Transcriptomic analysis of benign prostatic hyperplasia identifies critical pathways in prostatic overgrowth and 5-alpha reductase inhibitor resistance.

BACKGROUND: The medical therapy of prostatic symptoms (MTOPS) trial randomized men with symptoms of benign prostatic hyperplasia (BPH) and followed response of treatment with a 5α-reductase inhibitor (5ARI), an alpha-adrenergic receptor antagonist (α-blocker), the combination of 5ARI and α-blocker or no medical therapy (none). Medical therapy reduced risk of clinical progression by 66% but the reasons for nonresponse or loss of therapeutic response in some patients remains unresolved. Our previous work showed that prostatic glucocorticoid levels are increased in 5ARI-treated patients and that glucocorticoids can increased branching of prostate epithelia in vitro. To understand the transcriptomic changes associated with 5ARI treatment, we performed bulk RNA sequencing of BPH and control samples from patients who received 5ARI versus those that did not. Deconvolution analysis was performed to estimate cellular composition. Bulk RNA sequencing was also performed on control versus glucocorticoid-treated prostate epithelia in 3D culture to determine underlying transcriptomic changes associated with branching morphogenesis. METHOD: Surgical BPH (S-BPH) tissue was defined as benign prostatic tissue collected from the transition zone (TZ) of patients who failed medical therapy while control tissue termed Incidental BPH (I-BPH) was obtained from the TZ of men undergoing radical prostatectomy for low-volume/grade prostatic adenocarcinoma confined to the peripheral zone. S-BPH patients were divided into four subgroups: men on no medical therapy (none: n = 7), α-blocker alone (n = 10), 5ARI alone (n = 6) or combination therapy (α-blocker and 5ARI: n = 7). Control I-BPH tissue was from men on no medical therapy (none: n = 8) or on α-blocker (n = 6). A human prostatic cell line in 3D culture that buds and branches was used to identify genes involved in early prostatic growth. Snap-frozen prostatic tissue taken at the time of surgery and 3D organoids were used for RNA-seq analysis. Bulk RNAseq data were deconvoluted using CIBERSORTx. Differentially expressed genes (DEG) that were statistically significant among S-BPH, I-BPH, and during budding and branching of organoids were used for pathway analysis. RESULTS: Transcriptomic analysis between S-BPH (n = 30) and I-BPH (n = 14) using a twofold cutoff (p < 0.05) identified 377 DEG (termed BPH377) and a cutoff < 0.05 identified 3377 DEG (termed BPH3377). Within the S-BPH, the subgroups none and α-blocker were compared to patients on 5ARI to reveal 361 DEG (termed 5ARI361) that were significantly changed. Deconvolution analysis of bulk RNA seq data with a human prostate single cell data set demonstrated increased levels of mast cells, NK cells, interstitial fibroblasts, and prostate luminal cells in S-BPH versus I-BPH. Glucocorticoid (GC)-induced budding and branching of benign prostatic cells in 3D culture was compared to control organoids to identify early events in prostatic morphogenesis. GC induced 369 DEG (termed GC359) in 3D culture. STRING analysis divided the large datasets into 20-80 genes centered around a hub. In general, biological processes induced in BPH supported growth and differentiation such as chromatin modification and DNA repair, transcription, cytoskeleton, mitochondrial electron transport, ubiquitination, protein folding, and cholesterol synthesis. Identified signaling pathways were pooled to create a list of DEG that fell into seven hubs/clusters. The hub gene centrality was used to name the network including AP-1, interleukin (IL)-6, NOTCH1 and NOTCH3, NEO1, IL-13, and HDAC/KDM. All hubs showed connections to inflammation, chromatin structure, and development. The same approach was applied to 5ARI361 giving multiple networks, but the EGF and sonic hedgehog (SHH) hub was of particular interest as a developmental pathway. The BPH3377, 5ARI363, and GC359 lists were compared and 67 significantly changed DEG were identified. Common genes to the 3D culture included an IL-6 hub that connected to genes identified in BPH hubs that defined AP1, IL-6, NOTCH, NEO1, IL-13, and HDAC/KDM. CONCLUSIONS: Reduction analysis of BPH and 3D organoid culture uncovered networks previously identified in prostatic development as being reinitiated in BPH. Identification of these pathways provides insight into the failure of medical therapy for BPH and new therapeutic targets for BPH/LUTS.

Lysosome-dependent FOXA1 ubiquitination contributes to luminal lineage of advanced prostate cancer.

Changes in FOXA1 (forkhead box protein A1) protein levels are well associated with prostate cancer (PCa) progression. Unfortunately, direct targeting of FOXA1 in progressive PCa remains challenging due to variations in FOXA1 protein levels, increased FOXA1 mutations at different stages of PCa, and elusive post-translational FOXA1 regulating mechanisms. Here, we show that SKP2 (S-phase kinase-associated protein 2) catalyzes K6- and K29-linked polyubiquitination of FOXA1 for lysosomal-dependent degradation. Our data indicate increased SKP2:FOXA1 protein ratios in stage IV human PCa compared to stages I-III, together with a strong inverse correlation (r = -0.9659) between SKP2 and FOXA1 levels, suggesting that SKP2-FOXA1 protein interactions play a significant role in PCa progression. Prostate tumors of Pten/Trp53 mice displayed increased Skp2-Foxa1-Pcna signaling and colocalization, whereas disruption of the Skp2-Foxa1 interplay in Pten/Trp53/Skp2 triple-null mice demonstrated decreased Pcna levels and increased expression of Foxa1 and luminal positive cells. Treatment of xenograft mice with the SKP2 inhibitor SZL P1-41 decreased tumor proliferation, SKP2:FOXA1 ratios, and colocalization. Thus, our results highlight the significance of the SKP2-FOXA1 interplay on the luminal lineage in PCa and the potential of therapeutically targeting FOXA1 through SKP2 to improve PCa control.

Glucocorticoids are induced while dihydrotestosterone levels are suppressed in 5-alpha reductase inhibitor treated human benign prostate hyperplasia patients.

BACKGROUND: The development of benign prostatic hyperplasia (BPH) and medication-refractory lower urinary tract symptoms (LUTS) remain poorly understood. This study attempted to characterize the pathways associated with failure of medical therapy for BPH/LUTS. METHODS: Transitional zone tissue levels of cholesterol and steroids were measured in patients who failed medical therapy for BPH/LUTS and controls. Prostatic gene expression was measured using qPCR and BPH cells were used in organoid culture to study prostatic branching. RESULTS: BPH patients on 5-α-reductase inhibitor (5ARI) showed low levels of tissue dihydrotestosterone (DHT), increased levels of steroid 5-α-reductase type II (SRD5A2), and diminished levels of androgen receptor (AR) target genes, prostate-specific antigen (PSA), and transmembrane serine protease 2 (TMPRSS2). 5ARI raised prostatic tissue levels of glucocorticoids (GC), whereas alpha-adrenergic receptor antagonists (α-blockers) did not. Nuclear localization of GR in prostatic epithelium and stroma appeared in all patient samples. Treatment of four BPH organoid cell lines with dexamethasone, a synthetic GC, resulted in budding and branching. CONCLUSIONS: After failure of medical therapy for BPH/LUTS, 5ARI therapy continued to inhibit androgenesis but a 5ARI-induced pathway increased tissue levels of GC not seen in patients on α-blockers. GC stimulation of organoids indicated that the GC receptors are a trigger for controlling growth of prostate glands. A 5ARI-induced pathway revealed GC activation can serve as a master regulator of prostatic branching and growth.

Fetuin-A Promotes 3-Dimensional Growth in LNCaP Prostate Cancer Cells by Sequestering Extracellular Vesicles to Their Surfaces to Act as Signaling Platforms.

The present studies were conducted to evaluate key serum proteins and other components that mediate anchorage-independent growth (3-D growth) of LNCaP prostate cancer cells as spheroids. The cells were cultured on ultra-low attachment plates in the absence and presence of fetuin-A and with or without extracellular vesicles. The data show that fetuin-A (alpha 2HS glycoprotein) is the serum protein that mediates 3-D growth in these cells. It does so by sequestering extracellular vesicles of various sizes on the surfaces of rounded cells that grow as spheroids. These vesicles in turn transmit growth signals such as the activation of AKT and MAP kinases in a pattern that differs from the activation of these key growth signaling pathways in adherent and spread cells growing in 2-D. In the process of orchestrating the movement and disposition of extracellular vesicles on these cells, fetuin-A is readily internalized in adhered and spread cells but remains on the surfaces of non-adherent cells. Taken together, our studies suggest the presence of distinct signaling domains or scaffolding platforms on the surfaces of prostate tumor cells growing in 3-D compared to 2-D.

MicroRNA-21 deficiency suppresses prostate cancer progression through downregulation of the IRS1-SREBP-1 signaling pathway.

Sterol regulatory element-binding protein 1 (SREBP-1), a master transcription factor in lipogenesis and lipid metabolism, is critical for disease progression and associated with poor outcomes in prostate cancer (PCa) patients. However, the mechanism of SREBP-1 regulation in PCa remains elusive. Here, we report that SREBP-1 is transcriptionally regulated by microRNA-21 (miR-21) in vitro in cultured cells and in vivo in mouse models. We observed aberrant upregulation of SREBP-1, fatty acid synthase (FASN) and acetyl-CoA carboxylase (ACC) in Pten/Trp53 double-null mouse embryonic fibroblasts (MEFs) and Pten/Trp53 double-null mutant mice. Strikingly, miR-21 loss significantly reduced cell proliferation and suppressed the prostate tumorigenesis of Pten/Trp53 mutant mice. Mechanistically, miR-21 inactivation decreased the levels of SREBP-1, FASN, and ACC in human PCa cells through downregulation of insulin receptor substrate 1 (IRS1)-mediated transcription and induction of cellular senescence. Conversely, miR-21 overexpression increased cell proliferation and migration; as well as the levels of IRS1, SREBP-1, FASN, and ACC in human PCa cells. Our findings reveal that miR-21 promotes PCa progression by activating the IRS1/SREBP-1 axis, and targeting miR-21/SREBP-1 signaling pathway can be a novel strategy for controlling PCa malignancy.

The prostaglandin pathway is activated in patients who fail medical therapy for benign prostatic hyperplasia with lower urinary tract symptoms.

BACKGROUND: Little is known about how benign prostatic hyperplasia (BPH) develops and why patients respond differently to medical therapy designed to reduce lower urinary tract symptoms (LUTS). The Medical Therapy of Prostatic Symptoms (MTOPS) trial randomized men with symptoms of BPH and followed response to medical therapy for up to 6 years. Treatment with a 5α-reductase inhibitor (5ARI) or an alpha-adrenergic receptor antagonist (α-blocker) reduced the risk of clinical progression, while men treated with combination therapy showed a 66% decrease in risk of progressive disease. However, medical therapies for BPH/LUTS are not effective in many patients. The reasons for nonresponse or loss of therapeutic response in the remaining patients over time are unknown. A better understanding of why patients fail to respond to medical therapy may have a major impact on developing new approaches for the medical treatment of BPH/LUTS. Prostaglandins (PG) act on G-protein-coupled receptors (GPCRs), where PGE2 and PGF2 elicit smooth muscle contraction. Therefore, we measured PG levels in the prostate tissue of BPH/LUTS patients to assess the possibility that this signaling pathway might explain the failure of medical therapy in BPH/LUTS patients. METHOD: Surgical BPH (S-BPH) was defined as benign prostatic tissue collected from the transition zone (TZ) of patients who failed medical therapy and underwent surgical intervention to relieve LUTS. Control tissue was termed Incidental BPH (I-BPH). I-BPH was TZ obtained from men undergoing radical prostatectomy for low-volume, low-grade prostatic adenocarcinoma (PCa, Gleason score ≤ 7) confined to the peripheral zone. All TZ tissue was confirmed to be cancer-free. S-BPH patients divided into four subgroups: patients on α-blockers alone, 5ARI alone, combination therapy (α-blockers plus 5ARI), or no medical therapy (none) before surgical resection. I-BPH tissue was subgrouped by prior therapy (either on α-blockers or without prior medical therapy before prostatectomy). We measured prostatic tissue levels of prostaglandins (PGF2α , PGI2 , PGE2 , PGD2 , and TxA2 ), quantitative polymerase chain reaction levels of mRNAs encoding enzymes within the PG synthesis pathway, cellular distribution of COX1 (PTGS1) and COX2 (PTGS2), and tested the ability of PGs to contract bladder smooth muscle in an in vitro assay. RESULTS: All PGs were significantly elevated in TZ tissues from S-BPH patients (n = 36) compared to I-BPH patients (n = 15), regardless of the treatment subgroups. In S-BPH versus I-BPH, mRNA for PG synthetic enzymes COX1 and COX2 were significantly elevated. In addition, mRNA for enzymes that convert the precursor PGH2 to metabolite PGs were variable: PTGIS (which generates PGI2 ) and PTGDS (PGD2 ) were significantly elevated; nonsignificant increases were observed for PTGES (PGE2 ), AKR1C3 (PGF2α ), and TBxAS1 (TxA2 ). Within the I-BPH group, men responding to α-blockers for symptoms of BPH but requiring prostatectomy for PCa did not show elevated levels of COX1, COX2, or PGs. By immunohistochemistry, COX1 was predominantly observed in the prostatic stroma while COX2 was present in scattered luminal cells of isolated prostatic glands in S-BPH. PGE2 and PGF2α induced contraction of bladder smooth muscle in an in vitro assay. Furthermore, using the smooth muscle assay, we demonstrated that α-blockers that inhibit alpha-adrenergic receptors do not appear to inhibit PG stimulation of GPCRs in bladder muscle. Only patients who required surgery to relieve BPH/LUTS symptoms showed significantly increased tissue levels of PGs and the PG synthetic enzymes. CONCLUSIONS: Treatment of BPH/LUTS by inhibition of alpha-adrenergic receptors with pharmaceutical α-blockers or inhibiting androgenesis with 5ARI may fail because of elevated paracrine signaling by prostatic PGs that can cause smooth muscle contraction. In contrast to patients who fail medical therapy for BPH/LUTS, control I-BPH patients do not show the same evidence of elevated PG pathway signaling. Elevation of the PG pathway may explain, in part, why the risk of clinical progression in the MTOPS study was only reduced by 34% with α-blocker treatment.

Identification of Genes Required for Enzalutamide Resistance in Castration-Resistant Prostate Cancer Cells In Vitro.

Castration-resistant prostate cancer can be treated with the antiandrogen enzalutamide, but responses and duration of response are variable. To identify genes that support enzalutamide resistance, we performed a short hairpin RNA (shRNA) screen in the bone-homing, castration-resistant prostate cancer cell line, C4-2B. We identified 11 genes (TFAP2C, CAD, SPDEF, EIF6, GABRG2, CDC37, PSMD12, COL5A2, AR, MAP3K11, and ACAT1) whose loss resulted in decreased cell survival in response to enzalutamide. To validate our screen, we performed transient knockdowns in C4-2B and 22Rv1 cells and evaluated cell survival in response to enzalutamide. Through these studies, we validated three genes (ACAT1, MAP3K11, and PSMD12) as supporters of enzalutamide resistance in vitro Although ACAT1 expression is lower in metastatic castration-resistant prostate cancer samples versus primary prostate cancer samples, knockdown of ACAT1 was sufficient to reduce cell survival in C4-2B and 22Rv1 cells. MAP3K11 expression increases with Gleason grade, and the highest expression is observed in metastatic castration-resistant disease. Knockdown of MAP3K11 reduced cell survival, and pharmacologic inhibition of MAP3K11 with CEP-1347 in combination with enzalutamide resulted in a dramatic increase in cell death. This was associated with decreased phosphorylation of AR-Serine650, which is required for maximal AR activation. Finally, although PSMD12 expression did not change during disease progression, knockdown of PSMD12 resulted in decreased AR and AR splice variant expression, likely contributing to the C4-2B and 22Rv1 decrease in cell survival. Our study has therefore identified at least three new supporters of enzalutamide resistance in castration-resistant prostate cancer cells in vitro.

KDM5B Is Essential for the Hyperactivation of PI3K/AKT Signaling in Prostate Tumorigenesis.

KDM5B (lysine[K]-specific demethylase 5B) is frequently upregulated in various human cancers including prostate cancer. KDM5B controls H3K4me3/2 levels and regulates gene transcription and cell differentiation, yet the contributions of KDM5B to prostate cancer tumorigenesis remain unknown. In this study, we investigated the functional role of KDM5B in epigenetic dysregulation and prostate cancer progression in cultured cells and in mouse models of prostate epithelium-specific mutant Pten/Kdm5b. Kdm5b deficiency resulted in a significant delay in the onset of prostate cancer in Pten-null mice, whereas Kdm5b loss alone caused no morphologic abnormalities in mouse prostates. At 6 months of age, the prostate weight of Pten/Kdm5b mice was reduced by up to 70% compared with that of Pten mice. Pathologic analysis revealed Pten/Kdm5b mice displayed mild morphologic changes with hyperplasia in prostates, whereas age-matched Pten littermates developed high-grade prostatic intraepithelial neoplasia and prostate cancer. Mechanistically, KDM5B governed PI3K/AKT signaling in prostate cancer in vitro and in vivo. KDM5B directly bound the PIK3CA promoter, and KDM5B knockout resulted in a significant reduction of P110α and PIP3 levels and subsequent decrease in proliferation of human prostate cancer cells. Conversely, KDM5B overexpression resulted in increased PI3K/AKT signaling. Loss of Kdm5b abrogated the hyperactivation of AKT signaling by decreasing P110α/P85 levels in Pten/Kdm5b mice. Taken together, our findings reveal that KDM5B acts as a key regulator of PI3K/AKT signaling; they also support the concept that targeting KDM5B is a novel and effective therapeutic strategy against prostate cancer. SIGNIFICANCE: This study demonstrates that levels of histone modification enzyme KDM5B determine hyperactivation of PI3K/AKT signaling in prostate cancer and that targeting KDM5B could be a novel strategy against prostate cancer.

Prostatic osteopontin expression is associated with symptomatic benign prostatic hyperplasia.

BACKGROUND: Male lower urinary tract symptoms (LUTS) occur in more than half of men above 50 years of age. LUTS were traditionally attributed to benign prostatic hyperplasia (BPH) and therefore the clinical terminology often uses LUTS and BPH interchangeably. More recently, LUTS were also linked to fibrogenic and inflammatory processes. We tested whether osteopontin (OPN), a proinflammatory and profibrotic molecule, is increased in symptomatic BPH. We also tested whether prostate epithelial and stromal cells secrete OPN in response to proinflammatory stimuli and identified downstream targets of OPN in prostate stromal cells. METHODS: Immunohistochemistry was performed on prostate sections obtained from the transition zone of patients who underwent surgery (Holmium laser enucleation of the prostate) to relieve LUTS (surgical BPH, S-BPH) or patients who underwent radical prostatectomy to remove low-grade prostate cancer (incidental BPH, I-BPH). Images of stained tissue sections were captured with a Nuance Multispectral Imaging System and histoscore, as a measure of OPN staining intensity, was determined with inForm software. OPN protein abundance was determined by Western blot analysis. The ability of prostate cells to secrete osteopontin in response to IL-1ß and TGF-ß1 was determined in stromal (BHPrS-1) and epithelial (NHPrE-1 and BHPrE-1) cells by enzyme-linked immunosorbent assay. Quantitative polymerase chain reaction was used to measure gene expression changes in these cells in response to OPN. RESULTS: OPN immunostaining and protein levels were more abundant in S-BPH than I-BPH. Staining was distributed across all cell types with the highest levels in epithelial cells. Multiple OPN protein variants were identified in immortalized prostate stromal and epithelial cells. TGF-ß1 stimulated OPN secretion by NHPrE-1 cells and both IL-1ß and TGF-ß1 stimulated OPN secretion by BHPrS-1 cells. Interestingly, recombinant OPN increased the mRNA expression of CXCL1, CXCL2, CXCL8, PTGS2, and IL6 in BHPrS-1, but not in epithelial cell lines. CONCLUSIONS: OPN is more abundant in prostates of men with S-BPH compared to men with I-BPH. OPN secretion is stimulated by proinflammatory cytokines, and OPN acts directly on stromal cells to drive the synthesis of proinflammatory mRNAs. Pharmacological manipulation of prostatic OPN may have the potential to reduce LUTS by inhibiting both inflammatory and fibrotic pathways.

Foxa2 activates the transcription of androgen receptor target genes in castrate resistant prostatic tumors.

Prostate cancer (PCa) is the leading cancer among men. Androgen Deprivation Therapy (ADT) is a common treatment for advanced PCa. However, ADT eventually fails and PCa relapses, developing into castration-resistant prostate cancer (CRPCa). Although alternative pathways such as cancer stem-cell pathway and neuroendocrine differentiation bypass androgen receptor (AR) signaling, AR remains the central player in mediating CRPCa. In this study, we identified a mechanism that retains AR signaling after androgen deprivation. The TRAMP SV40 T antigen transgenic mouse is a model for PCa. The expression of SV40 T-antigen is driven by the androgen-responsive, prostate specific, Probasin promoter. It has been recognized that in this model, T-antigen is still expressed even after androgen ablation. It is unclear how the androgen-responsive Probasin promoter remains active and drives the expression of T-antigen in these tumors. In our study, we found that the expression of Foxa2, a forkhead transcription factor that is expressed in embryonic prostate and advanced stage prostate cancer, is co-expressed in T-antigen positive cells. To test if Foxa2 activates AR-responsive promoters and promotes the expression of T-antigen, we established the prostate epithelial cells that stably express Foxa2, NeoTag1/Foxa2 cells. Neotag1 cells were derived from the Probasin promoter driven SV40 T-antigen transgenic mouse. We found ectopic expression of Foxa2 drives the T-antigen expression regardless of the presence of androgens. Using this model system, we further explored the mechanism that activates AR-responsive promoters in the absence of androgens. Chromatin immunoprecipitation revealed the occupancy of both H3K27Ac, an epigenetic mark of an active transcription, and Foxa2 at the known AR target promoters, Probasin and FKBP5, in the absence of androgen stimulation. In conclusion, we have identified a mechanism that enables PCa to retain the AR signaling pathway after androgen ablation.

Role of Androgen Receptor Variants in Prostate Cancer: Report from the 2017 Mission Androgen Receptor Variants Meeting.

CONTEXT: Although a number of studies have demonstrated the importance of constitutively active androgen receptor variants (AR-Vs) in prostate cancer, questions still remain about the precise role of AR-Vs in the progression of castration-resistant prostate cancer (CRPC). OBJECTIVE: Key stakeholders and opinion leaders in prostate cancer convened on May 11, 2017 in Boston to establish the current state of the field of AR-Vs. EVIDENCE ACQUISITION: The meeting "Mission Androgen Receptor Variants" was the second of its kind sponsored by the Prostate Cancer Foundation (PCF). This invitation-only event was attended by international leaders in the field and representatives from sponsoring organizations (PCF and industry sponsors). Eighteen faculty members gave short presentations, which were followed by in-depth discussions. Discussions focused on three thematic topics: (1) potential of AR-Vs as biomarkers of therapeutic resistance; (2) role of AR-Vs as functionally active CRPC progression drivers; and (3) utility of AR-Vs as therapeutic targets in CRPC. EVIDENCE SYNTHESIS: The three meeting organizers synthesized this meeting report, which is intended to summarize major data discussed at the meeting and identify key questions as well as strategies for addressing these questions. There was a critical consensus that further study of the AR-Vs is an important research focus in CRPC. Contrasting views and emphasis, each supported by data, were presented at the meeting, discussed among the participants, and synthesized in this report. CONCLUSIONS: This article highlights the state of knowledge and outlines the most pressing questions that need to be addressed to advance the AR-V field. PATIENT SUMMARY: Although further investigation is needed to delineate the role of androgen receptor (AR) variants in metastatic castration-resistant prostate cancer, advances in measurement science have enabled development of blood-based tests for treatment selection. Detection of AR variants (eg, AR-V7) identified a patient population with poor outcomes to existing AR-targeting therapies, highlighting the need for novel therapeutic agents currently under development.

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.

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.

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

BACKGROUND: 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. METHODS: Tissue was collected from "Surgical" patients, treated specifically for lower urinary tract symptoms secondary to advanced BPH; and, cancer free transition zone from "Incidental" patients treated for low grade, localized peripheral zone prostate cancer. Clinical, molecular and histopathological profiles were analyzed. Human prostatic stromal and epithelial cell lines were genetically modified to regulate NF-κB activity, androgen receptor (AR) full length (AR-FL), and AR variant 7 (AR-V7) expression. RESULTS: SRD5A2 is upregulated in advanced BPH. SRD5A2 was significantly associated with prostate volume determined by Transrectal Ultrasound (TRUS), and with more severe lower urinary tract symptoms (LUTS) determined by American Urological Association Symptom Score (AUASS). Synthesis of androgens was seen in cells in which NF-κB was activated. AR-FL and AR-V7 expression increased SRD5A2 expression while forced activation of NF-κB increased all three SRD5A isoforms. Knockdown of SRD5A2 in the epithelial cells resulted in significant reduction in proliferation, AR target gene expression, and response to testosterone (T). In tissue recombinants, canonical NF-κB activation in prostatic epithelium elevated all three SRD5A isoforms and resulted in in vivo growth under castrated conditions. CONCLUSION: Increased BPH severity in patients correlates with SRD5A2 expression. We demonstrate that NF-κB and AR-V7 upregulate SRD5A expression providing a mechanism to explain failure of 5ARI therapy in BPH patients. Prostate 76:1004-1018, 2016. © 2016 Wiley Periodicals, Inc.

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.

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

BACKGROUND: 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. METHODS: NF-κB activation and androgen receptor variant (AR-V) expression were quantified in transition zone tissue samples from patients with a wide range of AUASS from incidental BPH in patients treated for low grade, localized peripheral zone prostate cancer to advanced disease requiring surgical intervention. To further investigate these pathways, human prostatic stromal and epithelial cell lines were transduced with constitutively active or kinase dead forms of IKK2 to regulate canonical NF-κB activity. The effects on AR full length (AR-FL) and androgen-independent AR-V expression as well as cellular growth and differentiation were assessed. RESULTS: Canonical NF-κB signaling was found to be upregulated in late versus early stage BPH, and to be strongly associated with non-insulin dependent diabetes mellitus. Elevated expression of AR-variant 7 (AR-V7), but not other AR variants, was found in advanced BPH samples. Expression of AR-V7 significantly correlated with the patient AUASS and TRUS volume. Forced activation of canonical NF-κB in human prostatic epithelial and stromal cells resulted in elevated expression of both AR-FL and AR-V7, with concomitant ligand-independent activation of AR reporters. Activation of NF-κB and over expression of AR-V7 in human prostatic epithelial cells maintained cell viability in the face of 5ARI treatment. CONCLUSION: Activation of NF-κB and AR-V7 in the prostate is associated with increased disease severity. AR-V7 expression is inducible in human prostate cells by forced activation of NF-κB resulting in resistance to 5ARI treatment, suggesting a potential mechanism by which patients may become resistant to 5ARI therapy.

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.

SKP2 inactivation suppresses prostate tumorigenesis by mediating JARID1B ubiquitination.

Aberrant elevation of JARID1B and histone H3 lysine 4 trimethylation (H3K4me3) is frequently observed in many diseases including prostate cancer (PCa), yet the mechanisms on the regulation of JARID1B and H3K4me3 through epigenetic alterations still remain poorly understood. Here we report that Skp2 modulates JARID1B and H3K4me3 levels in vitro in cultured cells and in vivo in mouse models. We demonstrated that Skp2 inactivation decreased H3K4me3 levels, along with a reduction of cell growth, cell migration and malignant transformation of Pten/Trp53 double null MEFs, and further restrained prostate tumorigenesis of Pten/Trp53 mutant mice. Mechanistically, Skp2 decreased the K63-linked ubiquitination of JARID1B by E3 ubiquitin ligase TRAF6, thus decreasing JARID1B demethylase activity and in turn increasing H3K4me3. In agreement, Skp2 deficiency resulted in an increase of JARID1B ubiquitination and in turn a reduction of H3K4me3, and induced senescence through JARID1B accumulation in nucleoli of PCa cells and prostate tumors of mice. Furthermore, we showed that the elevations of Skp2 and H3K4me3 contributed to castration-resistant prostate cancer (CRPC) in mice, and were positively correlated in human PCa specimens. Taken together, our findings reveal a novel network of SKP2-JARID1B, and targeting SKP2 and JARID1B may be a potential strategy for PCa control.

Activation of Wnt/ß-catenin signaling in a subpopulation of murine prostate luminal epithelial cells induces high grade prostate intraepithelial neoplasia.

BACKGROUND: Wnt/ß-catenin signaling is important for prostate development and cancer in humans. Activation of this pathway in differentiated luminal cells of mice induces high-grade prostate intraepithelial neoplasia (HGPIN). Though the cell of origin of prostate cancer has yet to be conclusively identified, a castration-resistant Nkx3.1-expressing cell (CARN) may act as a cell of origin for prostate cancer. METHODS: To activate Wnt/ß-catenin signaling in CARNs, we crossed mice carrying tamoxifen-inducible Nkx3.1-driven Cre to mice containing loxP sites in order to either conditionally knock out adenomatous polyposis coli (Apc) or constitutively activate ß-catenin directly. We then castrated and hormonally regenerated these mice to target the CARN population. RESULTS: Loss of Apc in hormonally normal mice induced HGPIN; however, after one or more rounds of castration and hormonal regeneration, Apc-null CARNs disappeared. Alternatively, when ß-catenin was constitutively activated under the same conditions, HGPIN was apparent. CONCLUSION: Activation of Wnt/ß-catenin signaling via Apc deletion is sufficient to produce HGPIN in hormonally normal mice. Loss of Apc may destabilize the CARN population under regeneration conditions. When ß-catenin is constitutively activated, HGPIN occurs in hormonally regenerated mice. A second genetic hit is likely required to cause progression to carcinoma and metastasis.