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。癌症干细胞 结肠中的肿瘤干细胞（CSC）是肿瘤起始、增殖、化学抗性和转移的来源， 并因此被认为对患者结果有不利影响。然而，目前还没有针对 用于治疗CRC的CSC。已经提出多能性因子来驱动CSC中的干性，但是 分子机制仍不清楚。了解使干性和 CSCs的致瘤性将鉴定新的治疗靶点。 多能性因子的作用在胚胎干细胞（ESCs）中得到了最好的理解， 已经被提出来驱动异质性。我们最近发现了一个潜在的转录回路， 我们假设多能性因子Klf4和Zfp281能够使ESCs在特定的干细胞之间转换， 状态，从而产生转录异质性。值得注意的是，在CRC中，Klf4和Zfp281也涉及 分别作为肿瘤抑制剂和促增殖剂。Klf4抑制细胞增殖和表达 Bmi1是我们以前鉴定为CRC肿瘤发生所需的表观遗传标记，而 Zfp281通常与Bmi1共表达。Klf4和Zfp281在ESC和CRC中显示相互表达 细胞，但它们与干细胞的相关性在两种情况下是相反的，因此Klf4与更多的干细胞相关。 干细胞样ESC但更分化的CRC细胞。多能性因子回路的作用尚未被证实。 在CSC中，Klf4和Zfp281之间的精确相互作用在ESC或CRC中是未知的。我 假设在胚胎和癌症发育过程中，Klf4和Zfp281相互对立， 形成一个调节干性和分化之间平衡的遗传回路。我进一步 提出扰乱这种遗传回路将限制干细胞和致瘤性。 在本研究中，我将研究Klf4和Zfp281如何相互作用，并在胚胎干细胞中调节干细胞性， 肿瘤发展目的1将定义Klf4和Zfp281的相互作用，其产生相反的干细胞 培养的ESC中的状态。目标2和3将研究Klf 4的作用和扰动的后果， Zfp281对大肠癌细胞干细胞性和致瘤性的调控具体而言，目标2将使用CRC线路， 类器官以确定Klf4和Zfp281敲除是否限制分化能力和表达 在CSCs中的Sterness项目。目的3将建立Klf4和Zfp281相互作用，并且 Bmi1，在CRC中，并确定这些如何有助于体内致瘤性。该项目将建立 Klf4和Zfp281在干细胞性和致瘤性中的作用以及它们之间回路的性质。的 这个项目的分子和机制的见解将进一步加深我们对CSCs如何启动和生长的理解 肿瘤，以及确定用于CRC治疗的新的治疗靶点。

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.