Deciphering mechanisms of melanoma metastasis with main focus on brain metastasis

黑色素瘤远处转移，特别是向脑的转移与治疗失败和复发相关，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的机制是至关重要的。因此，拟议项目的目的是确定黑色素瘤转移的新关键参与者和机制，主要关注脑转移。此外，基因/蛋白质和途径的特定特征的发现将能够对具有预后相关性的黑色素瘤转移的新亚组进行分类。神经生长因子受体CD 271的表达提出了一个有前途的候选分类器。为了验证CD 271，将通过免疫组织化学初步筛选匹配的颅外和脑转移瘤对的CD 271水平，并细分为CD 271高、CD 271低和CD 271阴性肿瘤。对来自柏林和德累斯顿两个中心的至少10对匹配的颅外和脑转移瘤以及肿瘤相邻或正常脑组织进行RNA测序，将提供关于差异调节的信号通路的见解，并将鉴定可能支持脑转移瘤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景观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，将对颅外和脑转移瘤的850 k CpG岛进行甲基化组分析。表达谱分析、甲基化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综合图谱，并将能够鉴定黑色素瘤转移中的共同或可逆调节基因。同时，我将研究小鼠模型中的转移机制，从而能够直接跟踪生物体中黑色素瘤细胞的途径，并跟踪器官中的遗传工程黑色素瘤细胞。为此，将内源性CD 271水平存在差异的GFP阳性黑素瘤细胞注射到受体小鼠的脾脏中。为了确定转移的程度，将采集血液样品并分析循环GFP和/或CD 271阳性肿瘤细胞，并且将检查所有器官，特别是脑、肺和肝，以确定肿瘤的存在。此外，已建立的颅外和脑转移瘤的RNA测序不仅将提供潜在的CD 271驱动的转移机制，而且还将提供独立于CD 271的机制。转移的候选驱动因素将通过CRISPR/CAS9介导的敲除和体外迁移测定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。

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.