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集合中询问超过600个候选基因，以富集EGFR信号传导的调节因子。该分析和随后的分析鉴定了SC 4 MOL（固醇C4-甲基氧化酶样），一种很少研究的固醇生物合成途径中间体，作为EGFR抑制剂厄洛替尼和西妥昔单抗治疗后细胞活力的有效调节剂。为了理解SC 4 MOL作用的机制，我们再次使用生物信息学策略来模拟SC 4 MOL及其伴侣蛋白NSDHL（NADP依赖性类固醇脱氢酶样）的进化上保守的直系同源物的相互作用网络。这预示着多个连接到外泌运输和蛋白质降解的监管机构。在网络预测的直接初步测试中，我们证明了SC 4 MOL或NSDHL的RNAi消耗导致EGFR从质膜上丢失，并增强其泛素化和降解。我们的假设是，SC 4 MOL和NSDHL与伴侣蛋白之间的相互作用的损失影响贩运，以加速EGFR降解，从而增强EGFR靶向药物。为了进一步验证该方法以鉴定靶向反应的修饰剂，并潜在地验证SC 4 MOL和NSDHL作为药物反应的生物标志物和/或药物开发的靶标，我们将进行以下两个目的： 目标1.研究SC 4 MOL和NSDHL调节EGFR转运的机制。我们将评估SC 4 MOL和NSDHL如何影响EGFR的运输和降解，以及SC 4 MOL和NSDHL的催化活性是否是其作用所必需的。我们将分析SC 4 MOL和一小部分预测的保守的相互作用伙伴之间的相互作用，这些相互作用伙伴在调节囊泡贩运中具有已知的作用，这些作用是通过对低等真核生物中的直系同源物进行生物信息学分析预测的。 目标二。研究SC 4 MOL和NSDHL与ERAD蛋白的相互作用。我们已经发现SC 4 MOL或NSDHL的沉默增强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）系统中的一些候选者，将测试其与SC 4 MOL和NSDHL的物理关联，以及在EGFR表达、运输和降解的SC 4 MOL和NSDHL依赖性调节中的作用。 考虑到EGFR作为癌症治疗靶点的重要性，这项工作对减少许多有临床价值的治疗药物的耐药性具有潜在的高度影响。 公共卫生相关性：为了实现我们将系统生物学方法整合到癌症耐药性分析和靶向治疗组合新策略中的长期目标，我们已经确定了SC 4 MOL（一种固醇代谢酶）与EGFR之间先前未知的合成致死性相互作用。对SC 4 MOL的保守直向同源物（潜在的互作同源物）和功能性连接的伴侣蛋白NSDHL的相互作用网络的分析已经确定了控制胞吐运输和降解的多个连接。在此基础上，我们提出了我们的新假设，即SC 4 MOL和NSDHL调节EGFR的运输和降解，是潜在的非常有价值的癌症治疗和预防的新靶点。

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.