BMP7 in melanoma niche morphogenesis and homeostasis

描述（由申请人提供）：黑色素瘤可以说是人类癌症中最致命的，部分原因是其转移倾向及其对常规抗癌疗法的抗性。 导致治疗失败的一个关键因素与肿瘤异质性有关，特别是具有干细胞样特性的亚群，称为黑色素瘤起始细胞（MIC）。 我们的长期目标是了解MIC及其基质协同促进生长和进展的分子机制，作为开发有效治疗策略的一种手段。 本研究的主要目的是阐明CD 133 +/ABCB 5 + MIC的微环境“生态位”，以阐明维持MIC代谢和复制完整性的复杂的细胞和分子相互作用。2）在VM中如此形成的血管状通道本身是CD 144 3）骨形态发生蛋白7（BMP 7）及其拮抗剂头蛋白的黑素瘤细胞表达与肿瘤进展相关，并且还共定位于VM区域; 4）BMP 7上调基质微环境中的血管生成因子VEGF，但同时诱导Noggin缺陷型黑素瘤细胞的选择性凋亡。 总的来说，我们的研究结果支持了中心假设，即CD 133 +/ABCB 5 + MIC相关的BMP 7不仅可以通过促进“小生境”形态发生（通过协调VM和血管生成）来支持MIC的维持，而且还可以平衡代谢需求（通过刺激VEGF依赖性血管生成并在竞争性、Noggin缺陷、非起始群体中诱导选择性凋亡）。 使用多标记免疫荧光和免疫导向激光捕获显微切割，然后进行qRT-PCR，结合功能循环分析，我们建议进一步描述体内黑色素瘤细胞CD 133、ABCB 5、BMP 7和CD 144表达与微循环类型/模式（例如黑色素瘤VM通道形成与血管生成）之间的时间/空间关系（目的1a）。 将在原位异种移植模型中使用不同节奏的诱导型RNAi介导的敲减来测试CD 133+黑色素瘤亚群在“小生境”形成（Aim 1b）中的动力学和功能影响。 最后，为了剖析“小生境”形态发生（Aim 2a）和肿瘤异质性/稳态（Aim 2b）中的CD 133 +/ABCB 5 + MIC相关的BMP 7分子信号，我们将采用功能丧失和获得的方法来评估BMP 7-VEGF/BMP 7-Noggin轴，在独特的三维（3D）器官型体外培养物和体内黑素瘤异种移植模型中的组织背景下。 确定MIC相关的BMP 7信号有助于“小生境”发展和维持的机制提供了一个新的机会，通过靶向其基质依赖性直接或间接地治疗性消除MIC。 公共卫生相关性：众所周知，黑素瘤对常规抗癌疗法具有抗性，这主要归因于具有干细胞样特性的细胞亚群，称为黑素瘤起始细胞（MIC）。 本研究的目的是阐明MIC及其组织环境合作，以确保生存和促进肿瘤生长的机制。 我们认为，定义这样的机制提供了一个新的机会，治疗消除MIC直接或间接针对他们的依赖组织环境。

DESCRIPTION (provided by applicant): 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 immunoguided 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. PUBLIC HEALTH RELEVANCE: Melanoma is notoriously resistant to conventional anti-cancer therapies, largely attributed to a subpopulation of cells with stem cell-like properties known as melanoma-initiating cells (MICs). The purpose of this study is to elucidate the mechanisms whereby MICs and their tissue environment collaborate to ensure survival and to promote tumor growth. We believe that defining such mechanisms offers a novel opportunity to therapeutically eliminate MICs directly or indirectly by targeting their dependency on tissue environment.