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.

Supplementary Figure 1 from The Nuclear Factor-κB Pathway Controls the Progression of Prostate Cancer to Androgen-Independent Growth

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.

You have accessJournal of UrologyCME1 May 2022PD16-10 5-ALPHA REDUCTASE INHIBITORS SUPPRESS DIHYDROTESTOSTERONE IN BENIGN PROSTATE HYPERPLASIA REQUIRING SURGERY, AND CAUSE STEROIDOGENESIS VIA 5ARI INDUCED PATHWAY Connor Forbes, Nicole L Miller, Thomas Case, Douglas Strand, Qi Liu, Marisol Ramirez-Solano, Justin M. Cates, Ned A. Porter, Hye-Young H. Kim, Phillip Wages, James L. Mohler, Robert J. Matusik, and Ren Jie Jin Connor ForbesConnor Forbes More articles by this author , Nicole L MillerNicole L Miller More articles by this author , Thomas CaseThomas Case More articles by this author , Douglas StrandDouglas Strand More articles by this author , Qi LiuQi Liu More articles by this author , Marisol Ramirez-SolanoMarisol Ramirez-Solano More articles by this author , Justin M. CatesJustin M. Cates More articles by this author , Ned A. PorterNed A. Porter More articles by this author , Hye-Young H. KimHye-Young H. Kim More articles by this author , Phillip WagesPhillip Wages More articles by this author , James L. MohlerJames L. Mohler More articles by this author , Robert J. MatusikRobert J. Matusik More articles by this author , and Ren Jie JinRen Jie Jin More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002548.10AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: 5-alpha reductase inhibitors (5ARIs) reduce the concentration of the active metabolite dihydrotestosterone (DHT) by inhibiting the conversion of testosterone (T) to DHT. Some patients have progression despite maximal medical therapy including with 5ARIs. How these patients escape 5ARI suppression of DHT is not previously established. METHODS: With Institutional Review Board approval, tissue from patients with BPH requiring surgery was collected (Surgical BPH; S-BPH). For controls, non-cancerous transitional zone tissue from patients who underwent radical prostatectomy for low grade prostate cancer was collected (Incidental BPH; I-BPH). Concentrations of androgens and androgen precursors were analyzed, as were other steroid levels. In vitro BPH cell lines were stimulated with the steroid dexamethasone based upon results and assessed for morphologic changes. RNA-sequencing was performed on patient and in vitro cells. RESULTS: Patients who underwent surgery for BPH who were taking 5ARIs had low DHT and elevated precursor T. Other DHT precursors dehydroepiandrosterone (DHEA) and androstenedione (ASD) were also elevated. A 5ARI induced pathway was noted in patients on this medicine, whereby certain glucocorticoids were elevated, through a suggested mechanism in Figure 1. Dexamethasone induced morphologic changes to in vitro cells lines; branching-type morphology was induced compared to controls in 4 BPH cell lines. RNA-sequencing of patient tissue and in vitro cells yielded a unique signature of upregulated genes. CONCLUSIONS: Patients on 5ARIs who progress to requiring surgery for BPH have elevated precursor T and lowered DHT. This confirms that there is no successful escape mechanism either through direct or backdoor pathways for DHT synthesis for patients on 5ARIs who fail medical therapy. A 5ARI-induced glucocorticoid pathway was observed. Glucocorticoids induce morphologic changes in vitro stimulating prostate branching. Source of Funding: The Bray Foundation, NIDDK 5R01 DK111554 to RJ, R01 DK115477 to DWS. This work was supported by National Cancer Institute (NCI) grant P30CA016056 and used the Roswell Park Comprehensive Cancer Center Bioanalytical, Metabolomics, and Pharmacokinetics Shared Resource (BMPK) © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e272 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Connor Forbes More articles by this author Nicole L Miller More articles by this author Thomas Case More articles by this author Douglas Strand More articles by this author Qi Liu More articles by this author Marisol Ramirez-Solano More articles by this author Justin M. Cates More articles by this author Ned A. Porter More articles by this author Hye-Young H. Kim More articles by this author Phillip Wages More articles by this author James L. Mohler More articles by this author Robert J. Matusik More articles by this author Ren Jie Jin More articles by this author Expand All Advertisement PDF DownloadLoading ...

Clinical management of castration-resistant prostate cancer (CRPC) resulting from androgen deprivation therapy (ADT) remains challenging. Many studies indicate that androgen receptor splice variants (ARVs) play a critical role in the development of CRPC, including resistance to the new generation of inhibitors of androgen receptor (AR) action. ARVs are constitutively active and lack the ligand-binding domain (LBD), thereby allowing prostate cancer (PC) to maintain AR activity despite therapies that target the AR (full-length AR; AR-FL). Previously, we have reported that long-term ADT increases the neuroendocrine (NE) hormone – Gastrin Releasing Peptide (GRP) and its receptor (GRP-R) expression in PC cells. Further, we demonstrated that activation of GRP/GRP-R signaling increases ARVs expression by activating NF-κB signaling, thereby promoting cancer progression to CRPC. Most importantly, as a cell surface protein, GRP-R is easily targeted by drugs to block GRP/GRP-R signaling. In this study, we tested if blocking GRP/GRP-R signaling by targeting GRP-R using GRP-R antagonist is sufficient to control CRPC progression. Our studies show that blocking GRP/GRP-R signaling by targeting GRP-R using RC-3095, a selective GRP-R antagonist, efficiently inhibits NF-κB activity and ARVs (AR-V7) expression in CRPC and therapy-induced NEPC (tNEPC) cells. In addition, blocking of GRP/GRP-R signaling by targeting GRP-R can sensitize CRPC cells to anti-androgen treatment (such as MDV3100). Further, preclinical animal studies indicate combination of GRP-R antagonist (targeting ARVs) with anti-androgen (targeting AR-FL) is sufficient to inhibit CRPC and tNEPC tumor growth.

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 5 alpha-reductase inhibitor (5ARI) or an alpha-adrenergic receptor antagonist (alpha-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 PGE(2) and PGF(2) 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 alpha-blockers alone, 5ARI alone, combination therapy (alpha-blockers plus 5ARI), or no medical therapy (none) before surgical resection. I-BPH tissue was subgrouped by prior therapy (either on alpha-blockers or without prior medical therapy before prostatectomy). We measured prostatic tissue levels of prostaglandins (PGF(2 alpha), PGI(2), PGE(2), PGD(2), and TxA(2)), 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 PGH(2) to metabolite PGs were variable: PTGIS (which generates PGI(2)) and PTGDS (PGD(2)) were significantly elevated; nonsignificant increases were observed for PTGES (PGE(2)), AKR1C3 (PGF(2 alpha)), and TBxAS1 (TxA(2)). Within the I-BPH group, men responding to alpha-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. PGE(2) and PGF(2 alpha) induced contraction of bladder smooth muscle in an in vitro assay. Furthermore, using the smooth muscle assay, we demonstrated that alpha-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 alpha-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 alpha-blocker treatment.

Abstract 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.

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 use LUTS and BPH interchangeably. More recently, LUTS were also linked to fibrogenic and inflammatory processes. We tested whether osteopontin (OPN), a pro-inflammatory and pro-fibrotic molecule, is increased in symptomatic BPH. We also tested whether prostate epithelial and stromal cells secrete OPN in response to pro-inflammatory stimuli and identified downstream targets of OPN in prostate stromal cells. Methods Immunohistochemistry was performed on prostate sections obtained from the transition zone (TZ) of patients who underwent surgery (Holmium laser enucleation of the prostate) to relieve LUTS i.e. 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. The ability of prostate cells to secrete osteopontin in response to IL-1β and TGF-β1 was determined in stromal (BHPrS-1) and epithelial (NHPrE-1 and BHPrE-1) cells by ELISA. qPCR was used to measure gene expression changes in these cells in response to OPN. Results OPN immunostaining (p=0.0107) and protein levels were more abundant in S-BPH than I-BPH. Staining was distributed across all cell types with highest levels in epithelial cells. Multiple OPN protein variants were identified in immortalized prostate stromal and epithelial cells. TGF-β1 stimulated OPN secretion by NHPrE-1 cells and both IL-1β and TGF-β1 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 pro-inflammatory cytokines, and OPN acts directly on stromal cells to drive the synthesis of pro-inflammatory mRNAs. Pharmacological manipulation of prostatic OPN may have the potential to reduce LUTS by inhibiting both inflammatory and fibrotic pathways.

You have accessJournal of UrologyBenign Prostatic Hyperplasia: Basic Research & Pathophysiology (MP06)1 Apr 2020MP06-06 INCREASED EXPRESSION OF OSTEOPONTIN IN THE PROSTATE IS ASSOCIATED WITH THE CLINICAL PROGRESSION OF BPH Petra Popovics*, Wisam N. Awadallah, Sarah Kohrt, Thomas C. Case, Nicole L. Miller, Emily Ricke, Wei Huang, Marisol Ramirez-Solano, Qi Liu, Robert J. Matusik, William A. Ricke, and Magdalena M. Grabowska Petra Popovics*Petra Popovics* More articles by this author , Wisam N. AwadallahWisam N. Awadallah More articles by this author , Sarah KohrtSarah Kohrt More articles by this author , Thomas C. CaseThomas C. Case More articles by this author , Nicole L. MillerNicole L. Miller More articles by this author , Emily RickeEmily Ricke More articles by this author , Wei HuangWei Huang More articles by this author , Marisol Ramirez-SolanoMarisol Ramirez-Solano More articles by this author , Qi LiuQi Liu More articles by this author , Robert J. MatusikRobert J. Matusik More articles by this author , William A. RickeWilliam A. Ricke More articles by this author , and Magdalena M. GrabowskaMagdalena M. Grabowska More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000820.06AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Approximately 30% of men with lower urinary tract symptoms (LUTS) are resistant to medical therapies and progress to surgical intervention. The pathological changes producing LUTS include prostatic proliferation identified histologically as benign prostatic hyperplasia (BPH). Fibrogenic and inflammatory processes are also implicated in LUTS. Identification of new molecular pathways that provoke inflammatory responses and fibrosis, which are not targeted by current therapies, are vital to improve treatments of LUTS. Our study compared the expression of osteopontin (OPN), a pro-inflammatory and fibrotic molecule, in the prostate of patients with BPH characterized as incidental BPH, or as progressed to surgery. We also identified stimulatory signals contributing to the prostatic expression of OPN, as well as, genes that are activated in response to its elevated levels in vitro. METHODS: Immunohistochemistry was performed on prostate sections obtained from patients who had either undergone surgery to relieve LUTS (S-BPH, n=30) or had incidental BPH (I-BPH). I-BPH refers to specimens isolated from the transition zone of prostates after radical prostatectomy for low grade prostate cancer (n=8). Stained slides were captured with a Nuance Multispectral Imaging system and scored with inForm software. Protein level of OPN was determined by Western blot. The ability of prostate cells to secrete osteopontin in response to IL-1β and TGF-β1 and the expressional changes in genes regulated by OPN were determined in stromal (BHPrS-1) and epithelial (NHPrE-1) cell lines by ELISA and qPCR, respectively. RESULTS: The S-BPH group had significantly elevated H-score (p=0.0107, Mann-Whitney U test) and increased protein levels of OPN. OPN expression showed a widespread tissue distribution with a visibly higher level in glandular cells. We found that multiple splice variants of OPN are expressed in stromal and epithelial cells and its secretion is stimulated by TGF-β1 in NHPrE-1 and IL-1β and TGF-β1 in BHPrS-1 cells. Interestingly, we observed an increase in the expression of inflammatory genes in response to OPN including CXCL1, CXCL2, CXCL8, PTGS2 and IL6 in BHPrS-1, but this effect was not replicated in epithelial cell lines. CONCLUSIONS: Increased OPN levels are associated with the progression of BPH which appears to be related to inflammatory processes since OPN secretion by prostate cells is stimulated by cytokines. Pharmacological manipulation of prostatic OPN may have the potential to reduce LUTS by inhibiting inflammatory and fibrotic pathways. Source of Funding: NIDDK K12 DK100022 to PP, CWRU Start-up fund to MMG, 5R01 DK111554-03 to RJM, U54 DK104310 to WAR © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e53-e53 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Petra Popovics* More articles by this author Wisam N. Awadallah More articles by this author Sarah Kohrt More articles by this author Thomas C. Case More articles by this author Nicole L. Miller More articles by this author Emily Ricke More articles by this author Wei Huang More articles by this author Marisol Ramirez-Solano More articles by this author Qi Liu More articles by this author Robert J. Matusik More articles by this author William A. Ricke More articles by this author Magdalena M. Grabowska More articles by this author Expand All Advertisement PDF downloadLoading ...

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.

Purpose Metastasis, the main cause of death from cancer, remains poorly understood at the molecular level. Experimental design Based on a pattern of reduced expression in human prostate cancer tissues and tumor cell lines, a candidate suppressor gene (SPARCL1) was identified. We used in vitro approaches to determine whether overexpression of SPARCL1 affects cell growth, migration, and invasiveness. We then employed xenograft mouse models to analyze the impact of SPARCL1 on prostate cancer cell growth and metastasis in vivo. Results SPARCL1 expression did not inhibit tumor cell proliferation in vitro. By contrast, SPARCL1 did suppress tumor cell migration and invasiveness in vitro and tumor metastatic growth in vivo, conferring improved survival in xenograft mouse models. Conclusions We present the first in vivo data suggesting that SPARCL1 suppresses metastasis of prostate cancer.

The oncoprotein stathmin 1 (STMN1) is upregulated in most, if not all, cancers of epithelial cell origin; therefore STMN1 is considered a target for cancer therapy. However, its role during metastasis has not been investigated. Here, we report for the first time that STMN1 strongly inhibits metastatic behavior in both normal epithelial and cancerous epithelial cells. Initially, loss-of-STMN1 compromises cell-cell adhesion. This is followed by epithelial-to-mesenchymal transition (EMT), increased cell migration, and metastasis via cooperative activation of p38 and through TGF-β-independent and -dependent mechanisms. In contrast, expressing STMN1 restores cell-cell adhesion and reverses the metastatic cascade. Primary prostate epithelial cell cultures from benign to undifferentiated adenocarcinoma (UA) clinical biopsies show that EMT-like cells arise while the cancer is still organ-confined and that their emergence is tumor-stage specific. Furthermore, primary EMT-like cells exhibit metastatic behavior both in vitro and in vivo as compared with their non-EMT counterpart. These observations predict that using STMN1 as a generic therapeutic target might accelerate metastasis. Instead, there may be a tumor stage-specific window-of-opportunity in which conserving STMN1 expression is required to inhibit emergence of metastatic disease.

BACKGROUND. The androgen-regulated probasin (PB) promoter has been used extensively to target transgenes to the prostate in transgenic mice; however, limited data exist on the mechanism that dictates prostate-specific gene expression. Tissue-specific gene expression involves synergistic effects among transcription factors associated in a complex bound to cis-acting DNA elements.METHODS. Using comprehensive linker scan mutagenesis, enzyme mobility shift and supershift assays, chromatin immunoprecipitation, and transgenic animal studies, we have extensively characterized the prostate-specific PB promoter.RESULTS. We identified a series of nonreceptor transcription factors that are bound to the prostate-specific rat PB promoter. These factors include several ubiquitously distributed proteins known to participate in steroid receptor-mediated transcription. In addition, we identified two tissue-specific DNA elements that are crucial in directing prostate-specific PB expression, and confirmed the functional importance of both elements in transgenic animal studies. These two elements are functionally interchangeable and can be bound by multiple protein complexes, including the forkhead transcription factor FoxA1, a "pioneer factor" that has a restricted distribution to some cells type that are ectoderm and endoderm in origin. Using transgenic mice, we further demonstrate that the minimal PB promoter region (-244/-96 bp) that encompasses these tissue-specific elements results in prostate-specific gene expression in transgenic mice, contains androgen receptor and FoxA1-binding sites, as well as ubiquitous transcription factor binding sites.CONCLUSION. We propose that these sequence-specific DNA-binding proteins, including tissue-restricted and ubiquitous factors, create the first level of transcriptional control, which responds to intracellular pathways that directs prostate-specific gene expression. Prostate 70: 934-951, 2010. (c) 2010 Wiley-Liss. Inc.

BACKGROUND. Hepsin is a cell surface protease that is over-expressed in more than 90% of human prostate cancer cases. The previously developed Probasin-hepsin/Large Probasin-T antigen (PB-hepsin/LPB-Tag) bigenic mouse model of prostate cancer demonstrates that hepsin promotes primary tumors that are a mixture of adenocarcinoma and neuroendocrine (NE) lesions, and metastases that are NE in nature. However, since the majority of human prostate tumors are adenocarcinomas, the contribution of hepsin in the progression of adenocarcinoma requires further investigation.METHODS. We crossed the PB-hepsin mice with PB-Hi-myc transgenic mouse model of prostate adenocarcinoma and characterized the tumor progression in the resulting PB-hepsin/PB-Hi-myc bigenic mice.RESULTS. We report that PB-hepsin/PB-Hi-myc bigenic mice develop invasive adenocarcinoma at 4.5 months. Further, histological analysis of the 12- to 17-month-old mice revealed that the PB-hepsin/PB-Hi-myc model develops a higher grade adenocarcinoma compared with age-matched tumors expressing only PB-Hi-myc. Consistent with targeting hepsin to the prostate, the PB-hepsin/PB-Hi-myc tumors showed higher hepsin expression as compared to the age-matched myc tumors. Furthermore, endogenous expression of hepsin increased in the PB-Hi-myc mice as the tumors progressed.CONCLUSIONS. Although we did not detect any metastases from the prostates in either the PB-hepsin/PB-Hi-myc or the PB-Hi-myc mice, our data suggests that hepsin and myc cooperate during the progression to high-grade prostatic adenocarcinoma. Prostate 70: 591-600, 2010. (C) 2009 Wiley-Liss, Inc.

BACKGROUND:Neuronal synaptic junction protein δ-catenin (CTNND2) is often overexpressed in prostatic adenocarcinomas but the mechanisms of its activation are unknown. To address this question, we studied the hypothesis that Hes1, human homolog of Drosophila Hairy and enhancer of split (Hes) 1, is a transcriptional repressor of δ-catenin expression and plays an important role in molecular carcinogenesis.RESULTS:We identified that, using a δ-catenin promoter reporter assay, Hes1, but not its inactive mutant, significantly repressed the upregulation of δ-catenin-luciferase activities induced by E2F1. Hes1 binds directly to the E-boxes on δ-catenin promoter and can reduce the expression of δ-catenin in prostate cancer cells. In prostate cancer CWR22-Rv1 and PC3 cell lines, which showed distinct δ-catenin overexpression, E2F1 and Hes1 expression pattern was altered. The suppression of Hes1 expression, either by γ-secretase inhibitors or by siRNA against Hes1, increased δ-catenin expression. γ-Secretase inhibition delayed S/G2-phase transition during cell cycle progression and induced cell shape changes to extend cellular processes in prostate cancer cells. In neuroendocrine prostate cancer mouse model derived allograft NE-10 tumors, δ-catenin showed an increased expression while Hes1 expression was diminished. Furthermore, E2F1 transcription was very high in subgroup of NE-10 tumors in which Hes1 still displayed residual expression, while its expression was only moderately increased in NE-10 tumors where Hes1 expression was completely suppressed.CONCLUSION:These studies support coordinated regulation of δ-catenin expression by both the activating transcription factor E2F1 and repressive transcription factor Hes1 in prostate cancer progression.

Activation of the androgen receptor is required for normal prostate physiology and in controlling the growth prostate cancer. However, the fact that multiple target organs express androgen receptor and are exposed to circulating androgens, yet fail to express prostate-specific markers and fail to develop androgen-dependent cancers, indicates that androgen receptor alone is not sufficient to dictate normal function and progression to cancer. Therefore, androgen action can be restricted in a given tissue by transcription factors that serve as co-regulators of androgen receptor. How androgen signaling acts in concert with other transcription factors, resulting in tissue-specific gene expression needs to be understood. The establishment of unique transcription factor regulatory networks is responsible, at least in part, to control androgen receptor action (1) in tissue-specific gene expression; (2) organ determination; and (3) cell differentiation. The identification of TF networks involved in these disparate events will allow researchers to elucidate the mechanisms that control prostate development, function, and pathology. Experimental evidence generated by our laboratory and others indicates that members of the Foxa subfamily of transcription factors play an important role in (1) normal prostate development; (2) the determination of prostatic cell fate; and (3) specific types of prostate pathology. This chapter reviews evidence generated by our laboratory and others regarding the important role of the Foxa transcription factors in the regulation of prostate-specific gene regulatory networks.

BACKGROUND:The purpose of this study was to identify candidate metastasis suppressor genes from a mouse allograft model of prostate cancer (NE-10). This allograft model originally developed metastases by twelve weeks after implantation in male athymic nude mice, but lost the ability to metastasize after a number of in vivo passages. We performed high resolution array comparative genomic hybridization on the metastasizing and non-metastasizing allografts to identify chromosome imbalances that differed between the two groups of tumors.RESULTS:This analysis uncovered a deletion on chromosome 2 that differed between the metastasizing and non-metastasizing tumors. Bioinformatics filters were employed to mine this region of the genome for candidate metastasis suppressor genes. Of the 146 known genes that reside within the region of interest on mouse chromosome 2, four candidate metastasis suppressor genes (Slc27a2, Mall, Snrpb, and Rassf2) were identified. Quantitative expression analysis confirmed decreased expression of these genes in the metastasizing compared to non-metastasizing tumors.CONCLUSION:This study presents combined genomics and bioinformatics approaches for identifying potential metastasis suppressor genes. The genes identified here are candidates for further studies to determine their functional role in inhibiting metastases in the NE-10 allograft model and human prostate cancer.

Abstract Typically, the initial response of a prostate cancer patient to androgen ablation therapy is regression of the disease. However, the tumor will progress to an “androgen-independent” stage that results in renewed growth and spread of the cancer. Both nuclear factor-κB (NF-κB) expression and neuroendocrine differentiation predict poor prognosis, but their precise contribution to prostate cancer progression is unknown. This report shows that secretory proteins from neuroendocrine cells will activate the NF-κB pathway in LNCaP cells, resulting in increased levels of active androgen receptor (AR). By blocking NF-κB signaling in vitro, AR activation is inhibited. In addition, the continuous activation of NF-κB signaling in vivo by the absence of the IκBα inhibitor prevents regression of the prostate after castration by sustaining high levels of nuclear AR and maintaining differentiated function and continued proliferation of the epithelium. Furthermore, the NF-κB pathway was activated in the ARR2PB-myc-PAI (Hi-myc) mouse prostate by cross-breeding into a IκBα+/− haploid insufficient line. After castration, the mouse prostate cancer continued to proliferate. These results indicate that activation of NF-κB is sufficient to maintain androgen-independent growth of prostate and prostate cancer by regulating AR action. Thus, the NF-κB pathway may be a potential target for therapy against androgen-independent prostate cancer. [Cancer Res 2008;68(16):6762–9]

Androgen receptor (AR) within prostatic mesenchymal cells, with the absence of AR in the epithelium, is still sufficient to induce prostate development. AR in the luminal epithelium is required to express the secretory markers associated with differentiation. Nkx3.1 is expressed in the epithelium in early prostatic embryonic development and expression is maintained in the adult. Induction of the mouse prostate gland by the embryonic mesenchymal cells results in the organization of a sparse basal layer below the luminal epithelium with rare neuroendocrine cells that are interdispersed within this basal layer. The human prostate shows similar glandular organization; however, the basal layer is continuous. The strong inductive nature of embryonic prostatic and bladder mesenchymal cells is demonstrated in grafts where embryonic stem (ES) cells are induced to differentiate and organize as a prostate and bladder, respectively. Further, the ES cells can be driven by the correct embryonic mesenchymal cells to form epithelium that differentiates into secretory prostate glands and differentiated bladders that produce uroplakin. This requires the ES cells to mature into endoderm that gives rise to differentiated epithelium. This process is control by transcription factors in both the inductive mesenchymal cells (AR) and the responding epithelium (FoxA1 and Nkx3.1) that allows for organ development and differentiation. In this review, we explore a molecular mechanism where the pattern of transcription factor expression controls cell determination, where the cell is assigned a developmental fate and subsequently cell differentiation, and where the assigned cell now emerges with it's own unique character.