The Oncogenic Effect of Sulfatase 2 in Human Hepatocellular Carcinoma Is Mediated in Part by Glypican 3–Dependent Wnt Activation

Abstract

Heparan sulfate proteoglycans (HSPGs) act as coreceptors or storage sites for growth factors and cytokines such as fibroblast growth factor and Wnts. Glypican 3 (GPC3) is the most highly expressed HSPG in hepatocellular carcinoma (HCC). Sulfatase 2 (SULF2), an enzyme with 6-O-desulfatase activity on HSPGs, is up-regulated in 60% of primary HCCs and is associated with a worse prognosis. We have previously shown that the oncogenic effect of SULF2 in HCC may be mediated in part through up-regulation of GPC3. Here we demonstrate that GPC3 stimulates the Wnt/b-catenin pathway and mediates the oncogenic function of SULF2 in HCC. Wnt signaling in vitro and in vivo was assessed in SULF2-negative Hep3B HCC cells transfected with SULF2 and in SULF2-expressing Huh7 cells transfected with short hairpin RNA targeting SULF2. The interaction between GPC3, SULF2, and Wnt3a was assessed by coimmunoprecipitation and flow cytometry. bcatenin-dependent transcriptional activity was assessed with the TOPFLASH (T cell factor reporter plasmid) luciferase assay. In HCC cells, SULF2 increased cell surface GPC3 and Wnt3a expression, stabilized b-catenin, and activated T cell factor transcription factor activity and expression of the Wnt/b-catenin target gene cyclin D1. Opposite effects were observed in SULF2-knockdown models. In vivo, nude mouse xenografts established from SULF2-transfected Hep3B cells showed enhanced GPC3, Wnt3a, and b-catenin levels. Conclusion: Together, these findings identify a novel mechanism mediating the oncogenic function of SULF2 in HCC that includes GPC3-mediated activation of Wnt signaling via the Wnt3a/glycogen synthase kinase 3 beta axis.

Activation of the Transcription Factor GLI1 by WNT Signaling Underlies the Role of SULFATASE 2 as a Regulator of Tissue Regeneration

“…The medium was changed daily. Four hours after the isolation of hepatocytes, the medium was changed, and cells were either transfected with control siRNA, siRNA targeting ␤-CATENIN or vectors expressing either non-targeting (NT) shRNA or shRNA targeting GLI1 (11,20). Transfection of isolated mouse hepatocytes was performed using Fugene 6 transfection reagent (Roche Diagnostics GmbH, Mannheim, Germany) following the manufacturer's protocol.…”

“…Each experiment was performed in six replicates at least three times. Total RNA and protein lysates were prepared from hepatocyte cultures as described previously (11).…”

“…Constructs expressing NT sequences or shRNA targeting GLI1 were previously described (20). TOPFLASH (TCF binding site TK-luciferase reporter) plasmids were obtained from Dr. Wanguo Liu as previously described (11,21). The GLI-luciferase reporter was kindly provided by Dr. Chi-chung Hui (University of Toronto, Toronto, Ontario, Canada), and the sleeping beauty and parental transposon vectors were provided by Dr. Karl Clark (Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN).…”

“…Immunohistochemical detection of BrdU was performed using anti-BrdU antibody as described previously (11). For ␤-CATENIN immunohistochemistry, rabbit polyclonal anti-␤-CATENIN antibody (1:600) was used.…”

“…Western Blot-Whole liver protein lysates were prepared in lysis buffer as previously described (11). Nuclear proteins were prepared using the NE-PER nuclear and cytoplasmic protein isolation kit (Thermo Scientific) according to the manufacturer's protocol.…”