Illuminating the Role of Oral Stem Cells in the Development of Oral Squamous Cell Carcinomas

总结 头颈部鳞状细胞癌（HNSCC）是全球第六大常见癌症。尽管 几十年的研究表明，受折磨的患者继续遭受惊人的~50%的五年死亡率。两 巨大的障碍阻碍了HNSCC更有效疗法的发展：1）对HNSCC的认识不足， 导致恶性肿瘤的途径和2）无法及早发现肿瘤。虽然很多事情 通过下一代测序，我了解了HNSCC的突变景观，这是一个巨大的 在将这些基因组信息转化为功能相关性方面仍然存在挑战。对动物模型的依赖 对于分离驾驶员和乘客突变至关重要，以确定协同作用的途径， 促进侵袭性肿瘤，并开发诊断/预后工具和治疗靶点。不幸的是， 开发和快速测试这些模型的能力受到大量时间和资源投入的阻碍 来产生转基因小鼠。本提案通过以下方式解决这些重大的知识差距： 协同实施两个主要开发的尖端遗传和成像技术， investigators.我们的长期目标是建立高通量口腔癌小鼠模型， 阐明促进肿瘤生长的神秘途径，为治疗提供新途径的见解。在这里， 我们采用我们的多功能子宫内慢病毒转导技术和新型LumiFluor生物发光技术， 共振能量转移（BRET）报告的一个有效的模型，口头取消，操纵未充分探索 全基因组HNSCC测序研究揭示的遗传途径，并可视化它们如何影响肿瘤 生长动力学我们的目标是了解自我更新和分化途径如何对正常 发展都是由癌细胞决定的。我们的理由是三分之二的HNSCC患者肿瘤显示 分化基因突变，这与患者生存率低相关。基于我们令人信服的 初步研究，我们将测试中心假设，对称和不对称细胞之间的不平衡， 细胞骨架控制纺锤体定向机制介导的肿瘤分裂和表观遗传 干细胞性调节在HNSCC肿瘤发生中起重要作用。我们的具体目标是：1） 确定纺锤体定向基因在口腔癌发生中的功能，因为该途径直接调节 自我更新/分化决定;和2）建立用于以下的高通量体内功能测定： 肿瘤发生的调节剂。这两个目标都使用了我们改进的Cre诱导的口腔癌小鼠模型， 由高危HPV 16 E6/E7和化学致癌物驱动的经验证的既定肿瘤发生方案 4-NQO。拟议的研究计划在技术和概念上都是创新的。纺锤体 定向是实体肿瘤生长动力学的重要调节因子，这一点尚未得到证实，我们拥有独特的 用于快速遗传操作的宫内慢病毒RNAi工具包，用于高级成像的LumiFluor报告基因，以及 HPV+ HNSCCs的诱导型模型，这将使我们能够对HNSCC生物学有重要的见解。

SUMMARY Head and neck squamous cell carcinomas (HNSCCs) are the sixth most common cancer worldwide. Despite decades of research, afflicted patients continue to suffer a staggering ~50% five-year mortality rate. Two enormous obstacles impede the development of more effective therapies for HNSCC: 1) poor understanding of the pathways that lead to malignancy and 2) inability to detect tumors early enough. While much has been learned about the mutational landscape of HNSCCs through next generation sequencing, a tremendous challenge remains in translating this genomic information into functional relevance. Reliance on animal models of cancer is critical for separating driver from passenger mutations, to determine pathways that synergize to promote aggressive tumors, and to develop diagnostic/prognostic tools and therapeutic targets. Unfortunately, the ability to develop and rapidly test these models is hampered by the large investment of time and resources required to generate transgenic mice. This proposal addresses these significant knowledge gaps through the synergistic implementation of cutting-edge genetic and imaging techniques developed by the two principal investigators. Our long-term goal is to establish high-throughput mouse models of oral cancer that will elucidate cryptic pathways that promote tumor growth, providing insights into new avenues for therapy. Here, we adopt our versatile in utero lentiviral transduction technique and novel LumiFluor bioluminescence resonance energy transfer (BRET) reporter to a validated model of oral cancel, to manipulate underexplored genetic pathways revealed by genome-wide HNSCC sequencing studies, and visualize how they affect tumor growth kinetics. Our objective is to understand how self-renewal and differentiation pathways critical to normal development are co-opted by cancer cells. Our rationale is that two-thirds of HNSCC patient tumors show mutations in differentiation genes, which is correlated with poor patient survival. Based on our compelling preliminary studies, we will test the central hypothesis that imbalance between symmetric and asymmetric cell divisions in tumors—mediated by cytoskeletal control of the spindle orientation machinery and epigenetic regulation of “stem-ness”—play important roles in HNSCC tumorigenesis. Our Specific Aims are to: 1) Determine the function of spindle orientation genes in oral carcinogenesis, as this pathway directly regulates self-renewal/differentiation decisions; and 2) Establish a high-throughput in vivo functional assay for modulators of tumorigenesis. Both aims use our improved Cre-inducible mouse model of oral cancer based on a validated, established tumorigenesis protocol driven by high-risk HPV16 E6/E7 and the chemical carcinogen 4-NQO. The proposed research plan is both technically and conceptually innovative. The idea that spindle orientation is an important regulator of solid tumor growth kinetics is untested, and we possess a unique toolkit—in utero lentiviral RNAi for rapid genetic manipulations, LumiFluor reporter for advanced imaging, and an inducible model of HPV+ HNSCCs—that will allow us to make important insights into HNSCC biology.