Structure-Function Analysis of Breast Cancer-Associated Mutations in ADAM12

描述(申请人提供)：金属蛋白酶去整合素ADAM12正在成为一个重要的乳腺癌相关基因和乳腺癌治疗的潜在靶点。在约10%的原发乳腺肿瘤中，存在两个体细胞突变，即金属蛋白酶结构域的D301H和去整合素域的G479E。我们发现，D/H和G/E突变导致新生的、非活性的ADAM12保留在内质网(ER)中，而活性ADAM12丢失在细胞表面。D/H和G/E突变对野生型ADAM12具有显性-负性效应。导致D/H和G/E突变突变的机制尚不清楚。这项建议的主要目标是了解为什么乳腺癌相关的ADAM12突变体被保留在ER中，以及ADAM12在癌细胞中错误定位的后果是什么。我们的主要假设是D/H和G/E突变体被错误折叠并被内质网质量控制系统保留，导致内质网应激和/或改变分泌/脱落到介质的蛋白质库和膜锚定蛋白质的模式。这项建议的具体目的是：1.确定D/H和G/E ADAM12突变体为什么保留在内质网中，并测试恢复这些突变体野生型ADAM12功能的方法。2.评估D/H和G/E ADAM12突变体对内质网功能的影响。3.比较表达野生型ADAM12和D/H、G/E突变型ADAM12的乳腺癌细胞的分泌体和细胞表面蛋白质组。这些研究将揭示乳腺癌相关突变改变癌细胞中ADAM12功能的机制。 公共卫生相关性：这个项目的重点是了解ADAM12突变的故障原因，这些突变经常在人类乳腺肿瘤中发现。我们的研究结果将对针对ADAM12基因突变患者量身定做的乳腺癌治疗产生重大影响。

DESCRIPTION (provided by applicant): Metalloprotease disintegrin ADAM12 is emerging as an important breast cancer-related gene and a potential target for breast cancer therapies. Two somatic mutations, D301H in the metalloprotease domain and G479E in the disintegrin domain, are present in ~10% of primary breast tumors. We found that the D/H and G/E mutations lead to the retention of the nascent, inactive ADAM12 in the endoplasmic reticulum (ER) and a loss of active ADAM12 at the cell surface. The D/H and G/E mutants have a dominant-negative effect on wild-type ADAM12. The mechanisms responsible for the altered trafficking of the D/H and G/E mutants are not known. The main objectives of this proposal are to understand why the breast cancer-associated ADAM12 mutants are retained in the ER and what the consequences are of ADAM12 mislocalization in cancer cells. Our main hypothesis is that the D/H and G/E mutants are misfolded and retained by the ER quality control system, leading to ER stress and/or changing the repertoire of proteins secreted/shed to medium and the pattern of membrane-anchored proteins. The Specific Aims of this proposal are: 1. Determine why the D/H and G/E ADAM12 mutants are retained in the endoplasmic reticulum, and test approaches to restore the wild-type ADAM12 functionality to these mutants. 2. Assess the effect of the D/H and G/E ADAM12 mutants on the functional performance of the ER. 3. Compare the secretome and cell surface proteome of breast cancer cells expressing the wild-type ADAM12 and the D/H and G/E ADAM12 mutants. These studies will uncover the mechanism by which the breast cancer associated mutations change the function of ADAM12 in cancer cells. PUBLIC HEALTH RELEVANCE: This project is focused on the understanding the cause of malfunction of ADAM12 mutants that are frequently found in human breast tumors. The results of our studies will have a strong impact on breast cancer therapies tailored to patients harboring mutations in the ADAM12 gene.