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

DNA 损伤反应在恶性睾丸生殖细胞肿瘤的发展和治疗敏感性中的作用。

• 批准号: 10304884
• 负责人: Amanda Loehr
• 金额: $ 4.32万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10304884
• 关键词: Aftercare; Alleles; Anchorage-Independent Growth; Apoptosis; Breast Carcinoma; Candidate Disease Gene; Cell Cycle Arrest; Cell Differentiation process; Cells; Chemoresistance; Colon Carcinoma; Comet Assay; DNA; DNA Damage; DNA Repair; DNA Replication Timing; DNA Sequence Alteration; DNA biosynthesis; Development; Embryo; Embryonal Carcinoma; Embryonal Carcinoma Cell; Embryonic Development; Enterobacteria phage P1 Cre recombinase; Event; Exercise; Fiber; Fibroblasts; Generations; Genes; Genetic Recombination; Genetic Risk; Genetically Engineered Mouse; Genome; Germ Cells; Human; In Situ; In Vitro; Lesion; Malignant - descriptor; Malignant Neoplasms; Measures; Modeling; Mus; Mutagens; Mutate; Mutation; Nature; Neoplasms; Oncogene Activation; Oncogenes; Oncogenic; Pathology; Pathway interactions; Phenotype; Pluripotent Stem Cells; Property; Risk; Role; Signal Transduction; Somatic Cell; Structure of primordial sex cell; System; Teratocarcinoma; Teratoma; Testicular Germ Cell Tumor; Testicular Neoplasms; Testicular malignant germ cell tumor; Therapeutic; Tissue-Specific Gene Expression; Tissues; Tumor Suppressor Genes; Tumorigenicity; Undifferentiated; cell transformation; cell type; chemotherapy; experience; experimental study; genotoxicity; improved; in vitro Model; in vivo; induced pluripotent stem cell; lung Carcinoma; mouse model; neoplastic cell; pressure; prevent; repaired; replication stress; response; stem cell biomarkers; stem cell therapy; therapy development; transcriptome sequencing; treatment response; tumor; tumor initiation; tumor progression; tumorigenesis; young man

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.

项目概要 睾丸生殖细胞肿瘤（TGCT）对传统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独特的 DNA 损伤反应 (DDR) 特征 出现。与体细胞不同，体细胞通常通过阻止细胞周期和传导 DNA 来响应 DNA 损伤 修复，生殖细胞以及长寿命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，降低后代遗传改变的风险。相似地， TGCT 前体病变，即原位生殖细胞肿瘤，不显示 DDR 的激活，而前体 大多数体细胞癌的病变都表达激活 DDR 的标记，以响应癌基因的激活， 作为肿瘤进展的屏障。为了研究 TGCT，我们的实验室开发了第一个基因工程 通过条件性激活癌基因 Kras 和灭活 Pten 建立恶性 TGCT 小鼠模型 生殖细胞中的肿瘤抑制基因。这些小鼠体内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 细胞在体内 体外培养，表达干细胞标志物，具有肿瘤增殖活性，并且在体外培养后很容易被杀死 化疗治疗。使用源自该模型的细胞，此处提出的实验将阐明 TGCT 及其产生细胞的 DDR 特性，将揭示其背后的机制 特殊的化学敏感性。具体来说，该提案旨在：了解细胞如何产生 TGCT 对致癌事件作出反应，显然避免了 DDR 激活（目标 1），并确定其机制 TGCT 胚胎癌成分的化疗敏感性和化疗耐药性的基础 他们的差异化对应物（目标 2）。目标 1 将通过生成原始生殖细胞样细胞进行研究 (PGCLC) 和源自诱导多能干细胞 (iPSC) 的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) 并评估恶性 转化、DNA 复制应激程度、DNA 损伤程度以及 DDR 的性质 未转化和转化的细胞。对于目标 2，我将分析 EC 和 基因毒性化疗治疗前后分化的细胞并研究 化学敏感性表型的差异调节途径。研究这种可治愈的疾病至关重要 因为了解 TGCT 化学敏感性的基础将广泛应用于癌症的开发 对传统化疗没有良好反应的许多其他癌症的治疗。此外， 了解多能细胞类型的恶性转化，包括胚胎生殖细胞和 iPSC， 对于改进基于干细胞的疗法非常重要，因为干细胞疗法具有致瘤性的风险。