Induction of Heparanase-1 Expression by Mutant B-Raf Kinase: Role of GA Binding Protein in Heparanase-1 Promoter Activation

Abstract

Heparanase-1 (HPR1), an endoglycosidase that specifically degrades heparan sulfate (HS) proteoglycans, is overexpressed in a variety of malignancies. Our present study sought to determine whether oncogene BRAF and RAS mutations lead to increased HPR1 expression. Reverse transcription-polymerase chain reaction analysis revealed that HPR1 gene expression was increased in HEK293 cells transiently transfected with a mutant BRAF or RAS gene. Flow cytometric analysis revealed that B-Raf activation led to loss of the cell surface HS, which could be blocked by two HPR1 inhibitors: heparin and PI-88. Cotransfection of a BRAF or RAS mutant gene with HPR1 promoter-driven luciferase reporters increased luciferase reporter gene expression in HEK293 cells. Knockdown of BRAF expression in a BRAF-mutated KAT-10 tumor cell line led to the suppression of HPR1 gene expression, subsequently leading to increased cell surface HS levels. Truncational and mutational analyses of the HPR1 promoter revealed that the Ets-relevant elements in the HPR1 promoter were critical for BRAF activation-induced HPR1 expression. Luciferase reporter gene expression driven by a four-copy GA binding protein (GABP) binding site was significantly lower in BRAF siRNA-transfected KAT-10 cells than in the control siRNA-transfected cells. We further showed that BRAF knockdown led to suppression of the expression of the GABPβ, an Ets family transcription factor involved in regulating HPR1 promoter activity. Taken together, our study suggests that B-Raf kinase activation plays an important role in regulating HPR1 expression. Increased HPR1 expression may contribute to the aggressive behavior of BRAF-mutated cancer.