Analysis of Possible Mechanisms Accounting for Raf-1 Kinase Inhibitor Protein Downregulation in Hepatocellular Carcinoma

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.