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HOXD13 methylation status is a prognostic indicator in breast cancer.

Homeobox protein Hox-D13 is encoded by HOXD13 gene which is frequently methylated in cancer and has been recognized as a tumor suppressor in pancreatic cancer. In this study, we examined HOXD13 mRNA expression in 40 pairs of breast cancers and corresponding normal breast tissues. Bisulfite sequencing of HOXD13 promoter was performed in 6 pairs of breast tumors and corresponding normal breast tissues to examine the potential HOXD13 CpG methylated sites. HOXD13 DNA methylation frequency analysis was performed using MethyLight in 196 pairs of breast cancers and corresponding normal breast samples. DNA methylation status and clinico-pathological features were investigated. Kaplan-Meier survival analysis and Cox proportional hazards models were utilized to assess the effect of methylation status on overall survival. We found that 60% (24/40) of breast cancers showed low HOXD13 mRNA expression when compared with corresponding normal breast tissue. The predicted CpG island was located in the -1325 bp to +675 bp region. Next, the -332 bp site in HOXD13 gene promoter was further examined and in 57.7% (113/196) samples methylation was detected at this site. HOXD13 methylation was correlated with larger tumor size (P = 0.004), but not with other clinico-pathological parameters. In addition, patients with methylated -HOXD13 promoter had worse overall survival (OS) (P = 0.005). Based on our results we conclude that HOXD13 methylation is a common event in primary breast cancer and is associated with poor survival of breast cancer patients. HOXD13 methylation could therefore potentially be used as a prognostic factor for breast cancer.