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

总结 一个单一的驱动突变BRAFV 600 E驱动了一半的黑色素瘤。然而，在大多数情况下， BRAFV 600 E的获得反而驱动良性肿瘤，如黑素细胞痣（普通痣）。我们寻求 破译阻止完全转化的细胞内机制，并利用这些知识， 制定候选的早期诊断和化学预防策略。我们发现了一个 微小RNA（miRNA）作为区别痣与正常痣或正常痣的最差异表达的转录本， 黑色素细胞或黑素瘤。我们已经报道了这些miRNAs的表达可以将活检的色素沉着的 诊断准确率高的肿瘤。为了确定这些miRNAs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 MIR 211 - 5 p的靶点。使用新鲜分离和CRISPR工程改造的人类 正常，痣和黑色素瘤黑素细胞，我们确定AURKB表达的抑制是关键的 在体外驱动BRAFV 600 E和MIR 211 - 5 p两者相关生长停滞的机制。因此，在目标1中， 目的是评估MIR 211 - 5 p/AURKB轴在体内痣形成和转化中的作用， 在黑色素瘤化学预防中破坏该轴的功效。其次，我们建立了一个非侵入性的检测方法， 用于活检前色素性病变的miRNA筛查。在一项小型试点研究中，我们发现 保留黑素细胞瘤形成的分类。在目标2中，我们的目标是验证使用 miRNA特征的非侵入性分析以筛选色素性皮肤病变。 我们的建议有三个特点。首先是我们的miRNA分类器的可重复性，目前 在六个独立的数据集上验证。其次，我们的模型系统，这种疾病工程的主要 人黑色素细胞、痣和黑色素瘤，结合体内系统， 黑色素瘤的遗传学和进展-使我们处于一个独特的位置，以控制特定的影响， 在研究这些重大事件时第三，我们开发了一种非侵入性的miRNA分析方法， 分子分析技术，这是非侵入性和病变特异性。我们的团队由以下领域的专家组成： miRNA和黑色素瘤的基础生物学，黑色素瘤的体内模型，局部药物递送，以及 黑色素瘤监测的日常实践，使我们能够全面应对这一项目。 本课题既有基础意义，又有临床应用潜力。我们的研究探索了痣的新解释 启动，由临床病变观察结果驱动。我们希望这些研究能直接导致 黑色素瘤的早期检测增加和局部给药策略的临床前验证 化学预防用于特别高风险的个体和/或解剖区域。

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.