CDNA Microarray Gene Expression Profiling of Hedgehog Signaling Pathway Inhibition in Human Colon Cancer Cells

Ting Shi,Mohammad Aziz,Tapati Mazumdar,Janet A Houghton,Zhong-Hui Duan,Akwasi Agyeman,Jennifer Devecchio

doi:10.1371/journal.pone.0013054

Abstract

BackgroundHedgehog (HH) signaling plays a critical role in normal cellular processes, in normal mammalian gastrointestinal development and differentiation, and in oncogenesis and maintenance of the malignant phenotype in a variety of human cancers. Increasing evidence further implicates the involvement of HH signaling in oncogenesis and metastatic behavior of colon cancers. However, genomic approaches to elucidate the role of HH signaling in cancers in general are lacking, and data derived on HH signaling in colon cancer is extremely limited.Methodology/Principal FindingsTo identify unique downstream targets of the GLI genes, the transcriptional regulators of HH signaling, in the context of colon carcinoma, we employed a small molecule inhibitor of both GLI1 and GLI2, GANT61, in two human colon cancer cell lines, HT29 and GC3/c1. Cell cycle analysis demonstrated accumulation of GANT61-treated cells at the G1/S boundary. cDNA microarray gene expression profiling of 18,401 genes identified Differentially Expressed Genes (DEGs) both common and unique to HT29 and GC3/c1. Analyses using GenomeStudio (statistics), Matlab (heat map), Ingenuity (canonical pathway analysis), or by qRT-PCR, identified p21Cip1 (CDKN1A) and p15Ink4b (CDKN2B), which play a role in the G1/S checkpoint, as up-regulated genes at the G1/S boundary. Genes that determine further cell cycle progression at G1/S including E2F2, CYCLIN E2 (CCNE2), CDC25A and CDK2, and genes that regulate passage of cells through G2/M (CYCLIN A2 [CCNA2], CDC25C, CYCLIN B2 [CCNB2], CDC20 and CDC2 [CDK1], were down-regulated. In addition, novel genes involved in stress response, DNA damage response, DNA replication and DNA repair were identified following inhibition of HH signaling.Conclusions/SignificanceThis study identifies genes that are involved in HH-dependent cellular proliferation in colon cancer cells, and following its inhibition, genes that regulate cell cycle progression and events downstream of the G1/S boundary.

Highlights

Hedgehog (HH) signaling plays a critical role in a variety of normal cellular processes
In HT29 and GC3/c1 cells treated with GANT61 (20 mM) for up to 48 hr, expression of the target genes GLI1 and GLI2 were both down-regulated, and the HH ligand receptor PTCH1, as determined by Quantitative Real-Time PCR (qRT-PCR) (Figure 1A)
To delineate the changes in gene expression in HT29 and GC3/c1 human colon carcinoma cell lines in response to treatment with the GLI1/GLI2 antagonist, GANT61, the expression of 18,401 human genes was profiled in control cells treated with vehicle (0.2% DMSO) and in cells treated with GANT61 (20 mM) for 24 hr

Summary

Introduction

Hedgehog (HH) signaling plays a critical role in a variety of normal cellular processes. It is pivotal in embryogenesis, regulation of the epithelial-to-mesenchymal transition, the patterning of a diverse range of vertebrate structures in a variety of organs, maintenance of adult tissue homeostasis, tissue repair, cellular proliferation, and in cell survival [1,2,3,4,5,6,7,8,9]. The canonical HH pathway is critical to normal mammalian gastrointestinal development, where it is involved in the coordinate regulation of differentiation of normal intestinal villi [10,11,12]. Hedgehog (HH) signaling plays a critical role in normal cellular processes, in normal mammalian gastrointestinal development and differentiation, and in oncogenesis and maintenance of the malignant phenotype in a variety of human cancers. Genomic approaches to elucidate the role of HH signaling in cancers in general are lacking, and data derived on HH signaling in colon cancer is extremely limited

Methods

Results

Conclusion