Functional Genomic Analysis Reveals Cross-talk between Peroxisome Proliferator-activated Receptor γ and Calcium Signaling in Human Colorectal Cancer Cells

Craig R Bush,Jennifer M Havens,Brian M Necela,Weidong Su,Lu Chen,Masahiro Yanagisawa,Panos Z Anastasiadis,Rudy Guerra,Bruce A Luxon,E Aubrey Thompson

doi:10.1074/jbc.m702708200

Abstract

Activation of PPARgamma in MOSER cells inhibits anchorage-dependent and anchorage-independent growth and invasion through Matrigel-coated transwell membranes. We carried out a longitudinal two-class microarray analysis in which mRNA abundance was measured as a function of time in cells treated with a thiazolidinedione PPARgamma agonist or vehicle. A statistical machine learning algorithm that employs an empirical Bayesian implementation of the multivariate HotellingT2 score was used to identify differentially regulated genes. HotellingT2 scores, MB statistics, and maximum median differences were used as figures of merit to interrogate genomic ontology of these targets. Three major cohorts of genes were regulated: those involved in metabolism, DNA replication, and migration/motility, reflecting the cellular phenotype that attends activation of PPARgamma. The bioinformatic analysis also inferred that PPARgamma regulates calcium signaling. This response was unanticipated, because calcium signaling has not previously been associated with PPARgamma activation. Ingenuity pathway analysis inferred that the nodal point in this cross-talk was Down syndrome critical region 1 (DSCR1). DSCR1 is an endogenous calcineurin inhibitor that blocks dephosphorylation and activation of members of the cytoplasmic component of nuclear factor of activated T cells transcription factors. Lentiviral short hairpin RNA-mediated knockdown of DSCR1 blocks PPARgamma inhibition of proliferation and invasion, indicating that DSCR1 is required for suppression of transformed properties of early stage colorectal cancer cells by PPARgamma. These data reveal a novel, heretofore unappreciated link between PPARgamma and calcium signaling and indicate that DSCR1, which has previously been thought to function by suppression of the angiogenic response in endothelial cells, may also play a direct role in transformation of epithelial cells.

Highlights

Peroxisome proliferator-activated receptor ␥ (PPAR␥),4 a member of the nuclear receptor family of transcription factors [1,2,3,4], is expressed as multiple splice variants, which arise because of alternative promoter utilization [5,6,7,8]
Because K-Ras mutations and loss of TGF␤ sensitivity commonly occur during the transition from adenoma to adenocarcinoma during colon carcinogenesis [80], we conclude that MOSER represents a very early stage colon cancer cell and is an appropriate model to study the effects of PPAR␥ on early stage colon carcinogenesis
Down syndrome critical region 1 (DSCR1) knockdown blocked PPAR␥ inhibition of MOSER cell invasion (Fig. 7C). These data indicate that DSCR1 is required for PPAR␥ suppression of the transformed properties of early stage colorectal cancer cells. Both pharmacological and naturally occurring PPAR␥ agonists inhibit experimental colon carcinogenesis in rodents, and a number of human colon cancer cell lines undergo growth arrest, differentiation, or apoptosis when treated with PPAR␥ agonists

Summary

Introduction

Peroxisome proliferator-activated receptor ␥ (PPAR␥), a member of the nuclear receptor family of transcription factors [1,2,3,4], is expressed as multiple splice variants, which arise because of alternative promoter utilization [5,6,7,8]. Reported no such effect [34, 35], and it has recently been shown that biallelic knock-out of PPAR␥ in colonic epithelial cells promotes colon carcinogenesis in APCϩ/Min mice [36]. These data strongly indicate that PPAR␥ blocks colon carcinogenesis in rodents. Because PPAR␥ is a transcription factor, it is likely that the tumor-suppressive effects of this receptor are because of changes in gene expression and that the mechanism of action of PPAR␥ might be inferred from a functional genomic study of the effects of thiazolidinediones on early stage colon cancer cells. PPAR␥ inhibits transformed properties of MOSER cells [56], making them an ideal model to study the mechanism whereby this receptor inhibits early steps in transformation of colonic epithelial cells

Methods

Results

Conclusion