Functional landscape of Eph receptor mutations in melanoma

描述（由申请人提供）：在所有癌症中，转移性黑色素瘤是最致命的癌症之一。在大多数情况下，黑色素瘤的发展是由于暴露在紫外线下皮肤黑色素细胞中诱导的多种基因突变，但这些突变很少被详细研究。大多数黑素瘤具有组成性激活RAS-RAF-MEK-ERK通路的突变，例如常见的NRAS和BRAF突变，众所周知，它们在恶性黑素瘤中起着关键作用。然而，其他途径如PTEN、TP53或CDKN2A肿瘤抑制因子的突变也是黑色素瘤发展所必需的。此外，最近的研究发现了其他导致恶性黑色素瘤的基因家族的新突变。本研究的目的是探索Eph受体突变在黑色素瘤发生和/或进展中发挥功能作用的新假设。最近对约300个黑色素瘤标本和细胞系的测序研究表明，大约一半的肿瘤在Eph受体酪氨酸激酶家族基因中携带一个或多个非同义突变。Eph受体调节许多基本的生物学和病理过程，如癌细胞的生长和侵袭，其中一些与黑色素瘤的发病有关。各种计算机分析预测，许多Eph受体突变有助于恶性肿瘤，与我们的初步研究一致，表明目前分析的几种EphA2和EphA3黑色素瘤突变严重影响受体功能特性。因此，我们提出了两个目的来探索Eph突变在黑色素瘤中的功能意义。在Aim 1中，我们将描述鉴定出的突变子集，以确定它们是否影响Eph受体执行其正常功能的能力，包括运输到细胞表面，结合ephrin配体，组装成受体信号簇，并履行其作为酪氨酸激酶的作用。我们将特别关注已知或很可能在黑色素瘤发病机制中发挥作用的Eph受体，以及最有可能是“驱动”突变的突变。在Aim 2中，我们将通过在黑色素细胞和黑色素瘤细胞中引入突变型和野生型受体，并测量细胞增殖/存活和迁移/侵袭性，研究具有代表性的Eph突变如何影响黑色素瘤细胞发育和/或恶性肿瘤。调查黑色素瘤中Eph受体突变的功能影响将为黑色素瘤细胞如何获得其恶性特性以及Eph受体家族在癌症发生和进展中的参与提供有价值的信息，这些信息目前尚不完全清楚。这些信息和研究工具的收集也将使未来更广泛的研究能够表征正常和突变的Eph受体在其他细胞培养和体内黑色素瘤模型中的重要性。对Eph基因突变的影响进行编目也可能有助于识别新的生物标志物以及基因组改变，以帮助黑色素瘤患者的预后和个性化治疗设计。

DESCRIPTION (provided by applicant): Among all cancers, metastatic melanoma is one of the most deadly. In most cases melanoma develops due to multiple gene mutations induced in skin melanocytes by exposure to UV light, but few of these mutations have been studied in detail. Most melanomas harbor mutations that constitutively activate the RAS-RAF-MEK-ERK pathway, such as the frequent NRAS and BRAF mutations, which are well known to play a critical role in melanoma malignancy. However, mutations in other pathways such as the PTEN, TP53 or CDKN2A tumor suppressors are also needed for melanoma development. Furthermore, recent studies have uncovered new mutations in other gene families that contribute to melanoma malignancy. The objective of the proposed research is to explore the novel hypothesis that Eph receptor mutations play a functional role in melanoma development and/or progression. Recent sequencing studies of ~300 melanoma specimens and cell lines have revealed that approximately half of the tumors carry one or more nonsynonymous mutations in genes of the Eph receptor tyrosine kinase family. The Eph receptors regulate many fundamental biological and pathological processes, such as the growth and invasiveness of cancer cells, and several of them have been implicated in melanoma pathogenesis. Various in silico analyses predict that many of the Eph receptor mutations contribute to malignancy, consistent with our preliminary studies showing that several EphA2 and EphA3 melanoma mutations analyzed so far drastically affect receptor functional properties. We therefore propose two aims to explore the functional significance of Eph mutations in melanoma. In Aim 1, we will characterize a subset of the mutations identified to determine if they affect the abilit of Eph receptors to perform their normal functions, including trafficking to the cell surface, binding ephrin ligands, assembling into receptor signaling clusters, and fulfilling their role as tyrosine kinases. We will especially focus on Eph receptors that are either known or very likely to play a role in melanoma pathogenesis and on the mutations most likely to be "driver" mutations. In Aim 2, we will investigate how representative Eph mutations affect melanoma cell development and/or malignancy by introducing mutant and wild-type receptors in melanocytes and melanoma cells, and measuring cell proliferation/survival and migration/invasiveness. Surveying the functional effects of Eph receptor mutations occurring in melanoma will provide valuable information on how melanoma cells acquire their malignant properties and on the involvement of the Eph receptor family in cancer development and progression, which is currently incompletely understood. This information, and the collection of research tools to be generated, will also enable future more extensive studies to characterize the importance of normal as well as mutated Eph receptors in additional cell culture and in vivo melanoma models. Cataloguing the effects of Eph gene mutations may also prove useful for identifying novel biomarkers as well as genomic alterations to help prognosis and the design of individualized therapies for melanoma patients.