Ras-MAPK inhibition induces AXIN1 loss in colorectal cancer by mTOR associated suppression of protein synthesis

BACKGROUND: AXIN1 is a central regulatory hub of many oncogenic pathways in colorectal cancer (CRC). As the main scaffold protein and least abundant component of the beta-catenin destruction complex, changes in AXIN1 levels tightly control Wnt signaling activity. How other cancer pathways beyond Wnt signaling regulate cellular AXIN1 levels is incompletely understood. METHODS: Colorectal cancer cell lines, murine and patient-derived intestinal and cancer organoids were used as model systems. Changes in AXIN1 levels upon drug perturbation were profiled by immunoblot, qPCR and RNA-seq. Ubiquitin-affinity immunoprecipitation assays and mass spectrometry were used to determine mechanisms of AXIN1 loss. To characterize effects on protein synthesis, we performed polysome and ribosome profiling (Ribo-seq). RESULTS: We show that targeting the Ras-MAPK pathway using clinically approved MEK1/2 inhibitors induces AXIN1 loss across a panel of CRC cell lines and patient-derived organoids. In contrast to GSK3 inhibitors, MEK1/2 inhibition neither affects protein stability nor post-translational modifications of AXIN1 and only caused a minor reduction of AXIN1 transcript levels. Co-treatment with tankyrase inhibitors could partially prevent loss of AXIN1 upon MEK1/2 inhibition. Using isogenic CRC cell lines and murine intestinal organoids, we show that APC truncations strongly reduce basal cellular AXIN1 levels, but do not alter dynamics of AXIN1 loss after MEK1/2 inhibition. Polysome profiling and Ribo-seq revealed that MEK1/2 inhibitors reduce global protein synthesis via an mTOR associated pathway. This translational repression is sufficient to cause significant AXIN1 loss, as treatment with mTOR or S6K inhibitors phenocopies the effect of MEK1/2 inhibitors. CONCLUSION: Our study demonstrates that AXIN1 protein homeostasis is critically controlled by Ras-MAPK signaling at the level of protein synthesis, and that MEK1/2 inhibitors cause AXIN1 loss by global translational repression.