Role of a Two-Factor Genetic Circuit Regulating Stemness in Colorectal Cancer

项目摘要 结直肠癌(CRC)是全球第三大癌症死亡原因。肿瘤干细胞 (CSCs)是肿瘤的起始、增殖、化疗耐药和转移的来源。 因此被认为会对患者的预后产生不利影响。然而，目前还没有针对性的治疗方法 CSCs用于治疗结直肠癌。多能性因子被提出用来驱动CSCs的干性，但 分子机制尚不清楚。了解分子调节器，使茎和 肿瘤干细胞的致瘤性将确定新的治疗靶点。 多能因子的作用在胚胎干细胞(ESCs)中被最好地理解，在那里它们相互作用 已经被提出用来驱动异质性。我们最近发现了一个潜在的转录回路 我们假设的多能因子Klf4和Zfp281使ESCs能够在特定的干细胞之间切换 状态，从而产生转录异质性。值得注意的是，在CRC中，KLF4和Zfp281也牵涉到 分别作为肿瘤抑制剂和促增殖剂。KLF4抑制血管内皮细胞增殖和表达 BMI1，一种表观遗传标记，我们之前发现它是结直肠癌发生所必需的，而 Zfp281通常与Bmi1共表达。KLF4和Zfp281在ESCs和CRC中的表达具有可逆性 细胞，但它们与茎的相关性在这两个上下文中是相反的，因此KLF4与更多 干细胞样但分化更多的CRC细胞。多能因子回路的作用尚未得到证实 KLF4和Zfp281之间的确切相互作用在ESCs和CRCs中都是未知的。我 假设在胚胎发育和癌症发展过程中，Klf4和Zfp281相互对立， 形成一个基因回路，调节茎和分化之间的平衡。我进一步 提出扰乱这一遗传回路将限制干性和致瘤性。 在这个提案中，我将研究Klf4和Zfp281是如何相互作用并调节胚胎和 肿瘤的发展。目标1将定义产生相反干细胞的Klf4和Zfp281的相互作用 在培养的胚胎干细胞中的状态。目标2和目标3将审查KLF4和KLF4的作用和扰动的后果 Zfp281对结直肠癌细胞干性和致瘤性的调节作用具体地说，AIM 2将使用CRC线路和 确定Klf4和Zfp281基因敲除是否限制分化能力和表达的有机化合物 在CSCS中的茎的程序。目标3将建立Klf4和Zfp281之间的相互作用，并 BMI1，并确定这些基因如何促进体内的致瘤性。该项目将建立 Klf4和Zfp281在干性和致瘤性中的作用以及它们之间的回路的性质。这个 这个项目的分子和机械洞察力将进一步加深我们对CSCs如何启动和生长的理解 以及为结直肠癌治疗寻找新的治疗靶点。

Project Summary Colorectal cancer (CRC) is the third leading cause of cancer death worldwide. Cancer stem cells (CSCs) in the colon are the source of tumor initiation, proliferation, chemoresistance, and metastasis, and are therefore considered to adversely impact patient outcome. However, there are no therapies targeting CSCs for treatment of CRC. Pluripotency factors have been proposed to drive stemness in CSCs, but the molecular mechanisms remain unclear. Understanding the molecular regulators that enable stemness and tumorigenicity in CSCs will identify novel therapeutic targets. The role of pluripotency factors is best understood in embryonic stem cells (ESCs), where their interplay has been proposed to drive heterogeneity. We recently identified a potential transcriptional circuit between pluripotency factors Klf4 and Zfp281 that we hypothesize enables ESCs to switch between specific stem cell states and thus generate transcriptional heterogeneity. Notably, in CRC, Klf4 and Zfp281 have also implicated as a tumor suppressor and pro-proliferative agent, respectively. Klf4 inhibits proliferation and expression of Bmi1, an epigenetic marker that we previously identified as required for CRC tumorigenesis, whereas Zfp281 is often co-expressed with Bmi1. Klf4 and Zfp281 display reciprocal expression in both ESCs and CRC cells, but their correlation with stemness is reversed in the two contexts, such that Klf4 is associated with more stem-like ESCs but more differentiated CRC cells. The role of pluripotency factor circuits has not been addressed in CSCs, and the precise interplay between Klf4 and Zfp281 is unknown in either ESCs or CRCs. I hypothesize that during both embryonic and cancer development, Klf4 and Zfp281 oppose each other, forming a genetic circuit that modulates the balance between stemness and differentiation. I further propose that perturbing this genetic circuit will restrict stemness and tumorigenicity. In this proposal, I will investigate how Klf4 and Zfp281 interact and regulate stemness in embryonic and tumor development. Aim 1 will define the interactions of Klf4 and Zfp281 that generate opposing stem cell states in cultured ESCs. Aims 2 and 3 will examine the role, and consequences of perturbation, of Klf4 and Zfp281 in regulating stemness and tumorgenicity of CRC cells. Specifically, Aim 2 will use CRC lines and organoids to determine whether Klf4 and Zfp281 knockout restricts the differentiation capacity and expression of stemness programs in CSCs. Aim 3 will establish the interplay of Klf4 and Zfp281 with each other, and also Bmi1, in CRC and determine how these contribute to tumorgenicity in vivo. This project will establish the role of Klf4 and Zfp281 in stemness and tumorigenicity and the nature of the circuit between them. The molecular and mechanistic insights of this project will further our understanding of how CSCs initiate and grow tumors, as well as identify novel therapeutic targets for CRC treatment.