肿瘤微环境中PKCζ / Calpain信号通路促进口腔黏膜癌变与转移的机理研究

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.

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。

在肿瘤微环境中蛋白激酶-蛋白酶交互作用促进/抑制肿瘤的增殖并影响免疫系统的活性。而蛋白激酶PKC与钙依赖性蛋白酶 Calpain 间的交互作用可能作为最关键的角色参与其中。我们在蛋白组学的结果分析中发现蛋白酶体激活因子PA28可能是PKC-Calpain 通路中的关键调控因子，本研究通过成功构建稳定沉默CAPN1基因的口腔鳞癌细胞株，及沉默/过表达PA28的口腔鳞癌细胞株，证实了Calpain1及PA28可作为口腔鳞癌预后的关键判定分子；发现Calpain1及PA28在口腔癌变过程中活性增强预示了肿瘤的进展；Calpain1还可以通过影响巨噬细胞的转化以及抑制T细胞的功能影响免疫系统对肿瘤细胞的监视及杀伤。后期我们将进一步研究PA28/PKC/Calpain在肿瘤微环境中是如何相互作用促进肿瘤生长及抑制免疫系统，为癌变阻断提供新的靶向。本项目明确了Calpain及其上游激活分子PA28在调控肿瘤上皮细胞癌变中的作用及机理，初步探求了Calpain调控巨噬细胞趋化与T淋巴细胞增殖的功能，获得预期理论成功；初步建立利用肿瘤微环境蛋白酶与激酶预测模型预后判断的方案与策略，并申请蛋白酶行口腔黏膜癌变诊断与阻断的发明专利一项（专利号201710002155.2），获得相关的应用成果；发表SCI收录论文4篇（含一篇proof），总IF值10分，其中一篇发表于EBioMedicine（Lancent新创刊杂志，暂无IF值，专注于临床转化）。同时培养毕业硕士生2名，博士生2名。