EVALUATION OF THE INDUCTION OF GENOMIC INSTABILITY AND METABOLIC ALTERATIONS BY SCD1 ENZYME IN HEPATOCYTE CELL LINES

Abstract

Hepatocellular carcinoma (HCC) is the sixth most common neoplasia in the world and the third cause of cancer mortality. The pathophysiology of HCC is an evolving issue and seems to be multifactorial. Similarly to other cancers, HCC originates from mutations in certain genes. Recent studies have suggested that the enzyme Stearoyl-CoA desaturase 1 (SCD1) overexpression is associated with genetic predisposition to hepatocarcinogenesis in mice and rats, whereas its suppression can reduce proliferation of HCC cells, in an AKT-dependent manner. Several studies have shown that SCD1 is increased in many types of cancers and in many metabolic disorders, such as diabetes, obesity, non-alcoholic hepatic steatosis and other diseases predisposing to the development of cancer. However, molecular mechanisms and understanding of the influence of SCD1 on genomic instability that may contribute to the development of HCC remain unknown. Therefore the present project aims to evaluate the potential of the enzyme SCD1 in promoting genomic instability and metabolic and morphological changes that cause malignant transformation in non-tumorigenic liver cell. For the assays, three cellular models of non-tumorigenic and tumorigenic human hepatocytes will be developed. The first two will be composed by primary human hepatocytes (PHH) and differentiated HepaRG (non-tumorigenic cells) overexpressing the SCD1 enzyme. The third will consist of human hepatocellular carcinoma (HepG2) cell line resistant to commercial SCD1 inhibitors,, which will be isolated and investigated accordingly and compared with parental cells sensitive to these compounds. Furthermore, metabolomics profile of SCD1-resistant HCC cancer cells will be determined in order to identify possible therapeutic targets. After the establishment these three cellular models, general biological assays will be performed for all cell lines, as for instance, the cell viability by the colorimetric MTT metabolic activity assays. Afterwards, some metabolic parameters will be evaluated by: analysis of the lipid profile by mass spectrometry, determination of oxygen consumption rates and evaluation of the acidification of the culture medium, in order to verify cell changes resulting from SCD1 overexpression and/or inhibition. To evaluate genomic instability in the cells lines, the following analysis will be carried out: cell cycle alterations; mitotic spindles formation and morphology, chromosomal distribution; induction of global DNA damages and production of reactive oxygen species (ROS). Furthermore, signaling pathways that likely could be modulated by high SCD1 expression or SCD1-resistance or not to their inhibitors: PIP3/Akt/PTEN; ²-catenin; ERK1/2; Cyclins D1 and B1; histones gH2AX and H2AX; CHK1 and CHK2 (DNA damage response via); LC3B; and p53. These proteins are involved in different processes, as such DNA repair, induction or inhibition of cell proliferation, autophagy and induction of some other type of genomic. Thus, the results obtained in this project will contribute to elucidation of the role of SCD1 in the onset of HCC and SCD1 resistance profile to commercial inhibitors. Consequently, these findings may culminate in the discovery of new molecular targets, as well as substantiate the validation of SCD-1 inhibitors in the prevention and treatment of HCCs.