人类表皮生长因子受体II（ERBB2）基因高频热点突变介导膀胱尿路上皮癌进展的分子机制研究

By analyzing the genomic data of bladder cancer, we identified an important hot mutation in erb-b2 receptor tyrosine kinase 2 (ERBB2). Publications suggested that ERBB2 mutations contribute to the tumor progression, whereas the mechanisms are still unknown. In our preliminary work, we successfully constructed a mutant T24 cell line that contained this specific hot mutation and identified that after mutation, the tumor cell growth and colony formation (CF) abilities were significantly increased. In addition, western blotting assay also identified that this special hot mutation increased the phosphorylation level of ERBB2. Based on these findings, we will try to construct another mutant cell line BIU-87, and apply cell proliferation, migration, invasion and CF assay to further confirm whether this specific mutation contributes to the bladder cancer progression. In addition, we try to predict the downstream targets of ERBB2 according to its phosphorylation conserve sequence, and apply western blotting, shRNA, Co-IP and functional assays to figure out one or more specific functional targets that can be directly activated by phosphorylating. In addition, we will also analyze the association between hot mutation status, coding protein phosphorylation level, protein or phosphorylation level of downstream targets and clinical stage, pathological grade, survival and prognosis, establishing theoretical foundation to identify novel targets for the diagnosis, therapy and prognosis prediction of bladder cancer patients.

申请团队通过分析膀胱癌组学数据，筛出一位于人类表皮生长因子受体II（ERBB2）基因上的高频热点突变。文献称该基因突变可影响肿瘤进展，但机制未明。团队前期研究已成功构建含有该热点突变的T24细胞系，并发现引入突变后的肿瘤细胞其增殖和克隆形成能力明显增强。此外，蛋白电泳揭示该热点突变可增加其编码蛋白磷酸化水平。据此，本项目拟在预实验基础上继续采用CRISPR/Cas9技术构建突变型BIU-87细胞系，利用增殖、迁移、侵袭和克隆形成实验进一步论证引入突变对肿瘤进展影响。并根据ERBB2激酶磷酸化保守序列预测下游靶蛋白，利用蛋白电泳、shRNA、Co-IP和功能实验方法，进一步筛选出能被其直接磷酸化激活且有功能的下游靶蛋白；此外，研究还将分析该热点突变、编码蛋白和靶蛋白磷酸化水平与患者临床分期、病理分级和生存预后关联，为达到从该基因作用中找到膀胱癌诊治及预后评估新靶标的终极目标奠定理论基础。

申请团队通过分析膀胱癌组学数据，筛出一位于人类表皮生长因子受体II（ERBB2）基因上的高频热点突变。文献称该基因突变可影响肿瘤进展，但机制未明。团队已成功构建含有该热点突变的T24细胞系，并发现引入突变后的肿瘤细胞其增殖和克隆形成能力明显增强。此外，课题组发现该基因突变后可增加其编码蛋白的磷酸化水平，进而激活Akt通路，促进肿瘤进展。而予以shERBB2或靶向抑制Akt磷酸化水平可显著抑制ERBB2高频热点突变诱导的膀胱肿瘤进展。受限于突变总体样本数的限制，当前课题并未发现ERBB2基因热点突变与膀胱肿瘤患者临床分期、病理分级和生存预后的关联，需要未来进一步大样本研究进一步探索。综上，本项目为携带ERBB2基因高频热点突变的膀胱癌患者提供新的治疗靶点。