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

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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.