Influence of Melanocyte Differentiation State on Melanoma Susceptibility

项目总结： 黑色素瘤是最致命的皮肤癌，美国每年有超过9万例新病例。存在以下风险 皮肤轻度色素沉着的人(在美国为1：38)患黑色素瘤的几率比那些 皮肤呈深色(美国为1：1000)。尽管这种差异通常归因于物理上的 黑色素的紫外线屏蔽作用，黑色素的防晒系数(SPF)只有2-3，这似乎 这不足以解释黑色素瘤发病率在深色和浅色皮肤中的40倍差异。我们的 初步数据显示，来自浅色皮肤的原代人类黑素细胞(MC)产生的数量较少 黑色素相对于来自深色皮肤的初级MC，这与MC分化降低有关 状态和增强的增殖能力。此外，细胞增殖和分化之间的差异 在具有医学相关黑色素瘤驱动因素的原发MC转化过程中，亮和暗MC持续存在 (BRAFV600E、CDK4R24C、P53R248W和hTERT)。来测试这些差异是否会转化为不同的黑色素瘤 在体内的表型，我设计了建筑上忠实的3-D人类皮肤，并原位异种移植 SCID小鼠身上的组织。我的初步数据显示，转化的浅色MC形成早期黑色素瘤，而 转变后的黑色MC则不会。这一观察结果让我们怀疑这种明显的差异是否是通过中介来实现的 黑色素合成本身，或黑色素合成的中间体，如二羟基苯丙氨酸(DOPA)。 我们发现，与浅色MC相比，深色MC含有大约300%的DOPA。 以前的工作和我的初步数据表明，DOPA作为信号分子发挥作用，并抑制 扩散。具体地说，我发现外源性供应的DOPA诱导轻度MC和 黑色素瘤细胞，但对原代暗色MC没有影响，可能含有内源性饱和水平 生产了DOPA。多巴酚A抗增殖作用的机制(S)目前尚不清楚，但我的 初步数据显示，DOPA抑制典型的GQ GPCR通路，导致MAPK和 PI3K/AKT信号通路。在与北卡罗来纳大学的布莱恩·罗斯博士的合作中， 我们进行了功能筛查，以测试DOPA是否与~320个非嗅觉GPCR中的任何一个结合 人类基因组。最热门的是GQ偶联的代谢性谷氨酸受体5(GRM5)，这是一种已知的 黑色素瘤司机。目标1将集中于确定DOPA的抗增殖作用的机制，通过 特别是它对GRM5的抑制作用，或我们GPCR筛查的其他热门药物。此外，我 确定DOPA在体内抑制医学上相关的BRAF驱动的同基因黑色素瘤的生长。目标2 目的：进一步了解DOPA在黑色素瘤抑制中的内源性和外源性作用。 体内与医学相关的黑色素瘤模型。总而言之，这些目标将有助于定义未被开发的 黑色素瘤病理生物学与浅色人黑色素瘤增加有关，并可能发现新的 使黑色素瘤患者受益的治疗目标。

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.