Evolutionary dynamics of melanoma metastasis

描述（由申请人提供）：转移性疾病仍然是晚期恶性肿瘤的定义特征，并且是绝大多数癌症死亡的原因。转移可以被定义为一个进化景观，由达尔文进化的关键要素组成：遗传（epi）基因型、地理分散和新的微环境选择压力。我假设，改变这些进化景观将为治疗癌症提供一种惊人的新方法，在这种方法中，癌细胞可以与宿主长期共存。为了实现这一目标，需要对肿瘤产生新基因型的方式以及微环境中的自然选择如何放大这些突变有深入的机制理解。我的研究利用了斑马鱼，这是一种小型脊椎动物，最近才被发现是一种重要的癌症模型。斑马鱼提供了几种独特的研究转移的能力：高通量转基因，无偏倚的遗传筛选，以及光学透明的成年鱼的单细胞成像。在本提案期间，我计划解决转移性黑色素瘤的三个主要问题：1)我们能否识别允许转移进展的早期遗传变化，它们是在原发肿瘤中还是在传播后出现的，2)转移是否需要适应性突变，这可以被修改吗？3)我们能否确定不利于转移进展的宿主微环境？为此，我将建立一个斑马鱼黑色素瘤模型，在p53功能丧失的背景下，BRAFV600E等位基因在黑素细胞祖细胞中表达（BRAFV600E；p53-/-模型）。通过将“彩虹”命运测绘系统与BRAFV600E相结合；p53-/- fish，我将使用外显子组测序来识别基于谱系，空间和时间的与转移相关的基因组病变。这些候选的变化可以功能化使用转移试验，我已经开发了透明的蓖麻菌株。我假设在转移过程中选择压力源导致了一种适应状态

DESCRIPTION (provided by applicant): Metastatic disease remains the defining feature of advanced malignancy, and is responsible for the vast majority of cancer deaths. Metastasis can be conceptualized as an evolutionary landscape, composed of key elements of Darwinian evolution: heritable (epi)genotypes, geographic dispersal, and novel microenvironmental selection pressures. I hypothesize that altering these evolutionary landscapes would provide a strikingly new method for treating cancer, in which the cancer cells can co-exist with the host over long periods of time. To achieve this requires a deep mechanistic understanding of the ways in which tumors generate novel genotypes, and how natural selection in the microenvironment amplifies these mutations. My work utilizes the zebrafish, a small vertebrate organism that has only recently come to light as an important cancer model. The zebrafish offers several unique capacities for studying metastasis: high-throughput transgenesis, unbiased genetic screens, and single cell imaging in the optically transparent casper adult fish. For the period of this proposal, I plan to address three primary questions in metastatic melanoma: 1) can we identify the incipient genetic changes that allow for metastatic progression, whether they arise in the primary tumor or after dissemination, 2) is adaptive mutation required for metastasis, and can this be modified?, and 3) can we identify host microenvironments that disfavor metastatic progression? To do this, I will build upon a zebrafish model of melanoma in which the BRAFV600E allele is expressed in melanocyte progenitors, in the context of p53 loss of function (the BRAFV600E;p53-/- model). By combining the "brainbow" fate mapping system with the BRAFV600E;p53-/- fish, I will use exome sequencing to identify genomic lesions associated with metastasis based on lineage, space and time. These candidate changes can be functionalized using a metastasis assay I have developed in the transparent casper strain. I hypothesize that selection stressors during metastasis lead to a state of adaptive mutation, in which the error rate of DNA replication is temporarily increased to find an evolutionary solution to that stress. To test this, I will generate zebrafish with mutation reportes, and then use this system to probe whether adaptive mutation mediated by error-prone Y family DNA polymerases promotes metastatic progression. Finally, since the host microenvironment provides the ultimate selection pressure, I will perform an unbiased genetic screen to identify novel stromal regulators of metastatic progression. Together, these studies provide a comprehensive framework that considers both tumor cell-intrinsic and microenvironmental dynamics of metastatic disease. The long-term goal of my laboratory is to utilize this information to identify therapies which can convert disseminated disease into a stable state, unable to further progress, leading to large improvements in long-term survival in patients with established metastases.