细胞表面GRP78与Src相互作用对肝细胞癌细胞侵袭和转移的调控作用研究

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. Its 5-year over survival is less than 5% and more than half of the patients appear recurrence or metastasis 5 years after surgical resection. The invasion and metastasis contributed largely to the high mortality and recurrence of HCC. Therefore, exploring the mechanisms regulating the invasion and metastasis is critical for searching new strategies to improve the outcome of HCC. .Cell surface GRP78 is a multifunctional protein, functions as a cell surface receptor and play critical roles in the regulation of cell proliferation, apoptosis, angiogenesis, tumor invasion and metastasis. Our previous studies showed that GRP78 is expressed at the cell surface of hepatocellular carcinoma cells and facilitates the invasion of tumor cells. Furthermore, we found that GRP78 directly interacted with Src at the cell surface. Inhibition of Src with PP2, a specific Src tyrosine kinase inhibitor, caused a marked decrease in the invasion and metastasis of tumor cells, indicating that Src may play critical role in GRP78 induced invasion and metastasis. In this proposal, the molecular mechanism by which GRP78 interacted with Src were analyzed using far-western, GST pulldown assay, co-transfection and anti-Flag or EGFP immunoprecipitation. Furthermore, the effect of this interaction between GRP78 and Src on tumor invasion and metastasis were investigated. For this purpose, we blocked the interaction by transfection of GRP78 mutants in which the critical amino acids were site directed mutated, the cell motility including the adhesion, spreading, cell polarity, invadopodia formation, cytoskeletal arrangement and extracellular matrix degradation were analyzed using transwell, wound healing assay, cell adhesion assay, cell spreading assay, gelatin zymography assay and laser confocal microscope observation in hepatocellular carcinoma. Finally, we analyzed whether the ATPase and PBD domain of GRP78 differentially regulated the FAK-Src/Cortactin and JNK/c-Jun/MMP-2 signaling pathways using the methods mentioned above. .Taken together, we tried to elucidate the role of Src in GRP78 induced invasion and metastasis of HCC and demonstrated that cell surface GRP78 may be a potential molecular target for inhibiting the invasion and metastasis of HCC.

细胞表面GRP78是一种具有多种功能的受体样蛋白质，在细胞表面可以与多种蛋白质相互作用，对肿瘤细胞的生存、增殖、侵袭和转移具有重要的调节作用。我们前期研究显示在肝细胞癌细胞表面存在GRP78表达，对肝细胞癌的侵袭和转移具有促进作用。初步研究发现在肝细胞癌中细胞表面GRP78可以与Src直接结合，提示Src在细胞表面GRP78促进肝细胞癌侵袭和转移过程可能发挥重要作用。本课题将应用蛋白质原核表达和纯化技术、GST pulldown、共转染、免疫共沉淀和小动物活体成像技术在细胞和实验动物模型中探讨细胞表面GRP78与Src互作的分子机制，并阻断GRP78与Src的相互作用探讨这种相互作用在肿瘤侵袭和转移过程中的调控作用及机制，深入阐明GRP78的ATPase和PBD结构域在GRP78促进肝细胞癌侵袭和转移中的作用及相关信号通路。

GRP78在肿瘤细胞增殖，远端转移等进程中发挥了重要作用，我们前期研究显示GRP78能够促进肝癌细胞侵袭转移，进一步研究发现肝癌细胞表面存在GRP78的表达，但其如何影响肝癌细胞侵袭转移及发挥作用的具体机制尚不清楚。本项目中，我们发现细胞表面GRP78是一种多功能的受体样蛋白质，α2M能够促进GRP78向细胞膜转位。首先我们应用免疫共沉淀等技术，发现细胞表面GRP78可以与Src直接结合，然后为探索Src在细胞表面GRP78促进肝细胞癌侵袭和转移的分子机制，我们构建了Src野生型、删除SH2和删除SH3的突变体载体，发现细胞表面GRP78对肝癌细胞侵袭转移的促进作用是通过与Src的SH3结构域结合实现的，GST-pulldown实验进一步证实了这种相互作用。进而，我们应用PP2阻断GRP78对Src的相互作用，发现PP2能够逆转α2M的对肝癌细胞侵袭转移的促进作用。为了深入阐明细胞表面GRP78的ATPase和PBD结构域在GRP78促进肝细胞癌侵袭和转移中的作用及相关信号通路，我们又构建了GRP78 delATPase和delPBD突变体，转染肝癌细胞，发现GRP78的ATPase和PBD结构域在GRP78促进肝细胞癌侵袭和转移中的作用及信号通路存在差异调节，ATPase结构域主要调节细胞运动，PBD结构域主要调节细胞外基质降解。我们的研究还发现肝癌细胞能够分泌GRP78，影响癌细胞对Sorafenib的耐药。在肝癌病人血液中GRP78明显高于正常人，而肝癌细胞系也能够分泌GRP78。Sorafenib能够诱导肝癌细胞分泌GRP78，这种分泌型GRP78能够促进肝癌细胞的增殖侵袭，降低Sorafenib诱导的调亡，说明分泌型GRP78对肝癌细胞Sorafenib耐药有重要的作用。基于GRP78在肝癌细胞Sorafenib耐药进程中的重要作用，我们又构建了Sorafenib耐药细胞系，应用BM-MSCs细胞作为工具，转染siGRP78后，检测含siGRP78的exosomes对于肝癌细胞的Sorafenib耐药的影响。发现含siGRP78的exosomes能够被肝癌耐药细胞吸收，并有效抑制肝癌细胞的Sorafenib耐药，抑制肝癌耐药细胞在体内及体外的增殖及侵袭转移。本课题研究结果为肝细胞癌的抗侵袭、转移提供新的线索为基于GRP78为靶点的临床诊断治疗提供理论依据。