Transforming growth factor beta-regulated gene expression in a mouse mammary gland epithelial cell line

BACKGROUND: Transforming growth factor beta (TGF-β) plays an essential role in a wide array of cellular processes. The most well studied TGF-β response in normal epithelial cells is growth inhibition. In some cell types, TGF-β induces an epithelial to mesenchymal transition (EMT). NMuMG is a nontransformed mouse mammary gland epithelial cell line that exhibits both a growth inhibitory response and an EMT response to TGF-β, rendering NMuMG cells a good model system for studying these TGF-β effects. METHOD: A National Institutes of Aging mouse 15,000 cDNA microarray was used to profile the gene expression of NMuMG cells treated with TGF-β1 for 1, 6, or 24 hours. Data analyses were performed using GenePixPro and GeneSpring software. Selected microarray results were verified by northern analyses. RESULTS: Of the 15,000 genes examined by microarray, 939 were upregulated or downregulated by TGF-β. This represents approximately 10% of the genes examined, minus redundancy. Seven genes previously not known to be regulated by TGF-β at the transcriptional level (Akt and RhoB) or not at all (IQGAP1, mCalpain, actinin α3, Ikki, PP2A-PR53), were identified and their regulation by TGF-β verified by northern blotting. Cell cycle pathway examination demonstrated downregulation of cyclin D(2), c-myc, Id2, p107, E2F5, cyclin A, cyclin B, and cyclin H. Examination of cell adhesion-related genes revealed upregulation of c-Jun, α-actinin, actin, myosin light chain, p120cas catenin (Catns), α-integrin, integrin β5, fibronectin, IQGAP1, and mCalpain. CONCLUSION: Using a cDNA microarray to examine TGF-β-regulated gene expression in NMuMG cells, we have shown regulation of multiple genes that play important roles in cell cycle control and EMT. In addition, we have identified several novel TGF-β-regulated genes that may mediate previously unknown TGF-β functions

Rapamycin Potentiates Transforming Growth Factor β-Induced Growth Arrest in Nontransformed, Oncogene-Transformed, and Human Cancer Cells

Transforming growth factor β (TGF-β) induces cell cycle arrest of most nontransformed epithelial cell lines. In contrast, many human carcinomas are refractory to the growth-inhibitory effect of TGF-β. TGF-β overexpression inhibits tumorigenesis, and abolition of TGF-β signaling accelerates tumorigenesis, suggesting that TGF-β acts as a tumor suppressor in mouse models of cancer. A screen to identify agents that potentiate TGF-β-induced growth arrest demonstrated that the potential anticancer agent rapamycin cooperated with TGF-β to induce growth arrest in multiple cell lines. Rapamycin also augmented the ability of TGF-β to inhibit the proliferation of E2F1-, c-Myc-, and (V12)H-Ras-transformed cells, even though these cells were insensitive to TGF-β-mediated growth arrest in the absence of rapamycin. Rapamycin potentiation of TGF-β-induced growth arrest could not be explained by increases in TGF-β receptor levels or rapamycin-induced dissociation of FKBP12 from the TGF-β type I receptor. Significantly, TGF-β and rapamycin cooperated to induce growth inhibition of human carcinoma cells that are resistant to TGF-β-induced growth arrest, and arrest correlated with a suppression of Cdk2 kinase activity. Inhibition of Cdk2 activity was associated with increased binding of p21 and p27 to Cdk2 and decreased phosphorylation of Cdk2 on Thr(160). Increased p21 and p27 binding to Cdk2 was accompanied by decreased p130, p107, and E2F4 binding to Cdk2. Together, these results indicate that rapamycin and TGF-β cooperate to inhibit the proliferation of nontransformed cells and cancer cells by acting in concert to inhibit Cdk2 activity

Activation of the Erk Pathway Is Required for TGF-β1-Induced EMT In Vitro

Transforming growth factor-β1 (TGF-β1) can be tumorsuppressive through the activation of the Smadmediated signaling pathway. TGF-β1 can also enhance tumor progression by stimulating epithelial-tomesenchymal transition (EMT) through additional pathways. EMT is characterized by the acquisition of a fibroblast-like cell morphology, dissolution of tight junctions, disruption of adherence junctions, and formation of actin stress fibers. There is evidence linking the activation of mitogen-activated protein kinase pathways to the induction of TGF-β1-mediated EMT. However, the role of Erk in the induction of TGF-β1-mediated EMT remains unclear. TGF-β1 treatment of normal murine mammary gland (NMuMG) epithelial cells resulted in increased gene expression of Ras, Raf, MEK1/2, and Erk1/2, as shown by microarray analysis and real-time polymerase chain reaction. Upon 24 and 48 hours of treatment with TGF-β1, NMuMG and mouse cortical tubule (MCT) epithelial cells underwent EMT as shown by changes in cell morphology, delocalization of zonula occludens-1 and E-cadherin from cell-cell junctions, and formation of actin stress fibers. TGF-β1 treatment also resulted in increased levels of phosphorylated Erk and Erk kinase activity. Treatment with an MEK inhibitor, U0126, inhibited increased Erk phosphorylation and kinase activity, and blocked TGF-β1-induced EMT in both cell lines. These data show that TGF-β1 induces the activation of the Erk signaling pathway, which is required for TGF-β1-mediated EMT in vitro

Aggressive pancreatic ductal adenocarcinoma in mice caused by pancreas-specific blockade of transforming growth factor-β signaling in cooperation with active Kras expression

Pancreatic ductal adenocarcinoma (PDAC) is an almost uniformly lethal disease in humans. Transforming growth factor-β (TGF-β) signaling plays an important role in PDAC progression, as indicated by the fact that Smad4, which encodes a central signal mediator downstream from TGF-β, is deleted or mutated in 55% and the type II TGF-β receptor (Tgfbr2) gene is altered in a smaller subset of human PDAC. Pancreas-specific Tgfbr2 knockout mice have been generated, alone or in the context of active Kras (Kras(G12D)) expression, using the Cre-loxP system driven by the endogenous Ptf1a (pancreatic transcription factor-1a) locus. Pancreas-selective Tgfbr2 knockout alone gave no discernable phenotype in 1.5 yr. Pancreas-specific Kras(G12D) activation alone essentially generated only intraepithelial neoplasia within 1 yr. In contrast, the Tgfbr2 knockout combined with Kras(G12D) expression developed well-differentiated PDAC with 100% penetrance and a median survival of 59 d. Heterozygous deletion of Tgfbr2 with Kras(G12D) expression also developed PDAC, which indicated a haploinsufficiency of TGF-β signaling in this genetic context. The clinical and histopathological manifestations of the combined Kras(G12D) expression and Tgfbr2 knockout mice recapitulated human PDAC. The data show that blockade of TGF-β signaling and activated Ras signaling cooperate to promote PDAC progression

Stromally Derived Lysyl Oxidase Promotes Metastasis of Transforming Growth Factor-β–Deficient Mouse Mammary Carcinomas

The tumor stromal environment can dictate many aspects of tumor progression. A complete understanding of factors driving stromal activation and their role in tumor metastasis is critical to furthering research with the goal of therapeutic intervention. Polyoma middle T-induced mammary carcinomas lacking the type II TGF-β receptor (PyMT(mgko)) are highly metastatic compared with control PyMT-induced carcinomas (PyMT(fl/fl)). We hypothesized that the PyMT(mgko)-activated stroma interacts with carcinoma cells to promote invasion and metastasis. We show that the extracellular matrix associated with PyMT(mgko) tumors is stiffer and has more fibrillar collagen and increased expression of the collagen crosslinking enzyme lysyl oxidase (LOX) compared with PyMT(fl/fl) mammary carcinomas. Inhibition of LOX activity in PyMT(mgko) mice had no effect on tumor latency and size, but significantly decreased tumor metastasis through inhibition of tumor cell intravasation. This phenotype was associated with a decrease in keratin 14-positive myoepithelial cells in PyMT(mgko) tumors following LOX inhibition as well as a decrease in focal adhesion formation. Interestingly, the primary source of LOX was found to be activated fibroblasts. LOX expression in these fibroblasts can be driven by myeloid cell-derived TGF-β, which is significantly linked to human breast cancer. Overall, stromal expansion in PyMT(mgko) tumors is likely caused through the modulation of immune cell infiltrates to promote fibroblast activation. This feeds back to the epithelium to promote metastasis by modulating phenotypic characteristics of basal cells. Our data indicate that epithelial induction of microenvironmental changes can play a significant role in tumorigenesis and attenuating these changes can inhibit metastasis. Cancer Res; 73(17); 5336-46. ©2013 AACR