Nevus associated microRNAs as mediators of BRAF-induced growth arrest and biomarkers of melanoma progression

摘要 一个单一的驱动程序突变BRAFV600E导致了一半的黑色素瘤。然而，在大多数情况下， BRAFV600E的获得反而会导致良性肿瘤，如黑色素细胞痣(常见的葡萄胎)。我们在寻找 破译阻止完全转化的细胞内机制，并利用这一知识 制定候选的早期诊断和化学预防策略。我们发现了一个签名 MicroRNAs(MiRNA)作为区分色素痣和正常皮肤的最具差异性的转录本 黑素细胞或黑色素瘤。我们已经报道，这些miRNAs的表达将活检后的色素 诊断准确率高的肿瘤。为了确定这些miRNA的知识是否有助于 黑色素瘤的预防：首先，我们对黑色素瘤进行了全面的鉴定和功能筛选 最具预测性的miRNA的靶点是MIR211-5p。使用新鲜分离的CRISPR工程人 在正常黑素细胞、黑色素瘤黑素细胞和黑色素瘤黑素细胞中，我们发现抑制AURKB的表达是一个关键 BRAFV600E和MIR211-5P共同抑制体外生长的机制。因此，在目标1中，我们的 目的探讨MIR211-5p/AURKB轴在体内痣形成和转化中的作用。 破坏这一轴在黑色素瘤化学预防中的有效性。其次，我们产生了一种非侵入性检测方法 用于在活检前对有色素性病变进行miRNA筛选。在一项小型的初步研究中，我们发现， 保留黑素细胞瘤的分类。在目标2中，我们的目标是验证使用 对miRNA签名的非侵入性分析以筛选有色素性皮肤病变。 我们的提案有三个突出之处。第一个是我们的miRNA分类器的重复性，目前 在六个独立的数据集上进行了验证。第二，我们针对这种疾病设计的初级模型系统 人类黑素细胞、痣和黑色素瘤，与体内系统相结合，概括了 黑色素瘤的遗传和进展--使我们处于独特的地位来控制特定于环境的影响 在研究这些关键事件时。第三，我们正在开发一种非侵入性的miRNA分析方法，一种 分子图谱技术既是非侵入性的，又是病变特异性的。我们的团队由以下领域的专家组成 MiRNA和黑色素瘤的基本生物学，黑色素瘤的体内模型，局部给药，以及 对黑色素瘤的日常监测，使我们能够全面应对这一项目。 本项目具有基础和临床潜在意义。我们的研究探索了对雀斑的新解释 启动，由临床病变的观察驱动。我们预计这些研究将直接导致 早期发现黑色素瘤的增加和局部治疗策略的临床前验证 针对特别高危个人和/或解剖区域的化学预防。

Summary A single driver mutation, BRAFV600E, drives half of all melanomas. However, in the majority of cases, acquisition of BRAFV600E instead drives benign tumors, such as melanocytic nevi (the common mole). We seek to decipher the intracellular mechanisms that prevent full transformation, and harness this knowledge to develop candidate early diagnosis and chemoprevention strategies. We have discovered a signature of microRNAs (miRNA) as the most differentially expressed transcripts distinguishing nevi from either normal melanocytes or melanomas. We have reported that expression of these miRNAs classifies biopsied pigmented neoplasms with high diagnostic accuracy. To determine whether knowledge of these miRNAs could aid in the prevention of melanoma: First, we conducted a comprehensive identification and functional screen of the targets of the most predictive miRNA, MIR211-5p. Using freshly isolated and CRISPR engineered human normal, nevus and melanoma melanocytes, we identified inhibition of AURKB expression as a critical mechanism driving both BRAFV600E and MIR211-5p associated growth arrest in vitro. Therefore, in Aim 1, our objective is to assess the roll of the MIR211-5p/AURKB axis in nevus formation and transformation in vivo, and the efficacy of disrupting this axis in melanoma chemoprevention. Second, we generated a non-invasive assay for miRNA screening of pigmented lesions prior to biopsy. In a small pilot study, we found the high accuracy of classification of melanocytic neoplasia was retained. In Aim 2, our objective is to validate the utility of using non-invasive profiling of the miRNA signature to screen pigmented skin lesions. Three advances distinguish our proposal. The first is the reproducibility of our miRNA classifier, currently validated on six independent datasets. Second, our model systems for this disease—engineered primary human melanocytes, nevi, and melanomas, combined with an in vivo system that recapitulates both the genetics and progression of melanoma—puts us in a unique position to control for context-specific effects when studying these critical events. Third, is our development of a non-invasive miRNA profiling assay, a molecular profiling technique that is both non-invasive and lesion-specific. Our team, consisting of experts in both the basic biology of miRNA and melanoma, in vivo models of melanoma, topical drug delivery, and the daily practice of melanoma surveillance, allows us to comprehensively tackle this project. This project has both basic and clinical potential significance. Our studies explore novel explanations for nevus initiation, driven by observations made from clinical lesions. We expect these studies to directly result in an increase in the early detection of melanomas and preclinical validation of a strategy for topical chemoprevention for particularly high-risk individuals and/oror anatomic areas.