Natural EGFR Antagonists and Cancer

描述（由申请人提供）：为了防止酪氨酸激酶受体（如表皮生长因子受体（EGFR））失控激活的可怕后果，在进化过程中设计了细胞外和细胞内的控制机制。EGFR调控的一种细胞外机制是由decorin提供的，decorin是一种富含亮氨酸的蛋白多糖，在体外和体内都能结合并下调EGFR活性。我们假设其他可溶形式的富含亮氨酸重复序列的蛋白质也可以类似地影响EGFR信号通路。因此，我们将注意力集中在LRIG1上，这是一种跨膜蛋白，在其外域含有15个富含亮氨酸的重复序列和3个igg样重复序列。我们生成了LRIG1的可溶性外结构域，仅包含富含亮氨酸的重复序列和侧翼富含cys的帽。我们发现，纳米摩尔浓度的LRIG1外结构域以剂量依赖性的方式抑制基础和配体依赖性EGFR激活和Erk1/2信号传导。反过来，这导致表达egfr的癌细胞的生长受到抑制，而缺乏受体表达的细胞则不会受到抑制。此外，我们提供了lrig1介导的功能需要EGFR的遗传证据，并证明A431细胞上存在高亲和力（Kd=10 nM）结合位点，其中绝大多数（高达75%）可以被EGF竞争性地取代。这些新结果表明，LRIG1的可溶性外结构域可能在组织重塑和肿瘤侵袭部位释放，可能作为EGFR活性的负调节因子。这项新资助申请的中心假设是，从癌细胞周围或癌细胞内部的细胞表面释放可溶性LRIG1外结构域可能代表一种对抗入侵肿瘤细胞的生物学机制，从而发挥天然EGFR拮抗剂的作用。具体而言，我们计划：

DESCRIPTION (provided by applicant): To prevent the dire consequences of uncontrolled activation of tyrosine kinase receptors, such as the epidermal growth factor receptor (EGFR), both extracellular and intracellular controlling mechanisms have been devised during evolution. One such extracellular mechanism of EGFR regulation is provided by decorin, a leucine-rich proteoglycan that binds to and downregulates EGFR activity both in vitro and in vivo. We hypothesized that other soluble forms of proteins harboring leucine-rich repeats could similarly affect the EGFR signaling pathway. Thus, we focused our attention on LRIG1, a transmembrane protein containing fifteen leucine-rich repeats and three Ig-like repeats in its ectodomain. We generated a soluble ectodomain of LRIG1 containing only the leucine-rich repeats and flanking Cys-rich caps. We discovered that nanomolar concentrations of LRIG1 ectodomain inhibit both basal and ligand-dependent EGFR activation and Erk1/2 signaling in a dose-dependent fashion. This, in turn, causes growth inhibition of EGFR-expressing carcinoma cells but not of cells lacking expression of the receptor. Furthermore, we provide genetic evidence for EGFR requirement in the LRIG1-mediated function, and demonstrate the existence of high-affinity (Kd=10 nM) binding sites on the A431 cells, the vast majority (up to 75%) of which can be competitively displaced by EGF. These novel results suggest that the soluble ectodomain of LRIG1, which could conceivably be released at sites of tissue remodeling and tumor invasion, might act as negative regulator of EGFR activity. The central hypothesis of this new grant application is that release of soluble LRIG1 ectodomain from the cell surface around or within carcinomas could represent a biological mechanism to counteract the invading tumor cells, thereby functioning as a natural EGFR antagonist. Specifically, we plan to: [1] Decipher the mechanism of action of LRIG1 ectodomain in suppressing EGFR activity, [2] Determine the mechanism of LRIG1-evoked signaling via the EGFR and its ability to inhibit cell growth , and [3] Investigate the in vivo function of LRIG1 ectodomain as an anti-oncogenic factor. These concerted research lines should firmly establish the functional roles of LRIG1 in tumorigenicity and shed light on its mechanism of action. The expected results could open novel perspectives for basic cancer research, and could lead to future approaches of cancer treatment by using a natural inhibitor of tumor cell growth.