The role of CADM1 in malignant melanoma

摘要：黑色素瘤是皮肤癌中最致命的一种，患者会出现转移性疾病 五年存活率只有2- 16%。然而，转移性播散前的早期临床干预 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能， 来揭示未来的治疗方法已知转录因子TWIST 1在人乳腺癌中升高， 在恶性肿瘤中，TWIST 1在转移性进展中起作用;但在黑色素瘤中，TWIST 1的作用知之甚少 调控基因对TWIST 1调节的转录组的微阵列分析发现， 调节粘附分子1。在其他癌症中，CADM 1已被证明是一种肿瘤， 是NK和CD 8 + T细胞的共刺激分子。CADM 1在黑色素瘤中的作用 大部分未知。我们的初步数据表明CADM 1抑制黑色素瘤侵袭/迁移， 促进失巢凋亡，并且至少部分被启动子甲基化抑制。此外，TCGA分析 研究表明，具有高CADM 1水平的患者对免疫疗法有更好的反应。这些数据 是进一步研究CADM 1在黑色素瘤中如何发挥作用的这项提议的基础。该提案将 解决以下具体目标，每个都有一个指导（K99）和一个独立（R 00）的组成部分：1。 确定CADM 1的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CADM 1促进失巢凋亡、减少侵袭和抑制血管外渗的能力。 体内模型（K99），并分析下游CADM 1效应蛋白（R 00）。2.）识别 TWIST 1介导的黑色素瘤中CADM 1调节的机制。组织微阵列将用于评估 CADM 1表达作为疾病阶段的函数，我们将定义TWIST 1在CADM 1启动子中的作用， 甲基化（K99）。我们还将鉴定TWIST 1相关的DNA甲基转移酶（R 00）。3.）第三章确定是否 CADM 1的缺失有助于黑色素瘤免疫逃避。黑色素瘤细胞具有调节表达的 CADM 1将进行体外细胞毒性测定以及体内肿瘤生长/肿瘤浸润测定。 淋巴细胞评估（K99）。此外，我将敲低假定的CADM 1共受体（CRTAM）， T细胞，并测试检查点阻断抑制剂对CADM 1调节的肿瘤（R 00）的功效。 成功完成这些研究与我将在本项目的指导阶段接受的培训有关。 奖为了协助这一进程，我召集了一组顾问，由导师安德鲁·阿普林博士领导。 （托马斯杰斐逊大学- TJU），沿着有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 并将支持我的培训工作，作为我向独立调查员过渡的一部分。完成 本提案的目标将定义一种未知的转移抑制因子在黑色素瘤中的功能， 我拥有成为一名成功的NIH资助的独立研究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.