Project B08

Mechanism of AXIN1 degradation after MEK inhibition

Project leader: Tianzuo Zhan

Abstract

The β-catenin destruction complex (DC) is a central regulatory nexus of the canonical Wnt pathway. AXIN1 is the main scaffold protein of the complex and its least abundant component. Protein levels of AXIN1 critically determine the stability of the DC, and are post-translationally regulated by many interaction partners. Recently, we discovered that MEK inhibitors induce canonical Wnt signaling in colorectal cancer (CRC) by downregulation of AXIN1. MEK inhibitors are highly specific inhibitors of Ras signaling and clinically used to treat different cancer types that harbor activating mutations of the Ras pathway. In contrast to melanoma or non-small cellular lung cancer, CRC is intrinsically resistant to MEK inhibitors. Our previous work suggests that activation of Wnt signaling is a potential feedback mechanism for CRC to overcome Ras pathway inhibition. Mechanistically, we observed that AXIN1 protein levels are rapidly reduced upon MEK inhibition, and that blocking de novo translation by cycloheximide enhances this loss, suggesting a process of active protein degradation. Understanding how protein stability of AXIN1 is controlled by MEK inhibition will provide insights into a novel intersection point of two key oncogenic pathways. In this project, we will investigate mechanisms that underlie the rapid degradation of AXIN1, using CRC cell lines as model systems. Specifically, we will address three topics: (1) Changes in post-translational modifications of AXIN1 upon MEK inhibition. Since protein stability of AXIN1 is critically determined by post-translational modifications, we will perform affinity purification and advanced proteomics analysis to identify specific modifications that are induced by MEK inhibition, with a focus on phosphorylation and ubiquitination. Furthermore, we will investigate the role of genes that are known to post-translationally modify AXIN1 in this process. (2) Identification of interaction partners involved in degradation of AXIN1 by MEK inhibition. We will determine how interactions of AXIN1 with core components of the DC (GSK3β, CKIα, β-catenin) and known binding partners (PP1, PP2A) rewire during treatment with MEK inhibitors, by co-immunoprecipitation and Western Blot. We will combine affinity purification of AXIN1 and mass spectrometry with focused RNAi screens to identify and functionally confirm novel interaction partners in AXIN1 degradation. (3) Effect of APC mutations on AXIN1 degradation by MEK inhibitors. The integrity of the DC can be modified by mutations of its key components and we previously showed that APC mutations enhance Wnt induction by MEK inhibitors. Hence, we will use CRISPR/Cas9 to generate isogenic cell lines harboring mutant APC that lack specific interaction domains. We will determine how these truncating mutations differentially affect Wnt induction and the kinetics of AXIN1 degradation by MEK inhibitors. In summary, these experiments will comprehensively characterize a novel mechanism of rapid AXIN1 degradation caused by Ras pathway inhibition.

Project-related publications

  • Zhan, T.*, V. Faehling*, B. Rauscher*, J. Betge, M.P. Ebert, and M. Boutros. 2020. Multi-omics integration identifies a selective vulnerability of colorectal cancer subtypes to YM155. Int. J. Cancer. ijc.33393. PMID: 33186476
  • Zhan, T.*, G. Ambrosi*, A.M. Wandmacher*, B. Rauscher, J. Betge, N. Rindtorff, R.S. Häussler, I. Hinsenkamp, L. Bamberg, B. Hessling, K. Müller-Decker, G. Erdmann, E. Burgermeister, M.P. Ebert, and M. Boutros. 2019. MEK inhibitors activate Wnt signalling and induce stem cell plasticity in colorectal cancer. Nat. Commun. 10:2197. PMID: 31097693
  • Zhan, T.*, N. Rindtorff*, J. Betge, M.P. Ebert, and M. Boutros. 2019. CRISPR/Cas9 for cancer research and therapy. Semin. Cancer Biol. 55:106–119. PMID: 29673923
  • Zhan, T.*, N. Rindtorff*, and M. Boutros. 2017. Wnt signaling in cancer. Oncogene. 36:1461–1473. PMID: 27617575
  • Heigwer, F.*, T. Zhan*, M. Breinig, J. Winter, D. Brügemann, S. Leible, and M. Boutros. 2016. CRISPRlibrary designer (CLD): software for multispecies design of single guide RNA libraries. Genome Biol. 17:55. PMID: 27013184