钙离子通道α2δ1亚基阳性(CACNA2D1+)肺癌细胞的放疗敏感性研究

Ionizing radiation is one of the major treatment methods for patients with unresectable lung cancer. However a certain proportion of tumors do not response effectively to radiation, or relapse after radiotherapy, which may be attributed to the existence of radio-resistant cancer stem cells. Although several markers for radio-resistant cancer stem cells have been characterized, specific drugs to target these cells are still to be identified. Our group have identified a specific marker, calcium channel α2δ1 subunit (CACNA2D1) isoform 5, for tumor-initiating cells in hepatocellular carcinoma. Its monoclonal antibody 1B50-1 had a therapeutic effect when combined with chemotherapy regimen on hepatocellular carcinoma engraftment by eliminating cancer stem cells. Recently, our experiments have confirmed its marking and therapeutic applications on lung cancer cells. Moreover, we found that CACNA2D1+ lung cancer cells are relatively radioresistant. Therefore, this study aims to further investigate the radio-sensitivity of CACNA2D1+ cells in non-small cell lung cancer cell lines and patient-derived tissues, and evaluate the therapeutic effect of 1B50-1 combined with radiotherapy on in vitro cell culture and tumor engraftment on mice. In addition, we will try to explain the relationship between the CACNA2D1 and cell radio-resistance. This study will get some insights into the mechanism of cell radio-resistance from a viewpoint of calcium ion channel, and the investigation the potential of 1B50-1 as a specific drug to target radio-resistance lung cancer stem cells may provide clues to enhance the therapeutic effect for radiotherapy of lung cancer.

放疗是肺癌治疗的主要方法之一。对放疗耐受的肿瘤干细胞是放疗后肿瘤复发的重要根源，但其特异性药物靶点研究相对较少。本课题组研究发现钙离子通道α2δ1亚基(CACNA2D1)5亚型是肝癌干细胞标志物，其单克隆抗体1B50-1与化疗药物联用可有效抑制小鼠肿瘤的生长，研究结果发表在Cancer Cell上；最近我们发现CACNA2D1+肺癌细胞致瘤性高，并对放疗相对耐受，提示CACNA2D1可作为肺癌干细胞的标志物和潜在的治疗靶点。因此本项目拟在非小细胞肺癌细胞系中研究CACNA2D1+细胞的放疗敏感性，检测1B50-1抗体联合放疗对肺癌细胞的杀伤作用，评价CACNA2D1的表达与肺癌病人放疗评效及预后的相关性，并对CACNA2D1与放疗耐受的机制进行初步探究。本研究将加深钙离子通道与细胞放疗敏感性相关机制的认识，并从特异性杀伤放疗耐受肿瘤干细胞的角度为提高肺癌放疗疗效提供新思路。

研究背景：放疗是治疗非小细胞肺癌的重要手段之一。对放疗耐受的肿瘤干细胞的存在与放疗后肿瘤的复发或较差的疗效密切相关。前期研究发现钙离子通道α2δ1亚基（CACNA2D1）5亚型是肿瘤干细胞标志物，其单克隆抗体1B50-1与化疗药物联用可有效抑制肿瘤在小鼠体内的生长。本工作在此基础上研究肿瘤细胞中CACNA2D1的表达与放疗敏感性的关系，以及1B50-1抗体联合放疗对肿瘤细胞的抑制作用。 ..主要研究内容：在非小细胞肺癌细胞系A549、H1975、H1299和PC9上开展实验。利用慢病毒感染建立CACNA2D1敲低和过表达细胞系。通过克隆形成实验检测细胞的放疗敏感性；通过球体形成实验检测细胞的自我更新能力；QPCR、western blog和免疫荧光检测与DNA损伤修复相关基因的表达情况。在体外培养细胞系和小鼠模型上检测CACNA2D1抗体1B50-1单用或与放疗联用对肿瘤的作用。..主要结果：分选CACNA2D1阳性及阴性细胞，阳性细胞的球体形成率高于阴性细胞，并且对放疗更耐受。在A549细胞中敲低CACNA2D1，细胞的放疗敏感性提高；在PC9和H1975中过表达CACNA2D1，细胞对放疗耐受，提示CACNA2D1导致了细胞对放疗的抵抗。CACNA2D1过表达或阳性的细胞具有更高效的DNA修复能力。CACNA2D1抗体1B50-1提高CACNA2D1阳性细胞的放疗敏感性；在A549移植瘤中，1B50-1联合放疗可有效抑制肿瘤的生长。..研究意义：钙离子通道α2δ1亚基提高非小细胞肺癌中肿瘤干细胞的放疗耐受。CACNA2D1的单克隆抗体对CACNA2D1阳性细胞起到放疗增敏作用，提示其可以与放疗联用提高放疗治疗非小细胞肺癌的疗效。