Key Laboratory of Metabolism and Molecular Medicine, Ministry of Education, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai, China.
Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai, China.
Br J Cancer. 2024 Feb;130(3):380-393. doi: 10.1038/s41416-023-02495-5. Epub 2023 Dec 18.
c-Met encoded by the proto-oncogene MET, also known as hepatocyte growth factor (HGF) receptor, plays a crucial role in cellular processes. MET exon 14 skipping alteration (METΔ14EX) is a newly discovered MET mutation. SMAD2 is an important downstream transcription factor in TGF-β pathway. Unfortunately, the mechanisms by which METΔ14EX leads to oncogenic transformation are scarcely understood. The relationship between METΔ14EX and SMAD2 has not been studied yet.
We generate METΔ14EX models by CRISPR-Cas9. In vitro transwell, wound-healing, soft-agar assay, in vivo metastasis and subcutaneous recurrence assay were used to study the role of METΔ14EX in tumour progression. RNA-seq, Western blotting, co-immunoprecipitation (CO-IP) and immunofluorescent were performed to explore the interaction between c-Met and SMAD2.
Our results demonstrated that METΔ14EX, independent of HGF, can prolong the constitutive activation of c-Met downstream signalling pathways by impeding c-Met degradation and facilitating tumour metastasis and recurrence. Meanwhile, METΔ14EX strengthens the interaction between c-Met and SMAD2, promoting SMAD2 phosphorylation. Therapeutically, MET inhibitor crizotinib impedes METΔ14EX-mediated tumour metastasis by decreasing SMAD2 phosphorylation.
These data elucidated the previously unrecognised role of METΔ14EX in cancer progression via activation of SMAD2 independent of TGF-β, which helps to develop more effective therapies for such patients. METΔ14EX alteration significantly triggers tumour progression via activation of SMAD2 signalling that are involved in activating tumour invasion, metastasis and recurrence. On the left, in the MET wild-type (METWT), the juxtamembrane (JM) domain is involved in the regulation of tyrosine kinase activity, receptor degradation, and caspase cleavage. On the right, the METΔ14EX mutation leads to the loss of the juxtamembrane domain, resulting in an abnormal MET protein lacking a CBL-binding site. This causes the accumulation of truncated MET receptors followed by constitutive activation of the MET signalling pathway. Thus, the METΔ14EX-mutated protein has strong binding and phosphorylation to SMAD2, which results in the phosphorylation of a large number of SMAD2/3 proteins that combine with SMAD4 to form a complex in the nucleus, activating downstream signalling pathways, such as EMT and ECM remodelling, resulting in tumour progression and recurrence. TF transcription factor.
原癌基因 MET 编码的 c-Met，也称为肝细胞生长因子（HGF）受体，在细胞过程中发挥着关键作用。MET 外显子 14 跳跃改变（METΔ14EX）是一种新发现的 MET 突变。SMAD2 是 TGF-β 通路中的一个重要下游转录因子。不幸的是，METΔ14EX 导致致癌转化的机制还知之甚少。METΔ14EX 与 SMAD2 之间的关系尚未得到研究。
我们通过 CRISPR-Cas9 生成 METΔ14EX 模型。体外 Transwell、划痕愈合、软琼脂测定、体内转移和皮下复发测定用于研究 METΔ14EX 在肿瘤进展中的作用。进行 RNA-seq、Western blotting、免疫共沉淀（CO-IP）和免疫荧光，以探讨 c-Met 和 SMAD2 之间的相互作用。
我们的结果表明，METΔ14EX 可以通过阻碍 c-Met 降解和促进肿瘤转移和复发，独立于 HGF 延长 c-Met 下游信号通路的组成性激活。同时，METΔ14EX 增强了 c-Met 和 SMAD2 之间的相互作用，促进了 SMAD2 的磷酸化。在治疗上，MET 抑制剂克唑替尼通过降低 SMAD2 磷酸化来抑制 METΔ14EX 介导的肿瘤转移。
这些数据阐明了 METΔ14EX 通过激活独立于 TGF-β 的 SMAD2 在癌症进展中的以前未被认识的作用，这有助于为这些患者开发更有效的治疗方法。METΔ14EX 改变通过激活参与激活肿瘤侵袭、转移和复发的 SMAD2 信号显著触发肿瘤进展。在左侧，在 MET 野生型（METWT）中，跨膜（JM）结构域参与酪氨酸激酶活性、受体降解和半胱氨酸蛋白酶切割的调节。在右侧，METΔ14EX 突变导致跨膜结构域缺失，导致缺乏 CBL 结合位点的异常 MET 蛋白。这导致截断的 MET 受体积累，随后 MET 信号通路的组成性激活。因此，METΔ14EX 突变蛋白与 SMAD2 具有很强的结合和磷酸化作用，导致大量磷酸化的 SMAD2/3 蛋白与 SMAD4 结合形成核内复合物，激活下游信号通路，如 EMT 和 ECM 重塑，导致肿瘤进展和复发。TF 转录因子。
