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

摘自亲本R01(CA205258) 头颈部鳞状细胞癌(HNSCC)发生于口腔、口咽部和喉部。 在发病率方面排名第六位的癌症，影响到全世界约60万患者。食道 鳞状细胞癌(ESCC)也很常见，据估计，2015年美国有1.7万新病例。 尽管强化治疗通常结合了手术、放射和化疗，但HNSCCs和 食管癌复发频繁，长期存活率低。在这里，我们建议使用一种方法，使用 对致癌物导致某些祖细胞/干细胞早期分子变化的假说进行谱系追踪检验 减少干细胞/祖细胞群体的多样性并随后导致两个领域的癌变的细胞 以及在我们的小鼠HNSCC和ESCC致癌模型中的肿瘤形成。因此，在这项提案中，我们 解决RFA-CA-15-008中的一个“挑衅性问题”：对于起源于癌前病变的肿瘤 领域，什么性质的细胞在这个领域可以被用来设计策略来抑制发展 未来的肿瘤？我们将通过实现两个具体目标来回答这个问题。简而言之，我们将双重使用 转基因Keratin14/cre-ERTAM；rosa26-lacZ小鼠永久标记个体正常干/祖细胞 口腔和食道中的细胞及其后代。依赖Cre的重组将仅被激活 在加入三苯氧胺()时表达K14的细胞中，三苯氧胺是cre-ERTAM蛋白所必需的 为我们提供了对cre重组酶活性的临时控制和特定细胞类型的控制，并且 使我们能够随着时间的推移跟踪这些干细胞/祖细胞及其后代的命运。在具体目标(1)中， 我们将进行谱系追踪、全基因组rna-seq分析和全外显子DNA测序。 这些永久标记的LacZ+细胞克隆来自个别上皮祖细胞/干细胞 在饮酒中使用烟草替代品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)口腔上皮的早期分子变化。 非常接近反映人HNSCC和食管鳞癌的小鼠模型中的食道上皮 发展。我们还将测量一些关键的表观遗传学变化。在具体目标(2)中，我们将识别关键 在癌变过程中较晚发生的分子变化，当可见的鳞状细胞存在时，通过执行 血统追踪、全基因组rna-seq分析和永久标记的全外显子DNA测序 来自个别上皮祖细胞/干细胞(HNSCCs和ESCCs)的细胞“克隆”。在AIM (2)我们还将把肿瘤与存在于干细胞附近的具有正常形态的细胞克隆进行比较 “外观正常”的上皮粘膜。完成这些目标将定义遗传和表观遗传变化 这是“野战癌化”的基础，提供了许多关于关键的、早期的分子变化的新信息 HNSCC与食道癌的关系，可用于开发化学防癌药物。

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.