Stromal expression of connective tissue growth factor promotes angiogenesis and prostate cancer tumorigenesis

Our previous studies have defined reactive stroma in human prostate cancer and have developed the differential reactive stroma (DRS) xenograft model to evaluate mechanisms of how reactive stroma promotes carcinoma tumorigenesis. Analysis of several normal human prostate stromal cell lines in the DRS model showed that some rapidly promoted LNCaP prostate carcinoma cell tumorigenesis and others had no effect. These differential effects were due, in part, to elevated angiogenesis and were transforming growth factor (TGF)-beta1 mediated. The present study was conducted to identify and evaluate candidate genes expressed in prostate stromal cells responsible for this differential tumor-promoting activity. Differential cDNA microarray analyses showed that connective tissue growth factor (CTGF) was expressed at low levels in nontumor-promoting prostate stromal cells and was constitutively expressed in tumor-promoting prostate stromal cells. TGF-beta1 stimulated CTGF message expression in nontumor-promoting prostate stromal cells. To evaluate the role of stromal-expressed CTGF in tumor progression, either engineered mouse prostate stromal fibroblasts expressing retroviral-introduced CTGF or 3T3 fibroblasts engineered with mifepristone-regulated CTGF were combined with LNCaP human prostate cancer cells in the DRS xenograft tumor model under different extracellular matrix conditions. Expression of CTGF in tumor-reactive stroma induced significant increases in microvessel density and xenograft tumor growth under several conditions tested. These data suggest that CTGF is a downstream mediator of TGF-beta1 action in cancer-associated reactive stroma and is likely to be one of the key regulators of angiogenesis in the tumor-reactive stromal microenvironment.     

Prostate tumor progression is mediated by a paracrine TGF-beta/Wnt3a signaling axis

Transforming growth factor (TGF)-beta is an important paracrine factor in tumorigenesis. Ligand binding of the type I and II TGF-beta receptors initiate downstream signaling. The role of stromal TGF-beta signaling in prostate cancer progression is unknown. In mice, the conditional stromal knockout of the TGF-beta type II receptor expression (Tgfbr2(fspKO)) resulted in the development of prostatic intraepithelial neoplasia and progression to adenocarcinoma within 7 months. Clinically, we observed a loss of TGF-beta receptor type II expression in 69% of human prostate cancer-associated stroma, compared to 15% of stroma associated with benign tissues (n=140, P-value <0.0001). To investigate the mechanism of paracrine TGF-beta signaling in prostate cancer progression, we compared the effect of the prostatic stromal cells from Tgfbr2(fspKO) and floxed TGF-beta type II receptor Tgfbr2(floxE2/floxE2) mice on LNCaP human prostate cancer cells in vitro and tissue recombination xenografts. Induction of LNCaP cell proliferation and tumorigenesis was observed by Tgfbr2(fspKO) prostate stroma as a result of elevated Wnt3a expression. Neutralizing antibodies to Wnt3a reversed LNCaP tumorigenesis. The TGF-beta inhibition of Wnt3a expression was in part through the suppression of Stat3 activity on the Wnt3a promoter. In conclusion, the frequent loss of stromal TGF-beta type II receptor expression in human prostate cancer can relieve the paracrine suppression of Wnt3a expression.     

Altered TGF-β signaling in a subpopulation of human stromal cells promotes prostatic carcinogenesis

Carcinoma-associated fibroblasts (CAF) play a critical role in malignant progression. Loss of TGF-β receptor II (TGFβR2) in the prostate stroma is correlated with prostatic tumorigenesis. To determine the mechanisms by which stromal heterogeneity because of loss of TGFβR2 might contribute to cancer progression, we attenuated transforming growth factor beta (TGF-β) signaling in a subpopulation of immortalized human prostate fibroblasts in a model of tumor progression. In a tissue recombination model, loss of TGFβR2 function in 50% of the stromal cell population resulted in malignant transformation of the nontumorigenic human prostate epithelial cell line BPH1. Mixing fibroblasts expressing the empty vector and dominant negative TGFβR2 increased the expression of markers of myofibroblast differentiation [coexpression of vimentin and alpha smooth muscle actin (αSMA)] through elevation of TGF-β1 and activation of the Akt pathway. In combination, these two populations of stromal cells recapitulated the tumor inductive activity of CAFs. TGFβR2 activity in mixed stromal cell populations cultured in vitro caused secretion of factors that are known to promote tumor progression, including TGF-β1, SDF1/CXCL12, and members of the fibroblast growth factor (FGF) and bone morphogenetic protein (BMP) families. In vivo, tissue recombination of fibroblasts overexpressing TGF-β1 and SDF1/CXCL12 not only induced transformation of BPH1 cells, but also promoted a robust growth of highly invasive cells, similar to effects produced by CAFs. While the precise nature and/or origin of the particular stromal cell populations in vivo remain unknown, these findings strongly link heterogeneity in TGF-β signaling to tumor promotion by tumor stromal cells.     

Fibroblast growth factor-2 mediates transforming growth factor-beta action in prostate cancer reactive stroma

Transforming growth factor-beta (TGF-beta) is overexpressed at sites of wound repair and in most adenocarcinomas including prostate cancer. In stromal tissues, TGF-beta regulates cell proliferation, phenotype and matrix synthesis. To address mechanisms of TGF-beta action in cancer-associated reactive stroma, we developed prostate stromal cells null for TGF-beta receptor II (TbetaRII) or engineered to express a dominant-negative Smad3 to attenuate TGF-beta signaling. The differential reactive stroma (DRS) xenograft model was used to evaluate altered stromal TGF-beta signaling on LNCaP tumor progression. LNCaP xenograft tumors constructed with TbetaRII null or dominant-negative Smad3 stromal cells exhibited a significant reduction in mass and microvessel density relative to controls. Additionally, decreased cellular fibroblast growth factor-2 (FGF-2) immunostaining was associated with attenuated TGF-beta signaling in stroma. In vitro, TGF-beta stimulated stromal FGF-2 expression and release. However, stromal cells with attenuated TGF-beta signaling were refractory to TGF-beta-stimulated FGF-2 expression and release. Re-expression of FGF-2 in these stromal cells in DRS xenografts resulted in restored tumor mass and microvessel density. In summary, these data show that TGF-beta signaling in reactive stroma is angiogenic and tumor promoting and that this effect is mediated in part through a TbetaRII/Smad3-dependent upregulation of FGF-2 expression and release.     

Characteristics of normal stromal components and their correlation with cancer occurrence in human prostate

Prostate cancer is one of the most common age-related malignancies. The occurrence frequency of prostate cancer is very different according to prostate zones. The prostate stroma is an important element in growth and differentiation of the normal prostate and also has a close relationship to the occurrence of benign prostatic hypertrophy and cancer. We examined 14 cases of normal prostate tissues obtained at autopsy and 11 cases of prostate cancer tissues at radical prostatectomy specimens with cancers for clarifying the characteristics of stromal components in the normal prostate and the correlation between the stroma and the occurrence of prostate cancers. Stromal cells, such as smooth muscle cells, myofibroblasts and fibroblasts were identified by immunohistochemistry (IHC). Connective tissue fibers were detected by Elastica van Gieson and also IHC stain. Quantitative analysis of the smooth muscle tissue and connective tissue fibers were performed using a computer image analyzer system. In the normal prostate, stromal components varied in each zone. Every zone of the prostate contained smooth muscle cells, myofibroblasts, fibroblasts and collagen fibers. Elastic fibers were clearly visible in the transition zone. Smooth muscle cells were the main stromal component but less numerous in the frequent occurrence zone (peripheral zone) of prostate cancer (p<0.05). Myofibroblasts and fibroblasts were found either in normal or cancer tissues, although a few in number. The increase of collagen fibers accompanied decrease of smooth muscle cells as prostate cancer grade increased (p<0.05). The characteristics of stromal components and their amounts in the normal prostate appear to correlate with a distinct predilection for cancer occurrence in the peripheral zone and a weak stromal reaction in prostate cancers.     

Promotion of angiogenesis by ps20 in the differential reactive stroma prostate cancer xenograft model

Human prostate cancer is associated with a reactive stroma typified by an increase in the proportion of myofibroblast type cells and elevated synthesis of extracellular matrix proteins. Increased vascular density has been identified in the reactive stroma compartment adjacent to both precancerous and cancerous prostate lesions. The differential reactive stroma (DRS) prostate cancer xenograft model has been developed to investigate the role of reactive stroma in prostate cancer progression. Using this model, we have shown that human prostate stromal cells promote angiogenesis and growth of LNCaP human prostate carcinoma cell tumors, and that these increases are transforming growth factor (TGF) beta1 regulated. Our laboratory isolated and identified previously the ps20 protein (WFDC1 gene) as a prostate stromal cell secreted protein. The ps20 protein contains a whey acidic protein-type four-disulfide core domain, which is a functional motif characterized by serine protease inhibition activity in a number of whey acidic protein domain-containing proteins. In the present study, we show ps20 expression by normal human prostate stromal smooth muscle cells and vascular smooth muscle cells indicating a possible role of ps20 in vessel wall biology. Using in vitro assays, we show that ps20 promotes endothelial cell motility but has no effect on endothelial cell proliferation. To address the potential effects of ps20 in a tumor microenvironment, we used the DRS model to evaluate both angiogenesis and tumorigenesis of tumors generated under either ps20 or control conditions. DRS tumors generated with LNCaP and human prostate stromal cells in the presence of ps20 showed a 67% increase in microvessel density compared with control tumors. Elevated DRS tumor growth in the ps20-treated tumors was reflected by a 29% increase in wet weight and a 58% increase in volume compared with controls. Similar tumors composed of GeneSwitch-3T3 cells engineered to express ps20-V5-His under mifepristone regulation showed a 129% increase in microvessel density after induction of ps20-V5-His. GeneSwitch-3T3 cells expressing ps20-V5-His were localized to vessel walls in a mural cell (pericyte) position indicating a possible direct stabilizing interaction with endothelial cells. In addition, we show that ps20 mRNA synthesis is induced by TGF-beta1, a known regulator of endothelial cell-pericyte interactions and of stromal cell-induced angiogenesis in DRS tumors. These findings suggest that ps20 may be a TGF-beta1-induced regulator of angiogenesis that functions by either promoting endothelial cell migration or by contributing to pericyte stabilization of newly formed vascular structures.     

Transforming growth factor-beta1 increases survival of human melanoma through stroma remodeling.

Transforming growth factor (TGF)-beta is growth inhibitory for normal epithelial cells and melanocytes but can stimulate mesenchymal cells. Resistance to its inhibitory effects is characteristic of human melanoma, the growth of which may instead be promoted by TGF-beta, because its production is increased with melanoma progression. Whether TGF-beta has an autocrine function for melanoma cells or is important for paracrine stimulation of the tumor stroma is not known. In this study, TGF-beta1 was expressed in melanoma cells via adenoviral gene transfer, and tumor growth was analyzed in vitro, in human skin grafts, and in mixtures with fibroblasts that were injected s.c. into immunodeficient mice. The TGF-beta1 produced by the melanoma cells activated the fibroblasts to produce matrix within and around the tumor mass, whereas control tumors showed less stroma and more cell death. High expression of collagen, fibronectin, tenascin, and alpha2 integrin was detected in the TGF-beta1-expressing tumors by immunohistochemistry. Number and size of lung metastases were significantly increased. cDNA expression array analysis of TGF-beta1-transduced fibroblasts embedded in type I collagen and of TGF-beta1-transduced melanoma cells demonstrated induction of types XV, XVIII, and VI collagens, tenascin, plasminogen activator inhibitor-I, vascular endothelial growth factor, cysteine-rich fibroblast growth factor receptor-1, and platelet-derived growth factor receptor-beta, which could be linked to promotion of growth and survival in melanoma. These data suggest that remodeling of the neighboring stroma, which provides a supporting scaffolding and a positive feedback stimulation of tumor growth, is an important function of TGF-beta1 in melanoma.     

Stromal Activation Associated with Development of Prostate Cancer in Prostate-Targeted Fibroblast Growth Factor 8b Transgenic Mice

Expression of fibroblast growth factor 8 (FGF-8) is commonly increased in prostate cancer. Experimental studies have provided evidence that it plays a role in prostate tumorigenesis and tumor progression. To study how increased FGF-8 affects the prostate, we generated and analyzed transgenic (TG) mice expressing FGF-8b under the probasin promoter that targets expression to prostate epithelium. Prostates of the TG mice showed an increased size and changes in stromal and epithelialmorphology progressing fromatypia and prostatic intraepithelial neoplasia (mouse PIN, mPIN) lesions to tumors with highly variable phenotype bearing features of adenocarcinoma, carcinosarcoma, and sarcoma. The development of mPIN lesions was preceded by formation of activated stroma containing increased proportion of fibroblastic cells, rich vasculature, and inflammation. The association between advancing stromal and epithelial alterations was statistically significant. Microarray analysis and validation with quantitative polymerase chain reaction revealed that expression of osteopontin and connective tissue growth factor was markedly upregulated in TG mouse prostates compared with wild type prostates. Androgen receptor staining was decreased in transformed epithelium and in hypercellular stroma but strongly increased in the sarcoma-like lesions. In conclusion, our data demonstrate that disruption of FGF signaling pathways by increased epithelial production of FGF-8b leads to strongly activated and atypical stroma, which precedes development of mPIN lesions and prostate cancer with mixed features of adenocarcinoma and sarcoma in the prostates of TG mice. The results suggest that increased FGF-8 in human prostate may also contribute to prostate tumorigenesis by stromal activation.     

Reactive stroma as a predictor of biochemical-free recurrence in prostate cancer.

Extensive scientific literature data point to reciprocal interactions between prostate stromal cells and prostate cancer cells that likely regulate tumor progression. To investigate whether these intratumoral-reactive stromal cells in human prostate cancer are predictive of survival, tumor stroma volume and specific stroma markers were quantitated by using tissue microarrays (index tumors of 847 patients), and the results were analyzed relative to the recurrence-free survival data set for these patients. Tumor tissue was evaluated with Masson's trichrome stains and by immunohistochemistry with antibody probes to smooth muscle alpha-actin, desmin, vimentin, pro-collagen type I, and calponin. The relative volume of intratumor stroma (5% stroma, grade 0; 5-15%, grade 1; 15-50%, grade 2; >50%, grade 3) and the expression index of stromal marker (staining intensity grade x percentage of positive cells per field) were quantitated and analyzed. Interpretable data were obtained from 545 patients. Statistical analysis of the survival data set showed that the volume of reactive stroma in the tumor was a significant predictor of disease-free survival. Stroma volume was most optimal as an independent predictor in tumors containing stroma, defined as Gleason 7 and lower grades. Of interest, tumors with either little to no stroma or tumors with abundant stroma each showed reduced recurrence-free survival. For specific stromal markers, reduced desmin and smooth muscle alpha-actin were hallmarks of cancer-associated reactive stroma relative to normal fibromuscular stroma. Quantitative analysis of desmin and smooth muscle alpha-actin expression showed both to be significant and independent predictors of recurrence-free survival. This is the first study to demonstrate that nonepithelial-reactive stroma elements in prostate cancer tumors can be used as prognostic indicators. These data also add to the concept that tumors are not purely epithelial and the tumor-reactive stroma must be considered an important biological component of the cancer.     

Induction of transforming growth factor beta in hormonally treated human prostate cancer.

Transforming growth factor beta-1 (TGF-beta 1) has been proposed as a mediator of tumour growth in a number of tumours and cell lines including prostate, and in a recent study was shown to be up-regulated in the stroma of breast cancer tissue following treatment with the anti-oestrogen tamoxifen. Immunolocalisation of the intracellular form of TGF-beta 1 confirmed that the source of the stromal TGF-beta 1 was the peritumoral fibroblasts. We present here the results of a study in which five patients with hormonally unresponsive prostatic carcinoma and seven patients responding to a luteinising hormone-releasing hormone analogue had prostate biopsies taken before and during treatment. These were stained for TGF-beta expression prior to treatment and at either relapse or 3 months later respectively. Six of seven clinically responding tumours and three of five relapsed tumours showed up-regulation of extracellular TGF-beta 1, again primarily in the stroma, with no apparent up-regulation of intracellular TGF-beta 1, TGF-beta 2 or TGF-beta 3. These data illustrate that the epithelial growth inhibitor TGF-beta 1 can be induced by hormonal manipulation in prostate cancer in vivo, and may continue to be up-regulated even after relapse. This suggests that relapse of hormonally treated prostate cancer may be associated with a failure of the epithelium to respond to stromal TGF-beta 1.     

Expression of transforming growth factor-beta 1 and growth in soft agar differentiate prostate carcinoma-associated fibroblasts from normal prostate fibroblasts

Carcinoma-associated fibroblasts (CAF) promote tumor progression of pre-neoplastic epithelial cells. To investigate the basis of this phenomenon, we compared the properties of fibroblasts cultured from normal human prostate (NHPF) to prostate CAF. NHPF and CAF were assayed for growth potential, cell death and proliferative capacity by measuring population doubling time, cell cycle distribution and capability to form colonies in soft agar. Resistance to genotoxic (UV radiation: 0-50 J/cm2) and chemotoxic (0-200 nM Taxol) agents were compared between CAF and NHPF by measuring cell viability and cell cycle analysis. Transforming growth factor beta1 (TGF-beta1) immunoreactivity was assessed in non-malignant and malignant prostatic tissue. No detectable differences were found when comparing CAF and NHPF with respect to population doubling time, cell cycle distribution and response to genotoxic and chemotoxic agents. The mean number of colonies in soft agar was 120.5 for CAF vs. 18.2 for NHPF (p < 0.05). Because TGF-beta1 and matrix metalloproteinase (MMP)-9 have been associated with growth of fibroblasts in soft agar and tumor promotion, we measured the expression of these factors in NHPF and CAF by ELISA. There was no difference in expression of MMP-9; however, TGF-beta1 was expressed in higher concentrations in CAF than in NHPF (p < 0.0014). Furthermore, TGF-beta1 expression was higher in the carcinoma-associated stroma of prostate cancer tissue than stroma of non-malignant prostatic tissue. Increased capability of CAF as compared to NHPF to form colonies in soft agar may be due to a higher expression of TGF-beta1 and correlates with the ability of CAF to promote malignant progression of prostate epithelial cells.     

Inducible prostate intraepithelial neoplasia with reversible hyperplasia in conditional FGFR1-expressing mice

Accurate determination of the contributions of oncogenes toward tumor progression requires their regulation. Herein, we created transgenic mice with prostate-specific expression of ligand-inducible FGFR1 or FGFR2, based on lipid-permeable dimerizing molecules, called chemical inducers of dimerization. Despite extensive homology and equivalent expression by both chimeric receptors in the ventral prostate gland, only FGFR1 triggers detectable nuclear translocation of Erk and progression to prostatic intraepithelial neoplasia (PIN). Induction of PIN grade I-II, indicated by multiple layers of atypical cells, is seen consistently by 12 weeks of chemical inducers of dimerization treatment. By 6 months, more extensive nuclear atypia, thickened "reactive" stroma, and basement membrane herniation occurs, corresponding to PIN IV. By timed removal of FGFR1 signaling, we show that induced hyperplasia is reversible until extensive intraductal vascularization occurs, but continued progression requires prolonged FGFR1 signaling. Additionally, by highlighting differences between the two receptors and creating the foundation for controlling FGFR1 signaling during prostate cancer progression, a model of early stage prostate cancer is established for developing targeted intervention directed toward the FGFR signaling axis.     

Cross-talk between paracrine-acting cytokine and chemokine pathways promotes malignancy in benign human prostatic epithelium

The present study explores the mechanisms by which human prostatic carcinoma-associated fibroblasts (CAF) induce tumorigenesis in initiated but nonmalignant human prostatic epithelial cells (BPH-1). CAF express elevated levels of both transforming growth factor-beta1 (TGF-beta1) and stromal cell-derived factor-1 (SDF-1/CXCL12). TGF-beta inhibits the growth of BPH-1 cells in vitro, but was found to be necessary for the tumorigenic response to CAF. This counterintuitive result suggested that the TGF-beta signaling system was involved in other processes relating to tumorigenesis. The SDF-1 receptor, CXCR4, is expressed at low levels in benign prostate tissue and in BPH-1 cells in culture. However, CXCR4 levels increase during prostate cancer progression. CXCR4 was found to be induced and localized to the cell membrane in BPH1 cells by CAF-conditioned medium and by CAF cells in tissue recombinants. TGF-beta was both necessary and sufficient to allow the detection of membrane-localized CXCR4 in BPH1 cells. Suppression of epithelial cell CXCR4 expression abrogated the tumorigenic response to CAF. SDF-1, secreted by CAF, acts via the TGF-beta-regulated CXCR4 to activate Akt in the epithelial cells. This mechanism elicits tumorigenesis and obviates the growth-inhibitory effects of TGF-beta. Thus, tumor stroma can contribute to carcinogenesis through synergism between TGF-beta, SDF-1, and CXCR4. These experiments suggest mechanisms by which TGF-beta can shift its role from an inhibitor to a promoter of proliferation during tumor progression. Both the TGF-beta and SDF-1 pathways are targets of drug discovery efforts; these data suggest potential benefits in the cotargeting of these pathways.     

Differential impact of TGF-beta and EGF on fibroblast differentiation and invasion reciprocally promotes colon cancer cell invasion

Several studies indicate that cancer-associated fibroblasts play a critical role in cancer cell invasion and metastasis, the hallmarks of malignancy. To better understand the mechanisms underlying such effects, we established a heterotypic model of human fibroblasts (primary colon fibroblasts and immortalized human dermal fibroblasts) in co-culture with human colon cancer cells (HCT-8/E11), using three-dimensional collagen type-I and Matrigel matrices. We report that TGF-beta is the unique and dominant factor to provide pro-invasive signals to HCT-8/E11 colon cancer cells from TGF-beta-treated human fibroblasts in three-dimensional collagen type I and Matrigel matrices. These effects are not mimicked or reversed by EGF or bFGF, and are associated with the TGF-beta-mediated induction of myofibroblast differentiation and functional markers, such as alpha-SMA, the haptotactic matrix molecule TNC, collagen type 1 maturation enzyme P4H, serine protease FAP, and myofibroblast contractility. Accordingly, TGF-beta induced a strong activation of RhoA and stress fiber formation in fibroblasts, with no impact on Rac1-GTP levels. In contrast, EGF down-regulated Rho-GTP levels in fibroblasts, giving permissive signals for Rac1 activation, fibroblast polarization, and invasion. Taken together, our data imply that TGF-beta and EGF exert invasive growth-promoting actions in human colon tumors through a differential and cumulative impact on the stromal and cancer cell compartments. Our data predict that inhibitors directed at this reciprocal molecular and cellular crosstalk will have therapeutic applications for targeting the invasive growth of human primary tumors and their metastatic spread.     

Down-regulation of CD9 expression during prostate carcinoma progression is associated with CD9 mRNA modifications.

PURPOSE: Cluster-of-differentiation antigen 9 (CD9) protein, a member of the tetraspanin family, has been implicated in carcinogenesis of various human tumors. Although decreased expression of the CD82 tetraspanin protein, a close CD9 relative, is associated with prostate cancer progression, CD9 expression has not been analyzed in this malignancy. EXPERIMENTAL DESIGN: CD9 expression in human prostatic adenocarcinoma was analyzed by immunohistochemistry on 167 primary tumors and 88 lymph node or bone metastases. CD9 cDNA was sequenced from two human prostate cancer cell lines, prostatic adenocarcinoma, high-grade prostatic intraepithelial neoplasia (PIN), and normal prostatic tissues. RESULTS: Although CD9 was detected in the epithelium of normal prostatic tissues, reduced or loss of CD9 expression within neoplastic cells was observed in 24% of 107 clinically localized primary adenocarcinomas, 85% of 60 clinically advanced primary adenocarcinomas, 85% of 65 lymph node metastases, and 65% of 23 bone metastases. Difference in CD9 expression between clinically localized and advanced diseases was highly significant (P < 1 x 10(-7)). Whereas there was no alteration of CD9 cDNA in normal tissues, all PC-3-derived cell lines, one PIN, and four prostatic adenocarcinomas harbored deletions in their CD9 cDNAs. Recurring CD9 point mutations were also found in PC-3M-LN4 cells, one PIN, and seven prostatic adenocarcinomas. CONCLUSIONS: CD9 expression is significantly reduced and even lost during prostate cancer progression. Moreover, deletions and mutations of the CD9 mRNA may be associated with loss of protein expression observed in tumor cells. Our data suggest that CD9 inactivation may play an important role in prostate cancer progression.     

Stromal androgen receptor regulates the composition of the microenvironment to influence prostate cancer outcome

Androgen receptor (AR) signaling in stromal cells is important in prostate cancer, yet the mechanisms underpinning stromal AR contribution to disease development and progression remain unclear. Using patient-matched benign and malignant prostate samples, we show a significant association between low AR levels in cancer associated stroma and increased prostate cancer-related death at one, three and five years post-diganosis, and in tissue recombination models with primary prostate cancer cells that low stromal AR decreases castration-induced apoptosis. AR-regulation was found to be different in primary human fibroblasts isolated from adjacent to cancerous and non-cancerous prostate epithelia, and to represent altered activation of myofibroblast pathways involved in cell cycle, adhesion, migration, and the extracellular matrix (ECM). Without AR signaling, the fibroblast-derived ECM loses the capacity to promote attachment of both myofibroblasts and cancer cells, is less able to prevent cell-matrix disruption, and is less likely to impede cancer cell invasion. AR signaling in prostate cancer stroma appears therefore to alter patient outcome by maintaining an ECM microenvironment inhibitory to cancer cell invasion. This paper provides comprehensive insight into AR signaling in the non-epithelial prostate microenvironment, and a resource from which the prognostic and therapeutic implications of stromal AR levels can be further explored.     

Inhibition of Nox4‐dependent ROS signaling attenuates prostate fibroblast activation and abrogates stromal‐mediated protumorigenic interactions

Carcinoma‐associated fibroblasts (CAFs) play a key onco‐supportive role during prostate cancer (PCa) development and progression. We previously reported that the reactive oxygen species (ROS)‐producing enzyme NADPH oxidase 4 (Nox4) is essential for TGFβ1‐mediated activation of primary prostate human fibroblasts to a CAF‐like phenotype. This study aimed to further investigate the functional relevance of prostatic Nox4 and determine whether pharmacological inhibition of stromal Nox4 abrogates paracrine‐mediated PCa‐relevant processes. RNA in situ hybridization revealed significantly elevated Nox4 mRNA levels predominantly in the peri‐tumoral stroma of clinical PCa with intense stromal Nox4 staining adjacent to tumor foci expressing abundant TGFβ protein levels. At pharmacologically relevant concentrations, the Nox1/Nox4 inhibitor GKT137831 attenuated ROS production, CAF‐associated marker expression and migration of TGFβ1‐activated but not nonactivated primary human prostate fibroblasts. Similar effects were obtained upon shRNA‐mediated silencing of Nox4 but not Nox1 indicating that GKT137831 primarily abrogates TGFβ1‐driven fibroblast activation via Nox4 inhibition. Moreover, inhibiting stromal Nox4 abrogated the enhanced proliferation and migration of PCa cell lines induced by TGFβ1‐activated prostate fibroblast conditioned media. These effects were not restricted to recombinant TGFβ1 as conditioned media from PCa cell lines endogenously secreting high TGFβ1 levels induced fibroblast activation in a stromal Nox4‐ and TGFβ receptor‐dependent manner. Importantly, GKT137831 also attenuated PCa cell‐driven fibroblast activation. Collectively, these findings suggest the TGFβ‐Nox4 signaling axis is a key interface to dysregulated reciprocal stromal–epithelial interactions in PCa pathophysiology and provide a strong rationale for further investigating the applicability of Nox4 inhibition as a stromal‐targeted approach to complement current PCa treatment modalities.     

Differential expression of neuropilin-1 in malignant and benign prostatic stromal tissue

Neuropilin-1 (NRP-1), a co-receptor for VEGF165, is overexpressed in various prostate cancer cell lines and in advanced prostate tumors. However, distribution of the NRP-1 in prostate tumors has not yet been evaluated. Using immunohistochemical analysis, we evaluated 21 archival prostate tumors and 5 benign glands for the expression of NRP-1. In addition, we utilized a quantitative RT-PCR method to examine mRNA expression in 9 additional prostate tumors obtained from radical prostatectomy specimens and compared this expression to the adjacent normal tissue. The RT-PCR analyses demonstrated overexpression of NRP-1 mRNA in malignant tissue samples by 10.0-fold as compared to adjacent normal tissue. By immunohistochemistry, NRP-1 protein was undetected or minimally detected in the epithelial tumor cells. However, NRP-1 immuno-reaction was detected in the surrounding tumor stroma. Variable immuno-reaction for NRP-1 was also seen in the adjacent normal tumor stroma and the stroma of the benign prostate samples. These observations suggest that neuropilin-1 is expressed in the prostatic stromal cells, not epithelial tumor cells, and this expression is significantly increased in the malignant phenotype.