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（TGCTs）对传统的遗传毒性化疗异常敏感。这 这可能是由于TGCT和生殖细胞的独特DNA损伤反应（DDR）特征， 起来。与体细胞不同，体细胞通常通过阻止细胞周期和传导DNA来对DNA损伤做出反应。 生殖细胞以及长寿的多能干细胞通常避免使用易错修复 这一机制有利于细胞凋亡，降低了后代遗传变异的风险。同样地， TGCT前体病变，即原位生殖细胞瘤形成，未显示DDR激活，而前体病变 大多数体细胞癌的病变表达激活的DDR标记物，其响应癌基因激活， 作为肿瘤进展的屏障。为了研究TGCT，我们的实验室已经开发出第一个基因工程 通过癌基因Kras的条件性激活和Pten的失活， 肿瘤抑制基因，在生殖细胞中。在这些小鼠中产生的恶性畸胎癌由以下组成： 多能性胚胎癌（EC）和分化的畸胎瘤组织。有趣的是，EC细胞， 并在体外培养，表达干细胞标志物，具有肿瘤增殖活性， 化疗使用来自该模型的细胞，本文提出的实验将阐明 TGCT及其产生细胞的DDR特性，这将告知其潜在的机制。 异常的化学敏感性具体来说，这项建议旨在：了解细胞如何产生 TGCT对致癌事件有反应，明显避免DDR激活（目的1），并确定其机制 TGCT的胚胎癌成分的化学敏感性和TGCT的化学抗性的基础。 其差异化的对应物（目标2）。目的1将通过产生原始生殖细胞样细胞进行研究 在一些实施方案中，本发明提供了来自诱导多能干细胞（iPSC）衍生的PGCLC和胚胎生殖细胞样细胞（EGCLC）。 来自具有条件Pten和Kras等位基因的小鼠胚胎成纤维细胞（MEF）， 转化，DNA复制应激的程度，DNA损伤的程度，以及DDR的性质。 未转化和转化的细胞。对于目标2，我将分析EC中的差异基因表达， 分化的细胞治疗前和治疗后的遗传毒性化疗，并研究的作用， 差异调节途径的化学敏感性表型。研究这种可治愈的 因为了解TGCT化学敏感性的基础将广泛应用于癌症的发展， 治疗许多其他对常规化疗反应不佳的癌症。此外，本发明还 了解多能细胞类型的恶性转化，包括胚胎生殖细胞和iPSC， 将是重要的改进干细胞为基础的治疗，这带来了致瘤性的风险。

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.