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））不受控制的激活的可怕后果，在进化过程中设计了细胞外和细胞内控制机制。一种这样的EGFR调节的细胞外机制由核心蛋白聚糖提供，核心蛋白聚糖是一种富含亮氨酸的蛋白聚糖，其在体外和体内结合并下调EGFR活性。我们假设，其他可溶性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LRIG 1上，LRIG 1是一种跨膜蛋白，在其胞外域含有15个富含亮氨酸的重复序列和3个Ig样重复序列。我们产生了一个可溶性胞外域LRIG 1只含有富含亮氨酸的重复序列和侧翼Cys丰富的帽。我们发现纳摩尔浓度的LRIG 1胞外域以剂量依赖性方式抑制基础和配体依赖性EGFR活化和Erk 1/2信号传导。反过来，这导致EGFR表达癌细胞的生长抑制，但不导致缺乏受体表达的细胞的生长抑制。此外，我们提供了LRIG 1介导的功能中EGFR需求的遗传证据，并证明了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竞争性取代。这些新的结果表明，LRIG 1的可溶性胞外域，可以想象，可以在组织重塑和肿瘤浸润的网站上释放，可能作为EGFR活性的负调节。这项新授权申请的中心假设是，从癌周围或癌内的细胞表面释放可溶性LRIG 1胞外域可能代表了抵抗入侵肿瘤细胞的生物学机制，从而作为天然EGFR拮抗剂发挥作用。具体而言，我们计划： [1]破译LRIG 1胞外域抑制EGFR活性的作用机制，[2]确定LRIG 1通过EGFR诱发的信号传导机制及其抑制细胞生长的能力，[3]研究LRIG 1胞外域作为抗癌因子的体内功能。 这些协调一致的研究路线应该牢固地建立LRIG 1在致瘤性中的功能作用，并阐明其作用机制。预期的结果可能为基础癌症研究开辟新的前景，并可能导致未来通过使用肿瘤细胞生长的天然抑制剂来治疗癌症的方法。

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.