Global analysis of gene expression changes during retinoic acid-induced growth arrest and differentiation of melanoma: comparison to differentially expressed genes in melanocytes vs melanoma

Abstract

Background: The incidence of malignant melanoma has significantly increased over the last decade. Some of these malignancies are susceptible to the growth inhibitory and pro-differentiating effects of all-trans-retinoic acid (RA). The molecular changes responsible for the biological activity of RA in melanoma are not well understood.

Results: In an analysis of sequential global gene expression changes during a 4-48 h RA treatment of B16 mouse melanoma cells, we found that RA increased the expression of 757 genes and decreased the expression of 737 genes. We also compared the gene expression profile (no RA treatment) between non-malignant melan-a mouse melanocytes and B16 melanoma cells. Using the same statistical test, we found 1495 genes whose expression was significantly higher in melan-a than in B16 cells and 2054 genes whose expression was significantly lower in melan-a than in B16 cells. By intersecting these two gene sets, we discovered a common set of 233 genes whose RNA levels were significantly different between B16 and melan-a cells and whose expression was altered by RA treatment. Within this set, RA treatment altered the expression of 203 (87%) genes toward the melan-a expression level. In addition, hierarchical clustering showed that after 48 h of RA treatment expression of the 203 genes was more closely related to the melan-a gene set than any other RA treatment time point. Functional analysis of the 203 gene set indicated that RA decreased expression of mRNAs that encode proteins involved in cell division/cell cycle, DNA replication, recombination and repair, and transcription regulation. Conversely, it stimulated genes involved in cell-cell signaling, cell adhesion and cell differentiation/embryonic development. Pathway analysis of the 203 gene set revealed four major hubs of connectivity: CDC2, CHEK1, CDC45L and MCM6.

Conclusion: Our analysis of common genes in the 48 h RA-treatment of B16 melanoma cells and untreated B16 vs. melan-a data set show that RA "normalized" the expression of genes involved in energy metabolism, DNA replication, DNA repair and differentiation. These results are compatible with the known growth inhibitory and pro-differentiating effects of RA. Pathway analysis suggests that CDC2, CHEK1, CDC45L and MCM6 are key players in mediating the biological activity of RA in B16 melanoma cells.

Figure 1

Hierarchical clustering of the 203 genes for which RA treatment of B16 cells reverts the expression towards that of melan-a cells. Average linkage was used with a Euclidean metric. The green-red color scale refers to log base two expression ratios with expression in untreated B16 cells as the denominator in all cases. Grey cells represent values which failed to pass the minimum expression level filter.

Figure 2

Pathway Studio Analysis of 203 Gene Set. Out of 203 genes, 74 genes were found to be involved in direct interactions. Protein hubs which have most significant interactions (CDC2, CHEK1, CDC45L and MCM6) are marked with an asterisk (*). Although there are relatively large numbers of interactions stemming from TP53, P21 and CMYC, these proteins were not among the most statistically significant hubs. Green color indicates a gene is upregulated in melan-a relative to B16 whereas red color indicates that a gene is downregulated in melan-a relative to B16. Color intensity reflects the expression ratio.