Dual-function antiandrogen/HDACi hybrids based on enzalutamide and entinostat.

The combination of androgen receptor antagonists with histone deacetylase inhibitors (HDACi) has been shown to be more effective than antiandrogens alone in halting growth of prostate cancer cell lines. Here we have designed, synthesized and assessed a series of antiandrogen/HDACi hybrids by combining structural features of enzalutamide with either SAHA or entinostat. The hybrids are demonstrated to maintain bifunctionality using a fluorometric HDAC assay and a bioluminescence resonance energy transfer (BRET) antiandrogen assay. Antiproliferative assays showed that hybrids bearing o-aminoanilide-based HDACi motifs outperformed hydroxamic acid based HDACi's. The hybrids demonstrated selectivity for epithelial cell lines vs. stromal cell lines, suggesting a potentially useful therapeutic window.

Development of a predictive model for stromal content in prostate cancer samples to improve signature performance.

Prostate cancer is heterogeneous in both cellular composition and patient outcome, and development of biomarker signatures to distinguish indolent from aggressive tumours is a high priority. Stroma plays an important role during prostate cancer progression and undergoes histological and transcriptional changes associated with disease. However, identification and validation of stromal markers is limited by a lack of datasets with defined stromal/tumour ratio. We have developed a prostate-selective signature to estimate the stromal content in cancer samples of mixed cellular composition. We identified stromal-specific markers from transcriptomic datasets of developmental prostate mesenchyme and prostate cancer stroma. These were experimentally validated in cell lines, datasets of known stromal content, and by immunohistochemistry in tissue samples to verify stromal-specific expression. Linear models based upon six transcripts were able to infer the stromal content and estimate stromal composition in mixed tissues. The best model had a coefficient of determination R2 of 0.67. Application of our stromal content estimation model in various prostate cancer datasets led to improved performance of stromal predictive signatures for disease progression and metastasis. The stromal content of prostate tumours varies considerably; consequently, deconvolution of stromal proportion may yield better results than tumour cell deconvolution. We suggest that adjusting expression data for cell composition will improve stromal signature performance and lead to better prognosis and stratification of men with prostate cancer. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Asporin is a stromally expressed marker associated with prostate cancer progression.

BACKGROUND: Prostate cancer shows considerable heterogeneity in disease progression and we propose that markers expressed in tumour stroma may be reliable predictors of aggressive tumour subtypes. METHODS: We have used Kaplan-Meier, univariate and multivariate analysis to correlate the expression of Asporin (ASPN) mRNA and protein with prostate cancer progression in independent cohorts. We used immunohistochemistry and H scoring to document stromal localisation of ASPN in a tissue microarray and mouse prostate cancer model, and correlated expression with reactive stroma, defined using Masson Trichrome staining. We used cell cultures of primary prostate cancer fibroblasts treated with serum-free conditioned media from prostate cancer cell lines to examine regulation of ASPN mRNA in tumour stromal cells. RESULTS: We observed increased expression of ASPN mRNA in a data set derived from benign vs tumour microdissected tissue, and a correlation with biochemical recurrence using Kaplan-Meier and Cox proportional hazard analysis. ASPN protein localised to tumour stroma and elevated expression of ASPN was correlated with decreased time to biochemical recurrence, in a cohort of 326 patients with a median follow up of 9.6 years. Univariate and multivariate analysis demonstrated that ASPN was correlated with progression, as were Gleason score, and clinical stage. Additionally, ASPN expression correlated with the presence of reactive stroma, suggesting that it may be a stromal marker expressed in response to the presence of tumour cells and particularly with aggressive tumour subtypes. We observed expression of ASPN in the stroma of tumours induced by p53 inhibition in a mouse model of prostate cancer, and correlation with neuroendocrine marker expression. Finally, we demonstrated that ASPN transcript expression in normal and cancer fibroblasts was regulated by conditioned media derived from the PC3, but not LNCaP, prostate cancer cell lines. CONCLUSIONS: Our results suggest that ASPN is a stromally expressed biomarker that correlates with disease progression, and is observed in reactive stroma. ASPN expression in stroma may be part of a stromal response to aggressive tumour subtypes.

Androgens and mammalian male reproductive tract development.

One of the main functions of androgen is in the sexually dimorphic development of the male reproductive tissues. During embryogenesis, androgen determines the morphogenesis of male specific organs, such as the epididymis, seminal vesicle, prostate and penis. Despite the critical function of androgens in masculinization, the downstream molecular mechanisms of androgen signaling are poorly understood. Tissue recombination experiments and tissue specific androgen receptor (AR) knockout mouse studies have revealed epithelial or mesenchymal specific androgen-AR signaling functions. These findings also indicate that epithelial-mesenchymal interactions are a key feature of AR specific activity, and paracrine growth factor action may mediate some of the effects of androgens. This review focuses on mouse models showing the interactions of androgen and growth factor pathways that promote the sexual differentiation of reproductive organs. Recent studies investigating context dependent AR target genes are also discussed. This article is part of a Special Issue entitled: Nuclear receptors in animal development.

Expression of pleiotrophin in the prostate is androgen regulated and it functions as an autocrine regulator of mesenchyme and cancer associated fibroblasts and as a paracrine regulator of epithelia.

BACKGROUND: Androgens and paracrine signaling from mesenchyme/stroma regulate development and disease of the prostate, and gene profiling studies of inductive prostate mesenchyme have identified candidate molecules such as pleiotrophin (Ptn). METHODS: Ptn transcripts and protein were localized by in situ and immunohistochemistry and Ptn mRNA was quantitated by Northern blot and qRT-PCR. Ptn function was examined by addition of hPTN protein to rat ventral prostate organ cultures, primary human fetal prostate fibroblasts, prostate cancer associated fibroblasts, and BPH1 epithelia. RESULTS: During development, Ptn transcripts and protein were expressed in ventral mesenchymal pad (VMP) and prostatic mesenchyme. Ptn was localized to mesenchyme surrounding ductal epithelial tips undergoing branching morphogenesis, and was located on the surface of epithelia. hPTN protein stimulated branching morphogenesis and stromal and epithelial proliferation, when added to rat VP cultures, and also stimulated growth of fetal human prostate fibroblasts, prostate cancer associated fibroblasts, and BPH1 epithelia. PTN mRNA was enriched in patient-matched normal prostate fibroblasts versus prostate cancer associated fibroblasts. PTN also showed male enriched expression in fetal human male urethra versus female, and between wt male and ARKO male mice. Transcripts for PTN were upregulated by testosterone in fetal human prostate fibroblasts and organ cultures of female rat VMP. Ptn protein was increased by testosterone in organ cultures of female rat VMP and in rat male urethra compared to female. CONCLUSIONS: Our data suggest that in the prostate Ptn functions as a regulator of both mesenchymal and epithelial proliferation, and that androgens regulate Ptn levels.

Identification of EphrinB1 expression in prostatic mesenchyme and a role for EphB-EphrinB signalling in prostate development.

Paracrine signalling from mesenchyme to epithelium plays a key role in regulating prostate organogenesis and it is important to identify the mesenchymally expressed molecules that regulate organ growth, though currently few such molecules are known. Tyrosine kinase signalling via EphB receptors has been characterised in many developmental processes, and EphB3 mRNA expression was detected in prostate inductive mesenchyme in previous gene profiling studies. This led us to examine the expression and function of EphrinB signalling in prostate development, to determine if EphrinB ligands might function as mesenchymal paracrine regulators of prostate growth. Using PCR, wholemount in situ hybridisation, and immunohistochemistry we examined the expression of EphB receptors and EphrinB ligands in rat prostate during development to determine which showed mesenchymal expression. EphB3 and EphrinB1 transcripts and proteins were expressed in the mesenchyme of developing prostate and in female urogenital mesenchyme and smooth muscle. The function of EphrinB signalling was examined using in vitro organ culture assays of ventral prostate (VP), which were treated with EphB3-Fc and EphrinB1-Fc proteins to inhibit or augment Ephrin signalling. Addition of recombinant EphB3-Fc resulted in a significant decrease in VP organ size, while recombinant EphrinB1-Fc resulted in a significant increase in VP organ size and epithelial proliferation. Additionally, EphrinB1-Fc reduced the degree of epithelial branching in VP organs and increased ductal tip size, though without disrupting normal differentiation. We have identified expression of EphrinB1 in prostatic mesenchyme and suggest that the EphrinB signalling system acts as a regulator of prostate growth. EphrinB-EphB signalling may function as an autocrine regulator of mesenchyme and/or as a paracrine regulator of epithelia.

Involvement of the SLIT/ROBO pathway in follicle development in the fetal ovary.

In humans and domestic mammals, pivotal processes in ovary development, including primordial follicle assembly, occur prenatally. These events are essential for determining fertility in adult life; however, they remain poorly understood at the mechanistic level. In mammals, the SLITs (SLIT1, SLIT2 and SLIT3) and their ROBO (ROBO1, ROBO2, ROBO3/RIG-1 and ROBO4/MAGIC ROBO) receptors regulate neural, leukocyte, vascular smooth muscle cell and endothelial cell migration. In addition, the SLIT/ROBO pathway has functional roles in embryonic development and in the adult ovary by inhibiting cell migration and promoting apoptosis. We therefore characterised follicle formation and investigated the expression and localisation of the ROBO/SLIT pathway in the ovine fetal ovary. Using RT-PCR, we identified SLIT2, SLIT3, ROBO1, ROBO2 and ROBO4 in sheep ovaries harvested across gestation. The real-time quantitative PCR results implied that ROBO2 expression and ROBO4 expression were elevated during the early stages of follicle formation and stayed abundant during primordial follicle maturation (P<0.05). Immunohistochemistry examination demonstrated that ROBO1 was localised to the pre-granulosa cells, while ROBO2, ROBO4 and SLIT2 were expressed in the oocytes of the developing primordial follicle. This indicates that in the fetal ovary, SLIT-ROBO signalling may require an autocrine and paracrine interaction. Furthermore, at the time of increased SLIT-ROBO expression, there was a significant reduction in the number of proliferating oocytes in the developing ovary (P<0.0001). Overall, these results suggest, for the first time, that the SLIT-ROBO pathway is expressed at the time of follicle formation during fetal ovary development.

Enzymically attaching oligosaccharide-linked 'cargoes' to cellulose and other commercial polysaccharides via stable covalent bonds.

The Equisetum enzyme hetero-trans-ß-glucanase (HTG) covalently grafts native plant cellulose (donor-substrate) to xyloglucan (acceptor-substrate), potentially offering a novel 'green' method of cellulose functionalisation. However, the range of cellulosic and non-cellulosic donor substrates that can be utilised by HTG is unknown, limiting our insight into its biotechnological potential. Here we show that HTG binds all celluloses tested (papers, tissues, hydrogels, bacterial cellulose) to radioactively- or fluorescently-labelled xyloglucan-heptasaccharide (XXXGol; acceptor-substrate). Glycol-chitin, glycol-chitosan and chitosan also acted as donor substrates but less effectively than cellulose. Cellulose-XXXGol conjugates were formed throughout the volume of a block of hydrogel, demonstrating penetration. Plant-derived celluloses (cellulose Iß) became more effective donor-substrates after 'mercerisation' in ≥3 M NaOH; the opposite was true for bacterial cellulose Iα. Cellulose-XXXGol bonds resisted boiling 6 M NaOH, demonstrating strong glycosidic bonding. In conclusion, HTG stably grafts native and processed celluloses to xyloglucan-oligosaccharides, which may carry valuable 'cargoes', exemplified by sulphorhodamine. We thus demonstrate HTG's biotechnological potential to modify various cellulose-based substrates such as textiles, pulps, papers, packaging, sanitary products and hydrogels.

A role for notch signaling in stromal survival and differentiation during prostate development.

Notch1 signaling is involved in epithelial growth and differentiation of prostate epithelia, and we have examined the role that notch signaling plays in the stroma of the developing prostate. We initially observed expression of delta-like 1 (Dlk1) and Notch2 in gene profiling studies of prostatic mesenchyme, and anticipated that they might be expressed in a key subset of inductive mesenchyme. Using quantitative RT-PCR, Northern blotting, and whole mount in situ hybridization, we confirmed that both Dlk1 and Notch2 mRNAs showed a restricted expression pattern within subsets of the stroma during prostate development. Localization of Dlk1 and Notch2 proteins mirrored the transcript expression, and showed both distinct and overlapping expression patterns within the stroma. Dlk1 and Notch2 were coexpressed in condensed inductive mesenchyme of the ventral mesenchymal pad (VMP), and were partially colocalized in the smooth muscle (SM) layer of the urethral stroma. In addition, Dlk1 was not expressed in SM adjacent to the VMP in female urethra. The function of notch signaling was examined using organ cultures of prostate rudiments and a small molecule inhibitor of notch receptor activity. Inhibition of notch signaling led to a loss of stromal tissue in both prostate and female VMP cultures, suggesting that this pathway was required for stromal survival. Inhibition of notch signaling also led to changes in both epithelial and stromal differentiation, which was evident in altered distributions of SM alpha-actin and p63 in prostates grown in vitro. The effects of notch signaling upon the stroma were only evident in the presence of testosterone, in contrast to effects upon epithelial differentiation.

Mesenchymal mechanisms in prostate organogenesis.

The development of the prostate is dependent upon androgens and stromal-epithelial interactions. Understanding the molecules and mechanisms by which androgens control prostate organogenesis has been a considerable challenge over the past few decades. Similarly, identifying the molecular signals passing between stromal and epithelial cells has been difficult, and consequently understanding how androgens and stromal-epithelial signalling interact is poorly understood. There remains significant uncertainty regarding how androgens control the growth of the prostate, although several pathways have been identified that are required for prostate development or which alter prostate growth. This review will summarize past findings relating to the pathways that might mediate the effects of androgens as well as molecules that act as stromal to epithelial signals in the prostate. It will also examine the approaches used to identify pathways of importance and the historical concepts that have informed these studies. In particular, the question of which mechanisms might be involved in early prostate organogenesis as well as anatomic aspects of organ induction will be described. Finally, models of prostatic development will be proposed and discussed.

Genome-wide analysis of androgen receptor binding and transcriptomic analysis in mesenchymal subsets during prostate development.

Prostate development is controlled by androgens, the eandrogen receptor (AR) and mesenchymal-epithelial signalling. We used chromatin immunoprecipitation sequencing (ChIP-seq) to define AR genomic binding in the male and female mesenchyme. Tissue- and single-cell-based transcriptional profiling was used to define mesenchymal AR target genes. We observed significant AR genomic binding in females and a strong enrichment at proximal promoters in both sexes. In males, there was greater AR binding to introns and intergenic regions as well as to classical AR binding motifs. In females, there was increased proximal promoter binding and involvement of cofactors. Comparison of AR-bound genes with transcriptomic data enabled the identification of novel sexually dimorphic AR target genes. We validated the dimorphic expression of AR target genes using published datasets and confirmed regulation by androgens using ex vivo organ cultures. AR targets showed variable expression in patients with androgen insensitivity syndrome. We examined AR function at single-cell resolution using single-cell RNA sequencing (scRNA-seq) in male and female mesenchyme. Surprisingly, both AR and target genes were distributed throughout cell subsets, with few positive cells within each subset. AR binding was weakly correlated with target gene expression.

Genome-wide analysis of AR binding and comparison with transcript expression in primary human fetal prostate fibroblasts and cancer associated fibroblasts.

The androgen receptor (AR) is a transcription factor, and key regulator of prostate development and cancer, which has discrete functions in stromal versus epithelial cells. AR expressed in mesenchyme is necessary and sufficient for prostate development while loss of stromal AR is predictive of prostate cancer progression. Many studies have characterized genome-wide binding of AR in prostate tumour cells but none have used primary mesenchyme or stroma. We applied ChIPseq to identify genomic AR binding sites in primary human fetal prostate fibroblasts and patient derived cancer associated fibroblasts, as well as the WPMY1 cell line overexpressing AR. We identified AR binding sites that were specific to fetal prostate fibroblasts (7534), cancer fibroblasts (629), WPMY1-AR (2561) as well as those common among all (783). Primary fibroblasts had a distinct AR binding profile versus prostate cancer cell lines and tissue, and showed a localisation to gene promoter binding sites 1 kb upstream of the transcriptional start site, as well as non-classical AR binding sequence motifs. We used RNAseq to define transcribed genes associated with AR binding sites and derived cistromes for embryonic and cancer fibroblasts as well as a cistrome common to both. These were compared to several in vivo ChIPseq and transcript expression datasets; which identified subsets of AR targets that were expressed in vivo and regulated by androgens. This analysis enabled us to deconvolute stromal AR targets active in stroma within tumour samples. Taken together, our data suggest that the AR shows significantly different genomic binding site locations in primary prostate fibroblasts compared to that observed in tumour cells. Validation of our AR binding site data with transcript expression in vitro and in vivo suggests that the AR target genes we have identified in primary fibroblasts may contribute to clinically significant and biologically important AR-regulated changes in prostate tissue.

Cell-lineage specificity and role of AP-1 in the prostate fibroblast androgen receptor cistrome.

Androgen receptor (AR) signalling in fibroblasts is important in prostate development and carcinogenesis, and is inversely related to prostate cancer mortality. However, the molecular mechanisms of AR action in fibroblasts and other non-epithelial cell types are largely unknown. The genome-wide DNA binding profile of AR in human prostate fibroblasts was identified by chromatin immunoprecipitation sequencing (ChIP-Seq), and found to be common to other fibroblast lines but disparate from AR cistromes of prostate cancer cells and tissue. Although AR binding sites specific to fibroblasts were less well conserved evolutionarily than those shared with cancer epithelia, they were likewise correlated with androgen regulation of fibroblast gene expression. Whereas FOXA1 is the key pioneer factor of AR in cancer epithelia, our data indicated that AP-1 likely plays a more important role in the AR cistrome in fibroblasts. The specificity of AP-1 and FOXA1 to binding in these cells is demonstrated using immunoblot and immunohistochemistry. Importantly, we find the fibroblast cistrome is represented in whole tissue/in vivo ChIP-seq studies at both genomic and resulting protein levels, highlighting the importance of the stroma in whole tissue -omic studies. This is the first nuclear receptor ChIP-seq study in prostatic fibroblasts, and provides novel insight into the action of fibroblast AR in prostate cancer.

Identification of genes expressed in a mesenchymal subset regulating prostate organogenesis using tissue and single cell transcriptomics.

Prostate organogenesis involves epithelial growth controlled by inductive signalling from specialised mesenchymal subsets. To identify pathways active in mesenchyme we used tissue and single cell transcriptomics to define mesenchymal subsets and subset-specific transcript expression. We documented transcript expression using Tag-seq and RNA-seq in female rat Ventral Mesenchymal Pad (VMP) as well as adjacent urethra comprised of smooth muscle and peri-urethral mesenchyme. Transcripts enriched in female VMP were identified with Tag-seq of microdissected tissue, RNA-seq of cell populations, and single cells. We identified 400 transcripts as enriched in the VMP using bio-informatic comparisons of Tag-seq and RNA-seq data, and 44 were confirmed by single cell RNA-seq. Cell subset analysis showed that VMP and adjacent mesenchyme were composed of distinct cell types and that each tissue contained two subgroups. Markers for these subgroups were highly subset specific. Thirteen transcripts were validated by qPCR to confirm cell specific expression in microdissected tissues, as well as expression in neonatal prostate. Immunohistochemical staining demonstrated that Ebf3 and Meis2 showed a restricted expression pattern in female VMP and prostate mesenchyme. We conclude that prostate inductive mesenchyme shows limited cellular heterogeneity and that transcriptomic analysis identified new mesenchymal subset transcripts associated with prostate organogenesis.

Reduced Contractility and Motility of Prostatic Cancer-Associated Fibroblasts after Inhibition of Heat Shock Protein 90.

BACKGROUND: Prostate cancer-associated fibroblasts (CAF) can stimulate malignant progression and invasion of prostatic tumour cells via several mechanisms including those active in extracellular matrix; METHODS: We isolated CAF from prostate cancer patients of Gleason Score 6-10 and confirmed their cancer-promoting activity using an in vivo tumour reconstitution assay comprised of CAF and BPH1 cells. We tested the effects of heat shock protein 90 (HSP90) inhibitors upon reconstituted tumour growth in vivo. Additionally, CAF contractility was measured in a 3D collagen contraction assay and migration was measured by scratch assay; RESULTS: HSP90 inhibitors dipalmitoyl-radicicol and 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) reduced tumour size and proliferation in CAF/BPH1 reconstituted tumours in vivo. We observed that the most contractile CAF were derived from patients with lower Gleason Score and of younger age compared with the least contractile CAF. HSP90 inhibitors radicicol and 17-DMAG inhibited contractility and reduced the migration of CAF in scratch assays. Intracellular levels of HSP70 and HSP90 were upregulated upon treatment with HSP90 inhibitors. Inhibition of HSP90 also led to a specific increase in transforming growth factor beta 2 (TGFß2) levels in CAF; CONCLUSIONS: We suggest that HSP90 inhibitors act not only upon tumour cells, but also on CAF in the tumour microenvironment.

Regulation of Fgf10 gene expression in the prostate: identification of transforming growth factor-beta1 and promoter elements.

Fibroblast growth factor 10 (FGF10) is a mesenchymal paracrine-acting factor that plays a key role in the organogenesis of the prostate, and Fgf10 transcripts exhibit a highly restricted expression pattern within prostatic mesenchyme. To study the regulation of Fgf10 we have used organ rudiments grown in vitro as well as a primary stromal cell system derived from the ventral mesenchymal pad (VMP), a condensed area of mesenchyme known to induce prostatic organogenesis. Characterization of VMP cells (VMPCs) showed that they retained expression of AR as well as transcripts for FGF10 and TGFbeta1, -2, and -3. We propose that VMPCs are a good model of specialized mesenchyme involved in prostatic organogenesis and are distinct from general urogenital sinus mesenchyme/stroma. Treatment of VMPCs with TGFbeta1 resulted in a rapid and transient decrease in Fgf10 transcript levels, which were reduced 9-fold at 3 h. TGFbeta1 also inhibited Fgf10 expression in VMP organ rudiments grown in vitro. To further analyze Fgf10 regulation, 6 kb of mouse genomic sequence 5' to the translation start site was characterized by promoter analysis. Deletion analysis of the Fgf10 promoter in VMPCs identified a region of the promoter that mediated a significant proportion of promoter activity as well as mediating promoter down-regulation by TGFbeta1. This element was located between nucleotides -182 and -172 and contained a consensus Sp1 binding site. Taken together, our data suggest that TGFbeta1 is a regulator of Fgf10 expression in prostatic mesenchyme and that a proximal element within the Fgf10 promoter plays an important role in its regulation and expression.

Differential effects of transforming growth factor-beta1 on cellular proliferation in the developing prostate.

TGFbeta1 plays an important role in the growth of the prostate and has been reported to stimulate or inhibit the proliferation of prostatic epithelia. We show here that Tgfbeta1, Tgfbeta2, and Tgfbeta3 mRNA expression correlated with developmental growth of the prostate. Recombinant TGFbeta1 inhibited the growth of the prostate when added to cultures of ventral prostate (VP) organs grown in vitro. Interestingly, TGFbeta1 had contrasting effects on cellular proliferation; it stimulated proliferation at the periphery of the organs (distal to urethra), but inhibited proliferation in the center of the organs (proximal to urethra). We speculate that differential effects on proliferation may be determined by the level of cellular differentiation, because cells at the periphery are undifferentiated whereas those in the center are more highly differentiated. TGFbeta1 also stimulated branching morphogenesis at growing ductal tips at the perimeter of the VP. To investigate potential mechanisms of TGFbeta1 action, we examined the three-dimensional distribution of smooth muscle in prostatic organs after treatment with TGFbeta1. TGFbeta1 showed a significant effect on the distribution of smooth muscle within VPs, which may mediate part of its effect on proliferation. Finally, we addressed how testosterone and TGFbeta1 might affect gene expression in our developmental system. Testosterone repressed the expression of Tgfbeta2 mRNA in the prostate, whereas TGFbeta1 showed a modest repression of fibroblast growth factor-10 mRNA. It appeared that the effects of these factors were more pronounced in a model of prostatic mesenchyme devoid of epithelia than in prostatic organs (containing epithelia).

Hormonal, cellular, and molecular regulation of normal and neoplastic prostatic development.

This review on normal and neoplastic growth of the prostate emphasizes the importance of epithelial-mesenchymal/stromal interactions. Accordingly, during prostatic development urogenital sinus mesenchyme (a) specifies prostatic epithelial identity, (b) induces epithelial bud formation, (c) elicits prostatic bud growth and regulates ductal branching, (d) promotes differentiation of a secretory epithelium, and (e) specifies the types of secretory proteins expressed. In reciprocal fashion, prostatic epithelium induces smooth muscle differentiation in the mesenchyme. Epithelial-mesenchymal interactions during development continue postnatally into adulthood as stromal-epithelial interactions which play a homeostatic role and in so doing reciprocally maintain epithelial and stromal differentiation and growth-quiescence. Prostatic carcinogenesis involves perturbation of these reciprocal homeostatic cell-cell interactions. The central role of mesenchyme in prostatic epithelial development has been firmly established through analysis of tissue recombinants composed of androgen-receptor-positive wild-type mesenchyme and androgen-receptor-negative epithelium. These studies revealed that at the very least ductal morphogenesis, epithelial cytodifferentiation, epithelial apoptosis and epithelial proliferation are regulated by stromal and not epithelial androgen receptors. Likewise, progression from non-tumorigenesis to tumorigenesis elicited by testosterone plus estradiol proceeds via paracrine mechanisms. Thus, stromal-epithelial interactions play critical roles in the hormonal, cellular, and molecular regulation of normal and neoplastic prostatic development.

Reduction of pro-tumorigenic activity of human prostate cancer-associated fibroblasts using Dlk1 or SCUBE1.

Human prostatic cancer-associated fibroblasts (CAFs) can elicit malignant changes in initiated but non-tumorigenic human prostate epithelium, demonstrating that they possess pro-tumorigenic properties. We set out to reduce the pro-tumorigenic activity of patient CAFs using the Dlk1 and SCUBE1 molecules that we had previously identified in prostate development. Our hypothesis was that mesenchymally expressed molecules might reduce CAF pro-tumorigenic activity, either directly or indirectly. We isolated primary prostatic CAFs and characterised their expression of CAF markers, expression of Notch2, Dlk1 and SCUBE1 transcripts, and confirmed their ability to stimulate BPH1 epithelial cell proliferation. Next, we expressed Dlk1 or SCUBE1 in CAFs and determined their effects upon tumorigenesis in vivo following recombination with BPH1 epithelia and xenografting in SCID mice. Tumour size was reduced by about 75% and BPH1 proliferation was reduced by about 50% after expression of Dlk1 or SCUBE1 in CAFs, and there was also a reduction in invasion of BPH1 epithelia into the host kidney. Inhibition of Notch signalling, using inhibitor XIX, led to a reduction in BPH1 cell proliferation in CAF-BPH1 co-cultures, whereas inhibition of Dlk1 in NIH3T3-conditioned media led to an increase in BPH1 growth. Our results suggest that pro-tumorigenic CAF activity can be reduced by the expression of developmental pathways.