van der Hoeven Dharini, Truong Thuy Nhu L, Naji Ali, Thapa Sabita, Hancock John F, van der Hoeven Ransome
Department of Diagnostic and Biomedical Sciences, School of Dentistry, University of Texas Health Science Center; Department of Integrative Biology and Pharmacology, McGovern Medical School, University of Texas Health Science Center.
Department of Diagnostic and Biomedical Sciences, School of Dentistry, University of Texas Health Science Center.
J Vis Exp. 2020 Oct 5(164). doi: 10.3791/61788.
The changes in the plasma membrane localization of the epidermal growth factor receptor (EGFR) and its downstream effector RAS have been implicated in several diseases including cancer. The free-living nematode C. elegans possesses an evolutionary and functionally conserved EGFR-RAS-ERK MAP signal cascade which is central for the development of the vulva. Gain of function mutations in RAS homolog LET-60 and EGFR homolog LET-23 induce the generation of visible nonfunctional ectopic pseudovulva along the ventral body wall of these worms. Previously, the multivulval (Muv) phenotype in these worms has been shown to be inhibited by small chemical molecules. Here we describe a protocol for using the worm in a liquid-based assay to identify inhibitors that abolish the activities of EGFR and RAS proteins. Using this assay, we show R-fendiline, an indirect inhibitor of K-RAS, suppresses the Muv phenotype expressed in the let-60(n1046) and let-23(sa62) mutant worms. The assay is simple, inexpensive, is not time consuming to setup, and can be used as an initial platform for the discovery of anticancer therapeutics.
表皮生长因子受体（EGFR）及其下游效应分子RAS的质膜定位变化与包括癌症在内的多种疾病有关。自由生活的线虫秀丽隐杆线虫拥有进化上和功能上保守的EGFR-RAS-ERK MAP信号级联，这对外阴发育至关重要。RAS同源物LET-60和EGFR同源物LET-23的功能获得性突变会导致这些线虫腹侧体壁沿线产生可见的无功能异位假外阴。此前，已证明这些线虫中的多外阴（Muv）表型会受到小分子的抑制。在这里，我们描述了一种在基于液体的检测中使用线虫来鉴定消除EGFR和RAS蛋白活性的抑制剂的方案。使用该检测方法，我们发现K-RAS的间接抑制剂R-芬地林可抑制let-60（n1046）和let-23（sa62）突变线虫中表达的Muv表型。该检测方法简单、成本低、设置不耗时，可作为发现抗癌治疗药物的初始平台。
