Geng Rong, Tan Xin, Zuo Zhixiang, Wu Jiangxue, Pan Zhizhong, Shi Wei, Liu Ranyi, Yao Chen, Wang Gaoyuan, Lin Jiaxin, Qiu Lin, Huang Wenlin, Chen Shuai
State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Sun Yat-sen University Cancer Center, 651 Dongfeng Road East, Guangzhou, 510507, Guangdong, P. R. China.
Department of Experimental Research, Sun Yat-sen University Cancer Center, Guangzhou, 510060, Guangdong, P. R. China.
Chin J Cancer. 2017 Jul 31;36(1):63. doi: 10.1186/s40880-017-0228-1.
The mitogen-activated extracellular signal-regulated kinase 1/2 (MEK1/2) inhibitor trametinib has shown promising therapeutic effects on melanoma, but its efficacy on colorectal cancer (CRC) is limited. Synthetic lethality arises with a combination of two or more separate gene mutations that causes cell death, whereas individual mutations keep cells alive. This study aimed to identify the genes responsible for resistance to trametinib in CRC cells, using a synthetic lethal short hairpin RNA (shRNA) screening approach.
We infected HT29 cells with a pooled lentiviral shRNA library and applied next-generation sequencing to identify shRNAs with reduced abundance after 8-day treatment of 20 nmol/L trametinib. HCT116 and HT29 cells were used in validation studies. Stable ring finger protein 183 (RNF183)-overexpressing cell lines were generated by pcDNA4-myc/his-RNF183 transfection. Stable RNF183-knockdown cell lines were generated by infection of lentiviruses that express RNF183 shRNA, and small interference RNA (siRNA) was used to knock down RNF183 transiently. Quantitative real-time PCR was used to determine the mRNA expression. Western blotting, immunohistochemical analysis, and enzyme-linked immunosorbent assay (ELISA) were used to evaluate the protein abundance. MTT assay, colony formation assay, and subcutaneous xenograft tumor growth model were used to evaluate cell proliferation.
In the primary screening, we found that the abundance of RNF183 shRNA was markedly reduced after treatment with trametinib. Trametinib induced the expression of RNF183, which conferred resistance to drug-induced cell growth repression and apoptotic and non-apoptotic cell deaths. Moreover, interleukin-8 (IL-8) was a downstream gene of RNF183 and was required for the function of RNF183 in facilitating cell growth. Additionally, elevated RNF183 expression partly reduced the inhibitory effect of trametinib on IL-8 expression. Finally, xenograft tumor model showed the synergism of RNF183 knockdown and trametinib in repressing the growth of CRC cells in vivo.
The RNF183-IL-8 axis is responsible for the resistance of CRC cells to the MEK1/2 inhibitor trametinib and may serve as a candidate target for combined therapy for CRC.
丝裂原活化的细胞外信号调节激酶1/2（MEK1/2）抑制剂曲美替尼已显示出对黑色素瘤有良好的治疗效果，但其对结直肠癌（CRC）的疗效有限。合成致死性是由两个或更多个单独的基因突变组合导致细胞死亡，而单个突变可使细胞存活。本研究旨在使用合成致死性短发夹RNA（shRNA）筛选方法，鉴定CRC细胞中对曲美替尼耐药的相关基因。
我们用一个汇集的慢病毒shRNA文库感染HT29细胞，并应用下一代测序来鉴定在20 nmol/L曲美替尼处理8天后丰度降低的shRNA。HCT116和HT29细胞用于验证研究。通过pcDNA4-myc/his-RNF183转染产生稳定的环指蛋白183（RNF183）过表达细胞系。通过感染表达RNF183 shRNA的慢病毒产生稳定的RNF183敲低细胞系，并使用小干扰RNA（siRNA）瞬时敲低RNF183。采用定量实时PCR测定mRNA表达。使用蛋白质印迹、免疫组织化学分析和酶联免疫吸附测定（ELISA）评估蛋白质丰度。采用MTT法、集落形成试验和皮下异种移植瘤生长模型评估细胞增殖。
在初次筛选中，我们发现用曲美替尼处理后RNF183 shRNA的丰度显著降低。曲美替尼诱导RNF183的表达，这赋予了对药物诱导的细胞生长抑制以及凋亡和非凋亡性细胞死亡的抗性。此外，白细胞介素-8（IL-8）是RNF183的下游基因，并且是RNF183促进细胞生长功能所必需的。此外，RNF183表达升高部分降低了曲美替尼对IL-8表达的抑制作用。最后，异种移植瘤模型显示RNF183敲低与曲美替尼在体内抑制CRC细胞生长方面具有协同作用。
RNF183-IL-8轴是CRC细胞对MEK1/2抑制剂曲美替尼耐药的原因，并且可能作为CRC联合治疗的候选靶点。
