Methylation and mutation of the inhibin-α gene in human melanoma cells and regulation of PTEN expression and AKT/PI3K signaling by a demethylating agent

Abstract

Inhibin suppresses the pituitary secretion of follicle-stimulating hormone and has been reported to act as a tumor suppressor gene in the gonad in mice. Epigenetic modifications, mutations, changes in the loss of heterozygosity (LOH) of the inhibin-alpha gene and regulation of gene expression in response to a demethylating agent [5-aza-2'-deoxycytidine (5-Aza-dC)] in human melanoma cells were assessed. In addition, the association between a mutation in the 5'-untranslated region (5'-UTR) of the inhibin-alpha subunit and the expression of phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 2 (PREX2) and phosphatase and tensin homolog (PTEN) as well as AKT/PI3K signaling was determined. The methylation status of the CpG sites of the inhibin-alpha promoter was analyzed by methylation-specific PCR in bisulfite-treated DNA. Cell viability was counted using the trypan blue assay, mRNA expression was examined via reverse transcription-quantitative PCR, and protein expression was examined via western blot analysis. The inhibin-alpha promoter was hypermethylated in G361, SK-MEL-3, SK-MEL-24 and SK-MEL-28 cells and moderately methylated in SK-MEL-5 cells. Inhibin-alpha gene mutations were observed in the 5'-UTR exon 1 of G361, SK-MEL-5, SK-MEL-24 and SK-MEL-28 cells as well as in exon 2 of SK-MEL-3 cells. Allelic imbalance, including LOH, in the inhibin-alpha gene was detected in human melanoma cells. Treatment with 5-Aza-dC increased inhibin-alpha mRNA and protein levels, inhibited cell proliferation, and delayed the doubling times of surviving melanoma cells. In 5-A za-dC-treated cells, PREX2 protein expression was slightly increased in G361 and SK-MEL-24 cells and decreased in SK-MEL3, SK-MEL-5 and SK-MEL-28 cells. However, the protein expression of PTEN was decreased in melanoma cells. In addition, AKT and PI3K protein phosphorylation levels increased in all melanoma cells, except of 6361 cells, demonstrating decreased PI3K protein phosphorylation. These data provided evidence that methylation, mutation and LOH are observed in the inhibin alpha-subunit gene and gene locus in human melanoma cells. Furthermore, the demethylating agent reactivated inhibin-alpha gene expression and regulated PREX2 expression. AKT/PI3K signaling increased as PTEN expression decreased. In addition, mutations in the tumor suppressor inhibin-alpha, PTEN and p.5.3 genes were not associated with transcriptional silencing, gene expression and cell growth as analyzed through experiments and literature reviews. These data demonstrated that methylation and mutations were associated with the inhibin-alpha gene in human melanoma cells and indicated the regulation of PTEN expression and AKT/PI3K signaling by a demethylating agent.