Influence of Melanocyte Differentiation State on Melanoma Susceptibility

项目摘要： 黑色素瘤是最致命的皮肤癌，在美国每年有超过90，000例新发病例。的风险 患有黑色素瘤的人与浅色皮肤的人（美国为1：38）相比， 深色皮肤（美国为1：1000）。虽然这种差异通常归因于物理 黑色素的紫外线屏蔽作用，黑色素的防晒系数（SPF）只有2-3， 不足以解释深色和浅色皮肤中黑色素瘤发病率的40倍差异。我们 初步数据显示，来自浅色皮肤的原代人黑素细胞（MC）产生的 黑色素相对于来自深色皮肤的原发性MC，这与MC分化降低有关 状态和增加增殖能力。此外，细胞增殖和分化的差异， 在具有医学相关黑色素瘤驱动因素的原发性MC转化后，亮和暗MC持续存在 （BRAFV600E、CDK4R24C、P53R248W和hTERT）。为了测试这些差异是否会转化为不同的黑素瘤 在体内的表型，我设计了建筑忠实的3-D人类皮肤和原位异种移植， SCID小鼠上的组织。我的初步数据显示，转化的浅色MC形成早期黑素瘤，而 转化的深色MC则没有。这一观察结果使我们怀疑，这种明显的差异是否是由 通过黑色素合成本身，或通过黑色素合成的中间体，如二羟基苯丙氨酸（DOPA）。 我们发现，深色色素的MC含有约300%以上的多巴，相比，轻MC。 先前的工作沿着我的初步数据表明，多巴作为一种信号分子， 增殖具体来说，我发现外源性多巴诱导轻MC黑色素合成， 黑色素瘤细胞，但对原发性暗MC没有影响，其可能含有饱和水平的内源性 生产DOPA。多巴抗增殖作用的机制目前尚不清楚，但我的研究表明， 初步数据表明，多巴抑制经典的Gq GPCR途径，导致MAPK的减少， PI 3 K/AKT信号通路。在与北卡罗来纳州大学的布莱恩·罗斯博士的合作中， 我们进行了一项功能性筛选，以测试DOPA是否结合到大脑中的~320个非嗅觉GPCR中的任何一个。 人类基因组最受欢迎的是Gq偶联的代谢型谷氨酸受体5（GRM 5），这是一种已知的 黑色素瘤司机。目的1将集中于确定多巴的抗增殖作用的机制， 特别关注其对GRM 5的抑制作用，或来自我们GPCR筛选的其他顶级命中。而且我 确定多巴抑制体内医学相关的同基因BRaf驱动的黑素瘤的生长。目的2 试图进一步了解多巴在黑色素瘤抑制中的内源性和外源性作用， 医学相关的体内黑素瘤模型。总之，这些目标将有助于确定尚未探索的领域， 黑色素瘤病理生物学负责增加黑色素瘤在浅色的人，并可能确定新的 治疗目标，以造福黑色素瘤患者。

Project Summary: Melanoma is the most lethal skin cancer with over 90,000 new cases in United States each year. The risk of developing melanoma is substantially greater for people with lightly pigmented skin (1:38 in the US) than those with darkly pigmented skin (1:1000 in the US). Although this discrepancy is generally attributed to the physical UV shielding effect of melanin pigment, the sun protective factor (SPF) of melanin is only 2-3, which seems insufficient to explain the 40-fold difference of melanoma incidence in darkly and lightly pigmented skin. Our preliminary data show that primary human melanocytes (MCs) from lightly pigmented skin produce less melanin relative to primary MCs from dark skin, and this is associated with a decreased MC differentiation state and increased proliferative capacity. Additionally, the proliferation and differentiation differences between light and dark MCs persist upon transformation of primary MCs with medically relevant melanoma drivers (BRAFV600E, CDK4R24C, P53R248W, and hTERT). To test if these differences translated into different melanoma phenotypes in vivo, I engineered architecturally faithful 3-D human skin and orthotopically xenografted the tissue on SCID mice. My preliminary data show that transformed light MCs form early melanomas, whereas transformed dark MCs do not. This observation led us to question whether this stark difference was mediated by melanin synthesis itself, or by intermediates of melanin synthesis, such as dihydroxyphenylalanine (DOPA). We found that darkly pigmented MCs contain approximately 300% more DOPA, as compared to light MCs. Previous work along with my preliminary data show that DOPA serves as a signaling molecule and inhibits proliferation. Specifically, I found that exogenously supplied DOPA induces melanin synthesis in light MCs and melanoma cells, but has no effect on primary dark MC, which likely contain saturating levels of endogenously produced DOPA. The mechanism(s) of the DOPA anti-proliferative effect are currently unknown, but my preliminary data show that DOPA inhibits canonical Gq GPCR pathways leading to a decrease in MAPK and PI3K/AKT signaling pathways. In a collaborative effort with Dr. Bryan Roth at the University of North Carolina, we conducted a functional screen to test whether DOPA binds to any of the ~320 non-olfactory GPCRs in the human genome. The top hit was the Gq-coupled metabotropic glutamate receptor 5 (GRM5), which is a known melanoma driver. Aim 1 will focus on determining the mechanism of DOPA’s anti-proliferative effect, by specifically focusing on its inhibitory role of GRM5, or other top hits from our GPCR screen. Additionally, I determined that DOPA inhibits growth of medically relevant syngeneic BRaf-driven melanoma in vivo. Aim 2 seeks to further understand the endogenous and exogenous roles of DOPA in melanoma inhibition in medically-relevant melanoma models in vivo. Together, these aims will help to define the underexplored melanoma pathobiology responsible for increased melanoma in lightly pigmented people, and may identify new therapeutic targets to benefit melanoma patients.