Oncogenic Ras and transforming growth factor-beta synergistically regulate AU-rich element-containing mRNAs during epithelial to mesenchymal transition

Abstract

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.

FIGURE 1

Ras and TGF-β activate their downstream effectors in RIE:iRas cells. RIE:iRas cells were pretreated for 15 min with (A) a TGF-β receptor kinase inhibitor (2 μmol/L LY364947/TRKi) or (B) a MEK inhibitor (10 μmol/L U0126) and then treated for 24 h with 5 mmol/L IPTG and/or 3 ng/mL TGF-β. Western blotting was done for phosphorylated Smad2 (P-Smad2), total Smad2, phosphorylated ERK (P-ERK), total ERK, COX-2, and β-actin. Blots shown are representative of three independent experiments.

FIGURE 2

Summary of microarray results and validation. A. Venn diagrams showing the distribution of genes up-regulated or down-regulated in RIE:iRas cells after treatment with 5 mmol/L IPTG to induce RasV12 expression, 3 ng/mL TGF-β treatment, or combined IPTG and TGF-β treatment for 72 h. The total number of genes with altered expression above 2-fold (P < 0.05) for each treatment is given in parenthesis. B. Quantitative real-time reverse transcription-PCR of Cox-2, Wnt5a, Spp1, and Cugbp2 in RIE:iRas cells treated for 72 h with 5 mmol/L IPTG, 3 ng/mL TGF-β, or IPTG and TGF-β together. Changes in gene expression under treated conditions were calculated relative to untreated samples and all values were normalized to the housekeeping gene Pmm1. Dotted lines show mean expression for each treatment. Significance was determined by ANOVA with Bonferroni correction. *, P < 0.02.

FIGURE 3

Oncogenic Ras and TGF-β synergistically increase VEGF expression in RIE:iRas and YAMC cells. A. RIE:iRas cells were treated with either vehicle, 5 mmol/L IPTG, 3 ng/mL TGF-β, or both IPTG and TGF-β for 24 h. VEGF protein levels were measured in conditioned medium by ELISA and normalized to total protein concentrations. Box plot shows data summarized from seven independently replicated experiments. *, P < 0.004, compared with untreated; **, P < 0.03, compared with untreated; #, P < 0.002, compared with all treatments. VEGF mRNA levels in RIE:iRas cells were visualized by Northern blot using a mouse VEGF₁₆₅ cDNA probe and 18S rRNA visualized with ethidium bromide. Northern blot is representative of six separate experiments. B. YAMC and YAMC-Ras cells were treated with or without 5 ng/mL TGF-β for 24 h and then VEGF levels were measured in conditioned medium by ELISA and normalized to total protein concentrations. Box plot shows data from eight independently replicated experiments. #, P < 0.0001, compared with all samples. C and D. RIE:iRas cells were treated with IPTG and/or TGF-β at varying doses for 24 h and VEGF levels were measured in conditioned medium by ELISA and normalized to total protein concentrations. Cells were treated with or without 5 mmol/L IPTG and 0, 0.04, 0.2, 1, 5, or 10 ng/mL of TGF-β or cells were treated with or without 3 ng/mL TGF-β and 0, 0.001, 0.01, 0.1, 1, or 5 mmol/L of IPTG. Points, average of three independent experiments; bars, SE. Dose dependence and synergistic interaction were confirmed via multiple linear regression (all P < 0.0001).

FIGURE 4

Ras-mediated and TGF-β–mediated increase in VEGF expression is COX-2 independent. RIE:iRas cells were pretreated with DMSO or 10 μmol/L NS398 for 15 min and then treated with vehicle, 5 mmol/L IPTG, 3 ng/mL TGF-β, or both IPTG and TGF-β for 24 h. A. VEGF protein in conditioned medium was measured by ELISA and normalized to total protein concentration. Box plots show data from four independently replicated experiments. B. Prostacyclin (6-keto-PGF_1α) levels in conditioned medium were measured by gas chromatography-mass spectrometry. Box plots show data from four independently replicated experiments. Significance between no inhibitor and NS398 treatment was determined by ANOVA with Bonferroni correction. *, P < 0.0001, compared with TGF-β; **, P = 0.09, compared with Ras; #, P < 0.0001, compared with Ras + TGF-β.

FIGURE 5

Oncogenic Ras and TGF-β cooperate to synergistically stabilize VEGF mRNA. Northern blot analysis of RIE:iRas cells after treatment for 24 h with vehicle, 5 mmol/L IPTG, 3 ng/mL TGF-β, or both IPTG and TGF-β and then treatment with 10 Ag/mL actinomycin D for up to 4 h. A. VEGF Northern blot and 18S rRNA, as detected by ethidium bromide staining. B. Percentage of VEGF mRNA, normalized to 18S rRNA levels, remaining after actinomycin D treatment. Data are representative of three separate experiments. □, untreated; ○, TGF-β; ■, RasV12; ●, RasV12 + TGF-β.