Arginine Methylation Initiates BMP-Induced Smad Signaling.

Kinase activation and substrate phosphorylation commonly form the backbone of signaling cascades. Bone morphogenetic proteins (BMPs), a subclass of TGF-ß family ligands, induce activation of their signaling effectors, the Smads, through C-terminal phosphorylation by transmembrane receptor kinases. However, the slow kinetics of Smad activation in response to BMP suggests a preceding step in the initiation of BMP signaling. We now show that arginine methylation, which is known to regulate gene expression, yet also modifies some signaling mediators, initiates BMP-induced Smad signaling. BMP-induced receptor complex formation promotes interaction of the methyltransferase PRMT1 with the inhibitory Smad6, resulting in Smad6 methylation and relocalization at the receptor, leading to activation of effector Smads through phosphorylation. PRMT1 is required for BMP-induced biological responses across species, as evidenced by the role of its ortholog Dart1 in BMP signaling during Drosophila wing development. Activation of signaling by arginine methylation may also apply to other signaling pathways.

Smad3-mediated recruitment of the methyltransferase SETDB1/ESET controls Snail1 expression and epithelial-mesenchymal transition.

During epithelial-mesenchymal transition (EMT), reprogramming of gene expression is accompanied by histone modifications. Whether EMT-promoting signaling directs functional changes in histone methylation has not been established. We show here that the histone lysine methyltransferase SETDB1 represses EMT and that, during TGF-ß-induced EMT, cells attenuate SETDB1 expression to relieve this inhibition. SETDB1 also controls stem cell generation, cancer cell motility, invasion, metastatic dissemination, as well as sensitivity to certain cancer drugs. These functions may explain the correlation of breast cancer patient survival with SETDB1 expression. At the molecular level, TGF-ß induces SETDB1 recruitment by Smad3, to repress Smad3/4-activated transcription of SNAI1, encoding the EMT "master" transcription factor SNAIL1. Suppression of SNAIL1-mediated gene reprogramming by SETDB1 occurs through H3K9 methylation at the SNAI1 gene that represses its H3K9 acetylation imposed by activated Smad3/4 complexes. SETDB1 therefore defines a TGF-ß-regulated balance between histone methylation and acetylation that controls EMT.

Arginine methylation of SMAD7 by PRMT1 in TGF-ß-induced epithelial-mesenchymal transition and epithelial stem-cell generation.

The epithelial-to-mesenchymal transdifferentiation (EMT) is crucial for tissue differentiation in development and drives essential steps in cancer and fibrosis. EMT is accompanied by reprogramming of gene expression and has been associated with the epithelial stem-cell state in normal and carcinoma cells. The cytokine transforming growth factor ß (TGF-ß) drives this program in cooperation with other signaling pathways and through TGF-ß-activated SMAD3 as the major effector. TGF-ß-induced SMAD3 activation is inhibited by SMAD7 and to a lesser extent by SMAD6, and SMAD6 and SMAD7 both inhibit SMAD1 and SMAD5 activation in response to the TGF-ß-related bone morphogenetic proteins (BMPs). We previously reported that, in response to BMP, protein arginine methyltransferase 1 (PRMT1) methylates SMAD6 at the BMP receptor complex, thereby promoting its dissociation from the receptors and enabling BMP-induced SMAD1 and SMAD5 activation. We now provide evidence that PRMT1 also facilitates TGF-ß signaling by methylating SMAD7, which complements SMAD6 methylation. We found that PRMT1 is required for TGF-ß-induced SMAD3 activation, through a mechanism similar to that of BMP-induced SMAD6 methylation, and thus promotes the TGF-ß-induced EMT and epithelial stem-cell generation. This critical mechanism positions PRMT1 as an essential mediator of TGF-ß signaling that controls the EMT and epithelial cell stemness through SMAD7 methylation.

ShcA Protects against Epithelial-Mesenchymal Transition through Compartmentalized Inhibition of TGF-ß-Induced Smad Activation.

Epithelial-mesenchymal transition (EMT) is a normal cell differentiation event during development and contributes pathologically to carcinoma and fibrosis progression. EMT often associates with increased transforming growth factor-ß (TGF-ß) signaling, and TGF-ß drives EMT, in part through Smad-mediated reprogramming of gene expression. TGF-ß also activates the Erk MAPK pathway through recruitment and Tyr phosphorylation of the adaptor protein ShcA by the activated TGF-ß type I receptor. We found that ShcA protects the epithelial integrity of nontransformed cells against EMT by repressing TGF-ß-induced, Smad-mediated gene expression. p52ShcA competed with Smad3 for TGF-ß receptor binding, and down-regulation of ShcA expression enhanced autocrine TGF-ß/Smad signaling and target gene expression, whereas increased p52ShcA expression resulted in decreased Smad3 binding to the TGF-ß receptor, decreased Smad3 activation, and increased Erk MAPK and Akt signaling. Furthermore, p52ShcA sequestered TGF-ß receptor complexes to caveolin-associated membrane compartments, and reducing ShcA expression enhanced the receptor localization in clathrin-associated membrane compartments that enable Smad activation. Consequently, silencing ShcA expression induced EMT, with increased cell migration, invasion, and dissemination, and increased stem cell generation and mammosphere formation, dependent upon autocrine TGF-ß signaling. These findings position ShcA as a determinant of the epithelial phenotype by repressing TGF-ß-induced Smad activation through differential partitioning of receptor complexes at the cell surface.

Smad4 Decreases the Population of Pancreatic Cancer-Initiating Cells through Transcriptional Repression of ALDH1A1.

Cancer progression involves a rare population of undifferentiated cancer-initiating cells that have stem cell-like properties for self-renewal capacity and high tumorigenicity. We investigated how maintenance of pancreatic cancer-initiating cells is influenced by Smad4, which is frequently deleted or mutated in pancreatic cancers cells. Smad4 silencing up-regulated the expression of aldehyde dehydrogenase 1A1 (ALDH1A1) mRNA, whereas forced expression of Smad4 in pancreatic cancer cells down-regulated it. Smad4 and ALDH1 expression inversely correlated in some human clinical pancreatic adenocarcinoma tissues, suggesting that ALDH1 in pancreatic cancer cells was associated with decreased Smad4 expression. We then examined whether ALDH1 served as a marker of pancreatic cancer-initiating cells. Pancreatic cancer cells contained ALDH1(hi) cells in 3% to 10% of total cells, with high tumorigenic potential. Because Smad4 is a major mediator of transforming growth factor (TGF)-ß family signaling, we investigated the regulatory mechanism of ALDH activity by TGF-ß and bone morphogenetic proteins. Treatment with TGF-ß attenuated ALDH1(hi) cells in several pancreatic cancer cells, whereas bone morphogenetic protein-4 was not as potent. Biochemical experiments revealed that TGF-ß regulated ALDH1A1 mRNA transcription through binding of Smad4 to its regulatory sequence. It appears that TGF-ß negatively regulates ALDH1 expression in pancreatic cancer cells in a Smad-dependent manner and in turn impairs the activity of pancreatic cancer-initiating cells.

TGF-ß family signaling in stem cells.

BACKGROUND: The diversity of cell types and tissue types that originate throughout development derives from the differentiation potential of embryonic stem cells and somatic stem cells. While the former are pluripotent, and thus can give rise to a full differentiation spectrum, the latter have limited differentiation potential but drive tissue remodeling. Additionally cancer tissues also have a small population of self-renewing cells with stem cell properties. These cancer stem cells may arise through dedifferentiation from non-stem cells in cancer tissues, illustrating their plasticity, and may greatly contribute to the resistance of cancers to chemotherapies. SCOPE OF REVIEW: The capacity of the different types of stem cells for self-renewal, the establishment and maintenance of their differentiation potential, and the selection of differentiation programs are greatly defined by the interplay of signaling molecules provided by both the stem cells themselves, and their microenvironment, the niche. Here we discuss common and divergent roles of TGF-ß family signaling in the regulation of embryonic, reprogrammed pluripotent, somatic, and cancer stem cells. MAJOR CONCLUSIONS: Increasing evidence highlights the similarities between responses of normal and cancer stem cells to signaling molecules, provided or activated by their microenvironment. While TGF-ß family signaling regulates stemness of normal and cancer stem cells, its effects are diverse and depend on the cell types and physiological state of the cells. GENERAL SIGNIFICANCE: Further mechanistic studies will provide a better understanding of the roles of TGF-ß family signaling in the regulation of stem cells. These basic studies may lead to the development of a new therapeutic or prognostic strategies for the treatment of cancers. This article is part of a Special Issue entitled Biochemistry of Stem Cells.

Prostate cancer cells and bone stromal cells mutually interact with each other through bone morphogenetic protein-mediated signals.

Functional interactions between cancer cells and the bone microenvironment contribute to the development of bone metastasis. Although the bone metastasis of prostate cancer is characterized by increased ossification, the molecular mechanisms involved in this process are not fully understood. Here, the roles of bone morphogenetic proteins (BMPs) in the interactions between prostate cancer cells and bone stromal cells were investigated. In human prostate cancer LNCaP cells, BMP-4 induced the production of Sonic hedgehog (SHH) through a Smad-dependent pathway. In mouse stromal MC3T3-E1 cells, SHH up-regulated the expression of activin receptor IIB (ActR-IIB) and Smad1, which in turn enhanced BMP-responsive reporter activities in these cells. The combined stimulation with BMP-4 and SHH of MC3T3-E1 cells cooperatively induced the expression of osteoblastic markers, including alkaline phosphatase, bone sialoprotein, collagen type II α1, and osteocalcin. When MC3T3-E1 cells and LNCaP cells were co-cultured, the osteoblastic differentiation of MC3T3-E1 cells, which was induced by BMP-4, was accelerated by SHH from LNCaP cells. Furthermore, LNCaP cells and BMP-4 cooperatively induced the production of growth factors, including fibroblast growth factor (FGF)-2 and epidermal growth factor (EGF) in MC3T3-E1 cells, and these may promote the proliferation of LNCaP cells. Taken together, our findings suggest that BMPs provide favorable circumstances for the survival of prostate cancer cells and the differentiation of bone stromal cells in the bone microenvironment, possibly leading to the osteoblastic metastasis of prostate cancer.

TGF-ß signaling and epithelial-mesenchymal transition in cancer progression.

PURPOSE OF REVIEW: TGF-ß acts as a potent driver of cancer progression through the induction of epithelial-mesenchymal transition (EMT), in which epithelial cells acquire mesenchymal phenotype, leading to enhanced motility and invasion. Recent reports highlight the fundamental roles of TGF-ß-induced EMT in multiple aspects of cancer progression. In this review, we focus on the novel insights into the roles of TGF-ß-induced EMT in cancer progression and the underlying mechanisms that enable TGF-ß to activate this epithelial plasticity response at transcription, translation, and posttranslational levels. RECENT FINDINGS: Smad-mediated transcription regulation is known to activate TGF-ß-induced EMT. More recently, novel mechanisms of epigenetic control, alternative splicing, miRNAs, translation control, and posttranslational modifications have been shown to play key roles in the control of EMT. In addition to initiating carcinoma cell invasion, TGF-ß-induced EMT can guide cancer cells to de-differentiate and gain cancer stem-cell-like properties. EMT also allows the generation of stromal cells that support and instruct cancer progression. SUMMARY: The differentiation plasticity of epithelial cells that mediates TGF-ß-induced EMT and reversion from mesenchymal to epithelial phenotype are increasingly seen as integral aspects of cancer progression that contribute to survival and dissemination of cancer cells. Further mechanistic insights under physiological conditions may lead to new therapeutic or prognostic strategies in cancer treatment.

Coordinated expression of REG4 and aldehyde dehydrogenase 1 regulating tumourigenic capacity of diffuse-type gastric carcinoma-initiating cells is inhibited by TGF-ß.

Aldehyde dehydrogenase 1 (ALDH1) has been shown to serve as a marker for cancer-initiating cells (CICs), but little is known about the regulation of the CIC functions of ALDH1+ cancer cells. We isolated ALDH1+ cells from human diffuse-type gastric carcinoma cells and characterized these cells using an Aldefluor assay. ALDH1+ cells constituted 5-8% of the human diffuse-type gastric carcinoma cells, OCUM-2MLN and HSC-39; were more tumourigenic than ALDH1- cells; and were able to self-renew and generate heterogeneous cell populations. Using gene expression microarray analyses, we identified REG4 (regenerating islet-derived family, member 4) as one of the genes up-regulated in ALDH1+ cells, and thus as a novel marker for ALDH1+ tumour cells. Induced expression of REG4 enhanced the colony-forming ability of OCUM-2MLN cells, while knockdown of REG4 inhibited the tumourigenic potential of ALDH1+ cells. We further found that TGF-ß signalling reduces the expression of ALDH1 and REG4, and the size of the ALDH1+ cell population. In human diffuse-type gastric carcinoma tissues, the expression of ALDH1 and REG4 correlated with each other, as assessed by immunohistochemistry, and ALDH1 expression correlated inversely with Smad3 phosphorylation as a measure of TGF-ß signalling. These findings illustrate that, in diffuse-type gastric carcinoma, REG4 is up-regulated in ALDH1+ CICs, and that the increased tumourigenic ability of ALDH1+ cells depends on REG4. Moreover, TGF-ß down-regulates ALDH1 and REG4 expression, which correlates with a reduction in CIC population size and tumourigenicity. Targeting REG4 in ALDH1+ CICs may provide a novel strategy in the treatment of diffuse-type gastric carcinoma.

Estrogen inhibits transforming growth factor beta signaling by promoting Smad2/3 degradation.

Estrogen is a growth factor that stimulates cell proliferation. The effects of estrogen are mediated through the estrogen receptors, ERalpha and ERbeta, which function as ligand-induced transcription factors and belong to the nuclear receptor superfamily. On the other hand, TGF-beta acts as a cell growth inhibitor, and its signaling is transduced by Smads. Although a number of studies have been made on the cross-talk between estrogen/ERalpha and TGF-beta/Smad signaling, whose molecular mechanisms remain to be determined. Here, we show that ERalpha inhibits TGF-beta signaling by decreasing Smad protein levels. ERalpha-mediated reductions in Smad levels did not require the DNA binding ability of ERalpha, implying that ERalpha opposes the effects of TGF-beta via a novel non-genomic mechanism. Our analysis revealed that ERalpha formed a protein complex with Smad and the ubiquitin ligase Smurf, and enhanced Smad ubiquitination and subsequent degradation in an estrogen-dependent manner. Our observations provide new insight into the molecular mechanisms governing the non-genomic functions of ERalpha.

Epithelial plasticity, epithelial-mesenchymal transition, and the TGF-ß family.

Epithelial cells repress epithelial characteristics and elaborate mesenchymal characteristics to migrate to other locations and acquire new properties. Epithelial plasticity responses are directed through cooperation of signaling pathways, with TGF-ß and TGF-ß-related proteins playing prominent instructive roles. Epithelial-mesenchymal transitions (EMTs) directed by activin-like molecules, bone morphogenetic proteins, or TGF-ß regulate metazoan development and wound healing and drive fibrosis and cancer progression. In carcinomas, diverse EMTs enable stem cell generation, anti-cancer drug resistance, genomic instability, and localized immunosuppression. This review discusses roles of TGF-ß and TGF-ß-related proteins, and underlying molecular mechanisms, in epithelial plasticity in development and wound healing, fibrosis, and cancer.

Chronic TGF-ß exposure drives stabilized EMT, tumor stemness, and cancer drug resistance with vulnerability to bitopic mTOR inhibition.

Tumors comprise cancer stem cells (CSCs) and their heterogeneous progeny within a stromal microenvironment. In response to transforming growth factor-ß (TGF-ß), epithelial and carcinoma cells undergo a partial or complete epithelial-mesenchymal transition (EMT), which contributes to cancer progression. This process is seen as reversible because cells revert to an epithelial phenotype upon TGF-ß removal. However, we found that prolonged TGF-ß exposure, mimicking the state of in vivo carcinomas, promotes stable EMT in mammary epithelial and carcinoma cells, in contrast to the reversible EMT induced by a shorter exposure. The stabilized EMT was accompanied by stably enhanced stem cell generation and anticancer drug resistance. Furthermore, prolonged TGF-ß exposure enhanced mammalian target of rapamycin (mTOR) signaling. A bitopic mTOR inhibitor repressed CSC generation, anchorage independence, cell survival, and chemoresistance and efficiently inhibited tumorigenesis in mice. These results reveal a role for mTOR in the stabilization of stemness and drug resistance of breast cancer cells and position mTOR inhibition as a treatment strategy to target CSCs.

Intracellular and extracellular TGF-ß signaling in cancer: some recent topics.

Transforming growth factor (TGF)-ß regulates a wide variety of cellular responses, including cell growth arrest, apoptosis, cell differentiation, motility, invasion, extracellular matrix production, tissue fibrosis, angiogenesis, and immune function. Although tumor-suppressive roles of TGF-ß have been extensively studied and well-characterized in many cancers, especially at early stages, accumulating evidence has revealed the critical roles of TGF-ß as a pro-tumorigenic factor in various types of cancer. This review will focus on recent findings regarding epithelial-mesenchymal transition (EMT) induced by TGF-ß, in relation to crosstalk with some other signaling pathways, and the roles of TGF-ß in lung and pancreatic cancers, in which TGF-ß has been shown to be involved in cancer progression. Recent findings also strongly suggested that targeting TGF-ß signaling using specific inhibitors may be useful for the treatment of some cancers. TGF-ß plays a pivotal role in the differentiation and function of regulatory T cells (Tregs). TGF-ß is produced as latent high molecular weight complexes, and the latent TGF-ß complex expressed on the surface of Tregs contains glycoprotein A repetitions predominant (GARP, also known as leucine-rich repeat containing 32 or LRRC32). Inhibition of the TGF-ß activities through regulation of the latent TGF-ß complex activation will be discussed.

Autocrine TGF-ß protects breast cancer cells from apoptosis through reduction of BH3-only protein, Bim.

Cancer cells undergo multi-step processes in obtaining the ability to metastasize, and are constantly exposed to signals that induce apoptosis. Acquisition of anti-apoptotic properties by cancer cells is important for metastasis, and recent studies suggest that transforming growth factor (TGF)-ß promotes the survival of certain types of cancer cells. Here, we found that in highly metastatic breast cancer cells, JygMC(A), JygMC(B) and 4T1, TGF-ß ligands were produced in autocrine fashion. Pharmacological inhibition of endogenous TGF-ß signalling by a TGF-ß type I receptor kinase inhibitor in serum-free conditions increased the expression of BH3-only protein, Bim (also known as Bcl2-like 11) in JygMC(A) and JygMC(B) cells, and caused apoptotic cell death. We also found that induction of Bim by TGF-ß was not observed in Foxc1 knocked-down cancer cells. These findings suggest that TGF-ß plays a crucial role in the regulation of survival of certain types of cancer cells through the TGF-ß-Foxc1-Bim pathway, and that specific inhibitors of TGF-ß signalling might be useful as apoptosis inducers in breast cancer cells.

Ki26894, a novel transforming growth factor-beta type I receptor kinase inhibitor, inhibits in vitro invasion and in vivo bone metastasis of a human breast cancer cell line.

Transforming growth factor (TGF)-beta signaling has been shown to promote tumor growth and metastasis in advanced cancer. Use of inhibitors of TGF-beta signaling may thus be a novel strategy for treatment of patients with such cancers. In this study, we investigated the effects of a novel TGF-beta type I receptor (TbetaR-I) kinase inhibitor, Ki26894, on bone metastasis of a highly bone-metastatic variant of human breast cancer MDA-MB-231 cells, termed MDA-MB-231-5a-D (MDA-231-D). Ki26894 blocked TGF-beta signaling in MDA-231-D cells, as detected by suppression of phosphorylation of Smad2 and inhibition of TGF-beta-responsive reporter activity. Moreover, Ki26894 decreased the motility and the invasion of MDA-231-D cells induced by TGF-beta in vitro. Ki26894 also suppressed transcription of plasminogen activator inhibitor-1 (PAI-1), parathyroid hormone-related protein (PTHrP), and interleukin-11 (IL-11) mRNA of MDA-231-D cells, which were stimulated by TGF-beta. X-ray radiography revealed that systemic Ki26894 treatment initiated 1 day before the inoculation of MDA-231-D cells into the left ventricle of BALB/cnu/nu female mice resulted in decreased bone metastasis of breast cancer cells. Moreover, Ki26894 prolonged the survival of mice inoculated with MDA-231-D cells compared to vehicle-treated mice. These findings suggest that TbetaR-I kinase inhibitors such as Ki26894 may be useful for blocking the progression of advanced cancers.