A Hepatocellular Carcinoma Tumor Marker and Hedgehog Pathway Mediator, Glypican-3, Inhibits Hepatic Stellate Cell Viability: A Novel Outcome of Hepatic Tumor-Stroma Interactions

Abstract

Introduction: Human hepatocellular carcinoma (HCC) is one of the deadliest cancers worldwide, and its incidence is rapidly increasing in the U.S. HCC is truly 2 diseases in 1 as it often arises in the setting of cirrhosis. We previously identified dysregulation of the Hedgehog (Hh) pathway as a novel mechanism for hepatocarcinogenesis and identified a role for Hh signaling in maintaining the viability and regulating the fibrinogenic capacity of hepatic stellate cells (HSC), which are implicated in cirrhosis. It is increasingly recognized that HSCs and HCCs can communicate via paracrine signaling, but Hh signaling has not been studied in this process. We hypothesized that a secreted HCC tumor marker and Hh mediator, Glypican-3 (GPC3), may play a role in regulating HCC (tumor)-HSC (stroma) interactions. Methods: We developed an in vitro co-culture (CC) model system with Transwell plates to recapitulate HCC-HSC interactions in order to assess HCC effects upon HSC viability, apoptosis, and gene transcription. We employed 2 Hh-responsive human HCC lines [PLC/PRF/5 (PLC) and SK-Hep1 (SK)] and 1 human HSC line (LX-2). Assays for cell viability (WST) and apoptosis (caspase-3/7 activity) were performed (48-72 h, N=3-9). Using real-time RT-PCR, mRNA expression of Hh target genes was studied. Western analysis was used to study GPC3 protein expression during CC. Recombinant human GPC3, as well as other Hh ligand inhibitors were analyzed for effects. Results: Using our CC system, both HCC lines had no effect upon HSC viability but caused a reduction in HSC apoptosis by 2% (SK) and 50% (PLC). This suggested that HCCs secrete paracrine factor(s) that increase HSC apoptosis and decrease HSC proliferation. Concomitantly, CC resulted in transcriptional downregulation of the Hh target gene, Gli1 (SK: 1.5-fold, PLC: 2.7-fold). at baseline, real-time RT-PCR showed that PLC expressed >73,600-fold more Gpc3 mRNA than SK. CC led to a mixed response in Gpc3 mRNA levels with a 7.3-fold decrease in PLC mRNA but 4.5-fold increase in SK mRNA. at the protein level, GPC3 was strongly expressed in both lines after CC. to determine if GPC3 affects HSCs, we treated LX-2 with GPC3 (5 μg/ml). This resulted in an 83.4% reduction in HSC viability at 48 h. Similar effects were seen with 2 other anti-Hh treatments [5E1 (anti-Hh antibody) and HIP (Hh-interacting protein)]. Conclusions: For the first time, we show that HCC-HSC interactions lead to downregulation of Hh signaling in HSC. in part, this appears to be mediated by HCC's paracrine secretion of GPC3, an HCC biomarker, which inhibits Hh signaling and HSC viability. the underlying reasons for this inhibition remain unclear but may be related to GPC3 regulation of peritumoral angiogenesis. in summary, we expand upon data suggesting a role for tumor-stroma interactions in hepatic carcinogenesis-fibrinogenesis. These findings may have clinical implications, as a trial is underway to investigate the efficacy of anti-GPC3 therapy for HCC.