Beyond Prostate Cancer: An Androgen Receptor Splice Variant Expression in Multiple Malignancies, Non-Cancer Pathologies, and Development.

Multiple studies have demonstrated the importance of androgen receptor (AR) splice variants (SVs) in the progression of prostate cancer to the castration-resistant phenotype and their utility as a diagnostic. However, studies on AR expression in non-prostatic malignancies uncovered that AR-SVs are expressed in glioblastoma, breast, salivary, bladder, kidney, and liver cancers, where they have diverse roles in tumorigenesis. AR-SVs also have roles in non-cancer pathologies. In granulosa cells from women with polycystic ovarian syndrome, unique AR-SVs lead to an increase in androgen production. In patients with nonobstructive azoospermia, testicular Sertoli cells exhibit differential expression of AR-SVs, which is associated with impaired spermatogenesis. Moreover, AR-SVs have been identified in normal cells, including blood mononuclear cells, neuronal lipid rafts, and the placenta. The detection and characterization of AR-SVs in mammalian and non-mammalian species argue that AR-SV expression is evolutionarily conserved and that AR-SV-dependent signaling is a fundamental regulatory feature in multiple cellular contexts. These discoveries argue that alternative splicing of the AR transcript is a commonly used mechanism that leads to an expansion in the repertoire of signaling molecules needed in certain tissues. Various malignancies appropriate this mechanism of alternative AR splicing to acquire a proliferative and survival advantage.

Pesticides and Bladder Cancer: Mechanisms Leading to Anti-Cancer Drug Chemoresistance and New Chemosensitization Strategies.

Multiple risk factors have been associated with bladder cancer. This review focuses on pesticide exposure, as it is not currently known whether agricultural products have a direct or indirect effect on bladder cancer, despite recent reports demonstrating a strong correlation. While it is known that pesticide exposure is associated with an increased risk of bladder cancer in humans and dogs, the mechanism(s) by which specific pesticides cause bladder cancer initiation or progression is unknown. In this narrative review, we discuss what is currently known about pesticide exposure and the link to bladder cancer. This review highlights multiple pathways modulated by pesticide exposure with direct links to bladder cancer oncogenesis/metastasis (MMP-2, TGF-ß, STAT3) and chemoresistance (drug efflux, DNA repair, and apoptosis resistance) and potential therapeutic tactics to counter these pesticide-induced affects.

Androgen receptor transcriptional activity is required for heregulin-1ß-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 in vivo studies showed that castration suppressed ErbB3 nuclear localization in HSPC, but not CRPC tumors. In vitro treatment with the ErbB3 ligand heregulin-1ß (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-3 cells 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.

Cisplatin-induced increase in heregulin 1 and its attenuation by the monoclonal ErbB3 antibody seribantumab in bladder cancer.

Cisplatin-based combination chemotherapy is the foundation for treatment of advanced bladder cancer (BlCa), but many patients develop chemoresistance mediated by increased Akt and ERK phosphorylation. However, the mechanism by which cisplatin induces this increase has not been elucidated. Among six patient-derived xenograft (PDX) models of BlCa, we observed that the cisplatin-resistant BL0269 express high epidermal growth factor receptor, ErbB2/HER2 and ErbB3/HER3. Cisplatin treatment transiently increased phospho-ErbB3 (Y1328), phospho-ERK (T202/Y204) and phospho-Akt (S473), and analysis of radical cystectomy tissues from patients with BlCa showed correlation between ErbB3 and ERK phosphorylation, likely due to the activation of ERK via the ErbB3 pathway. In vitro analysis revealed a role for the ErbB3 ligand heregulin1-ß1 (HRG1/NRG1), which is higher in chemoresistant lines compared to cisplatin-sensitive cells. Additionally, cisplatin treatment, both in PDX and cell models, increased HRG1 levels. The monoclonal antibody seribantumab, that obstructs ErbB3 ligand-binding, suppressed HRG1-induced ErbB3, Akt and ERK phosphorylation. Seribantumab also prevented tumor growth in both the chemosensitive BL0440 and chemoresistant BL0269 models. Our data demonstrate that cisplatin-associated increases in Akt and ERK phosphorylation is mediated by an elevation in HRG1, suggesting that inhibition of ErbB3 phosphorylation may be a useful therapeutic strategy in BlCa with high phospho-ErbB3 and HRG1 levels.

Androgen receptor-dependent regulation of metabolism in high grade bladder cancer cells.

The observed sex disparity in bladder cancer (BlCa) argues that androgen receptor (AR) signaling has a role in these malignancies. BlCas express full-length AR (FL-AR), constitutively active AR splice variants, including AR-v19, or both, and their depletion limits BlCa viability. However, the mechanistic basis of AR-dependence is unknown. Here, we depleted FL-AR, AR-v19, or all AR forms (T-AR), and performed RNA-seq studies to uncover that different AR forms govern distinct but partially overlapping transcriptional programs. Overlapping alterations include a decrease in mTOR and an increase of hypoxia regulated transcripts accompanied by a decline in oxygen consumption rate (OCR). Queries of BlCa databases revealed a significant negative correlation between AR expression and multiple hypoxia-associated transcripts arguing that this regulatory mechanism is a feature of high-grade malignancies. Our analysis of a 1600-compound library identified niclosamide as a strong ATPase inhibitor that reduces OCR in BlCa cells, decreased cell viability and induced apoptosis in a dose and time dependent manner. These results suggest that BlCa cells hijack AR signaling to enhance metabolic activity, promoting cell proliferation and survival; hence targeting this AR downstream vulnerability presents an attractive strategy to limit BlCa.

Comparative Cancer Cell Signaling in Muscle-Invasive Urothelial Carcinoma of the Bladder in Dogs and Humans.

Muscle-invasive urothelial carcinoma (MIUC) is the most common type of bladder malignancy in humans, but also in dogs that represent a naturally occurring model for this disease. Dogs are immunocompetent animals that share risk factors, pathophysiological features, clinical signs and response to chemotherapeutics with human cancer patients. This review summarizes the fundamental pathways for canine MIUC initiation, progression, and metastasis, emerging therapeutic targets and mechanisms of drug resistance, and proposes new opportunities for potential prognostic and diagnostic biomarkers and therapeutics. Identifying similarities and differences between cancer signaling in dogs and humans is of utmost importance for the efficient translation of in vitro research to successful clinical trials for both species.

Depletion of androgen receptor low molecular weight isoform reduces bladder tumor cell viability and induces apoptosis.

Bladder cancer (BlCa) exhibits a gender disparity where men are three times more likely to develop the malignancy than women suggesting a role for the androgen receptor (AR). Here we report that BlCa cells express low molecular weight (LMW) AR isoforms that are missing the ligand binding domain (LBD). Isoform expression was detected in most BlCa cells, while a few express the full-length AR. Immunofluorescence studies detect AR in the nucleus and cytoplasm, and localization is cell dependent. Cells with nuclear AR expression exhibit reduced viability and increased apoptosis on total AR depletion. A novel AR-LMW variant, AR-v19, that is missing the LBD and contains 15 additional amino acids encoded by intron 3 sequences was detected in most BlCa malignancies. AR-v19 localizes to the nucleus and can transactivate AR-dependent transcription in a dose dependent manner. AR-v19 depletion impairs cell viability and promotes apoptosis in cells that express this variant. Thus, AR splice variant expression is common in BlCa and instrumental in ensuring cell survival. This suggests that targeting AR or AR downstream effectors may be a therapeutic strategy for the treatment of this malignancy.

The Androgen Receptor in Prostate Cancer: Effect of Structure, Ligands and Spliced Variants on Therapy.

The androgen receptor (AR) plays a predominant role in prostate cancer (PCa) pathology. It consists of an N-terminal domain (NTD), a DNA-binding domain (DBD), a hinge region (HR), and a ligand-binding domain (LBD) that binds androgens, including testosterone (T) and dihydrotestosterone (DHT). Ligand binding at the LBD promotes AR dimerization and translocation to the nucleus where the DBD binds target DNA. In PCa, AR signaling is perturbed by excessive androgen synthesis, AR amplification, mutation, or the formation of AR alternatively spliced variants (AR-V) that lack the LBD. Current therapies for advanced PCa include androgen synthesis inhibitors that suppress T and/or DHT synthesis, and AR inhibitors that prevent ligand binding at the LBD. However, AR mutations and AR-Vs render LBD-specific therapeutics ineffective. The DBD and NTD are novel targets for inhibition as both perform necessary roles in AR transcriptional activity and are less susceptible to AR alternative splicing compared to the LBD. DBD and NTD inhibition can potentially extend patient survival, improve quality of life, and overcome predominant mechanisms of resistance to current therapies. This review discusses various small molecule and other inhibitors developed against the DBD and NTD-and the current state of the available compounds in clinical development.

Non-circadian aspects of BHLHE40 cellular function in cancer.

While many genes specifically act as oncogenes or tumor suppressors, others are tumor promoters or suppressors in a context-dependent manner. Here we will review the basic-helix-loop-helix (BHLH) protein BHLHE40, (also known as BHLHB2, STRA13, DEC1, or SHARP2) which is overexpressed in gastric, breast, and brain tumors; and downregulated in colorectal, esophageal, pancreatic and lung cancer. As a transcription factor, BHLHE40 is expressed in the nucleus, where it binds to target gene promoters containing the E-box hexanucleotide sequence, but can also be expressed in the cytoplasm, where it stabilizes cyclin E, preventing cyclin E-mediated DNA replication and cell cycle progression. In different organs BHLHE40 regulates different targets; hence may have different impacts on tumorigenesis. BHLHE40 promotes PI3K/Akt/mTOR activation in breast cancer, activating tumor progression, but suppresses STAT1 expression in clear cell carcinoma, triggering tumor suppression. Target specificity likely depends on cooperation with other transcription factors. BHLHE40 is activated in lung and esophageal carcinoma by the tumor suppressor p53 inducing senescence and suppressing tumor growth, but is also activated under hypoxic conditions by HIF-1α in gastric cancer and hepatocellular carcinomas, stimulating tumor progression. Thus, BHLHE40 is a multi-functional protein that mediates the promotion or suppression of cancer in a context dependent manner.

The p14ARF tumor suppressor restrains androgen receptor activity and prevents apoptosis in prostate cancer cells.

Prostate cancer (PCa) is characterized by a unique dependence on optimal androgen receptor (AR) activity where physiological androgen concentrations induce proliferation but castrate and supraphysiological levels suppress growth. This feature has been exploited in bipolar androgen therapy (BAT) for castrate resistant malignancies. Here, we investigated the role of the tumor suppressor protein p14ARF in maintaining optimal AR activity and the function of the AR itself in regulating p14ARF levels. We used a tumor tissue array of differing stages and grades to define the relationships between these components and identified a strong positive correlation between p14ARF and AR expression. Mechanistic studies utilizing CWR22 xenograft and cell culture models revealed that a decrease in AR reduced p14ARF expression and deregulated E2F factors, which are linked to p14ARF and AR regulation. Chromatin immunoprecipitation studies identified AR binding sites upstream of p14ARF. p14ARF depletion enhanced AR-dependent PSA and TMPRSS2 transcription, hence p14ARF constrains AR activity. However, p14ARF depletion ultimately results in apoptosis. In PCa cells, AR co-ops p14ARF as part of a feedback mechanism to ensure optimal AR activity for maximal prostate cancer cell survival and proliferation.

XPO1 inhibition by selinexor induces potent cytotoxicity against high grade bladder malignancies.

Treatment options for high grade urothelial cancers are limited and have remained largely unchanged for several decades. Selinexor (KPT-330), a first in class small molecule that inhibits the nuclear export protein XPO1, has shown efficacy as a single agent treatment for numerous different malignancies, but its efficacy in limiting bladder malignancies has not been tested. In this study we assessed selinexor-dependent cytotoxicity in several bladder tumor cells and report that selinexor effectively reduced XPO1 expression and limited cell viability in a dose dependent manner. The decrease in cell viability was due to an induction of apoptosis and cell cycle arrest. These results were recapitulated in in vivo studies where selinexor decreased tumor growth. Tumors treated with selinexor expressed lower levels of XPO1, cyclin A, cyclin B, and CDK2 and increased levels of RB and CDK inhibitor p27, a result that is consistent with growth arrest. Cells expressing wildtype RB, a potent tumor suppressor that promotes growth arrest and apoptosis, were most susceptible to selinexor. Cell fractionation and immunofluorescence studies showed that selinexor treatment increased nuclear RB levels and mechanistic studies revealed that RB ablation curtailed the response to the drug. Conversely, limiting CDK4/6 dependent RB phosphorylation by palbociclib was additive with selinexor in reducing bladder tumor cell viability, confirming that RB activity has a role in the response to XPO1 inhibition. These results provide a rationale for XPO1 inhibition as a novel strategy for the treatment of bladder malignancies.

miR-148a dependent apoptosis of bladder cancer cells is mediated in part by the epigenetic modifier DNMT1.

Urothelial cell carcinoma of the bladder (UCCB) is the most common form of bladder cancer and it is estimated that ~15,000 people in the United States succumbed to this disease in 2013. Bladder cancer treatment options are limited and research to understand the molecular mechanisms of this disease is needed to design novel therapeutic strategies. Recent studies have shown that microRNAs play pivotal roles in the progression of cancer. miR-148a has been shown to serve as a tumor suppressor in cancers of the prostate, colon, and liver, but its role in bladder cancer has never been elucidated. Here we show that miR-148a is down-regulated in UCCB cell lines. We demonstrate that overexpression of miR-148a leads to reduced cell viability through an increase in apoptosis rather than an inhibition of proliferation. We additionally show that miR-148a exerts this effect partially by attenuating expression of DNA methyltransferase 1 (DNMT1). Finally, our studies demonstrate that treating cells with both miR-148a and either cisplatin or doxorubicin is either additive or synergistic in causing apoptosis. These data taken together suggest that miR-148a is a tumor suppressor in UCCB and could potentially serve as a novel therapeutic for this malignancy.

The emerging role of the androgen receptor in bladder cancer.

Men are three to four times more likely to get bladder cancer than women. The gender disparity characterizing bladder cancer diagnoses has been investigated. One hypothesis is that androgen receptor (AR) signaling is involved in the etiology and progression of this disease. Although bladder cancer is not typically described as an endocrine-related malignancy, it has become increasingly clear that AR signaling plays a role in bladder tumors. This review summarizes current findings regarding the role of the AR in bladder cancer. We discuss work demonstrating AR expression in bladder cancer and its role in promoting formation and progression of tumors. Additionally, we discuss the therapeutic potential of targeting the AR in this disease.

Dicer ablation promotes a mesenchymal and invasive phenotype in bladder cancer cells.

Dicer expression is frequently altered in cancer and affects a wide array of cellular functions acting as an oncogene or tumor suppressor in varying contexts. It has been shown that Dicer expression is also deregulated in urothelial cell carcinoma of the bladder (UCCB) but the nature of this deregulation differs between reports. The aim of the present study was to gain a better understanding of the role of Dicer in bladder cancer to help determine its contribution to the disease. The results showed that Dicer transcript levels were decreased in UCCB tumor tissues as compared to normal tissues, suggesting that Dicer is a tumor suppressor. However, consistent with previous results, we demonstrated that knockdown of Dicer decreases cell viability and increases the induction of apoptosis, suggesting that Dicer is an oncogene. To resolve this discrepancy, we assessed the effects of decreased Dicer expression on epithelial-to­mesenchymal transition, migration and invasion. We showed that decreased Dicer levels promoted a mesenchymal phenotype and increased migration. Additionally, the results showed that Dicer protein ablation leads to increased cell invasion, higher levels of matrix metalloproteinase-2, and decreased levels of key miRNAs shown to inhibit invasion. The results of this study suggest that decreased Dicer levels may portend a more malignant phenotype.

Transcription of Nrdp1 by the androgen receptor is regulated by nuclear filamin A in prostate cancer.

Prostate cancer (PCa) progression is regulated by the androgen receptor (AR); however, patients undergoing androgen-deprivation therapy (ADT) for disseminated PCa eventually develop castration-resistant PCa (CRPC). Results of previous studies indicated that AR, a transcription factor, occupies distinct genomic loci in CRPC compared with hormone-naïve PCa; however, the cause of this distinction was unknown. The E3 ubiquitin ligase Nrdp1 is a model AR target modulated by androgens in hormone-naïve PCa but not in CRPC. Using Nrdp1, we investigated how AR switches transcription programs during CRPC progression. The proximal Nrdp1 promoter contains an androgen response element (ARE); we demonstrated AR binding to this ARE in androgen-sensitive PCa. Analysis of hormone-naive human prostatectomy specimens revealed correlation between Nrdp1 and AR expression, supporting AR regulation of NRDP1 levels in androgen-sensitive tissue. However, despite sustained AR levels, AR binding to the Nrdp1 promoter and Nrdp1 expression were suppressed in CRPC. Elucidation of the suppression mechanism demonstrated correlation of NRDP1 levels with nuclear localization of the scaffolding protein filamin A (FLNA) which, as we previously showed, is itself repressed following ADT in many CRPC tumors. Restoration of nuclear FLNA in CRPC stimulated AR binding to Nrdp1 ARE, increased its transcription, and augmented NRDP1 protein expression and responsiveness to ADT, indicating that nuclear FLNA controls AR-mediated androgen-sensitive Nrdp1 transcription. Expression of other AR-regulated genes lost in CRPC was also re-established by nuclear FLNA. Thus, our results indicate that nuclear FLNA promotes androgen-dependent AR-regulated transcription in PCa, while loss of nuclear FLNA in CRPC alters the AR-regulated transcription program.

The role of EGFR family inhibitors in muscle invasive bladder cancer: a review of clinical data and molecular evidence.

PURPOSE: Conventional platinum based chemotherapy for advanced urothelial carcinoma is plagued by common resistance to this regimen. Several studies implicate the EGFR family of RTKs in urothelial carcinoma progression and chemoresistance. Many groups have investigated the effects of inhibitors of this family in patients with urothelial carcinoma. This review focuses on the underlying molecular pathways that lead to urothelial carcinoma resistance to EGFR family inhibitors. MATERIALS AND METHODS: We performed a PubMed® search for peer reviewed literature on bladder cancer development, EGFR family expression, clinical trials of EGFR family inhibitors and molecular bypass pathways. Research articles deemed to be relevant were examined and a summary of original data was created. Meta-analysis of expression profiles was also performed for each EGFR family member based on data sets accessible via Oncomine®. RESULTS: Many clinical trials using inhibitors of EGFR family RTKs have been done or are under way. Those that have concluded with results published to date do not show an added benefit over standard of care chemotherapy in an adjuvant or second line setting. However, a neoadjuvant study using erlotinib before radical cystectomy demonstrated promising results. CONCLUSIONS: Clinical and preclinical studies show that for reasons not currently clear prior treatment with chemotherapeutic agents rendered patients with urothelial carcinoma with muscle invasive bladder cancer resistant to EGFR family inhibitors as well. However, EGFR family inhibitors may be of use in patients with no prior chemotherapy in whom EGFR or ERBB2 is over expressed.

On the origins of the androgen receptor low molecular weight species.

Prostate cancer (CaP), a commonly diagnosed malignancy, is readily treated by androgen ablation. This treatment temporarily halts the disease, but castration-resistant neoplasms that are refractory to current therapies emerge. While these neoplasms are no longer dependent on physiological levels of androgens, they remain reliant on the expression of the androgen receptor (AR). There are multiple mechanisms by which CaP cells circumvent androgen ablation therapies. These include AR mutations that broaden ligand specificity, AR overexpression, AR activation by growth factors and cytokines, overexpression of AR co-activators, altered steroid metabolism, and a locus-wide histone transcriptional activation of some AR targets. This review focuses on a more recently described mechanism: the expression of low molecular weight AR species that are missing the ligand-binding domain and function independently of ligand to drive proliferation. The etiology, biological activity, unique features, predictive value, and therapeutic implication of these androgen receptor isoforms are discussed in depth.

Enhancing the effectiveness of androgen deprivation in prostate cancer by inducing Filamin A nuclear localization.

As prostate cancer (CaP) is regulated by androgen receptor (AR) activity, metastatic CaP is treated with androgen deprivation therapy (ADT). Despite initial response, patients on ADT eventually progress to castration-resistant CaP (CRPC), which is currently incurable. We previously showed that cleavage of the 280 kDa structural protein Filamin A (FlnA) to a 90 kDa fragment, and nuclear localization of the cleaved product, sensitized CRPC cells to ADT. Hence, treatment promoting FlnA nuclear localization would enhance androgen responsiveness. Here, we show that FlnA nuclear localization induced apoptosis in CRPC cells during ADT, identifying it as a treatment tool in advanced CaP. Significantly, the natural product genistein combined polysaccharide (GCP) had a similar effect. Investigation of the mechanism of GCP-induced apoptosis showed that GCP induced FlnA cleavage and nuclear localization and that apoptosis resulting from GCP treatment was mediated by FlnA nuclear localization. Two main components of GCP are genistein and daidzein: the ability of GCP to induce G2 arrest was due to genistein whereas sensitivity to ADT stemmed from daidzein; hence, both were needed to mediate GCP's effects. FlnA cleavage is regulated by its phosphorylation; we show that ADT enhanced FlnA phosphorylation, which prevented its cleavage, whereas GCP inhibited FlnA phosphorylation, thereby sensitizing CaP cells to ADT. In a mouse model of CaP recurrence, GCP, but not vehicle, impeded relapse following castration, indicating that GCP, when administered with ADT, interrupted the development of CRPC. These results demonstrate the efficacy of GCP in promoting FlnA nuclear localization and enhancing androgen responsiveness in CaP.

The interleukin 6 receptor is a direct transcriptional target of E2F3 in prostate tumor derived cells.

BACKGROUND: The E2F/RB pathway is frequently disrupted in multiple human cancers. E2F3 levels are elevated in prostate tumors and E2F3 overexpression independently predicts clinical outcome. The goals of this study were to identify direct transcriptional targets of E2F3 in prostate tumor derived cells. METHODS: Expression array studies identified the interleukin 6 receptor (IL-6R) as an E2F3 target. E2F3-dependent expression of IL-6R was analyzed by real time PCR and Western immunoblot analysis in several cell lines. Chromatin immunoprecipitation (ChIP) and IL-6R-luciferase reporter plasmid studies were used to characterize the IL-6R promoter. RESULTS: Expression array studies identified genes that were regulated by E2F3 in prostate tumor derived cell lines. The network most significantly associated with E2F3-regulated transcripts was cytokine signaling and the IL-6R was a component of several of the most prominent E2F3-regulated pathways. The transcriptional regulation of IL-6R by E2F3 knockdown was validated in several prostate tumor-derived cell lines at the RNA level and protein level. The IL-6R regulatory region containing ChIP-identified E2F3 binding sites was cloned into a reporter and co-transfected with an E2F3a expression plasmid. The luciferase assay showed that E2F3a transactivated the IL-6R promoter in a dose dependent manner. The functional consequence of IL-6R decrease was a reduction in the levels of ERK1/2 phosphorylation, indicating that IL-6R initiated signaling was altered. CONCLUSION: These studies connect the E2F and IL-6 signaling cascade, thus providing the mechanistic link between two major regulatory networks that are perturbed during prostate tumorigenesis.