.30±0.031.001.65±0.011.001.35±0.061.05±0.02AIPC31.001.001.001.001.001.00PC3-AR V71.001.001.001.001.001.00 Citation Format: Maryam Ghashghaei, Thierry Muanza, Moulay Alaoui-Jamali, Miltiadis Paliouras, Mohammad Tamim Niazi. Enzalutamide versus abiraterone as a radiosensitizer in hormone-sensitive prostate cancer cells [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 858.

MP29-09 NOVEL SELECTIVE LYSINE SPECIFIC DEMETHYLASE 1 INHIBITORS EFFECTIVELY IMPAIR CASTRATION RESISTANT PROSTATE CANCER GROWTH

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology I1 Apr 2018MP29-09 NOVEL SELECTIVE LYSINE SPECIFIC DEMETHYLASE 1 INHIBITORS EFFECTIVELY IMPAIR CASTRATION RESISTANT PROSTATE CANCER GROWTH Toshiki Etani, Taku Naiki, Takashi Nagai, Keitaro Iida, Ryosuke Ando, Noriyasu Kawai, Keiichi Tozawa, Takayoshi Suzuki, and Takahiro Yasui Toshiki EtaniToshiki Etani More articles by this author , Taku NaikiTaku Naiki More articles by this author , Takashi NagaiTakashi Nagai More articles by this author , Keitaro IidaKeitaro Iida More articles by this author , Ryosuke AndoRyosuke Ando More articles by this author , Noriyasu KawaiNoriyasu Kawai More articles by this author , Keiichi TozawaKeiichi Tozawa More articles by this author , Takayoshi SuzukiTakayoshi Suzuki More articles by this author , and Takahiro YasuiTakahiro Yasui More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.929AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Lysine-specific demethylase 1 (LSD1), the first-identified histone demethylase, is a novel target for prostate cancer therapy. We examined the anticancer effects of NCL1 and NCD38, selective inhibitors of LSD1 (LSD1i) that were first discovered at our university. METHODS Various tests using LNCaP (a hormone-sensitive prostate cancer cell line) and PCai1 (a castration-resistant prostate cancer [CRPC] cell line established in our institute), PC3 and 22Rv1 (CRPC cell line) cells were used to confirm the anticancer effects of NCL1 and NCD38. Cell viability was assessed by performing a WST assay. Invasion and migration assay were also performed to assess the status of cell invasion and cell migration. For autophagy analysis, cells were incubated with or without chloroquine (CQ) in the presence or absence of LSD1i. Subsequently, transmission electron microscopy (TEM), fluorescent immunocytochemistry, and WST assay were performed with the treated and non-treated cells. A combination index analysis was performed to assess the effects of LSD1i and CQ. Finally, the effect on xenograft tumors in CRPC mice models was measured by subcutaneously injecting castrated nude mice with PCai1 cells. Mice were injected intraperitoneally with LSD1i or the vehicle, and their subsequent growth was recorded. RESULTS The WST assay revealed reduced number of viable cells after LSD1i treatment. A dose-dependent induction of apoptosis by LSD1i was noted using flow cytometry. LSD1i inhibited invasion and migration. Autophagosomes were observed in the LNCaP cells treated with NCL1. The expression level of LC3-II was significantly increased in the cells treated with LSD1i and CQ. Moreover, the combination of NCL1 and CQ significantly decreased cell growth in vitro; however, it had no synergistic effect in vivo. Xenograft tumor volume was reduced in the NCL1 and NCD38-treated mice compared to the controls; no adverse effects were observed. CONCLUSIONS Castration-resistant prostate cancer growth was effectively suppressed with NCL1 and NCD38 both, in vitro and in vivo, without any adverse events via apoptosis regulation and autophagy, indicating the potential use of LSD1 inhibitors as therapeutic agents for prostate cancer. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e369 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Toshiki Etani More articles by this author Taku Naiki More articles by this author Takashi Nagai More articles by this author Keitaro Iida More articles by this author Ryosuke Ando More articles by this author Noriyasu Kawai More articles by this author Keiichi Tozawa More articles by this author Takayoshi Suzuki More articles by this author Takahiro Yasui More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

MicroRNA-Dependent Regulation of IGF1R Gene Expression in Hormone-Sensitive and Hormone-Resistant Prostate Cancer Cells.

Using multiple parallel sequencing on Illumina platform, we identified eight microRNAs that showed significant opposite changes of gene expression in cells of the hormone-sensitive LNCaP prostate cancer cell line and in cells of the hormone-resistant DU-145 cell line, in comparison to the microRNA expression in the normal prostate tissue cells. We found that the insulin-like growth factor 1 receptor (IGF1R) gene is a target of five microRNAs whose expression is increased in LNCaP cells and reduced in DU-145 cells.

MP83-03 NOVEL SELECTIVE LYSINE SPECIFIC DEMETHYLASE 1 INHIBITORS EFFECTIVELY IMPAIR CASTRATION RESISTANT PROSTATE CANCER GROWTH

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology II1 Apr 2017MP83-03 NOVEL SELECTIVE LYSINE SPECIFIC DEMETHYLASE 1 INHIBITORS EFFECTIVELY IMPAIR CASTRATION RESISTANT PROSTATE CANCER GROWTH TOSHIKI ETANI, Taku Naiki, Takayoshi Suzuki, Takashi Nagai, Keitaro Iida, Ryosuke Ando, Noriyasu Kawai, Keiichi Tozawa, Tohru Mogami, and Takahiro Yasui TOSHIKI ETANITOSHIKI ETANI More articles by this author , Taku NaikiTaku Naiki More articles by this author , Takayoshi SuzukiTakayoshi Suzuki More articles by this author , Takashi NagaiTakashi Nagai More articles by this author , Keitaro IidaKeitaro Iida More articles by this author , Ryosuke AndoRyosuke Ando More articles by this author , Noriyasu KawaiNoriyasu Kawai More articles by this author , Keiichi TozawaKeiichi Tozawa More articles by this author , Tohru MogamiTohru Mogami More articles by this author , and Takahiro YasuiTakahiro Yasui More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.2571AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Lysine-specific demethylase 1 (LSD1), the first identified histone demethylase, is a novel target for prostate cancer therapy. Herein we examined the anticancer effects of NCL1 and NCD38, the selective inhibitors of LSD1 that were first discovered at our university. METHODS Various tests using LNCaP (a hormone-sensitive prostate cancer cell line) and PCai1 (a castration-resistant prostate cancer [CRPC] cell line established in our institute) cells were used to confirm the anticancer effects of NCL1 and NCD38. Cell viability was assessed by performing a WST assay in the presence of NCL1, NCD38, or a standard vehicle (control). Chromatin immunoprecipitation (ChIP) and PCR were used to confirm the methylation status. For autophagy analysis, LNCaP and PCai1 cells were incubated with or without CQ in the presence or absence of NCL1. Subsequently, transmission electron microscopy (TEM), fluorescent immunocytochemistry, and WST assay were performed with the treated and non-treated cells. A combination index analysis was performed to assess the effects of NCL1 and CQ. Lastly, the effect on xenograft tumors in CRPC mice models was measured by subcutaneously injecting castrated nude mice with PCai1 cells. Mice were injected intraperitoneally with NCL1, NCD38, CQ, or the vehicle, and subsequent growth was recorded. RESULTS WST assay revealed a reduction in the number of viable cells after NCL1 and NCD38 treatment. ChIP showed NCL1-induced H3K9me2 accumulation at the ELK4 and KLK2 promoters, whereas a dose-dependent induction of apoptosis by NCL1 was noted by flow cytometry. Autophagosomes were observed in LNCaP cells treated with NCL1. The expression level of LC3-II was significantly increased in cells treated with NCL1 and CQ. Furthermore, the combination of NCL1 and CQ significantly decreased cell growth in vitro, but had no synergistic effect in vivo. Xenograft tumor volume was reduced in the NCL1 and NCD38-treated mice when compared with the controls; no adverse effects were observed. CONCLUSIONS Castration resistant prostate cancer growth was effectively suppressed with NCL1 and NCD38 both in vitro and in vivo, without any adverse events, via regulation of apoptosis and autophagy; thus, indicating the potential use of LSD1 inhibitors as therapeutic agents for prostate cancer. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e1106-e1107 Advertisement Copyright & Permissions© 2017MetricsAuthor Information TOSHIKI ETANI More articles by this author Taku Naiki More articles by this author Takayoshi Suzuki More articles by this author Takashi Nagai More articles by this author Keitaro Iida More articles by this author Ryosuke Ando More articles by this author Noriyasu Kawai More articles by this author Keiichi Tozawa More articles by this author Tohru Mogami More articles by this author Takahiro Yasui More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

The combined effect of USP7 inhibitors and PARP inhibitors in hormone-sensitive and castration-resistant prostate cancer cells.

Purpose of the studyReduced levels of the tumor suppressor protein CCDC6 sensitize cancer cells to the treatment with PARP-inhibitors. The turnover of CCDC6 protein is regulated by the de-ubiquitinase USP7, which also controls the androgen receptor (AR) stability. Here, we correlated the expression levels of CCDC6 and USP7 proteins in primary prostate cancers (PC). Moreover, we tested the efficacy of the USP7 inhibitors, in combination with PARP-inhibitors as a novel therapeutic option in advanced prostate cancer.Experimental techniquesPC cells were exposed to USP7 inhibitor, P5091, together with cycloheximide, to investigate the turnover of the USP7 substrates, AR and CCDC6. As outcome of the AR downregulation, transcription targets of AR and its variant V7 were examined by qPCR. As a result of CCDC6 degradation, the induction of PARP inhibitors sensitivity was evaluated by analyzing PC cells viability and foci formation. We scored and correlated CCDC6 and USP7 expression levels in a prostate cancer tissue microarray (TMA).ResultsP5091 accelerated the degradation of AR and V7 isoform affecting PSA, UBE2C, CDC20 transcription and PC cells proliferation. Moreover, P5091 accelerated the degradation of CCDC6 sensitizing the cells to PARP-inhibitors, that acted sinergistically with genotoxic agents. The immunohistochemical analysis of both CCDC6 and USP7 proteins exhibited significant correlation for the intensity of staining (p ≤ 0.05).Data interpretationThus, CCDC6 and USP7 represent predictive markers for the combined treatment of the USP7-inhibitors and PARP-inhibitors in advanced prostate cancer.