The role of the DNA damage response in the development and therapeutic sensitivity of malignant testicular germ cell tumors.

项目摘要 睾丸生殖细胞肿瘤(TGCT)对常规的基因毒性化疗特别敏感。这 可能是由于TGCT及其来源的生殖细胞具有不同的DNA损伤反应(DDR)特征 站起来。与体细胞不同，体细胞通常通过阻止细胞周期和引导DNA来响应DNA损伤 修复、生殖细胞以及长寿的多能干细胞通常避免使用容易出错的修复 机制，并有利于细胞凋亡，降低后代发生基因改变的风险。同样， TGCT的前驱病变，原位生殖细胞肿瘤，没有显示出DDR的激活，而前驱病变 大多数体细胞癌的皮损表达活化的DDR标记，以响应癌基因的激活 是肿瘤进展的障碍。为了研究TGCT，我们的实验室开发了第一个基因工程 癌基因Kras的条件性激活和Pten、a的失活建立小鼠恶性TGCT模型 生殖细胞中的肿瘤抑制基因。在这些小鼠身上产生的恶性畸胎癌由 多能胚胎癌(EC)和分化的畸胎瘤组织。有趣的是，EC细胞，无论是在体内 并在体外培养，表达干细胞标记，具有肿瘤增殖活性，并容易在以下情况下被杀伤 化疗。使用来自该模型的细胞，这里提出的实验将阐明 TGCT及其产生的细胞的DDR特性，这将揭示其潜在的机制 具有特殊的化学敏感性。具体地说，这项提议的目的是：了解细胞是如何产生 TGCT对致癌事件作出反应，明显避免DDR激活(目标1)，并确定其机制 TGCT胚胎癌成分的化疗敏感性及其耐药性的研究 它们的差异化对应物(目标2)。目标1将通过产生原始生殖细胞样细胞来研究 诱导多能干细胞(IPSCs)来源的胚胎生殖细胞样细胞(EGCLCs)和PGCLCs 携带条件性Pten和Kras等位基因的小鼠胚胎成纤维细胞及其恶性程度的评估 转化、DNA复制胁迫程度、DNA损伤程度以及DDR的性质 未转化细胞和转化细胞。对于目标2，我将分析在EC和 分化细胞在基因毒性化疗治疗前后的作用及探讨 化疗敏感性表型中的差异调控通路。研究这种可治愈的方法是至关重要的。 癌症，因为了解TGCT化疗敏感性的基础将广泛应用于肿瘤的发展 对许多其他癌症的治疗，这些癌症对传统化疗不起作用。另外， 了解多能细胞类型的恶性转化，包括胚胎生殖细胞和IPSCs， 对于基于干细胞的疗法的改进将是重要的，因为干细胞疗法具有致瘤风险。

Project Summary Testicular germ cell tumors (TGCTs) are exceptionally sensitive to conventional genotoxic chemotherapy. This is likely due to the distinct DNA damage response (DDR) features of TGCTs and the germ cells from which they arise. Unlike somatic cells which often respond to DNA damage by arresting the cell cycle and conducting DNA repair, germ cells as well as long-lived pluripotent stem cells typically avoid the use of error-prone repair mechanisms and favor apoptosis, reducing the risk of genetic alterations in subsequent generations. Similarly, the TGCT precursor lesion, germ cell neoplasia in situ, does not show activation of a DDR, whereas precursor lesions of most somatic cancers express markers of an activated DDR in response to oncogene activation which serves as a barrier to tumor progression. To study TGCTs, our lab has developed the first genetically engineered mouse model of malignant TGCTs by conditional activation of Kras, an oncogene, and inactivation of Pten, a tumor suppressor gene, in germ cells. The malignant teratocarcinomas generated in these mice are composed of pluripotent embryonal carcinoma (EC) and differentiated teratoma tissue. Interestingly, EC cells, both in vivo and cultured in vitro, express stem cell markers, have tumor propagating activity, and are readily killed following chemotherapy treatment. Using cells derived from this model, the experiments proposed here will elucidate the DDR properties of TGCTs and the cells from which they arise, which will inform the mechanisms underlying their exceptional chemosensitivity. Specifically, this proposal aims to: understand how the cells that give rise to TGCTs respond to oncogenic events, apparently avoiding DDR activation (Aim 1), and determine the mechanism underlying the chemosensitivity of the embryonal carcinoma components of TGCTs and the chemoresistance of their differentiated counterparts (Aim 2). Aim 1 will be investigated by generating primordial germ cell-like cells (PGCLCs) and embryonic germ cell-like cells (EGCLCs) from induced pluripotent stem cells (iPSCs) derived from mouse embryonic fibroblasts (MEFs) with conditional Pten and Kras alleles and assessing malignant transformation, the degree of DNA replication stress, the extent of DNA damage, and the nature of the DDR in untransformed and transformed cells. For Aim 2, I will analyze differential gene expression in EC and differentiated cells before and after treatment with genotoxic chemotherapy and investigate the role of differentially regulated pathways in the chemosensitivity phenotype. It is critically important to study this curable cancer because understanding the basis of TGCT chemosensitivity will apply broadly to the development of treatments for the many other cancers that do not respond favorably to conventional chemotherapy. Additionally, understanding the malignant transformation of pluripotent cell types, including embryonic germ cells and iPSCs, will be important for the improvement of stem-cell based therapies, which carry the risk of tumorgenicity.