The Role of EGFR Negative Regulators in GI Neoplasia

EGFR 负调节因子在胃肠道肿瘤中的作用

• 批准号: 8510385
• 负责人: Robert J. Coffey
• 金额: --
• 依托单位: VETERANS HEALTH ADMINISTRATION
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8510385
• 关键词: Affect; Age; Alleles; Brunner Glands; Cancer Etiology; Cancer Patient; Cancer cell line; Cell Culture System; Cell surface; Cessation of life; Cetuximab; Clinical; Colon; Colorectal Cancer; Epidermal Growth Factor Receptor; Epithelium; FDA approved; Generations; Health; Histologic; Human; Immunohistochemistry; In Vitro; Incidence; Injection of therapeutic agent; Intestinal Neoplasms; Intestines; Knockout Mice; Lead; Link; Malignant Neoplasms; Metastatic Neoplasm to the Liver; Modeling; Monoclonal Antibodies; Mus; Neoplasm Metastasis; Neoplasms; Outcome; Pathogenesis; Patients; Phenotype; Play; Primary Carcinoma; Protein Tyrosine Kinase; Proteins; Receptor Signaling; Rectal Cancer; Rectum; Regulation; Role; Sampling; Signal Transduction; Staging; Stem cells; Tamoxifen; Tissue Microarray; Tumor Burden; United States; Veterans; Woman; base; clinically relevant; comparative efficacy; in vivo; in vivo Model; insight; men; novel therapeutics; outcome forecast; overexpression; progenitor; prognostic; tumor; tumor progression; tumorigenesis

DESCRIPTION (provided by applicant): EGF receptor (EGFR) overexpression occurs in over 50% of colorectal cancers (CRC) and is linked to metastasis and poor prognosis. Cetuximab, a monoclonal antibody against the EGFR, is approved by the FDA for the treatment of advanced CRC, but is effective in only 10% of CRC patients. We have shown that EGFR signaling plays an iterative role in intestinal neoplasia, acting at a post-initiation, establishment stage and again later during tumor progression. We recently found that Lrig1, a cell surface EGFR negative regulator that accelerates EGFR degradation, marks intestinal stem cells and that Lrig1 null mice develop intestinal tumors. These insights emerged from our generation and analysis of Lrig1-CreERT2 mice. Mig-6 is another EGFR negative regulator that is also expressed in the intestinal stem cell zone; it is a cytosolic protein that acts by blocking EGFR tyrosine kinase activity. Constitutive Mig-6 null mice develop rectal carcinoma but die at an early age because of severe rheumatological abnormalities. We have obtained mice with a conditional floxed allele of Mig-6. By crossing homozygous Lrig1-CreERT2 driver mice to conditional Mig-6 mice, we will selectively eliminate Lrig1 and Mig-6 in the intestine following injection of tamoxifen. Levels of both LRIG1 and MIG-6 are decreased in a number of human cancers, although immunohistochemical analysis has not been performed in CRC. We hypothesize that 1) targeted disruption of Lrig1 and Mig-6 in the intestinal progenitor compartment will lead to heightened and sustained Egfr signaling that predisposes to intestinal neoplasia and 2) their combined loss will result in more advanced tumors. We propose the following three specific aims to examine how loss of Lrig1 and Mig-6 contributes to intestinal neoplasia. Aim 1. Determine the mechanism of intestinal neoplasia in Lrig1 null mice. We will explore a two- compartment model of tumorigenesis based on dynamic crosstalk between the expanded Brunner's glands and the overlying epithelium. We will assess the importance of Egfr signaling in the tumor phenotype by crossing Lrig1 null mice to homozygous Wa2 mice, an Egfr hypomorph with markedly reduced Egfr activity. We predict these mice will not form tumors. Aim 2. Examine regulation of Lrig1 and Mig-6 in CRC cell lines in vitro and determine whether loss of both Lrig1 and Mig-6 in vivo enhances intestinal neoplasia. We will compare the efficacy of cetuximab in CRC cell lines that express (or not) these two EGFR negative regulators. We predict that restoring expression of these EGFR negative regulators will enhance the anti-tumor efficacy of cetuximab. By crossing homozygous Lrig1-CreERT2 mice to conditional Mig-6 mice, we will eliminate both Mig-6 and Lrig1 in the intestine upon tamoxifen-induced Cre activation. We predict there will be increased tumor burden in the colon and rectum. Aim 3. Determine whether LRIG1 and MIG-6 levels are decreased in human CRC and whether this decrease is clinically relevant. We will perform immunohistochemistry (IHC) for LRIG1, MIG-6 and total and phospho (p)-EGFR on two clinically and histologically annotated CRC tissue arrays: 1) primary human CRC and 2) matched primary carcinoma and liver metastasis. We predict that loss or reduced levels of LRIG1 and MIG-6 will correlate with increased levels of total and p-EGFR and that their combined loss will portend poor clinical outcomes. Analysis of these in vivo models, combined with in vitro cell culture systems and clinical samples, will provide new insights into pathogenesis of intestinal neoplasia and may lead to novel therapeutic strategies for CRC.

描述（由申请人提供）： EGF 受体 (EGFR) 过度表达发生在超过 50% 的结直肠癌 (CRC) 中，并与转移和不良预后相关。西妥昔单抗是一种针对 EGFR 的单克隆抗体，已被 FDA 批准用于治疗晚期 CRC，但仅对 10% 的 CRC 患者有效。我们已经证明，EGFR 信号传导在肠道肿瘤中发挥着反复作用，在肿瘤进展过程中的启动后、建立阶段和后期再次发挥作用。我们最近发现 Lrig1（一种细胞表面 EGFR 负调节因子，可加速 EGFR 降解）标记肠道干细胞，并且 Lrig1 缺失小鼠会出现肠道肿瘤。这些见解来自我们对 Lrig1-CreERT2 小鼠的生成和分析。 Mig-6 是另一种 EGFR 负调节因子，也在肠道干细胞区表达；它是一种胞质蛋白，通过阻断 EGFR 酪氨酸激酶活性发挥作用。 Mig-6 缺失小鼠会患上直肠癌，但由于严重的风湿病异常而早年死亡。我们获得了具有 Mig-6 条件性 floxed 等位基因的小鼠。通过将纯合 Lrig1-CreERT2 驱动小鼠与条件 Mig-6 小鼠杂交，我们将在注射他莫昔芬后选择性地消除肠道中的 Lrig1 和 Mig-6。尽管尚未在结直肠癌中进行免疫组织化学分析，但在许多人类癌症中，LRIG1 和 MIG-6 的水平均下降。我们假设 1) 肠道祖细胞室中 Lrig1 和 Mig-6 的靶向破坏将导致 Egfr 信号增强和持续，从而诱发肠道肿瘤；2) 它们的联合缺失将导致更晚期的肿瘤。我们提出以下三个具体目标来研究 Lrig1 和 Mig-6 的缺失如何导致肠道肿瘤。目的 1. 确定 Lrig1 缺失小鼠肠道肿瘤形成的机制。我们将探索基于扩张的布伦纳腺和上皮细胞之间动态串扰的肿瘤发生的两室模型。我们将通过将 Lrig1 缺失小鼠与纯合子 Wa2 小鼠杂交来评估 Egfr 信号传导在肿瘤表型中的重要性，Wa2 小鼠是一种 Egfr 亚型，其 Egfr 活性显着降低。我们预测这些小鼠不会形成肿瘤。目标 2. 在体外检查 CRC 细胞系中 Lrig1 和 Mig-6 的调节，并确定体内 Lrig1 和 Mig-6 的缺失是否会增强肠肿瘤。我们将比较西妥昔单抗在表达（或不表达）这两种 EGFR 负调节因子的 CRC 细胞系中的疗效。我们预测恢复这些 EGFR 负调节因子的表达将增强西妥昔单抗的抗肿瘤功效。通过将纯合 Lrig1-CreERT2 小鼠与条件性 Mig-6 小鼠杂交，我们将在他莫昔芬诱导的 Cre 激活后消除肠道中的 Mig-6 和 Lrig1。我们预测结肠和直肠的肿瘤负荷将会增加。目标 3. 确定人类 CRC 中 LRIG1 和 MIG-6 水平是否降低以及这种降低是否具有临床相关性。我们将对两个临床和组织学注释的 CRC 组织芯片进行 LRIG1、MIG-6 以及总和磷酸 (p)-EGFR 的免疫组织化学 (IHC)：1) 原发性人类 CRC 和 2) 匹配的原发性癌和肝转移。我们预测 LRIG1 和 MIG-6 水平的缺失或降低将与总EGFR和p-EGFR水平的升高相关，并且它们的综合缺失将预示着不良的临床结果。对这些体内模型的分析，结合体外细胞培养系统和临床样本，将为肠肿瘤的发病机制提供新的见解，并可能带来新的结直肠癌治疗策略。