Department of Biochemistry and Molecular Biology.
BACKGROUND & AIMS:
The function of cytoplasmic AFP as a regulatory factor in the growth of tumor cells has been well defined. However, its precise mechanism of action and its clinical significance remains to be worked out.
Specimens from HCC patients were analyzed by using immunohistochemistry, coimmunoprecipitation (CoIP) and chromatin immunoprecipitation (ChIP) assays for evaluating the role of AFP in RAR signaling-mediated carcinogenesis. Quantitative real-time reverse transcription PCR, Western blotting, confocal microscopy, CoIP, GST pull-down, siRNA, gene transfection and ChIP assays were also used for analysis of cell lines.
RAR is able to interact with cytoplasmic AFP and binds to the element of the regulatory region of the Fn14 gene in neoplastic tissue in HCC patients. Assay of hepatocyte cell lines of differing AFP expression showed that cytoplasmic AFP is able to block ATRA induced nuclear translocation of RAR and expression of the Fn14 gene. Knockdown of AFP in siRNA-transfected HepG2 and Bel7402 cells led to greater binding of RAR to its response element. The expression of the Fn14 gene was therefore up-regulated as reflected by increases in mRNA and protein levels. Conversely, transfection of HLE and L02 cells (AFP negative) with the afp gene resulted in apparent reduction of RAR binding to DNA and Fn14 protein.
Demonstration of the involvement of cytoplasmic AFP in RAR-mediated expression of the Fn14 gene strongly indicates AFP plays a signal molecule-like role in the regulation of hepatocellular carcinoma growth.