HSP90通过调控PKM2磷酸化促进肝癌细胞糖酵解及增殖侵袭转移的作用机制研究

Hepatocellular carcinoma (HCC) is one of the most common malignant tumor in China. Since our previous study indicates that HSP90 is an independent prognostic marker in HCC and HSP90 promotes cell proliferation and induces apoptosis in HCC cells, but the mechanisms is still unclear. The abnormal activation and accumulation of PKM2 protein in the tumor is a key factor for Tumor metabolism , but the mechanisms of PKM2 in non-metabolic pathway is still unknown. Our previous research shown that HSP90 was positively correlated with HSP90 protein in HCC tissues and HSP90 regulated the expression of PKM2 protein in HCC cells. We found that there was interaction between HSP90 and PKM2 by using co-immunoprecipitation and phosphorylation sites recognized in PKM2 protein when overexpression of HSP90 by using mass spectrometric analysis. In the present project, we try to determine whether HSP90 functions as a oncogene by phosphorylation PKM2 protein. Furthermore, we attempt to verify the mechanisms involved in phosphorylation and regulation of PKM2 protein by HSP90 through gene mutagenesis, and find out if there are any other proteins, such as GSK3, taking part in this process. The aim of this project is to investigate the molecular mechanisms of HSP90 regulates PKM2 protein in HCC , in order to provide the scientific basis to exploit novel targeting agents and predictive biomarkers for HCC clinically.

肝细胞癌（HCC）是我国常见的恶性肿瘤之一。我们前期证实HSP90是一个独立的HCC患者预后标记物，HSP90具有促进HCC细胞增殖和凋亡的作用，但是具体机制不清；PKM2在肿瘤中调控肿瘤代谢，但是其在肿瘤非代谢途径中的具体分子机制仍不明确。课题组前期研究发现HCC组织中HSP90与PKM2蛋白表达正相关，HCC细胞中HSP90正向调控PKM2蛋白，免疫共沉淀发现HSP90与PKM2蛋白存在相互作用，进一步质谱分析发现过表达HSP90可以使PKM2发生磷酸化，本研究是个既往研究的深入，拟通过体内外实验证明HSP90通过磷酸化PKM2发挥其致癌作用，进而通过基因突变等技术探讨HSP90磷酸化PKM2的具体分子机制，并明确在这一调控过程中是否有其他蛋白如GSK3β的参与。本项目旨在阐明HSP90调控PKM2在肝细胞癌中的具体作用机制，为临床上开发新的靶向治疗药物和预后标志物提供依据。

热休克蛋白90 (Heat shock protein 90, HSP90)作为一种常见的致癌蛋白，具有调节蛋白构象、稳定性和降解的作用。丙酮酸激酶M2 (PKM2)是促进肿瘤细胞代谢、生长转移的关键调节因子，目前已被证实包括肝癌在内的多种人类癌症中过表达。然而，HSP90和PKM2过表达在肝癌中促癌功能的分子机制尚不清楚。本课题采用免疫印迹法和免疫染色法检测肝癌标本和细胞中HSP90和PKM2的表达；通过串联亲和纯化、共免疫沉淀和GST-pull down实验证实了HSP90与PKM2的相互作用。本研究证实HSP90可以与PKM2相互作用，从而增加了肝癌细胞中PKM2的蛋白含量。免疫组化(IHC)染色显示肝癌组织中HSP90表达与PKM2表达呈正相关。HSP90可以增加PKM2在Thr-328位点的磷酸化。蛋白激酶糖原合酶kinase-3β(GSK-3β)可以与HSP90、PKM2形成蛋白复合物，并直接介导HSP90调控PKM2的Thr-328磷酸化。Thr-328磷酸化对于维持PKM2的稳定性及其调节糖酵解、线粒体呼吸、增殖和凋亡的生物学功能至关重要。同时，本研究发现HSP90通过调控PKM2促进肝癌细胞的糖酵解和增殖，抑制肝癌细胞的凋亡。体内实验表明，HSP90对小鼠肝癌细胞生长的促进作用需要PKM2的参与。此外，本研究通过临床病理资料分析证实HSP90和PKM2阳性表达与高-甲胎蛋白(AFP)水平、大肿瘤、门静脉肿瘤血栓(PVTT)、晚期肿瘤结节转移(TNM)等不良的临床病理特征相关，HSP90表达阳性及PKM2表达阳性与肝癌患者预后不良显著相关。此外，本项目发现中药林蛙油生物活性肽（ORPH）能有效降低HCC细胞中PKM2的表达，ORPH通过上调miR-491-5p在转录后机制上降低PKM2的表达，其通过调节miR-491-5p/PKM2轴对HCC细胞产生抑制作用。综述所述，本研究得出结果HSP90可以通过磷酸化PKM2从而促进肝癌细胞糖酵解和增殖，减少细胞凋亡，ORPH通过靶向miR-491-5p/PKM2轴抑制肝癌细胞的生长、转移和糖酵解，ORPH可能是一种潜在有效的肝癌抗肿瘤药物。