Davis et al. BMC Cancer 2011, 11:173 http://www.biomedcentral.com/1471-2407/11/173 expresses high levels of MAP2K4. Expression of MAP2K4 in transfected cells was evaluated at 48 h post- transfection by QPCR in each of the assays performed (Additional file 5, Figure S3). In each case, highly effi- cient gene silencing was observed, with at least 75% silencing of MAP2K4 expression in both cell lines ana- lyzed. Cellular proliferation and metabolism was mea- sured across a seven-day period. Both JAM and HOSE6.3 showed some reduction in cell number over this time course in the cells treated with MAP2K4 siRNA, however this was only significant for the JAM cells (P = 0.001, 2-way ANOVA). A recent study used a stable RNAi system to knock down expression of MAP2K4, and was able to select clones with reduced MAP2K4 expression, suggesting that loss of the gene may not always adversely affect cell viability or prolifera- tion [32]. In this system the study found that MAP2K4 inactivation correlated with increased invasiveness through the induction of the epithelial to mesenchymal transition (EMT). Another recent study found that ecto- pic expression of MAP2K4 mutants increased ancho- rage-independent growth in NIH3T3 cells [33]. The effect of MAP2K4 gene knockdown may therefore be dependent on the in vitro experimental system used, perhaps not surprisingly given the role of the protein in such a complex signalling network. Conclusions In summary, we have conducted a comprehensive analy- sis of mutation, methylation, expression and gene knockdown of MAP2K4 in ovarian cancer. We did not detect any methylation at the MAP2K4 promoter in ovarian tumour tissue, consistent with one other study to date [16]. We did find genetic alterations confirming MAP2K4 as a tumour suppressor gene for ovarian can- cer in a subset of high grade serous and endometrioid cases, including a specific 16 bp homozygous deletion in exon 7 as well as larger homozygous deletions. In addi- tion, we found that MAP2K4 was frequently targeted by hemizygous deletion and that this correlated with decrease in gene expression in several independent ovar- ian cancer data sets. Additional material Additional file 1: Table S1 Clinical samples analyzed in the mutation and methylation screens Additional file 2: Table S2 Oligonucleotide primer sequences Additional file 3: Figure S1 Correlation of copy number with expression for MAP2K4. Three data sets are shown: Ramakrishna Gene1.0ST/SNP6.0 data [29], TCGA U133A/SNP6.0, and Etemadmoghadam U133Plus2/50K data [30]. For the U133 platforms probeset 203266_s_at is shown. Copy number is taken from the average copy number value of SNPs within the segment of lowest copy number intersecting with the MAP2K4 gene. R and p values for Pearson’s correlation. Page 6 of 7 Additional file 4: Figure S2 Overall survival of ovarian cancer patients relative to MAP2K4 expression. A. Partek output for Cox regression analysis in the Tothill data set of MAP2K4 expression probeset 203266_s_at, including patient age and residual disease as co-factors. B. Kaplan-Meier curves, showing difference in survival of patients with low MAP2K4 expression (bottom quartile of cases, red solid line) compared to all remaining cases (blue dashed line) from Tothill et al., 2008 and The Cancer Genome Atlas (TCGA). Graphs truncated at 150 months. P value shown is the log rank (Mantel-Cox) test. Additional file 5: Figure S3 Reduction in MAP2K4 expression following siRNA knockdown Acknowledgements This study was funded by the Victorian Breast Cancer Research Consortium, Australia (VBCRC) and the Australian National Health and Medical Research Council (NHMRC). MR is supported by a Cancer Council of Victoria Postgraduate Scholarship. GLR is supported by an Australian Postgraduate Award. Author details 1VBCRC Cancer Genetics Laboratory, Peter MacCallum Cancer Centre, St. Andrew’s Pl., East Melbourne, Victoria, Australia. 2Department of Pathology, University of Melbourne, Parkville, Victoria, Australia. 3Centre for Cancer Research, Monash Institute of Medical Research, Monash University, Clayton, Victoria, Australia. Authors’ contributions IGC and KLG conceived of the study, participated in its design and coordination and helped to draft the manuscript. SJD performed the mutation, methylation and functional analyses and drafted the manuscript. DYHC and GLR assisted in the mutation and methylation analyses. MR assisted in the functional analyses. All authors read and approved the final manuscript. Competing interests The authors declare that they have no competing interests. Received: 24 August 2010 Accepted: 17 May 2011 Published: 17 May 2011 References 1. Chang L, Karin M: Mammalian MAP kinase signalling cascades. Nature 2001, 410(6824):37-40. 2. Derijard B, Raingeaud J, Barrett T, Wu IH, Han J, Ulevitch RJ, Davis RJ: Independent human MAP-kinase signal transduction pathways defined by MEK and MKK isoforms. Science 1995, 267(5198):682-685. 3. Johnson GL, Lapadat R: Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science 2002, 298(5600):1911-1912. 4. Davis RJ: Signal transduction by the JNK group of MAP kinases. Cell 2000, 103(2):239-252. 5. Ip YT, Davis RJ: Signal transduction by the c-Jun N-terminal kinase (JNK)– from inflammation to development. Curr Opin Cell Biol 1998, 10(2):205-219. 6. Xia Z, Dickens M, Raingeaud J, Davis RJ, Greenberg ME: Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 1995, 270(5240):1326-1331. 7. Yang D, Tournier C, Wysk M, Lu HT, Xu J, Davis RJ, Flavell RA: Targeted disruption of the MKK4 gene causes embryonic death, inhibition of c- Jun NH2-terminal kinase activation, and defects in AP-1 transcriptional activity. Proc Natl Acad Sci USA 1997, 94(7):3004-3009. 8. Xu X, Raber J, Yang D, Su B, Mucke L: Dynamic regulation of c-Jun N- terminal kinase activity in mouse brain by environmental stimuli. Proc Natl Acad Sci USA 1997, 94(23):12655-12660. 9. Teng DH, Perry WL, Hogan JK, Baumgard M, Bell R, Berry S, Davis T, Frank D, Frye C, Hattier T, Hu R, Jammulapati S, Janecki T, Leavitt A, Mitchell JT, Pero R, Sexton D, Schroeder M, Su PH, Swedlund B, Kyriakis JM, Avruch J, Bartel P, Wong AK, Tavtigian SV, et al: Human mitogen-activated protein