Department of Gastrointestinal Medical Oncology, Harbin Medical University Cancer Hospital, Harbin, China.
Key Laboratory of Tumor Immunology in Heilongjiang, Harbin, China.
BMC Med. 2023 Sep 25;21(1):366. doi: 10.1186/s12916-023-03066-4.
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.
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.
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.
多靶点酪氨酸激酶抑制剂（mTKIs）如瑞戈非尼和索拉非尼已被批准用于治疗多种实体瘤。然而，mTKIs 在结直肠癌（CRC）中的疗效有限，其潜在机制仍很大程度上难以捉摸。本研究旨在探讨 mTKIs 在 CRC 中的耐药机制。
采用 RNA 测序鉴定 mTKIs 治疗下激活素 A 受体样 1（ACVRL1）的表达。通过 gain/loss-of-function 实验评估 ACVRL1 在 mTKIs 耐药中的生物学功能。通过液相色谱-质谱联用（LC-MS）、免疫共沉淀（Co-IP）、染色质免疫沉淀（ChIP）、泛素化实验、双荧光素酶报告基因实验等方法探讨 ACVRL1 介导 mTKI 耐药的潜在机制。
RNA 测序鉴定出 CRC 细胞中 mTKIs 治疗下 ACVRL1 的激活。ACVRL1 敲低和过表达显著影响 CRC 细胞对 mTKIs 的体外和体内敏感性。机制上，我们发现β-catenin/TCF-1-KCNQ1OT1/miR-7-5p 轴介导了 ACVRL1 的激活。此外，LC-MS 实验表明 ACVRL1 与谷胱甘肽过氧化物酶 2（GPX2）蛋白相互作用。免疫沉淀实验定义了 ACVRL1 截断（282-503aa）可与 GPX2 相互作用，并用 ACVRL1 截断进行的挽救实验证实了这种相互作用在驱动 mTKI 耐药中的重要性。免疫共沉淀实验证实 ACVRL1 与泛素特异性肽酶 15（USP15）相关，USP15 可直接在 K187（K，赖氨酸）位点去泛素化 GPX2，导致 GPX2 蛋白积累。在 GPX2 CRISPR 敲除细胞模型的赖氨酸突变体挽救实验中证实了 GPX2 K187 突变体的重要性。结果，ROS 清除增加和细胞凋亡减少最终导致 CRC 中 mTKI 耐药。
我们的研究结果表明，Wnt/β-catenin/KCNQ1OT1/miR-7-5p/ACVRL1/GPX2 生物学轴在 CRC 中起着至关重要的作用，针对该轴可能是克服 mTKI 耐药的有效方法。
