ACVRL1 drives resistance to multitarget tyrosine kinase inhibitors in colorectal cancer by promoting USP15-mediated GPX2 stabilization

Abstract

Background: Multitarget tyrosine kinase inhibitors (mTKIs) such as Regorafenib and Sorafenib have already been approved for the treatment of many solid tumours. However, the efficacy of mTKIs in colorectal cancer (CRC) is limited; the underlined mechanism remains largely elusive. Our study was aimed to find out the resistance mechanism of mTKIs in CRC.

Methods: RNA sequencing was used to identify the expression of Activin A receptor-like type 1 (ACVRL1) under the treatment of mTKIs. Gain/loss-of-function experiments were performed to assess the biological function of ACVRL1 in resistance to mTKIs. The underlying mechanisms of ACVRL1-mediated mTKI resistance were investigated by using liquid chromatography-mass spectrometry assays (LC-MS), co-immunoprecipitation assays (Co-IP), chromatin immunoprecipitation assays, ubiquitination assays, dual luciferase reporter assays, etc. RESULTS: RNA sequencing identified the activation of ACVRL1 under the treatment of mTKIs in CRC cells. ACVRL1 knockdown and overexpression significantly affects the sensitivity of CRC cells to mTKIs both in vitro and vivo. Mechanistically, we found the β-catenin/TCF-1-KCNQ1OT1/miR-7-5p axis mediated the activation of ACVRL1. Furthermore, LC-MS assays indicated the interaction between ACVRL1 and glutathione peroxidase 2(GPX2) protein. IP assay defined ACVRL1 truncation (282-503aa) could be responsible for interacting with GPX2, and rescue experiments with ACVRL1 truncations confirmed the importance of this interaction in driving mTKI resistance. Co-IP assays confirmed that ACVRL1 associates with ubiquitin-specific peptidase 15(USP15) which directly deubiquinates GPX2 at the K187(K, lysine) site, leading to the accumulation of GPX2 protein. Rescue experiments performed with the lysine mutants in GPX2 CRISPR knockout cell model confirmed the importance of GPX2 K187 mutant. As a result, the increased ROS clearance and decreased cell apoptosis eventually lead to mTKI resistance in CRC.

Conclusions: Our results demonstrate that the Wnt/β-catenin/KCNQ1OT1/miR-7-5p/ACVRL1/GPX2 biological axis plays a vital role in CRC, targeting which may be an effective approach for overcoming mTKI resistance.

Keywords: Activin A receptor-like type 1; Colorectal cancer; Drug resistance; Glutathione peroxidase 2; Multitarget tyrosine kinase inhibitors; Reactive oxygen species.