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

摘要自PAGER 01（CA 205258） 头颈部鳞状细胞癌（HNSCC）发生在口腔、口咽和喉中， 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）也很常见，2015年美国估计有17，000例新发病例。 尽管通常结合手术、放疗和化疗的强化治疗，但HNSCC和 食管鳞癌复发频繁，长期生存率低。在这里，我们建议使用一种方法， 谱系追踪，以检验致癌物引起某些祖细胞/干细胞早期分子变化的假设 减少干/祖细胞群多样性并随后导致两种区域癌变的细胞 和肿瘤形成。因此，在本提案中，我们 解决RFA-CA-15-008的一个“挑衅性问题”：对于癌前病变引起的肿瘤 领域，什么属性的细胞在这一领域可以用来设计策略，以抑制发展 未来的肿瘤？我们将通过执行两个具体目标来回答这个问题。简单地说，我们将使用双 转基因角蛋白14/cre-ERTAM; Rosa 26-lacZ小鼠永久标记个体正常干/祖细胞 细胞及其后代在口腔和食道中的作用。依赖于Cre的重组只有在 在加入cre-ERTAM蛋白所需的他莫昔芬（Tam）时表达K14的细胞中， 是活跃的，给我们时间控制和细胞类型特异性控制cre重组酶活性， 使我们能够跟踪这些干/祖细胞及其后代随时间的命运。具体目标（1）， 我们将进行谱系追踪，全基因组RNA-seq分析和全外显子组DNA测序， 这些永久标记的lacZ+细胞“克隆”来源于个体上皮祖细胞/干细胞， 在用致癌物4-硝基喹啉氧化物（4-NQO）治疗期间， 水这一目标将描绘发生在（a）口腔上皮和（B）口腔粘膜中的早期分子变化。 非常接近地反映人类HNSCC和食管SCC的小鼠模型中的食管上皮 发展我们还将测量一些关键的表观遗传变化。在具体目标（2）中，我们将识别关键 分子变化发生在癌变过程中，当可见SCC存在时， 谱系追踪、全基因组RNA-seq分析和全外显子组DNA测序， 细胞的“克隆”，其来源于单个上皮祖细胞/干细胞（HNSCC和ESCC两者）。在Aim中 (2)我们还将比较肿瘤与SCC附近正常形态的细胞克隆， “外观正常”的上皮粘膜。这些目标的完成将定义遗传和表观遗传变化 这是“实地癌变”的基础，提供了许多关于关键的早期分子变化的新信息， HNSCC和食管癌的发生，可用于开发癌症化学预防药物。

ABSTRACT FROM PARENT R01 (CA205258) 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 proposal 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.