Investigation of signaling pathways that regulate human colon crypt maturation and their dysregulation in tumorigenesis

Abstract

Intestinal epithelial cell polarity and lineages along the crypt-villus axis provide an excellent model in studying genetic programs controlling cellular proliferation and differentiation. Multipotent stem cells residing at the bottom of the intestinal crypts undergo cell division, where daughter cells will migrate upwards and undergo cell cycle arrest and differentiation. Although the biological and genetic regulation of these processes still remains unclear, there has been report on several key pathways regarding this regulation, including Wnt, Notch and TGF signaling pathways. This study is based on the hypothesis that perturbation of the signaling pathways that regulate cellular polarity and maturation along the colon crypt axis is closely related to tumor development and progression in gastrointestinal tract cancers. To study the differential gene expression between upper and bottom part of colon crypt, a total of 18 cDNA microarray experiments were performed using RNA extracted from 9 paired upper and bottom parts of the colon crypts. Gene expression data were extracted, and Significance Analysis of Microarrays (SAM) was performed to identify genes with significant correlating expression at bottom compartment of the crypts. 969 cDNA clones with significant differential expression were identified, in which 367 and 602 cDNA clones were preferentially upregulated and downregulated respectively in bottom proliferative compartment of the crypts. A significant correlation of these 969 SAM list genes with WNT target genes (obtained from inducible dominant negative TCF4 transfection in CRC cell lines by van de Watering et al) was observed, demonstrating that colon development is tightly regulated by WNT-signaling pathway through TCF4 transcription regulation. Biological pathway analysis using GenMapp illustrated that the colon crypt development involved multiple molecular pathways, including cell cycle, apoptosis, MAP kinase, WNT and BMP pathways etc. Strikingly, we have identified a set of genes that code for secretory proteins of several key pathways where most of them have been previously demonstrated to be expressed in connective tissues, postulating that intestinal myofibroblasts were playing important roles in maintaining the microenvironment for epithelial cells development. With the help of the colon top/bottom array data set obtained here, we hope to provide a comprehensive picture for understanding the global regulation in colonic epithelial cells maturation by in depth analysis of the gene expression profile.