Oral Cancer Initiating Cells: Characterization

描述（由申请人提供）：尽管强化治疗通常结合手术、放疗和化疗，口腔鳞状细胞癌（OSCCs）的长期生存率仅为15-50%。因此，在OSCCs的药物治疗/化疗方面有很大的改进需求。目前的一种理论认为，常规治疗之所以失败，是因为它不能充分治疗致癌细胞（CICs），也称为癌症干细胞（CSCs）。本实验室建立了小鼠口腔癌的4-NQO（4-硝基喹啉氧化物）癌变模型，这是目前应用最广泛的研究OSCC发生的小鼠模型。当我们在饮用水中提供4-NQO（一种致癌物质和吸烟引起的肿瘤病变的替代物）时，小鼠的口腔出现了模仿人类的病变，包括增生、不典型增生、白斑、乳头状瘤和侵袭性鳞状细胞癌（SCCs）；此外，该小鼠模型中OSCC的分子标记与人类OSCC中的许多分子标记相同。在此，我们建议在这个4-NQO口腔癌发生模型中使用细胞谱系追踪方法来验证参与染色质调节的某些Polycomb蛋白，特别是Bmi1，也在口腔中假定的CICs中起主要作用的假设。在添加他莫昔芬时，通过使用具有他莫昔芬调控的、由Bmi1启动子驱动的creER（TAM）融合蛋白基因的转基因小鼠，将表达Bmi1的细胞永久标记，并将其与Rosa26“五彩纸”报告基因转基因小鼠杂交。这些小鼠将在癌变过程中进行随访，以确定随时间发展的OSCCs中表达Bmi1的细胞后代和Bmi1+标记细胞的表达。我们还将通过在小鼠口腔癌变过程中使用强力霉素调节的表达载体，通过过表达Bmi1，特别是在口腔上皮中，以一种受调节的方式，来表征Bmi1在OSCC中的功能。这些目标的完成将为我们提供许多关于Bmi1基因的新信息，Bmi1基因被认为是人类OSCCs中形成CICs所需的关键基因。此外，本研究中使用和进一步发展的技术将为我们提供有用的、强大的工具，用于识别和研究OSCC中的CICs，包括它们的自我更新能力、分化能力和表型流动性。这些知识对于发现新疗法至关重要

DESCRIPTION (provided by applicant): Despite intensive treatment that generally combines surgery, radiation, and chemotherapy, oral squamous cell carcinomas (OSCCs) have a long-term survival rate of only 15-50%. Thus, there is a great need for improvements in pharmacologic treatments/chemotherapeutics for OSCCs. One current theory is that conventional treatment fails because it does not adequately treat cancer-initiating cells (CICs), also called cancer stem cells (CSCs). Our laboratory developed the 4-NQO (4-nitroquinoline oxide) carcinogenesis model of oral cancer for mice, now the most widely used murine model for the study of the development of OSCC. When we provide 4-NQO, a carcinogen and a surrogate for the neoplastic lesions caused by smoking, in the drinking water, mice develop lesions in their oral cavities that mimic those in humans, including hyperplasia, dysplasia, leukoplakia, papilloma, and invasive squamous cell carcinomas (SCCs); moreover, the molecular markers of OSCC in this murine model are the same as many of those in human OSCCs. Here we propose to use a cell lineage-tracing approach in this 4-NQO oral carcinogenesis model to test the hypothesis that certain Polycomb proteins which are involved in chromatin regulation, specifically Bmi1, also play a major role in putative CICs in the oral cavity. Bmi1-expressing cells will be permanently marked at the time of tamoxifen addition by using transgenic mice that have a tamoxifen-regulated, creER(TAM) fusion protein gene driven by the Bmi1 promoter, and crossing them with Rosa26 "confetti" reporter transgenic mice. These mice will be followed during the carcinogenesis process to determine the Bmi1-expressing cell progeny and the expression of Bmi1+ marked cells in OSCCs that develop over time. We will also characterize the functions of Bmi1 in OSCC by over- expressing Bmi1, specifically in the oral cavity epithelium and in a regulated manner, through the use of a doxycycline-regulated expression vector in mice during oral cavity carcinogenesis. Completion of these aims will provide us with much new information about the Bmi1 gene, which is thought to be a key gene required for formation of CICs in human OSCCs. Moreover, the techniques used and further developed in this proposed research will provide us with useful, powerful tools with which to identify and study CICs in OSCC, including their ability to self-renew, their abilityto differentiate, and their phenotypic fluidity. This knowledge is essential to discover new therapies and to screen for drugs that target CICs in human OSCCs.