Original Articles Frequent Alteration of the Yin Yang 1/Raf-1 Kinase Inhibitory Protein Ratio in Hepatocellular Carcinoma Monica Notarbartolo, 1 Lydia Giannitrapani, 2 Nicoletta Vivona, 1 Paola Poma, 1 Manuela Labbozzetta, 1 Ada Maria Florena, 3 Rossana Porcasi, 3 Vito Michele Rosario Muggeo, 4 Luigi Sandonato, 5 Melchiorre Cervello, 6 Giuseppe Montalto, 2 and Natale D’Alessandro 1 Abstract 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-kB pathways and promotes drug- induced apoptosis. Mutual influences between YY1 and RKIP may exist, and there are already separate evi- dences that relevant increases in YY1 and reductions in RKIP occur in hepatocellular carcinoma (HCC). How- ever, 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. Introduction H epatocellular carcinoma (HCC) is one of the most common malignant diseases worldwide, reflecting the epidemiology of chronic hepatitis B or C infections as major risk factors for its development. Its prognosis remains un- favorable for the high percentage of patients with advanced disease at the first diagnosis or who relapse after resection: in these cases HCC is deadly aggressive and responds poorly to drug therapy or radiotherapy emphasizing the need of new approaches for its prevention and treatment (Montalto et al., 2002; Walzer et al., 2008; Wong et al., 2008). It is well recognized that the adverse characteristics of HCC are due to multifactorial, although still only partly understood, molecular processes: here we focus on two relevant factors dysregulated in HCC and on their possible interrelationship. In this dimension, many studies have shown the key role that the multifunctional transcription factor Yin Yang 1 (YY1) and the Raf-1 Kinase Inhibitor Protein (RKIP) can have in cancer biology (Gordon et al., 2006; Klysik et al., 2008; Odabei et al., 2004; Wang et al., 2006; Yeung et al., 1999). YY1 acts as a repressor, activator, or initiator element binding protein. It controls several critical genes (including p53, c-myc, c-fos, cyclin D1, Fas, DDR5, and VEGF) and its upregulation favors different aspects of tumorigenesis, including cell proliferation, inhibition of apoptosis, invasion, metastasis, and angiogene- sis. Whether YY1 acts as an activator or repressor on a gene depends on the promoter context and interactions with dif- ferent other regulatory proteins, which include the coactivator YY1AP (Gordon et al., 2006; Ohtomo et al., 2007; Wang et al., 2004, 2006). RKIP, a member of the phosphatidylethanolamine binding protein (PEBP) family, inhibits different kinases in the MAPK and NF-kB activation pathways, thereby abrogating their oncogenic activities; both these signalling pathways are fre- quently upregulated in HCC. RKIP may also be involved in regulating the spindle checkpoint and the progression through mitosis (Eves et al., 2006; Klysik et al., 2008; Odabei et al., 2004; Tang et al., 2010). Information that RKIP may be an 1 Dipartimento di Scienze Farmacologiche Pietro Benigno, 2 Dipartimento di Medicina Clinica e delle Patologie Emergenti, 3 Dipartimento di Patologia Umana, 4 Dipartimento di Scienze Statistiche e Matematiche Silvio Vianelli, 5 Dipartimento di Discipline Chirurgiche ed Oncolo- giche, Universita ` degli Studi di Palermo, Palermo, Italy. 6 IBIM, C.N.R, Palermo, Italy. OMICS A Journal of Integrative Biology Volume 15, Number 5, 2011 ª Mary Ann Liebert, Inc. DOI: 10.1089/omi.2010.0096 267