Abstract 5620: Therapeutic targeting of glypican-3 using TGF-β2 as a novel treatment approach for HCC

Abstract

Abstract Objective Glypican-3 (GPC3) is a valuable diagnostic marker and a potential therapeutic target in hepatocellular carcinoma (HCC). It is over-expressed in more than 60% of the HCC tumors. As a histological marker, GPC3 can be used to distinguish HCC cells from cirrhotic, non-tumorous liver. As a diagnostic marker, GPC3 be used to complement the diagnostic potential of AFP for screening of HCC patients. Previously we found that GPC3 knockdown can suppress HCC tumor growth both in vitro and in vivo. Gene expression analysis revealed that GPC3 suppression significantly up-regulated the expression of TGF-β2 at both RNA and protein levels. TGF-β2 itself possesses anti-proliferative effect in GPC3 positive HCC cell lines (HepG2 and Huh7). This study aims at evaluating the use of TGF-β2 as a novel treatment approach for HCC by targeting the GPC3/TGFβ signaling axis. Materials and methods To study the effects of TGF-β2 on HCC cells, cell lines with different level of GPC3 expression (HepG2, Huh7, Hep3B, Hep40, PLC/PRF/5, SNU-398, SNU-182, SNU-449, SNU-475) were treated with 1 or 5 ng/ml of rhTGF-β2 for 48 hours in their normal culture media supplemented with 10% FBS. Cell proliferation was determined by using MTT assay and colony formation assay. Cell cycle progression was measured by using flow cytometry. The presence of self-renewing cancer cells was determined by spheroid formation assay. Gene expression analysis was performed to identify the genes associated with TGF-β2 treatment in HCC cells. For in vivo study, orthotopic xenografts of HCC patient tumors in mice were established, and mice were injected with 100 ng of rhTGF-β2 every week via i.p. injection. Tumor growth was monitored by using non-invasive bioluminescence imaging. Results TGF-β2 effectively inhibited the growth of GPC3 positive cells as shown by the decreased cell proliferation and colony formation (HepG2, Huh7, Hep40, PLC.PRF/5, Hep3B and SNU-398). Additionally, GPC3 positive cells responded to TGF-β2 treatment with down-regulation of anti-apoptotic genes (Bcl-2, Bcl-xL and Mcl-1) and cell cycle arrest at G0/G1 phase. Cells with no endogenous GPC3 expression did not respond to TGF-β2 treatment. Moreover, TGF-β2 treatment inhibited self-renewal of GPC3 positive HCC cell lines as shown by inhibition of spheroid formation (P<0.01 compared to untreated cells). Gene expression analysis revealed that TGF-β2 treatment down-regulated the expression of GPC3 (72%, P<0.05), NDRG1 (56%, P<0.05) and CDC37 (56%, P<0.05), three genes that were reported to promote HCC tumor progression. In vivo, TGF-β2 inhibited HCC xenograft growth, after 3 weeks of treatment (P<0.05 compared to PBS control), as shown by the bioluminescence imaging. Conclusions In conclusion, molecular targeting of GPC3 by using rhTGF-β2 offers a novel treatment option for the clinical management of GPC3-positive HCC patients by targeting the GPC3/TGFβ signaling axis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5620. doi:1538-7445.AM2012-5620