ABSTRACT
Title
Possible mechanisms of Raf-1 Kinase Inhibitor Protein down-regulation in hepatocellular carcinoma
Authors
M. Notarbartolo1, P. Poma1 , N. Vivona1, M. Labbozzetta1, R. Porcasi2, N. D’Alessandro1
1Sezione di Farmacologia “P. Benigno”, 2Sezione di Anatomia Patologica “P. Craxì”, Dip. di Scienze per la Promozione della Salute ”G. D’Alessandro”, Università degli Studi di Palermo, Italia.
1Sezione di Farmacologia “P. Benigno”, 2Sezione di Anatomia Patologica “P. Craxì”, Dip. di Scienze per la Promozione della Salute ”G. D’Alessandro”, Università degli Studi di Palermo, Italia.
Abstract
RKIP (Raf-1 Kinase Inhibitor Protein) is a tumor and metastasis suppressor and is down-regulated in many cancer types. It inhibits the oncogenic activities of MAPK and NF-κB pathways and promotes drug induced apoptosis. We and others have shown significantly reduced RKIP expression, both at mRNA and protein level, in human hepatocellular carcinomas (HCCs) compared to adjacent peritumoral cirrhotic tissues or to healthy livers (1, 2). However, little is known about the mechanisms of RKIP down-regulation, especially in HCC. We have sequenced the promoter and coding regions, including the exon/intron boundaries, of the RKIP gene in human HCC cell lines (HA22T/VGH, HepG2, Hep3B) and in five clinical HCC samples and have ruled out that DNA mutations could be responsible for the lowered RKIP expression.
It is known that epigenetic changes, like histone deacetylation or DNA methylation, can alter gene expression. In HA22T/VGH and HepG2 cells the histone deacetylase inhibitor (HDACi) trichostatin (TSA) induced antiproliferative and apoptotic effects and exhibited antitumor synergy with doxorubicin. TSA caused also histone H3 hyperacetylation and modified the expression of different relevant genes (like β-catenin, cyclin D1, hTERT, XIAP and IL-6). Nevertheless, TSA did not modify RKIP mRNA and protein levels in the two representative HCC cell lines.
Further, we have analyzed RKIP gene promoter methylation in the HCC cell lines and in the five tumor samples; only Hep3B cells showed a moderate methylation of the gene, which was reverted by treatment with the demethylating agent 5-aza-2’-deoxycytidine. The same treatment caused also up-regulation of RKIP at mRNA but not at protein level. No methylation of the gene was found in the tumor samples.
It has been shown that in prostate cancer RKIP expression is negatively regulated by SNAIL, a transcriptional repressor activated by NF-κB (3). However, we could not show any relationship between the expression of SNAIL (mRNA or protein) and the levels of RKIP in the HCC cell lines; the same analysis is still in progress for the clinical tumor samples.
Up-regulation of microRNAs (miRNAs) has been associated to cancer progression, due to inhibition of tumor suppressor genes expression. Our preliminary data have shown up-regulation of different miRNAs (miR-1, miR-7, miR-18a, miR-21, miR-34b, miR-221, miR-224) in clinical HCC samples; according to computational analysis, RKIP is one of the targets of miR-224, which thus might cause RKIP mRNA reduction.
Overall, RKIP is likely to be down-regulated both by transcriptional and post-transcriptional mechanisms in human HCCs. Further studies are necessary to better identify these mechanisms, also to find therapeutic approaches able to overcome RKIP dysregulation. Our results underline also the therapeutic potential of HDACis in HCC.
1. Notarbartolo et al. (2011). OMICS. In press.
2. Schuierer et al. (2006). Oncol Rep. 16, 451–456.
3. Wu K et al. (2009). Crit Rev Immunol. 29: 241–54.
It is known that epigenetic changes, like histone deacetylation or DNA methylation, can alter gene expression. In HA22T/VGH and HepG2 cells the histone deacetylase inhibitor (HDACi) trichostatin (TSA) induced antiproliferative and apoptotic effects and exhibited antitumor synergy with doxorubicin. TSA caused also histone H3 hyperacetylation and modified the expression of different relevant genes (like β-catenin, cyclin D1, hTERT, XIAP and IL-6). Nevertheless, TSA did not modify RKIP mRNA and protein levels in the two representative HCC cell lines.
Further, we have analyzed RKIP gene promoter methylation in the HCC cell lines and in the five tumor samples; only Hep3B cells showed a moderate methylation of the gene, which was reverted by treatment with the demethylating agent 5-aza-2’-deoxycytidine. The same treatment caused also up-regulation of RKIP at mRNA but not at protein level. No methylation of the gene was found in the tumor samples.
It has been shown that in prostate cancer RKIP expression is negatively regulated by SNAIL, a transcriptional repressor activated by NF-κB (3). However, we could not show any relationship between the expression of SNAIL (mRNA or protein) and the levels of RKIP in the HCC cell lines; the same analysis is still in progress for the clinical tumor samples.
Up-regulation of microRNAs (miRNAs) has been associated to cancer progression, due to inhibition of tumor suppressor genes expression. Our preliminary data have shown up-regulation of different miRNAs (miR-1, miR-7, miR-18a, miR-21, miR-34b, miR-221, miR-224) in clinical HCC samples; according to computational analysis, RKIP is one of the targets of miR-224, which thus might cause RKIP mRNA reduction.
Overall, RKIP is likely to be down-regulated both by transcriptional and post-transcriptional mechanisms in human HCCs. Further studies are necessary to better identify these mechanisms, also to find therapeutic approaches able to overcome RKIP dysregulation. Our results underline also the therapeutic potential of HDACis in HCC.
1. Notarbartolo et al. (2011). OMICS. In press.
2. Schuierer et al. (2006). Oncol Rep. 16, 451–456.
3. Wu K et al. (2009). Crit Rev Immunol. 29: 241–54.