The Wnt signaling pathway, mediated through active beta-catenin, is responsible for initiating the majority of cases of human colorectal cancer (CRC), and we have previously shown that hyperactivation of this pathway by histone deacetylase inhibitors (HDACis), such as butyrate, can induce the death of CRC cells. An important cellular switch that mediates the effects of Wnt-signaling activation is variation in the association between beta-catenin and the transcriptional coactivators cAMP response element binding (CREB) binding protein (CBP) and p300. Association of CBP with beta-catenin is thought to activate a set of genes linked to cell proliferation, while the p300-mediated Wnt genetic program is believed to promote cell differentiation. Small molecule agents have been discovered that modulate CBP/p300 Wnt transcriptional programs by altering the association of CBP and p300 to beta-catenin. ICG-001 and ICG-427 inhibit CBP- and p300-mediated Wnt activity, respectively, while IQ-1 prevents the shift from CBP-mediated to a p300-mediated Wnt activity.
Aim 1 of this proposal is designed to determine the role of CBP- and p300-mediated Wnt signaling in the response of CRC cells to HDACis. Aim 2 is to determine the role of CBP and p300 in the maintenance of high- and low-Wnt fractions in CRC cell line. Aim 3 will compare the effects of CBP- and p300-mediated Wnt activity on CRC initiation and progression.
In Aim 1, cells will be cotreated with HDACis and ICG-001, ICG-427, or IQ-1 and the levels of Wnt activity, apoptosis, proliferation, differentiation, and CBP- or p300-beta-catenin binding measured. Aim 2 of this proposal may mirror similar heterogeneity observed in human tumors and which may be of clinical significance. Aim 3 will use CRC cell line model systems of initiation and progression: the normal colon cell lines CCD-841CoN, the adenoma line LT97, the primary colon carcinoma cell line SW480, and the lymph node metastasis cell line SW620. Cells will be treated with HDACis and the small molecule agents, and assayed as described above.
We will also attempt to use changes in CBP- and p300-mediated Wnt signaling to shift colonic cells between cell type, modifying CBP- and p300-mediated gene expression in the LT97 adenoma line to shift the adenoma phenotype to more characteristic of the CCD-841CoN normal cells, or the SW480 carcinoma cells. We will use microarray analyses to determine the patterns of gene expression responsible for these CBP- or p300-mediated changes in colonic neoplastic phenotype.