RUNX1 is essential for mesenchymal stem cell proliferation and myofibroblast differentiation.

Myofibroblasts are a key cell type in wound repair, cardiovascular disease, and fibrosis and in the tumor-promoting microenvironment. The high accumulation of myofibroblasts in reactive stroma is predictive of the rate of cancer progression in many different tumors, yet the cell types of origin and the mechanisms that regulate proliferation and differentiation are unknown. We report here, for the first time to our knowledge, the characterization of normal human prostate-derived mesenchymal stem cells (MSCs) and the TGF-ß1-regulated pathways that modulate MSC proliferation and myofibroblast differentiation. Human prostate MSCs combined with prostate cancer cells expressing TGF-ß1 resulted in commitment to myofibroblasts. TGF-ß1-regulated runt-related transcription factor 1 (RUNX1) was required for cell cycle progression and proliferation of progenitors. RUNX1 also inhibited, yet did not block, differentiation. Knockdown of RUNX1 in prostate or bone marrow-derived MSCs resulted in cell cycle arrest, attenuated proliferation, and constitutive differentiation to myofibroblasts. These data show that RUNX1 is a key transcription factor for MSC proliferation and cell fate commitment in myofibroblast differentiation. This work also shows that the normal human prostate gland contains tissue-derived MSCs that exhibit multilineage differentiation similar to bone marrow-derived MSCs. Targeting RUNX1 pathways may represent a therapeutic approach to affect myofibroblast proliferation and biology in multiple disease states.

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.

Recruitment of CD34(+) fibroblasts in tumor-associated reactive stroma: the reactive microvasculature hypothesis.

Reactive stroma co-evolves with cancer, exhibiting tumor-promoting properties. It is also evident at sites of wound repair and fibrosis, playing a key role in tissue homeostasis. The specific cell types of origin and the spatial/temporal patterns of reactive stroma initiation are poorly understood. In this study, we evaluated human tumor tissue arrays by using multiple labeled, quantitative, spectral deconvolution microscopy. We report here a novel CD34/vimentin dual-positive reactive fibroblast that is observed in the cancer microenvironment of human breast, colon, lung, pancreas, thyroid, prostate, and astrocytoma. Recruitment of these cells occurred in xenograft tumors and Matrigel plugs in vivo and was also observed in stromal nodules associated with human benign prostatic hyperplasia. Because spatial and temporal data suggested the microvasculature as a common site of origin for these cells, we analyzed microvasculature fragments in organ culture. Interestingly, fibroblasts with identical phenotypic properties and markers expanded radially from microvasculature explants. We propose the concept of reactive microvasculature for the evolution of reactive stroma at sites of epithelial disruption common in both benign and malignant disorders. Data suggest that the reactive stroma response is conserved among tissues, in normal repair, and in different human cancers. A more clear understanding of the nature and origin of reactive stroma is needed to identify novel therapeutic targets in cancer and fibrosis.

The WFDC1 gene: role in wound response and tissue homoeostasis.

The present evaluates the key features of the WFDC1 [WAP (whey acidic protein) four disulfide core 1] gene that encodes ps20 (20 kDa prostate stromal protein), a member of the WAP family. ps20 was first characterized as a growth inhibitory activity that was secreted by fetal urogenital sinus mesenchymal cells. Purified ps20 exhibited several activities that centre on cell adhesion, migration and proliferation. The WFDC1 gene was cloned, contained seven exons, and was mapped to chromosome 16q24, suggesting that it may function as a tumour suppressor; however, direct evidence of this has not emerged. In vivo, ps20 stimulated angiogenesis, although expression of WFDC1/ps20 was down-regulated in the reactive stroma tumour microenvironment in prostate cancer. WFDC1 expression is differential in other cancers and inflammatory conditions. Recent studies point to a role in viral infectivity. Although mechanisms of action are not fully understood, WFDC1/ps20 is emerging as a secreted matricellular protein that probably affects response to micro-organisms and tissue repair homoeostasis.

Keratinocyte-derived chemokine induces prostate epithelial hyperplasia and reactive stroma in a novel transgenic mouse model.

BACKGROUND: Interleukin-8 (IL-8) is upregulated in fibrotic and malignant diseases and is a key mediator of proliferative responses. Elevated IL-8 was recently correlated with benign prostatic hyperplasia epithelium and a myofibroblast reactive stroma. Thus, we sought to determine whether overexpressed IL-8 and keratinocyte-derived chemokine (KC), the functional murine homolog of IL-8, induce prostate epithelial hyperplasia and a reactive phenotype. METHODS: Transgenic mice that overexpress KC within prostate epithelia and xenograft models with engineered human cells that overexpress IL-8 were developed. RESULTS: Overexpression of KC in transgenic mice produced hyperplastic prostate epithelial acini associated with a periacinar reactive stroma. KC induced an altered epithelial/stroma proliferation index ratio, increased acini diameter, epithelial infolding, and expression of prototypical reactive stroma markers. Overexpression of IL-8 in normal human prostate epithelial xenografts correlated with elevated epithelial proliferation index and altered morphology. Elevated human prostate stromal and epithelial cell proliferation, nodule-like morphology and increased xenograft survival were observed in IL-8-overexpressing orthotopic xenografts. CONCLUSIONS: Together, these data demonstrate that overexpression of IL-8/KC results in a prostate epithelial hyperplasia with an associated reactive stroma phenotype. The novel transgenic mouse and human xenograft models described here may be useful in dissecting key mechanisms of IL-8 induced prostate hyperplasia and reactive stroma.

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.

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.

TGF-ß induction of FGF-2 expression in stromal cells requires integrated smad3 and MAPK pathways.

Transforming Growth Factor-ß (TGF-ß) regulates the reactive stroma microenvironment associated with most carcinomas and mediates expression of many stromal derived factors important for tumor progression, including FGF-2 and CTGF. TGF-ß is over-expressed in most carcinomas, and FGF-2 action is important in tumor-induced angiogenesis. The signaling mechanisms of how TGF-ß regulates FGF-2 expression in the reactive stroma microenvironment are not understood. Accordingly, we have assessed key signaling pathways that mediate TGF-ß1-induced FGF-2 expression in prostate stromal fibroblasts and mouse embryo fibroblasts (MEFs) null for Smad2 and Smad3. TGF-ß1 induced phosphorylation of Smad2, Smad3, p38 and ERK1/2 proteins in both control MEFs and prostate fibroblasts. Of these, Smad3, but not Smad2 was found to be required for TGF-ß1 induction of FGF-2 expression in stromal cells. ChIP analysis revealed a Smad3/Smad4 complex was associated with the -1.9 to -2.3 kb upstream proximal promoter of the FGF-2 gene, further suggesting a Smad3-specific regulation. In addition, chemical inhibition of p38 or ERK1/2 MAPK activity also blocked TGF-ß1-induced FGF-2 expression in a Smad3-independent manner. Conversely, inhibition of JNK signaling enhanced FGF-2 expression. Together, these data indicate that expression of FGF-2 in fibroblasts in the tumor stromal cell microenvironment is coordinately dependent on both intact Smad3 and MAP kinase signaling pathways. These pathways and key downstream mediators of TGF-ß action in the tumor reactive stroma microenvironment, may evolve as putative targets for therapeutic intervention.

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.

WFDC1/ps20: a host factor that influences the neutrophil response to murine hepatitis virus (MHV) 1 infection.

The whey acidic protein family member, WFDC1/ps20 is a permissivity factor in HIV infection. Herein we describe a contrasting role for ps20 in limiting MHV-1 infection. Intranasal MHV-1 infection produces a respiratory infection in mice. Using ps20 knockout mice we provide evidence that intranasal MHV-1 infection results in increased lung viral titers in ps20(-/-) compared to ps20(+/+) mice. Accompanying MHV-1 infection we observe an increase in the number of neutrophils infiltrating the BAL and an increase in the percentage of neutrophils in the lung draining lymph nodes of ps20(-/-) compared with ps20(+/+) mice. Gene expression levels for the neutrophil chemoattractants CXCL1 and CXCL2 are elevated in the lungs of ps20(-/-) mice post-MHV-1 infection. Characterization of the immune cell profile in naïve ps20(-/-) mice revealed an increase in circulating neutrophils compared to ps20(+/+) mice. No notable differences in other immune cell profiles were observed between the ps20(+/+) and ps20(-/-) mice. Accordingly, we examined MHV-1 infection of neutrophils and provide evidence that neutrophils isolated from ps20(-/-) mice are more susceptible to MHV-1 infection than neutrophils isolated from ps20(+/+) mice. These data suggest roles for ps20 in regulating expression of neutrophil-specific chemotactic factors, thereby potentially modulating neutrophil migration, and in modulating neutrophil susceptibility to MHV-1 infection.

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.

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.

Decreased stromal expression and increased epithelial expression of WFDC1/ps20 in prostate cancer is associated with reduced recurrence-free survival.

BACKGROUND: WAP-type four disulfide core (WFDC1)/ps20 is a member of the whey acidic protein family, which includes several serine protease inhibitors. Expression of WFDC1/ps20 was previously demonstrated in the normal human prostate stromal compartment. To further current understanding of the role of WFDC1/ps20 in prostate cancer, altered expression of WFDC1/ps20 protein in prostate cancer was evaluated. METHODS: Immunohistochemical staining for WFDC1/ps20 was performed using tissue microarrays. Quantitation was based on the percentage of positive-staining stromal or epithelial cells and staining intensity. Resulting data was analyzed relative to the recurrence-free survival data and additional information for this patient set. RESULTS: Decreased stromal expression of WFDC1/ps20 predicted shorter recurrence-free survival time by univariate analysis. Decreased stromal WFDC1/ps20 expression correlated with higher radical prostatectomy Gleason scores, positive surgical margins, extracapsular extension, higher clinical stage, and higher preoperative prostate specific antigen levels. Increased epithelial expression of WFDC1/ps20 also predicted shorter recurrence-free survival times by univariate analysis. Increased epithelial expression of WFDC1/ps20 correlated with higher biopsy and radical prostatectomy Gleason scores, and higher clinical stage. CONCLUSIONS: Decreased stromal WFDC1/ps20 expression reflects the evolution of a prostate cancer reactive stroma, while increased epithelial WFDC1/ps20 expression may indicate progression to a more aggressive epithelial phenotype and may indicate an epithelial mesenchymal transition (EMT) process. Further evaluation of WFDC1/ps20 biologic functions will aid in the understanding of this interesting expression profile.