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: 8243750
• 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/8243750
• 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. PUBLIC HEALTH RELEVANCE: Colorectal cancer (CRC) is the second leading cause of cancer deaths in the United States. The incidence of CRC increases with age and men are more often affected than women. Thus, it poses a major health problem for Veterans. Our lab has previously shown that EGFR plays an iterative role in the pathogenesis of CRC. Cetuximab, a monoclonal antibody against the EGFR, is approved by the FDA for the treatment of advanced CRC; however, it is effective in only 10% of patients. This proposal will study two EGFR negative regulators, Lrig1 and Mig-6, which are frequently lost in a number of cancers. Targeted loss of both Lrig1 and Mig-6 in the mouse intestine leads to intestinal tumors. A detailed understanding of their role in intestinal neoplasia may lead to novel therapeutic strategies that may ultimately enhance the action of cetuximab. These studies will determine whether LRIG1 and MIG-6 levels in CRC patients correlate to levels and activity of the EGFR and whether this information has prognostic significance.

描述（由申请人提供）： 表皮生长因子受体（EGFR）过度表达发生在超过50%的结直肠癌（CRC）中，并与转移和预后不良有关。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。我们已经表明，EGFR信号在肠肿瘤形成中起着反复的作用，作用于启动后的建立阶段，并在肿瘤进展过程中再次发挥作用。我们最近发现，Lrig 1是一种细胞表面EGFR负调控因子，可加速EGFR降解，标志着肠道干细胞，Lrig 1缺失小鼠发生肠道肿瘤。这些见解来自我们对Lrig 1-CreERT 2小鼠的一代和分析。Mig-6是另一种EGFR负调控因子，也在肠干细胞区表达;它是一种通过阻断EGFR酪氨酸激酶活性起作用的胞质蛋白。组成型Mig-6基因敲除小鼠发生直肠癌，但由于严重的血液流变学异常而在早期死亡。我们获得了具有Mig-6条件性floxed等位基因的小鼠。通过将纯合子Lrig 1-CreERT 2驱动小鼠与条件性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Lrig 1和Mig-6。尽管尚未在CRC中进行免疫组织化学分析，但许多人类癌症中LRIG 1和MIG-6的水平均下降。我们假设：1）肠祖细胞隔室中Lrig 1和Mig-6的靶向破坏将导致增强和持续的Egfr信号传导，从而易患肠肿瘤; 2）它们的联合丢失将导致更晚期的肿瘤。我们提出了以下三个具体目标，以检查Lrig 1和Mig-6的损失如何有助于肠肿瘤。目标1.确定Lrig 1基因敲除小鼠中肠肿瘤形成的机制。我们将探索一个基于扩张的Brunner腺和上覆上皮之间动态串扰的肿瘤发生的二室模型。我们将通过将Lrig 1缺失小鼠与纯合子Wa 2小鼠杂交来评估Egfr信号在肿瘤表型中的重要性，纯合子Wa 2小鼠是Egfr活性显著降低的Egfr亚型。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Lrig 1和Mig-6的调节，并确定体内Lrig 1和Mig-6的缺失是否会增强肠肿瘤形成。我们将比较西妥昔单抗在表达（或不表达）这两种EGFR阴性调节因子的CRC细胞系中的疗效。我们预测，恢复这些EGFR负调控因子的表达将增强西妥昔单抗的抗肿瘤疗效。通过将纯合子Lrig 1-CreERT 2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Lrig 1。我们预测结肠和直肠的肿瘤负荷会增加。目标3.确定人类CRC中LRIG 1和LRIG 6水平是否降低，以及这种降低是否具有临床意义。我们将对两种临床和组织学注释的CRC组织阵列进行LRIG 1、EGFR-6和总EGFR和磷酸化EGFR的免疫组织化学（IHC）：1）原发性人CRC和2）匹配的原发性癌和肝转移。我们预测LRIG 1和EGFR-6的丢失或水平降低将与总EGFR和p-EGFR水平升高相关，并且它们的联合丢失将预示不良的临床结局。结合体外细胞培养系统和临床样本，对这些体内模型的分析将为肠道肿瘤的发病机制提供新的见解，并可能导致新的CRC治疗策略。 公共卫生关系： 结直肠癌（CRC）是美国癌症死亡的第二大原因。CRC的发病率随着年龄的增长而增加，男性比女性更经常受到影响。这对退伍军人来说是一个严重的健康问题。我们的实验室先前已经表明EGFR在CRC的发病机制中起着反复的作用。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;然而，它仅对10%的患者有效。该提案将研究两种EGFR负调控因子Lrig 1和Mig-6，它们在许多癌症中经常丢失。小鼠肠道中Lrig 1和Mig-6的靶向缺失导致肠道肿瘤。详细了解它们在肠肿瘤中的作用可能会导致新的治疗策略，最终可能增强西妥昔单抗的作用。这些研究将确定CRC患者中LRIG 1和EGFR-6水平是否与EGFR水平和活性相关，以及该信息是否具有预后意义。