BMP7 in melanoma niche morphogenesis and homeostasis

摘要 黑色素瘤可以说是人类癌症中最致命的，部分原因是它容易转移， 以及它对传统抗癌疗法的抵抗力。导致治疗失败的一个关键因素是 肿瘤的异质性，特别是具有干细胞样特性的亚群，称为黑色素瘤 启动电池(MIC)。我们的长期目标是了解MICS和MICS的分子机制 它们的基质共同促进生长和进展，作为开发有效治疗方法的一种手段 战略。在当前的项目中，我们重点定义了CD133+/ABCB5+的微环境“生态位” MICS作为一扇大门，阐明维持新陈代谢和 麦克风的复制完整性。我们的初步数据表明：1)CD133+/ABCB5+MICs在空间上是 以与血管生成拟态(Vm)相同的模式排列；2)如此形成的管状通道 VM本身就是CD144(VE-钙粘蛋白)+，并与真实的内皮结构密切相关，在 3)骨形态发生蛋白7在黑色素瘤细胞中的表达 (Bmp7)及其拮抗剂Noggin与肿瘤进展相关，并共同定位于VM区域； 4)BMP7上调间质微环境中血管生成因子血管内皮生长因子的表达 诱导Noggin缺陷黑色素瘤细胞选择性凋亡。总体而言，我们的调查结果支持中央 假设CD133+/ABCB5+MIC相关的BMP7可能不仅通过以下方式支持MIC的维持 促进“利基”形态发生(通过协调的VM和血管生成)，但也平衡代谢 需求(通过刺激血管内皮生长因子依赖的血管生成和诱导选择性细胞凋亡 竞争、诺金缺陷、非启蒙人群)。利用多标记免疫荧光和免疫- 引导激光捕获显微切割和qRT-PCR结合功能循环分析，我们 建议进一步描绘黑色素瘤细胞CD133、ABCB5、BMP7之间的时空关系 和CD144的表达，以及微循环的类型/模式(例如黑色素瘤VM通道形成与 体内血管生成)(目标1a)。“生态位”中CD133+黑色素瘤亚群的动态变化及功能影响 形成(目标1b)将在使用可诱导的RNAi介导的敲除的原位异种移植模型中进行测试 以不同的速度。最后，剖析了CD133+/ABCB5+MIC相关的BMP7分子信号。 形态发生(目标2a)和肿瘤异质性/内稳态(目标2b)，我们将使用- 功能方法评估BMP7-VEGF/BMP7-Noggin轴，在组织环境中独特的三个- 体外三维器官培养和体内黑色素瘤异种移植模型。定义机制 通过它，与MIC相关的BMP7信号有助于“利基”开发和维护提供 通过针对MIC的间质依赖，直接或间接地通过治疗消除MIC的新机会。

Summary Melanoma is arguably the most virulent among human cancers, in part due to its propensity to metastasize, and its resistance to conventional anti-cancer therapies. One key factor responsible for treatment failure relates to tumor heterogeneity, particularly subpopulations that possess stem cell-like properties, known as melanoma initiating cells (MICs). Our long-term goal is to understand the molecular mechanisms, whereby MICs and their stroma collaborate to promote growth and progression, as a means to develop effective therapeutic strategies. In the current project, we focus on defining the microenvironmental "niche", of CD133+/ABCB5+ MICs as a gateway to elucidate the complex cellular and molecular interplay that maintains the metabolic and replicative integrity of MICs. Our preliminary data indicate that : 1) the CD133+/ABCB5+ MICs are spatially arranged in a pattern identical to that of "vasculogenic mimicry (VM)"; 2) the vessel-like channels so formed in VM are themselves CD144 (VE-cadherin)+ and intimately associated with authentic endothelial structures, in keeping with so-called "perivascular niches"; 3) melanoma cell expression of bone morphogenetic protein 7 (BMP7) and its antagonist Noggin is associated with tumor progression and also co-localizes to areas of VM; and 4) BMP7 upregulates the angiogenic factor VEGF in the stromal microenvironment, but at the same time induces selective apoptosis in Noggin-deficient melanoma cells. Collectively, our findings support the central hypothesis that CD133+/ABCB5+ MIC-associated BMP7 may not only support the maintenance of MICs by facilitating "niche" morphogenesis (through coordinated VM and angiogenesis), but also balance metabolic demands (through stimulating VEGF-dependent angiogenesis and inducing selective apoptosis in the competitive, Noggin-deficient, non-initiating population). Using multi-label immunofluorescence and immuno- guided laser capture microdissection followed by qRT-PCR, together with functional circulation analysis, we propose to further delineate the temporal/spatial relationship between melanoma cell CD133, ABCB5, BMP7 and CD144 expression, and the types/ patterns of microcirculation (e.g. melanoma VM channel formation vs. angiogenesis) in vivo (Aim 1a). The dynamics and functional impacts of CD133+ melanoma subsets in "niche" formation (Aim 1b) will be tested in an orthotopic xenograft model using inducible RNAi-mediated knockdown at varying tempos. Finally, to dissect the CD133+/ ABCB5+ MIC-associated BMP7 molecular signals in "niche" morphogenesis (Aim 2a) and tumor heterogeneity/ homeostasis (Aim 2b), we will employ loss- and gain-of- function approaches to assess the BMP7-VEGF/ BMP7-Noggin axes, in a tissue context in unique three- dimensional (3D) organotypic cultures in vitro and melanoma xenograft models in vivo. Defining mechanisms through which MIC-associated BMP7 signals contribute to "niche" development and maintenance offers a novel opportunity to therapeutically eliminate MICs directly or indirectly by targeting their stromal dependency.