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Metaplastic epithelial cells of Barrett's esophagus transformed by the combination of p53-knockdown and oncogenic Ras expression are known to activate signal transducer and activator of transcription 3 (STAT3). When phosphorylated at tyrosine 705 (Tyr705), STAT3 functions as a nuclear transcription factor that can contribute to oncogenesis. STAT3 phosphorylated at serine 727 (Ser727) localizes in mitochondria, but little is known about mitochondrial STAT3's contribution to carcinogenesis in Barrett's esophagus, which is the focus of this study. We introduced a constitutively active variant of human STAT3 (STAT3CA) into the following: 1) non-neoplastic Barrett's (BAR-T) cells; 2) BAR-T cells with p53 knockdown; and 3) BAR-T cells that express oncogenic H-Ras(G12V). STAT3CA transformed only the H-Ras(G12V)-expressing BAR-T cells (evidenced by loss of contact inhibition, formation of colonies in soft agar, and generation of tumors in immunodeficient mice), and did so in a p53-independent fashion. The transformed cells had elevated levels of both mitochondrial (Ser727) and nuclear (Tyr705) phospho-STAT3. Introduction of a STAT3CA construct with a mutated tyrosine phosphorylation site into H-Ras(G12V)-expressing Barrett's cells resulted in high levels of mitochondrial phospho-STAT3 (Ser727) with little or no nuclear phospho-STAT3 (Tyr705), and the cells still formed tumors in immunodeficient mice. Thus tyrosine phosphorylation of STAT3 is not required for tumor formation in Ras-expressing Barrett's cells. We conclude that mitochondrial STAT3 (Ser727) can contribute to oncogenesis in Barrett's cells that express oncogenic Ras. These findings suggest that agents targeting STAT3 might be useful for chemoprevention in patients with Barrett's esophagus.
Copyright © 2015 the American Physiological Society.
Little research has addressed the role of membrane trafficking and recycling in the regulation of the transformed phenotype of neoplastic cells. The small GTPase Rab25 is an epithelial-specific modulator of membrane recycling. Recent studies have demonstrated that Rab25 expression is up-regulated in a number of epithelial cancers and overexpression may increase the aggressive phenotype of certain cancers. We have utilized the nontransformed RIE cell line to examine the influence of Rab25 on transformation. Overexpression of Rab25 in RIE cells leads to morphological transformation as well as growth in soft agar, tumor formation in nude mice, disruption of integrin-based focal adhesions, and alteration in modified microtubule subsets. Although the predominance of recent cancer research has focused on the manipulation of the actin-based cytoskeleton, recycling trafficking relies on microtubules. Transformation of RIE cells through overexpression of Rab25, but not with H-Ras(V12) , was reversed by inhibitors of microtubule polymerization. These results suggest that up-regulation of Rab25 in RIE cells leads to microtubule-dependent transformation. Thus, depolymerization of microtubules may be a potent therapeutic target for cancer therapy through the reversal of the invasive phenotype of certain cancer cells.
Copyright © 2010 Wiley-Liss, Inc.
Overexpression of Ras(V12) in MCF10A cells, an immortalized mammary epithelial cell line, leads to transformation of these cells. We demonstrate that this is accompanied by degradation of C/EBPbeta1. C/EBPbeta is a transcription factor in which three protein isoforms exist because of alternative translation at three in-frame methionines. When C/EBPbeta1 is expressed in MCF10A-Ras(V12) cells, immunoblot analysis reveals that C/EBPbeta1 is degraded in these cells. Treatment of MCF10A-Ras(V12)-C/EBPbeta1 cells with the cdk inhibitor roscovitine leads to stabilization of C/EBPbeta1. It has been previously shown that cdk2 phosphorylates C/EBPbeta on Thr235. We demonstrate that mutation of Thr235 to alanine in C/EBPbeta1 is sufficient to restore the stability of C/EBPbeta1 expression in MCF10A-Ras(V12) cells. Overexpression of Ras(V12) in primary cells induces senescence rather than transformation, thus suppressing tumorigenesis. C/EBPbeta is required for Ras(V12)-induced senescence in primary mouse embryonic fibroblasts. Upregulation of interleukin-6 (IL6) by C/EBPbeta has been shown to be necessary for oncogene-induced senescence, but the specific isoform of C/EBPbeta has not been investigated. We show that the C/EBPbeta1 isoform upregulates IL6 when introduced into normal fibroblasts. In addition, we show that C/EBPbeta1 induces senescence. Taken together, degradation of C/EBPbeta1 by Ras activation may represent a mechanism to bypass OIS.
Transforming growth factor-beta (TGF-beta) cooperates with oncogenic members of the Ras superfamily to promote cellular transformation and tumor progression. Apart from the classic (H-, K-, and N-) Ras GTPases, only the R-Ras subfamily (R-Ras, R-Ras2/TC21, and R-Ras3/M-Ras) has significant oncogenic potential. In this study, we show that oncogenic R-Ras transformation of EpH4 cells requires TGF-beta signaling. When murine EpH4 cells were stably transfected with a constitutively active R-Ras(G38V) mutant, they were no longer sensitive to TGF-beta-mediated growth inhibition and showed increased proliferation and transformation in response to exogenous TGF-beta. R-Ras/EpH4 cells require TGF-beta signaling for transformation to occur and they produce significantly elevated levels of endogenous TGF-beta, which signals in an autocrine fashion. The effects of TGF-beta are independent of Smad2/3 activity and require activation of TGF-beta-associated kinase 1 (TAK1) and its downstream effectors c-Jun NH(2)-terminal kinase and p38 mitogen-activated protein kinase as well as the phosphoinositide 3-kinase/Akt and mammalian target of rapamycin pathways. Thus, TAK1 is a novel link between TGF-beta signaling and oncogenic R-Ras in the promotion of tumorigenesis.
Colon cancer progression is characterized by activating mutations in Ras and by the emergence of the tumor-promoting effects of transforming growth factor-beta (TGF-beta) signaling. Ras-inducible rat intestinal epithelial cells (RIE:iRas) undergo a well-described epithelial to mesenchymal transition and invasive phenotype in response to H-RasV12 expression and TGF-beta treatment, modeling tumor progression. We characterized global gene expression profiles accompanying Ras-induced and TGF-beta-induced epithelial to mesenchymal transition in RIE:iRas cells by microarray analysis and found that the regulation of gene expression by the combined activation of Ras and TGF-beta signaling was associated with enrichment of a class of mRNAs containing 3' AU-rich element (ARE) motifs known to regulate mRNA stability. Regulation of ARE-containing mRNA transcripts was validated at the mRNA level, including genes important for tumor progression. Ras and TGF-beta synergistically increased the expression and mRNA stability of vascular endothelial growth factor (VEGF), a key regulator of tumor angiogenesis, in both RIE:iRas cells and an independent cell culture model (young adult mouse colonocyte). Expression profiling of human colorectal cancers (CRC) further revealed that many of these genes, including VEGF and PAI-1, were differentially expressed in stage IV human colon adenocarcinomas compared with adenomas. Furthermore, genes differentially expressed in CRC are also significantly enriched with ARE-containing transcripts. These studies show that oncogenic Ras and TGF-beta synergistically regulate genes containing AREs in cultured rodent intestinal epithelial cells and suggest that posttranscriptional regulation of gene expression is an important mechanism involved in cellular transformation and CRC tumor progression.
Cyclooxygenase-2 (COX-2) expression appears to be important in colorectal carcinogenesis. Elevated COX-2 expression and activity have been observed in several different transformed cell types. Prior studies implicating involvement of the Ras oncogene and growth factors on COX-2 expression were largely derived from rat small intestinal cell lines. We have investigated whether mouse colonocyte COX-2 levels are regulated by oncogenic Ras or transforming growth factor-beta(1) (TGF-beta(1)), and whether these factors also serve to regulate cellular invasiveness. Young adult mouse colonocyte cells are colonocytes derived from the "Immortomouse" and immortalized by the SV40 large T antigen. Young adult mouse colonocyte Ras cells were derived by transfection of young adult mouse colonocyte cells with oncogenic Ha-Ras and are known to be tumorigenic. We found that the induction of COX-2 and eicosanoid release were augmented in the presence of activated Ras and that TGF-beta(1) caused a further increase in COX-2 in the Ras-transformed mouse colonocytes. Increased COX-2 expression was correlated with increased release of prostaglandins E(2) and I(2). Activated Ras and TGF-beta increased the invasiveness of the young adult mouse colonocyte cells, but treatment with a COX-2 inhibitor did not inhibit invasiveness. Thus we found that transforming growth factor-beta collaborates to increase COX-2 expression, protaglandin release, and invasiveness in mouse colonocytes, but the increased COX-2 activity does not appear to contribute to the invasive response.
Invasion is a defining event in carcinoma progression. In general, invasive carcinoma is characterized by an epithelial-fibroblastoid conversion associated with loss of cell-cell adhesion receptors such as E-cadherin and beta-catenin. We report here that TGF-beta1 promotes the invasiveness by modulating the alterations of cellular plasticity including a loss of cell-cell contact in Ras-transformed epithelial cells. In order to examine the role of TGF-beta1 in the Ras-induced responses, intestinal epithelial cells expressing a conditionally activated Ha-Ras(Val12) (RIE-iRas cells) were used in this study. Induced expression of activated Ha-Ras(Val12) caused morphologic transformation of the RIE-iRas cells with an increase in vimentin expression and a decrease of E-cadherin levels. There was also redistribution of beta-catenin from the cytoplasm to the nucleus after the induction of Ras. TGF-beta1 treatment enhanced both the decrease in E-cadherin levels and the redistribution of beta-catenin. Interestingly, the activation of Ras markedly decreased the level of TGF-beta receptor type II (TbetaRII) in RIE-iRas cells. However, the expression of plasminogen activator inhibitor-1, which is known to be transcriptionally induced by TGF-beta1, was strongly induced by TGF-beta1 despite the marked downregulation of TbetaRII. The induction of Ha-Ras(Val12) markedly increased the invasiveness in RIE-iRas cells, as evaluated by a collagen type I-coated Boyden-chamber assay, and the Ras-mediated invasiveness was significantly enhanced by TGF-beta1 treatment. Expression of a dominant-negative form of TbetaRII in the RIE-iRas cells abrogated both growth-inhibitory and invasion responses to TGF-beta1. Collectively, these results suggest that TGF-beta1 and oncogenic Ras collaborate in promoting cellular invasiveness in intestinal epithelial cells. The enhancement of invasiveness was correlated with decreased E-cadherin levels and subcellular distribution of beta-catenin. The enhancement of oncogenic Ras-mediated cell transformation by TGF-beta1 occurs via TbetaRII.
Copyright 2001 Academic Press.
The cellular responses to activated Ras vary depending on cell type. Normal cells are often induced into pathways that lead to cell growth arrest, senescence, and/or apoptosis in response to activated Ras expression. These are important protective anti-tumorigenic responses that restrict the propagation of cells bearing activated oncogenes. Here we show that induction of Ha-Ras(Val-12) in Rat-1 fibroblasts resulted in G(1) growth arrest and apoptosis with loss of viable cells that is accompanied by a marked decrease in cyclin D1 levels via increased ubiquitin-proteasome-dependent cyclin D1 turnover. This is in contrast with a rat intestinal epithelial cell line in which induction of Ha-Ras(Val-12) results in transformation associated with sustained proliferation and increased levels of cyclin D1, that is not accompanied by anoikis or apoptosis. Expression of the cyclin D1 mutant (T286A) that contains an alanine for threonine 286 substitution and is resistant to ubiquitin-proteasome degradation in the Ha-Ras(Val-12) expressing Rat-1 cells resulted in a sustained transformed phenotype with no accumulation of cells in G(1). Inhibition of mitogen-activated protein kinase (MEK1/2) pathway partially reversed the Ras-mediated decrease in cyclin D1. Induction of Ha-Ras(Val-12) resulted in activation of Akt kinase and inactivation of glycogen-synthase-3beta kinase that are associated with reduction of cyclin D1 protein. These results suggest that Ras-mediated cyclin D1 degradation in Rat-1 cells appears to be partially dependent on activation of mitogen-activated protein kinase pathway and independent of glycogen-synthase-3beta kinase pathway.
The trkB gene codes for a receptor tyrosine kinase, which is essential for the development of the peripheral nervous system. This receptor can be activated by three different neurotrophins: BDNF, NT-4/5 and NT-3. The extracellular domain of trkB was found to be encoded in 10 exons corresponding to receptor subdomains previously identified on the basis of protein sequence comparisons. Exon 9 was skipped in a novel tyrosine kinase transcript of the trkB gene, designated ctrkB-Short (ctrkB-S). While the previously described trkB receptor ctrkB-Long (ctrkB-L) and trkB-S receptors were activated similarly by BDNF, trkB-S interacted poorly with NT-4/5 and NT-3 as measured by ligand binding, ligand-induced autophosphorylation and ligand-dependent activation of p21ras. Efficient activation of ctrkB-S by NT-3 was restored by a single amino acid replacement in NT-3 (D15A). Both trkB-L and trkB-S transcripts were detected in embryonic neurons.
H-Ras, the protein product of the cellular homologue of the Harvey ras oncogene, undergoes a complex series of post-translational modifications that include C-terminal isoprenylation, proteolysis, methylation, and palmitoylation. Palmitoylation has been shown to enhance the transformation efficiency of H-Ras about 10-fold in vivo. A recent study (Magee, A. I., Gutierrez, L., McKay, I. A., Marshall, C. J., and Hall, A. (1987) EMBO J. 6, 3353-3357) has provided strong evidence that the palmitate undergoes a dynamic acylation-deacylation cycle, but details concerning the enzymology of this process and its regulation are lacking. To begin to dissect this event, we have developed an assay for the enzymatic removal of palmitate from [3H]palmitate-labeled H-Ras. This substrate was produced in a baculovirus expression system and was used to purify to homogeneity a novel 37-kDa enzyme from bovine brain cytosol that removes the radiolabeled palmitate. The purified enzyme is sensitive to diethyl pyrocarbonate and insensitive to phenylmethylsulfonyl fluoride and N-ethylmaleimide. Interestingly, the thioesterase recognizes H-Ras as a substrate only when H-Ras is in its native conformation (bound to Mg2+ and guanine nucleotide). The palmitoylated alpha subunits of the heterotrimeric G proteins are also substrates for the enzyme.