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

项目摘要 阐明RUNX 1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，在乳腺癌中启动是有意义的，并且具有临床相关性的潜力。 RUNX 1与信号传导通路、染色质重塑因子和染色质组织的介质相互作用 在基因调控区的蛋白质，以协调细胞增殖，生长和分化。我们最近的成果 表明RUNX 1功能是减少肿瘤生长，阻止上皮向间质转化，介导 高级染色质组织，并抑制乳腺癌干细胞（BCSC）中涉及关键基因 病理我的初步结果表明，RUNX 1抑制PI 3 K和MTOR通路基因（例如，PIK 3R1， PIK 3C 2A、S 0 S1）、细胞因子（例如，IL 1a，CXCL 8，JAK 2）和其他关键基因（例如，HIF 1a、UPAR和ZEB 1）， 通过比较BCSC与非BCSC的RNA-seq鉴定的新基因子集。BCSC能够 自我更新，驱动肿瘤生长，抵抗化疗，并导致癌症复发。与假定的 RUNX 1在这些过程中的作用，乳腺肿瘤中RUNX 1的低表达与预后不良相关。 我的中心假设是RUNX 1调节BrCa中失调的靶基因的表达， 它在介导高级染色质组织中的作用对这些细胞的增殖和分化至关重要。 功能协调发展的我建议确定RUNX 1在基底乳腺癌中发挥作用的机制， 强调其对BCSC（CD 24-低/CD 44+高）相对于非BCSC（CD 24+高/CD 44-低）的影响。的 将通过流式分选，随后通过免疫荧光法， qPCR和ChIP-qPCR检测这些基因附近的RUNX 1结合位点。RUNX 1丢失对早期的影响 乳腺癌进展和BCSC将使用鼠乳腺脂肪垫注射来确定。接下来我们就 研究RUNX 1在介导增强子调节和这些细胞的高级染色质组织中的作用。 BCSC中的关键基因，以及这种组织如何通过消耗正常和早期乳腺癌中的RUNX 1而改变 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呈现出改变的高级染色质组织，与 它们的干样表达谱，并且RUNX 1的缺失诱导更高级的染色质组织， 对这些关键基因来说更像茎。对基因组结构和基础基因表达的机制性洞察 乳腺癌细胞和BCSC将增强选择性靶向乳腺肿瘤的能力， 对常规治疗难治的BCSC。这些研究将为我未来的职业生涯打下坚实的基础 专注于癌症发生和发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.