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

Abstract B47: Identification of novel E2F3 transcriptional targets expands the role of the RB/E2F axis beyond cell cycle control

Abstract The RB/E2F axis is frequently disrupted in multiple tumors. Classical studies reported that the RB tumor suppressor exerts its transcriptional repressor function through an interaction with the E2F family of transcription factors (E2F1-3) to control the timely expression of genes required for DNA replication and cell cycle progression. The E2F3 locus encodes two E2F3 isoforms, a and b, which have unique N-terminal sequences. Multiple studies indicate that E2F3, rather than E2F1 or 2, is elevated in various cancers, including prostate. In prostate tumors elevated E2F3 expression is an independent prognostic factor of clinical outcome, but the direct transcriptional targets of E2F3a and E2F3b have not been characterized. We found that E2F3a and b expression was increased in prostate tumor derived cell lines when compared to non-transformed controls. An expression array analysis following knockdown (k/d) of total E2F3 identified several unexpected targets including Interleukin 6 receptor (IL-6R), a critical component of the IL6 signaling cascade and calpain 2, a protease that is essential in cellular migration and has been shown to be elevated during prostate tumorigenesis. Further studies showed that the two genes were regulated by different E2F3 isoforms- IL6R was a target of E2F3a, while calpain 2 was regulated by E2F3b. Expression of both genes was enhanced by RB ablation. E2F3a-dependent IL-6R regulation was apparent in PC3, LNCaP, CWR-R1, and 22Rv1 cells. Chromatin immunoprecipitation (ChIP) studies identified sequences in the IL-6R promoter that were bound by E2F3. Transient co-transfection studies using an E2F3a expression plasmid showed that E2F3a transactivated the IL-6R promoter in a dose dependent manner. The IL-6R initiated signaling cascade was perturbed following a k/d of E2F3a since the levels of ERK1/2 phosphorylation was reduced. An siRNA-mediated ablation of each isoform revealed that E2F3b, not E2Fa, was effective is reducing calpain 2 levels. ChIP studies indicated that E2F3 binds to the endogenous calpain 2 promoter, and in transient transfection studies E2F3b transactivated the calpain 2 promoter in a dose dependent manner. An analysis of the calpain 2 promoter identified an androgen receptor (AR) half site. Additional transfection studies showed that the AR can cooperate with E2F3b in transactivation of the calpain 2 promoter. Moreover, reduced expression of E2F3b impaired cellular migration in a wound assay. The expression of the E2F3 isoforms has not been previously analyzed in the TRAMP prostate tumor model. Western immunoblot studies of five TRAMP tumors and three age and strain matched prostates showed that TRAMP tumors had highly elevated expression of both E2F3 isoforms. These results indicate that increased expression of both E2F3 isoforms is a feature of human tumor-derived cell lines and the TRAMP mouse model of prostate tumorigenesis. Calpain 2, expression was readily detected in all of the TRAMP tumors, but not in the control tissue. Hence E2F3 overexpression and increased calpain levels are features of human and mouse tumors. This analysis broadens the role of E2F3 in prostate tumorigenesis beyond the regulation of cell cycle progression. E2F3a is a link between the E2F/RB and the IL-6 signaling cascade, while E2F3b regulates expression of a protease that is essential in cell adhesion and migration. Therefore E2F3 deregulation affects multiple signaling networks to promote tumorigenesis. Citation Format: Stephen J. Libertini, Maria Mudryj, Alan P. Lombard, Honglin Chen, Veronica Rodriguez, Carlos Perez-Stable, Bushra al-Bataina, Tilak Koilvaram, Michael George, Allen C. Gao. Identification of novel E2F3 transcriptional targets expands the role of the RB/E2F axis beyond cell cycle control [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr B47.</jats:p

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

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 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