人端粒酶逆转录酶(hTERT)线粒体转位在肝癌化疗耐药中的作用机制研究

The literature and our previous studies have shown that telomerase activation is involved in chemotherapy resistance in cancers. However, the detail mechanism is not clarified. Other studies and our preliminary results found that the human telomerase reverse transcriptase (hTERT) existed mitochondrial localization. After mutating mitochondrial localization sequence of hTERT, hepatocellular carcinoma cells to cisplatin resistance was reduced. These results suggest that mitochondrial hTERT may be involved in chemotherapy resistance. Further studies demonstrated that plasmid of hTERT without the reverse transcriptase activity (DNhTERT) increased expression of stemness related factor (such as Oct4 et al), decreased ROS and ATP in transfected hepatocellular carcinoma cells. Meanwhile we also found that hTERT and mitochondrial respiratory chain complex I were colocalized by immunofluorescence. Based on the above results, we hypothesize that hTERT protein translocates to mitochondrial and interact with respiratory chain complex I, then inhibite the function of mitochondrial respiratory chain, and further promote the stemness of hepatocellular carcinoma cells by decreasing the prodution of ROS and ATP, lead to cancer chemotherapy resistance at last. Based on the above hypothesis, we would intend to use various molecular biology technology to clarified the molecular basis of interaction between mitochondrial hTERT and respiratory chain complex I in hepatocellular carcinoma cells. We would further confirm the effects of hTERT interating with complex I on stemness and the drug resistance of hepatocellular carcinoma by different levels from cells, animals to clinic. We hope to enrich the molecular mechanism of drug resistance based on hTERT in hepatocellular carcinoma. These results will provide a theoretical basis for the design of anticancer drugs targeting hTERT.

文献及课题组既往研究表明端粒酶活化参与了肿瘤化疗耐药过程,但机制不清.文献及课题组前期发现人端粒酶逆转录酶(hTERT)存在线粒体定位,突变线粒体定位序列后,肝癌细胞对顺铂耐药明显减弱,提示线粒体hTERT参与肝癌化疗耐药.进一步研究发现无逆转录活性的hTERT(DNhTERT)转染的肝癌细胞干性相关因子Oct4等的表达增高, ROS和ATP生成减少,免疫荧光证实hTERT与线粒体呼吸链复合物Ⅰ存在共定位.据此提出“hTERT可能通过线粒体转位,与呼吸链复合物Ⅰ相互作用,抑制线粒体呼吸链功能,ROS、ATP生成减少,促进肝癌细胞干性化,导致肝癌化疗耐药”的假说.课题组拟采用分子生物学技术,解析线粒体hTERT与呼吸链复合物I互作的分子基础,并从细胞、动物及临床等不同层面验证hTERT与复合物Ⅰ结合影响肝癌耐药,以丰富hTERT肝癌耐药的分子机制,为靶向hTERT抗癌药物设计提供理论依据.

化疗抵抗是临床肿瘤治疗中亟待解决的难题。包括瓦伯格效应在内的异常糖代谢是肿瘤代谢的标志之一，可显著促进肿瘤耐药。靶向异常线粒体代谢为克服肿瘤化疗耐药提供了新的思途径。人端粒酶逆转录酶hTERT与肿瘤发生发展密切相关，新近文献报道hTERT亦可定位于线粒体，但其与肿瘤代谢、耐药的关系及相应机制尚不明确。本项目证实了hTERT线粒体定位可显著促进肿瘤细胞化疗耐药，且与肿瘤的临床疗效呈负相关。线粒体hTERT可不依赖于其经典的逆转录酶功能促进瓦伯格效应进而加强化疗耐药。线粒体定位hTERT通过干扰呼吸链复合物Complex I的功能从而抑制肿瘤细胞氧化磷酸化，降低氧耗率；同时促进肿瘤细胞细胞外酸化、乳酸生成及葡萄糖消耗。通过质谱联合Co-IP筛选，鉴定了可与hTERT互作的线粒体蛋白ND1，进一步通过不同位点突变发现可能介导hTERT-ND1互作的功能区域。基于上述结果设计的穿膜肽TAT-Pep可显著抑制hTERT与ND1的蛋白互作，增强Complex I的活性，促进肿瘤细胞氧化磷酸化（增强OCR及最大呼吸）、抑制肿瘤细胞有氧糖酵解（抑制ECAR、糖酵解能力、降低乳酸生成及葡萄糖消耗）。在PDX动物模型中，抑制hTERT与ND1互作可增敏CDDP的抗肿瘤效果。综上所述，本项目揭示了线粒体hTERT通过结合ND1抑制Comple I功能，增强肿瘤糖酵解进而促进肿瘤化疗耐药。靶向线粒体hTERT重塑肿瘤代谢为增强化疗效果提供了新的思路。