Epigenetic changes and nuclear factor-κB activation, but not microRNA-224, downregulate Raf-1 kinase inhibitor protein in triple-negative breast cancer SUM 159 cells

Raf-1 kinase inhibitor protein (RKIP) is a tumor suppressor and metastasis inhibitor, which enhances drug-induced apoptosis of cancer cells. Downregulation of RKIP may be significant in the biology of highly aggressive and drug-resistant tumors, for example triple-negative breast cancers (TNBCs). Potential causes for the low levels of RKIP expressed by SUM 159 TNBC cells were investigated in the present study. Bisulphite modification, methylation specific-polymerase chain reaction (PCR) and a TransAM NF-κB assay were performed and the results suggested that various mechanisms, including methylation of the gene promoter, histone deacetylation and nuclear factor-κB (NF-κB) activation, but not targeting by microRNA-224 (miR/miRNA-224), as determined by transfection of pre-miR-224 miRNA precursor or anti-miR-224 miRNA inhibitor, may downregulate RKIP in these cells. Furthermore, reverse transcription-quantitative PCR, western blotting, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium cell growth assay and flow cytometry revealed that in SUM 159 cells, the demethylating agent 5-aza-2'-deoxycytidine (5-AZA), the histone deacetylase inhibitor trichostatin A (TSA) and the NF-κB inhibitor dehydroxymethylepoxyquinomicin (DHMEQ) enhanced RKIP expression and resulted in significant cell growth inhibition and induction of apoptosis. 5-AZA and TSA mainly produced additive antitumor effects, while the combination of DHMEQ and TSA exhibited significant synergy in cell growth inhibition and induction of apoptosis assays. Increasing evidence that aberrant activation of NF-κB signaling is a frequent characteristic of TNBC highlights the fact that this transcription factor may be a useful target for treatment of such tumors. In addition to DHMEQ, proteasome inhibitors may also represent valuable therapeutic resources in this context. Notably, proteasome inhibitors, in addition to the inhibition of NF-κB activation, may also restore RKIP levels by inhibiting proteasome degradation of the ubiquitinated protein. The current results contribute to the understanding of the molecular mechanisms of RKIP downregulation in TNBC and suggest possible novel therapeutic approaches for the treatment of these types of cancer.