STYK1通过ATG14L-Beclin1-VPS34复合体促进细胞自噬及其对胰腺癌发展进程的调控研究

细胞自噬是肿瘤抵抗缺氧、缺血等微环境，适应恶劣环境生存的重要机制。自噬水平的高低与胰腺癌的恶性程度密切相关。我们发现，原癌基因STYK1能够促进胰腺癌细胞基础及诱导条件下的自噬水平，且能够与ATG14L、Beclin1及VPS34直接相互作用。据此推测，STYK1有可能通过影响ATG14L-Beclin1-VPS34自噬复合体的组装而调控胰腺癌的发展进程。本课题拟在前期工作的基础上，从以下三个方面进行研究：①在细胞水平研究STYK1调控胰腺癌细胞自噬的分子机制；②AMPK磷酸化STYK1并促进自噬的分子机制；③在斑马鱼模型中研究STYK1对自噬的影响。通过以上研究，以期阐明STYK1促进胰腺癌自噬活性的分子机制，也为临床上胰腺癌的有效治疗及创新药物的研发提供全新的思路。

Autophagy is an important mechanism for cancer to adapt to, survive in or fight against severe environments such as hypoxia and ischemia. Activation of oncogenic KRAS in pancreatic cancer generates a metabolic stress characterized by a temporary deficit in energy, which must be compensated by increasing metabolic resources through the activation of autophagy. Indeed, autophagy is highly correlated with the malignancy of pancreatic cancer. We recently found in our preliminary experiments that oncogene serine/threonine/tyrosine kinase 1 (STYK1, also called novel oncogene with kinase domain, NOK) promotes autophagy in pancreatic cancer cells in basal, starvation, and hydrogen peroxide induced conditions without altering the activity of mammalian target of rapamycin (mTOR) or AMP-activated protein kinase (AMPK). We further found by co-immunoprecipitation and GST pull-down assays that STYK1 directly interacts with ATG14L, Beclin1 and VPS34. However, the detailed mechanisms of how STYK1 promotes autophagy remain unclear. Based on these preliminary results,in this proposal, we will use cultured cell and zebrafish model, in combination with molecular biology, mutagenesis, morpholino knockdown, CRISPR-Cas9 knockout, and over-expression to determine how STYK1 regulates autophagy induction and its role in pancreatic cancer progression. For this purpose we will perform the following experiments in three Aims. In Aim 1, we propose to investigate whether the regulation of autophagy by STYK1 is through the interaction with ATG14L, Beclin1 and VPS34, and promotion of the assembly of autophagy initiation complex in both basal and induced conditions. Further, we will identify ~6 short blocking peptides that disrupt the STYK1-ATG14L, STYK1-Beclin1 and/or STYK1-VPS34 interactions. In Aim 2, we propose to examine the role of AMPK in STYK1 phosphorylation, and the cross-talk between AMPK signaling and RPTK in autophagy initiation. In Aim 3, we propose to examine whether STYK1 affects autophagy activation in zebrafish and investigated the effect of the peptides through peritoneal injection of membrane permeable blocking peptides. Successful completion of experiments outlined in this proposal will enhance our current understanding of the mechanism of how STYK1 promotes autophagy and pancreatic cancer tumorigenesis, and also provide novel therapeutic strategies and targets for pancreatic cancer.