Table 4_A rare germline mutation reverses the suppressive effect of GPC5 thereby promoting lung adenocarcinoma development and tumorigenesis.xlsx

Background and ObjectiveGlypican-5 (GPC5) has been well-characterized as a tumor suppressor in lung adenocarcinoma (LUAD); however, the functional implications of its germline mutations in cancer pathogenesis remain largely unexplored. In this study, we identified and characterized a pathogenic GPC5 variant (c.776C>T, p.Pro259Leu) within a Chinese LUAD pedigree, systematically investigating its oncogenic mechanisms through comprehensive molecular and cellular analyses.
MethodsOur investigation employed a multifaceted approach beginning with the recruitment of a LUAD-affected family cohort (n=4 patients, 1 healthy control), followed by exome sequencing of matched blood and FFPE tumor samples. Through rigorous rare variant analysis, we prioritized the GPC5 c.776C>T variant, subsequently validating its pathogenicity via integrated computational modeling and immunohistochemical profiling. Mechanistic studies in A549 and H2009 LUAD cell lines encompassed: (1) comprehensive proliferation and apoptosis assessment using CCK-8, colony formation, EdU incorporation, and flow cytometry; (2) migration and invasion evaluation through Transwell and wound healing assays; (3) EMT/Wnt pathway interrogation via Western blot analysis of E-cadherin, N-cadherin, Vimentin, and β-catenin expression patterns; and (4) definitive functional validation through GPC5 overexpression and knockdown experiments.
ResultsGenetic analysis revealed the GPC5 c.776C>T variant exhibited complete cosegregation with LUAD phenotype in the pedigree while being absent in control populations (gnomAD frequency: 0.000003989), accompanied by significantly reduced GPC5 expression in tumor tissues. Functional characterization demonstrated that compared to wild-type, the mutant variant conferred aggressive oncogenic properties: significantly enhanced proliferative capacity, impaired apoptosis induction, and markedly increased migratory potential. Molecular analyses revealed the mutant promoted EMT activation through nuclear β-catenin accumulation and subsequent upregulation of mesenchymal markers. Crucially, siRNA-mediated GPC5 knockdown phenocopied these oncogenic effects, providing definitive evidence of its tumor-suppressive function.
DiscussionOur findings establish that the GPC5 c.776C>T mutation drives LUAD progression through a novel molecular mechanism involving impaired β-catenin degradation, subsequent nuclear translocation, and consequent EMT activation. These results position GPC5 as a critical nodal regulator of Wnt/β-catenin signaling in LUAD pathogenesis and suggest its germline mutations may serve as valuable biomarkers for hereditary LUAD risk assessment. Therapeutically, these findings highlight the potential utility of Wnt pathway inhibitors in managing GPC5-mutant LUAD cases, while also providing a molecular framework for future investigations into glypican family members in cancer biology.

背景与目的
磷脂酰肌醇蛋白聚糖-5（Glypican-5, GPC5）作为肺腺癌（lung adenocarcinoma, LUAD）的抑癌基因已得到充分验证，但其生殖系突变在癌症发生发展中的功能意义仍有待深入探索。本研究在一个中国肺腺癌家系中鉴定并表征了一处致病性GPC5变异（c.776C>T, p.Pro259Leu），并通过全面的分子与细胞实验系统解析其致癌机制。

方法
本研究采用多维度研究策略：首先招募了受累肺腺癌家系队列（共4例患者，1例健康对照），随后对匹配的血液样本与福尔马林固定石蜡包埋（formalin-fixed paraffin-embedded, FFPE）肿瘤样本进行外显子测序。通过严格的罕见变异分析筛选出GPC5 c.776C>T变异，随后通过整合计算建模与免疫组化分析验证其致病性。在A549与H2009肺腺癌细胞系中开展的机制研究包括：(1) 采用CCK-8法、集落形成实验、EdU掺入实验与流式细胞术全面评估细胞增殖与凋亡水平；(2) 通过Transwell小室实验与划痕愈合实验检测细胞迁移与侵袭能力；(3) 通过蛋白质免疫印迹分析E-钙粘蛋白、N-钙粘蛋白、波形蛋白与β-连环蛋白的表达模式，解析上皮间质转化（epithelial-mesenchymal transition, EMT）与Wnt信号通路的激活情况；(4) 通过GPC5过表达与敲低实验进行最终的功能验证。

结果
遗传分析显示，GPC5 c.776C>T变异在该家系中与肺腺癌表型完全共分离，且在对照人群中极为罕见（gnomAD数据库频率为0.000003989），同时肿瘤组织中GPC5的表达水平显著降低。功能表征实验表明，与野生型GPC5相比，该突变体赋予细胞更强的致癌表型：增殖能力显著增强、凋亡诱导受损、迁移潜能显著提升。分子机制分析显示，该突变通过促进β-连环蛋白入核积累，进而上调间质标志物表达，激活上皮间质转化过程。至关重要的是，小干扰RNA（small interfering RNA, siRNA）介导的GPC5敲低可重现上述致癌效应，为GPC5的抑癌功能提供了确凿证据。

讨论
本研究结果证实，GPC5 c.776C>T突变通过一种全新的分子机制驱动肺腺癌进展：即破坏β-连环蛋白的降解途径，促进其核转位并最终激活上皮间质转化。上述结果明确了GPC5作为肺腺癌发生中Wnt/β-连环蛋白信号通路的关键节点调控因子，同时提示其生殖系突变可作为遗传性肺腺癌风险评估的潜在生物标志物。在治疗层面，本研究结果凸显了Wnt通路抑制剂在治疗GPC5突变型肺腺癌中的应用潜力，同时也为未来癌症生物学中磷脂酰肌醇蛋白聚糖家族成员的研究提供了分子框架。