Rab25抑制非小细胞肺癌细胞自噬的机制研究

Macroautophagy (hereafter referred to as autophagy) is essential for the metastasis of solid tumors. Targeting tumor cell autophagy potentially benefits the clinical therapeutics of metastatic cancers. However, how to particularly and precisely manipulate the autophagy of cancerous cells but not, or at least minimally, affecting that of adjacent normal cells, still needs to be explored. . Our previous studies mainly focused on the regulatory mechanisms of adaptor protein p66Shc on starvation or extracellular matrix detachment induced non-small cell lung cancer (NSCLC) cell autophagy. Recently, by Co-IP, HPLC-MS and Duolink analysis, we identified Rab25, a small GTPase involved in apical recycling endosome trafficking, as a binding partner of p66Shc. Further study showed that Rab25 expression is remarkably high in clinical NSCLC samples and cultured NSCLC cell lines. The shRNA knockdown of Rab25 elevates autophagy, and inhibits cell migration and invasion. These results together suggested Rab25 as a possible tumor promoter of NSCLC; and that autophagy may play a key role in Rab25-mediated cancer progression. Accordingly, we propose that Rab25 binds to p66Shc and suppresses autophagy to promote NSCLC metastasis with unknown mechanism(s). . In the current study, first, we will explore the binding sites of Rab25 and p66Shc. We then will analyze how the mutants that lack of Rab25-p66Shc interaction respond to starvation induced autophagy. Next, we will determine that whether the deficit GTPase activity of Rab25 affects autophagy. Besides, as a classic marker of apical recycling endosome, how Rab25 balances the endomembrane trafficking and autophagy still needs to be addressed. Moreover, the reported binding partners and effectors of Rab25, such as Rab11FIP2 and Rab11FIP3, are predicted as LC3 interacting proteins by online autophagy database iLIR. Their possible roles in regulating autophagy will be analyzed. Third, we will reveal the role of Rab25 on cell invasion, migration and proliferation by using transwell assay, wound healing assay and BrdU assay, respectively. We will estimate the function of Rab25-p66Shc mediated autophagy in the metastasis of human tumor xenograft of SCID (severe combined immuno deficiency) mice by subcutaneous implantation in the right chest or tail vein injection and measuring the weight and size of formed tumor. Last, we will determine the correlation between Rab25, p66Shc and autophagy in tissue array by using immunohistology staining. . Based on these studies, we will decipher the regulatory mechanism(s) of Rab25 on NSCLC cell autophagy and tumor metastasis. This investigation may provide new insights in the intervention and treatment of lung cancer metastasis.

细胞自噬在实体肿瘤转移中发挥重要功能，但如何实现靶向调控肿瘤细胞自噬函待解决。我们前期工作发现：Rab25在非小细胞肺癌（NSCLC）临床样本和细胞系中特异性高表达；敲低Rab25抑制NSCLC细胞迁移和侵袭，并促进细胞自噬；饥饿诱导时Rab25直接结合信号衔接蛋白p66Shc。我们的已发表论文证实p66Shc参与调控肿瘤细胞自噬。由此，本项目将围绕“Rab25结合p66Shc抑制NSCLC细胞自噬并促进肿瘤转移”这一科学假设，展开如下研究：（1）阐明Rab25和p66Shc的相互作用机制；（2）从互作蛋白、细胞囊泡内膜定位及GTP酶活性三方面考察Rab25调控自噬的机制；（3）通过细胞实验、动物实验和组织芯片分析，揭示Rab25调控的细胞自噬与NSCLC转移特性的内在联系。本研究将阐明Rab25在NSCLC转移中的功能和作用机制，为靶向肿瘤细胞自噬的NSCLC临床防治新策略提供理论基础。

细胞自噬在实体肿瘤转移中发挥重要功能，但如何实现靶向调控肿瘤细胞自噬函待解决。我们前期工作发现：Rab家族及转录调控因子SYK、SPIB等在非小细胞肺癌（NSCLC）临床样本和细胞系中特异性高表达；敲低则抑制NSCLC细胞迁移和侵袭，并促进细胞自噬；饥饿诱导时则直接结合信号衔接蛋白p66Shc。我们的已发表论文证实p66Shc参与调控肿瘤细胞自噬。由此，本项目将围绕“Rab25结合p66Shc抑制NSCLC细胞自噬并促进肿瘤转移”这一科学假设，展开如下研究：阐明Rab25和p66Shc的相互作用机制；从互作蛋白、细胞囊泡内膜定位及GTP酶活性三方面考察Rab25调控自噬的机制；通过细胞实验、动物实验和组织芯片分析，揭示Rab25调控的细胞自噬与NSCLC转移特性的内在联系。本研究阐明了细胞自噬相关蛋白NSCLC转移中的功能和作用机制，为靶向肿瘤细胞自噬的NSCLC临床防治新策略提供理论基础。