cDNA microarray gene expression profiling of hedgehog signaling pathway inhibition in human colon cancer cells

Abstract

Background: 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. 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 findings: To 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 p21(Cip1) (CDKN1A) and p15(Ink4b) (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/significance: This 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.

Figure 1. Expression of GLI1, GLI2, PTCH1 and cell cycle distribution of HT29 and GC3/c1 cells.

Cells were treated for up to 48 hr with GANT61 (20 µM) or with 0.2% DMSO (vehicle control). (A) qRT-PCR of GLI1, GLI2 and PTCH1 genes from time 0 to 48 hr. (B) DNA was extracted at 24 hr after treatment, stained with propidium iodide, and subsequently analyzed for the effects of inhibition of HH signaling on phase distribution of cells within the cell cycle by flow cytometry.

Figure 2. Venn diagram summarizing Differentially Expressed Genes (DEGs) in GANT61-treated HT29 and GC3/c1 cells.

Cells were treated with GANT61 (20 µM) or vehicle alone (0.2% DMSO) for 24 hr, and total RNA extracted as described in Materials and Methods. Changes in gene expression were determined by cDNA microarray gene profiling using the Illumina Human-ref8 V3.0 Bead-Chip array. Genes with a False Discovery Rate (FDR)-adjusted p-value (p<0.001) and fold change >1.5 were considered DEGs. Upper panel: up-regulated genes. Lower panel: down-regulated genes. Differential expression for the total, unique up-regulated, unique down-regulated, common up-regulated, and common down-regulated DEGs, are shown.

Figure 3. The top 15 canonical signaling pathways influenced by inhibition of GLI1/GLI2 function in HT29 and GC3/c1 cells.

The top 15 canonical signaling pathways, determined by IPA, that were significantly up-regulated or down-regulated by GANT61 treatment in HT29 and GC3/c1 cells, are shown. The 1,368 DEGs in HT29 and 1,002 DEGs in GC3/c1 were mapped to the IPA- defined network. The significance p-values that determine the probability that the association between the genes in the dataset and the canonical pathway is by chance alone were calculated by Fisher's exact test, and are expressed as –log (p-value). A. Pathways with enriched down-regulated genes. B. Pathways with enriched up-regulated genes. Blue: Pathways common to both HT29 and GC3/c1. Red: Pathways unique to either HT29 or GC3/c1. Yellow squares: Ratio of the number of DEGs that map to a specific canonical pathway divided by total number of genes that make up that pathway.

Figure 4. Heat map showing fold change patterns of most highly DEGs in GANT61-treated human colon carcinoma cell lines.

The heat map was generated in Matlab (Mathworks), and compares fold change patterns of the most highly DEGs in HT29 and GC3/c1 cells after GANT61 treatment. The most highly DEGs demonstrated a differential expression p-value of p<0.001 between vehicle control (0.2% DMSO) and GANT61-treated cells. Left panel (red): up-regulated genes. Right panel (green): down-regulated genes. Genes denoted with asterisks define those genes with specific roles in G1/S transition, S-phase progression, DNA replication or repair, or regulation of the G2- or M- phase transitions. Fold changes of all down-regulated DEGs and all but one up-regulated DEG are ≤8 (central color spectrum bar).

Figure 5. Selected DEGs from cDNA array gene expression profiling analyzed by qRT- PCR in HT29 (A) or GC3/c1 (B).

Cells were treated with vehicle alone (0.2% DMSO) or GANT61 (20 µM) for 16 hr, 24 hr, 38 hr, or 48 hr. Total RNA was extracted and qRT-PCR was performed as described in Materials and Methods using the primer sets listed in Table 2. Data represent the mean±SD of 4 determinations, and GAPDH was used to normalize the relative mRNA levels.

Figure 6. Schematic representation of genes involved in GANT61-induced inhibition of cell cycle progression.

From cDNA microarray, heat map, and qRT-PCR analyses, genes involved at different phases of the cell cycle including the G1/S transition, and progression through S- G2- and M- phases, are shown. The genes identified include CDK inhibitors, members of the CDK and CDC families, cyclins, genes involved in DNA replication and repair, and genes that regulate the mitotic spindle, and involve 5 of the 12 common signaling pathways determined by IPA analysis. Red: Up-regulated genes. Green: Down-regulated genes. Light shade→dark shade, increasing differential expression.