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）是最常见的癌症类型，起源于分层的鳞状细胞癌。 口腔颌面部上皮尽管通过高通量表征HNSCC做出了巨大努力， 尽管使用了多组学方法，但患者预后仍有待显著改善。这主要是由于 对癌症相关病变如何重新编程上皮细胞以扩张的不完全理解， 在细胞和分子水平上转化。复层上皮是一种组织，其特征在于（a）高度 增殖的基础祖细胞，（B）祖细胞分化，其伴随着 有丝分裂潜能，和（c）干细胞在增殖祖细胞和分化的有丝分裂后细胞之间的命运选择。 states.因此，复层上皮细胞的生长速率由更新和分化的平衡速率控制。 在我最近发表的研究中，我发现Pik 3ca的致癌激活， HNSCC中的癌基因，导致复层上皮生长不利。使用直接测量细胞命运 选择在体内，我发现致癌PI 3 K信号诱导分化。这有助于平衡 加速细胞周期独立的衰老或凋亡，并作为一个主导的细胞机制 以限制克隆扩张。在这项研究的基础上，我目前的建议将测试遗传学的假设， PI 3 K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，我在体内进行了多轮基因筛选， 在约500例与PIK 3CA突变相关的患者源性病变中，通过这个大- 规模努力I确定了克服PI 3 K诱导分化所需的最小病变组合 并启动HNSCC形成。在这些病变中，Trp 53的缺失作为主要的更新启动子。使用 典型的p53功能缺陷型Trp 533 KR/3 KR基因敲入动物，我发现Trp 53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、衰老和凋亡。通过比较 WT、Trp 533 KR/3 KR和Trp 53-/-上皮细胞之间的染色体可及性 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。与此同时，我鉴定了HNSCC中特异性表达的分泌因子， 祖先生态位我使用遗传筛选来测试它们克服PI 3 K诱导的分化的潜力。与 新的胎盘内慢病毒注射策略，我能够感染一系列的基质细胞存在于龛 复层上皮细胞因此，我将在HNSCC期间对基质细胞中的关键促更新因子进行功能检测， 启动以揭示介导上皮更新的潜在分子途径。总之，我的 初步数据确定了关键的遗传病变和分泌的生态位因子，这些因子可能通过以下方式驱动HNSCC的形成： 克服PI 3 K诱导的分化。我的建议将继续测试这一模式，最终目标是 揭示PI 3 K诱导的分化屏障在HNSCCs中如何被突破的分子机制。

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.