Steven J. Ressler

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.

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.

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.

RUNX1 is essential for mesenchymal stem cell proliferation and myofibroblast differentiation

Significance Recruitment, proliferation, and differentiation of myofibroblasts are common in many disease states. Mechanisms that regulate proliferation and differentiation are poorly understood, although TGF-β is a key inducer of differentiation. Here, we report, for the first time to our knowledge, that runt-related transcription factor 1 (RUNX1) regulates mesenchymal stem cell (MSC) biology and progenitor cell commitment to myofibroblasts. In this work, we describe the first identification, to our knowledge, of tissue-resident MSCs from adult normal human prostate gland and the role of these MSCs as myofibroblast precursors. We also pinpoint the role of RUNX1 in regulating proliferation and differentiation in both marrow-derived and tissue-resident MSCs. Perturbation of RUNX1 activity may provide insights for developing antifibrotic and anticancer therapies via targeting the reactive stroma microenvironment. 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-β1–regulated pathways that modulate MSC proliferation and myofibroblast differentiation. Human prostate MSCs combined with prostate cancer cells expressing TGF-β1 resulted in commitment to myofibroblasts. TGF-β1–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.

Keratinocyte-Derived Chemokine Induces Prostate Epithelial Hyperplasia and Reactive Stroma in a Novel Transgenic Mouse Model

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.

WFDC1/ps20 is a novel innate immunomodulatory signature protein of human immunodeficiency virus (HIV)-Permissive CD4(+) CD45RO(+) memory T cells that promotes infection by upregulating CD54 integrin expression and is elevated in HIV type 1 infection

ABSTRACT Understanding why human immunodeficiency virus (HIV) preferentially infects some CD4+ CD45RO+ memory T cells has implications for antiviral immunity and pathogenesis. We report that differential expression of a novel secreted factor, ps20, previously implicated in tissue remodeling, may underlie why some CD4 T cells are preferentially targeted. We show that (i) there is a significant positive correlation between endogenous ps20 mRNA in diverse CD4 T-cell populations and in vitro infection, (ii) a ps20+ permissive cell can be made less permissive by antibody blockade- or small-interference RNA-mediated knockdown of endogenous ps20, and (iii) conversely, a ps20low cell can be more permissive by adding ps20 exogenously or engineering stable ps20 expression by retroviral transduction. ps20 expression is normally detectable in CD4 T cells after in vitro activation and interleukin-2 expansion, and such oligoclonal populations comprise ps20positive and ps20low/negative isogenic clones at an early differentiation stage (CD45RO+/CD25+/CD28+/CD57−). This pattern is altered in chronic HIV infection, where ex vivo CD4+ CD45RO+ T cells express elevated ps20. ps20 promoted HIV entry via fusion and augmented CD54 integrin expression; both of these effects were reversed by anti-ps20 antibody. We therefore propose ps20 to be a novel signature of HIV-permissive CD4 T cells that promotes infection in an autocrine and paracrine manner and that HIV has coopted a fundamental role of ps20 in promoting cell adhesion for its benefit. Disrupting the ps20 pathway may therefore provide a novel anti-HIV strategy.

TGF-β1 induces an age-dependent inflammation of nerve ganglia and fibroplasia in the prostate gland stroma of a novel transgenic mouse.

TGF-β1 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-β1 is known to modulate several stromal responses; however there are few transgenic models to study its integrated biology. To address the actions of TGF-β1 in prostate disorders, we targeted expression of an epitope tagged and constitutively active TGF-β1 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-β1 induced fibrosis and altered matrix deposition concurrent with inflammation in the stromal compartment. Together, these data suggest that elevated TGF-β1 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.

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.

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

Abstract 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.