Pro-metastasis function of TGFbeta mediated by the Smad pathway

The transforming growth factor beta (TGFbeta) signaling pathway plays a vital role in the development and homeostasis of normal tissues. Abnormal function of this pathway contributes to the initiation and progression of cancer. Smad proteins are key signal transducers of the TGFbeta pathway and are essential for the growth suppression function of TGFbeta. Smads are bona fide tumor suppressors whose mutation, deletion, and silencing are associated with many types of human cancer. However, the involvement and functional mechanism of Smad proteins in cancer metastasis are poorly defined. Recent studies using genetically modified cancer cells and mouse tumor models have provided concrete evidence for a Smad-dependent mechanism for metastasis promotion by TGFbeta. Understanding the dual roles of Smad proteins in tumor initiation and progression has important implications for cancer therapeutics.     

Autocrine TGFbeta signaling mediates vitamin D3 analog-induced growth inhibition in breast cells

In this study, we address whether TGFbeta signaling mediates vitamin D3 analog-induced growth inhibition in nonmalignant and malignant breast cells. Normal mammary epithelial cells (184), immortalized nonmalignant mammary epithelial cells (184A1 and MCF10A), and breast cancer cells (early passage MCF7: MCF7E) were sensitive to the inhibitory effects of vitamin D3 analogs (EB1089 and MC1288) while late passage MCF7 breast cancer (MCF7L) cells were relatively resistant. A similar pattern of sensitivity to TGFbeta was observed with these cells. Thus, the sensitivity to the vitamin D3 analogs correlated with the sensitivity to TGFbeta. MCF7L TGFbetaRII-transfected cells, which have autocrine TGFbeta activity, were more sensitive to EB1089 than MCF7L cells. TGFbeta neutralizing antibody was found to block the inhibitory effects of these analogs. These results are consistent with the idea that autocrine TGFbeta signaling mediates the anti-proliferative effects of the vitamin D3 analogs in these cells. The expression of TGFbeta isoforms and/or TGFbeta receptors was induced by the analogs in the vitamin D3 and TGFbeta sensitive cells. Vitamin D3 analogs did not induce TGFbeta or TGFbeta receptor expression in the resistant MCF7L cells. Therefore, EB1089 induces autocrine TGFbeta activity through increasing expression of TGFbeta isoforms and/or TGFbeta receptors. In addition, EB1089 induced nuclear VDR protein levels in the sensitive 184A1 cells but not in the resistant MCF7L cells. 184A1 cells were more sensitive to EB1089-induced VDR-dependent transactivation than MCF7L cells as measured by a luciferase reporter construct containing the VDRE, indicating a defect of VDR signaling in MCF7L cells. Smad3, a TGFbeta signaling mediator, coactivated VDR-dependent transactivation in 184A1 cells but not in MCF7L cells. These results indicate that Smad3 coactivates VDR to further enhance TGFbeta signaling and vitamin D3 signaling in the sensitive 184A1 cells. The results also indicate that Smad3 is not of itself sufficient to coactivate VDR in TGFbeta/vitamin D3 resistant MCF7L cells and other factors are required. We found that the PI 3-kinase pathway inhibitor LY29004 inhibited the synergy of TGFbeta and EB1089 on VDR-dependent transactivation activity. This indicates that the crosstalk between TGFbeta and vitamin D signaling is also PI 3-kinase pathway dependent.     

Abrogation of TGFbeta signaling induces apoptosis through the modulation of MAP kinase pathways in breast cancer cells

Transforming growth factor beta (TGFbeta) can modulate the activity of various MAP kinases. However, how this pathway may mediate TGFbeta-induced malignant phenotypes remains elusive. We investigated the role of autocrine TGFbeta signaling through MAP kinases in the regulation of cell survival in breast carcinoma MCF-7 cells and untransformed human mammary epithelial cells (HMECs). Our results show that abrogation of autocrine TGFbeta signaling with the expression of a dominant negative type II TGFbeta receptor (DNRII) or the treatment with a TGFbeta type I receptor inhibitor significantly increased apoptosis in MCF-7 cell, but not in HMEC. The expression of DNRII markedly decreased activated/phosphorylated Erk, whereas increased activated/phosphorylated p38 in MCF-7 cells. In contrast, there was no or little change of phosphorylated Erk and p38 in HMECs after the expression of DNRII. Inhibition of Erk activity in MCF-7 control cell induced apoptosis whereas restoration of Erk activity in MCF-7 DNRII cell reduced apoptosis. Similarly, inhibition of p38 activity also inhibited apoptosis in MCF-7 DNRII cell. Thus, autocrine TGFbeta signaling can enhance the survival of MCF-7 cells by maintaining the level of active Erk high and the level of active p38 low. Furthermore, the survival properties of TGFbeta pathway appear related to transformation supporting the notion that it may be a potential target for cancer therapy.     

Endoglin null endothelial cells proliferate faster and are more responsive to transforming growth factor beta1 with higher affinity receptors and an activated Alk1 pathway

Endoglin is an accessory receptor for transforming growth factor beta (TGFbeta) in endothelial cells, essential for vascular development. Its pivotal role in angiogenesis is underscored in Endoglin null (Eng-/-) murine embryos, which die at mid-gestation (E10.5) from impaired yolk sac vessel formation. Moreover, mutations in endoglin and the endothelial-specific TGFbeta type I receptor, ALK1, are linked to hereditary hemorrhagic telangiectasia. To determine the role of endoglin in TGFbeta pathways, we derived murine endothelial cell lines from Eng+/+ and Eng-/- embryos (E9.0). Whereas Eng+/+ cells were only partially growth inhibited by TGFbeta, Eng-/- cells displayed a potent anti-proliferative response. TGFbeta-dependent Smad2 phosphorylation and Smad2/3 translocation were unchanged in the Eng-/- cells. In contrast, TGFbeta treatment led to a more rapid activation of the Smad1/5 pathway in Eng null cells that was apparent at lower TGFbeta concentrations. Enhanced activity of the Smad1 pathway in Eng-/- cells was reflected in higher expression of ALK1-dependent genes such as Id1, Smad6, and Smad7. Analysis of cell surface receptors revealed that the TGFbeta type I receptor, ALK5, which is required for ALK1 function, was increased in Eng-/- cells. TGFbeta receptor complexes were less numerous but displayed a higher binding affinity. These results suggest that endoglin modulates TGFbeta signaling in endothelial cells by regulating surface TGFbeta receptors and suppressing Smad1 activation. Thus an altered balance in TGFbeta receptors and downstream Smad pathways may underlie defects in vascular development and homeostasis.     

Preferential production of latent transforming growth factor beta-2 by primary prostatic epithelial cells and its activation by prostate-specific antigen

Three mammalian isoforms of transforming growth factor-beta (TGFbeta) are known, TGFbeta1, 2, and 3, that have non-overlapping functions during development. However, their specific roles in cancers such as prostate cancer are less clear. Here we show that primary cultures of prostatic epithelial cells preferentially produce and activate the latent TGFbeta2 isoform. Paired cultures of normal and malignant prostate cells from prostate cancer patients produced predominantly the TGFbeta2 isoform, with 30- to 70-fold less TGFbeta1. By mono-Q ion exchange chromatography, three major peaks of latent TGFbeta2 activity were observed corresponding to the known small latent TGFbeta2 complex, the known large latent TGFbeta2 complex and a novel eluting peak of latent TGFbeta2. Although prostate cells are known to activate latent TGFbeta, the mechanism for activation is currently unclear. We investigated whether prostate specific antigen (PSA), a serine protease used as a clinical marker for prostate cancer, could play a role in the activation of latent TGFbeta. Unlike plasmin, a known activator of both latent TGFbeta1 and 2, PSA specifically activated the recombinant small latent form of TGFbeta2, but not TGFbeta1. Prostate epithelial cells, therefore, preferentially produce the TGFbeta2 isoform and PSA, a protease produced by the prostate, specifically targets the activation of this TGFbeta isoform. PSA-mediated activation of latent TGFbeta2 may be an important mechanism for autocrine TGFbeta regulation in the prostate and may potentially contribute to the formation of osteoblastic lesions in bone metastatic prostate cancer.     

Ras and TGF[beta] cooperatively regulate epithelial cell plasticity and metastasis: dissection of Ras signaling pathways.

Multistep carcinogenesis involves more than six discrete events also important in normal development and cell behavior. Of these, local invasion and metastasis cause most cancer deaths but are the least well understood molecularly. We employed a combined in vitro/in vivo carcinogenesis model, that is, polarized Ha-Ras-transformed mammary epithelial cells (EpRas), to dissect the role of Ras downstream signaling pathways in epithelial cell plasticity, tumorigenesis, and metastasis. Ha-Ras cooperates with transforming growth factor beta (TGFbeta) to cause epithelial mesenchymal transition (EMT) characterized by spindle-like cell morphology, loss of epithelial markers, and induction of mesenchymal markers. EMT requires continuous TGFbeta receptor (TGFbeta-R) and oncogenic Ras signaling and is stabilized by autocrine TGFbeta production. In contrast, fibroblast growth factors, hepatocyte growth factor/scatter factor, or TGFbeta alone induce scattering, a spindle-like cell phenotype fully reversible after factor withdrawal, which does not involve sustained marker changes. Using specific inhibitors and effector-specific Ras mutants, we show that a hyperactive Raf/mitogen-activated protein kinase (MAPK) is required for EMT, whereas activation of phosphatidylinositol 3-kinase (PI3K) causes scattering and protects from TGFbeta-induced apoptosis. Hyperactivation of the PI3K pathway or the Raf/MAPK pathway are sufficient for tumorigenesis, whereas EMT in vivo and metastasis required a hyperactive Raf/MAPK pathway. Thus, EMT seems to be a close in vitro correlate of metastasis, both requiring synergism between TGFbeta-R and Raf/MAPK signaling.     

The TGFbeta/Smad 3-signaling pathway is involved in butyrate-mediated vitamin D receptor (VDR)-expression

Previously, we demonstrated the pivotal role of the vitamin D receptor (VDR) in mediating the butyrate-induced differentiation in colon cancer cells. Smad 3, a downstream component of transforming growth factor-beta (TGFbeta) signaling, has been shown to act as a coactivator of VDR and to possibly regulate the vitamin D signaling pathway. In this study, we demonstrate a distinct impact of the TGFbeta/Smad 3-signaling pathway in the butyrate-mediated VDR expression and induction of differentiation. Butyrate treatment resulted in a significant induction of the phosphorylation level of Smad 3, while the combination of butyrate and a specific TGFbeta1-antibody or a TGFbeta-receptor inhibitor considerably diminished the butyrate-induced upregulation of VDR expression. Using a specific inhibitor, we were also able to demonstrate an involvement of the p38 MAPK in the increase of Smad 3 phosphorylation following butyrate treatment, thus opening the view to further elucidate possible mechanisms mediating the upregulation of VDR expression following butyrate treatment in colon cancer cells.     

TGFbeta primes breast tumors for lung metastasis seeding through angiopoietin-like 4

Cells released from primary tumors seed metastases to specific organs by a nonrandom process, implying the involvement of biologically selective mechanisms. Based on clinical, functional, and molecular evidence, we show that the cytokine TGFbeta in the breast tumor microenvironment primes cancer cells for metastasis to the lungs. Central to this process is the induction of angiopoietin-like 4 (ANGPTL4) by TGFbeta via the Smad signaling pathway. TGFbeta induction of Angptl4 in cancer cells that are about to enter the circulation enhances their subsequent retention in the lungs, but not in the bone. Tumor cell-derived Angptl4 disrupts vascular endothelial cell-cell junctions, increases the permeability of lung capillaries, and facilitates the trans-endothelial passage of tumor cells. These results suggest a mechanism for metastasis whereby a cytokine in the primary tumor microenvironment induces the expression of another cytokine in departing tumor cells, empowering these cells to disrupt lung capillary walls and seed pulmonary metastases.     

Cigarette smoke concentrate increases 8-epi-PGF2alpha and TGFbeta1 secretion in rat mesangial cells

Epidemiological studies have shown that cigarette smoke, an oxidant agent, is a risk factor for the development of diabetic nephropathy (DN), in which pathogenesis transforming growth factor beta(1) (TGFbeta(1)) plays a key role. In our experimental model we exposed mesangial cell cultures to cigarette smoke concentrate (CSC) to study the effect of smoking on the pathogenesis of DN. Thus, we analyzed the effect of CSC on TGFbeta(1) and lipid peroxidation (8-epi-PGF(2alpha)) in rat mesangial cells. Furthermore, since the protein kinase C (PKC) pathway appears to be a key factor for the enhanced production of TGFbeta(1), we also analyzed the effect of the selective PKCbeta inhibitor LY379196 on TGFbeta(1) response to CSC. CSC induced an increase of both TGFbeta(1) and 8-epi-PGF(2) compared to basal conditions (5 mM glucose). The CSC-induced increase in TGFbeta(1) secretion was significantly suppressed by LY379196. These data suggest that smoking could increase TGFbeta(1) production, probably due to oxidative stress and PKCbeta activation. This finding supports the concept that smoking is a risk factor for DN development.     

TGFβ signaling in male germ cells regulates gonocyte quiescence and fertility in mice

During testis development, proliferation and death of gonocytes are highly regulated to establish a standard population of adult stem spermatogonia that maintain normal spermatogenesis. As Transforming Growth Factor beta (TGFbeta) can regulate proliferation and apoptosis, we investigated its expression and functions during testis development. We show that TGFbeta2 is only expressed in quiescent gonocytes and decreases gonocyte proliferation in vitro. To study the functions of TGFbeta2, we developed conditional mice that invalidate the TGFbeta receptor type II in germ cells. Most of the knock-out animals die during fetal life, but the surviving adults show a reduced pool of spermatogonial stem/progenitor cells and become sterile with time. Using an organ culture system mimicking in vivo development, we show higher proportions of proliferating and apoptotic gonocytes from 13.5 dpc until 1 dpp, suggesting a reduction of germinal quiescence in these animals. Conversely, a 24-hour TGFbeta2-treatment of explanted wild-type testes, isolated every day from 13.5 dpc until 1 dpp, increased the duration of quiescence.These data show that the TGFbeta signaling pathway plays a physiological role during testis development by acting directly as a negative regulator of the fetal and neonatal germ cell proliferation, and indicate that the TGFbeta signaling pathway might regulate the duration of germ cell quiescence and is necessary to maintain adult spermatogenesis.     

Roles of TGFbeta signaling in epidermal/appendage development

The transforming growth factor beta (TGFbeta) superfamily encompasses a number of structurally related proteins that can be divided into several subfamilies including TGFbetas, activins/inhibins and bone morphogenetic proteins (BMPs). The Smads are major intracellular mediators in transducing the signals of TGFbeta superfamily members, and are abundantly expressed in the developing epidermis and epidermal appendages. Moreover, the phenotypes of transgenic/knockout mice with altered components of the TGFbeta superfamily signaling pathway suggest that TGFbeta superfamily signaling is required for epidermal/appendage development. TGFbeta superfamily members are involved in most events during epidermal/appendage development through the TGFbeta signal transduction pathway and through cross talk with other signaling pathways. Future studies will be instrumental in defining the precise roles for TGFbeta superfamily signaling in epidermal/appendage development.     

Transforming growth factor beta2 is a neuronal death-inducing ligand for amyloid-beta precursor protein

APP, amyloid beta precursor protein, is linked to the onset of Alzheimer's disease (AD). We have here found that transforming growth factor beta2 (TGFbeta2), but not TGFbeta1, binds to APP. The binding affinity of TGFbeta2 to APP is lower than the binding affinity of TGFbeta2 to the TGFbeta receptor. On binding to APP, TGFbeta2 activates an APP-mediated death pathway via heterotrimeric G protein G(o), c-Jun N-terminal kinase, NADPH oxidase, and caspase 3 and/or related caspases. Overall degrees of TGFbeta2-induced death are larger in cells expressing a familial AD-related mutant APP than in those expressing wild-type APP. Consequently, superphysiological concentrations of TGFbeta2 induce neuronal death in primary cortical neurons, whose one allele of the APP gene is knocked in with the V642I mutation. Combined with the finding indicated by several earlier reports that both neural and glial expression of TGFbeta2 was upregulated in AD brains, it is speculated that TGFbeta2 may contribute to the development of AD-related neuronal cell death.     

Divergence of epidermal growth factor - transforming growth factor beta signaling in embryonic orofacial tissue

The epidermal growth factor (EGF) and transforming growth factor beta (TGFbeta) families of signaling molecules play a major role in growth and development of embryos. Abrogation of either signaling pathway results in defects in embryogenesis, including cleft palate. In the developing palate, both EGF and TGFbeta regulate cellular proliferation, extracellular matrix synthesis, and cellular differentiation but often in an opposing manner. Evidence from various adult cell types suggests the existence of cross talk between the EGF and TGFbeta signaling pathways, although it is unclear whether such cross talk exists in murine embryonic maxillary mesenchymal cells, from which the developing palate is derived. In this study, embryonic maxillary mesenchymal cells in culture were treated with EGF and TGFbeta, either singly or in combination, and the cells were subsequently examined for signaling interactions between these two pathways. Immunoblot analyses of nuclear extracts of embryonic maxillary mesenchymal cells revealed that TGFbeta-induced nuclear translocation of Smad 2 and Smad 3 proteins was not affected by EGF. Conversely, immunoblot analyses of whole-cell extracts of these cells indicated that EGF-induced phosphorylation of extracellular signal-regulated kinase proteins, ERK1 and ERK2, was not affected by TGFbeta. Expression of a transfected luciferase reporter gene driven by a promoter with Smad binding elements was induced by TGFbeta in these cells but was not affected by EGF. Last, TGFbeta was found to induce expression of the endogenous gelatinase B gene in embryonic maxillary mesenchymal cells; however, this effect was independent of any interaction of EGF. Collectively, data from this study suggest that the EGF and TGFbeta signal transduction pathways do not converge in murine embryonic maxillary mesenchymal cells.     

Critical involvement of ILK in TGFbeta1-stimulated invasion/migration of human ovarian cancer cells is associated with urokinase plasminogen activator system

The present study investigated the role of integrin-linked kinase (ILK) in TGFbeta1-stimulated invasion/migration of human ovarian cancer cells. We investigated TGFbeta1 regulation of ILK, and effects of ILK knockdown on TGFbeta1-stimulated invasion/migration and the associated proteinase systems, urokinase plasminogen activator (uPA) and matrix metalloproteinases (MMPs) in SKOV3 cells. TGFbeta1 stimulated ILK kinase activity, and had no effect on ILK protein/mRNA levels. Transient transfection of an ILK-specific siRNA (ILK-H) reduced ILK protein level, mRNA level and kinase activity. ILK knockdown by ILK-H suppressed the basal and TGFbeta1-stimulated invasion and migration. Further, ILK-H reduced the basal and TGFbeta1-stimulated secretion of uPA, and increased the secretion of its inhibitor (PAI-1). Conversely, ILK-H did not affect TGFbeta1-stimulated secretion of MMP2 and its cell-associated activator MT1-MMP. Additionally, TGFbeta1 activated Smad2 phosphorylation, and this was not affected by ILK knockdown. Earlier reports indicate that Smad2 activation increased the expression of MMP2 and MT1-MMP. Thus, TGFbeta1 may act through ILK-independent and Smad2-dependent signaling in regulating MMP2 and MT1-MMP in SKOV3 cells. Collectively, this study suggests that ILK serves as a key mediator in TGFbeta1 regulation of uPA/PAI-1 system critical for the invasiveness of human ovarian cancer cells. And ILK is a potential target for cancer therapy.     

Disabled-2 (Dab2) mediates transforming growth factor beta (TGFbeta)-stimulated fibronectin synthesis through TGFbeta-activated kinase 1 and activation of the JNK pathway

The multifunctional cytokine transforming growth factor beta (TGFbeta) exerts many of its effects through its regulation of extracellular matrix components, including fibronectin (FN). Although expression of both TGFbeta and FN are essential for embryonic development and wound healing in the adult, overexpression leads to excessive deposition of extracellular matrix observed in many fibroproliferative disorders. We previously have demonstrated that TGFbeta-stimulated FN induction requires activation of the c-Jun N-terminal kinase (JNK) pathway; however, the signaling molecules that link the TGFbeta receptors to the JNK pathway remain unknown. We show here that the cytosolic adaptor protein disabled-2 (Dab2) directly stimulates JNK activity, whereas stable small interfering RNA-mediated ablation of Dab2 in NIH3T3 mouse fibroblasts and A10 rat aortic smooth muscle cells demonstrates that its expression is required for TGFbeta-mediated FN induction. We demonstrate that TGFbeta treatment stimulates the association of Dab2 with the mitogen-activated protein kinase kinase kinase, TAK1. Attenuation of cellular TAK1 levels by transient double-stranded RNA oligonucleotide transfection as well as overexpression of kinase-deficient TAK1 leads to abrogation of TGFbeta-stimulated FN induction. Furthermore, cell migration, another JNK-dependent response, is attenuated in NIH3T3-siDab2-expressing clones. We, therefore, delineate a signaling pathway proceeding from the TGFbeta receptors to Dab2 and TAK1, leading to TGFbeta-stimulated JNK activation, FN expression, and cell migration.