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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.
The 5'-untranslated region of the ornithine decarboxylase (ODC) mRNA contains an internal ribosomal entry site (IRES). Mutational analysis of the ODC IRES has led to the identification of sequences necessary for cap-independent translation of the ODC mRNA. To discover novel IRES trans-acting factors (ITAFs), we performed a proteomics screen for proteins that regulate ODC translation using the wild-type ODC mRNA and a mutant version with an inactive IRES. We identified two RNA-binding proteins that associate with the wild-type ODC IRES but not the mutant IRES. One of these RNA-binding proteins, PCBP2, is an established activator of viral and cellular IRESs. The second protein, ZNF9 (myotonic dystrophy type 2 protein), has not been shown previously to bind IRES-like elements. Using a series of biochemical assays, we validated the interaction of these proteins with ODC mRNA. Interestingly ZNF9 and PCBP2 biochemically associated with each other and appeared to function as part of a larger holo-ITAF ribonucleoprotein complex. Our functional studies showed that PCBP2 and ZNF9 stimulate translation of the ODC IRES. Importantly these results may provide insight into the normal role of ZNF9 and why ZNF9 mutations cause myotonic dystrophy.