Signaling by the Wnt family of secreted proteins through transcription coactivator ?-catenin (the Wnt/?-catenin pathway) plays central roles in regulation of intestinal stem cells (ISCs) and homeostasis of the gastrointestinal (GI) tract. R-spondin (Rspo) proteins are secreted molecules that enhance Wnt/?-catenin signaling through stabilizing Wnt receptors, and they exhibit potent stimulation effect on self-renewal and proliferation of ISCs. Anomaly of Wnt/Rspo signaling leads to GI diseases including colorectal cancer (CRC). Wnt/?-catenin signaling controls ISCs through an ISC-specific gene expression program, which is driven by the DNA-bound TCF/LEF (T cell factor/Lymphoid enhancer factor) family of transcription factors in complex with ?- catenin. TCF/?-catenin-mediated transcriptional regulation has been a cornerstone for our understanding of the Wnt/?-catenin pathway including in ISC regulation and CRC pathogenesis. To better understand Wnt/Rspo signaling and search for additional potential therapeutic target for CRC treatment, we performed a functional cDNA expression screen and identified a Zinc-finger (Znf) transcription factor as a potent stimulators of TCF/?-catenin-dependent transcription. Our preliminary data suggest that this Znf is required for (i) Wnt/Rspo stimulation of ISC expansion in mouse intestinal organoids; (ii) for TCF/?- catenin-mediated Wnt target genes/stem cell signature genes in human CRC cell lines; and (iii) for proliferation of CRC cell lines. Our preliminary data further suggest that the Znf binds to TCF, and co-occupies with TCF/?- catenin on enhancers/promoters of Wnt target genes in chromatin. Our preliminary findings identify a novel critical component of Wnt/Rspo signaling in ISCs and CRC cells, and reveal an unappreciated complexity in the mechanism by which TCF/?-catenin-mediated gene activation is achieved. We propose three specific aims in this application to investigate the Znf in Wnt/Rspo signaling in ISC regulation and CRC pathogenesis.
In Aim 1 we will define the Znf requirement in TCF/?-catenin-driven transcription via genome-wide RNA-seq and CHIP-seq techniques, thereby addressing whether this factor is required for all or a subset of TCF/?-catenin target genes;
In Aim 2 we will examine the Znf requirement in human intestinal organoids and primary CRC organoids, attempting to validate its critical role in human GI and cancer biology; and in Aim 3 we will generate conditional Znf deletion mutant mice, thereby studying its role in intestinal tissue homeostasis and tumor formation in vivo.
This project aims to understand how a major cell-cell communication pathway, initiated by Secreted WNT/RSPO proteins, governs intestinal stem cells and homeostasis of the gastrointestinal (GI) tract and participates in pathogenesis of human colorectal cancer (CRC). Our studies will likely provide critical insights into GI biology and GI cancer therapeutics.
