GALNT1介导的Hedgehog信号调控膀胱癌干细胞自我更新机制研究

Bladder cancer is one of the most common malignancies, whose tumorigenesis have not been elucidated. We developed two novel monoclonal antibody BCMab1 and BCMab8, which could separate bladder cancer stem cells（BCSCs）.We found that Hedgehog signaling was activated in BCSCs. We also found that glycosyl transferase GALNT1 was highly expressed in BCSCs, which deduced that glycosylation modification mediated by GALNT1 participated in activation of Hedgehog signaling. We will identify the new surface markers in BCSCs, and verify their biological characteristics. We will explore the effects of Hedgehog signaling on self-renewal of BCSCs. We will elucidate SHh glycosylation modification mediated by GALNT1 is required for activation of SHh protein, and verify the Hedgehog signaling mediated by GALNT1 plays the most important role in maintaining self-renewal for BCSCs. We will identify the peptide inhibitors of GALNT1 and investigate its intervention effect on self-renewal of BCSCs. Our study will reveal the mechanism of self-renewal for BCSCs regulated by Hedgehog signaling, and provide theoretical foundations for designing drugs, which suppress self-renewal of BCSCs.

膀胱癌为泌尿系最常见的恶性肿瘤，其发病机制尚未明了。我们鉴定了两株抗膀胱癌特异性单抗BCMab1和BCMab8，能分离膀胱癌干细胞（BCSCs）。前期发现Hedgehog信号在BCSCs中高度活化。还发现BCSCs中高度表达糖基转移酶GALNT1，推断GALNT1介导的糖基化修饰参与Hedgehog信号活化。在此基础上，我们将鉴定BCSCs新的标志物，探明其生物学特征。探究Hedgehog信号介导膀胱癌干细胞自我更新的调控作用，明确GALNT1介导的SHh糖基化修饰是SHh蛋白活化所必须的，GALNT1介导的Hedgehog信号是膀胱癌干细胞自我更新维持的重要调控机制。鉴定GALNT1的多肽抑制剂，明确对BCSCs自我更新的干预作用。本课题将揭示Hedgehog信号对BCSCs自我更新调控的机理，为干预BCSCs设计抑制自我更新的药物提供理论基础。

通过该项目的实施，我们筛选鉴定了识别膀胱癌干细胞的新标志物单抗BCMab1，成功分选了膀胱癌干细胞，揭示了半乳糖基转移酶GALNT1调控膀胱癌干细胞的分子机制。主要进展总结如下：1. 发现膀胱癌干细胞中Hedgehog信号通路高度活化，膀胱癌干细胞中GALNT1高表达，表明GALNT1基因启动子更开放表达。2. GALNT1对膀胱癌干细胞的干性维持是必须的，在BCMab1+CD44+膀胱癌干细胞中敲低GALNT1，使其成球能力显著下降，肿瘤细胞在小鼠中的致瘤性降低。3. GALNT1介导了膀胱癌干细胞中Hh信号激活，维持了BCMab1+CD44+膀胱癌干细胞干性。4.利用单细胞外显子测序，分析体细胞突变等位基因频率谱，探讨了BCMab1+CD44+膀胱癌干细胞起源问题。申请人在研究中做了卓越的贡献，发表了大量高水平的研究论文20余篇，其中影响因子大于10的论文16篇，包括Cell、Cell Stem Cell、 Nat Cell Biol、Immunity、J Clin Invest、Nat Struct Mol Biol、Eur Urol、J Exp Med （3篇)、J Heptol（2篇)、Nat Commun（2篇)等，相关成果申请了3项发明专利。项目负责人范祖森2014年荣获谈家桢生命科学创新奖，2018年北京市科技进步一等奖。综上，本研究得到了全面而深入的探索，完全实现了预期的研究目标。在终期结题会评综合评价A，结论为“本项目全面完成研究计划，取得优异成绩”。