Abstract
Interactions between the Wnt/beta-catenin and the extracellular signal-regulated kinase (ERK) pathways have been posited, but the molecular mechanisms and cooperative roles of such interaction in carcinogenesis are poorly understood. In the present study, the Raf-1, MEK, and ERK activities were concomitantly decreased in fibroblasts, which inhibit morphological transformation and proliferation by Axin induction. The inhibition of the components of the ERK pathway by Axin occurred in cells retaining wild-type beta-catenin, including primary hepatocytes, but not in cells retaining non-degradable mutant beta-catenin. Axin inhibits cellular proliferation and ERK pathway activation induced by either epidermal growth factor or Ras, indicating a role of Axin in the regulation of growth induced by ERK pathway activation. ERK pathway regulation by Axin occurs at least partly via reduction of the protein level of Ras. Both wild-type and mutant Ras proteins are subjected to regulation by Axin, which occurs in cells retaining wild-type but not mutant beta-catenin gene. The role of beta-catenin in the regulation of the Ras-ERK pathway was further confirmed by Ras reduction and subsequent inhibitions of the ERK pathway components by knock down of mutated form of beta-catenin. The Ras regulation by Axin was blocked by treatment of leupeptin, an inhibitor of the lysosomal protein degradation machinery. Overall, Axin inhibits proliferation of cells at least partly by reduction of Ras protein level via beta-catenin. This study provides evidences for the role of the Ras-ERK pathway in carcinogenesis caused by mutations of the Wnt/beta-catenin pathway components.
Highlights
The Wnt/-catenin signaling pathway plays a crucial role in carcinogenesis as well as development [1,2,3,4]
Ras Regulation by Axin via -Catenin finding of which was revealed in primary hepatocytes and several cancer cells. -Catenin was identifiable as a mediator in ERK pathway regulation by Axin, for which regulation was investigated by measurements of Axin overexpression on activation of the ERK pathway components in different types of cells that retained either a mutated or functionally active wildtype -catenin gene
Simultaneous introduction of -catenin gene and ras mutations into mice resulted in the development of hepatocellular carcinoma in 100% of tested animals, neither ras nor -catenin gene alone was sufficient for hepatocarcinogenesis [23]
Summary
Plasmids—The expression vector for Axin, pCS2-MT-Axin, and inducible expression vectors for GFP (pBI-EGFP) and GFPAxin (pBI-EGFP-Axin) were described in previous studies [14, 25]. For quantitative analyses of proliferation, L929-GFP-Axin cells were transfected with pZIP-Raf-CCAX [26] for 24 h followed by subsequent induction of Axin with 0.5 g/ml Dox for 24 h at 37 °C in 5% CO2. The cells were seeded into 6-well plates at 2 ϫ 104 cells/well, and the medium was replaced every day with fresh DMEM containing 10% FBS with or without 0.5 g/ml Dox. Where required, 20 ng/ml EGF was added. The cells were treated with 0.5 g/ml Dox. Induced L929-Axin-GFP cells were fixed for 15 min at room temperature with 3.7% paraformaldehyde in PBS at pH 7.4, per-. L929-GFPAxin cells that failed to express GFP-Axin upon Dox treatment (marked by arrows) retained the typical polygonal morphology of L929 cells (Fig. 2). A minimum of The increase was abolished after introduction of GFP-Axin three 0.5-m Z slices were acquired for each cell
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