Regulation of EGFR by SC4MOL- and NSDHL-Dependent Trafficking

描述(由申请人提供)：我们的目标是将系统生物学方法整合到癌症耐药机制的分析中，从而开发和评估有效选择最佳治疗组合的新策略。在我们最近的工作中，我们使用siRNA筛选从我们通过网络建模提名的一组候选基因中询问了600多个基因，以丰富EGFR信号的调节。这项研究和随后的分析确定，SC4MOL(类固醇C4-甲基氧化酶)，一种鲜有研究的固醇生物合成途径的中间体，在使用EGFR抑制剂erlotinib和西妥昔单抗治疗后，是细胞活力的有效调节因子。为了了解SC4MOL的作用机制，我们再次使用生物信息学策略来模拟SC4MOL及其伙伴蛋白NSDHL(NADP依赖的类固醇脱氢酶)在进化上保守的同源物的相互作用网络。这预示着与胞外运输和蛋白质降解的调节因素有多种联系。在网络预测的直接初步检验中，我们证明了SC4MOL或NSDHL的RNAi耗尽导致质膜上EGFR的丢失，并增强了其泛素化和降解。我们的假设是，SC4MOL和NSDHL之间失去与伴侣蛋白的相互作用会影响转运，从而加速EGFR的降解，从而增强EGFR靶向药物。为了进一步验证这种识别靶向反应修饰物的方法，并潜在地验证SC4MOL和NSDHL作为药物反应的生物标志物和/或药物开发的靶点，我们将实现以下两个目标： 目的1.探讨SC4MOL和NSDHL调控EGFR转运的机制。我们将评估SC4MOL和NSDHL如何影响EGFR的贩运和降解，以及它们的行动是否需要SC4MOL和NSDHL的催化活性。我们将分析SC4MOL与一小组预测的保守相互作用伙伴之间的相互作用，这些相互作用伙伴在调控泡运输方面具有已知的作用，这是通过对低等真核生物中的同源基因的生物信息学分析预测的。 目的2.研究SC4MOL和NSDHL与ERAD蛋白的相互作用。我们发现，沉默SC4MOL或NSDHL可增强EGFR泛素化和溶酶体降解。我们的分析确定了ER相关蛋白降解(ERAD)系统中的一些候选对象，将测试该系统与SC4MOL和NSDHL的物理关联，以及在SC4MOL和NSDHL依赖的EGFR表达、运输和降解调节中的作用。 鉴于EGFR作为肿瘤治疗靶点的重要性，这项工作对降低与许多临床有价值的治疗药物相关的耐药性具有潜在的高影响。

DESCRIPTION (provided by applicant): Our goal is to integrate systems biology approaches into the analysis of cancer drug resistance mechanisms, and thus to develop and evaluate new strategies for the efficient selection of optimal therapeutic combinations. In our recent work, we used siRNA screening to interrogate over 600 candidate genes from a set we nominated by network modeling to be enriched for regulators of EGFR signaling. This and subsequent analysis identified SC4MOL (sterol C4-methyl oxidase-like), a little-studied intermediate in the sterol biosynthesis pathway, as a potent regulator of cell viability following treatment with the EGFR inhibitors erlotinib and cetuximab. To understand the mechanism of SC4MOL action, we again used a bioinformatics strategy to model an interaction network for evolutionarily conserved orthologs of SC4MOL and its partner protein, NSDHL (NADP-dependent steroid dehydrogenase-like). This predicted multiple connections to regulators of exocytic trafficking and protein degradation. In direct preliminary test of the network predictions, we demonstrated that RNAi depletion of SC4MOL or NSDHL resulted in loss of EGFR from the plasma membrane, and enhanced its ubiquitination and degradation. Our hypothesis is that loss of interactions between SC4MOL and NSDHL with partner proteins influences trafficking so as to accelerate EGFR degradation, thereby potentiating EGFR-targeting drugs. To further validate this approach to identifying modifiers of targeted response, and potentially validate SC4MOL and NSDHL as biomarkers of drug response and/or targets for drug development, we will perform the following two Aims: Aim 1. Investigate the mechanism by which SC4MOL and NSDHL regulate EGFR trafficking. We will assess how SC4MOL and NSDHL influence EGFR trafficking and degradation, and whether the catalytic activity of SC4MOL and NSDHL is required for their actions. We will analyze interactions between SC4MOL and a small set of predicted conserved interaction partners with known roles in regulating vesicular trafficking predicted by bioinformatics analysis of the orthologs in lower eukaryotes. Aim 2. Investigate the interaction of SC4MOL and NSDHL with ERAD proteins. We have found silencing of SC4MOL or NSDHL enhances EGFR ubiquitination and lysosomal degradation. Our analysis identified a number of candidates within the ER-associated protein degradation (ERAD) system, which will be tested for physical association with SC4MOL and NSDHL, and for a role in SC4MOL- and NSDHL-dependent regulation of EGFR expression, trafficking, and degradation. Given the prominence of EGFR as a target in cancer therapy, this work has potentially high impact to reduce drug resistance relevant to many clinically valuable therapeutic agents.