BRSK2调控自噬促进非小细胞肺癌细胞转移的机制

Tumor metastasis is the main cause of death in patients with lung cancer. Autophagy can help tumor cells resist the adverse environmental conditions so as to promote tumor metastasis. Protein kinase BRSK2 has been shown to be related to the regulation of autophagy pathway and tumor metastasis with less clear mechanism. We found that BRSK2 is expressed in various non-small cell lung cancer (NSCLC) cell lines and clinical lung cancer samples. shRNA-mediated knocking down BRSK2 inhibits invasion, migration and autolysosomal exocytosis in NSCLC cells, and the inhibition effect of autophagic inhibitors on migration and invasion cannot be superimposed on the effect produced by BRSK2 knockdown. Through Co-immunoprecipitation and mass spectrum, we found that BRSK2 can bind and phosphorylate SNAP25, which forms a complex with the lysosomal SNARE-associated protein, vesicle associated membrane protein 8 (VAMP8) and modulates autolysosomal exocytosis. The crystal structure of BRSK2 homologous protein suggests BRSK2 is able to modulate autophagy through other pathways. These findings indicate a significant and novel function of BRSK2 as a potent regulator of autophagy and metastasis. Along with our preliminary data of the important modulation function of SNAP25 in autolysosomal exocytosis, we propose here that inactivation of BRSK2 pathway might be a promising strategy to inhibit lung cancer metastasis. To test this hypothesis, we will find the interacting protein partners and the kinase substrate protein of BRSK2 by Nano-HPLC/mass spectrometric analysis and their validation by PLA (Proximity Ligation Assay), co-immunoprecipitation and kinase assay, respectively. Then, we will use clinical lung cancer samples, lung cancer cell lines as well as mouse model to investigate the crosstalk between autophagy and metastasis mediated by BRSK2 and its binding partners as well as its substrate protein in vitro and in vivo. Through “In Situ Capture of Chromatin Interactions by Biotinylated dCas9” and promoter methylation assay, we will elucidate the mechanism of the ectopic expression of BRSK2 in NSCLC cells. Based on our crystal structure of BRSK2 homologous protein, we will find the inhibitor of BRSK2 through molecular docking, and test the effects of the inhibitor through molecular, cell and animal experiments. Hopefully, we may gain significant insight into the autophagic events associated with lung cancer metastasis and add to means of assessing suppression of BRSK2 function and impaired autophagy for prevention of NSCLC metastasis. Also, a better understanding of the role of BRSK2 underlying autophagy and metastasis including the determination of the upstream regulators and downstream effectors of BRSK2 functional pathways may lead to the development of novel anti-tumor strategies.

肿瘤转移是导致肺癌患者死亡的主要原因，自噬可以帮助肿瘤细胞抵御转移过程中的不利环境条件。前期研究表明，肺癌细胞系及临床样本中蛋白激酶BRSK2高表达，knockdown BRSK2可以导致细胞侵袭、迁移能力降低以及自噬溶酶体外排受阻，抑制自噬对于上述现象的抑制不能与knockdown BRSK2的效果相叠加。通过Co-IP和质谱检测发现BRSK2能够结合并磷酸化介导自噬溶酶体外排的SNAP25。BRSK2同源蛋白晶体结构暗示其可能通过其他途径调控自噬，这些都提示BRSK2调控自噬促进肿瘤转移。本课题将深入研究BRSK2调控自噬的磷酸化底物蛋白及其影响肿瘤转移的机制，进而研究在改变BRSK2表达水平后肿瘤细胞在体外、体内生理特性的变化，并在小鼠模型及肺癌临床组织中验证，再结合结构生物学研究探索BRSK2抑制剂，为发展针对BRSK2的非小细胞肺癌治疗新途径提供实验基础。

(一)研究背景 . 在肿瘤发展的过程中，自噬可以通过缓解肿瘤细胞转移过程中遭遇的外界压力来促进肿瘤转移和发展的进程。在肿瘤发生发展的过程中，自噬的调控途径是当前研究的热点之一。. 我们前期的研究表明，非小细胞肺癌细胞系及临床样本中BRSK2高表达，下调BRSK2引起细胞自噬失调，且细胞增殖变慢，转移、侵袭能力减弱，提示BRSK2参与调控非小细胞肺癌细胞自噬途径从而影响其他生物学功能，但BRSK2行使功能的分子机制未知。.(二)主要研究内容.1.检测不同分期的非小细胞肺癌组织样本中BRSK2的表达情况，分析BRSK2与肺癌表型的关系。 .2.通过Co-IP结合质谱的方法找到BRSK2调控的与自噬相关的蛋白，阐明其调控自噬的机制。 .3.研究BRSK2介导的自噬失调对非小细胞肺癌细胞体外成克隆、转移、侵袭能力的影响，阐明其通过自噬影响肺癌表型的机制。 .4.利用结构生物学手段找到BRSK2发挥功能的关键位点并进行验证，阐明其发挥自噬调控功能的分子机制。. (三)重要结果及其科学意义.1.证实了BRSK2的表达与肺癌患者的低生存期相关。为肺癌的分子诊断提供了新基础。 .2.揭示了BRSK2通过调控SNAP25来调控自噬溶酶体的外排过程，并且这个过程与细胞增殖，转移、侵袭能力密切相关。证实了BRSK2通过调控自噬溶酶体的代谢过程参与肿瘤的发展进程。 .3.解析了BRSK2同源蛋白的晶体结构，通过结构分析阐明BRSK2通过磷酸化SNAP25发挥自噬调控功能的详细分子机制。.