Zhao Tangliang, Bao Yi, Gan Xinxin, Wang Jie, Chen Qiong, Dai Zhihui, Liu Bing, Wang Anbang, Sun Shuhan, Yang Fu, Wang Linhui
Department of Urology, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China.
Department of Medical Genetics, Second Military Medical University, Shanghai 200433, China.
Theranostics. 2019 Aug 14;9(21):6175-6190. doi: 10.7150/thno.35572. eCollection 2019.
: Although sunitinib has been shown to improve the survival rate of advanced renal cell carcinoma (RCC) patients, poor drug response is a major challenge that reduces patient benefit. It is important to elucidate the underlying mechanism so that the therapeutic response to sunitinib can be restored. : We used an Illumina HumanMethylation 850K microarray to find methylation-differentiated CpG sites between sunitinib-nonresponsive and -responsive RCC tissues and Sequenom MassARRAY methylation analysis to verify the methylation chip results. We verified glutaminyl peptide cyclotransferase (QPCT) expression in sunitinib-nonresponsive and -responsive RCC tissues via qRT-PCR, western blot and immunohistochemical assays. Then, cell counting kit 8 (CCK-8), plate colony formation and flow cytometric assays were used to verify the function of QPCT in RCC sunitinib resistance after QPCT intervention or overexpression. Chromatin immunoprecipitation (ChIP) was performed to clarify the upstream regulatory mechanism of QPCT. A human proteome microarray assay was used to identify downstream proteins that interact with QPCT, and co-immunoprecipitation (co-IP) and confocal laser microscopy were used to verify the protein chip results. : We found that the degree of methylation in the QPCT promoter region was significantly different between sunitinib-nonresponsive and -responsive RCC tissues. In the sunitinib-nonresponsive tissues, the degree of methylation in the QPCT promoter region was significantly reduced, and the expression of QPCT was upregulated, which correlated with a clinically poor response to sunitinib. A knockdown of QPCT conferred sunitinib sensitivity traits to RCC cells, whereas an overexpression of QPCT restored sunitinib resistance in RCC cells. Mechanistically, reducing the methylation degree of the QPCT promoter region by 5-aza-2'-deoxycytidine (decitabine) in RCC cells could increase the expression of QPCT and NF-κB (p65) bound to the QPCT promoter region, positively regulating its expression, while the hypermethylation in the QPCT promoter region could inhibit the binding of NF-κB (p65). QPCT could bind to HRAS and attenuate the ubiquitination of HRAS, thus increasing its stability and leading to the activation of the ERK pathway in RCC cells. : QPCT may be a novel predictor of the response to sunitinib therapy in RCC patients and a potential therapeutic target.
尽管舒尼替尼已被证明可提高晚期肾细胞癌（RCC）患者的生存率，但药物反应不佳是一个重大挑战，会降低患者获益。阐明其潜在机制对于恢复对舒尼替尼的治疗反应很重要。我们使用Illumina HumanMethylation 850K芯片来寻找舒尼替尼无反应和有反应的RCC组织之间甲基化差异的CpG位点，并使用Sequenom MassARRAY甲基化分析来验证甲基化芯片结果。我们通过qRT-PCR、蛋白质免疫印迹和免疫组织化学分析验证了舒尼替尼无反应和有反应的RCC组织中谷氨酰胺基肽环化转移酶（QPCT）的表达。然后，使用细胞计数试剂盒8（CCK-8）、平板集落形成和流式细胞术分析来验证QPCT干预或过表达后QPCT在RCC舒尼替尼耐药中的功能。进行染色质免疫沉淀（ChIP）以阐明QPCT的上游调控机制。使用人类蛋白质组芯片分析来鉴定与QPCT相互作用的下游蛋白质，并使用免疫共沉淀（co-IP）和共聚焦激光显微镜来验证蛋白质芯片结果。我们发现舒尼替尼无反应和有反应的RCC组织中QPCT启动子区域的甲基化程度存在显著差异。在舒尼替尼无反应的组织中，QPCT启动子区域的甲基化程度显著降低，QPCT的表达上调，这与对舒尼替尼的临床反应不佳相关。敲低QPCT赋予RCC细胞舒尼替尼敏感性特征，而QPCT过表达恢复了RCC细胞对舒尼替尼的耐药性。机制上，用5-氮杂-2'-脱氧胞苷（地西他滨）降低RCC细胞中QPCT启动子区域的甲基化程度可增加QPCT和与QPCT启动子区域结合的NF-κB（p65）的表达，正向调节其表达，而QPCT启动子区域的高甲基化可抑制NF-κB（p65）的结合。QPCT可与HRAS结合并减弱HRAS的泛素化，从而增加其稳定性并导致RCC细胞中ERK通路的激活。QPCT可能是RCC患者对舒尼替尼治疗反应的新型预测指标和潜在治疗靶点。
