口腔黏膜癌变中不同类型蛋白酶激酶相互协作，通过磷酸化和蛋白水解影响癌变进程，而PKCζ与钙依赖性蛋白酶Calpain交互作用可能作为最关键的角色参与其中，前期已初步预测出PKCζ磷酸化激活Calpain的位点，而其调控癌变的机理值得我们进一步探求。 提出假设：肿瘤微环境中，PKCζ磷酸化激活Calpain1,2从而影响口腔鳞癌细胞增殖、凋亡、迁移、侵袭与血管生成，并影响肿瘤相关巨噬细胞TAM趋化，活化后的TAM分泌细胞因子正反馈于PKCζ，形成环形通路调控口腔黏膜癌变及转移。研究将利用质谱鉴定和点突变实验确立磷酸化位点，利用大样本临床随访队列明确PKCζ/Calpain通路与黏膜癌变预后关系，并通过阻断PKCζ/ Calpain通路信号转导，观察此通路对肿瘤细胞与TAM 细胞促癌变表型的影响，并明确其靶向底物及作用机制，为癌变阻断提供新的靶向。

Our previous proteomic studies have highlighted elaborate interplay between both post-translational regulatory systems "phosphorylation and proteolysis" in oral cancer initiation and development. The functional consequences of this regulatory crosstalk are especially relevant in the different stages of cancer progression, while PKCζ and calpain (a calcium dependent cysteine proteinases) interplay could play a key role in it. What are the clinical implications and regulatory mechanism derived from the fertile dialogue between PKCζ and calpain in oral cancer? Based on the prelimilary data we hypothesis: Phosphorylation of calpain1,2 by PKCζ could induce inhibition of apoptosis, increased proliferation, migration, adhesion, invasion and angiognenesis,moreover,could induce the chemotaxis of tumor related macrophage.Further, the activated macrophage could secret some cytokine which promote activation of PKCζ/calpain pathway thereby generating a positive feedback loop to promote the carcinogenesis and invasion of oral cancer. In the present project, since our prelimilary study has forecasted the phosphorylation site of calpain, further we will validate the sites with mass spectrum identification and site mutation assay. The association of calpain with prognosis will be determined in a large cohort with regular follow-up. The activation of calpain1,2 by PKCζ Phosphorylation on tumor cell and tumor related macrophage could be determined by using PKCζand calpain specific siRNA, protein inhibitor, peudo-substrate in OSCC cells, node mice and metastasis models. Finally, the potential of combined PKCζand calpain specific inhibitor and siRNA will be determined in targer therapy.