Department of Biotherapy, Cancer Center and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, No.17, Section 3 of Renmin South Road, 610041, Chengdu, People's Republic of China.
Cell Commun Signal. 2023 Aug 1;21(1):189. doi: 10.1186/s12964-023-01217-x.
The Annexin A6 (AnxA6) protein is known to inhibit the epidermal growth factor receptor (EGFR)-extracellular signal regulated kinase (ERK)1/2 signaling upon EGF stimulation. While the biochemical mechanism of AnxA6 inactivating phosphorylation of EGFR and ERK1/2 is not completely explored in cancer cells.
Cells were transiently co-transfected with pFlag-AnxA6, pHA-UBC9 and pHis-SUMO1 plasmids to enrich the SUMOylated AnxA6 by immunoprecipitation, and the modification level of AnxA6 by SUMO1 was detected by Western blot against SUMO1 antibody. The SUMOylation level of AnxA6 was compared in response to chemical SUMOylation inhibitor treatment. AnxA6 SUMOylation sites were further identified by LC-MS/MS and amino acid site mutation validation. AnxA6 gene was silenced through AnxA6 targeting shRNA-containing pLKO.1 lentiviral transfection in HeLa cells, while AnxA6 gene was over-expressed within the Lenti-Vector carrying AnxA6 or mutant AnxA6 plasmid in A431 cells using lentiviral infections. Moreover, the mutant plasmid pGFP-EGFR was constructed to test AnxA6 regulation on EGFR mutation-induced signal transduction. Moreover, cell proliferation, migration, and gefitinib chemotherapy sensitivity were evaluated in HeLa and A431 cells under AnxA6 konckdown or AnxA6 overexpression by CCK8, colony form and wound healing assays. And tumorigenicity in vivo was measured in epithelial cancer cells-xenografted nude mouse model.
AnxA6 was obviously modified by SUMO1 conjugation within Lys (K) residues, and the K299 was one key SUMOylation site of AnxA6 in epithelial cancer cells. Compared to the wild type AnxA6, AnxA6 knockdown and its SUMO site mutant AnxA6 showed less suppression of dephosphorylation of EGFR-ERK1/2 under EGF stimulation. The SUMOylated AnxA6 was prone to bind EGFR in response to EGF inducement, which facilitated EGFR-PKCα complex formation to decrease the EGF-induced phosphorylation of EGFR-ERK1/2 and cyclin D1 expression. Similarly, AnxA6 SUMOylation inhibited dephosphorylation of the mutant EGFR, thereby impeding EGFR mutation-involved signal transduction. Moreover, AnxA6 knockdown or the K299 mutant AnxA6 conferred AnxA6 inability to suppress tumor progression, resulting in drug resistance to gefitinib in epithelial cancer cells. And in epithelial cancer cells-xenografted nude mouse model, both the weight and size of tumors derived from AnxA6 knockdown or AnxA6 mutation-expressing cells were much greater than that of AnxA6-expressing cells.
Besides EGFR gene mutation, protein SUMOylation modification of EGFR-binding protein AnxA6 also functions pivotal roles in mediating epithelial cancer cell growth and gefitinib drug effect. Video Abstract.
已知膜联蛋白 A6（AnxA6）蛋白可抑制表皮生长因子受体（EGFR）-细胞外信号调节激酶（ERK）1/2 信号通路在表皮生长因子（EGF）刺激下的磷酸化。然而，在癌细胞中，AnxA6 使 EGFR 和 ERK1/2 磷酸化失活的生化机制尚未完全阐明。
瞬时转染 pFlag-AnxA6、pHA-UBC9 和 pHis-SUMO1 质粒，通过免疫沉淀富集 SUMO 化的 AnxA6，并用 SUMO1 抗体检测 AnxA6 的修饰水平。通过 LC-MS/MS 鉴定和氨基酸位点突变验证，比较化学 SUMO 化抑制剂处理后 AnxA6 的 SUMO 化水平。用针对 SUMO1 抗体的 Western blot 检测 AnxA6 的 SUMO 化水平。AnxA6 基因通过靶向 shRNA 的 pLKO.1 慢病毒转染在 HeLa 细胞中沉默，而 AnxA6 基因在 A431 细胞中通过携带 AnxA6 或突变 AnxA6 质粒的 Lenti-Vector 过表达，使用慢病毒感染。此外，构建了突变型 pGFP-EGFR 质粒，以测试 AnxA6 对 EGFR 突变诱导的信号转导的调节作用。此外，通过 CCK8、集落形成和划痕愈合试验在 HeLa 和 A431 细胞中评估 AnxA6 敲低或过表达对细胞增殖、迁移和吉非替尼化疗敏感性的影响。并通过上皮癌细胞-裸鼠异种移植模型测量体内肿瘤生成情况。
AnxA6 明显被 SUMO1 修饰，在赖氨酸（K）残基上发生共轭，并且在上皮癌细胞中，K299 是 AnxA6 的一个关键 SUMO 化位点。与野生型 AnxA6 相比，AnxA6 敲低及其 SUMO 位点突变型 AnxA6 在 EGF 刺激下对 EGFR-ERK1/2 的去磷酸化抑制作用减弱。SUMO 化的 AnxA6 易于在 EGF 诱导下与 EGFR 结合，这有利于 EGFR-PKCα 复合物的形成，从而减少 EGF 诱导的 EGFR-ERK1/2 和细胞周期蛋白 D1 的磷酸化。同样，AnxA6 SUMO 化抑制了突变型 EGFR 的去磷酸化，从而阻碍了 EGFR 突变参与的信号转导。此外，AnxA6 敲低或 K299 突变型 AnxA6 使 AnxA6 无法抑制肿瘤进展，导致上皮癌细胞对吉非替尼产生耐药性。在上皮癌细胞-裸鼠异种移植模型中，AnxA6 敲低或表达突变型 AnxA6 的细胞来源的肿瘤的重量和大小均明显大于表达 AnxA6 的细胞。
除了 EGFR 基因突变外，EGFR 结合蛋白 AnxA6 的蛋白 SUMO 化修饰在介导上皮癌细胞生长和吉非替尼药物作用方面也起着关键作用。
