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.

Analysis of possible mechanisms accounting for raf-1 kinase inhibitor protein downregulation in hepatocellular carcinoma.

Abstract Raf-1 kinase inhibitor protein (RKIP) is a tumor and metastasis suppressor that promotes drug-induced apoptosis in cancer cells. It is frequently downregulated, both at the mRNA and protein level, in hepatocellular carcinoma (HCC), but the mechanisms leading to this reduction are obscure. We sequenced the whole RKIP gene in three human HCC cell lines (HA22T/VGH, HepG2, and Hep3B), and in five clinical HCC samples, but could not find any gene variant that might account for their low RKIP levels. We also examined whether gene methylation may be responsible for the altered RKIP expression. No methylation of the RKIP gene was found in the tumor samples, while among the cell lines only Hep3B showed methylation of the gene, which was reduced by treatment with 5-aza-2'-deoxycytidine (5-AZA). The same treatment caused upregulation of RKIP at the mRNA, but not at the protein level, indicating that gene methylation is not a principal mechanism of the decrease in RKIP in the Hep3B cells. Furthermore, different elements consistently suggested that RKIP may be a target repressed by miR-224, a miRNA that is frequently and specifically upregulated in HCC, but our results excluded that this occurs, at least in the HCC cell lines. Factors like Snail, EZH2, and HDAC, have been implicated in the RKIP downregulation present in breast and prostate tumors, though some of our results from the cell lines do not support that they play such a role in HCC; however, this aspect is worthy of further study. However, recent results of ours and others suggest a significant involvement of proteosomal degradation and of its pharmacological inhibition. In conclusion, the causes of RKIP downregulation in HCC remain incompletely understood. However, we think that the present observations will be useful to generate further research, with the ultimate possible goal of devising specific approaches to restore the relevant antitumor function of the factor.

Frequent alteration of the Yin Yang 1/Raf-1 kinase inhibitory protein ratio in hepatocellular carcinoma.

The transcription factor Yin Yang 1 (YY1) can favor several aspects of tumorigenesis. In turn, Raf-1 Kinase Inhibitor Protein (RKIP) inhibits the oncogenic activities of MAPK and NF-κB pathways and promotes drug-induced apoptosis. Mutual influences between YY1 and RKIP may exist, and there are already separate evidences that relevant increases in YY1 and reductions in RKIP occur in hepatocellular carcinoma (HCC). However, the levels of the two factors have never been concomitantly examined in HCC. We evaluated by RT-PCR the mRNA levels of YY1, YY1AP, RKIP, and survivin in 35 clinical HCCs (91% HCV-related), in their adjacent cirrhotic tissues and in 6 healthy livers. Immunohistochemical analyses were also performed. The ratio of YY1 to RKIP mRNA was constantly profoundly inverted in the tumors compared with the adjacent nontumoral tissues. A similar result occurred frequently at protein level. Hyperactivation of YY1 in tumors was corroborated by its nuclear localization and the finding that in the tumors there were also increases in YY1AP, a YY1 coactivator not expressed in normal liver, and in survivin, as a possible target of YY1. The frequent alteration in the YY1-RKIP balance might represent a marker of malignant progression and be exploited for therapeutic interventions in HCC.

C-reactive protein but not soluble CD40 ligand and homocysteine is associated to common atherosclerotic risk factors in a cohort of coronary artery disease patients.

OBJECTIVES: One third to one half of the variation in vascular disease occurrence remains unexplained by traditional risk factors. Since atherosclerosis may, in part, be an inflammatory disease, circulating factors related to inflammation may be predictors of cardiovascular disease. The aim of this study was to evaluate the association between common atherosclerotic risk factors and markers of inflammation. DESIGN AND METHODS: Serum levels of soluble CD40 (sCD40L), high-sensitive C-reactive protein (hs-CRP) and homocysteine (Hcy) were measured in 251 patients selected from a series of 438 subjects affected by previous myocardial infarction, angina or other cardiovascular diseases. RESULTS: sCD40L levels were lower in patients with previous myocardial infarction while no association was observed between sCD40L and Hcy levels and other risk factors. Only hs-CRP levels positively correlated with increased number of risk factors. CONCLUSION: In a setting of patients affected with coronary artery disease no association between sCD40L and homocysteine levels and atherosclerotic risk factors was observed; only hs-CRP showed increased levels according to the number of risk factors. Future studies using larger cohorts will be needed to validate the clinical use of markers of inflammation in the prediction of cardiovascular events.