Truong D. Dang

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.

Purification of a Novel Protein (ps20) from Urogenital Sinus Mesenchymal Cells with Growth Inhibitory Properties in Vitro

Our previous studies have characterized mesenchyme-derived proteins to identify biologically active proteins and novel markers for stromal cell paracrine action relative to stromal-epithelial interactions. Previous reports have characterized properties of a growth inhibitory activity (to bladder and prostatic epithelial cells), secreted by U4F fetal rat urogenital sinus mesenchymal cells, not cross-reactive with antibodies to known cytokines, and provisionally termed UGIF. The present study reports the characterization, purification, and biological properties of a 20-21-kDa protein responsible for UGIF activity. The 20-21-kDa protein (termed ps20) was purified to near homogeneity, the amino-terminal sequence was determined, and biological properties were characterized in vitro. Amino-terminal sequence analysis indicated no direct matches or regions of homology with known proteins. Purified ps20 induced a linear and saturable inhibition of thymidine incorporation in PC-3 prostatic carcinoma cells (half-maximal activity at 2.6 nM), inhibited cell proliferation (increased population doubling time from 19.8 to 25.8 h), and induced a 210% stimulation in the synthesis of secreted proteins. These data suggest that ps20 may be a candidate paracrine effector protein and may play a role in stromal-epithelial cell interactions in the prostate gland.

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.

Transforming Growth Factor-β1 Induces Nuclear to Cytoplasmic Distribution of Androgen Receptor and Inhibits Androgen Response in Prostate Smooth Muscle Cells1

Stromal-epithelial interactions in the prostate gland are dependent on androgen regulation of prostate stromal cells, yet little is known about androgen action in these cell types. Recent reports have demonstrated that androgen-regulated gene transcription can be stimulated or inhibited by certain growth factors, indicating cross-talk mechanisms. To address potential cross-talk in signaling pathways between androgen and transforming growth factor-beta1 (TGFbeta1) in prostate stromal cells, the PS-1 prostate smooth muscle cell line was examined. In the presence of physiological concentrations of androgen, PS-1 cell proliferation was stimulated, and androgen receptor (AR) exhibited a nuclear localization pattern. The addition of TGFbeta1 (25 pM) was capable of blocking androgen-induced proliferation, but had no direct effect in cultures without androgen. Immunocytochemistry to localize AR subcellular distribution showed that TGFbeta1 (5-100 pM) altered the distribution of AR from the nucleus to the cytoplasm. Other growth factors, including fibroblast growth factor-2, epidermal growth factor, and TGFbeta2 had no effect on AR distribution. The TGFbeta1-induced nuclear to cytoplasmic change in receptor localization was rapid (initiated within 30 min), was neutralized by TGFbeta1 antibodies, did not require new protein synthesis, and was complete by 6 h. Removal of TGFbeta1 from the culture medium resulted in a rapid redistribution of AR to the nucleus, indicating reversible mechanisms. Northern analysis of the ddp17 marker transcript for androgen action in PS-1 cells showed that androgen-stimulated ddp17 expression was inhibited in the presence of TGFbeta1 (25 pM). TGFbeta1 induced a similar nuclear to cytoplasmic distribution of AR in primary cultures of rat prostate stromal cells. TGFbeta1, however, had no effect on AR distribution in either the LNCaP prostatic carcinoma cell line or the DDT1MF-2 leiomyosarcoma cell line. Specific cross-talk between TGFbeta1 and AR signaling pathways in prostate stromal cells may play a significant role in prostate development and stromal cell response in carcinoma progression.

ADENOVIRUS-MEDIATED SUICIDE GENE THERAPY FOR EXPERIMENTAL BLADDER CANCER

OBJECTIVES To determine the feasibility, safety, and efficacy of suicide gene therapy using adenoviral-mediated herpes simplex virus thymidine kinase gene (HSV-tk) and the prodrug ganciclovir (GCV) in a murine model of human transitional cell carcinoma. METHODS We used a replication-defective adenoviral construct containing the beta-galactosidase gene (ADV/Rous sarcoma virus [RSV]-beta-gal) as a control or ADV/RSV-tk as the therapeutic vector under the transcriptional control of the RSV long-terminal repeat promoter. Transduction efficiency was assessed in vitro by infection of MBT-2 cells with ADV/RSV-beta-gal at various multiplicities of infection (MOI) utilizing 5-bromo-4-chlor-3-indolyl-beta-D-galactoside (X-gal) staining. Sensitivity of MBT-2 cells to the therapeutic vector was determined after infection with ADV/RSV-tk with or without GCV. Subcutaneous tumors were established in syngeneic C3H/He female mice with 5 x 10(5) MBT-2 cells. Optimal dosing of ADV/RSV-tk was determined by direct percutaneous tumor injection with increasing viral doses and treatment with GCV. Treatment efficacy, long-term survival, and toxicity were determined in separate, similar, controlled experiments. RESULTS In vitro studies indicated greater than 95% transduction 96 hours after inoculation at an MOI of 3000 and a greater than 95% cell death rate with RSV-tk + GCV at an MOI of 61 or greater. In vivo experiments demonstrated an optimal viral dose of 3 x 10(8) plaque-forming units (pfu) and a greater than fourfold reduction in tumor growth for the animals treated with ADV/RSV-tk compared with control animals (P = 0.0013). Toxicity was limited to histologic evidence of hepatitis with ADV/RSV-tk doses greater than 3 x 10(8) pfu + GCV. Long-term survival of treatment animals was significantly increased over that of control animals (59%, P = 0.0001). CONCLUSIONS ADV/RSV-tk with GCV treatment results in efficient gene transfer in vitro and provides effective therapy in experimental murine bladder cancer by significantly inhibiting tumor growth and improving host survival.

Localization of Transforming Growth Factor-β1 and Type II Receptor in Developing Normal Human Prostate and Carcinoma Tissues

Transforming growth factor-β1 (TGF-β1) is implicated in prostate development, and elevated expression of TGF-β1 has been correlated with prostate carcinogenesis. In this study, cell type specificity of TGF-β1 and TGF-β receptor Type II (RcII) protein expression was determined by immunocytochemistry in human normal prostate and compared to prostate carcinoma tissues. Heterogeneous localization patterns of LAP-TGF-β1 (TGF-β1 precursor) and RcII were observed in both epithelial and mesenchymal cells in fetal prostate, with LAP-TGF-β1 localizing to more basal epithelial cells. Homogeneity of LAP-TGF-β1 staining was increased in neonatal, prepubertal, and adult prostate, with elevated immunoreac-tivity noted in epithelial acini relative to stromal tissue for both LAP-TGF-β1 and RcII proteins. In stromal tissues, RcII cell localization exhibited staining patterns nearly identical to smooth muscle α-actin. In prostate carcinoma, LAP-TGF-β1 localized to carcinoma cells with an increased staining heterogeneity relative to normal prostate. In contrast to normal epithelial cells, carcinoma epithelial cells exhibited low to nondetectable RcII staining. Stromal cell staining patterns for LAP-TGF-β1 and RcII in carcinoma, however, were identical to those of normal prostate stromal cells. These studies implicate both epithelial and stromal cells as sites of TGF-β1 synthesis and RcII localization in the developing and adult normal human prostate. In addition, these data indicate a loss of epithelial expression of RcII concurrent with altered LAP-TGF-β1 expression in human prostate carcinoma cells.

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.

Characterization of tumor-derived pancreatic stellate cells.

BACKGROUND Pancreatic stellate cells (PSCs) are key mediators of the desmoplastic reaction that characterizes pancreatic adenocarcinoma. We sought to isolate and characterize tumor-derived pancreatic stellate (TDPS) cells to further understand how these stromal cells influence pancreatic cancer behavior. METHODS We established a stable line of non-immortalized PSCs from a patient with pancreatic adenocarcinoma using a modified prolonged outgrowth method. Cell staining for cytokeratin, vimentin, and alpha smooth muscle actin (alphaSMA) was performed. Total RNA was harvested from TDPS and panc-1 cells and gene expression determined by microarray analysis. RESULTS TDPS cells contain lipid droplets in the cytoplasm, and later stain positive for both vimentin and alphaSMA, indicative of activated myofibroblasts. Microarray analysis revealed a distinct gene expression profile compared with pancreatic cancer cells, including expression of proteases that facilitate cancer cell invasion and growth factors known to activate pancreatic cancer cells. Additionally, TDPS cells expressed many of the key components of the pancreatic tumor stroma, including collagen, fibronectin, and S100A4, confirming their importance in the tumor microenvironment. CONCLUSIONS Characterization of tumor-derived PSCs will facilitate further studies to determine how the tumor microenvironment promotes the aggressive behavior of pancreatic cancer.