Roles and regulation of transcriptional reprogramming in squamous carcinogenesis

总结 激活原癌基因或抑癌基因的突变是肿瘤发生的根本原因 虽然这些突变在基因组学上并不常见，但最近的基因组分析也在健康组织的许多细胞中检测到了这些突变。这些数据 经典的皮肤癌发生研究表明，原癌基因或肿瘤抑制基因的突变 在表皮祖细胞（EPCs）中耐受并维持，直到难以捉摸的机制将这些细胞转化为 - 将癌前细胞转化为干细胞样肿瘤增殖细胞（TPC），其可以支持肿瘤形成， 增长我们在鳞状细胞癌（SCC）模型中对这些TPC进行了转录谱分析，并定义了一个基因， 表达特征，区分TPC和EPC。在这个签名中，我们识别出了 PITX 1和SOX 2因子在>60%的小鼠和人SCC中，即使它们在表观遗传学上受到抑制， 而在正常皮肤上皮细胞中检测不到。我们发现PITX 1和SOX 2是SCC生长所必需的， 小鼠和患者来源的SCC模型高度富集TPC中可获得的基因调节增强子， 而不是EPCs，并负责SCC标志基因的表达。虽然PITX 1和SOX 2是关键 对于鳞状细胞癌的发生，目前尚不清楚是什么事件触发了它们在TPC中的从头表达，以及它们是否 它们结合浓缩的（失活的）染色质并打开染色质以激活SCC特异性基因表达的能力 将EPC重新编程为TPC。在这里，我们建议测试假设，致癌RAS和 炎症共同或独立地激活RUNX 1，RUNX 1启动PITX 1的表达， SOX 2，使它们能够将EPC转录重编程为TPC， 致癌作用为了验证这一假设，我们提出：1）确定是否以及如何炎症诱导 RUNX 1活性促进正常和癌前皮肤上皮细胞中PITX 1的从头表达;和2） 确定异位表达的RUNX 1、PITX 1和/或SOX 2是否以及如何将EPC重编程为TPC， 促进SCC启动。为了实现这些目标，我们已经建立了基因功能的获得和丧失 在本地小鼠和人SCC模型中的方法沿着全基因组分析（ATAC-seq， ChIP-seq、4C-seq、RNA-seq）和荧光转录报告基因测定。我们有独特的优势， 揭示了分子事件，解释：1）PITX 1和SOX 2如何在小鼠中重新表达， 人SCC，2）SOX 2和PITX 1是否建立或使用SCC定义基因调节增强子来控制 SCC标记基因的表达，以及3）基因表达的这些变化如何转化癌前病变 细胞转化为TPC，促进鳞状细胞癌发生。我们提出的研究将提供分子见解 这些承诺将指导开发预防或治疗患者SCC的方法和/或定义 可能促进其他癌症（包括但不限于肺癌、食管癌和 胰腺SCC是最常见的致命癌症之一。

SUMMARY Mutations that activate proto-oncogenes or inactivate tumor suppressor genes are the root causes of tumor initiation, but recent genomic analyses also detect these mutations in many cells of healthy tissues. These data and classic skin carcinogenesis studies suggest that mutations in proto-oncogenes or tumor-suppressor genes are tolerated and maintained in epidermal progenitor cells (EPCs) until elusive mechanisms transform these precancerous cells into stem-cell-like tumor propagating cells (TPCs) that can support tumor formation and growth. We transcriptionally profiled these TPCs in squamous cell carcinoma (SCC) models and defined a gene expression signature that distinguishes TPCs from EPCs. Within this signature, we identified the transcription factors PITX1 and SOX2 in >60% of mouse and human SCCs, even though they are epigenetically repressed and not detectable in normal skin epithelial cells. We showed PITX1 and SOX2 are required for SCC growth in mouse and patient-derived SCC models highly enriched on gene regulatory enhancers accessible in TPCs but not EPCs, and responsible for the expression of SCC signature genes. Although PITX1 and SOX2 are pivotal for squamous carcinogenesis, it is still unclear what events trigger their de novo expression in TPCs, and whether their ability to bind condensed (inactive) chromatin and open it to activate SCC-specific gene expression reprograms EPCs into TPCs. Here, we propose to test the hypothesis that oncogenic RAS and inflammation together or independently activate RUNX1, which initiates expression of PITX1 and then SOX2, allowing them to transcriptionally reprogram EPCs into TPCs that promote squamous carcinogenesis. To test this hypothesis, we propose to: 1) determine whether and how inflammation-induced RUNX1 activity promotes de novo PITX1 expression in normal and pre-cancerous skin epithelial cells; and 2) determine whether and how ectopically expressed RUNX1, PITX1, and/or SOX2 reprogram EPCs into TPCs to promote SCC initiation. To accomplish these aims, we already established genetic gain- and loss-of-function approaches in autochthonous mouse and human SCC models along with genome-wide analyses (ATAC-seq, ChIP-seq, 4C-seq, RNA-seq) and fluorescent transcriptional reporter assays. We are uniquely positioned to reveal the molecular events that explain: 1) how PITX1 and SOX2 become expressed de novo in mouse and human SCCs, 2) whether SOX2 and PITX1 establish or use SCC-defining gene regulatory enhancers to control the expression of SCC signature genes, and 3) how these changes in gene expression transform precancerous cells into TPCs that promote squamous carcinogenesis. Our proposed research will provide molecular insights that promise to guide the development of approaches to prevent or treat SCCs in patients and/or define mechanisms that may promote the initiation of other cancers including but not limited to lung, esophageal, and pancreatic SCCs, which are among the most common deadly cancers.