Regulation of the metastasis suppressor gene MKK4 in ovarian cancer

Objectives.MKK4 is a metastasis suppressor that is downregulated in some ovarian cancers. We sought to investigate whether promoter methylation, loss of heterozygosity, or changes in phosphorylation are involved in MKK4 dysregulation during ovarian carcinogenesis.Methods.Bisulfite sequencing was used to determine MKK4 promoter methylation. PCR analysis of tumor/normal DNA was performed to determine LOH at the MKK4 locus. Normal human ovarian surface epithelium (HOSE) and SKOV-3 cells were serum starved and treated with EGF, TGFβ, or wortmannin. Western blotting was performed using antibodies that detect total and phosphorylated MKK4.Results.No MKK4 promoter hypermethylation was detected in 21 ovarian cancers. LOH was detected at the MKK4 intragenic marker D17S969 in 35% of cases and at D17S1303 in 20%. MKK4 protein was detected in 97% of ovarian tumors. The inactivated phosphoserine 80 (ser-80) form comprised 62% of phosphorylated MKK4 protein in ovarian tumors. Treatment of HOSE or SKOV-3 cells with EGF induced a 1.7- to 4.2-fold increase in phosphorylation of ser-80 MKK4 without altering total MKK4 protein. TGFβ increased MKK4 ser-80 phosphorylation by 5.4-fold above baseline. The PI3K/Akt pathway inhibitor wortmannin decreased the amount of ser-80 MKK4 by 50%, and inhibited EGF stimulation of MKK4 ser-80 phosphorylation by 60%.Conclusions.LOH of MKK4 occurs in some ovarian cancers, but without loss of MKK4 protein. MKK4 expression does not appear to be downregulated by promoter methylation. Peptide growth factors induce MKK4 ser-80 phosphorylation, which downregulates its activity. PI3K/Akt pathway inhibitors can partially block ser-80 phosphorylation and this may have therapeutic implications.