Our lab is interested in understanding how Wnt/ß-catenin and Hippo/YAP signaling pathways control intestinal proliferation and how these pathways contribute to colorectal cancer when de-regulated. We use molecular biology and genetic approaches in human tissue culture and mouse model systems to dissect how these pathways regulate cellular proliferation.
Project 1: Wnt/ß-catenin signaling, Myc and intestinal proliferation
The Wnt signaling pathway is critical to establish and maintain the proliferative compartment of the intestines. Wnt instructs intestinal stem cells to self-renew and to produce transit-amplifying progenitor cells. The key effector protein in the canonical Wnt signaling pathway is the ß-catenin transcriptional co-activator. In the cytoplasm, ß-catenin associates with a multiprotein destruction complex that contains the adenomatous polyposis coli (APC), Axis inhibitor (Axin), glycogen synthase kinase beta (GSK3ß), and casein kinase I (CKI) proteins. Here ß-catenin is phosphorylated and subsequently targeted for degradation by the proteasome. When Wnt glycoproteins bind Frizzled and LRP cell surface receptors, the destruction complex disassembles, and stabilized ß-catenin translocates into the nucleus. In the nucleus, ß-catenin binds to members of the T-cell factor (TCF) family of transcription factors to activate target gene transcription. Mutations in components of the Wnt/ß-catenin signaling pathway, most often in the APC gene, are found in 85-90% of sporadic colon cancer cases and in individuals with Familial Adenomatous Polyposis (FAP). These mutations lead to heightened accumulation of ß-catenin in the nucleus and inappropriate activation of target genes controlled by ß-catenin/TCF complexes. Therefore identifying direct Wnt/ß-catenin targets in colon cancer cells is required to understand colorectal carcinogenesis.
Our lab used ChIP-Seq to identify ß-catenin binding regions in human colorectal cancer genomes. A recent screen found over 2,000 high confidence targets, including ß-catenin binding regions around the c-Myc proto-oncogene (MYC). MYC is required for Wnt/ß-catenin induced cellular proliferation in the intestine and Myc is commonly overexpressed in colorectal cancers. Wnt/ß-catenin-activation of MYC expression was thought to be controlled solely through a 5′ Wnt responsive element (WRE) that maps to the proximal promoter region. However, we identified a more robust ß-catenin binding region that maps 1.4-kb downstream from the MYC transcription stop site in our genome-wide screen. We have shown that this binding region demarcates a functional enhancer element which we call the MYC 3′ WRE. We have shown that this MYC 3′ WRE is the principal regulatory element that controls MYC expression in response to Wnt/ß-catenin and mitogen signaling. We have also shown that ß-catenin/TCF4 complexes assemble a chromatin loop at MYC that integrates the MYC 5′ WRE and 3′ WREs.
Model for Wnt/ß-catenin regulation of MYC gene expression in CRC cells.
Three projects in the lab are aimed at understanding how the MYC 3′ WRE controls MYC expression and the role for the MYC 3′ WRE in regulating intestinal homeostasis and intestinal diseases.
1. Is the MYC 3′ WRE required for intestinal homeostasis and colorectal cancer? We have generated a mouse model that lacks the MYC 3′ WRE and are using genetic approaches to answer this question.
2. Which trans acting factors bind to and are required for regulation of MYC expression through the 3′ WRE? We are using biochemical and molecular biological approaches to first identify proteins that associate with the 3′ WRE in human colorectal cancer (CRC) cells. We will then determine how these factors regulate MYC expression in CRC cell lines.
3. What factors mediate the MYC 5’3′ chromatin loop? ß-Catenin and TCF4 are required for mediating the MYC 5’3′ chromatin loop in CRC cells. However, as these proteins cannot dimerize we are searching for factors that provide the “molecular glue” that stabilizes the loop.
Project 2: Cross-talk between Wnt/ß-catenin and Hippo/YAP signaling pathways
The Hippo signaling pathway controls organ size in D. melanogaster and higher eukaryotes. Yes-associated protein (YAP) is a transcriptional co-activator and it is key effector protein in the Hippo pathway. The subcellular localization of YAP is controlled by phosphorylation. When the Hippo pathway is engaged, activation of a core kinase complex containing Hippo (Mst1/2 in mammals), Lats 1/2 kinases, Mob, and Salvador, leads to YAP phosphorylation and its retention in the cytoplasm through interactions with 14-3-3 proteins. When the Hippo pathway is disengaged, YAP is translocated to the nucleus where it associates with members of the TEA domain-containing transcription factors (TEADs). YAP/TEAD complexes activate expression of growth-promoting genes including connective tissue growth factor (CTGF) and the EGF family member amphiregulin (AREG). We have recently identified a Wnt responsive element within the first intron of YAP and have demonstrated that ß-catenin/TCF4 complexes regulate YAP gene expression in colorectal cancer cell lines. Furthermore, we have found that YAP promotes CRC growth in soft agar and CRC growth in mouse xenografts. These results implicate YAP as an oncogene whose expression is driven by abberant Wnt/ß-catenin signaling in human CRC cells.
We are using molecular biological, biochemical and functional genomics approaches to address the following questions.
1. How does YAP function to increase target gene expression? Does YAP regulate epigenetic signatures and the chromatin architecture at target genes?
2. Do Wnt/ß-catenin targets also bind YAP/TEAD complexes?
3. What are the direct YAP targets in human CRC cells?