背景:Cingulin(CGN)是位于紧密连接的关键细胞骨架衔接蛋白。本研究通过遗传和机制分析研究了CGN突变与癌症易感性增加之间的联系,并提出了一种潜在的靶向治疗方法。
方法:在一个没有已知致病变异的高癌症密度家族中,我们进行了肿瘤靶向和种系全基因组测序,以鉴定新的癌症相关变异.随后,这些变异在222名癌症患者队列中得到验证,CGNc.3560C>T被鉴定为潜在的癌症风险等位基因。将野生型(WT)(c.3560C>C)和变体(c.3560C>T)转染到癌细胞系中,并掺入原位异种移植小鼠模型中,以评估其对癌症进展的影响。蛋白质印迹,免疫荧光分析,迁移和入侵测定,双向凝胶电泳与质谱,免疫沉淀试验,和siRNA应用用于探索CGNc.3560C>T的生物学后果。
结果:在癌细胞系和原位动物模型中,与CGNWT相比,CGNc.3560C>T增强了肿瘤进展,对奥沙利铂的敏感性降低。该变体诱导上皮标志物下调,间充质标记和转录因子的上调,其会聚以启动上皮-间质转化(EMT)。进行蛋白质组学分析以研究CGNc.3560C>T中驱动EMT的元素。这项探索揭示了由变体诱导的IQGAP1的过表达,对比CGNWT中观察到的水平。免疫沉淀测定证实了CGN和IQGAP1之间的直接相互作用。IQGAP1作为多种GTP酶的调节因子,尤其是Rho家族.这种过表达的IQGAP1始终与Rac1的激活相关,如通过对具有CGNc.3560C>T的癌细胞系和临床样品的分析所证明的。siRNA下调IQGAP1后,激活的Rac1被抑制。用NSC23766治疗,一种Rac1-GEF相互作用的选择性抑制剂,导致Rac1失活。这种干预缓解了携带CGNc.3560C>T的癌细胞中的EMT程序。具有WTCGN的异种移植肿瘤对NSC23766治疗没有敏感性,但NSC23766显示出减弱肿瘤生长的能力,c.3560C>T。
结论:CGNc.3560C>T导致IQGAP1过表达,随后触发Rac1依赖性EMT。靶向激活的Rac1是阻止携带这种特定变体的癌症进展的策略。
BACKGROUND: Cingulin (CGN) is a pivotal cytoskeletal adaptor protein located at tight junctions. This study investigates the link between CGN mutation and increased cancer susceptibility through genetic and mechanistic analyses and proposes a potential targeted therapeutic approach.
METHODS: In a high-cancer-density family without known pathogenic variants, we performed tumor-targeted and germline whole-genome sequencing to identify novel cancer-associated variants. Subsequently, these variants were validated in a 222 cancer patient cohort, and CGN c.3560C > T was identified as a potential cancer-risk allele. Both wild-type (WT) (c.3560C > C) and variant (c.3560C > T) were transfected into cancer cell lines and incorporated into orthotopic xenograft mice model for evaluating their effects on cancer progression. Western blot, immunofluorescence analysis, migration and invasion assays, two-dimensional gel electrophoresis with mass spectrometry, immunoprecipitation assays, and siRNA applications were used to explore the biological consequence of CGN c.3560C > T.
RESULTS: In cancer cell lines and orthotopic animal models, CGN c.3560C > T enhanced tumor progression with reduced sensitivity to oxaliplatin compared to the CGN WT. The variant induced downregulation of epithelial marker, upregulation of mesenchymal marker and transcription factor, which converged to initiate epithelial-mesenchymal transition (EMT). Proteomic analysis was conducted to investigate the elements driving EMT in CGN c.3560C > T. This exploration unveiled overexpression of IQGAP1 induced by the variant, contrasting the levels observed in CGN WT. Immunoprecipitation assay confirmed a direct interaction between CGN and IQGAP1. IQGAP1 functions as a regulator of multiple GTPases, particularly the Rho family. This overexpressed IQGAP1 was consistently associated with the activation of Rac1, as evidenced by the analysis of the cancer cell line and clinical sample harboring CGN c.3560C > T. Notably, activated Rac1 was suppressed following the downregulation of IQGAP1 by siRNA. Treatment with NSC23766, a selective inhibitor for Rac1-GEF interaction, resulted in the inactivation of Rac1. This intervention mitigated the EMT program in cancer cells carrying CGN c.3560C > T. Consistently, xenograft tumors with WT CGN showed no sensitivity to NSC23766 treatment, but NSC23766 demonstrated the capacity to attenuate tumor growth harboring c.3560C > T.
CONCLUSIONS: CGN c.3560C > T leads to IQGAP1 overexpression, subsequently triggering Rac1-dependent EMT. Targeting activated Rac1 is a strategy to impede the advancement of cancers carrying this specific variant.