Regulation of the metastasis suppressor gene MKK4 in ovarian cancer.
OBJECTIVE
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, TGFbeta, 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. TGFbeta 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.
Spillman
Monique A
MA
Division of Gynecologic Oncology, Duke University Medical Center, Box 3079, Durham, NC 27710, USA.
Lacy
Judith
J
Murphy
Susan K
SK
Whitaker
Regina S
RS
Grace
Lisa
L
Teaberry
Vanessa
V
Marks
Jeffrey R
JR
Berchuck
Andrew
A
D001483:Base Sequence; D045744:Cell Line, Tumor; D018899:CpG Islands; D019175:DNA Methylation; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D016147:Genes, Tumor Suppressor; D006801:Humans; D007150:Immunohistochemistry; D019656:Loss of Heterozygosity; D048670:MAP Kinase Kinase 4 / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D020935:MAP Kinase Signaling System; D008969:Molecular Sequence Data; D009367:Neoplasm Staging; D010051:Ovarian Neoplasms / Q000201:enzymology* / Q000235:genetics* / Q000473:pathology; D010766:Phosphorylation; D011401:Promoter Regions, Genetic
D048670:MAP Kinase Kinase 4; C482559:MAP2K4 protein, human