(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）发生在口腔、口咽和喉，是发病率排名第6的癌症，影响全世界约600，000例患者。食管鳞状细胞癌（ESCC）也很常见，2015年美国估计有17，000例新发病例。尽管强化治疗通常结合手术，放疗和化疗，但HNSCC和ESCC经常复发，长期生存率很低。在这里，我们建议使用一种使用谱系追踪的方法来测试致癌物引起某些祖细胞/干细胞的早期分子变化的假设，这些变化降低了干细胞/祖细胞群体的多样性，随后导致我们的小鼠HNSCC和ESCC致癌模型中的实地癌变和肿瘤形成。因此，在本申请中，我们解决了RFA-CA-15-008中的一个“挑衅性问题”：对于从癌前区域产生的肿瘤，该区域中细胞的哪些特性可用于设计抑制未来肿瘤发展的策略？我们将通过执行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Keratin 14/cre-ERTAM; Rosa 26-lacZ小鼠永久标记口腔和食管中的单个正常干/祖细胞及其后代。Cre依赖性重组将仅在加入他莫昔芬（Tam）时表达K14的细胞中激活，这是Cre-ERTAM蛋白激活所必需的，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，并使我们能够跟踪这些干/祖细胞及其后代的命运。 时间在具体目标（1）中，我们将对这些永久标记的lacZ+细胞“克隆”进行谱系追踪，全基因组RNA测序分析和全外显子组DNA测序，这些细胞来自个体上皮祖细胞/干细胞，在饮用水中使用致癌物4-硝基喹啉氧化物（4-NQO）（烟草替代物）处理期间。这一目标将描绘 在非常接近地反映人HNSCC和食管SCC发展的小鼠模型中，在（a）口腔上皮和（B）食管上皮中发生的早期分子变化。我们还将测量一些关键的表观遗传变化。在具体目标（2）中，我们将通过进行谱系追踪，全基因组RNA-seq分析和永久标记的细胞“克隆”的全外显子组DNA测序，识别在致癌过程后期发生的关键分子变化，当可见SCC存在时，来自单个上皮祖细胞/干细胞（HNSCC和ESCC）。在目标（2）中，我们还将比较肿瘤与“正常外观”上皮粘膜中SCC附近存在的具有正常形态的细胞克隆。这些目标的完成将定义遗传和表观遗传的变化，基础上的“现场癌变”，提供了许多新的信息，关键的，早期的分子变化HNSCC和食管癌，可以利用开发癌症化学预防药物。

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.