Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
The International Cooperation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai 200438, China.
Theranostics. 2020 Apr 6;10(12):5384-5397. doi: 10.7150/thno.41616. eCollection 2020.
: The existence of primary and acquired drug resistance is the main obstacle for the effect of multi-kinase inhibitor sorafenib and regorafenib in advanced hepatocellular carcinoma (HCC). However, plenty of patients did not significantly benefit from sorafenib treatment and little is known about the mechanism of drug resistance. : Laser capture microdissection was used to acquire matched normal liver and tumor tissues on formalin-fixed paraffin-embedded specimens collected before sorafenib therapy from the first surgery of 119 HCC patients. Ultra-deep sequencing (~1000×) targeting whole exons of 440 genes in microdissected specimens and siRNA screen in 7 cell lines were performed to find mutations associated with differential responses to sorafenib. Patient-derived xenograft models were employed to determine the role of in response to sorafenib. Lentiviruses harboring wild-type and c.G52C-mutant were applied to explore the function of in resistance to sorafenib. ChIP-PCR assay for analysis of OCT4 transcriptional activity was performed to explore the affinity with the promoter. Statistical analyses were used to associate levels of p53 and OCT4 with tumor features and patient outcomes. : Total 1,050 somatic mutations and 26 significant driver genes were identified. SiRNA screening in 7 HCC cell lines was further performed to identify mutations associated with differential responses to sorafenib. A recurrent nonsynonymous mutation c.G52C in () was strongly associated with good response to sorafenib, whereas the stop-gain mutation in showed the opposite outcome both in vitro and in vivo. -induced stem cell factor (encoded by gene, SCF) expression and cross-activation of c-KIT/FLT3-BRAF signals were identified indispensably for sorafenib resistance, which could be reversed by the combination of c-KIT tyrosine kinase inhibitors or neutralizing antibody against SCF. Mechanistically, an OCT4 binding site in upstream of promoter was identified with a higher affinity to wildtype of OCT4 rather than G52C-mutant form, which is indispensable for OCT4-induced expression of and sorafenib resistance. : Our study reported a novel somatic mutation in (c.G52C) responsible for the sorafenib effect, and also shed new light on the treatment of HCC through the combination of specific tyrosine kinase inhibitors according to individual genetic patterns.
：原发性和获得性药物耐药性的存在是多激酶抑制剂索拉非尼和regorafenib在晚期肝细胞癌（HCC）中疗效的主要障碍。然而，大量患者并未从索拉非尼治疗中显著获益，且药物耐药性的机制知之甚少。
：从 119 例 HCC 患者首次手术时福尔马林固定石蜡包埋标本中采集的索拉非尼治疗前的匹配正常肝组织和肿瘤组织，采用激光捕获显微切割。对微切割标本中 440 个基因的全外显子进行超深度测序（~1000×），并在 7 种细胞系中进行 siRNA 筛选，以寻找与索拉非尼反应差异相关的突变。患者来源的异种移植模型用于确定 在对索拉非尼的反应中的作用。携带野生型和 c.G52C-突变 的慢病毒被应用于探索 对索拉非尼耐药的功能。ChIP-PCR 分析用于分析 OCT4 转录活性，以探索与 启动子的亲和力。统计分析用于将 p53 和 OCT4 的水平与肿瘤特征和患者结局相关联。
：共鉴定出 1050 个种系突变和 26 个显著的驱动基因。进一步在 7 种 HCC 细胞系中进行 siRNA 筛选，以鉴定与索拉非尼反应差异相关的突变。（）中强烈与索拉非尼良好反应相关的复发性非同义突变 c.G52C，而 中的无义突变则表现出相反的结果，无论是在体外还是体内。诱导的干细胞因子（由 基因编码，SCF）表达和 c-KIT/FLT3-BRAF 信号的交叉激活被鉴定为索拉非尼耐药所必需的，这可以通过 c-KIT 酪氨酸激酶抑制剂或针对 SCF 的中和抗体的组合来逆转。从机制上讲，在 启动子的上游鉴定到一个 OCT4 结合位点，与野生型 OCT4 而不是 G52C 突变体形式具有更高的亲和力，这对于 OCT4 诱导的 表达和索拉非尼耐药性是必不可少的。
：我们的研究报道了一个负责索拉非尼效应的新的体细胞突变 （c.G52C），并通过根据个体遗传模式结合特定的酪氨酸激酶抑制剂为 HCC 的治疗提供了新的思路。
