Davis et al. BMC Cancer 2011, 11:173 http://www.biomedcentral.com/1471-2407/11/173 Page 4 of 7 Figure 1 Somatic alteration of MAP2K4 in ovarian cancer. A. Homozygous deletions (red bars) over the MAP2K4 region detected by Affymetrix SNP array. B. An aberrant shift was detected by HRM (red trace) compared with the matching normal DNA and the other tumor samples (blue traces). C. The sequence trace of re-amplified, non-WGA tumor DNA (IC489T) compared to normal (IC489N) shows a 16 bp homozygous deletion in exon 7 (red box), which causes a frameshift alteration leading to premature termination of the protein as indicated by the sequence below. We analyzed promoter methylation by MS-SSCP and bisulfite sequencing to assess whether this reduction in expression was mediated by epigenetic mechanisms (Figure 2). We did not detect any electrophoretogram shifts in any tumor DNA that would indicate methyla- tion in this region, in contrast to the positive SssI trea- ted control DNA. This was confirmed by selecting a few samples for sequencing. Consequently, methylation does not appear to be a mechanism by which MAP2K4 is silenced in ovarian cancer. In contrast, there was a high proportion of samples exhibiting LOH in conjunction with copy number loss at the MAP2K4 locus [18]. 56% of samples with LOH (38% of samples overall) were sub- ject to copy number loss, indicating that MAP2K4 may be targeted by this genetic mechanism. In a recent copy number analysis [28] we found that 164/398 (41%) ovar- ian carcinomas of primarily serous/endometrioid sub- type showed at least hemizygous copy number loss at MAP2K4. In addition, there was a strong correlation between copy number and gene expression in three microarray data sets of high grade serous/endometrioid ovarian carcinoma (Additional file 3, Figure S1): our own ([29]; r = 0.46, p = 0.001, n = 47), Etemadmogha- dam et al., ([30]; r = 0.59, p < 0.0001, n = 79), and TCGA (r = 0.51, p < 0.0001, n = 157). Therefore, the loss of MAP2K4 expression may be largely attributable to genomic loss, rather than epigenetic mechanisms of gene regulation. To assess the impact of reduced MAP2K4 expression we used the full TCGA data (n = 400 with outcome data) and another published data set ([31], n = 207) to investigate any association with clinical outcome in inva- sive ovarian carcinoma (Additional file 4, Figure S2). Both data sets were comprised of primarily high-grade serous carcinomas. In a Cox regression analysis includ- ing age and residual disease as co-factors in the model, low expression of MAP2K4 was associated with improved overall survival in the Tothill data set (P = 0.031, hazards ratio = 1.7), but was not significant in the TCGA data set (P = 0.75). Expression of MAP2K4 was not significant in either data set when progression free survival was considered. When viewing this data using a Kaplan-Meier plot having split the samples based on quartiles of expression values, the difference in survival was associated only with the bottom quartile (low expressors) in the Tothill data set. To measure the effect of reduced MAP2K4 expression, we knocked down MAP2K4 in two cell lines using siRNA and assessed cellular proliferation (Figure 3). We used an immortalized cell line derived from human ovarian surface epithelium (HOSE6.3 [24]). While this line is not normal, it is non-tumorigenic and should have fewer disrupted pathways or additional genetic lesions than the cancer cell lines used previously. For comparison, we selected a cell line established in culture from an ovarian tumor xenograft, JAM [23], which