Prostate Cell Growth Research Articles

Loss of Par3 promotes prostatic tumorigenesis by enhancing cell growth and changing cell division modes.

Although cell polarity plays an important role in epithelial tumorigenesis, the consequence of polarity protein loss in prostatic tumorigenesis and the underlying mechanisms remain unclear. Using conditional knockout mouse models, we found in the current study that loss of polarity protein Par3 increases prostatic epithelial cell growth, elevates symmetrical cell divisions in basal cells, and randomizes spindle orientation in luminal cells, causing the development of high-grade prostatic intraepithelial neoplasia (PIN). Mechanistically, loss of Par3 dissociates the Par3/merlin/Lats1 complex, consequently inhibiting phosphorylation of Lats1 to attenuate the Hippo pathway. Furthermore, attenuated Hippo pathway enhances nuclear translocation of Yes-associated protein (YAP), which promotes cell proliferation and symmetrical cell divisions through transcriptional activation of Ki-67 and Sox2. In addition, Lats1 dephosphorylation impairs its interaction with G protein signaling modulator 2 (GPSM2, which is also known as LGN) that causes randomization of spindle orientation in luminal cells. Interestingly, co-deletion of Par3 and Lats1 for complete blockade of the Hippo pathway in mice results in prostate tumor initiation, whereas co-deletion of Par3 and YAP for disrupting YAP nuclear translocation reverses the phenotypes to a relatively normal state. Therefore, our findings highlight combination of Par3 loss and blockade of the Hippo pathway as a novel mechanism for prostatic tumorigenesis.

Observations on spontaneous tumor formation in mice overexpressing mitotic kinesin Kif14

The KIF14 locus is gained and overexpressed in various malignancies, with prognostic relevance. Its protein product, a mitotic kinesin, accelerates growth of normal mammary epithelial cells in vitro and retinoblastoma tumours in a mouse model, while KIF14 knockdown blocks growth of brain, liver, ovarian, breast, prostate, and other tumour cells and xenografts. However, the tumour-initiating effects of Kif14 overexpression have not been studied. We aged a cohort of Kif14-overexpressing transgenic mice and wild-type littermates and documented survival, cause of death, and tumour burden. The Kif14 transgene was expressed in all tissues examined, and was associated with increased proliferation marker expression. Neither mouse weights nor overall survival differed between genotypes. However, Kif14 transgenic mice showed a higher incidence of fatal lymphomas (73 vs. 50%, p = 0.03, Fisher’s exact test), primarily follicular and diffuse B-cell lymphomas. Non-tumour findings included a bilateral ballooning degeneration of lens in 12% of Kif14 transgenic mice but no wild-type mice (p = 0.02). Overall, this work reveals a novel association of Kif14 overexpression with lymphoma but suggests that Kif14 does not have as prominent a role in initiating cancer in other cell types as it does in accelerating tumour development in response to other oncogenic insults.

Protein Palmitoylation Plays an Important Role in Trichomonas vaginalis Adherence

The flagellated protozoan parasite Trichomonas vaginalis is the etiologic agent of trichomoniasis, the most common non-viral sexually transmitted infection worldwide. As an obligate extracellular pathogen, adherence to epithelial cells is critical for parasite survival within the human host and a better understanding of this process is a prerequisite for the development of therapies to combat infection. In this sense, recent work has shown S-acylation as a key modification that regulates pathogenesis in different protozoan parasites. However, there are no reports indicating whether this post-translational modification is a mechanism operating in T. vaginalis In order to study the extent and function of S-acylation in T. vaginalis biology, we undertook a proteomic study to profile the full scope of S-acylated proteins in this parasite and reported the identification of 363 proteins involved in a variety of biological processes such as protein transport, pathogenesis related and signaling, among others. Importantly, treatment of parasites with the palmitoylation inhibitor 2-bromopalmitate causes a significant decrease in parasite: parasite aggregation as well as adherence to host cells suggesting that palmitoylation could be modifying proteins that are key regulators of Trichomonas vaginalis pathogenesis.

Blebbistatin modulates prostatic cell growth and contrapctility through myosin II signaling.

To investigate the effect of blebbistatin (BLEB, a selective myosin inhibitor) on regulating contractility and growth of prostate cells and to provide insight into possible mechanisms associated with these actions. BLEB was incubated with cell lines of BPH-1 and WPMY-1, and intraprostatically injected into rats. Cell growth was determined by flow cytometry, and in vitro organ bath studies were performed to explore muscle contractility. Smooth muscle (SM) myosin isoform (SM1/2, SM-A/B, and LC17a/b) expression was determined via competitive reverse transcriptase PCR. SM myosin heavy chain (MHC), non-muscle (NM) MHC isoforms (NMMHC-A and NMMHC-B), and proteins related to cell apoptosis were further analyzed via Western blotting. Masson's trichrome staining was applied to tissue sections. BLEB could dose-dependently trigger apoptosis and retard the growth of BPH-1 and WPMY-1. Consistent with in vitro effect, administration of BLEB to the prostate could decrease rat prostatic epithelial and SM cells via increased apoptosis. Western blotting confirmed the effects of BLEB on inducing apoptosis through a mechanism involving MLC20 dephosphorylation with down-regulation of Bcl-2 and up-regulation of BAX and cleaved caspase 3. Meanwhile, NMMHC-A and NMMHC-B, the downstream proteins of MLC20, were found significantly attenuated in BPH-1 and WPMY-1 cells, as well as rat prostate tissues. Additionally, BLEB decreased SM cell number and SM MHC expression, along with attenuated phenylephrine-induced contraction and altered prostate SMM isoform composition with up-regulation of SM-B and down-regulation of LC17a, favoring a faster contraction. Our novel data demonstrate BLEB regulated myosin expression and functional activity. The mechanism involved MLC20 dephosphorylation and altered SMM isoform composition.

Abstract A058: Integrative prostate cancer tissue proteomics dissects clear and distinct proteomes for human prostate cancer and benign prostatic hyperplasia

Abstract Current approaches in proteomics techniques and MS systems have revived the quest for novel biomarkers in prostate cancer (PCa) boosting the molecular characterization of the disease. To reveal fundamental differences between benign and malignant growth of prostate cells, we combined a new protein extraction procedure that disrupts the crosslinked proteins from the previously formaldehyde-fixed, paraffin-embedded tissue samples with in-depth proteomic analysis (ESI-MS/MS). Human PCa and benign prostatic hyperplasia (BPH) tissue samples were obtained using phase-transfer surfactant-aided extraction/tryptic digestion of formalin-fixed and paraffin-embedded sections mounted on microscope slides. Data analysis was based on label-free spectral counting, identifying with a minimum of two peptides, 1331 and 1239 proteins in PCa and BPH tissue proteomes, respectively. 71 proteins were exclusively present in PCa samples, while 122 proteins where exclusively present in BPH samples. In order to prioritize candidate markers for PCa, we compared protein expression based on normalized spectral counts between tissue samples. We set as cut-offs proteins that were found with a minimum of three peptides within the PCa and BPH proteomes. This filter resulted in the selection of two clusters of 11 and 16 proteins, respectively. The data sets highlighted distinct proteins that were previously studied in the context of prostate cancer progression, including SSBP1, GDF15, NDRG1, C4A, and APOE for PCa and DUSP3, MME, SRI, and DSG1 for BPH, thus providing further confirmation for the robustness of our quantification method. We next subjected our candidate list to bioinformatics analysis (Oncomine). Accordingly, the 5 proteins mentioned for PCa were significantly upregulated (fold change >1.5, P≤0.05) in prostate adenocarcinoma vs. normal prostate gland. Whole-exome analysis (cBioportal) revealed amplification as the most frequent genetic alteration and RNASeq data also confirmed a significant upregulation for these proteins (P≤0.05). Strikingly, proteins associated with BPH were significantly downregulated (fold change >1.5, P≤0.05) across the same comparison and RNA-seq data also confirmed a significant downregulation for these proteins (P≤0.05). This report showcases significant and extensive differences in protein expression patterns between BPH and prostate carcinoma. Proteome analysis of prostate tissues should help to disclose the molecular mechanisms underlying prostate malignant growth, resulting in new sets of biomarkers for diagnostic, prognostic, and therapeutic use. Citation Format: Sofia Lage Vickers, Juan Antonio Bizzotto, Alejandra Paez, Javier Cotignola, Carlos Scorticati, Osvaldo Mazza, Pia Valacco, Geraldine Gueron, Elba Vazquez. Integrative prostate cancer tissue proteomics dissects clear and distinct proteomes for human prostate cancer and benign prostatic hyperplasia [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A058.

Abstract A078: Towards understanding noncanonical phosphatidylinositol kinases in the maintenance of prostate metabolism

Abstract An estimated 1 in 7 men will develop prostate cancer (PCa) with many progressing to advanced castrate-resistant disease. Unlike other tissue types, normal prostate cell growth and development is heavily dependent on the androgen receptor (AR) signaling pathway. While the introduction of novel AR antagonists for clinical treatment has improved outcomes, most castration-resistant prostate cancer (CRPC) patients ultimately develop resistance to these therapies. A need exists to better understand the mechanisms that control the transition of prostate cells from a hormone-dependent to castrate-resistant state. Androgens strongly influence the metabolic state of PCa cells to favor sustained cellular growth. We hypothesize there are effectors working in conjunction with AR to coordinate alterations to androgen-dependent metabolism that are linchpins in the orchestration of the transition to CRPC. Leading candidates are members of phosphoinositol (PI) pathways, which have a high frequency of alteration in PCa (i.e phosphoinositide 3-kinase (PI3K)). Herein we explore a family of poorly understood lipid kinases called the type II phosphatidylinositol-5-phosphate 4-kinases (PI5P4Ks) and predict them to be critical regulators of cancer cell survival. PI5P4Ks are druggable targets that act by phosphorylating the lipid phosphatidylinositol-5-phosphate (PI 5-P) at the 4 position of the inositol ring to generate phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2; PIP2). We implicate the three PI5P4K isoforms (PI5P4Kα, PI5P4Kβ, and PI5P4Kγ) encoded by the genes PIP4K2A, B, and C, to be important regulators of cancer metabolism that play a role in the maintenance of prostate biology and oncogenesis. Analysis of transcript data revealed expression of PIP4K2A, B, and C in primary PCa patient samples, which was correlated with an AR activation gene signature and hotspot tumor suppressor deletion. As well, isoform expression was assessed for differential expression in relation to an integrated neuroendocrine prostate cancer mRNA score (TCGA; n=333). PI5P4Kα and PI5P4Kβ protein was detected in primary and advanced prostate cancer using optimized antibodies of patient tissue TMAs (n= 72). Using in vitro LNCaP cell models, siRNA knockdown systems were tested to evaluate the molecular consequence of targeting PIP4K2A and PIP4K2B in androgen-dependent systems. Stable knockdown using fluorescently labeled lentiviral shRNA constructs significantly reduced proliferation of shPIP4K2 treated cells. As well, we have produced a prostate-specific PI5P4K knockout mouse model by expressing probasin-driven Cre in a homozygous 129/SvEv Pip4k2aflx/flx murine strain. Finally, implementation of a discovery-based metabolomic platform (Metabolon HD4) was used to profile the overall shift in metabolite species that results from downregulating the expression of PIP4K2A in androgen-dependent cell models. In summary, we have developed novel insights into the role of a family of noncanonical PI kinases in prostate biology. There are a growing number of PI3K/AKT inhibitors being tested in combination with androgen deprivation therapy in clinical trials, but there is still almost nothing known about the potential crosstalk of the greater PI kinase network. These data convincingly implicate a fundamental role for PI5P4Ks in PCa androgen signaling and metabolism, as well as lay the foundation of phenotypic understanding of what PI5P4K is responsible for in the prostate. Citation Format: Joanna Triscott, Matteo Benelli, Verena Sailer, Davide Prandi, Brooke Emerling, Francesca Demichelis, Lewis Cantley, Mark A. Rubin. Towards understanding noncanonical phosphatidylinositol kinases in the maintenance of prostate metabolism [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A078.

Biological role of cytoplasmic transducin beta like related protein 1-induced proliferation and tumorigenicity in prostate cancer

Androgen receptor mediated transcription and function in prostate cancer and critical for prostate cell growth and gland differentiation. Transducin beta like related protein 1 (TBLR1) primarily localizes in the nucleus in benign prostate tissue and is significantly reduced in prostate cancer. The objective of this study is investigated the role of cytoplasmic TBLR1in prostate cancer. Real-time PCR, Western blotting and immunocytochemistry were used to evaluate Transducin beta like related protein 1 (TBLR1) expression in prostate cancer cell lines and normal prostate cells, tissue samples and adjacent nontumor tissues, and in 142 paraffin-embedded specimens. Immunohistochemical analysis revealed high expression of TBLR1 in 89 of 214 paraffin-embedded archival prostate cancer. The expression level of TBLR1 was significantly increased in prostate cancer both in vivo and in vitro and correlated with clinical stage (P<0.05) and metastasis (P<0.001). Over all the study showed that the TBLR1 plays a key role in the development of prostate cancer cells and TBLR1 may be prognostic marker and a potential therapeutic target in the treatment human prostate cancer.

Androgenic modulation of AR-Vs.

The importance of androgen receptor variants (AR-Vs) is recognized in prostate cancer. AR-Vs have been the focus of many studies. Expression of AR-Vs has been proposed as a biomarker for resistance to androgen deprivation therapy for metastatic disease. Herein, we show dynamic changes in AR-Vs expression in response to androgen modulation. The C4-2B cell line was exposed to low (10-13 M) and high (10-8 M) androgen (dihydrotestosterone, DHT) levels, with or without flutamide. mRNA and protein expression levels were assessed by qPCR and immunohistochemistry, respectively. We demonstrated that high levels of DHT downregulate AR-FL and AR-Vs. Even though AR-Vs did not present ligand-binding domain, thus were not capable of binding to DHT, they present dynamic changes under androgen treatment. Treatment with flutamide alone or in association with low levels of DHT stimulates growth of prostatic cells. Importantly, we provide evidence that AR-Vs respond differently to androgenic modulation. These findings have implications for a better understanding of the role of AR-Vs in prostate carcinogenesis.

Abstract 5900: Inhibition of CYP3A5 down-regulates cancer drug resistance genes and induces synthetic lethality in prostate cancer cells

Abstract Background and objective: Androgen deprivation therapy (ADT) is among the most effective of all cancer therapies for prostate cancer; however, resistance to therapy leading to castrate resistant prostate cancer (CRPC) is inevitable in most patients. Recently we demonstrated that CYP3A5 expressed in prostate cancer cells facilitates the nuclear translocation of AR, directly impacting the transcriptional regulation of AR-dependent genes promoting prostate cancer cell growth. Our preliminary data show that CYP3A5 is a part of the HSP90-AR complex, known to regulate AR nuclear localization and activation. Additionally, we observed that CYP3A5 inhibition also blocks growth of prostate cancer cells lacking AR ligand binding site (22RV1). This observation suggested presence of an alternative mechanism of regulation of prostate cell growth by CYP3A5, independent of its AR activation. We hypothesize that CYP3A5 inhibition can also sensitize prostate cancer cells to therapy by downregulating expression of drug resistance genes independent of its AR regulation. Our aim is to decipher the role of CYP3A5 in therapeutic resistance, by screening for CYP3A5 regulated cancer drug resistance genes and to further understand the underlying mechanism of this regulation. Method: We used cancer drug resistance PCR array after CYP3A5 siRNA knockdown to identify CYP3A5 regulated genes involved in therapeutic resistance. Further we evaluated the role of CYP3A5 in inducing synthetic lethality in combination with DNA damage repair inhibiting drugs by imagining the γ-H2AX foci. Results: CYP3A5 siRNA knockdown downregulates 11 genes involved in cancer drug resistance: TOP2A, BRCA1/2, CCNE1, CDK2/4, DHFR, MVP, MYC, RARB and HPRT1 with a p≤0.005 value. Of the list of downregulated genes, the three genes TOP2A, BRCA2 and BRCA1 have the most fold change and their loss is known to impair homologous recombination (HR), leading to synthetic lethality. HR defects in CRPC, sensitizes these tumors to PARP and topoisomerase inhibitors. We tested the effect of CYP3A5 inhibition in inducing HR defects by analyzing γ-H2AX foci counts in combination with topoisomerase inhibitor etoposide. Increased number of γ-H2AX foci suggest that CYP3A5 siRNA may induce synthetic lethality in prostate cancer cells when used in combination with DNA damage repair inhibiting drugs by impairing HR and downregulating TOP2A and BRCA1/2 expression. Conclusions: CYP3A5 inhibition downregulates DNA damage repair genes (TOP2A and BRCA1/2) and cell cycle progression genes (CCNE1, CDK 2/4) and impairs HR. CYP3A5 inhibition can sensitize CRPC to PARP and topoisomerase inhibitors as it induces synthetic lethality by downregulating TOP2A and BRCA1/2 expression. CYP3A5 inhibition can be potentially exploited therapeutically in combination with PARP inhibitors as it functionally impairs HR and causes synthetic lethality. Citation Format: Priyatham Gorjala, Ranjana Mitra, Oscar Goodman. Inhibition of CYP3A5 down-regulates cancer drug resistance genes and induces synthetic lethality in 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 5900.

Effect of ASP6432, a Novel Type 1 Lysophosphatidic Acid Receptor Antagonist, on Urethral Function and Prostate Cell Proliferation.

Current pharmacotherapies for lower urinary tract symptoms associated with benign prostate hyperplasia (LUTS/BPH) are in need of improvement. Lysophosphatidic acid (LPA) is a phospholipid with various biologic functions. However, its exact role in the lower urinary tract and its target receptor subtype have not been fully elucidated. We investigated the role of LPA and the type 1 LPA receptor (LPA1) in urethral/prostatic contractile function and prostate cell proliferation by pharmacologically characterizing ASP6432 (potassium 1-(2-{[3,5-dimethoxy-4-methyl-N-(3-phenylpropyl)benzamido]methyl}-1,3-thiazole-4-carbonyl)-3-ethyl-2,2-dioxo-2λ6-diazathian-1-ide), a novel LPA1 antagonist. ASP6432 exhibited potent and selective antagonistic activity against LPA1 in cells expressing LPA receptor subtypes. In isolated rat tissue strips and anesthetized rats, ASP6432 concentration-/dose-dependently inhibited LPA-induced urethra and prostate contractions. In addition, in anesthetized rats, ASP6432 maximally decreased the urethral perfusion pressure (UPP) in the absence of exogenous LPA stimulation by 43% from baseline, whereas tamsulosin, an α1-adrenoceptor antagonist, reduced UPP by 22%. Further, in human prostate stromal cells, ASP6432 significantly and concentration-dependently suppressed LPA-induced bromodeoxyuridine incorporation. These results demonstrate a pivotal role for LPA and LPA1 in the regulation of urethral tonus and prostate cell proliferation. The potent urethral relaxation and inhibition of prostatic stromal cell growth indicate the potential of ASP6432 as a novel therapeutic agent for LUTS/BPH.

Ophiopogonin D', a Natural Product From Radix Ophiopogonis, Induces in Vitro and in Vivo RIPK1-Dependent and Caspase-Independent Apoptotic Death in Androgen-Independent Human Prostate Cancer Cells.

Objective: The purpose of this study was to evaluate the anticancer effects of Ophiopogonin D′ (OPD′, a natural product extracted from a traditional Chinese medicine (Radix Ophiopogonis) against androgen-independent prostate cancer cells and to explore the underlying molecular mechanism(s) of action.Methods: The CCK-8 assay was used to assess the viability of prostate cancer cells. The cell morphology was examined by an ultrastructural analysis via transmission electron microscopy. Cells in apoptosis (early and late stages) were detected using an Annexin V-FITC/propidium iodide kit with a FACSCaliber flow cytometer. JC-1, a cationic lipophilic probe, was employed to measure the mitochondrial membrane potential (MMP) of PC3 cells. Changes in the protein expression of RIPK1, C-RIPK1, caspase 8, cleaved-caspase 8, Bim, Bid, caspase 10, and cleaved-caspase 10 were evaluated by Western blotting. The mRNA expression of Bim was examined by quantitative real-time reverse transcription polymerase chain reaction. Z-VAD-FMK (a caspase inhibitor) and necrostatin-1 (a specific inhibitor of RIPK1) were utilized to determine whether the cell death was mediated by RIPK1 or caspases. PC3 and DU145 xenograft models in BALB/c nude mice were used to evaluate the anticancer activity of OPD′ in vivo.Results: OPD′ was shown to exert potent anti-tumor activity against PC3 cells. It induced apoptosis via a RIPK1-related pathway, increased the protein expression levels of RIPK1 and Bim, and decreased the levels of cleaved-RIPK1, caspase 8, cleaved-caspase 8, Bid, caspase 10, and cleaved-caspase 10. OPD′ also increased the mRNA expression of Bim. The protein expression of Bim was decreased when cells were pre-treated with necrostatin-1. Treatment with OPD′ inhibited the growth of PC3 and DU145 xenograft tumors in BALB/c nude mice.Conclusion: OPD′ significantly inhibited the in vitro and in vivo growth of prostate cells via RIPK1, suggesting that OPD′ may be developed as a potential anti-prostate cancer agent.

The Inhibitory Effect of Roscovitine on Prostate Cancer Cell Proliferation and Androgen Receptor Phosphorylation

It has been reported that ccyclin-dependent kinases (CDKs) play important roles in modulating androgen receptor (AR) function and prostate cancer cell growth. Roscovitine is a CDK inhibitor and had been applied in clinical trials in some types of cancer. The aim of this study is to investigate the role of AR phosphorylation in Roscovitine-induced inhibition of prostate cancer cell growth. The results indicated that Roscovitine treatment significantly inhibited the growth of both prostate cancer cell lines and normal prostatic cells. The result of xenografted tumor growth also support the finding. The induces of AR activation, including subcellular localization, promoter activity, and prostate specific antigen (PSA) production/secretion, were all inhibited after Roscovitine treatment. Besides, there was no additive effect of Roscovitine with AR inhibitor (Bicalutamide) treatment on LNCaP cell growth. It suggests that Roscovitine and AR inhibitor might share similar mechanism on inhibiting prostate cancer cell growth. In summary, AR might be one of the pharmaceutical targets of Roscovitine on inhibiting prostate cancer cells and can be used in future basic research as well as clinical application.

A phase Ib open-label, dose escalation and expansion study to investigate the safety, pharmacokinetics, pharmacodynamics and clinical activity of GSK525762 in combination with abiraterone or enzalutamide in metastatic castrate-resistant prostate cancer.

TPS391 Background: Metastatic castrate-resistant prostate cancer (mCRPC) remains an incurable illness as resistance develops after androgen deprivation therapy (ADT) and/or androgen receptor (AR) axis targeted therapies. The bromodomain (BRD) and extraterminal (BET) proteins are critical for transcription. Preclinically, one of these proteins, BRD4, acts in complex with AR to mediate androgen signaling that leads to prostate cell growth and proliferation. GSK525762 is an oral pan-BET inhibitor that suppresses BET dependent activated AR-driven transcription. Combined with androgen production or receptor targeted agents like abiraterone or enzalutamide, GSK525762 may enhance efficacy of or overcome resistance to either agent. Methods: This is a Phase Ib open-label, dose-escalation study to evaluate the safety and efficacy of oral administration of GSK525762 in combination with either abiraterone plus prednisone (Arm A) or enzalutamide (Arm B) in mCRPC patients whose disease has progressed on prior abiraterone or enzalutamide. Patients must have documented prostate cancer progression as assessed by rising PSA or radiographic progression of soft tissue by PCWG3-modified RECIST 1.1 criteria or bone metastasis. Dose escalation is designed to identify safe doses to move into dose expansion. Dose expansion will explore safety and efficacy in patients who failed in first line (L2 population) or after multiple lines of prior therapy (LX population). Primary objectives include defining the safety, tolerability and clinical activity of GSK525762 when combined with products in Arm A or Arm B. Primary clinical activity endpoint is defined as the response rate of subjects achieving a 50% or more reduction from baseline of PSA at 12 weeks or thereafter. Dose escalation will employ a modified Toxicity Probability Interval (mTPI) design. Dose expansion will use a Bayesian adaptive design, which will calculate posterior probability that utility of the dose is clinically significant at interim futility analysis for each dose level. Funding: GSK Clinical trial information: NCT03150056.

Sarcosine influences apoptosis and growth of prostate cells via cell-type specific regulation of distinct sets of genes.

Sarcosine is a widely discussed oncometabolite of prostate cells. Although several reports described connections between sarcosine and various phenotypic changes of prostate cancer (PCa) cells, there is still a lack of insights on the complex phenomena of its effects on gene expression patterns, particularly in non-malignant and non-metastatic cells. To shed more light on this phenomenon, we performed parallel microarray profiling of RNA isolated from non-malignant (PNT1A), malignant (22Rv1), and metastatic (PC-3) prostate cell lines treated with sarcosine. Microarray results were experimentally verified using semi-quantitative-RT-PCR, clonogenic assay, through testing of the susceptibility of cells pre-incubated with sarcosine to anticancer agents with different modes of actions (inhibitors of topoisomerase II, DNA cross-linking agent, antimicrotubule agent and inhibitor of histone deacetylases) and by evaluation of activation of executioner caspases 3/7. We identified that irrespective of the cell type, sarcosine stimulates up-regulation of distinct sets of genes involved in cell cycle and mitosis, while down-regulates expression of genes driving apoptosis. Moreover, it was found that in all cell types, sarcosine had pronounced stimulatory effects on clonogenicity. Except of an inhibitor of histone deacetylase valproic acid, efficiency of all agents was significantly (P < 0.05) decreased in sarcosine pre-incubated cells. Our comparative study brings evidence that sarcosine affects not only metastatic PCa cells, but also their malignant and non-malignant counterparts and induces very similar changes in cells behavior, but via distinct cell-type specific targets.

Epigallocatechin-3-gallate reduces the proliferation of benign prostatic hyperplasia cells via regulation of focal adhesions

AimsBenign prostatic hyperplasia (BPH) is the most common urological disease that is characterized by the excessive growth of prostatic epithelial and stromal cells. Pharmacological therapy for BPH has limited use due to the many side effects so there is a need for new agents including natural compounds such as epigallocatechin-3-gallate (EGCG). This study was undertaken to assess the role of EGCG, suppressing the formation of BPH by reducing inflammation and oxidative stress, in cytoskeleton organization and ECM interactions via focal adhesions. Main methodsWe performed MTT assay to investigate cell viability of BPH-1 cells, wound healing assay to examine cell migration, immunofluorescence assay for F-actin organization and paxillin distribution and finally immunoblotting to investigate focal adhesion protein levels in the presence and absence of EGCG. Key findingsWe found that EGCG inhibits cell proliferation at the concentration of 89.12μM, 21.2μM and 2.39μM for 24, 48 and 72h, respectively as well as inhibitory effects of EGCG on BPH-1 cell migration were observed in a wound healing assay. Furthermore, it was determined by immunofluorescence labeling that EGCG disrupts F-actin organization and reduces paxillin distribution. Additionally, EGCG decreases the activation of FAK (Focal Adhesion Kinase) and the levels of paxillin, RhoA (Ras homolog gene family, member A), Cdc42 (cell division cycle 42) and PAK1 (p21 protein-activated kinase 1) in a dose-dependent manner. SignificanceFor the first time, by this study, we found evidence that BPH-1 cell proliferation could be inhibited with EGCG through the disruption of cytoskeleton organization and ECM interactions. Consequently, EGCG might be useful in the prevention and treatment of diseases characterized by excessive cell proliferation such as BPH.

Anticarcinogenic Effect of Spices Due to Phenolic and Flavonoid Compounds-In Vitro Evaluation on Prostate Cells.

This study shows the effects of spices, and their phenolic and flavonoid compounds, on prostate cell lines (PNT1A, 22RV1 and PC3). The results of an MTT assay on extracts from eight spices revealed the strongest inhibitory effects were from black pepper and caraway seed extracts. The strongest inhibitory effect on prostatic cells was observed after the application of extracts of spices in concentration of 12.5 mg·mL−1. An LC/MS analysis identified that the most abundant phenolic and flavonoid compounds in black pepper are 3,4-dihydroxybenzaldehyde and naringenin chalcone, while the most abundant phenolic and flavonoid compounds in caraway seeds are neochlorogenic acid and apigenin. Using an MTT assay for the phenolic and flavonoid compounds from spices, we identified the IC50 value of ~1 mmol·L−1 PNT1A. The scratch test demonstrated that the most potent inhibitory effect on PNT1A, 22RV1 and PC3 cells is from the naringenin chalcone contained in black pepper. From the spectrum of compounds assessed, the naringenin chalcone contained in black pepper was identified as the most potent inhibitor of the growth of prostate cells.

AB053. Labeling of prostate tumor-specific replication-selective oncolytic adenoviruses with radioactive 125I: inhibitory effects on prostate cancer cell

BackgroundThe authors established a 125I-labeled replication-selective oncolytic adenovirus and human telomerase reverse transcriptase/prostate-specific antigen (125I-RSOAds-hTERT/PSA) oncolytic adenovirus marker and investigated the effects of different labeling conditions. This study also explored the possible mechanism whereby 125I-RSOAds-hTERT/PSA inhibited the proliferation of prostate cancer cells.MethodsN-bromosuccinimide (NBS) was used as an oxidant for 125I labeling, and various concentrations of oncolytic viruses and NBS were prepared to determine the optimal conditions for labeling. The effects of the amount of 125I, reaction time, pH, and reaction volume on the labeling rate of the 125I-RSOAds-hTERT/PSA oncolytic adenovirus marker were measured. Radioactive oncolytic adenoviruses were isolated and purified by column chromatography; the radiochemical purities of the 125I-RSOAds-hTERT/PSA marker at different times were detected by paper chromatography. After the addition of radioactive iodine-labeled prostate cancer-specific oncolytic adenoviruses to prostate cancer cells, changes in the inhibitory rate were measured by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assays.ResultsThe radiochemical purity of the 125I-RSOAds-hTERT/PSA marker was >95%, and the marker was stable (93–94%) after storage at 4 °C for 7 days. The optimal conditions were 0.5 µL of 125I (about 0.2 mCi, 7.4 MBq), 25 µg of NBS, 100 µL of 8×109 viral protein (VP)/mL 125I-RSOAds-hTERT/PSA virus solution, 3 min of reaction time, pH 7.5, and 120 µL PBS. Radioactive iodine-labeled prostate cancer-specific oncolytic adenoviruses inhibited the proliferation of prostate cancer cells significantly.ConclusionsRadioactive 125I labeling of the hTERT/PSA dual-regulated prostate cancer-specific oncolytic adenovirus is feasible, and the radiochemical purity of the marker was stable under defined conditions. Radioactive iodine-labeled prostate cancer-specific replication-selective oncolytic adenoviruses significantly inhibited prostate cell growth.

Abstract 1584: Wnt/beta-catenin and Foxa2 axis activates AR signaling in castration resistant prostate cancer

Abstract Background: Prostate cancer (PCa) is the leading cancer among men in the world. Androgen deprivation therapy is a common treatment to cease prostate growth. However, androgen deprivation eventually fails and PCa relapses, evolving into castration resistant prostate cancer (CRPCa). It has been established that androgen receptor (AR) continues to be the central player in mediating CRPCa. However, the mechanism by which the AR signaling remains active is unclear. We have reported that activation of Wnt/beta-catenin signaling pathway promotes castrate-resistant progression. Our study has also shown that activation of the Wnt/beta-catenin pathway induces the expression of Foxa2, a fork-head transcription factor that is expressed in embryonic prostate &amp; advanced stage PCa. Here, we study the Wnt pathway and the role of Foxa2 in retaining active AR signaling in advanced PCa in the absence of androgens. TRAMP mice, SV40 T-antigen transgenic mouse models for PCa, were used. It has been recognized that in this model, T-antigen, which is driven by androgen-responsive probasin promoter, remains expressed following androgen ablation. In this study, we identified a mechanism that sustains an active AR signaling in CRPCa. Methods: To study whether the Wnt/beta-catenin pathway is active in advanced stage PCa, we crossed the TRAMP mice with GFP-Wnt reporter mice. Some of the TRAMP/Wnt-reporter mice were castrated to study how androgen deprivation affects Wnt/beta-Catenin signaling and promotes PCa progression. Prostates were analyzed using IHC and IF staining. We also established a prostate epithelial cell line that stably expresses Foxa2. Cell proliferation was examined with IncuCyte Zoom software. The expression of AR target genes was assessed by using quantitative RT-PCR and Western blotting. ChIP was conducted to assess the recruitment of transcriptional machinery to the promoters of AR-regulated genes. Results: Utilizing the TRAMP/Wnt mouse model, we show that Wnt/beta-Catenin signaling is active in advanced stage PCa. Additionally, our data showed that Foxa2, the downstream target of Wnt/beta-Catenin signaling, drives the expression of androgen-responsive genes, regardless of androgen deprivation. Furthermore, cell proliferation assays revealed that the expression of Foxa2 enabled an androgen-independent growth of prostatic cells. Using this model system, we further explored the mechanism that activates the transcription of AR-responsive genes in the absence of androgens. Our data indicated that Foxa2 is capable of recruiting H3k27Ac to AR-responsive promoters. Conclusion: Together, these data indicate that Wnt signaling is activated in advanced stage PCa, driving Foxa2 expression, and promoting AR signaling to persist. This represents a novel mechanism that enables advanced PCa to retain AR signaling pathway following androgen ablation. Targeting both Wnt signaling and Foxa2 may pose as novel therapeutics for CRPCa. Citation Format: Zachary M. Connelly, Shu Yang, Jiahe Li, Robert Matusik, Xiuping Yu. Wnt/beta-catenin and Foxa2 axis activates AR signaling in castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1584. doi:10.1158/1538-7445.AM2017-1584

Mitochondrion-associated protein peroxiredoxin 3 promotes benign prostatic hyperplasia through autophagy suppression and pyroptosis activation.

Benign prostatic hyperplasia (BPH) is one of the most common diseases in the senior men and age plays an important role in the initiation and development of BPH. Mammalian cells primarily use the autophagy-lysosome system to degrade misfolded/aggregated proteins and dysfunctional organelles such as mitochondria and suppress pyroptosis, a type of cell death that stimulates inflammatory responses and growth of other cells around. Peroxiredoxin 3 (PRDX3) is the only mitochondrion-associated member of peroxiredoxin family enzymes that exert their protective antioxidant role in cells through their peroxidase activity. We hypothesized that PRDX3 may inhibit autophagy to activate pyroptosis to induce growth of prostatic epithelial cells. Here we show that PRDX3 maintained the integrity of mitochondria and its depletion led to an enhancement of oxidative stresses. PRDX3-associated and PRDX3-free mitochondria co-existed in the same cells. PRDX3 expressed at higher levels in prostatic epithelial cells in prostate tissues from BPH patients and BPH-representative cell line than in prostate tissues from healthy donors and a cell line representing normal epithelial cells. PRDX3 suppressed autophagy flux and activated pyroptosis to induce inflammatory responses and stimulate the over-growth of prostate tissues. Therefore, higher levels of PDRX3 in prostatic epithelial cells may promote the initiation and development of BPH through autophagy inhibition and pyroptosis activation.

Is vitamin D associated with testosterone in benign prostate hyperplasia?

Introduction. Benign prostate hyperplasia (BPH) affects about 50% of male population between 51-60 years and almost 90% of 81-90 aged males. It is considered that BPH pathogenesis involves epithelial cells and stromal tissue proliferation inside prostate gland and testosterone is one of the promoting factors of prostate cell growth. Evidences about the antiproliferative effects of vitamin D and the widespread vitamin D deficiency and insufficiency among Bulgarian population suggest a possible relation between vitamin D and testosterone in BPH patients. Aim. To evaluate the vitamin D status and total testosterone (TT) levels in BPH patients and their associations with laboratory parameters such as prostate specific antigen (PSA) for prostate growth. Materials and methods. A total of 37 male BPH patients (mean age 67,14±7,77 years) were enrolled in the study. In all patients, BPH was histologically proven. PSA and ТТ levels were analyzed immunochemically. The circulating form of vitamin D, 25-hydroxyvitamin D (25OHD) was assayed by liquid chromatography with mas-spectrometry detection (LC-MS/MS). Other covariates (BMI, age,) were collected by interview at the time of hospitalization. Classical biochemical parameters were assayed by routine spectrophotometric tests. Descriptive statistics, variation and non-parametric correlation analysis were used. The level of significance was set at p<0.05. Results. The mean level of 25OHD for BPH patients was close to the lower reference limit of 50nmol/L recommended by the US Endocrine Society Guideline. The majority of BPH patients (56.8%) display 25OHD levels above 50nmol/L, 43.2% of them were vitamin D deficient (25OHD < 50nmol/L), 8.1% - with severe vitamin D deficiency (25OHD <25nmol/L), and only 6 patients (16.2%) had optimal 25OHD levels above the limit of 75 nmol/L. The mean serum TT levels of BPH patients were 10.74 ± 4.026 nmol/L, close to the lower limit of 10.4 nmol/L for normal TT, according to the recommendations of the Endocrine Society. A significant seasonal variations were found for 25OHD levels (p<0.05) between the cold and warm season. Similar seasonality was not established for TT. Two-thirds of BPH patients (62.9%) were with PSA values below the upper limit of the reference interval of 4 ng/ml. Higher 25OHD levels (59.21 ± 3.756 nmol/l, p= 0.06) were established for the group with PSA below the threshold of 4ng/ml. A moderate negative correlation (Spearman r= -0.6707, p<0.01) was found only for the vitamin D deficient group. In case of vitamin D sufficiency, a weak positive trend was detected. Conclusion. Our study indicated vitamin D insufficiency in BPH patients according to the criteria of the Endocrine Society. Strong negative correlation between 25OHD and TT levels was found for vitamin D deficient BPH patients. Higher 25OHD were associated with lower PSA values indicating a potential favorable effect of 25OHD on slackening of BPH.