(PQ1) Characterization of Premalignant Fields in a Murine Model of Head and Neck and Esophageal Cancers

描述（由申请人提供）：头颈部鳞状细胞癌（HNSCC）发生于口腔、口咽和喉部，是发病率第六大癌症，全球约有60万患者。食管鳞状细胞癌（ESCC）也很常见，2015年美国估计有17000例新病例。尽管强化治疗通常结合手术、放疗和化疗，但HNSCCs和escc经常复发，长期生存率较低。在这里，我们建议使用谱系追踪的方法来验证这样的假设：在我们的小鼠HNSCC和ESCC癌变模型中，致癌物会导致一些祖细胞/干细胞的早期分子变化，从而减少干细胞/祖细胞群体的多样性，并随后导致现场癌变和肿瘤形成。因此，在本应用中，我们解决了RFA-CA-15-008的一个“具有争议性的问题”：对于从恶性肿瘤前区域产生的肿瘤，该区域的细胞的哪些特性可用于设计抑制未来肿瘤发展的策略？我们将通过实现两个具体目标来回答这个问题。简而言之，我们将使用双转基因Keratin14/cre - ERTAM；Rosa26 - lacZ小鼠在口腔和食道中永久标记单个正常干细胞/祖细胞及其后代。Cre依赖性重组仅在添加他莫昔芬（Tam）时表达K14的细胞中被激活，这是Cre - ERTAM蛋白激活所必需的，这使我们能够对Cre重组酶活性进行时间控制和细胞类型特异性控制，并允许我们跟踪这些干细胞/祖细胞及其后代的命运

DESCRIPTION (provided by applicant): Head and neck squamous cell carcinoma (HNSCC) occurs in the oral cavity, oropharynx, and larynx and is the 6th leading cancer in terms of incidence, affecting ~600,000 patients throughout the world. Esophageal squamous cell carcinoma (ESCC) is also quite common, with 17,000 new cases estimated in 2015 in the USA. Despite intensive treatment that generally combines surgery, radiation, and chemotherapy, HNSCCs and ESCCs relapse frequently and have poor long‑term survival rates. Here we propose to use an approach using lineage‑tracing to test the hypothesis that carcinogens cause early molecular changes in some progenitor/stem cells that reduce the diversity of the stem/progenitor cell population and subsequently result in both field cancerization and tumor formation in our murine HNSCC and ESCC carcinogenesis models. Thus, in this application we address one of the "Provocative Questions" of RFA-CA-15-008: For tumors that arise from a pre-malignant field, what properties of cells in this field can be used to design strategies to inhibit the development of future tumors? We will answer this question by performing two specific aims. Briefly, we will use doubly transgenic Keratin14/cre‑ERTAM; Rosa26‑lacZ mice to permanently mark individual normal stem/ progenitor cells and their progeny in the oral cavity and esophagus. Cre‑dependent recombination will be activated only in cells that express K14 at the time of addition of tamoxifen (Tam), which is required for the cre‑ERTAM protein to be active, giving us both temporal control and cell type‑specific control over the cre recombinase activity and allowing us to follow the fates of these stem/ progenitor cells and their progeny over time. In Specific Aim (1), we will perform lineage‑tracing, whole genome RNA‑seq analyses, and whole exome DNA sequencing of these permanently marked lacZ+ "clones" of cells derived from individual, epithelial progenitor/stem cells during treatment with the carcinogen 4‑nitroquinoline oxide (4‑NQO), a tobacco surrogate, in the drinking water. This aim will delineate early molecular changes that occur in (a) the oral epithelium, and (b) the esophageal epithelium in a mouse model that very closely reflects human HNSCC and esophageal SCC development. We will also measure some key epigenetic changes. In Specific Aim (2) we will discern the key molecular changes that occur much later in the carcinogenesis process, when visible SCCs are present, by performing lineage tracing, whole genome RNA‑seq analyses, and whole exome DNA sequencing of permanently marked "clones" of cells, derived from individual epithelial progenitor/ stem cells (both HNSCCs & ESCCs). In Aim (2) we will also compare tumors to clones of cells with normal morphology that are present near SCCs in "normal appearing" epithelial mucosa. Completion of these aims will define genetic and epigenetic changes that underlie 'field cancerization,' providing much new information about critical, early molecular changes in HNSCC and esophageal carcinogenesis that could be exploited to develop cancer chemopreventive drugs.