Androgen receptor variant-7 regulation by tenascin-c induced src activation.

BACKGROUND: Bone metastatic prostate cancer does not completely respond to androgen-targeted therapy and generally evolves into lethal castration resistant prostate cancer (CRPC). Expression of AR-V7- a constitutively active, ligand independent splice variant of AR is one of the critical resistant mechanisms regulating metastatic CRPC. TNC is an extracellular matrix glycoprotein, crucial for prostate cancer progression, and associated with prostate cancer bone metastases. In this study, we investigated the mechanisms that regulate AR-V7 expression in prostate cancer cells interacting with osteogenic microenvironment including TNC. METHODS: Prostate cancer/preosteoblast heterotypical organoids were evaluated via immunofluorescence imaging and gene expression analysis using RT-qPCR to assess cellular compartmentalization, TNC localization, and to investigate regulation of AR-V7 in prostate cancer cells by preosteoblasts and hormone or antiandrogen action. Prostate cancer cells cultured on TNC were assessed using RT-qPCR, Western blotting, cycloheximide chase assay, and immunofluorescence imaging to evaluate (1) regulation of AR-V7, and (2) signaling pathways activated by TNC. Identified signaling pathway induced by TNC was targeted using siRNA and a small molecular inhibitor to investigate the role of TNC-induced signaling activation in regulation of AR-V7. Both AR-V7- and TNC-induced signaling effectors were targeted using siRNA, and TNC expression assessed to evaluate potential feedback regulation. RESULTS: Utilizing heterotypical organoids, we show that TNC is an integral component of prostate cancer interaction with preosteoblasts. Interaction with preosteoblasts upregulated both TNC and AR-V7 expression in prostate cancer cells which was suppressed by testosterone but elevated by antiandrogen enzalutamide. Interestingly, the results demonstrate that TNC-induced Src activation regulated AR-V7 expression, post-translational stability, and nuclear localization in prostate cancer cells. Treatment with TNC neutralizing antibody, Src knockdown, and inhibition of Src kinase activity repressed AR-V7 transcript and protein. Reciprocally, both activated Src and AR-V7 were observed to upregulate autocrine TNC gene expression in prostate cancer cells. CONCLUSION: Overall, the findings reveal that prostate cancer cell interactions with the cellular and ECM components in the osteogenic microenvironment plays critical role in regulating AR-V7 associated with metastatic CRPC. Video Abstract.

Stromal TGF-ß signaling induces AR activation in prostate cancer.

AR signaling is essential for the growth and survival of prostate cancer (PCa), including most of the lethal castration-resistant PCa (CRPC). We previously reported that TGF-ß signaling in prostate stroma promotes prostate tumor angiogenesis and growth. By using a PCa/stroma co-culture model, here we show that stromal TGF-ß signaling induces comprehensive morphology changes of PCa LNCaP cells. Furthermore, it induces AR activation in LNCaP cells in the absence of significant levels of androgen, as evidenced by induction of several AR target genes including PSA, TMPRSS2, and KLK4. SD-208, a TGF-ß receptor 1 specific inhibitor, blocks this TGF-ß induced biology. Importantly, stromal TGF-ß signaling together with DHT induce robust activation of AR. MDV3100 effectively blocks DHT-induced, but not stromal TGF-ß signaling induced AR activation in LNCaP cells, indicating that stromal TGF-ß signaling induces both ligand-dependent and ligand-independent AR activation in PCa. TGF-ß induces the expression of several growth factors and cytokines in prostate stromal cells, including IL-6, and BMP-6. Interestingly, BMP-6 and IL-6 together induces robust AR activation in these co-cultures, and neutralizing antibodies against BMP-6 and IL-6 attenuate this action. Altogether, our study strongly suggests tumor stromal microenvironment induced AR activation as a direct mechanism of CRPC.

WFDC1 is a key modulator of inflammatory and wound repair responses.

WFDC1/ps20 is a whey acidic protein four-disulfide core member that exhibits diverse growth and immune-associated functions in vitro. In vivo functions are unknown, although WFDC1 is lower in reactive stroma. A Wfdc1-null mouse was generated to assess core functions. Wfdc1-null mice exhibited normal developmental and adult phenotypes. However, homeostasis challenges affected inflammatory and repair processes. Wfdc1-null mice infected with influenza A exhibited 2.75-log-fold lower viral titer relative to control mice. Wfdc1-null infected lungs exhibited elevated macrophages and deposition of osteopontin, a potent macrophage chemokine. In wounding studies, Wfdc1-null mice exhibited an elevated rate of skin closure, and this too was associated with elevated deposition of osteopontin and macrophage recruitment. Wfdc1-null fibroblasts exhibited impaired spheroid formation, elevated adhesion to fibronectin, and an increased rate of wound closure in vitro. This was reversed by neutralizing antibody to osteopontin. Osteopontin mRNA and cleaved protein was up-regulated in Wfdc1-null cells treated with lipopolysaccharide or polyinosinic-polycytidylic acid coordinate with constitutively active matrix metallopeptidase-9 (MMP-9), a protease that cleaves osteopontin. These data suggest that WFDC1/ps20 modulates core host response mechanisms, in part, via regulation of osteopontin and MMP-9 activity. Release from WFDC1 regulation is likely a key component of inflammatory and repair response mechanisms, and involves the processing of elevated osteopontin by activated MMP-9, and subsequent macrophage recruitment.

FGFR1 is essential for prostate cancer progression and metastasis.

The fibroblast growth factor receptor 1 (FGFR1) is ectopically expressed in prostate carcinoma cells, but its functional contributions are undefined. In this study, we report the evaluation of a tissue-specific conditional deletion mutant generated in an ARR2PBi(Pbsn)-Cre/TRAMP/fgfr1(loxP/loxP) transgenic mouse model of prostate cancer. Mice lacking fgfr1, in prostate cells developed smaller tumors that also included distinct cancer foci still expressing fgfr1 indicating focal escape from gene excision. Tumors with confirmed fgfr1 deletion exhibited increased foci of early, well-differentiated cancer and phyllodes-type tumors, and tumors that escaped fgfr1 deletion primarily exhibited a poorly differentiated phenotype. Consistent with these phenotypes, mice carrying the fgfr1 null allele survived significantly longer than those without fgfr1 deletion. Most interestingly, all metastases were primarily negative for the fgfr1 null allele, exhibited high FGFR1 expression, and a neuroendocrine phenotype regardless of fgfr1 status in the primary tumors. Together, these results suggest a critical and permissive role of ectopic FGFR1 signaling in prostate tumorigenesis and particularly in mechanisms of metastasis.

TGF-ß1 induces an age-dependent inflammation of nerve ganglia and fibroplasia in the prostate gland stroma of a novel transgenic mouse.

TGF-ß1 is overexpressed in wound repair and in most proliferative disorders including benign prostatic hyperplasia and prostate cancer. The stromal microenvironment at these sites is reactive and typified by altered phenotype, matrix deposition, inflammatory responses, and alterations in nerve density and biology. TGF-ß1 is known to modulate several stromal responses; however there are few transgenic models to study its integrated biology. To address the actions of TGF-ß1 in prostate disorders, we targeted expression of an epitope tagged and constitutively active TGF-ß1 via the enhanced probasin promoter to the murine prostate gland epithelium. Transgenic mice developed age-dependent lesions leading to severe, yet focal attenuation of epithelium, and a discontinuous basal lamina. These changes were associated with elevated fibroplasia and frequency of collagenous micronodules in collapsed acini, along with an induced inflammation in nerve ganglia and small vessels. Elevated recruitment of CD115+ myeloid cells but not mature macrophages was observed in nerve ganglia, also in an age-dependent manner. Similar phenotypic changes were observed using a human prostate epithelium tissue recombination xenograft model, where epithelial cells engineered to overexpress TGF-ß1 induced fibrosis and altered matrix deposition concurrent with inflammation in the stromal compartment. Together, these data suggest that elevated TGF-ß1 expression induces a fibroplasia stromal response associated with breach of epithelial wall structure and inflammatory involvement of nerve ganglia and vessels. The novel findings of ganglia and vessel inflammation associated with formation of collagenous micronodules in collapsed acini is important as each of these are observed in human prostate carcinoma and may play a role in disease progression.

Elevated epithelial expression of interleukin-8 correlates with myofibroblast reactive stroma in benign prostatic hyperplasia.

OBJECTIVES: Numerous inflammatory diseases display elevated interleukin (IL)-8, and most are associated with a reactive stroma. IL-8 expression is also elevated in benign prostatic hyperplasia (BPH), yet little is known about reactive stroma in BPH. Whether a reactive stroma response exists in BPH, whether this correlates with elevated IL-8, and whether IL-8 can induce a reactive stroma phenotype have not been determined. This study was designed to specifically address these issues. METHODS: Normal prostate transition zone tissue and BPH specimens, as identified by the Baylor College of Medicine pathology department, were examined by quantitative immunohistochemistry to correlate IL-8, smooth muscle alpha-actin, vimentin, calponin, and tenascin-C. In addition, human prostate stromal cell cultures were used to evaluate the effect of IL-8 on the expression of reactive stroma biomarkers. RESULTS: BPH nodules exhibited elevated epithelial IL-8 immunoreactivity, and this correlated with elevated smooth muscle alpha-actin, reduced calponin, and altered deposition of tenascin-C, relative to the normal prostate transition zone tissue (P <0.05). Multiple vimentin-positive prostate stromal fibroblast cultures were induced by IL-8 to also co-express smooth muscle alpha-actin and tenascin-C, typical of a reactive stroma myofibroblast phenotype. CONCLUSIONS: These data show that BPH reactive stroma is fundamentally different from normal prostate fibromuscular stroma and is typified by the emergence of a reactive stroma myofibroblast phenotype. This reactive stroma pattern correlated spatially with IL-8 elevation in adjacent epithelium. Additionally, IL-8 induced expression of myofibroblast markers in human prostate fibroblasts in vitro. These results suggest that IL-8 acts as a regulator of BPH reactive stroma and is therefore a potential therapeutic target.

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.

Transforming growth factor-beta1 induced myofibroblasts regulate LNCaP cell death.

PURPOSE: Reactive stroma represents a generic wound response phenomenon, which has been identified in areas of tissue injury and carcinogenesis. To determine whether reactive stroma influences prostate tumor cell growth 3 primary prostate stromal cell lines were treated with transforming growth factor-beta1 (TGF-beta1) to induce the reactive stroma phenotype and then co-cultured with LNCaP cells. MATERIALS AND METHODS: Flow cytometry was performed in LNCaP cells that had been co-cultured with induced reactive stroma or control stroma and an index of cell death and proliferation was obtained. Using the previously described 3 way differential reactive stroma xenograft tumor model consisting of LNCaP cells, stromal cells and Matrigel (Collaborative Research, Bedford, Massachusetts) LNCaP cell apoptosis was evaluated using TUNEL staining in a background of varying degrees of reactive stroma. RESULTS: Flow cytometric analysis revealed that LNCaP cells co-cultured with TGF-beta1 induced stromal cells demonstrated a significantly decreased rate of cell death compared with controls (p <0.001). In an animal model LNCaP cells of the 3 way xenograft constructs treated with TGF-beta1 latency associated peptide, an inhibitor of TGF-beta1, showed increased apoptosis by TUNEL staining (p <0.001). Double label immunohistochemistry analysis demonstrated that TGF-beta1 induced stromal cells had an increased proportion of myofibroblasts, the identifying cell type of reactive stroma. Furthermore, the degree of reactive stroma inversely corresponded to the degree of LNCaP cell death. CONCLUSIONS: These findings indicate that reactive stroma influences prostate cancer cell growth and warrant investigation of the regulatory mechanisms between reactive stroma and prostate cancer cells.

Regulation of rat prostate stromal cell myodifferentiation by androgen and TGF-beta1.

BACKGROUND: Myodifferentiation of stromal cells is a key step in prostate development and is a hallmark of reactive stroma in prostate cancer. Little is known about regulatory mechanisms, however, prostate stromal cells are androgen-regulated and TGF-beta1 is a known stimulator of stromal myodifferentiation. The PS-1 rat prostate stromal cell line expresses androgen receptor, and exhibits androgen-regulated gene expression and proliferation. TGF-beta1 inhibits androgen action in PS-1 cells through translocation of androgen receptor from the nucleus to the cytoplasm. The present study was conducted to determine whether myodifferentiation of PS-1 cells is regulated by androgen and TGF-beta1, and how myodifferentiation affects androgen receptor localization and cell proliferation. METHODS: PS-1 cell cultures were exposed to physiological concentrations of dihydrotestosterone, TGF-beta1, and combinations of both in chemically defined medium. Immunocytochemistry and Western blotting for smooth muscle alpha-actin filament formation, smooth muscle alpha-actin protein levels, calponin expression, PCNA index, and androgen receptor localization were performed. RESULTS: Dihydrotestosterone (DHT) and TGF-beta1 each separately promoted PS-1 myodifferentiation. A combination did not affect the rate of differentiation, however, the level of alpha-actin protein was elevated and PCNA was decreased in co-stimulated conditions. TGF-beta1 induction resulted in a transient translocation of androgen receptor from the nucleus to the cytoplasm during differentiation followed by a resumed nuclear localization in myodifferentiated cells. CONCLUSIONS: These data indicate that a complex cross-talk mechanism exists between androgen and TGF-beta1 signaling in prostate stromal cells that affects cell proliferation and myodifferentiation. These findings also suggest that androgen and TGF-beta1 interactions may cooperatively regulate myodifferentiation of stromal cells in the stromal response in prostate cancer.

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.

Inhibition of transforming growth factor-beta activity decreases angiogenesis in a human prostate cancer-reactive stroma xenograft model.

We have shown previously that reactive stroma promotes angiogenesis and growth of LNCaP human prostate tumors in the differential reactive stroma xenograft model. Regulators of reactive stroma are not known, but transforming growth factor (TGF)-beta1 is a likely candidate. Three-way differential reactive stroma tumors were generated in the presence of TGF-beta1 latency-associated peptide (LAP) or TGF-beta1 neutralizing antibody. Tumors treated with either of those TGF-beta inhibitors exhibited a reduction in blood vessels, and blood lakes were observed in some areas. The microvessel density of LAP-treated tumors was decreased 3.5-fold relative to control tumors. Moreover, the average wet-weight of LAP-treated tumors was reduced 46% compared with control tumors. The results of this study suggest that TGF-beta regulates reactive stroma and its ability to promote angiogenesis and tumor growth.

Reactive stroma in human prostate cancer: induction of myofibroblast phenotype and extracellular matrix remodeling.

PURPOSE: Generation of a reactive stroma environment occurs in many human cancers and is likely to promote tumorigenesis. However, reactive stroma in human prostate cancer has not been defined. We examined stromal cell phenotype and expression of extracellular matrix components in an effort to define the reactive stroma environment and to determine its ontogeny during prostate cancer progression. EXPERIMENTAL DESIGN: Normal prostate, prostatic intraepithelial neoplasia (PIN), and prostate cancer were examined by immunohistochemistry. Tissue samples included radical prostatectomy specimens, frozen biopsy specimens, and a prostate cancer tissue microarray. A human prostate stromal cell line was used to determine whether transforming growth factor beta1 (TGF-beta1) regulates reactive stroma. RESULTS: Compared with normal prostate tissue, reactive stroma in Gleason 3 prostate cancer showed increased vimentin staining and decreased calponin staining (P < 0.001). Double-label immunohistochemistry revealed that reactive stromal cells were vimentin and smooth muscle alpha-actin positive, indicating the myofibroblast phenotype. In addition, reactive stroma cells exhibited elevated collagen I synthesis and expression of tenascin and fibroblast activation protein. Increased vimentin expression and collagen I synthesis were first observed in activated periacinar fibroblasts adjacent to PIN. Similar to previous observations in prostate cancer, TGF-beta1-staining intensity was elevated in PIN. In vitro, TGF-beta1 stimulated human prostatic fibroblasts to switch to the myofibroblast phenotype and to express tenascin. CONCLUSIONS: The stromal microenvironment in human prostate cancer is altered compared with normal stroma and exhibits features of a wound repair stroma. Reactive stroma is composed of myofibroblasts and fibroblasts stimulated to express extracellular matrix components. Reactive stroma appears to be initiated during PIN and evolve with cancer progression to effectively displace the normal fibromuscular stroma. These studies and others suggest that TGF-beta1 is a candidate regulator of reactive stroma during prostate cancer progression.

Stromal cells promote angiogenesis and growth of human prostate tumors in a differential reactive stroma (DRS) xenograft model.

Reactive stroma has been reported in many cancers, including breast, colon,and prostate. Although changes in stromal cell phenotype and extracellular matrix have been reported, specific mechanisms of how reactive stroma affects tumor progression are not understood. To address the role of stromal cells in differential regulation of tumor incidence, growth rate, and angiogenesis, LNCaP xenograft tumors were constructed in nude mice with five different human prostate stromal cell lines as well as GeneSwitch-3T3 cells engineered to express lacZ under mifepristone regulation. Alone, LNCaP prostate carcinoma cells were essentially nontumorigenic, whereas combinations of LNCaP cells with three different human prostate stromal cell lines (L/S tumors) resulted in a tumor incidence (50-63%) similar to that of control LNCaP plus Matrigel (L/M) tumors over a 9-week period. In contrast, LNCaP combinations with two other human prostate stromal cell lines were nontumorigenic, illustrating that stromal cell effects are differential. L/S tumors exhibited well-developed blood vessels at 2 weeks, whereas control L/M tumors were avascular at 2 weeks and exhibited blood lakes in lieu of extensive vessels at later time points. Xenografts constructed under three-way conditions (LNCaP, Matrigel, and stromal cells; L/M/S tumors) exhibited a 100% tumor incidence and showed rapid blood vessel formation as early as day 7 with mature vessels formed by day 10. L/M/S tumors exhibited a 10.3-fold increase in microvessel density, and the corresponding hemoglobin:tumor weight ratio was increased 2-fold relative to L/M control tumors at day 10. L/M/S tumor wet weight and volume increased by 1.6- and 2.4-fold, respectively, by day 21, compared with control L/M tumors. L/M/S tumors made with LNCaP cells plus GeneSwitch-3T3-pGene/lacZ stromal cells showed similar results. Mifepristone-regulated gene expression was observed in stromal cells immediately adjacent to clusters of carcinoma cells and in vessel walls in a mural cell (pericyte) position. This study shows that regulation of angiogenesis is one mechanism through which stromal cells affect LNCaP tumor incidence and growth rate. This regulation may be mediated through direct recruitment and interactions of stromal cells with endothelial cells. Furthermore, this study describes for the first time a model system with regulated transgene expression in the stromal compartment of an experimental carcinoma. These findings point to the stromal compartment as a potential source of new prognostic markers and therapeutic targets and show the utility of the carcinoma-stromal xenograft model system in dissecting specific mechanisms of reactive stroma.