Does Runx1 Suppress Breast Cancer Stemness by mediating higher order chromatin organization?

项目概要 阐明 RUNX1 转录因子在控制染色质结构和基因组调控中的作用 从生物学角度来看，乳腺癌的发生是有意义的，并且有可能具有临床相关性。 RUNX1 与信号通路介质、染色质重塑因子和染色质组织相互作用 基因调控区域的蛋白质协调细胞增殖、生长和分化。我们最近的结果 表明 RUNX1 的功能是减少肿瘤生长、防止上皮细胞向间质细胞转化、介导 高阶染色质组织，并抑制与乳腺癌干细胞 (BCSC) 相关的关键基因 病理学。我的初步结果表明 RUNX1 抑制 PI3K 和 MTOR 通路基因（例如 PIK3R1、 PIK3C2A、SOS1）、细胞因子（例如 IL1a、CXCL8、JAK2）和其他关键基因（例如 HIF1a、UPAR 和 ZEB1）和 通过 RNA-seq 比较 BCSC 和非 BCSC 鉴定出的一个新的基因子集。 BCSC 有能力 自我更新、驱动肿瘤生长、抵抗化疗并导致癌症复发。与推测一致 由于 RUNX1 在这些过程中的作用，乳腺肿瘤中 RUNX1 的低表达与不良预后相关。 我的中心假设是 RUNX1 调节 BrCa 中失调的靶基因的表达 进展，并且它在介导高级染色质组织中的作用对于这些过程至关重要 功能。我建议确定 RUNX1 在基底乳腺癌中发挥作用的机制， 强调其对 BCSC（CD24-低/CD44 高）与非 BCSC（CD24 高/CD44-低）的影响。的 将通过流式分选建立对非 BCSC 或 BCSC 内关键基因的调控，然后 qPCR 和 ChIP-qPCR 用于检测这些基因附近的 RUNX1 结合位点。 RUNX1缺失对早期的影响 将使用小鼠乳腺脂肪垫注射来确定乳腺癌进展和 BCSC。接下来，我们将 检查 RUNX1 在介导增强子调节和高级染色质组织中的作用 BCSC 中的关键基因，以及如何通过消耗正常和早期乳房中的 RUNX1 来改变该组织 癌细胞。这些研究将采用高通量、高分辨率捕获染色质构象捕获 （捕获 Hi-C）。我假设 BCSC 呈现出一种改变的高阶染色质组织，与 它们的茎样表达谱，并且 RUNX1 的缺失会诱导更高阶的染色质组织， 这些关键基因更像茎。对基因组结构和基础表达的机制洞察 乳腺癌细胞和 BCSC 将增强选择性靶向乳腺肿瘤的能力，以及 对常规治疗无效的 BCSC。这些学习将为我未来的职业生涯打下坚实的基础 专注于癌症发生和进展中的基因组组织和表达。

PROJECT SUMMARY Elucidating the role of the RUNX1 transcription factor in control of chromatin architecture and genomic regulation in breast cancer initiation is meaningful from a biological perspective, and has potential to be clinically relevant. RUNX1 interacts with mediators of signaling pathways, chromatin remodeling factors, and chromatin organizing proteins at gene regulatory regions to coordinate cell proliferation, growth and differentiation. Our recent results indicate that RUNX1 functions to decrease tumor growth, prevent epithelial to mesenchymal transition, mediate higher-order chromatin organization, and suppress key genes implicated in breast cancer stem cell (BCSC) pathology. My preliminary results suggest that RUNX1 suppresses PI3K and MTOR pathway genes (eg. PIK3R1, PIK3C2A, SOS1), cytokines (eg. IL1a, CXCL8, JAK2) and other key genes (eg. HIF1a, UPAR, and ZEB1) and a novel subset of genes that were identified by RNA-seq comparing BCSCs to non-BCSCs. BCSCs are capable of self-renewal, drive tumor growth, resist chemotherapy, and cause cancer relapse. Consistent with the putative role of RUNX1 in these processes, low RUNX1 expression in breast tumors is associated with poor prognosis. My central hypothesis is that RUNX1 regulates expression of target genes that are dysregulated in BrCa progression, and that its role in mediating higher-order chromatin organization is essential to these functions. I propose to determine the mechanisms by which RUNX1 functions in basal breast cancer, with an emphasis on its influence on BCSCs (CD24-low/CD44+high) versus non-BCSCs (CD24+high/CD44-low). The regulation of key genes specifically within non-BCSCs or BCSCs will be established by flow sorting followed by qPCR, and ChIP-qPCR for RUNX1 binding sites nearby these genes. The impact of RUNX1 loss on early stage breast cancer progression and BCSCs will be determined using murine mammary fat pad injection. Next, we will examine the role of RUNX1 in mediating enhancer regulation and higher order chromatin organization of these key genes in BCSCs, and how this organization is altered by depleting RUNX1 in normal and early-stage breast cancer cells. These studies will employ high-throughput, high resolution capture chromatin conformation capture (capture Hi-C). I hypothesize that BCSCs present an altered higher order chromatin organization, consistent with their stem-like expression profile, and that the loss of RUNX1 induces higher order chromatin organization that is more stem-like for these key genes. Mechanistic insight into genome architecture and expression in basal breast cancer cells and BCSCs will enhance capabilities for selectively targeting breast tumors, as well as BCSCs that are refractory to conventional treatments. These studies will form a solid basis for my future career focus on genomic organization and expression in cancer initiation and progression.