Modeling the Transcriptional Consequences of Epidermal Growth Factor Receptor Ablation in Ras-Initia

EGFR targeted therapy is in clinical use to treat squamous cell carcinoma of the head and neck and other cancers of lining epithelium. Activating Ras mutations are a negative prognostic factor for response to EGFR ablation therapy in multiple cancer types. Furthermore, the major adverse consequence of this therapy is an acneiform papulopustular eruption on normal skin. To better understand Ras signaling in an EGFR depleted environment, we performed gene expression profiling on oncogenic Ras transformed and wildtype mouse keratinocytes with EGFR ablated chronically by genetic deletion or acutely by drug treatment. We were able to identify a 25 gene signature specific to the Ras-EGFR ablation interaction and a distinct 19 gene EGFR ablation signature on normal keratinocytes. EGFR ablation in the context of wildtype Ras reduces ontologies favoring cell cycle control and transcription while ablation in the context of oncogenic Ras enriches ontologies for ion channels and membrane transporters, particularly focused on calcium homeostasis. The study showed substantial differences in ontologies between chronic EGFR ablation and acute pharmacological ablation, both with and without Ras activation. Pathway and biochemical analysis indicates that activation of p38α is a response to both acute and chronic abrogation of EGFR signaling under conditions of Ras activation. This was confirmed in oncogenic RAS transformed human keratinocytes and in tumors developing in orthografts of EGFR ablated mouse keratinocytes transformed by oncogenic Ras. EGFR ablation in the absence of oncogenic Ras revealed Erk and IL-1β related pathways. These findings reveal previously unrecognized interactions between Ras and EGFR signaling in squamous tumor cells that could influence the therapeutic response to EGFR ablation therapy. Keratinocyte samples were collected from CD-1 EGFR total wild type and knock-out mice. Each of these were treated with 3 different EGFR tyrosine kinase inhibitors (AG1478, Erlotinib, PD153035) in combination with and without ras transduction. Control samples included untreated keratinocyte samples. Universal mouse reference RNA was used as the reference sample. Most groups contained 5 biological replicates, one being a reverse fluoro for a total of 63 samples.

表皮生长因子受体（Epidermal Growth Factor Receptor, EGFR）靶向疗法已获批临床应用，用于治疗头颈部鳞状细胞癌及其他被覆上皮来源恶性肿瘤。致癌性Ras突变在多种癌种中均为EGFR消融治疗应答的不良预后因素。此外，该疗法的主要不良反应为正常皮肤出现痤疮样脓疱丘疹疹。为深入阐明EGFR缺失环境中的Ras信号调控机制，本研究对经长期遗传敲除或急性药物处理实现EGFR消融的致癌性Ras转化小鼠角质形成细胞与野生型小鼠角质形成细胞开展了基因表达谱分析。研究成功鉴定出与Ras-EGFR消融互作特异性相关的25个基因特征标签，以及正常角质形成细胞中EGFR消融独有的19个基因特征标签。在野生型Ras背景下，EGFR消融会削弱与细胞周期调控、转录相关的基因本体富集条目；而在致癌性Ras背景下，EGFR消融则会富集离子通道与膜转运蛋白相关的基因本体富集条目，尤其聚焦于钙稳态调控。本研究还发现，无论是否存在Ras激活，长期EGFR消融与急性药物消融的基因本体富集条目均存在显著差异。通路与生化分析显示，在Ras激活条件下，p38α丝裂原活化蛋白激酶（p38α）的激活是对EGFR信号通路被急性或慢性阻断的共同应答反应，该结果在致癌性RAS转化的人角质形成细胞以及经致癌性Ras转化、EGFR消融的小鼠角质形成细胞原位移植瘤模型中得到了验证。在无致癌性Ras存在的情况下，EGFR消融会激活Erk（Extracellular Regulated Protein Kinase）与白细胞介素1β（Interleukin 1β, IL-1β）相关通路。上述研究结果揭示了鳞状肿瘤细胞中此前未被认知的Ras与EGFR信号通路互作模式，该互作或可影响EGFR消融治疗的临床应答效果。本研究的样本采集自CD-1品系EGFR全野生型及EGFR敲除小鼠，各组样本分别经3种EGFR酪氨酸激酶抑制剂（AG1478、厄洛替尼（Erlotinib）、PD153035）单独或联合Ras基因转染处理，同时设置未处理的角质形成细胞作为对照。本研究采用通用小鼠参考RNA作为参考样本，大部分实验组包含5个生物学重复，其中1个为反向荧光标记样本，最终共计63个样本。