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/β-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. β-catenin-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 β-catenin, and activated T cell factor transcription factor activity and expression of the Wnt/β-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 β-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.

Figure 1. SULF2 increases Wnt3a expression and enhances Wnt/β-catenin signaling in HCC cells

(A) Hep3B Vector and Hep3B SULF2-H cells were treated with 0, 2, and 10 ng/ml of Wnt3a ligand for 24 hours, washed and lysed, and Western immunoblotting performed using antibodies against Wnt3a and actin (loading control). SULF2 increased basal and Wnt3a-induced Wnt3a expression. (B and C) TOPFLASH luciferase assay showing the effect of SULF2 on Wnt3a-induced Tcf/Lef transcriptional activity in HCC cells. Hep3B cells were transfected with a TOPFLASH reporter construct and either SULF2-expressing construct or an empty vector. After serum-starvation, cells were treated with 5 ng/ml Wnt3a and TOPFLASH luciferase activity was measured after 6 hours (B) or 24 hours (C). SULF2 enhanced Wnt3a-induced luciferase activity as early as 6 hours and the effect was sustained over 24 hours (p<0.0002).

Figure 2. Wnt3a binding to HCC cells is heparan sulfate-dependent and mediated by GPC3

(A) Wnt3a binding to Hep3B Parental, Hep3B Vector and Hep3B SULF2 cells was assessed by flow cytometry. Cells were incubated with 5 ng of biotinylated Wnt3a without or with 10 or 50 µg heparan sulfate. After staining with streptavidin, 20,000 cells were counted by flow cytometry. There was dose-dependent inhibition of Wnt3a binding by heparan sulfate in all cells. (D) Wnt3a binding to Hep3B SULF2-H cells is GPC3-dependent. Hep3B SULF2-H cells were transiently transfected with a GFP-coexpressing plasmid encoding either a scrambled control shRNA or an shRNA targeting the GPC3 mRNA. GPC3 suppression decreased Wnt3a binding to Hep3B SULF2-H cells. Addition of heparan sulfate (HS) decreased binding further.

Figure 3. SULF2, GPC3 and Wnt3a associate at the cell surface in a possible ternary complex

(A and B) Lysates from Hep3B Vector and Hep3B SULF2 cells were used for immunoprecipitation using anti-SULF2 and anti-GPC3 antibodies. Western immunoblotting was performed using antibody against GPC3 and SULF2. The HC and LC bands refer to immunoglobulin heavy chains and light chains, respectively. The blots were stripped and reprobed with antibody against Wnt3a (lower panels of Figure 2A and 2B). (C) Immunocytochemistry showing co-localization of SULF2 and GPC3 in Hep3B cells transiently transfected with SULF2. Nuclei were counterstained with DAPI.

Figure 4. Expression of SULF2 up-regulates cell surface Wnt3a and activates the Wnt/β-catenin signaling pathway

(A) Western immunoblotting was performed on whole cell lysates from Hep3B Vector (control), low-expressing Hep3B SULF2-L and high-expressing Hep3B SULF2-H cells using antibodies against Wnt3a, phospho-GSK3β, β-catenin and actin. SULF2 expression resulted in increases in Wnt3a, phospho-GSK3β, and β-catenin (B) Immunocytochemistry showed that SULF2 increased expression of GPC3, Wnt3a and β-catenin. (C) Transient transfection with TOPFLASH and FOPFLASH plasmids. SULF2 enhanced Tcf-mediated transcriptional activity in Hep3B cells (p=0.025); (D) SULF2 increased cyclin D1 expression; conversely, downregulation of SULF2 decreased cyclin D1 levels.

Figure 5. Knockdown of SULF2 down-regulates GPC3 expression and inhibits Wnt signaling in Huh7 cells

(A) Whole cell lysates of Huh7 Vector cells and two stable clones of Huh7 cells transfected with shRNA targeting SULF2, Huh7 SULF2 shRNA-3 and Huh7 SULF2 shRNA-4, were prepared. Western immunoblotting was performed using antibodies against Wnt3a, phospho-GSK3β, β-catenin and actin (loading control). Downregulation of SULF2 decreased Wnt3a, phospho-GSK3β, and β-catenin (B) Huh7 cells were transiently transfected with GFP-expressing plasmids carrying either a control scrambled shRNA sequence or shRNA targeting SULF2 mRNA (shRNA-a). After 24 hours, cells were immunostained for β-catenin. Cells expressing the scrambled shRNA (left column, green fluorescent cells) showed no difference in β-catenin levels; in contrast, cells expressing the SULF2 shRNA (right column, green fluorescence) showed substantially decreased β-catenin as compared to the untransfected cells without green fluorescence. (C) Huh7 Vector and Huh7 SULF2 shRNA stable clones were transiently transfected with TOPFLASH and FOPFLASH plasmids. Downregulation of SULF2 inhibited Tcf-mediated transcriptional activity in Huh7 cells (p=0.002). (D) Suppression of SULF2 expression decreased cellular levels of cyclin D1 as detected by Western immunoblotting.

Figure 6. SULF2 up-regulates GPC3 and Wnt3a and promotes tumor growth in HCC nude mouse xenografts mainly through increased cell proliferation

Hep3B Vector and Hep3B SULF2-H clones were subcutaneously inoculated into the flanks of ten nude mice. SULF2 significantly enhanced tumor growth in vivo (11). (A) Successive sections from paraffin-embedded xenografts from Hep3B Vector (upper panel) and Hep3B SULF2-H cells (lower panel) were immunostained with antibodies against SULF2, GPC3 and Wnt3a respectively; nuclei were counterstained with hematoxylin. SULF2 expression was associated with increases in tumor cell GPC3, Wnt3a, and β-catenin. (B) Ki-67 staining from Hep3B SULF2-derived xenografts compared with Hep3B Vector. Xenografts from Hep3 SULF2 cells showed smaller sized but increased number of brown-staining proliferative cells (arrows) (magnification ×200). (C) Graph quantitating increased cell proliferation in Hep3B SULF2-derived xenografts as compared to Hep3B Vector xenografts (average of 6 high power fields).

Figure 7. SULF2 does not decrease apoptosis in HCC mouse xenografts

Sections from paraffin embedded xenografts from Hep3B Vector and Hep3B SULF2-H cells were stained for apoptotic cells (arrows). (A) Caspase-3 assay showed few apoptotic cells in both Hep3B Vector- and Hep3B SULF2-derived xenograft sections (magnification ×200). (B) There was no significant difference in active caspase-3 between the 2 groups. (C) TUNEL staining also showed no difference between the groups. Values represent the mean of TUNEL-positive cells in 6 high power fields.

Figure 8. Working model for the effect of SULF2 on Wnt/β-catenin signaling in HCC cells

Expression of SULF2 in HCC cells up-regulates cell surface GPC3 and Wnt3a. SULF2 desulfates GPC3 HSGAGs, leading to release of Wnt, binding and activation of the Wnt receptor Frizzled and phosphorylation of GSK3β. Phosphorylation of GSK3β results in the dissolution of the complex responsible for degradation of β-catenin, thus allowing β-catenin to accumulate in the cytosol and translocate to the nucleus. This results in increased Tcf/Lef transcription and up-regulation of target genes, including cyclin D1, with consequent promotion of cell proliferation in vitro and tumor growth in vivo.