FA pathway activities in the normal and transformed epidermis

摘要 人的表皮是抵御机械、化学、辐射和传染性侮辱的关键屏障。 外部环境。角质形成细胞是这种复层组织中的主要细胞类型，并被组织成 基本干细胞和祖细胞，以及较浅分化的后代。表皮动态平衡和 诚信受到严格监管，对人类健康至关重要。鳞状细胞癌可能是一种 表皮动态平衡出现问题的最好例子。散发性和遗传性鳞状细胞癌都有关联 参与DNA修复的Fanconi贫血(FA)途径的21个基因发生突变 交联剂。FA途径和鳞状细胞癌发展之间的密切联系表明了这一点的重要作用 角质形成细胞和表皮中的通路--这些作用尚未被研究，但将在这里进行研究 利用FA患者群体和工具研究角质形成细胞生物学。这些工具包括人类IPSC衍生的 角质形成细胞和3D器官型上皮筏的FA基因同源，以及FA的皮肤活检 辛辛那提儿童医院独有的患者(与对照组)。我们的翻译研究 根据最新数据显示FA缺乏患者的细胞黏附、细胞骨架和膜缺陷 角质形成细胞、皮肤活检和3D器官型木筏。有缺陷的角质形成细胞也具有高运动性， 侵袭性，尤其是在转化时--侵袭性表型取决于DNA的活性 损伤感受器DNA-PK和小的GTP酶rac1，以及神经节苷脂的积聚。这个 最重要的假说是FA途径的缺失导致膜上的rac1信号小体的组装 富含神经节苷脂的结构域(如脂筏)，以及由此产生的让人想起的表型 上皮间充质转化(EMT样)。目的1验证FA途径调节的假设 通过神经节苷脂的积累和DNA-DNA在正常表皮中的黏附/完整性和细胞运动 PK/rac1信号转导。目的2直接测试FA损失对转化的影响，定义FA途径- 神经节苷脂生物合成的依赖通量及其在维持鳞状细胞癌生长和进展中的作用。结果是 将确定a)信号和基于脂质的机制，通过这些机制，核DNA修复机制控制 人类表皮的完整性，以及b)预防和治疗红斑狼疮的生物标志物和治疗靶点 获得性和遗传性FA通路缺陷患者的鳞状细胞癌发生。

ABSTRACT Human epidermis serves as a critical barrier against mechanical, chemical, radiation and infectious insults in the external environment. Keratinocytes are the main cell type in this stratified tissue, and are organized into basal stem and progenitor cells, and the more superficial differentiated progeny. Epidermal homeostasis and integrity are tightly regulated and critical for human health. Squamous cell carcinomas (SCCs) are perhaps a prime example of epidermal homeostasis gone awry. Both sporadic and inherited forms of SCC are associated with mutations in the 21 genes of the Fanconi anemia (FA) pathway, which participate in the repair of DNA crosslinks. Close associations between the FA pathway and SCC development point to important roles for this pathway in keratinocytes and epidermis – roles that have not yet been studied, but will be investigated here using FA patient populations and tools to study keratinocyte biology. These tools include human iPSC-derived keratinocytes and 3D organotypic epithelial rafts isogenic for FA genes, as well as skin biopsies from FA patients (vs. controls) that are uniquely available at Cincinnati Children's Hospital. Our translational studies build on recent data demonstrating cell adhesion, cytoskeletal and membrane defects in FA-deficient keratinocytes, skin biopsies, and 3D organotypic rafts. The defective keratinocytes were also hyper-motile, and invasive particularly when transformed – and the invasive phenotype depended on the activities of the DNA damage sensor kinase DNA-PK and the small GTPase Rac1, as well as ganglioside accumulation. The overarching hypothesis is that FA pathway loss leads to the assembly of a Rac1 signalosome in membrane domains enriched for gangliosides (eg lipid rafts), and consequent phenotypes that are reminiscent of epithelial-mesenchymal-transition (EMT-like). Aim 1 tests the hypothesis that the FA pathway regulates adhesion/integrity and cell motility in the normal epidermis through ganglioside accumulation and DNA- PK/Rac1 signaling. Aim 2 directly tests the impact of FA loss on transformation, defines the FA-pathway- dependent flux of ganglioside biosynthesis and its role in sustaining SCC growth and progression. The results will identify a) signaling and lipid-based mechanisms whereby nuclear DNA repair machineries control the integrity of human epidermis, and b) biomarkers and therapeutic targets for the prevention and treatment of SCC development in patients with acquired and inherited FA pathway deficiencies.