SB-431542 and Gleevec inhibit transforming growth factor-beta-induced proliferation of human osteosarcoma cells

Transforming growth factor-beta (TGF-beta) has growth-stimulating effects on mesenchymal cells and several tumor cell lines. The signaling pathway for this effect is, however, not well understood. We examined how TGF-beta stimulates proliferation of MG63 human osteosarcoma cells. Two distinct type I receptors for TGF-beta, ALK-1 and ALK-5, were expressed and functional in MG63 cells. Of these two receptors, ALK-5 appears to be responsible for the growth stimulation because expression of constitutively active ALK-5, but not ALK-1, stimulated proliferation of MG63 cells. SB-431542 (0.3 microM), a novel inhibitor of ALK4/5/7 kinase, suppressed TGF-beta-induced growth stimulation. DNA microarray analysis as well as quantitative real-time PCR analysis of RNAs from TGF-beta-treated cells demonstrated that several growth factors, including platelet-derived growth factor AA, were induced in response to TGF-beta in MG63 cells. Gleevec (1 microM) as well as AG1296 (5 microM) inhibited TGF-beta-induced growth stimulation of MG63 cells, suggesting that platelet-derived growth factor AA was mainly responsible for the growth-stimulatory effect of TGF-beta. We also examined the mechanisms of perturbation of growth-suppressing signaling in MG63 cells. We found that expression of c-Myc, which is down-regulated by TGF-beta in many other cells, was up-regulated in MG63 cells, suggesting that up-regulation of c-Myc expression may be the mechanism canceling growth-suppressing signaling of TGF-beta in MG63 cells.     

TGF-beta-induced upregulation of MMP-2 and MMP-9 depends on p38 MAPK, but not ERK signaling in MCF10A human breast epithelial cells

Transforming growth factor (TGF)-beta has been reported to exert growth inhibitory activity in normal epithelial cells whereas it induces cell proliferation and invasive phenotypes in advanced carcinomas. Our previous study showed that MCF10A, a spontaneously immortalized "normal" breast epithelial cell line, is resistant to TGF-beta-induced growth inhibition, suggesting that conversion of TGF-beta growth inhibitory signaling into an oncogenic pathway may occur at the early stage of tumor development/progression. To address this issue, we investigated the TGF-beta signaling pathway and its role in phenotypic transformation of MCF10A cells. TGF-beta treatment induced changes in the MCF10A cell morphology from cuboidal to an elongated spindle-like shape, accompanied with down-regulation of epithelial cell marker E-cadherin. TGF-beta treatment was sufficient to induce migrative and invasive phenotypes in these cells, an important phenotypic conversion during tumor progression. We also showed that TGF-beta treatment rapidly activated ERK-1/2 and p38 MAPK leading to upregulation of matrix metalloproteinase (MMP)-2 and MMP-9. Using chemical inhibitors and dominant negative mutants of MAPKs, we provide evidence that while both p38 MAPK and ERKs are required for TGF-beta-induced MCF10A cell migration and invasion, TGF-beta-induced MMP-2 and MMP-9 expression depends on p38 MAPK signaling, but is independent of ERK activity. This study demonstrates the roles of TGF-beta signaling pathways for induction of oncogenic signaling in preneoplastic human breast epithelial cells and will deepen our understanding of TGF-beta signaling in the progress of breast cancer.     

Duel nature of TGF-beta signaling: tumor suppressor vs. tumor promoter

PURPOSE OF REVIEW: Transforming growth factor beta type I (TGF-beta) is a ubiquitous cytokine that is well known for its ability to inhibit epithelial cell proliferation. Somatic mutations abrogating the TGF-beta signal transduction pathway are found in many gastrointestinal cancers, confirming its importance as a tumor suppressor. In contrast, many nongastrointestinal epithelial malignancies lack these somatic alterations, yet these cancers still acquire resistance to the growth-inhibitory effects of TGF-beta. In many instances, this resistance is part of a signaling switch whereby TGF-beta loses its growth inhibitory effects and is then used by the epithelial cell in a growth-promoting fashion. The mechanisms that underlie this change in the phenotypic growth response to TGF-beta are now being elucidated. This review focuses on recent advances in understanding the dual nature of the TGF-beta pathway as it relates to human carcinogenesis. RECENT FINDINGS: Elucidating the molecular basis that enables epithelial cells to change from a growth-suppressive to growth-stimulatory phenotype on TGF-beta exposure is an area of active research. Besides enhancing cancer cell growth, TGF-beta is also thought to promote a malignant cell's ability to metastasize by mediating changes in the cytoskeletal architecture, known as an epithelial-to-mesenchymal transition. This process enables a cancer cell to invade and spread to distal sites. Strong evidence has now emerged suggesting that the ability of a cell to use TGF-beta as a growth-promoting/invasive cytokine is a result of a number of different cellular and nuclear factors, including the absence or disruption of cyclin-dependent kinase inhibitors. This imbalance in cell cycle regulators may be the key element that dictates a cell's response to TGF-beta as growth-inhibitory versus growth-stimulatory, thus explaining the dual nature of TGF-beta signaling. SUMMARY: Current studies are beginning to shed light on the mechanisms that allow some nongastrointestinal epithelial cancers to evade the growth inhibitory effects of TGF-beta while simultaneously using this cytokine for growth advantage. By dissecting this phenotypic switch during tumor development, important genes, proteins, and pathways that are involved with TGF-beta signaling continue to be discovered. Knowledge of how premalignant cells and tumor cells respond to the growth promoting effects of TGF-beta and the genes that regulate this process will aid in the development of novel therapeutics and treatment strategies.     

Integration of Ras subeffector signaling in TGF-beta mediated late stage hepatocarcinogenesis

Immortalized p19(ARF) null hepatocytes (MIM) feature a high degree of functional differentiation and are susceptible to transforming growth factor (TGF)-beta driven growth arrest and apoptosis. In contrast, polarized MIM hepatocytes expressing hyperactive Ha-Ras continue proliferation in cooperation with TGF-beta, and adopt an invasive phenotype by executing an epithelial to mesenchymal transition (EMT). In this study, we analyzed the involvement of Ras subeffectors in TGF-beta mediated hepatocellular EMT by employing MIM hepatocytes, which express Ras mutants allowing selective activation of either mitogen-activated protein kinase (MAPK) signaling (V12-S35) or phosphoinositide 3-OH (PI3)3 kinase (PI3K) signaling (V12-C40). We found that MAPK signaling in MIM-S35 hepatocytes was necessary and sufficient to promote resistance to TGF-beta mediated inhibition of proliferation in vitro and in vivo. MIM-S35 hepatocytes showed also PI3K activation during EMT, however, MAPK signaling on its own protected hepatocytes from apoptosis. Yet, MIM-C40 hepatocytes failed to form tumors and required additional MAPK stimulation to overcome TGF-beta mediated growth arrest. In vivo, the collaboration of MAPK signaling and TGF-beta activity drastically accelerated the cell-cycle progression of the hepatocytes, leading to vast tumor formation. From these data we conclude that MAPK is crucial for the cooperation with TGF-beta to regulate the proliferation as well as the survival of hepatocytes during EMT, and causes the fatal increase in hepatocellular tumor progression.     

Resistance to gefitinib in PTEN-null HER-overexpressing tumor cells can be overcome through restoration of PTEN function or pharmacologic modulation of constitutive phosphatidylinositol 3'-kinase/Akt pathway signaling.

PURPOSE: Tyrosine kinase (TK) inhibitors are emerging as a promising new approach to the treatment of HER overexpressing tumors, however optimal use of these agents awaits further definition of the downstream signaling pathways that mediate their effects. We reported previously that both EGFR- and Her2-overexpressing tumors are sensitive to the new EGFR-selective TK inhibitor gefitinib (ZD1839, "Iressa"), and sensitivity to this agent correlated with its ability to down-regulate Akt. However, EGFR-overexpressing MDA-468 cells, which lack PTEN function, are resistant to ZD1839, and ZD1839 is unable to down-regulate Akt activity in these cells. EXPERIMENTAL DESIGN: To study the role of PTEN function, we generated MDA468 cells with tet-inducible PTEN expression. RESULTS: We show here that the resistance of MDA-468 cells to ZD1839 is attributable to EGFR-independent constitutive Akt activation caused by loss of PTEN function in these cells. Reconstitution of PTEN function through tet-inducible expression restores ZD1839 sensitivity to these cells and reestablishes EGFR-stimulated Akt signaling. Although restoration of PTEN function to tumors is difficult to implement clinically, much of the effects of PTEN loss are attributable to overactive PI3K/Akt pathway signaling, and this overactivity can be modulated by pharmacologic approaches. We show here that pharmacologic down-regulation of constitutive PI3K/Akt pathway signaling using the PI3K inhibitor LY294002 similarly restores EGFR-stimulated Akt signaling and sensitizes MDA-468 cells to ZD1839. CONCLUSIONS: Sensitivity to ZD1839 requires intact growth factor receptor-stimulated Akt signaling activity. PTEN loss leads to uncoupling of this signaling pathway and results in ZD1839 resistance, which can be reversed with reintroduction of PTEN or pharmacologic down-regulation of constitutive PI3K/Akt pathway activity. These data have important predictive and therapeutic clinical implications.     

Collaborative activities of macrophage-stimulating protein and transforming growth factor-beta1 in induction of epithelial to mesenchymal transition: roles of the RON receptor tyrosine kinase

Epithelial to mesenchymal transition (EMT) is a process occurring during embryonic development and cancer progression. Using recepteur d'origine nantais (RON)-expressing epithelial cells as a model, we showed that RON activation causes spindle-shaped morphology with increased cell motilities. These activities resemble those observed in EMT induced by transforming growth factor (TGF)-beta1 or by Ras-Raf signaling. By immunofluorescent and Western blot analyses, we found that constitutive RON expression results in diminished expression of E-cadherin, redistribution of beta-catenin, reorganization of actin cytoskeleton, and increased expression of vimentin, a mesenchymal filament. RON expression is also essential for TGF-beta1-induced expression of alpha-smooth muscle actin (alpha-SMA), a specialized mesenchymal marker. In the study of signaling pathways responsible for RON-mediated EMT, it was found that PD98059, a MAP kinase inhibitor, blocks the collaborative activities of RON and TGF-beta1 in induction of alpha-SMA expression and restores epithelial cells to their original morphology. Moreover, we showed that RON expression increases Smad2 gene promoter activities and protein expression, which significantly lowers TGF-beta1 threshold for EMT induction. These results suggest that persistent RON expression and activation cause the loss of epithelial phenotypes. These changes, collaborating with TGF-beta1 signaling, could play a critical role in epithelial transdifferentiation towards invasiveness and metastasis of certain cancers.     

Differential expression of and responsiveness to transforming growth factor-beta (TGF-beta) isoforms in hormone-dependent and independent lines of mouse mammary tumors.

Transforming growth factor-beta2 (TGF-beta2) and -beta3 mRNA expressions were studied in ductal hormone-dependent (HD) and -independent (HI) in vivo lines of the medroxyprogesterone acetate (MPA)-induced mammary tumor model in Balb/c mice. MPA treatment of HD tumors induced a significant decrease in TGF-beta2 and -beta3 mRNA levels. Progression to an HI phenotype of ductal tumors was associated with reduced TGF-beta2 and -beta3 expressions, as compared with their HD counterparts. Exogenously added TGF-beta1, -beta2, and -beta3 (1 ng/ml) inhibited the proliferation of primary cultures of epithelial cells from ductal HD and HI tumors. In addition, TGF-beta expression and effects were studied in the other type of MPA-induced mammary tumors, which are of lobular origin and lack steroid hormone receptors and evidence an HI behavior. These lobular HI lines showed TGF-beta2 levels similar to those found in HD lines growing in MPA-treated mice. In contrast, TGF-beta3 mRNA levels were 12- to 20-fold higher than in HD tumors. Primary cultures of lobular HI epithelial cells required either TGF-beta concentrations of 10 ng/ml to show an inhibitory response, or were completely resistant to TGF-beta inhibition. Studies of the molecular mechanisms involved in reduction or loss of TGF-beta responsiveness in lobular HI tumors showed that cell surface type II TGF-beta receptor levels were lower in these tumors than those present in HD tumors. Our results support the hypothesis that TGF-beta could play a role as an autocrine growth inhibitor in HD and HI ductal tumors. Autonomous growth of lobular HI tumors could be favored by undetectable or low TGF-beta1 and -beta2 expressions and by reduced or lost sensitivity of epithelial cells to TGF-beta's antiproliferative effects. However, the extremely high levels of TGF-beta3 expression in lobular HI tumors, in spite of reduced sensitivity to TGF-beta3 inhibitory growth effect in tumor epithelial cells, suggest a net positive role for TGF-beta3 in these tumors.     

Transforming growth factor-beta promotes invasion in tumorigenic but not in nontumorigenic human prostatic epithelial cells

Transforming growth factor-beta (TGF-beta) is a pleiotropic growth factor with actions that are dependent on circumstances, including dose, target cell type, and context. TGF-beta can elicit both growth-promoting and growth-suppressive activities. In normal tissues, TGF-beta generally acts to restrict growth and maintain differentiation. However, during tumorigenesis, changes in TGF-beta expression and cellular responses can promote tumorigenesis. The present study examines the effects of TGF-beta on the nontumorigenic human prostatic epithelial cell line BPH1 and on three derivative tumorigenic sublines BPH1(CAFTD)1, BPH1(CAFTD)3, and BPH1(CAFTD)5. The data show that TGF-beta has different effects on the nontumorigenic and tumorigenic cells. The nontumorigenic cells are growth inhibited by TGF-beta. In contrast, the tumorigenic sublines are not growth inhibited but instead undergo an epithelial to mesenchymal transformation (EMT) in response to TGF-beta. The tumorigenic lines show constitutively elevated levels of phosphorylated Akt, which modulates their response to TGF-beta by blocking Smad3 and p21 nuclear translocation. On TGF-beta stimulation of the tumorigenic sublines, the activated Akt allows the cell to escape cell cycle arrest. The phosphatidylinositol 3-kinase/Akt pathway is also involved in TGF-beta-induced EMT, defined here by induction of vimentin expression and enhanced cellular motility. In vivo, tumorigenic cells with constitutively active TGF-beta signaling show increased invasion with EMT, which express vimentin, located specifically at the invasive front of the tumor. These data indicate that following malignant transformation TGF-beta can play a direct role in promoting prostatic cancer and further that these responses are context specific in vivo.     

GLI inhibitor GANT-61 diminishes embryonal and alveolar rhabdomyosarcoma growth by inhibiting Shh/AKT-mTOR axis

Rhabdomyosarcoma (RMS) typically arises from skeletal muscle. Currently, RMS in patients with recurrent and metastatic disease have no successful treatment. The molecular pathogenesis of RMS varies based on cancer sub-types. Some embryonal RMS but not other sub-types are driven by sonic hedgehog (Shh) signaling pathway. However, Shh pathway inhibitors particularly smoothened inhibitors are not highly effective in animals. Here, we show that Shh pathway effectors GLI1 and/or GLI2 are over-expressed in the majority of RMS cells and that GANT-61, a specific GLI1/2 inhibitor dampens the proliferation of both embryonal and alveolar RMS cells-derived xenograft tumors thereby blocking their growth. As compared to vehicle-treated control, about 50% tumor growth inhibition occurs in mice receiving GANT-61 treatment. The proliferation inhibition was associated with slowing of cell cycle progression which was mediated by the reduced expression of cyclins D1/2/3 & E and the concomitant induction of p21. GANT-61 not only reduced expression of GLI1/2 in these RMS but also significantly diminished AKT/mTOR signaling. The therapeutic action of GANT-61 was significantly augmented when combined with chemotherapeutic agents employed for RMS therapy such as temsirolimus or vincristine. Finally, reduced expression of proteins driving epithelial mesenchymal transition (EMT) characterized the residual tumors.     

Loss of TGF-beta type II receptor in fibroblasts promotes mammary carcinoma growth and invasion through upregulation of TGF-alpha-, MSP- and HGF-mediated signaling networks

Stromal fibroblasts regulate epithelial cell behavior through direct and indirect cell-cell interactions. To clarify the role of TGF-beta signaling in stromal fibroblasts during mammary development and tumorigenesis, we conditionally knocked out the TGF-beta type II receptor gene in mouse mammary fibroblasts (Tgfbr2(fspKO)). Tgfbr2(fspKO) mice exhibit defective mammary ductal development, characterized in part by increased ductal epithelial cell turnover associated with an increase in stromal fibroblast abundance. Tgfbr2(fspKO) mammary fibroblasts transplanted with mammary carcinoma cells promote growth and invasion, which is associated with increased activating phosphorylation of the receptors: erbB1, erbB2, RON, and c-Met. Furthermore, the increased receptor phosphorylation correlates with increased secretion of the cognate ligands by Tgfbr2(fspKO) fibroblasts. Treatment of tumor cells with fibroblast-conditioned medium leads to increased tumor cell proliferation and motility, which are blocked by addition of pharmacologic inhibitors of TGF-alpha signaling or neutralizing antibodies to macrophage-stimulating protein (MSP), HGF, or c-Met. These studies characterize a significant role for stromal TGF-beta signaling in mammary tissue homeostasis and mammary tumor progression via regulation of TGF-alpha, MSP, and HGF signaling pathways.     

Disrupted transforming growth factor-beta signaling and deregulated growth in human biliary tract cancer cells

Biliary tract carcinoma is a common neoplasm in Japan, and its treatment is difficult because it tends to promote fibrosis and easily invades surrounding tissues. To better characterize the biological features of this carcinoma, we investigated abnormalities in the transforming growth factor-beta (TGF-beta) signaling pathway in five human biliary tract cancer cell lines: RBE, KMBC, SK-ChA-1, Mz-ChA-1, and Mz-ChA-2. We stably transfected into these cells the luciferase reporter plasmid carrying promoter of the plasminogen activator inhibitor-1 gene, the expression of which is stimulated by TGF-beta1. Treating the KMBC and Mz-ChA-1 cells with TGF-beta1 neither inhibited cell growth nor stimulated luciferase activity. In contrast, the RBE and Mz-ChA-2 cells responded well to TGF-beta1 treatment. TGF-beta1-treated SK-ChA-1 cells exhibited attenuated luciferase activity and their growth was not inhibited. Smad4 mRNA was not detected in SK-ChA-1 and Mz-ChA-1 cells by Northern blot analysis. Genetic analysis disclosed a nonsense mutation in the Mad homologue 2a domain of the Smad4 gene in the SK-ChA-1 cells and a heterozygous deletion in the TGF-beta type II receptor gene in the KMBC cells. Expression of the exogenous Smad4 gene in the Mz-ChA-1 cells by transient transfection restored their luciferase activity. When these TGF-beta1-insensitive and less-TGF-beta1-sensitive cell lines were xenografted into nude mice, they developed tumors that had more prominent, intervening fibrosis (desmoplasia) than the tumors caused by TGF-beta1-sensitive cells. Thus, a tight correlation between disruption of the TGF-beta signaling pathway and deregulated growth of cancer cells has been demonstrated in biliary tract carcinoma. This seems to be a critical event in this carcinoma and may also be correlated with stromal cell reaction in cancer invasion.