Deciphering mechanisms of melanoma metastasis with main focus on brain metastasis

破译黑色素瘤转移机制，重点关注脑转移

• 批准号: 392534127
• 负责人: Dr. Torben Redmer
• 金额: --
• 依托单位: Institut für Medizinische Biochemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2017
• 资助国家: 德国
• 起止时间: 2016-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/392534127?language=en
• 关键词: Deciphering; mechanisms; melanoma; metastasis; main

Melanoma distant metastasis especially to the brain is associated with treatment failure and relapse, hence insight in mechanisms of how melanoma cells acquire a metastatic phenotype and escape therapeutic regimens is essential. Therefore the aim of the proposed project is the identification of new key players and mechanisms of melanoma metastasis with main focus on metastasis to the brain. Furthermore, the uncovering of specific signatures of genes/proteins and pathways will enable the classification of new sub-groups of melanoma metastases with prognostic relevance. Expression of the nerve growth factor receptor CD271 presents a promising candidate classifier. For validation of CD271, matched pairs of extracranial and brain metastases will be initially screened by immunohistochemistry for the levels of CD271 and sub-divided into CD271high, CD271low and CD271neg tumors. RNA-sequencing of at least 10 matched pairs of extracranial and brain metastases as well as of tumor-adjacent or normal brain tissue received from two centers in Berlin and Dresden will provide insight about differentially regulated signaling pathways and will identify growth factors and neurotrophic factors potentially supporting the establishment of brain metastases. In addition, the exploration of the mutational landscape of tumors by exome-sequencing will foster the uncovering of intrinsically regulated metastasis mechanisms and genetic relationships among metastases. For exploration of epigenetic regulatory mechanisms, a methylome analysis of 850k CpG islands of extracranial and brain metastases will be performed. The integrated analysis of expression profiling, methylome analysis and exome sequencing data will provide a comprehensive map of each tumor and will enable the identification of commonly or reversibly regulated genes among melanoma metastases. In parallel, I will study mechanisms of metastasis in mouse models enabling the direct following of the routes of melanoma cells in the organism and tracking of e.g. genetically engineered melanoma cells in organs. To this end, GFP-positive melanoma cells with differences in endogenous CD271 levels will be injected into the spleen of recipient mice. To ascertain the extent of metastasis, blood samples will be taken and analyzed for circulating GFP and/or CD271-positive tumor cells and all organs especially, brain, lungs and liver will be inspected for presence of tumors. RNA-sequencing of established extracranial and brain metastases will in addition provide inside not only into a potential CD271-driven metastasis mechanism but also into mechanisms independent of CD271. Candidate drivers of metastasis will be functionally validated by CRISPR/CAS9 mediated knock-out and in vitro migration assays as well as in vivo models. The results of this study will help to understand the molecular basis of melanoma brain metastasis and will foster the development of new therapeutic strategies.

黑色素瘤的远处转移尤其是脑转移与治疗失败和复发有关，因此了解黑色素瘤细胞如何获得转移表型和逃避治疗方案是至关重要的。因此，该项目的目标是确定黑色素瘤转移的新的关键因素和机制，主要集中在脑转移上。此外，基因/蛋白质和通路的特定特征的发现将使具有预后相关性的新的黑色素瘤转移亚组的分类成为可能。神经生长因子受体CD271的表达是一种有希望的候选分类器。为了验证CD271的有效性，将通过免疫组织化学对匹配的颅外和脑转移瘤进行CD271水平的初步筛选，并将其细分为CD271高、CD271低和CD271neg肿瘤。从柏林和德累斯顿的两个中心收到的至少10对匹配的颅外和脑转移以及肿瘤邻近或正常脑组织的RNA测序将提供对不同调控信号通路的洞察，并将识别可能支持脑转移建立的生长因子和神经营养因子。此外，通过外显子组测序探索肿瘤的突变图景将有助于揭示内在调节的转移机制和转移之间的遗传关系。为了探索表观遗传调控机制，将对850k个颅外和脑转移的CpG岛进行甲基组分析。表达谱分析、甲基组分析和外显子组测序数据的综合分析将提供每个肿瘤的全面图谱，并将能够在黑色素瘤转移中识别常见或可逆调节的基因。同时，我将研究小鼠模型中的转移机制，使之能够直接跟踪黑色素瘤细胞在生物体中的路线，并跟踪例如器官中的基因工程黑色素瘤细胞。为此，将内源性CD271水平不同的GFP阳性黑色素瘤细胞注射到受体小鼠的脾中。为了确定转移的程度，将采集血液样本并分析循环中的GFP和/或CD271阳性的肿瘤细胞，并将检查所有器官，特别是脑、肺和肝脏是否存在肿瘤。此外，对已建立的颅外和脑转移瘤进行RNA测序将不仅提供潜在的CD271驱动的转移机制，而且还提供独立于CD271的机制。转移的候选驱动因素将通过CRISPR/Cas9介导的基因敲除和体外迁移试验以及体内模型进行功能验证。这项研究的结果将有助于了解黑色素瘤脑转移的分子基础，并将促进新的治疗策略的发展。