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中筛选600多个候选基因，以丰富EGFR信号传导的调节因子。这项研究和随后的分析确定了SC4MOL（甾醇c4 -甲基氧化酶样），这是一种在甾醇生物合成途径中很少研究的中间体，在使用EGFR抑制剂埃洛替尼和西妥昔单抗治疗后，作为细胞活力的有效调节剂。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及其伴侣蛋白NSDHL （nadp依赖性类固醇脱氢酶样）的进化保守同源物的相互作用网络。这预测了与胞外运输和蛋白质降解调节因子的多种联系。在网络预测的直接初步测试中，我们证明了SC4MOL或NSDHL的RNAi缺失导致质膜上EGFR的丢失，并增强了其泛素化和降解。我们的假设是，SC4MOL和NSDHL与伴侣蛋白之间相互作用的丧失影响了运输，从而加速了EGFR降解，从而增强了EGFR靶向药物的作用。为了进一步验证该方法识别靶向反应修饰因子，并潜在地验证SC4MOL和NSDHL作为药物反应的生物标志物和/或药物开发靶标，我们将执行以下两个目标：

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. PUBLIC HEALTH RELEVANCE: In pursuit of our long-term goal of integration of systems biology approaches into the analysis of cancer drug resistance and of new strategies for targeted therapeutic combinations, we have identified a previously unknown synthetic lethality interaction between SC4MOL, a sterol metabolism enzyme, and EGFR. Analysis of interaction network for conserved orthologs (potential interologs) of SC4MOL and a functionally linked partner protein, NSDHL, has identified multiple connections to control exocytic trafficking and degradation. On this basis, we formulated our new hypothesis that SC4MOL and NSDHL regulate EGFR traffic and degradation, and are potentially highly valuable new targets for cancer therapy and prevention.