Study of APE1/Ref-1 expression in human hepatocellular carcinoma. Evaluation of its prognostic significance

ABSTRACT: Human Hepatocellular Carcinoma (HCC) is the fifth most frequent neoplasm worldwide and its incidence is rising in Western countries. HCC is frequently diagnosed at advanced stage and, until today, there are no effective treatments of this late phase. Therefore, research focused on finding new markers of progression and new molecular targets for therapy for HCC is of the utmost importance. Unquestionably, HCC is the most serious complication of long-standing chronic disease representing generally the final event of a liver disease usually originated decades earlier. Any chronic liver disease that causes cirrhosis is considered a risk factor for HCC development and this disease is characterized by chronic inflammation involving repeated rounds of necrosis and regeneration associated with sustained oxidative stress. Apurinic apyrimidinic endonuclease/redox effector factor 1 (APE1/Ref-1) is a multifunctional protein involved in redox regulation of transcription factors and acting as an endonuclease of the base excision repair (BER) pathway of DNA lesions. APE1/Ref-1 has been found to be up-regulated and abnormally localized in the cytoplasmic compartment of several cancer cells. Of notice is the finding that cytoplasmic localization is associated with a poor prognosis. Since at the time I started my thesis no data were available about the possible involvement of APE1/Ref-1 in HCC, the aim of this work was to investigate the expression and sub-cellular localization of APE1/Ref-1 in HCC. In addition, my aim was also to analyze the correlation of these parameters with the progression of the disease. To accomplish these points I analyzed human samples of HCC and cirrhosis (in vivo model) and an in vitro model constituted by HCC cell lines at different degree of differentiation. I also assessed the protective role of APE1/Ref-1 to oxidative damage as a function of its sub-cellular localization over-expressing, in normal hepatocytes, wild type and mutated forms of the protein that force its distribution to different cellular compartments. Using these approaches, we found that HCC is characterized by increased APE1/Ref-1 mRNA and protein levels and, that this up-regulation increases accordingly with tumor differentiation grading showing an association between tumor progression and APE1/Ref-1 levels. The increased amount of APE1/Ref-1 induces its nuclear and cytoplasmic accumulation in HCC. The cytoplasmic localization is peculiar of HCC and more frequently observed in poorly-differentiated cancers. The fact that APE1/Ref-1 accumulates in the cytoplasm of poorly-differentiated tumors and correlates with a shorter survival time after HCC resection points to a possible role of the APE1/Ref-1 sub-cellular localization as a prognostic marker of HCC aggressiveness. Thereafter, I demonstrated that APE1/Ref-1 over-expression in the normal hepatocyte cell line (IHH) enhances survival after oxidative damage induced by exposure to H2O2 and UV radiation although the different forms of the protein behave in a different ways. Wild type and truncated forms of APE1/Ref-1, that localize mainly in the cytoplasm and mitochondria, confer protection to H2O2 damage while the non-cleavable form, localized in the nucleus, does not. Conversely, all the mutants protect IHH from UV induced apoptosis. These data demonstrate for the first time that APE1/Ref-1 expression is deregulated in HCC, that its over-expression has a protective role in vitro and that APE1/Ref-1 cytoplasmic localization has a prognostic significance in vivo. We favor the conclusion that APE1/Ref-1 may be considered as a new prognostic marker for HCC aggressiveness. Its possible involvement in HCC development offer a new molecular key to elucidate the role of this protein in oxidative stress related hepatocarcinogenesis.