Analysis of members of the SLIT-ROBO pathway as diagnostic and prognostic tools in hepatocellular carcinoma with special focus on ROBO2-associated cellular phenotype

Abstract

Hepatocellular carcinoma is the sixth most common cancer in the world, with an annual incidence exceeding half a million. It is associated mainly with hepatitis B and C viral infections; and is the main cause of death among cirrhotic patients. Aflatoxin B1 exposure, chronic alcohol consumption and virtually all cirrhosis-inducing conditions are of the other etiologies. For early diagnosis of HCC, surveillance of the risk groups is a crucial task requiring the development of novel markers for HCC with stronger sensitivity and specificity. In addition, description of biomarkers specific to hepatocellular carcinoma subtypes could identify novel targets for therapy. In this study, we analyzed members of the SLIT-ROBO gene families as novel diagnostic and prognostic markers in hepatocellular carcinoma. We defined an expression signature for members of the SLIT-ROBO gene families in HCC cell lines and tissues by real-time quantitative RT-PCR analysis. We showed that ROBO1 was overexpressed as stage and differentiation of the HCC proceeds. Furthermore, ROBO4 downregulation and SLIT2 overexpression marked late stage and poorly differentiated HCCs. Our results suggest that the expression of ROBO1 and ROBO4 can be used in early diagnosis of HCC. As another focus, we stably knockdowned ROBO2 expression in a model AFP positive cell line Huh7 and characterized the associated cellular phenotype. ROBO2 downregulation caused a significant decrease in proliferation rate whereas in wound-healing assay no significant difference in migration rate was observed. In addition, we performed a microarray experiment and found the differentially expressed genes between stable ROBO2 knockdown and negative clones. In this analysis, we found an overexpression of CK19, CD44, ABCG2/ BCRP1 hepatic progenitor cell markers and CD133 that is also a putative cancer stem cell marker of HCC, in stable ROBO2 knockdown clones. In addition KLF4 expression was augmented in these ROBO2 knockdown clones. We propose a genetic association between SLIT-ROBO pathway and CD133 at transcriptional level.