FAP通过整联蛋白信号途径促进口腔鳞癌生长和转移的分子机制

The fibroblast activation protein alpha(FAP) is one of the membrane-bound serine protease, which up-regulated in cancer cells and stromal calls. It is a target of therapy for its important role of promoting tumor growth,invasion and metastasis. Our previous studies demonstrated that FAP is high expression in the tissue of oral squamous cell carcinoma (OSCC). Down-regulation of FAP suppresses cell proliferation and metastasis through PI3K/AKT,Ras-ERK and β-catenin signaling in oral squamous cell carcinoma. However, the substrates and the extracellular or intracellular mechanisms of FAP are still unclear. The recombinant wide FAP (wFAP) or no activity mutant FAP (mFAP) from cultured mammalian cells are used to separate the substrates by affinity chromatography co-precipitation and identified with liquid chromatography tandem mass spectrometry (LC-MS). The OSCC cells and fibroblast cells are treated with recombinant wFAP or mFAP to analyze the extracellular signaling by integrin and PI3K/AKT,Ras-ERK and β-catenin signaling pathway. Cells transfected with tFAP (transmembrane domain deletion FAP) are used to determining whether FAP exert its role in the cytoplasm. The transplanted tumor with stable wFAP, mFAP and tFAP cDNA transfected Cells are used to investigate the role of FAP in promoting tumor growth,invasion and metastasis. The important innovations of this project are that (1) the recombinant wFAP or mFAP are used to separate and identify the substrates by affinity chromatography co-precipitation and LC-MS; (2) the cells transfected with tFAP are used to determining whether FAP exert its role in the cytoplasm. This project will elucidate the molecular mechanisms of FAP in extra-cell or intra-cell, and find the new molecular target for OSCC therapy.

成纤维激活蛋白（FAP）是一种跨膜丝氨酸蛋白酶，在恶性肿瘤细胞及基质中高表达。我们前期研究显示，FAP可通过PI3K-Akt、Ras-ERK和β-catenin通路促进肿瘤生长和转移。但FAP在细胞内外直接作用的分子及机制尚不清楚。本项目拟用真核表达的野生FAP(wFAP)和无活性突变FAP（mFAP）通过亲和纯化方法分离FAP结合蛋白，用液相色谱-质谱联用仪鉴定FAP底物，酶化学实验进一步验证。重组FAP处理体外培养OSCC细胞探讨FAP胞外通过整联蛋白进行信号转导的分子机制。缺失跨膜区的FAP(tFAP)转染成纤维细胞评判FAP能否直接在细胞内发挥作用。wFAP、mFAP和tFAP稳定转染细胞小鼠移植瘤实验分析FAP能否在胞内、外促进肿瘤生长、侵袭和转移。采用共沉淀及亲和纯化法结合液-质联用仪，批量分离并鉴定FAP底物，tFAP基因转染评判FAP能否在细胞内发挥作用是本项目的主要创新点

成纤维激活蛋白（FAP）是一种跨膜丝氨酸蛋白酶，在恶性肿瘤细胞及基质中高表达。我们前期研究显示，FAP可通过PI3K-Akt、Ras-ERK和β-catenin通路促进口腔鳞癌（OSCC）的生长和转移。但FAP在细胞内外直接作用的分子及机制尚不清楚。本研究采用免疫共沉淀及质谱分析方法筛选出FAP的互作蛋白DPP9，并证实低表达DPP9能促进OSCC的生长､迁移和侵袭；过表达FAP能下调DPP9表达，促进OSCC的生长､迁移和侵袭，回复实验证实上调DPP9表达能逆转FAP促进OSCC的生长､迁移和侵袭。证实过表达FAP通过下调DPP9，促进OSCC的生长､迁移和侵袭。本研究还初步证实，过表达FAP，β-catenin表达上调，促进OSCC生长､迁移和侵袭；使用β-catenin抑制剂XAV-939后，可以反转过表达FAP对OSCC细胞生物学特性的影响。证实FAP可通过β-catenin信号轴促进OSCC的生长､迁移和侵袭。本研究将为OSCC的靶向治疗提供新的靶点，为OSCC的发病机制提供新的思路。