Functional exploration of progenitor renewal and differentiation in oral epithelial homeostasis and cancer

项目摘要 头颈部鳞状细胞癌(HNSCC)是最常见的起源于分层的癌症类型 口腔颌面部上皮细胞。尽管付出了巨大的努力通过高吞吐量来表征HNSCC 多组学方法，对患者预后的显著改善尚未取得。这在很大程度上是因为 对癌症相关病变如何重新编程上皮细胞以扩张和 转变，在细胞和分子水平上。复层上皮是具有(A)高度特征的组织 增殖的基础祖细胞，(B)伴随着完全丧失的祖细胞分化 有丝分裂潜能，以及(C)在增殖性祖细胞和分化后有丝分裂后干细胞之间的命运选择 各州。因此，复层上皮的生长速度由更新和分化的平衡速度控制。 在我最近发表的研究中，我发现最常见的突变基因PIK3CA的致癌激活 HNSCC中的癌基因导致复层上皮细胞生长不良。使用细胞命运的直接测量 在体内的选择中，我发现致癌的PI3K信号诱导分化。这起到了平衡作用 加速细胞周期，独立于衰老或凋亡，并作为主要的细胞机制 以限制克隆扩张。在这项研究的基础上，我目前的提议将检验这样一个假设：基因 在PI3K突变型HNSCC中发现的病变和生态位因子通过克服癌基因- 诱导分化。在我的初步工作中，我在体内使用了多轮基因筛查来识别 与PIK3CA突变相关的约500个患者来源的皮损中的更新启动子。穿过这么大的- 规模努力I确定克服PI3K诱导分化所需的最小病变组合 并启动HNSCC编队。在这些病变中，TrP53的缺失是主要的更新启动子。vbl.使用 一个典型的p53功能缺陷的Trp533KR/3KR敲入动物，我发现Trp53缺失通过以下方式驱动HNSCC 促进更新，不依赖于细胞周期停滞、衰老和细胞凋亡。通过比较 WT、Trp533KR/3KR和TrP53-/-上皮间的染色体可及性我发现P53可以 转录抑制关键的更新基因，我建议研究这些基因对p53的重要性- 介导的祖细胞分化。同时，我确定了在HNSCC中特异表达的分泌因子 先祖利基。我使用基因筛查来测试它们克服PI3K诱导分化的潜力。使用一个 新的胎盘内慢病毒注射策略，我能够感染存在于壁龛中的一系列基质细胞 复层上皮。因此，我将在HNSCC期间对基质细胞中的关键促更新因子进行功能测试 启动以揭示介导上皮更新的潜在分子途径。总而言之，我的 初步数据确定了可能通过以下途径推动HNSCC形成的关键遗传损伤和分泌的生态位因子 克服PI3K诱导分化。我的提案将继续测试这一模式，最终目标是 揭示PI3K诱导HNSCCs分化障碍被打破的分子机制。

Project Summary Head and neck squamous cell carcinoma (HNSCC) is the most common cancer type initiated from stratified epithelium of oral and maxillofacial region. Despite great effort to characterize HNSCC via high throughput multi-omics approaches, significant improvements in patient prognosis are yet to be made. This is largely due to incomplete understanding of how cancer-associated lesions reprogram epithelial cells to expand and transform, at cellular and molecular levels. Stratified epithelium is a tissue characterized by (a) highly proliferative basal progenitor cells, (b) progenitor differentiation which is accompanied by complete loss of mitotic potential, and (c) stem cell fate choice between the proliferative progenitor and differentiated postmitotic states. As such, growth rate of stratified epithelia is controlled by balancing rates of renewal and differentiation. In my recently published work, I found that oncogenic activation of Pik3ca, the most commonly mutated oncogene in HNSCC, results in growth disadvantage in stratified epithelia. Using direct measure of cell fate choice in vivo, I found that oncogenic PI3K signaling induces differentiation. This serves to counterbalance accelerated cell cycle independently of senescence or apoptosis, and acts as a dominant cellular mechanism to restrict clonal expansion. Building on that study, my current proposal will test the hypothesis that genetic lesions and niche factors found in PI3K mutant HNSCC promote tumorigenesis by overcoming oncogene- induced differentiation. In my preliminary work I used multiple rounds of genetic screens in vivo to identify renewal promoters among ~500 patient-derived lesions associated with PIK3CA mutations. Through this large- scale effort I identified the minimal combination of lesions required to overcome PI3K induced differentiation and initiate HNSCC formation. Among these lesions, loss of Trp53 acts as a primary renewal promoter. Using a canonical p53 function deficient Trp533KR/3KR knock-in animal, I found that Trp53 loss drives HNSCC by promoting renewal, and independent of cell cycle arrest, senescence and apoptosis. By comparing chromosome accessibility between WT, Trp533KR/3KR and Trp53-/- epithelium I found that p53 can transcriptionally suppress key renewal genes, and I propose to study the importance of these genes for p53- mediated progenitor differentiation. In parallel, I identified secretory factors specifically expressed in HNSCC progenitor niche. I used a genetic screen to test their potential to overcome PI3K induced differentiation. With a novel intra-placenta lentivirus injection strategy, I am able to infect a spectrum of stromal cells present in niche of stratified epithelium. Thus, I will functional test key pro-renewal factors in stromal cells during HNSCC initiation to uncover the underlying molecular pathways mediating epithelial renewal. In summary, my preliminary data identify key genetic lesions and secreted niche factors that may drive HNSCC formation by overcoming PI3K induced differentiation. My proposal will continue to test this model, with the ultimate goal of uncovering molecular mechanism of how PI3K induced differentiation barrier is breached in HNSCCs.