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

描述（由申请人提供）：头颈鳞状细胞癌 (HNSCC) 发生在口腔、口咽和喉部，是发病率排名第六的癌症，影响全世界约 600,000 名患者。食管鳞状细胞癌 (ESCC) 也相当常见，2015 年美国估计有 17,000 例新病例。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，且长期生存率较低。在这里，我们建议使用谱系追踪方法来检验这样的假设：致癌物会导致某些祖细胞/干细胞的早期分子变化，从而降低干细胞/祖细胞群的多样性，并随后导致我们的小鼠头颈部鳞状细胞癌和食管鳞癌致癌模型中的局部癌化和肿瘤形成。因此，在本申请中，我们解决了 RFA-CA-15-008 的“挑衅性问题”之一：对于源自恶变前区域的肿瘤，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个体正常干/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，为我们提供了对 cre 重组酶活性的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“克隆”细胞进行谱系追踪、全基因组 RNA 序列分析和全外显子组 DNA 测序，这些细胞源自个体上皮祖细胞/干细胞，在饮用水中接受致癌物质 4-硝基喹啉氧化物 (4-NQO)（一种烟草替代品）处理期间。这个目标将勾勒出 小鼠模型中 (a) 口腔上皮和 (b) 食管上皮发生的早期分子变化非常密切地反映了人类 HNSCC 和食管鳞状细胞癌的发展。我们还将测量一些关键的表观遗传变化。在特定目标 (2) 中，我们将通过对源自个体上皮祖细胞/干细胞（HNSCC 和 ESCC）的永久标记的细胞“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，来辨别癌发生过程后期（当存在可见 SCC 时）发生的关键分子变化。在目标 (2) 中，我们还将肿瘤与存在于“正常出现”上皮粘膜中的鳞状细胞癌附近的具有正常形态的细胞克隆进行比较。这些目标的完成将定义“现场癌化”背后的遗传和表观遗传变化，提供有关 HNSCC 和食管癌发生的关键早期分子变化的许多新信息，可用于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.