Natural EGFR Antagonists and Cancer

描述(申请人提供)：为了防止酪氨酸激酶受体失控激活的可怕后果，如表皮生长因子受体(EGFR)，在进化过程中设计了细胞外和细胞内的控制机制。一种这样的EGFR调节的细胞外机制是由核心蛋白聚糖提供的，它是一种富含亮氨酸的蛋白多糖，在体外和体内都能结合和下调EGFR的活性。我们假设，含有富含亮氨酸重复序列的其他可溶形式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，在其胞外结构域中包含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LRIG1的可溶性胞外区，只包含富含亮氨酸的重复序列和侧翼富含半胱氨酸的帽子。我们发现，纳米分子浓度的LRIG1胞外结构域以剂量依赖的方式抑制基础和配体依赖的EGFR激活和ERK1/2信号转导。这反过来会抑制表达EGFR的癌细胞的生长，但不会抑制缺乏该受体表达的细胞的生长。此外，我们提供了在LRIG1介导的功能中需要EGFR的遗传学证据，并证明了A431细胞上存在高亲和力(Kd=10 nM)的结合位点，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，LRIG1的可溶性胞外结构域可能在组织重塑和肿瘤侵袭部位释放，可能是EGFR活性的负调节因子。这一新的拨款申请的中心假设是，从癌周围或癌内的细胞表面释放可溶性LRIG1胞外结构域可能代表了一种生物学机制，以对抗侵袭的肿瘤细胞，从而发挥天然的EGFR拮抗剂的作用。具体来说，我们计划： [1]破译LRIG1胞外结构域抑制EGFR活性的作用机制，[2]确定LRIG1通过EGFR诱导的信号转导机制及其抑制细胞生长的能力，[3]研究LRIG1胞外结构域作为抗肿瘤因子的体内功能。 这些协调一致的研究路线应该坚定地确立LRIG1在肿瘤发生中的功能作用，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一种天然的肿瘤细胞生长抑制物来治疗癌症的方法。

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.