Hironobu Yamashita

Inhibition of NF-kappa B signaling restores responsiveness of castrate-resistant prostate cancer cells to anti-androgen treatment by decreasing androgen receptor-variant expression

Androgen receptor splicing variants (ARVs) that lack the ligand-binding domain (LBD) are associated with the development of castration-resistant prostate cancer (CRPC), including resistance to the new generation of high-affinity anti-androgens. However, the mechanism by which ARV expression is regulated is not fully understood. In this study, we show that the activation of classical nuclear factor-kappa B (NF-κB) signaling increases the expression of ARVs in prostate cancer (PCa) cells and converts androgen-sensitive PCa cells to become androgen-insensitive, whereas downregulation of NF-κB signaling inhibits ARV expression and restores responsiveness of CRPC to anti-androgen therapy. In addition, we demonstrated that combination of anti-androgen with NF-κB-targeted therapy inhibits efficiently tumor growth of human CRPC xenografts. These results indicate that induction of ARVs by activated NF-κB signaling in PCa cells is a critical mechanism by which the PCa progresses to CRPC. This has important implications as it can prolong the survival of CRPC patients by restoring the tumors to once again respond to conventional androgen-deprivation therapy (ADT).

NFI transcription factors interact with FOXA1 to regulate prostate-specific gene expression

Androgen receptor (AR) action throughout prostate development and in maintenance of the prostatic epithelium is partly controlled by interactions between AR and forkhead box (FOX) transcription factors, particularly FOXA1. We sought to identity additional FOXA1 binding partners that may mediate prostate-specific gene expression. Here we identify the nuclear factor I (NFI) family of transcription factors as novel FOXA1 binding proteins. All four family members (NFIA, NFIB, NFIC, and NFIX) can interact with FOXA1, and knockdown studies in androgen-dependent LNCaP cells determined that modulating expression of NFI family members results in changes in AR target gene expression. This effect is probably mediated by binding of NFI family members to AR target gene promoters, because chromatin immunoprecipitation (ChIP) studies found that NFIB bound to the prostate-specific antigen enhancer. Förster resonance energy transfer studies revealed that FOXA1 is capable of bringing AR and NFIX into proximity, indicating that FOXA1 facilitates the AR and NFI interaction by bridging the complex. To determine the extent to which NFI family members regulate AR/FOXA1 target genes, motif analysis of publicly available data for ChIP followed by sequencing was undertaken. This analysis revealed that 34.4% of peaks bound by AR and FOXA1 contain NFI binding sites. Validation of 8 of these peaks by ChIP revealed that NFI family members can bind 6 of these predicted genomic elements, and 4 of the 8 associated genes undergo gene expression changes as a result of individual NFI knockdown. These observations suggest that NFI regulation of FOXA1/AR action is a frequent event, with individual family members playing distinct roles in AR target gene expression.

Thrombospondin-1 acts as a ligand for CD148 tyrosine phosphatase

CD148 is a receptor-type protein tyrosine phosphatase that is expressed in several cell types, including vascular endothelial cells and duct epithelial cells. Growing evidence demonstrates a prominent role for CD148 in negative regulation of growth factor signals, suppressing cell proliferation and transformation. However, its extracellular ligand(s) remain unknown. To identify the ligand(s) of CD148, we introduced HA-tagged CD148 into cultured endothelial cells and then isolated its interacting extracellular protein(s) by biotin surface labeling and subsequent affinity purifications. The binding proteins were identified by mass spectrometry. Here we report that soluble thrombospondin-1 (TSP1) binds to the extracellular part of CD148 with high affinity and specificity, and its binding increases CD148 catalytic activity, leading to dephosphorylation of the substrate proteins. Consistent with these findings, introduction of CD148 conferred TSP1-mediated inhibition of cell growth to cells which lack CD148 and TSP1 inhibition of growth. Further, we demonstrate that TSP1-mediated inhibition of endothelial cell growth is antagonized by soluble CD148 ectodomain as well as by CD148 gene silencing. These findings provide evidence that CD148 functions as a receptor for TSP1 and mediates its inhibition of cell growth.

Loss of FOXA1 Drives Sexually Dimorphic Changes in Urothelial Differentiation and Is an Independent Predictor of Poor Prognosis in Bladder Cancer.

We previously found loss of forkhead box A1 (FOXA1) expression to be associated with aggressive urothelial carcinoma of the bladder, as well as increased tumor proliferation and invasion. These initial findings were substantiated by The Cancer Genome Atlas, which identified FOXA1 mutations in a subset of bladder cancers. However, the prognostic significance of FOXA1 inactivation and the effect of FOXA1 loss on urothelial differentiation remain unknown. Application of a univariate analysis (log-rank) and a multivariate Cox proportional hazards regression model revealed that loss of FOXA1 expression is an independent predictor of decreased overall survival. An ubiquitin Cre-driven system ablating Foxa1 expression in urothelium of adult mice resulted in sex-specific histologic alterations, with male mice developing urothelial hyperplasia and female mice developing keratinizing squamous metaplasia. Microarray analysis confirmed these findings and revealed a significant increase in cytokeratin 14 expression in the urothelium of the female Foxa1 knockout mouse and an increase in the expression of a number of genes normally associated with keratinocyte differentiation. IHC confirmed increased cytokeratin 14 expression in female bladders and additionally revealed enrichment of cytokeratin 14-positive basal cells in the hyperplastic urothelial mucosa in male Foxa1 knockout mice. Analysis of human tumor specimens confirmed a significant relationship between loss of FOXA1 and increased cytokeratin 14 expression.

FOXA1, GATA3 and PPARɣ Cooperate to Drive Luminal Subtype in Bladder Cancer: A Molecular Analysis of Established Human Cell Lines

Discrete bladder cancer molecular subtypes exhibit differential clinical aggressiveness and therapeutic response, which may have significant implications for identifying novel treatments for this common malignancy. However, research is hindered by the lack of suitable models to study each subtype. To address this limitation, we classified bladder cancer cell lines into molecular subtypes using publically available data in the Cancer Cell Line Encyclopedia (CCLE), guided by genomic characterization of bladder cancer by The Cancer Genome Atlas (TCGA). This identified a panel of bladder cancer cell lines which exhibit genetic alterations and gene expression patterns consistent with luminal and basal molecular subtypes of human disease. A subset of bladder cancer cell lines exhibit in vivo histomorphologic patterns consistent with luminal and basal subtypes, including papillary architecture and squamous differentiation. Using the molecular subtype assignments, and our own RNA-seq analysis, we found overexpression of GATA3 and FOXA1 cooperate with PPARɣ activation to drive transdifferentiation of a basal bladder cancer cells to a luminial phenotype. In summary, our analysis identified a set of human cell lines suitable for the study of molecular subtypes in bladder cancer, and furthermore indicates a cooperative regulatory network consisting of GATA3, FOXA1, and PPARɣ drive luminal cell fate.

Bone Metastasis of Prostate Cancer Can Be Therapeutically Targeted at the TBX2–WNT Signaling Axis

Identification of factors that mediate visceral and bone metastatic spread and subsequent bone remodeling events is highly relevant to successful therapeutic intervention in advanced human prostate cancer. TBX2, a T-box family transcription factor that negatively regulates cell-cycle inhibitor p21, plays critical roles during embryonic development, and recent studies have highlighted its role in cancer. Here, we report that TBX2 is overexpressed in human prostate cancer specimens and bone metastases from xenograft mouse models of human prostate cancer. Blocking endogenous TBX2 expression in PC3 and ARCaPM prostate cancer cell models using a dominant-negative construct resulted in decreased tumor cell proliferation, colony formation, and invasion in vitro Blocking endogenous TBX2 in human prostate cancer mouse xenografts decreased invasion and abrogation of bone and soft tissue metastasis. Furthermore, blocking endogenous TBX2 in prostate cancer cells dramatically reduced bone-colonizing capability through reduced tumor cell growth and bone remodeling in an intratibial mouse model. TBX2 acted in trans by promoting transcription of the canonical WNT (WNT3A) promoter. Genetically rescuing WNT3A levels in prostate cancer cells with endogenously blocked TBX2 partially restored the TBX2-induced prostate cancer metastatic capability in mice. Conversely, WNT3A-neutralizing antibodies or WNT antagonist SFRP-2 blocked TBX2-induced invasion. Our findings highlight TBX2 as a novel therapeutic target upstream of WNT3A, where WNT3A antagonists could be novel agents for the treatment of metastasis and for skeletal complications in prostate cancer patients. Cancer Res; 77(6); 1331-44. ©2017 AACR.

On a FOX hunt: functions of FOX transcriptional regulators in bladder cancer

Genomic and transcriptional studies have identified discrete molecular subtypes of bladder cancer. These observations could be the starting point to identify new treatments. Several members of the forkhead box (FOX) superfamily of transcription factors have been found to be differentially expressed in the different bladder cancer subtypes. In addition, the FOXA protein family are key regulators of embryonic bladder development and patterning. Both experimental and clinical data support a role for FOXA1 and FOXA2 in urothelial carcinoma. FOXA1 is expressed in embryonic and adult urothelium and its expression is altered in urothelial carcinomas and across disparate molecular bladder cancer subtypes. FOXA2 is normally absent from the adult urothelium, but developmental studies identified FOXA2 as a marker of a transient urothelial progenitor cell population during bladder development. Studies also implicate FOXA2 in bladder cancer and several other FOX proteins might be involved in development and/or progression of this disease; for example, FOXA1 and FOXO3A have been associated with clinical patient outcomes. Future studies should investigate to what extent and by which mechanisms FOX proteins might be directly involved in bladder cancer pathogenesis and treatment responses.

Repression of Transcription Factor AP-2 Alpha by Peroxisome Proliferator Activated Receptor Gamma Reveals a Novel Transcriptional Circuit in basal-squamous Bladder Cancer

The discovery of bladder cancer transcriptional subtypes provides an opportunity to identify high risk patients, and tailor disease management. Recent studies suggest tumor heterogeneity contributes to “plasticity” of molecular subtype during progression and following treatment. Nonetheless, the transcriptional drivers of the aggressive basal-squamous subtype remain unidentified. As PPARγ has been repeatedly implicated in the luminal subtype of bladder cancer, we hypothesized inactivation of this transcriptional master regulator during progression results in increased expression of basal-squamous specific transcription factors (TFs) which act to drive aggressive behavior. We initiated a pharmacologic and RNA-seq-based screen to identify PPARγ-repressed, basal-squamous specific TFs. Hierarchical clustering of RNA-seq data following treatment of a panel of human bladder cancer cell lines with a PPARγ agonist identified a number of TFs regulated by PPARγ activation, several of which are implicated in urothelial and squamous differentiation. One PPARγ-repressed TF implicated in squamous differentiation identified is Transcription Factor Activating Protein 2 alpha (TFAP2A). We show TFAP2A and its paralog TFAP2C are overexpressed in basal-squamous bladder cancer and in squamous areas of cystectomy samples, and that overexpression is associated with increased lymph node metastasis and distant recurrence, respectively. Biochemical analysis confirmed the ability of PPARγ activation to repress TFAP2A, while PPARγ antagonist studies indicate the requirement of a functional receptor. In vivo tissue recombination studies show TFAP2A and TFAP2C promote tumor growth in line with the aggressive nature of basal-squamous bladder cancer. Our findings suggest PPARγ inactivation, as well as TFAP2A and TFAP2C overexpression cooperate with other TFs to promote the basal-squamous transition.

MP54-01 TRANSCRIPTION FACTOR ACTIVATOR PROTEIN 2 FAMILY MEMBERS ARE MARKERS OF THE BASAL MOLECULAR SUBTYPE OF HUMAN BLADDER CANCER AND REPRESSED BY LUMINAL TRANSCRIPTIONAL MASTER REGULATORS

INTRODUCTION AND OBJECTIVES: Recent analysis identified luminal and basal gene expression subtypes of human bladder cancer (BLCa) with prognostic significance. We reported that loss of the luminal transcription factor FOXA1 is associated with poor clinical outcome and that FOXA1 cooperates with GATA3 and PPARg to reprogram human basal BLCa cells into cells expressing a luminal gene expression pattern. Interestingly, RNA-seq analysis showed that expression of the transcription factors TFAP2A and TFAP2C, which are involved in squamous differentiation (SqD), are repressed following PPARg activation. Therefore, in this study we investigated the role of TFAP2A and TFAP2C in basal human bladder cancers. METHODS: Computational data analysis using TCGA data was used to determine if TFAP2A and TFAP2C expression is correlated with gene expression subtype. Immunohistochemistry (IHC) of clinical bladder cancer samples was performed to determine the correlation of TFAP2A and TFAP2C expression with SqD and clinical attributes of aggressive disease. Western blotting was also performed to investigate the expression of TFAP2A and TFAP2C in human BLCa cell lines. Finally, we performed tissue recombination xenografting using TFAP2C-overexpressing BLCa cells to assess in vivo tumorigenicity. RESULTS: Computational analysis showed increased expression of TFAP2AandTFAP2C is significantly associatedwith a basal gene expression subtype in BLCa. In addition, IHC of human BLCa tissue revealed TFAP2A expression is significantly associated with SqD and lymph node metastasis, while TFAP2C expression was significantly associated with SqD and distant recurrence. Western blotting of 10 human BLCa cell lines showed that TFAP2A and TFAP2C are highly expressed in cells identified by us as robust models of basal disease. Activation of the luminal master regulator PPARg by rosiglitazone induced a reduction in TFAP2Aexpression in SW780,UMUC1, and 5637 cell lines. Moreover, reduction in TFAP2A expression following rosiglitazone treatment was prevented by treatment with a PPARg antagonist. Finally, tissue recombination xenografting using T24 and UMUC3 cells stably overexpressing TFAP2C enhanced tumor formation in vivo. CONCLUSIONS: TFAP2A and TFAP2C are novel molecular markers of basal BLCa and SqD. Expression of these transcription factors is an indicator of poor clinical outcome. In addition, TFAP2A is a PPARg repressed gene and overexpression of TFAP2C increases in vivo tumorigenicity.

MP98-12 FOXA1 KNOCKOUT IS ASSOCIATED WITH INCREASED CARCINOGENIC SUSCEPTIBILITY AND ANDROGEN RECEPTOR EXPRESSION IN MURINE BLADDER CANCER

INTRODUCTION AND OBJECTIVES: Reactive oxygen species (ROS) have been identified as important chemical mediators in cell growth and differentiation. The glutathione redox system is the main mechanism protecting against damage caused by ROS in the human body. In this study, we investigated the role and therapeutic potential of the glutathione redox system in bladder cancer. METHODS: The expression levels of glutathione peroxidase 2 (GPX2) and Ki-67 proteins were analyzed in human transurethral resection (TUR) specimens by immunohistochemistry; correlations between the GPX2 expression and prognosis were also analyzed. In addition, male F344 rats were given 0.05% BBN in drinking water and 0.1% Phenyl isothiocyanate in their diet for 36 weeks. Bladder tissue samples were collected from each animal for analyses. Furthermore, the rat cell line, BC31, and human cell lines, T24, RT4, TCC-SUP, and 5637, were transfected with GPX2 siRNA and negative control siRNA (NC). Subsequently, cell proliferation rates and ROS levels were investigated by cell counting, DCFH assay, western blotting, and flow cytometry. siRNAor NCtransfected BC31 cells were subcutaneously implanted into nude mice. RESULTS: GPX2 was strongly expressed in low grade and low MIB-1 index cancers. PFS and CSS rates were significantly better in patients with higher GPX2 than in those with lower GPX2. Furthermore, GPX2 expression was significantly lower in the normal epithelium of the control group of animals with bladder cancer when compared with those in the treated group, and GPX2 expression was significantly higher in urothelial cancer than in the normal epithelium. BC31 and RT4 cells strongly expressed GPX2 when compared with the other cell lines. Silencing of GPX2 caused significant growth inhibition, and the DCFH assay revealed significant reductions in ROS levels in the siRNAtreated cells. Caspase-dependent apoptosis was fund to be the cause for the decrease in proliferation rates in the siRNA group. Interestingly, tumor growth was significantly inhibited in the BC31-implanted nude mice using the siRNA strategy for Gpx2. CONCLUSIONS: Our findings demonstrated that GPX2 plays several important roles in carcinogenesis through the regulation of apoptosis against intracellular ROS, and may be considered as a novel marker or therapeutic target in bladder cancer.