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

描述(申请人提供)：头颈部鳞状细胞癌(HNSCC)发生在口腔、口咽和喉部，是发病率第六大的癌症，影响全球约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、放疗和化疗，但HNSCC和ESCC经常复发，长期存活率很低。在我们的小鼠HNSCC和ESCC致癌模型中，我们建议使用一种使用谱系追踪的方法来检验这一假说，即致癌物导致某些祖细胞/干细胞群体的早期分子变化，从而降低干细胞/祖细胞群体的多样性，从而导致野生性癌变和肿瘤形成。因此，在这一应用中，我们解决了RFA-CA-15-008的一个“挑衅性问题”：对于来自癌前领域的肿瘤，该领域中的细胞的哪些特性可以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小鼠永久标记口腔和食道中的单个正常干细胞/祖细胞及其后代。只有在加入三苯氧胺()时表达K14的细胞中才会激活依赖于Cre的重组，这是cre-ERTAM蛋白激活所必需的，这让我们能够对Cre重组酶活性进行暂时控制和特定细胞类型的控制，并允许我们跟踪这些干/祖细胞及其后代的命运。 时间到了。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-SEQ分析和全外显子DNA测序，这些克隆来自个体、上皮祖细胞/干细胞，在饮用水中使用致癌物质4-硝基喹啉氧化物(4-NQO)处理期间。这一目标将描绘出 早期分子变化发生在(A)口腔上皮，和(B)小鼠模型中的食道上皮，非常接近地反映人类HNSCC和食道SCC的发展。我们还将测量一些关键的表观遗传学变化。在特定的目标(2)中，我们将通过执行谱系追踪、全基因组RNA-SEQ分析和永久标记的来自个别上皮祖细胞/干细胞(HNSCCs和ESCCs)的细胞克隆的全外显子DNA测序，来辨别在癌症发生过程中发生的更晚的关键分子变化。在AIM(2)中，我们还将把肿瘤与形态正常的克隆细胞进行比较，这些克隆细胞出现在“正常外观”的上皮粘膜中的SCC附近。这些目标的完成，将定义作为“现场癌变”基础的遗传和表观遗传变化，提供许多关于HNSCC和食道癌发生的关键的、早期的分子变化的新信息，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.