Rhophilin家族成员RHPN2促进c-Myc蛋白稳定性的机制及其在肺癌生长和维持肺癌肿瘤干细胞特性中的作用

C-Myc is an important transcription factor that regulates tumor cell metabolism, growth, and cancer stem cell properties, and its post-translational level is regulated by various modifications such as phosphorylation, ubiquitination, and SUMOylation. Our previous work has shown that the cytoskeletal reconstructor RHPN2 improves the stability of c-Myc protein and the expression of the target protein glutamine synthetase (GS), promotes tumor proliferation in vivo as well as the clonogenic sphere formation ability of lung cancer cells, and the expression level of RHPN2 correlates with the prognosis of lung adenocarcinoma patients. However, it is unclear how RHPN2 regulates the stability of c-Myc protein. Several factors that may regulate c-Myc protein stability have been screened by iTRAQ. Our proposal is to screen and identify the factors involved in the regulation of c-Myc protein stability by RHPN2 on the basis of previous studies, and to elucidate its binding to c-Myc and the mechanism of c-Myc protein modification at the molecular and cellular levels, so as to further study its effect on the growth of lung cancer and CSC characteristics at the cellular and animal levels, and its effect on tumor growth in vivo and its clinical significance. This helps to understand the role of the RHPN2-Myc axis on the development of lung cancer and is expected to provide new ideas and therapeutic strategies for targeting Myc.

c-Myc是调控肿瘤细胞代谢、生长和肿瘤干细胞特性的重要转录因子，其翻译后水平受到磷酸化、泛素化和SUMO化等多种修饰调控。我们前期工作表明，细胞骨架重建因子RHPN2提高c-Myc蛋白的稳定性和靶蛋白谷氨酰胺合成酶（GS）的表达，促进肿瘤体内增殖以及肺癌细胞克隆球形成能力，RHPN2的表达水平与肺腺癌患者预后相关。但RHPN2如何调节c-Myc蛋白的稳定性尚不清楚。我们已经通过iTRAQ已经筛选得到数个可能调控c-Myc蛋白稳定性的因子。本研究计划在前期的基础上，筛选并确认参与RHPN2调控c-Myc蛋白稳定性的因子，并在分子和细胞水平上阐明其与c-Myc结合，以及修饰c-Myc蛋白的作用机制，进而在细胞和动物水平研究其对肺癌生长和CSC特性的影响，对体内肿瘤生长的作用以及临床意义。这有助于了解RHPN2-Myc通路对肺癌的发生发展的作用，有望为靶向针对Myc提供新的思路和治疗策略。

RHPN2是rho结合蛋白rhophilin家族成员之一，能够调节肌动蛋白细胞骨架和囊泡运输，促进癌症的间质转化。我们前期发现，RHPN2在肺腺癌（LUAD）中发生突变或表达异常，然而，RHPN2在肺癌中的作用尚不完全清楚。本项目围绕着探寻RHPN2在肺癌发生发展中的功能和机制这一问题，大致按预定的内容进行研究。利用自己实验室以及公共数据库的表达谱数据证实，相较于正常组织，RHPN2 mRNA在肺腺癌及其它多种肿瘤组织高表达，而且RHPN2高表达与肺腺癌患者的总体生存期（OS）和无病生存期（DFS）等临床预后指标较差密切相关； RHPN2是肺癌细胞增殖和侵袭所必须的，同时在动物模型体内促进肺癌的形成；机制研究发现，RHPN2增强GLUL基因编码的谷氨酰胺合成酶（GS）的表达，促进肺癌肿瘤干细胞的形成，从而更加耐受谷氨酰胺缺乏的生长条件；进而发现RHPN2促进c-Myc蛋白的磷酸化增强c-Myc的稳定性从而促进其靶蛋白GS的表达；同时我们采用免疫沉淀-质谱分析（IP-MS）和iTRAQ蛋白质组学技术分别分析了RHPN2蛋白相互作用的蛋白以及下游调控的蛋白，以及这些蛋白之间的相互关系，发现RHPN2调控多种细胞骨架、细胞周期相关蛋白。同时我们也发现RHPN2抑制RhoA的激活，进而激活Hippo通路，抑制YAP活性，根据文献推测这可能与RHPN2结合STRIPAK复合物抑制磷酸酶PP2AC有关；这些发现为深入揭示RHPN2的功能打下了基础，同时也可能为肺癌的靶向治疗提供了新的思路和策略。