Magdalena M. Grabowska

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.

FOXA1 deletion in luminal epithelium causes prostatic hyperplasia and alteration of differentiated phenotype

The forkhead box (Fox) superfamily of transcription factors has essential roles in organogenesis and tissue differentiation. Foxa1 and Foxa2 are expressed during prostate budding and ductal morphogenesis, whereas Foxa1 expression is retained in adult prostate epithelium. Previous characterization of prostatic tissue rescued from embryonic Foxa1 knockout mice revealed Foxa1 to be essential for ductal morphogenesis and epithelial maturation. However, it is unknown whether Foxa1 is required to maintain the differentiated status in adult prostate epithelium. Here, we employed the PBCre4 transgenic system and determined the impact of prostate-specific Foxa1 deletion in adult murine epithelium. PBCre4/Foxa1loxp/loxp mouse prostates showed progressive florid hyperplasia with extensive cribriform patterning, with the anterior prostate being most affected. Immunohistochemistry studies show mosaic Foxa1 KO consistent with PBCre4 activity, with Foxa1 KO epithelial cells specifically exhibiting altered cell morphology, increased proliferation, and elevated expression of basal cell markers. Castration studies showed that, while PBCre4/Foxa1loxp/loxp prostates did not exhibit altered sensitivity in response to hormone ablation compared with control prostates, the number of Foxa1-positive cells in mosaic Foxa1 KO prostates was significantly reduced compared with Foxa1-negative cells following castration. Unexpectedly, gene expression profile analyses revealed that Foxa1 deletion caused abnormal expression of seminal vesicle-associated genes in KO prostates. In summary, these results indicate Foxa1 expression is required for the maintenance of prostatic cellular differentiation.

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.

SOX2 expression in the developing, adult, as well as, diseased prostate.

Background:SOX2 is a member of SOX (SRY-related high mobility group box) family of transcription factors.Methods:In this study, we examined the expression of SOX2 in murine and human prostatic specimens by immunohistochemistry.Results:We found that SOX2 was expressed in murine prostates during budding morphogenesis and in neuroendocrine (NE) prostate cancer (PCa) murine models. Expression of SOX2 was also examined in human prostatic tissue. We found that SOX2 was expressed in 26 of the 30 BPH specimens. In these BPH samples, expression of SOX2 was limited to basal epithelial cells. In contrast, 24 of the 25 primary PCa specimens were negative for SOX2. The only positive primary PCa was the prostatic NE tumor, which also showed co-expression of synaptophysin. Additionally, the expression of SOX2 was detected in all prostatic NE tumor xenograft lines. Furthermore, we have examined the expression of SOX2 on a set of tissue microarrays consisting of metastatic PCa tissues. Expression of SOX2 was detected in at least one metastatic site in 15 of the 24 patients with metastatic castration-resistant PCa; and the expression of SOX2 was correlated with synaptophysin.Conclusions:SOX2 was expressed in developing prostates, basal cells of BPH, as well as prostatic NE tumors.

NF‐κB and androgen receptor variant expression correlate with human BPH progression

Benign prostatic hyperplasia (BPH) is a common, chronic progressive disease. Inflammation is associated with prostatic enlargement and resistance to 5α‐reductase inhibitor (5ARI) therapy. Activation of the nuclear factor‐kappa B (NF‐κB) pathway is linked to both inflammation and ligand‐independent prostate cancer progression.

Nfib Regulates Transcriptional Networks That Control the Development of Prostatic Hyperplasia

A functional complex consisting of androgen receptor (AR) and forkhead box A1 (FOXA1) proteins supports prostatic development, differentiation, and disease. In addition, the interaction of FOXA1 with cofactors such as nuclear factor I (NFI) family members modulates AR target gene expression. However, the global role of specific NFI family members has yet to be described in the prostate. In these studies, chromatin immunoprecipitation followed by DNA sequencing in androgen-dependent LNCaP prostate cancer cells demonstrated that 64.3% of NFIB binding sites are associated with AR and FOXA1 binding sites. Interrogation of published data revealed that genes associated with NFIB binding sites are predominantly induced after dihydrotestosterone treatment of LNCaP cells, whereas NFIB knockdown studies demonstrated that loss of NFIB drives increased AR expression and superinduction of a subset of AR target genes. Notably, genes bound by NFIB only are associated with cell division and cell cycle. To define the role of NFIB in vivo, mouse Nfib knockout prostatic tissue was rescued via renal capsule engraftment. Loss of Nfib expression resulted in prostatic hyperplasia, which did not resolve in response to castration, and an expansion of an intermediate cell population in a small subset of grafts. In human benign prostatic hyperplasia, luminal NFIB loss correlated with more severe disease. Finally, some areas of intermediate cell expansion were also associated with NFIB loss. Taken together, these results show a fundamental role for NFIB as a coregulator of AR action in the prostate and in controlling prostatic hyperplasia.

Activation of GRP/GRP-R signaling contributes to castration-resistant prostate cancer progression

Numerous studies indicate that androgen receptor splice variants (ARVs) play a critical role in the development of castration-resistant prostate cancer (CRPC), including the resistance to the new generation of inhibitors of androgen receptor (AR) action. Previously, we demonstrated that activation of NF-κB signaling increases ARVs expression in prostate cancer (PC) cells, thereby promoting progression to CRPC. However, it is unclear how NF-κB signaling is activated in CRPC. In this study, we report that long-term treatment with anti-androgens increases a neuroendocrine (NE) hormone — gastrin-releasing peptide (GRP) and its receptor (GRP-R) expression in PC cells. In addition, activation of GRP/GRP-R signaling increases ARVs expression through activating NF-κB signaling. This results in an androgen-dependent tumor progressing to a castrate resistant tumor. The knock-down of AR-V7 restores sensitivity to antiandrogens of PC cells over-expressing the GRP/GRP-R signaling pathway. These findings strongly indicate that the axis of Androgen-Deprivation Therapy (ADT) induces GRP/GRP-R activity, activation NF-κB and increased levels of AR-V7 expression resulting in progression to CRPC. Both prostate adenocarcinoma and small cell NE prostate cancer express GRP-R. Since the GRP-R is clinically targetable by analogue-based approach, this provides a novel therapeutic approach to treat advanced CRPC.

Androgen receptor differentially regulates the proliferation of prostatic epithelial cells in vitro and in vivo

Androgens regulate the proliferation and differentiation of prostatic epithelial cells, including prostate cancer (PCa) cells in a context-dependent manner. Androgens and androgen receptor (AR) do not invariably promote cell proliferation; in the normal adult, endogenous stromal and epithelial AR activation maintains differentiation and inhibits organ growth. In the current study, we report that activation of AR differentially regulates the proliferation of human prostate epithelial progenitor cells, NHPrE1, in vitro and in vivo. Inducing AR signaling in NHPrE1 cells suppressed cell proliferation in vitro, concomitant with a reduction in MYC expression. However, ectopic expression of AR in vivo stimulated cell proliferation and induced development of invasive PCa in tissue recombinants consisting of NHPrE1/AR cells and rat urogenital mesenchymal (UGM) cells, engrafted under renal capsule of adult male athymic mice. Expression of MYC increased in the NHPrE1/AR recombinant tissues, in contrast to the reduction seen in vitro. The inhibitory effect of AR signaling on cell proliferation in vitro were reduced by co-culturing NHPrE1/AR epithelial cells with prostatic stromal cells. In conclusion, these studies revealed that AR signaling differentially regulates proliferation of human prostatic epithelia cells in vitro and in vivo through mechanisms involving stromal/epithelial interactions.

NF-κB and androgen receptor variant 7 induce expression of SRD5A isoforms and confer 5ARI resistance.

Benign prostatic hyperplasia (BPH) is treated with 5α‐reductase inhibitors (5ARI). These drugs inhibit the conversion of testosterone to dihydrotestosterone resulting in apoptosis and prostate shrinkage. Most patients initially respond to 5ARIs; however, failure is common especially in inflamed prostates, and often results in surgery. This communication examines a link between activation of NF‐κB and increased expression of SRD5A2 as a potential mechanism by which patients fail 5ARI therapy.

Abstract B23: Neuroendocrine differentiation in prostate cancer and development

Introduction: It is now recognized that androgen deprivation therapy (ADT) can induce neuroendocrine (NE) differentiation (NED) in prostate cancer (PCa) as well as histologically distinct NE cancer in >25% of castrate resistant prostate cancer (CRPCa). Neuroendocrine cells, which are present but rare in the normal prostate, secrete growth factors but the mechanism of normal NED in the developing prostate is largely unknown. We hypothesize that the neuroendocrine phenotype, seen in CRPCa, recaptures events that occurs during normal prostatic development in response to the androgens. The purpose of these experiments is to determine if NED occurs in the development of the normal mouse prostate in response to androgen. Methods: 8-10 week old male mice (n=7 per group) were castrated and 2 weeks post-castration were treated with androgens (DHT) or oil (control), and then euthanized 1, 2 or 4 hours after treatment. Prostate lobes were then dissected, analyzed by immunohistochemistry for synaptophysin, a marker of NED, and scored blindly (Table 1). Intensity and distribution were then summed and analyzed using the Mann-Whitney test. ScoreIntensityDistribution 0 No expression No expression 1 Weak Antibody expression in 0-33% of epithelial cells 2 Mild Antibody expression in 33%-66% of epithelial cells 3 Strong Antibody expression in 66%- 100% of epithelial cells Table 1: Immunohistochemistry scoring system Results: No significant difference in synaptophysin staining was found in the epithelium between oil and DHT treated samples in the anterior, dorsal, and lateral prostate. There was a significant difference in synaptophysin staining found between oil and DHT treated samples at the 1 hour time point in the ventral prostate (p Conclusion: Androgens induce rapid and transient NED in the murine ventral prostate epithelium, indicated by a spike in synaptophysin at the 1 hour time point. By 2 hours, there is no significant difference in synaptophysin staining between the oil and DHT treated samples. This transient elevation in a NED marker is only observed in the ventral lobe. This indicates that CRPCa may recapture events that occur in the growth of the normal prostate. Citation Format: Danielle N. Sanders, Magdalena Grabowska, Robert Matusik. Neuroendocrine differentiation in prostate cancer and development. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr B23.