BMP7 in melanoma niche morphogenesis and homeostasis

描述(申请人提供)：黑色素瘤可以说是人类癌症中最致命的，部分原因是它容易转移，而且对传统的抗癌疗法具有抵抗力。治疗失败的一个关键因素与肿瘤的异质性有关，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MIC及其基质协作促进生长和进展的分子机制，以此作为开发有效治疗策略的手段。在目前的项目中，我们专注于定义CD133+/ABCB5+MICS的微环境“生态位”，作为阐明维持MICS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+MICs的空间排列模式与“血管生成拟态”相同；2)在血管生成拟态中形成的血管样通道本身就是CD144(VE-cadherin)+，并与真实的内皮结构密切相关，与所谓的“血管周围生态位”一致；3)黑色素瘤细胞表达骨形态发生蛋白7(BMP7)及其拮抗剂Noggin与肿瘤的进展有关，也共定位于VM区域；以及4)BMP7上调间质微环境中的血管生成因子VEGF，但同时诱导Noggin缺陷的黑色素瘤细胞选择性凋亡。综上所述，我们的发现支持一个中心假设，即CD133+/ABCB5+MIC相关的BMP7不仅可能通过促进“小生境”形态发生(通过协调的VM和血管生成)支持MICs的维持，而且还可能平衡代谢需求(通过在竞争性、Noggin缺陷、非启动的人群中刺激依赖于VEGF的血管生成和诱导选择性的细胞凋亡)。利用多标记免疫荧光和免疫引导激光捕获显微切割技术，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和CD144表达之间的时空关系，以及体内微循环的类型/模式(例如黑色素瘤VM通道的形成与血管生成)(目标1a)。CD133+黑色素瘤亚群在“小生境”形成(目标1b)中的动力学和功能影响将在异种原位移植模型中进行测试，该模型使用可诱导的RNAi介导的不同节奏的敲除。最后，为了分析CD133+/ABCB5+MIC相关的BMP7分子信号在“利基”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器官培养和体内黑色素瘤异种移植模型中，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相关的BMP7信号促进“小生境”发育和维持的机制提供了一种新的机会，通过针对MIC的间质依赖，直接或间接地从治疗上消除MIC。 公共卫生相关性：众所周知，黑色素瘤对传统抗癌疗法具有耐药性，这主要归因于具有干细胞样特性的细胞亚群，即黑色素瘤启动细胞(MICs)。本研究的目的是阐明MICS及其组织环境协同作用以确保生存和促进肿瘤生长的机制。我们认为，定义这些机制提供了一个新的机会，通过靶向MIC对组织环境的依赖，直接或间接地从治疗上消除MIC。

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.