细胞自噬异常显著影响细胞内外环境的稳态和自噬性存活,而自噬溶酶体途径对早熟性衰亡的调控机制还未知。在研究核蛋白因子NUPR1对自噬的转录调节机制中，我们发现细胞特异性表达的SNAP25（自噬性SNARE复合物中的成员）是调控细胞自噬途径的关键蛋白之一，介导自噬溶酶体的外排；当下调该基因表达时，导致不可逆的早熟性细胞衰亡，提示SNARE复合物参与的内膜识别途径是自噬溶酶体流量关键的调控靶点，据此我们提出：抑制SNARE复合物介导的细胞自噬流量，可能是操控自噬性存活与衰亡的分子基础。故本课题将以NUPR1调控SNAP25的转录，进而介导自噬溶酶体的外排为切入点，深入研究SNARE复合物调控自噬性存活的分子机制，并在非小细胞肺癌样品中加以验证。本项目的研究结果将有助于了解肿瘤细胞自噬调控途径的分子机制，同时探讨基于自噬性SNARE复合物介导的生物学效应来抑制肺癌细胞体内存活的可行性。

Macroautophagy (hereafter called autophagy) is an evolutionarily conserved pathway of lysosomal-mediated cellular self-digestion. It involves the formation of a double-membrane vesicle, the autophagosome, which engulfs cytoplasmic components and delivers them to the lysosome for degradation. Under a variety of stressful conditions, autophagy is an obligate requirement for cancer cell survival. A aberrantly balance between autophagic survival and cell death is believed to drive all stages of cancer progression. However, the mechanisms of autophagic survival and cell death regulation in cancer cells which distinctly modulate cells to allow autophagic survival remain largely elusive. Our data showed that nuclear protein 1, NUPR1 (also called p8, or Com1), a transcriptional coregulator mediates autophagic survival in non-small cell lung cancer cells (NSCLCs) and plays an important role in NSCLC progression. Experimental validation shows that NUPR1 regulates the late stage of autolysosome processing through the induction of the SNARE protein SNAP25, which forms a complex with the lysosomal SNARE-associated protein, vesicle associated membrane protein 8 (VAMP8). Mechanistically, inactivation of SNAP25 by shRNA causes enhanced autophagic flux alongside a persistent reduction in autolysosomal exocytosis and consequently triggers premature senescent cell death with autolysosomal vacuolization. These findings indicate a significant and novel function of SNAP25 as a potent regulator of autolysosomal process in diverse cancer cell demise decision. Based on these experimental data, we propose here that inactivation of SNARE pathway with the enhanced autophagic flux might be a promising strategy to inhibit lung cancer survival. To test this hypothesis, we will use clinical lung cancer samples, lung cancer cell lines as well as mouse model to investigate the crosstalk between autophagic survival and autophagic cell death mediated by SNARE complex in vitro and in vivo. The on-going project of SNARE functional investigation will be initially focused on the interacting protein partners with SNAP25 by Nano-HPLC/mass spectrometric analysis and their validation by Duolink and co-immunoprecipitation, respectively. The regulatory pathways of autophagic survival and premature senescence will also be elucidated and evaluated in clinical NSCLC samples as new candidates for tumor suppression. For example, the combination of autophagic inhibition with cytotoxic drugs and deficeint Cas9(dCas9) fusion proteins guided by gene-specific sgRNAs to either repress (CRISPRi) or activate (CRISPRa) transcription of target genes is an attracting manipulation to overcome resistance to antitumor therapy. Hopefully, the strategy proposed here may gap in our knowledge of the autophagic survival and premature senescent cell death events associated with lung cancer progression and may add to means of assessing the suppression of SNARE function and the impaired autophagy for prevention of NSCLC incidence.