Transforming growth factor-ß-induced lncRNA-Smad7 inhibits apoptosis of mouse breast cancer JygMC(A) cells.

Transforming growth factor (TGF)-ß exhibits both pro-apoptotic and anti-apoptotic effects on epithelial cells in a context-dependent manner. The anti-apoptotic function of TGF-ß is mediated by several downstream regulatory mechanisms, and has been implicated in the tumor-progressive phenotype of breast cancer cells. We conducted RNA sequencing of mouse mammary gland epithelial (NMuMG) cells and identified a long non-coding RNA, termed lncRNA-Smad7, which has anti-apoptotic functions, as a target of TGF-ß. lncRNA-Smad7 was located adjacent to the mouse Smad7 gene, and its expression was induced by TGF-ß in all of the mouse mammary gland epithelial cell lines and breast cancer cell lines that we evaluated. Suppression of lncRNA-Smad7 expression cancelled the anti-apoptotic function of TGF-ß. In contrast, forced expression of lncRNA-Smad7 rescued apoptosis induced by a TGF-ß type I receptor kinase inhibitor in the mouse breast cancer cell line JygMC(A). The anti-apoptotic effect of lncRNA-Smad7 appeared to occur independently of the transcriptional regulation by TGF-ß of anti-apoptotic DEC1 and pro-apoptotic Bim proteins. Small interfering RNA for lncRNA-Smad7 did not alter the process of TGF-ß-induced epithelial-mesenchymal transition, phosphorylation of Smad2 or expression of the Smad7 gene, suggesting that the contribution of this lncRNA to TGF-ß functions may be restricted to apoptosis. Our findings suggest a complex mechanism for regulating the anti-apoptotic and tumor-progressive aspects of TGF-ß signaling.

Basolateral BMP signaling in polarized epithelial cells.

Bone morphogenetic proteins (BMPs) regulate various biological processes, mostly mediated by cells of mesenchymal origin. However, the roles of BMPs in epithelial cells are poorly understood. Here, we demonstrate that, in polarized epithelial cells, BMP signals are transmitted from BMP receptor complexes exclusively localized at the basolateral surface of the cell membrane. In addition, basolateral stimulation with BMP increased expression of components of tight junctions and enhanced the transepithelial resistance (TER), counteracting reduction of TER by treatment with TGF-ß or an anti-tumor drug. We conclude that BMPs maintain epithelial polarity via intracellular signaling from basolaterally localized BMP receptors.

RB1CC1 protein positively regulates transforming growth factor-beta signaling through the modulation of Arkadia E3 ubiquitin ligase activity.

Transforming growth factor-ß (TGF-ß) signaling is controlled by a variety of regulators, of which Smad7, c-Ski, and SnoN play a pivotal role in its negative regulation. Arkadia is a RING-type E3 ubiquitin ligase that targets these negative regulators for degradation to enhance TGF-ß signaling. In the present study we identified a candidate human tumor suppressor gene product RB1CC1/FIP200 as a novel positive regulator of TGF-ß signaling that functions as a substrate-selective cofactor of Arkadia. Overexpression of RB1CC1 enhanced TGF-ß signaling, and knockdown of endogenous RB1CC1 attenuated TGF-ß-induced expression of target genes as well as TGF-ß-induced cytostasis. RB1CC1 down-regulated the protein levels of c-Ski but not SnoN by enhancing the activity of Arkadia E3 ligase toward c-Ski. Substrate selectivity is primarily attributable to the physical interaction of RB1CC1 with substrates, suggesting its role as a scaffold protein. RB1CC1 thus appears to play a unique role as a modulator of TGF-ß signaling by restricting substrate specificity of Arkadia.

[Encounter of cancer cells with bone. Epithelial-mesenchymal transition in cancer].

Acquisition of invasive phenotypes of cancer cells is one of the key steps to promote metastasis. It has been reported that fibroblastic cells are involved in enhancement of proliferation and invasion of cancer cells. Thus, it is important to know the molecular mechanisms of EMT (epithelial-mesenchymal transition) for developing diagnosis and cancer therapy. Since TGF-ßis a key mediator of EMT and frequently expressed in various tumors, it may regulate not only the EMT of cancer cells as they acquire metastatic properties, but also the EMT of normal epithelial cells that are adjacent to tumors. In this review, we will discuss the EMT induced by TGF-ß.

TGF-ß regulates isoform switching of FGF receptors and epithelial-mesenchymal transition.

The epithelial-mesenchymal transition (EMT) is a crucial event in wound healing, tissue repair, and cancer progression in adult tissues. Here, we demonstrate that transforming growth factor (TGF)-ß induced EMT and that long-term exposure to TGF-ß elicited the epithelial-myofibroblastic transition (EMyoT) by inactivating the MEK-Erk pathway. During the EMT process, TGF-ß induced isoform switching of fibroblast growth factor (FGF) receptors, causing the cells to become sensitive to FGF-2. Addition of FGF-2 to TGF-ß-treated cells perturbed EMyoT by reactivating the MEK-Erk pathway and subsequently enhanced EMT through the formation of MEK-Erk-dependent complexes of the transcription factor δEF1/ZEB1 with the transcriptional corepressor CtBP1. Consequently, normal epithelial cells that have undergone EMT as a result of combined TGF-ß and FGF-2 stimulation promoted the invasion of cancer cells. Thus, TGF-ß and FGF-2 may cooperate with each other and may regulate EMT of various kinds of cells in cancer microenvironment during cancer progression.

Thyroid transcription factor-1 inhibits transforming growth factor-beta-mediated epithelial-to-mesenchymal transition in lung adenocarcinoma cells.

Thyroid transcription factor-1 (TTF-1) is expressed in lung cancer, but its functional roles remain unexplored. TTF-1 gene amplification has been discovered in a part of lung adenocarcinomas, and its action as a lineage-specific oncogene is highlighted. Epithelial-to-mesenchymal transition (EMT) is a crucial event for cancer cells to acquire invasive and metastatic phenotypes and can be elicited by transforming growth factor-beta (TGF-beta). Mesenchymal-to-epithelial transition (MET) is the inverse process of EMT; however, signals that induce MET are largely unknown. Here, we report a novel functional aspect of TTF-1 that inhibits TGF-beta-mediated EMT and restores epithelial phenotype in lung adenocarcinoma cells. This effect was accompanied by down-regulation of TGF-beta target genes, including presumed regulators of EMT, such as Snail and Slug. Moreover, silencing of TTF-1 enhanced TGF-beta-mediated EMT. Thus, TTF-1 can exert a tumor-suppressive effect with abrogation of cellular response to TGF-beta and attenuated invasive capacity. We further revealed that TTF-1 down-regulates TGF-beta2 production in A549 cells and that TGF-beta conversely decreases endogenous TTF-1 expression, suggesting that enhancement of autocrine TGF-beta signaling accelerates the decrease of TTF-1 expression and vice versa. These findings delineate potential links between TTF-1 and TGF-beta signaling in lung cancer progression through regulation of EMT and MET and suggest that modulation of TTF-1 expression can be a novel therapeutic strategy for treatment of lung adenocarcinoma.

Role of Ras signaling in the induction of snail by transforming growth factor-beta.

The epithelial-mesenchymal transition (EMT) is a crucial morphological event that occurs during the progression of epithelial tumors. EMT can be induced by transforming growth factor (TGF)-beta in some tumor cells. Here, we demonstrate the molecular mechanism whereby Snail, a key regulator of EMT, is induced by TGF-beta in tumor cells. Snail induction by TGF-beta was highly dependent on cooperation with active Ras signals, and silencing of Ras abolished Snail induction by TGF-beta in pancreatic cancer Panc-1 cells. Transfection of constitutively active Ras into HeLa cells led to induction of Snail by TGF-beta, while representative direct targets of TGF-beta, including Smad7 and PAI-1, were not affected by Ras signaling. Using mitogen-activated protein kinase inhibitors or Smad3 or Smad2 mutants, we found that phosphorylation at the linker region of Smad2/3 was not required for the induction of Snail by TGF-beta. Taken together, these findings indicate that Ras and TGF-beta-Smad signaling selectively cooperate in the induction of Snail, which occurs in a Smad-dependent manner, but independently of phosphorylation at the linker region of R-Smads by Ras signaling.

Smurf2 induces ubiquitin-dependent degradation of Smurf1 to prevent migration of breast cancer cells.

Ubiquitin-dependent protein degradation is involved in various biological processes, and accumulating evidence suggests that E3 ubiquitin ligases play important roles in cancer development. Smad ubiquitin regulatory factor 1 (Smurf1) and Smurf2 are E3 ubiquitin ligases, which suppress transforming growth factor-beta (TGF-beta) family signaling through degradation of Smads and receptors for TGF-beta and bone morphogenetic proteins. In addition, Smurf1 has been reported to promote RhoA ubiquitination and degradation and regulate cell motility, suggesting the involvement of Smurf1 in cancer progression. However, the regulation and biological function of Smurf1 and Smurf2 in cancer development remain to be elucidated. In the present study, we show the post-translational regulation of Smurf1 by Smurf2 and the functional differences between Smurf1 and Smurf2 in the progression of breast cancer cells. Smurf2 interacted with Smurf1 and induced its ubiquitination and degradation, whereas Smurf1 failed to induce degradation of Smurf2. Knockdown of Smurf2 in human breast cancer MDA-MB-231 cells resulted in increases in the levels of Smurf1 protein, and enhancement of cell migration in vitro and bone metastasis in vivo. Of note, knockdown of Smurf1, but not of Smurf2, enhanced TGF-beta signaling in MDA-MB-231 cells, suggesting that increased an protein level of Smurf1 offsets the effect of Smurf2 knockdown on TGF-beta signaling. These results indicate that two related E3 ubiquitin ligases, Smurf1 and Smurf2, act in the same direction in TGF-beta family signaling but play opposite roles in cell migration.

Repopulating activity of ex vivo-expanded murine hematopoietic stem cells resides in the CD48-c-Kit+Sca-1+lineage marker- cell population.

A better understanding of the biology of cultured hematopoietic stem cells (HSCs) is required to achieve ex vivo expansion of HSCs. In this study, clonal analysis of the surface phenotype and repopulating activity of ex vivo-expanded murine HSCs was performed. After 7 days of culture with stem cell factor, thrombopoietin, fibroblast growth factor-1, and insulin-like growth factor-2, single CD34-/lowc-Kit+Sca-1+lineage marker- (CD34-KSL) cells gave rise to various numbers of cells. The proportion of KSL cells decreased with increasing number of expanded cells. Transplantation studies revealed that the progeny containing a higher percentage of KSL cells tended to have enhanced repopulating potential. We also found that CD48 was heterogeneously expressed in the KSL cell population after culture. Repopulating activity resided only in the CD48-KSL cell population, which had a relatively long intermitotic interval. Microarray analysis showed surprisingly few differences in gene expression between cultured CD48-KSL cells (cycling HSCs) and CD48+KSL cells (cycling non-HSCs) compared with freshly isolated CD34-KSL cells (quiescent HSCs), suggesting that the maintenance of stem cell activity is controlled by a relatively small number of genes. These findings should lead to a better understanding of ex vivo-expanded HSCs.

c-Ski activates MyoD in the nucleus of myoblastic cells through suppression of histone deacetylases.

c-Ski, originally identified as an oncogene product, induces myogenic differentiation in nonmyogenic fibroblasts through transcriptional activation of muscle regulatory factors. Although c-Ski does not bind to DNA directly, it binds to DNA through interaction with Smad proteins and regulates signaling activities of transforming growth factor-beta (TGF-beta). In the present study, we show that c-Ski activates the myogenin promoter independently of regulation of endogenous TGF-beta signaling. Expression of myogenin is regulated by a transcription factor complex containing proteins of the MyoD family and the myocyte enhancer factor 2 (MEF2) family. c-Ski acts on the MyoD-MEF2 complex and modulates the activity of MyoD in myogenin promoter regulation. Interestingly, histone deacetylase (HDAC) inhibitors up-regulated basal activity of transcription from a MyoD-responsive reporter, although c-Ski failed to further augment this transcription in the presence of HDAC inhibitors. c-Ski is observed both in the cytoplasm and in the nucleus, but its nuclear localization is required for myogenic differentiation. We conclude that c-Ski induces myogenic differentiation through acting on MyoD and inhibiting HDAC activity in the nucleus of myogenic cells.