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.     

Calcitriol-induced prostate-derived factor: autocrine control of prostate cancer cell growth

Calcitriol (1alpha,25-dihydroxycholecalciferol) seems to play an important role in the complex control of prostate cell growth. It inhibits proliferation and induces differentiation and apoptosis in prostate cancer cells. However, the molecular mechanisms of the antiproliferative activity of calcitriol are not completely understood. The expression of prostate-derived factor (PDF), a member of the transforming growth factor-beta (TGF-beta) superfamily, has been shown to be associated with proapoptotic and antimitotic activities. We show that calcitriol induces PDF expression in LNCaP human prostate cancer cells in a concentration- and time-dependent manner. In LNCaP cells, the suppression of cell growth by calcitriol is accompanied by stimulation of PDF mRNA and protein synthesis. Human recombinant PDF inhibits LNCaP cell growth. We do not detect any effect of PDF-specific antibody on the basal growth of LNCaP cells, but this antibody partially reverses the suppression of LNCaP cell growth by calcitriol, suggesting that the effect of calcitriol on cell growth is at least partially mediated by PDF. In PC-3 cells, which are less responsive to the growth-inhibitory effect of calcitriol, it has no effect on PDF expression. We do not detect an effect of recombinant PDF on SMAD phosphorylation in LNCaP cells, but ERK1/2 kinases are transiently phosphorylated in response to PDF, which suggests that in LNCaP cells PDF may exert its action through pathway alternative to the classical TGF-beta signaling pathway. The present study describes the regulation of PDF, the proapoptotic protein of the TGF-beta superfamily, by calcitriol in androgen-responsive prostate cancer cells. This is a new link between calcitriol and growth factors of TGF-beta superfamily in the control of prostate cell growth.     

Cancer-associated transforming growth factor beta type II receptor gene mutant causes activation of bone morphogenic protein-Smads and invasive phenotype

Transforming growth factor beta (TGFbeta) plays a key role in maintaining tissue homeostasis by inducing cell cycle arrest, differentiation and apoptosis, and ensuring genomic integrity. Furthermore, TGFbeta orchestrates the response to tissue injury and mediates repair by inducing epithelial to mesenchymal transition and by stimulating cell motility and invasiveness. Although loss of the homeostatic activity of TGFbeta occurs early on in tumor development, many advanced cancers have coopted the tissue repair function to enhance their metastatic phenotype. How these two functions of TGFbeta become uncoupled during cancer development remains poorly understood. Here, we show that, in human keratinocytes, TGFbeta induces phosphorylation of Smad2 and Smad3 as well as Smad1 and Smad5 and that both pathways are dependent on the kinase activities of the type I and II TGFbeta receptors (T beta R). Moreover, cancer-associated missense mutations of the T beta RII gene (TGFBR2) are associated with at least two different phenotypes. One type of mutant (TGFBR2(E526Q)) is associated with loss of kinase activity and all signaling functions. In contrast, a second mutant (TGFBR2(R537P)) is associated with high intrinsic kinase activity, loss of Smad2/3 activation, and constitutive activation of Smad1/5. Furthermore, this TGFBR2 mutant endows the carcinoma cells with a highly motile and invasive fibroblastoid phenotype. This activated phenotype is T beta RI (Alk-5) independent and can be reversed by the action of a dual T beta RI and T beta RII kinase inhibitor. Thus, identification of such activated T beta RII receptor mutations in tumors may have direct implications for appropriately targeting these cancers with selective therapeutic agents.     

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.     

Effects of RNAi-mediated Smad4 silencing on growth and apoptosis of human rhabdomyosarcoma cells

Transforming growth factor-beta (TGF-beta) signals through membrane-bound heteromeric serine/threonine kinase receptors. Upon ligand binding, TGF-beta activates intracellular Smad proteins and regulates proliferation and apoptosis in various cell types. To demonstrate the effects of TGF-beta/Smad signal on growth and apoptosis of human embryonal rhabdomyosarcoma (RMS) cells, a strategy of RNAi-mediated 'gene silencing' of Smad4 was used to interrupt endogenous TGF-beta/Smad signaling in an RMS cell line, RD, and the regulation of exogenous TGF-beta1 to growth and apoptosis of the cells was also determined. Physiologically, TGF-beta/Smad signaling was essential for the normal growth of RD. The interruption of endogenous TGF-beta/Smad signaling by RNAi significantly suppressed the growth of RD cells and dramatically induced apoptosis of RD cells. Exogenous TGF-beta1 also inhibited the growth of RD cells, but had no effect on apoptosis. It also partially counteracted the growth inhibition and apoptosis induced by Smad4 silencing in RD cells. These findings provide a new insight into how TGF-beta/Smad signaling regulates the growth and apoptosis of cancer cells. Moreover, as a powerful tool, shRNA interference suppresses endogenous Smad4 gene expression and subsequently modulates cell growth and apoptosis, which may provide a novel basis for the development of rational intervention strategies in RMS therapy.     

Prostatic carcinoma cell migration via alpha(v)beta3 integrin is modulated by a focal adhesion kinase pathway

The highly invasive human prostate cancer PC3 cell line was found to express the alpha(v)beta3 integrin; in contrast, the noninvasive LNCaP prostate cancer cell line did not express alpha(v)beta3. PC3 cells adhered to and migrated on vitronectin (VN), an alpha(v)beta3 ligand expressed in mature bone where prostate cancer cells preferentially metastasize. In contrast, LNCaP cells did not adhere to or migrate on VN. Analysis of primary human prostate cancer cells isolated from 16 surgical specimens, showed that these cells expressed alpha(v)beta3, whereas normal prostate epithelial cells did not. In addition, only primary prostate cancer cells adhered to and migrated on VN. The role of alpha(v)beta3 in mediating prostate epithelial cell migration was confirmed using LNCaP cell transfectants expressing beta3 (beta3-LNCaP). Exogenous expression of alpha(v)beta3 induced LNCaP cells to adhere to and migrate on VN. In response to alpha(v)beta3 engagement, increased tyrosine phosphorylation of focal adhesion kinase (FAK), a signaling molecule activated by integrins and able to modulate cell migration, was detected. Transfection of FAK-related nonkinase, known to compete with FAK for its correct localization and phosphorylation, caused inhibition of beta3-LNCaP cell migration, specifically on VN. These data indicate that de novo expression of alpha(v)beta3 integrin in prostate cancer cells generates a migratory phenotype that is modulated by a FAK signaling pathway. This study points to alpha(v)beta3 as potential target in prostate cancer cell invasion and metastasis.     

Effects of transforming growth factor beta (TGF-beta) receptor on lung carcinogenesis

Transforming growth factor beta (TGF-beta) type-II receptor mutations have been reported in several epithelial-type human malignancies. To elucidate the role of TGF-beta RII in lung cancer progression, we prepared gene-modified clones of the human lung cancer cell line NCI-H23. NCI-H23, a human non-small-cell lung adenocarcinoma cell line which has a frameshift mutation in, and reduced expression of, the TGF-beta type-II receptor (TGF-beta RII), exhibits resistance to growth inhibition by TGF-beta(1) in vitro. Transfection of NCI-H23 with a retroviral vector expressing wild-type TGF-beta RII restored the responsiveness of cells to exogenous TGF-beta(1) with reduced cell proliferation. Immunocytochemical analysis demonstrated nuclear translocation of Smad3 after TGF-beta(1) treatment in RII-restored NCI-H23 cells. Underphosphorylation of the retinoblastoma protein accompanying p21 up-regulation was observed after TGF-beta(1) treatment of NCI-H23-RII cells. Receptor restoration also changed the levels of VEGF mRNA induced by TGF-beta(1). However, impairment of TGF-beta signalling did not alter microvessel formation in vivo in transplanted tumours. Instead, in vivo tumorigenesis experiments revealed a remarkable difference in the number and sizes of the tumours derived from NCI-H23-RII cells and dominant negative NCI-H23-dnRII cells (P < 0.01). Collectively, these observations suggest that impairment of TGF-beta signal transduction contributes significantly to tumour progression, mainly by cell proliferation rather than by modulation of angiogenesis in human NCI-H23 lung carcinoma cells.     

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.     

Relation of type II transforming growth factor-beta receptor to hepatic fibrosis and hepatocellular carcinoma

Hepatocarcinogenesis is closely related to hepatic fibrosis. In this study, we investigated the relationship of type II transforming growth factor-beta receptor (T beta RII) to hepatic fibrosis and hepatocellular carcinoma (HCC). In vivo: liver tissues were obtained from 30 patients (10 chronic hepatitis, 7 cirrhosis, 13 HCC). Protein expression and immunolocalization of T beta RII were examined by Western blot analysis and immunohistochemistry. In vitro: T beta RII protein expression in hepatoma cell lines (HepG2, Hep3B, HLE, HLF and Huh7) was examined by Western blot analysis. Next, we transfected T beta RII cDNA to Huh7, and compared the change of cell number and observed the induction of apoptosis after TGF-beta1 treatment using a FACScan flow cytometer. In vivo: T beta RII immunolocalization in liver tissues was significantly decreased in patients with HCC compared with that of patients with chronic hepatitis or liver cirrhosis. In Western blot analysis, T beta RII expression in tissues attenuated in comparison with that in non-tumor tissues in some patients with HCC. In vitro: T beta RII protein expression in HLE, HLF and Huh7 cells was weaker than that in HepG2 and Hep3B cells. In Huh7 cells transfected T beta RII cDNA, cell arrest and apoptosis were obviously induced. These results indicated that human HCC has a reduced expression of T beta RII for TGF-beta1. This may provide a selective growth advantage to HCC to escape the inhibitory growth signals of TGF-beta1, and may be linked with critical steps in the growth of hepatoma cells.     

Transforming growth factor-beta regulates mammary carcinoma cell survival and interaction with the adjacent microenvironment

Transforming growth factor (TGF)-beta signaling has been associated with early tumor suppression and late tumor progression; however, many of the mechanisms that mediate these processes are not known. Using Cre/LoxP technology, with the whey acidic protein promoter driving transgenic expression of Cre recombinase (WAP-Cre), we have now ablated the type II TGF-beta receptor (T beta RII) expression specifically within mouse mammary alveolar progenitors. Transgenic expression of the polyoma virus middle T antigen, under control of the mouse mammary tumor virus enhancer/promoter, was used to produce mammary tumors in the absence or presence of Cre (T beta RII((fl/fl);PY) and T beta RII((fl/fl);PY;WC), respectively). The loss of TGF-beta signaling significantly decreased tumor latency and increased the rate of pulmonary metastasis. The loss of TGF-beta signaling was significantly correlated with increased tumor size and enhanced carcinoma cell survival. In addition, we observed significant differences in stromal fibrovascular abundance and composition accompanied by increased recruitment of F4/80(+) cell populations in T beta RII((fl/fl);PY;WC) mice when compared with T beta RII((fl/fl);PY) controls. The recruitment of F4/80(+) cells correlated with increased expression of known inflammatory genes including Cxcl1, Cxcl5, and Ptgs2 (cyclooxygenase-2). Notably, we also identified an enriched K5(+) dNp63(+) cell population in primary T beta RII((fl/fl);PY;WC) tumors and corresponding pulmonary metastases, suggesting that loss of TGF-beta signaling in this subset of carcinoma cells can contribute to metastasis. Together, our current results indicate that loss of TGF-beta signaling in mammary alveolar progenitors may affect tumor initiation, progression, and metastasis through regulation of both intrinsic cell signaling and adjacent stromal-epithelial interactions in vivo.     

A dominant negative mutation of transforming growth factor-beta receptor type II gene in microsatellite stable oesophageal carcinoma

Recent investigations revealed microsatellite instability in colon cancers are associated with mutations of the transforming growth factor-beta receptor type II gene (TGF-beta RII) that encodes a transmembrane protein containing an intracellular serine/threonine kinase domain. Activation of TGF-beta receptor type I (RI) and RII by TGF-beta induces nuclear translocation of Smad proteins including Smad2 and Smad4 that have been originally identified as tumour suppressor genes. We have previously reported six cases with microsatellite instability in 32 oesophageal carcinomas. In this study, we analysed genetic mutations of TGF-beta RII, Smad2 and Smad4 in these oesophageal carcinoma tissues and established 16 cell lines. No genetic mutation was detected in any tissues or cell lines except one tissue sample of microsatellite stable oesophageal carcinoma, that is, a mis-sense mutation of glutamic acid to glutamine at codon 526 (E526Q) in the TGF-beta RII serine/threonine kinase domain. Interestingly, the mutant TGF-beta RII E526Q can completely inhibit TGF-beta-induction of nuclear translocation of Smad4 protein in oesophageal carcinoma cells. This mutation of TGF-beta RII that is not associated with microsatellite instability might make a dominant negative effect on TGF-beta signal transduction in oesophageal carcinoma.     

Smad3 is overexpressed in advanced human prostate cancer and necessary for progressive growth of prostate cancer cells in nude mice.

PURPOSE: The purpose of this study was to investigate the potential role of Smad3, a key mediator of transforming growth factor-beta signaling, in progression of prostate cancer. EXPERIMENTAL DESIGN: Expression of Smad proteins was determined in human prostate cancer tissue array and cell lines. Growth and metastasis of cells overexpressing dominant-negative Smad3 (Smad3D) were studied to determine its role in tumor progression in mice. Cell growth, apoptosis, and expression of angiogenic molecules in tumor lesions were studied to determine potential pathways that Smad3 promotes tumor progression. RESULTS: Smad3 was overexpressed in human prostate cancer, which correlated with Gleason score and expression of proliferating cell nuclear antigen. Androgen-independent PC-3MM2 and DU145 cells expressed much higher levels of Smad3 than did androgen-dependent LNCaP, 22Rv1, and LAPC-4 cells. Overexpression of Smad3D in PC-3MM2 cells (PC-3MM2-Smad3D) had minimal direct effects on cell growth but attenuated effects of transforming growth factor-beta1 on gene expression and cell growth. Overexpression of Smad3D did not significantly alter tumor incidence but reduced tumor growth rate and metastasis incidence. Most cells in the control tumors, but not PC-3MM2-Smad3D tumors, were positively stained by an antibody to proliferating cell nuclear antigen. Microvessels and expression of angiogenic molecule interleukin-8 were significantly reduced in tumors from PC-3MM2-Smad3D cells. PC-3MM2-Smad3D tumors also expressed lower levels of vascular endothelial growth factor and platelet-derived growth factor. CONCLUSIONS: These data suggest that Smad3, through regulating angiogenic molecule expression in tumor cells, is critical for progression of human prostate cancer.     

PI3K-Akt signaling is involved in the regulation of p21(WAF/CIP) expression and androgen-independent growth in prostate cancer cells

The purpose of this study is to investigate the role of PI3K-Akt signaling in prostate cancer cell growth and androgen receptor (AR)-mediated gene expression. Androgen-dependent LNCaP cells and their androgen-independent counterpart, LNCaP-AI cells, were used. We found that PI3K-Akt signaling is elevated in LNCaP-AI cells compared to that in LNCaP cells and is involved in androgen-independent growth. More importantly, PI3K-Akt signaling enhances AR activity and is involved in the induction of AR target genes, such as p21(WAF/CIP), a gene with anti-apoptosis activity and associated with androgen-independent growth in human prostate cancer. A receptor tyrosine kinase inhibitor also inhibits the PI3K-Akt signaling and compromises AR activity and cell growth. These findings suggest that the PI3K-Akt cell growth survival pathway and its downstream-regulated gene, p21(WAF/CIP), are targets for developing novel therapies against prostate cancer, especially those androgen-independent diseases.