Original Article
Copyright ©2012 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Gastroenterol. Jul 14, 2012; 18(26): 3389-3399
Published online Jul 14, 2012. doi: 10.3748/wjg.v18.i26.3389
Osteopontin increases hepatocellular carcinoma cell growth in a CD44 dependant manner
Renee J Phillips, Karla J Helbig, Kylie H Van der Hoek, Devanshi Seth, Michael R Beard
Renee J Phillips, Karla J Helbig, Kylie H Van der Hoek, Michael R Beard, School of Molecular and Biomedical Science, The University of Adelaide, 5000 Adelaide, Australia
Renee J Phillips, Karla J Helbig, Kylie H Van der Hoek, Michael R Beard, Centre for Cancer Biology SA Pathology, 5000 Adelaide, Australia
Devanshi Seth, Drug Health Services, Royal Prince Alfred Hospital, 2050 Camperdown, Australia
Author contributions: Helbig KJ and Beard MR designed the research; Phillips RJ, Helbig KJ, Van der Hoek KH and Seth D performed the experiments and analysed data; Phillips RJ, Helbig KJ and Beard MR wrote the paper.
Supported by The NH and MRC of Australia
Correspondence to: Michael R Beard, PhD, School of Molecular and Biomedical Science, The University of Adelaide, North Terrace, 5005 Adeleide, Australia. michael.beard@adelaide.edu.au
Telephone: +61-8-83035522 Fax: +61-8-83037532
Received: January 5, 2012
Revised: March 23, 2012
Accepted: March 29, 2012
Published online: July 14, 2012

AIM: To investigate the role of osteopontin (OPN) and its splice variants in the proliferation of hepatocellular carcinoma (HCC).

METHODS: The expression of OPN variants in HCC cell lines as well as HCC tissue samples and non-tumour tissue was studied using polymerase chain reaction. OPN variant cDNAs were cloned into a mammalian expression vector allowing both transient expression and the production of stable OPN expressing cell lines. OPN expression was studied in these cells using Western blotting, immunofluoresnce and enzyme linked immunosorbent assay. A CD44 blocking antibody and siRNA targeting of CD44 were used to examine the role of this receptor in the OPN stimulated cell growth observed in culture. Huh-7 cells stably expressing either OPN-A, -B or -C were injected subcutaneously into the flanks of nude mice to observe in vivo tumour growth. Expression of OPN mRNA and protein in these tumours was examined using reverse transcription-polymerase chain reaction and immunohistochemistry.

RESULTS: OPN is expressed in HCC in 3 forms, the full length OPN-A and 2 splice variants OPN-B and -C. OPN variant expression was noted in HCC tissue as well as cognate surrounding cirrhotic liver tissue. Expression of these OPN variants in the HCC derived cell line Huh-7 resulted in secretion of OPN into the culture medium. Transfer of OPN conditioned media to naïve Huh-7 and HepG2 cells resulted in significant cell growth suggesting that all OPN variants can modulate cell proliferation in a paracrine manner. Furthermore the OPN mediated increase in cellular proliferation was dependent on CD44 as only CD44 positive cell lines responded to OPN conditioned media while siRNA knockdown of CD44 blocked the proliferative effect. OPN expression also increased the proliferation of Huh-7 cells in a subcutaneous nude mouse tumour model, with Huh-7 cells expressing OPN-A showing the greatest proliferative effect.

CONCLUSION: This study demonstrates that OPN plays a significant role in the proliferation of HCC through interaction with the cell surface receptor CD44. Modulation of this interaction could represent a novel strategy for the control of HCC.

Keywords: Osteopontin, Hepatocellular carcinoma, CD44 antigen, Nude mice, Xenograft