BMP7 in melanoma niche morphogenesis and homeostasis

描述（由申请人提供）：黑色素瘤被认为是人类癌症中最致命的，部分原因是它的转移倾向，以及对传统抗癌疗法的耐药性。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，即黑色素瘤起始细胞（MICs）。我们的长期目标是了解MICs及其基质协同促进生长和进展的分子机制，以此作为制定有效治疗策略的手段。在当前的项目中，我们专注于定义CD133+/ABCB5+ mic的微环境“生态位”，作为阐明维持mic代谢和复制完整性的复杂细胞和分子相互作用的门户。我们的初步数据表明：1)CD133+/ABCB5+ mic在空间上的排列模式与“血管源性模仿（VM）”相同；2) VM中形成的血管样通道本身是CD144 (VE-cadherin)+，与真实的内皮结构密切相关，与所谓的“血管周围壁龛”保持一致；3)黑色素瘤细胞中骨形态发生蛋白7 （bone morphogenetic protein 7, BMP7）及其拮抗剂Noggin的表达与肿瘤进展相关，并可共定位于VM区域；4) BMP7上调基质微环境中血管生成因子VEGF，同时诱导noggin缺陷黑色素瘤细胞选择性凋亡。总的来说，我们的研究结果支持了CD133+/ABCB5+ mic相关的BMP7可能不仅通过促进“生态位”形态发生（通过协调VM和血管生成）来支持mic的维持，而且还平衡代谢需求（通过刺激vegf依赖性血管生成和诱导竞争性、noggin缺陷、非初始化群体的选择性凋亡）。利用多标记免疫荧光和免疫引导激光捕获显微解剖以及qRT-PCR，结合功能循环分析，我们建议进一步描述黑色素瘤细胞CD133、ABCB5、BMP7和CD144表达与体内微循环类型/模式（例如黑色素瘤VM通道形成与血管生成）之间的时空关系（Aim 1a）。CD133+黑色素瘤亚群在“生态位”形成（Aim 1b）中的动力学和功能影响将在原位异种移植模型中进行测试，使用不同节奏的rnai介导的诱导敲除。最后，为了解剖CD133+/ABCB5+ mic相关的BMP7分子信号在“生态位”形态发生（Aim 2a）和肿瘤异质性/稳态（Aim 2b）中的作用，我们将在体外独特的三维（3D）器官型培养和体内黑色素瘤异种移植模型的组织背景下，采用功能丧失和功能获得的方法来评估BMP7- vegf /BMP7- noggin轴。明确mic相关BMP7信号促进“生态位”发展和维持的机制，为通过靶向基质依赖性直接或间接消除mic提供了新的机会。

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.