肾癌新基因PBRM1的表达缺失在肾癌进展及抗血管新生药物耐药发生中的作用

Anti-angiogenic drugs are the preferred systemic treatment for advanced renal cell carcinoma. Just like the cytotoxic drugs, anti-angiogenic agents will eventually step to treatment failure due to drug resistance, in which the specific mechanism is largely unknown. PBRM1 kidney cancer gene is a newly discovered gene from whole genome sequence analysis in kidney cancer. It is recognized as a tumor suppressor gene, and its inactivating mutations can increase the hypoxia tolerance and promote cell proliferation of renal cell carcinoma, which may be novel mechanism for the resistance to anti-angiogenic drugs. In this study, we will firstly detect the expression pattern of PBRM1 protein and PBRM1 gene mutations in clinical tumor specimens and peripheral blood samples, to verify the specific value of PBRM1 in the progression and treatment of renal cell carcinoma. Then we will establish a renal tumor cell model resistant to sunitinib, and control PBRM1 gene expression, using PBRM1 mimics and inhibitors respectively, to investigate their impact on the proliferation and drug resistance of renal cell carcinoma. Furthermore, interactions between PBRM1 and VHL-HIF-VEGFR pathway will also be surveyed on both gene and protein levels. At last, we will further survey the resistance reversal effect of PBRM1 agonist in nude mice model. This study helps to clarify novel mechanism in anti-angiogenesis resistance of renal cell carcinoma, and provide ideas for reversing the drug resistance.

抗血管新生药物是晚期肾癌的首选治疗，与细胞毒药物一样，也会因发生耐药而失效，其中的具体机制不明。PBRM1基因是肾癌全基因组序列分析中最新发现的基因，作为抑癌基因，PBRM1的突变失活可提高肾癌细胞对缺氧的耐受，促进细胞的增殖，这可能是抗血管生成药物耐药的新机制。本研究先在临床肾癌标本和外周血中检测PBRM1蛋白表达和基因突变情况，证明PBRM1基因表达在肾癌临床进展中的意义。随后建立肾癌耐药的细胞模型，从控制PBRM1的表达入手，分别采用PBRM1模拟物和抑制剂研究其对肾癌增殖和耐药的影响，同时研究PBRM1激动剂对耐药的逆转作用；分别从基因和蛋白水平研究PBRM1和VHL-HIF-VEGFR通路的交互作用。最后建立肾癌耐药的动物模型，进一步验证PBRM1在逆转抗血管新生药物耐药中的价值。通过本研究有助于阐明肾癌对抗血管新生药物产生耐药的新机制，并为寻找逆转耐药的方法提供新思路。

抗血管新生药物是目前晚期肾癌的首选治疗，与细胞毒药物一样，也会因发生耐药而失效，其中的具体机制不明。PBRM1基因在ACHN细胞株中的表达高于7860细胞株，敲除PBRM1基因后，ACHN细胞的增殖、迁移、克隆形成增加，细胞周期G1期减少、M期增多，凋亡显著减少，说明PBRM1可以作为肾癌的抑癌基因。随后通过差异基因高内涵筛选，发现PBRM1基因的下游基因ITGB3BP和RPL31的增殖抑制阳性。同时我们建立肾癌细胞株的舒尼替尼耐药模型，采用PBRM1模拟物过表达PBRM1基因，研究其对肾癌增殖和耐药的影响，同时研究PBRM1激动剂对耐药的逆转作用。ACHN舒尼替尼耐药细胞过表达PBRM1基因后，细胞的增殖、迁移、克隆形成下降，细胞周期G0期增加、G1和M期减少，凋亡显著增加。随后通过裸鼠成瘤实验，接种高表达PBRM1基因ACHN-R细胞的小鼠与空载体对照相比，瘤体生长减慢，提示PBRM1基因可能具有抑制sunitinib耐药发生的作用。最后，通过基因芯片筛选PBRM1敲除介导的下游通路变化，发现细胞因子-细胞因子受体相互作用通路的表达差异最显著，PBRM1在肾癌耐药细胞中通过调控IL6、CXCL2、IL6ST、IL8基因的表达，发挥抑制耐药发生的作用。通过本研究有助于阐明肾癌对抗血管新生药物产生耐药的机制，并为寻找逆转耐药的方法提供新思路。