Regulation and Role of miR-183-96-182 in Melanocyte Differentiation and Melanoma

描述（由申请人提供）：黑色素瘤是最具侵袭性的皮肤癌，其发病率和死亡率在全球范围内迅速上升。低生存率源于这些肿瘤的高转移性和化疗耐药行为，但这种侵袭性表型的分子基础尚不清楚。我们和其他人已经证明黑色素瘤细胞表现出干细胞样的特性：它们通常表达发育基因，具有多分化潜力，似乎唤起了黑色素细胞产生的神经嵴干细胞的迁移性质。这些观察结果表明，黑素细胞谱系中发育程序的改变可能是这些细胞向转移性黑色素瘤恶性进化的基础。事实上，转移可以被认为是正常的迁移程序出错，我们的实验室已经发现，在黑色素瘤细胞系和组织中，一组特定的mirna（通常调节发育、分化和组织稳态）经常过度表达。我们已经证明，这种miR-183-96-182簇通过控制MITF的表达来促进体外迁移和体内转移，MITF是黑素细胞分化的主要调节剂。有趣的是，我们还发现miR-183-96-182在胚胎干细胞（ES）中高度表达，并且在体外黑素细胞分化过程中沉默，与MITF水平呈负相关。此外，我们的初步研究表明，组蛋白乙酰化和krupple样因子-4 (KLF4)，一种典型的多能转录因子，控制miR-183-96-182在hESCs和黑色素瘤细胞中的表达。我们假设miR-183-96-182抑制是黑素细胞分化所必需的，并且该簇的改变可能通过赋予黑素细胞干细胞特性来促进黑素瘤转移。为了验证这些假设，我们将首先确定miR-183-96-182在生理和病理背景下的转录和表观遗传调控，从人类胚胎干细胞阶段到黑素细胞分化和黑色素瘤细胞（目的1）。在Aim 2中，我们将通过几种基于细胞的测定来测试miR-183-96-182和/或KLF4是否赋予黑色素瘤细胞干细胞样特性（例如，自我更新、多分化能力）。在Aim 3中，我们将确定该miRNA簇和/或KLF4的调节如何影响小鼠黑色素瘤细胞的转移倾向。了解调节miR-183-96-182过表达的机制，这显然与转移有关，可以为改变这种极具侵袭性的癌症的侵袭性提供一个立足点，并可能为其他顽固性癌症类型的类似研究提供一个框架。

DESCRIPTION (provided by applicant): The incidence and mortality of melanoma, the most aggressive form of skin cancer, are rapidly increasing worldwide. The abysmal survival rates stem from the highly metastatic and chemoresistant behavior of these tumors, but the molecular basis of this aggressive phenotype remains unclear. We and others have shown that melanoma cells display stem-cell-like properties: they often express developmental genes, have multi-differentiation potential, and seem to evoke the migratory nature of neural crest stem cells from which melanocytes arise. These observations suggest that alterations in developmental programs within the melanocytic lineage might underlie the malignant evolution of these cells into metastatic melanoma. In fact, metastasis can be conceived as a normal migration program gone awry, and our laboratory has found that a particular cluster of miRNAs (which in general regulate development, differentiation, and tissue homeostasis) is frequently overexpressed in melanoma cell lines and tissues. We have shown that this miR-183-96-182 cluster promotes migration in vitro and metastasis in vivo in part by controlling the expression of MITF, a master regulator of melanocyte differentiation. Intriguingly, we have also found that miR-183-96-182 is highly expressed in embryonic stem cells (ES), and is silenced during in vitro melanocyte differentiation, inversely correlating with MITF levels. Furthermore, our preliminary studies indicate that histone acetylation and Krupple-like factor-4 (KLF4), a canonical pluripotency transcription factor, govern miR-183-96-182 expression in both hESCs and melanoma cells. We hypothesize that miR-183-96-182 repression is required for melanocyte differentiation, and that alterations in this cluster promote melanoma metastasis, perhaps by conferring stem cell properties to melanoma cells. To test these hypotheses, we will first determine the transcriptional and epigenetic regulation of the miR-183-96-182 cluster in physiological and pathological contexts, from human embryonic stem cell stage through melanocyte differentiation and in melanoma cells (Aim 1). In Aim 2 we will test whether miR-183-96-182 and/or KLF4 confer stem-cell-like properties (e.g., self-renewal, multi-differentiation capacity) on melanoma cells through several cell-based assays. In Aim 3, we will determine how modulation of this miRNA cluster and/or KLF4 influence the propensity of melanoma cells for metastasis in mice. Understanding the mechanisms that modulate miR-183-96-182 overexpression, which is clearly involved in metastasis, could provide a foothold for altering the aggressiveness of this very aggressive cancer, and perhaps provide a framework for similar studies in other recalcitrant cancer types.