ADAM12的水解功能在小细胞肺癌增殖和转移中的分子机制研究

It has recently been revealed that A Disintegrin And Metalloproteinase 12 (ADAM12) is highly expressed in samll cell lung cancer （SCLC） cells from patients. ADAM12 can be used as a diagnostic marker for the proliferation, invasion, and metastasis of SCLC cells and its protease acitivity is highly correlated with disease status in cancer. Up to now the identification of its substrates is based on the educated guess and further validation in cells and is lack of systematic investigation. Therefore, it is difficult to connect the protease activity of ADAM12 with the proliferation and metastasis of lung cancer cells at the molecular level. In the past few years, we have developed two robust proteomic approaches that label the N-terminus and C-terminus, respectively, of proteins for the efficient identification of their terminal peptides in a high-throughput manner. In addition, using these approaches, we have identified dozens of new substrates and their cleavage sites in the apoptosis of a cancer cell line. In this application, we will use these two terminal labeling approaches, in combination with quantitative proteomics, to investigate the spectrum of ADAM12 substrates and their cleavage sites in SCLC cell lines. Then we will use genetic knockdown, site-directed mutagenesis of ADAM12 substrates and cleavage sites, and biological approaches to investigate the function of the substrates and their cleavage. We will further elucidate the excat molecular mechanisms by which ADAM12 modulates the proliferation and metastasis of SCLC cells. Through this study, we would like to provide insight in the discovery of molecular pathways and therapeutic targets for the treatment of this cancer.

最近对临床样本的分析发现去整合素金属蛋白酶12（ADAM12）在小细胞肺癌中表达过高，是癌细胞增殖、侵袭和转移的诊断标志物，其水解功能与肿瘤的发展密切相关。迄今为止对ADAM12水解底物的研究仅限于猜测其可能的底物并进行验证但缺乏系统的研究，从而很难把它的水解功能与肺癌细胞的增殖和转移在分子水平上联系起来。我们在过去几年里建立了两种互补的蛋白质端基标记方法对蛋白质水解进行了高通量鉴定，在肿瘤细胞凋亡过程中发现了凋亡酶的多个新水解底物，找到其水解位点。在本申请中，我们拟通过这两种端基标记方法和定量蛋白质组学技术，对ADAM12在小细胞肺癌中的水解底物进行高通量定量分析，获得其水解底物谱及水解位点；进而通过基因敲低、定点突变水解底物等方法对其功能进行研究；并阐明ADAM12调控肺癌细胞增殖和转移的确切分子机制。这一研究将为小细胞肺癌的治疗提供新的理论依据，并为药物研发提供新的靶点。

肺癌是全球发病率和死亡率最高的癌症，而小细胞肺癌由于恶性程度高、倍增时间短、转移早而广极易发生耐药和复发。因此，阐明小细胞肺癌增殖、转移和侵袭的分子机制，极有可能找到新的潜在的治疗靶点。已有研究发现去整合素金属蛋白水解酶ADAM12在小细胞肺癌病人组织样本中高表达，其高表达与患者的生存率呈负相关，且ADAM12促进小细胞肺癌的增殖和转移，但其作用机制不清。本课题通过非标记定量蛋白质组学方法、细胞培养稳定同位素标记（SILAC）技术、点击糖富集分泌组蛋白的方法（SPECS）分别筛选与ADAM12相互作用蛋白、调控胞内下游蛋白和分泌体蛋白。首先，我们通过谱图计数和MaxLFQ非标记定量分析获得28个与跨膜型ADAM12L和16个与分泌型ADAM12S相互作用的蛋白。生化实验发现myoferlin增加ADAM12L前体蛋白稳定性，从而上调ADAM12L成熟型蛋白水平，增加其水解酶活性，降低其底物蛋白E-Cadherin水平。质谱和生化实验发现GRP94、P4HB、PDIA6等参与蛋白质折叠的蛋白与分泌型ADAM12S特异性结合，但不与跨膜型ADAM12L作用；敲低GRP94下调胞内ADAM12S蛋白，并降低其分泌；过表达GRP94上调ADAM12S及增加其分泌。这些结果预示GRP94可能通过调控ADAM12S的分泌促进肿瘤细胞的转移和侵袭。然后，我们应用SILAC定量蛋白质组学和生化实验发现在H1688细胞中ADAM12调控多种与代谢相关基因的表达，如HK1、MDH1、MVD和G6PD。当敲低糖酵解代谢关键限速酶HK1后，ADAM12S促进小细胞肺癌增殖、迁移、侵袭、克隆形成的作用几乎被完全抑制。其次，我们通过细胞内标记糖蛋白、点击化学反应、定量蛋白质组学筛选等方法获得了65个受ADAM12S调控的分泌蛋白，其中CD146、SPP1等蛋白与肿瘤细胞的增殖、迁移和侵袭密切相关。另外，我们优化了蛋白质羧基端生物素标记方法，结合质谱技术筛选获得了药物诱导肿瘤细胞凋亡过程中凋亡酶水解底物及其位点，发现凋亡酶可以激活其它水解酶或促进底物蛋白其它位点的水解，这些水解蛋白形成一个相互作用网络，决定细胞凋亡过程中的形态变化。本研究在细胞水平上基本阐明了ADAM12促进小细胞肺癌增殖、迁移和侵袭的分子机制。研究结果共发表署项目号论文11篇，其中SCI论文9篇，二区论文8篇，培养研究生7名。