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，是绝大多数癌症死亡的原因。转移可以被概念化为一个进化景观，由达尔文进化的关键要素组成：可遗传（epi）基因型，地理扩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，其中癌细胞可以与宿主长期共存。为了实现这一目标，需要深入了解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。我的工作利用了斑马鱼，一种小型脊椎动物，最近才被发现是一种重要的癌症模型。斑马鱼提供了几个独特的能力，研究转移：高通量转基因，无偏见的遗传筛选，并在光学透明的卡斯珀成鱼单细胞成像。在本提案期间，我计划解决转移性黑色素瘤中的三个主要问题：1）我们能否识别允许转移进展的初始遗传变化，无论它们是在原发肿瘤中还是在传播后出现，2）适应性突变是转移所需的吗？和3）我们能识别不利于转移进展的宿主微环境吗？为此，我将建立一个黑色素瘤的斑马鱼模型，其中BRAFV 600 E等位基因在黑素细胞祖细胞中表达，在p53功能丧失的背景下（BRAFV 600 E;p53-/-模型）。通过将“brainbow”命运定位系统与BRAFV 600 E;p53-/- fish相结合，我将使用外显子组测序来基于谱系、空间和时间鉴定与转移相关的基因组病变。这些候选的变化可以使用我在透明卡斯珀菌株中开发的转移测定来功能化。我假设，在转移过程中的选择压力导致了一种适应性的状态， 突变，其中DNA复制的错误率暂时增加，以找到一个进化的解决方案，这种压力。为了验证这一点，我将产生具有突变基因的斑马鱼，然后使用这个系统来探测由易错Y家族DNA聚合酶介导的适应性突变是否促进转移进展。最后，由于宿主微环境提供了最终的选择压力，我将进行无偏倚的遗传筛选，以确定转移进展的新基质调节因子。总之，这些研究提供了一个全面的框架，考虑了肿瘤细胞内在和转移性疾病的微环境动力学。我实验室的长期目标是利用这些信息来确定可以将播散性疾病转化为稳定状态的治疗方法，无法进一步发展，从而大大改善已确定转移患者的长期生存率。

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.