ALK融合基因在肾透明细胞癌中的表达及功能研究

发布时间：2018-10-16 20:43

【摘要】：肾透明细胞癌是肾癌最常见的病理类型，约占肾癌的80%～85%。分子靶向药物如贝伐单抗，舒尼替尼，索拉非尼等已经成为晚期转移性肾癌治疗的一二线药物，虽然在治疗上带来令人鼓舞的成绩，但是达到完全缓解无病生存的患者还是很少。ALK重排和突变在多种肿瘤的发生发展中起着至关重要的作用，针对ALK的靶向治疗成为肿瘤治疗研究的热点。临床上已经批准ALK抑制剂用于NSCLC、ACLC、IMT的治疗。目前在肾髓质癌、肾未分类癌和乳头状肾细胞癌也发现ALK基因重排形成的融合基因，使得ALK有望成为肾癌新的治疗靶点。由于肾透明细胞癌是最常见肾癌，因此，，本研究目的是探讨肾透明细胞癌患者中是否存在ALK基因重排的现象，探索ALK在肾透明细胞癌发生、发展中的作用。首先应用新型的ALK抗体筛查87例肾透明细胞癌中ALK表达情况，4例为ALK蛋白表达阳性，阳性率为4.59%。ALK蛋白主要表达于细胞质，部分胞核也有表达。通过5'-RACE技术和DNA测序结果表明，其中2例标本有ALK重排，形成EML4-ALK融合基因，都是1型变异体基因（variant1）。FISH结果同样证实这两例肾透明细胞癌中存在EML4-ALK融合基因。利用分子克隆方法构建EML4-ALKvariant1融合基因慢病毒表达系统，感染人肾近曲小管上皮细胞HK-2，探讨EML4-ALK在肾癌中的作用。功能学研究表明，EML4-ALK variant1融合基因转染到HK-2中，能增强HK-2细胞的生长和增殖的能力。ALK特异性小分子抑制剂TAE684能选择性抑制ALK激酶活性，从而抑制稳定表达EML4-ALKvariant1基因的HK-2细胞的增殖。体内实验结果显示，稳定转染表达EML4-ALK variant1基因能使非致瘤性的HK-2细胞在体内成瘤，证明EML4-ALK variant1融合基因在体内具有致瘤活性。更重要是，ALK抑制剂Crizotinib能够显著抑制ALK驱动的HK-2肿瘤的生长。本研究首次报道在人肾透明细胞癌中存在EML4-ALK融合基因突变，体内外功能学实验证明，Crizotinib有望成为肾透明细胞癌治疗的新靶向药物。
[Abstract]:Renal clear cell carcinoma (RCC) is the most common pathological type of RCC, accounting for 80% of RCC. Molecular targeted drugs such as bevacizumab, sunitinib, sorafenil have become first-line and second-line drugs in the treatment of advanced metastatic renal cell carcinoma, although there have been encouraging results in the treatment. ALK rearrangement and mutation play an important role in the occurrence and development of many kinds of tumors. Targeted therapy for ALK has become a hot topic in tumor therapy. ALK inhibitors have been approved clinically for the treatment of NSCLC,ACLC,IMT. At present, fusion gene of ALK gene rearrangement has been found in renal medullary carcinoma, renal unclassified carcinoma and papillary renal cell carcinoma, which makes ALK a new therapeutic target for renal cell carcinoma. Since renal clear cell carcinoma is the most common renal cell carcinoma, the aim of this study was to investigate whether ALK gene rearrangement exists in patients with renal clear cell carcinoma, and to explore the role of ALK in the occurrence and development of renal clear cell carcinoma. The new ALK antibody was used to screen the expression of ALK in 87 cases of renal clear cell carcinoma, 4 cases were positive for ALK protein expression, the positive rate was mainly expressed in the cytoplasm, and some of the nuclei were also expressed. The results of 5'-RACE and DNA sequencing showed that two of them had ALK rearrangement and formed EML4-ALK fusion gene. Both of them were variant 1 genes (variant1). FISH results also confirmed the existence of EML4-ALK fusion gene in these two cases of renal clear cell carcinoma). The expression system of EML4-ALKvariant1 fusion gene lentivirus was constructed by molecular cloning to investigate the role of EML4-ALK in renal cell carcinoma (RCC) by infecting human proximal tubule epithelial cells (HK-2,). Functional studies showed that transfection of EML4-ALK variant1 fusion gene into HK-2 enhanced the ability of HK-2 cells to grow and proliferate. TAE684, a small molecular inhibitor of ALK, selectively inhibited the activity of ALK kinase and thus inhibited the proliferation of HK-2 cells stably expressing EML4-ALKvariant1 gene. The results of in vivo experiments showed that stable transfection of EML4-ALK variant1 gene could induce tumorigenesis of non-tumorigenic HK-2 cells in vivo, which proved that EML4-ALK variant1 fusion gene had tumorigenic activity in vivo. More importantly, Crizotinib, a ALK inhibitor, significantly inhibited the growth of HK-2 tumors driven by ALK. In this study, we first reported the existence of EML4-ALK fusion gene mutation in human renal clear cell carcinoma. Functional experiments in vitro and in vivo demonstrated that Crizotinib may be a new target drug for the treatment of renal clear cell carcinoma.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.11

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