The role of CADM1 in malignant melanoma

摘要：黑色素瘤是皮肤癌中最致命的一种，患者表现为转移性疾病。 五年存活率只有2%-16%。然而，转移前的早期临床干预 十年存活率约为92%。我们的目标是研究黑色素瘤细胞如何获得转移潜力，希望 以揭示未来的治疗途径。已知转录因子Twist1在 恶性肿瘤在转移进展中起作用；然而，在黑色素瘤中，人们对Twist1知之甚少 受调控的基因。对Twist1调节转录组的微阵列分析发现了一种阴性 调节黏附分子，CADM1。在其他癌症中，CADM1已被证明是一种肿瘤 抑制因子，是NK和CD8+T细胞的共刺激分子。CADM1在黑色素瘤中的作用 很大程度上是未知的。我们的初步数据表明，CADM1抑制黑色素瘤的侵袭/迁移， 促进失巢凋亡，并至少部分受到启动子甲基化的抑制。此外，TCGA分析 证明CADM1水平高的患者对免疫疗法有更好的反应。这些数据 是这项提案的基础，该提案进一步研究了CADM1在黑色素瘤中的作用。这项提议将 解决以下具体目标，每个目标都有一个指导(K99)和一个独立的(R00)组件：1. 确定CADM1的结构域和翻译后修饰，这些结构域和翻译后修饰有助于其抗转移功能。 我们将检测突变的CADM1在促进细胞失巢、减少侵袭和抑制细胞外渗方面的能力。 体内模型(K99)，并分析下游的CADM1效应蛋白(R00)。2.)找出 Twist1介导的CADM1在黑色素瘤中的调控机制组织微阵列将用于评估 CADM1的表达是疾病分期的函数，我们将定义Twist1在CADM1启动子中的作用 甲基化(K99)。我们还将鉴定Twist1相关的DNA甲基转移酶(S)(R00)。3.)确定是否 CADM1的缺失有助于黑色素瘤的免疫逃避。黑色素瘤细胞的调控表达 CADM1将接受体外细胞毒性测试以及体内肿瘤生长/肿瘤浸润性检测 淋巴细胞检测(K99)。此外，我将敲除可能的CADM1共同受体(CRTAM)在 T细胞，并检测检查点阻断抑制剂对CADM1调节的肿瘤(R00)的疗效。 成功完成这些研究与我将在指导阶段接受的培训有关 获奖。为了协助这一过程，我召集了一个由导师安德鲁·阿普林博士领导的顾问小组 (托马斯·杰斐逊大学-TJU)，以及Meenhard Herlyn博士(Wistar研究所)，Mauricio Reginato (德雷克塞尔大学)、克里斯托弗·斯奈德(TJU)和保罗·福蒂纳(TJU)。这个小组将帮助指导我的研究 并将支持我的培训努力，作为我向独立调查员过渡的一部分。已完成的 该提案中的目标将定义黑色素瘤中未知转移抑制因子的功能，并提供 我拥有成为一名成功的NIH资助的独立调查员所需的技能和培训。

ABSTRACT: Melanoma is the deadliest form of skin cancer as patients presenting with metastatic disease have a five-year survival of only 2-16%. However, early clinical intervention prior to metastatic dissemination yields ten-year survival rates of ~92%. We aim to study how melanoma cells gain metastatic potential, hoping to expose future therapeutic avenues. The transcription factor, TWIST1 is known to be elevated in malignancies and play a role in metastatic progression; yet in melanoma little is known about TWIST1 regulated genes. Microarray analysis of the TWIST1 modulated transcriptome uncovered a negatively regulated adhesion molecule, CADM1. In other cancers, CADM1 has been shown to act as a tumor suppressor and is a co-stimulatory molecule for NK and CD8+ T-cells. How CADM1 functions in melanoma is largely unknown. Our preliminary data suggest that CADM1 suppresses melanoma invasion/migration, promotes anoikis, and is repressed at least in part by promoter methylation. Additionally, TCGA analysis demonstrates that patients with high CADM1 levels have a better response to immunotherapies. These data are the basis of this proposal which further examines how CADM1 functions in melanoma. The proposal will address the following specific aims, each having a mentored (K99) and an independent (R00) component: 1.) Identify domains and post-translational modifications of CADM1 that contribute to its anti-metastatic function. We will assay the ability of mutant CADM1 to promote anoikis, reduce invasion, and suppress extravasation in an in vivo model (K99), and analyze the downstream CADM1 effector proteins (R00). 2.) Identify the mechanism of TWIST1 mediated CADM1 regulation in melanoma. Tissue microarrays will be used to evaluate CADM1 expression as a function of disease stage, and we will define a role for TWIST1 in CADM1 promoter methylation (K99). We will also identify TWIST1 associated DNA methyltransferase(s) (R00). 3.) Determine if loss of CADM1 contributes to melanoma immune evasion. Melanoma cells with modulated expression of CADM1 will be subjected to in vitro cytotoxicity assays as well as in vivo tumor growth/tumor infiltrating lymphocyte assessments (K99). Furthermore, I will knock-down the putative CADM1 co-receptor (CRTAM) on T-cells, and test the efficacy of checkpoint blockade inhibitors against CADM1 modulated tumors (R00). Successful completion of these studies is linked to the training I will receive during the mentored phase of this award. To assist with this process, I have assembled a group of advisors led by mentor Dr. Andrew Aplin (Thomas Jefferson University - TJU), along with Drs. Meenhard Herlyn (Wistar Institute), Mauricio Reginato (Drexel University), Christopher Snyder (TJU), and Paolo Fortina (TJU). This group will help guide my research and will support my training efforts as part of my transition to an independent investigator. Completion of the goals in this proposal will define the function of an unknown metastatic suppressor in melanoma and provide me with the skills and training necessary to be a successful NIH funded independent investigator.