Davis et al. BMC Cancer 2011, 11:173 http://www.biomedcentral.com/1471-2407/11/173 deletions and mutations reported in a variety of cancer types [9]. In recent high-throughput sequencing and copy number studies, MAP2K4 appears to be only very rarely targeted by base pair level mutation but is consis- tently identified as a common target for deletion, includ- ing homozygous deletion [10-12]. While some of these deletions may be a consequence of LOH targeting the nearby TP53 gene on 17q12, in a few instances there are homozygous deletions that appear to target only MAP2K4. Despite this genetic support for a tumor sup- pressor role, functional studies have generated mixed results. One experimental model demonstrated that over- expression of MAP2K4 could reduce the incidence and onset of metastasis in ovarian and prostate cancer cell lines [13]. However, in contrast, overexpression and silencing of the gene in a different cellular context was found to support a pro-oncogenic role for MAP2K4 [14]. In ovarian cancer, in addition to the apparent role of MAP2K4 in metastasis suppression, there have been limited data showing loss of expression and genetic aberrations in several small cohorts [15-17]. Gene expression by immunohistochemistry and quantitative PCR was found to be reduced in cancer samples com- pared to normal or benign ovarian tissue. The only study to examine epigenetic control of MAP2K4 in any cancer type was performed on a small number of ovar- ian cancer cases, and did not find any evidence for pro- moter methylation [16]. We have previously reported that recurrent homozygous deletions appear to specifi- cally target MAP2K4 in primary ovarian tumors, and the gene lay in a minimal region of loss of heterozygosity (LOH) in 68% (72/106) of the cases, the second most frequent locus targeted by LOH [18]. Despite this accu- mulating evidence, the status of MAP2K4 as a tumor suppressor in ovarian cancer is equivocal and therefore in this study we have undertaken a comprehensive ana- lysis of mutation, methylation and gene knockdown. Methods Clinical specimens One hundred and sixty one tumor tissue samples (77 serous, 21 mucinous, 33 endometrioid, 8 clear cell and 22 other, Additional file 1, Table S1) were obtained from patients presenting to hospitals in the south of England, UK or at the Peter MacCallum Cancer Centre, Australia. DNA was extracted either from whole tissue or from samples that were manually needle microdis- sected to ensure they contained >85% epithelial tumor cells. RNA was extracted from microdissection of subse- quent 10 μm sections using the mirVana miRNA Isola- tion Kit (Ambion Inc, Austin, TX). Normal DNA was extracted from matching peripheral blood samples as described previously [19]. DNA for mutation screening underwent whole genome amplification (WGA) using Page 2 of 7 the Repli-G Phi-mediated amplification system as described previously [20]. Institutional ethics commit- tees approved this study. Mutation screening of MAP2K4 using High Resolution Melt (HRM) Primers were designed to amplify each exon and the intron/exon boundaries of the MAP2K4 coding sequence using the software packages ExonPrimer and Primer3 [21], except for exon 11, which used previously published primers [12]. Exon 7 was covered by two overlapping pri- mer pairs. Exon 1 was not analyzed as the PCR could not be optimized to amplify a specific product, despite multi- ple different primer sets, most likely because of the high GC content. Exon 1 contains 114 bp of coding sequence and has similarly been excluded from other mutation screening studies [12,22]. Primer sequences and amplifi- cation conditions are listed in Additional file 2, Table S2. HRM using PCR products amplified from WGA template DNA and DNA sequencing were carried out as described previously [20]. Somatic alterations were confirmed by re-sequencing from non-WGA tumor DNA and the matching normal DNA. MAP2K4 promoter methylation analysis by Methylation- Specific Single Strand Conformation Polymorphism (MS- SSCP) and bisulfite sequencing The MAP2K4 CpG island was amplified using previously published oligonucleotides [16]. DNA samples were bisulphite treated using the MethylEasy Xceed Rapid DNA Bisulphite Modification kit (Human Genetic Signa- tures, Sydney, Australia) following the manufacturer’s instructions. After PCR amplification, products were ana- lyzed by SSCP using the ABI 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA) as described pre- viously [20], with SssI methylase-treated normal DNA used as a positive control for CpG island methylation. Normal DNA samples were treated using CpG methyl- transferase SssI (New England Biolabs, Ipswich, MA), which methylates all CpGs before bisulfite treatment. Samples showing a shift in mobility were sequenced as described above. Knock-down of MAP2K4 in cell culture Reagents for transient gene knockdown were obtained from Dharmacon (Thermo Fisher Scientific, Lafayette, CO), comprising the siRNA SMARTpools for MAP2K4 (L-003574-00) and a non-targeting control siRNA (D-001810-0X). This analysis was performed using the JAM and HOSE6.3 ovarian cell lines. The JAM cell line is a derivative of a poorly differentiated serous cystade- nocarcinoma [23], while the HOSE6.3 cell line has been established by E6/E7 immortalisation of human ovarian surface epithelial cells [24].