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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

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. (HEPATOLOGY 2010;)

Hepatocellular carcinoma (HCC) is the third most frequent cause of cancer death worldwide.1 The survival of HCC patients is poor, and only 10% to 20% of HCCs are detected at an early enough stage for potentially curative therapy. Locoregional therapies are usually palliative, and there are limited options for chemotherapy. Therefore, new agents are needed for the effective treatment of the majority of HCCs.2

The Wnt/Frizzled/β-catenin pathway is activated in approximately 50% of HCCs. Wnt ligands (Wnt3, Wnt3a, Wnt4, and Wnt5a) and Frizzled receptors (Frizzled 3, Frizzled 6, and Frizzled 7) have been implicated in the development of HCC, and up to 95% of HCCs show potential Wnt/Frizzled activating events.3-5 The Wnt/β-catenin pathway is regulated by heparan sulfate proteoglycans (HSPGs), which modulate cell surface signaling by acting as coreceptors or storage sites for Wnt proteins. HSPGs consist of a protein core to which heparan sulfate glycosaminoglycan (HSGAG) chains are attached; these are variably sulfated at the 2-O, 3-O, and 6-O positions of their component disaccharides. Glypicans are cell surface–anchored HSPGs that regulate the activity of Wnts.6, 7 In particular, glypican 3 (GPC3) is highly overexpressed in HCCs and is being developed as a target for HCC therapy.8, 9 Wnt3a has been shown to mediate the GPC3-induced growth of HCCs via the canonical Wnt/β-catenin pathway.5, 10

Sulfated HSGAG chains of GPC3 and other HSPGs are potential substrates for desulfation at the 6-O position by human sulfatase 2 (SULF2). The sulfation state of HSGAGs is critical for growth factor binding; hence, SULF2 may regulate tumor growth by releasing growth factors from HSGAG storage sites at the cell surface and in the extracellular matrix and thus may increase the local concentration of growth factors available to bind to cell surface receptors and enhance cell signaling. We have shown that SULF2 up-regulates fibroblast growth factor (FGF) signaling in a heparan sulfate (HS)–dependent and GPC3-dependent manner in HCC cells both in vitro and in vivo.11

Because GPC3 activates Wnt signaling and is a potential substrate for desulfation by SULF2, we hypothesized that desulfation by SULF2 releases stored Wnts from HSGAG sites on GPC3. Released Wnt then binds to Frizzled receptors and activates the Wnt/β-catenin pathway. We investigated the roles of SULF2 and GPC3 in Wnt3a signaling by addressing the following questions:

  • 1
    Does SULF2 enhance Wnt/β-catenin activation in HCC cells?
  • 2
    Are Wnt3a binding to HCC cells and Wnt/β-catenin activation dependent on HS and GPC3?
  • 3
    Does Wnt3a associate with SULF2 and GPC3?
  • 4
    Does SULF2 drive Wnt/β-catenin signaling in the absence of exogenous Wnt?
  • 5
    Does knockdown of SULF2 decrease GPC3 and Wnt3a expression and inhibit Wnt/β-catenin signaling?
  • 6
    Is the association between SULF2, GPC3, and Wnt3a demonstrable in vivo?

We show that SULF2 activates Wnt/β-catenin signaling in HCC cells and that this process is GPC3-dependent and can be independent of exogenous Wnts.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Chemicals and Antibodies

HS, anti-actin antibody, horseradish peroxidase–conjugated mouse immunoglobulin G, and rabbit immunoglobulin G were purchased from Sigma Chemical Co. (St Louis, MO); anti-GPC3 antibody was obtained from BioMosaics (Burlington, VT); and recombinant human Wnt3a and anti-Wnt3a antibody were acquired from R&D Systems (Minneapolis, MN). The plasmid vectors pSS-H1p and pG-SUPER were gifts from Dr. Daniel D. Billadeau and Dr. Shin-Ichiro Kojima, respectively. TOPFLASH and FOPFLASH (mutant Tcf binding site TK-luciferase reporter) plasmids were obtained from Dr. Wanguo Liu. The rabbit polyclonal anti-SULF2 antibody was previously reported.11

HCC Cell Lines

The Hep3B, PLC/PRF/5, and HepG2 cell lines were obtained from the American Type Culture Collection (Manassas, VA) and were cultured as recommended. Huh7 was from Dr. Gregory Gores.

SULF2 Stable Transfectant Clones

SULF2-negative Hep3B cells were stably transfected with SULF2-expressing plasmids. SULF2-expressing Huh7 cells were stably transfected with plasmids expressing short hairpin RNA (shRNA) sequences targeting SULF2.11 Two SULF2-transfected Hep3B clones were selected for the experiments: a low–SULF2-expressing clone (Hep3B-SULF2-L) and a high–SULF2-expressing clone (Hep3B-SULF2-H). Similarly, two SULF2-knockdown Huh7 clones, Huh7 SULF2 shRNA-3 and Huh7 SULF2 shRNA-4, were selected. The target sequences used for SULF2 shRNA constructs (shRNA-a and shRNA-b) were reported previously.11

Wnt3a Binding by Flow Cytometry

Wnt3a was biotinylated with EZ-Link Sulfo-NHS-LC-Biotin (Pierce). Hep3B cells (100,000) were transiently transfected with control pcDNA3.1 (Invitrogen) or full-length hSULF2 in pcDNA3.1 for 48 hours with FuGENE 6; they were then pelleted and resuspended at a concentration of 4 × 106 cells/mL in phosphate-buffered saline (PBS). Cells were treated with PBS (control), 10 μg of HS, or 50 μg of HS for 5 minutes on ice. Biotinylated Wnt3a (5 ng) was added (a background control with no added Wnt3a was used for each condition), cells were incubated on ice for 30 minutes, and 5 μL of a 1:10 dilution of streptavidin phycoerythrin [in PBS and 0.1% bovine serum albumin (BSA); Jackson Immunoresearch] was added before reincubation on ice for another 30 minutes in the dark. Cells were washed with PBS and 2% BSA, pelleted, and resuspended in 0.4 mL of 4% paraformaldehyde. Flow cytometry was performed with a FACScan analyzer (BD Biosciences, San Jose, CA). Similar experiments used Hep3B SULF2-H transfected with shRNA against GPC3 or control scrambled shRNA and 20 ng/mL HS.

Immunocytochemistry and Confocal Microscopy

SULF2-positive or SULF2-negative Huh7 and Hep3B cells were seeded on glass cover slips in six-well plates and were incubated for 24 hours. Immunocytochemistry and confocal microscopy were performed with antibodies against SULF2, GPC3, Wnt3a, and β-catenin.12

Western Immunoblotting

SULF2-positive or SULF2-negative Huh7 and Hep3B cells were cultured for 24 hours, and whole-cell lysates were prepared.12 The protein (20 μg/lane) was separated by electrophoresis and transferred onto a polyvinylidene fluoride membrane. Western immunoblotting was performed with antibodies against SULF2, GPC3, Wnt3a, β-catenin, phospho-β-catenin, glycogen synthase kinase 3 beta (GSK3β), phospho-GSK3β, and cyclin D1 with β-actin as the loading control.

Immunoprecipitation Using Anti-SULF2 and Anti-GPC3 Antibodies

Hep3B vector and Hep3B SULF2-H cells in 10-cm dishes were washed twice with ice-cold PBS and lysed on ice for 30 minutes in 1 mL of a modified radio immunoprecipitation assay lysis buffer supplemented with the Complete Mini protease inhibitor mixture. After the determination of the protein concentration and dilution of the lysate to approximately 2 μg/μL of total cell protein with PBS, the lysate was precleared by the addition of 20 μL of a Protein G Sepharose bead slurry per milliliter of lysate and by incubation at 4°C for 1 hour on a rocker. SULF2 and GPC3 proteins were immunoprecipitated by the incubation of the precleared lysate with a rabbit anti-SULF2 antibody or a mouse anti-GPC3 antibody and Protein G Sepharose (40 μL) overnight at 4°C. Immune complexes were pelleted by centrifugation for 1 minute at 14,000g, washed three times with a lysis buffer, and released from the beads by 5 minutes of boiling in 40 μL of a 2× sample buffer. The beads were collected by centrifugation, and the supernatants were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Western immunoblot analysis was performed as described previously.

Luciferase Assay for TOPFLASH/FOPFLASH Reporter Activity

TOPFLASH.

Hep3B and PLC/PRF/5 cells, plated onto 24-well plates at a density of 6 × 104 cells per well, were allowed to adhere overnight. On the following day, the cells were transfected with the TOPFLASH reporter construct (0.025 μg/well) and either the SULF2-expressing construct or an empty vector with Lipofectamine (0.1 μg/well). After 5 hours, serum-containing medium was added, and the cells were cultured overnight. The cells were then serum-starved in a medium containing 0.5% BSA overnight, and this was followed by treatment with the Wnt3a ligand (R&D Systems) for the indicated times. Cell lysates were assayed with a luciferase assay system (Promega). The luciferase activity was normalized to the total protein content.

TOPFLASH/FOPFLASH.

SULF2-positive or SULF2-negative Hep3B and Huh7 cells were plated onto 12-well plates and cultured to 60% to 70% confluency. The cells were transfected with either 0.5 μg of the TOPFLASH plasmid [a T cell factor (Tcf) reporter plasmid] or 0.5 μg of the FOPFLASH plasmid (with mutant Tcf binding sites) with Lipofectamine Plus.13, 14 To adjust for the transfection efficiency, 10 ng of the pRL-CMV vector (Promega) was cotransfected. Twenty-four hours later, cell lysates were prepared, and firefly and Renilla luciferase activities were quantitated with a dual-luciferase reporter assay system (Promega).

Mouse Xenografts and Immunohistochemistry

BALB/c nu/nu nude mouse xenografts were derived from SULF2-negative Hep3B vector and SULF2-positive Hep3B SULF2-5 cells.11 Immunohistochemistry was performed with an antibody against SULF2, GPC3, Wnt3a, or β-catenin.11 The primary antibody was replaced with 1% BSA/trishydroxymethylaminomethane-buffered saline for negative controls. The institutional animal care and use committee approved the protocols.

Ki-67 and Caspase-3 Assays.

Tissue sections were stained with antibodies against Ki-67 (Dako; 1:100) and caspase-3 (Cell Signaling; 1:800) with a Dako Autostainer Plus and were counterstained with hematoxylin.

Terminal Deoxynucleotidyl Transferase–Mediated Deoxyuridine Triphosphate Nick-End Labeling (TUNEL) Assay.

Liver sections were TUNEL-stained with a peroxidase in situ cell death detection kit (Roche Diagnosis GmbH, Mannheim, Germany). The number of TUNEL-positive cells per 6 high-power fields (HPFs) was quantified.

Statistical Analysis

All data represent at least three independent experiments and are expressed as means and standard errors of the mean. Differences between groups were compared with an unpaired, two-tailed t test.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Wnt3a-Induced Activation of the Wnt Pathway Is Both SULF2-Dependent and GPC3-Dependent

Wnt3a is an important regulator of HCC growth.5 Desulfation of cell surface HSPGs by quail sulfatase 1 has been proposed to release sequestered Wnt ligands bound to HSPGs at the cell surface and thus enhance the binding of released Wnts to their Frizzled receptors.15 We investigated (1) the effects of SULF2 on Wnt signaling in HCC cells upon exposure to exogenous Wnt3a and (2) whether SULF2 activation of Wnt signaling is dependent on HS. Hep3B vector and Hep3B-SULF2-H cells were treated with the Wnt3a ligand (0, 2, or 10 ng/mL) for 24 hours and washed extensively. Wnt3a levels in cell lysates were then compared by western immunoblotting. In Hep3B vector cells, there was a small increase in Wnt3a when cells were treated with 2 ng/mL Wnt3a, but there was no further increase at 10 ng/mL. In Hep3B-SULF2-H cells, the basal level of Wnt3a was higher. Treatment with 2 ng/mL Wnt3a did not increase Wnt3a; however, 10 ng/mL Wnt3a led to a substantial increase in Wnt3a, and this suggested that SULF2 increased endogenous Wnt3a levels (Fig. 1A). Moreover, the TOPFLASH luciferase reporter assay showed that Wnt3a stimulation of transiently transfected Hep3B-SULF2 cells induced significant Wnt/β-catenin pathway activity (P < 0.0002) as early as 6 hours after transfection and was sustained over 24 hours (Fig. 1B,C). Similar SULF2 enhancement of Wnt3a-induced TOPFLASH expression occurred in PLC/PRF/5 cells, which also have low SULF2 expression (P < 0.03; Supporting Fig. 1).

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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 the Wnt3a ligand (0, 2, or 10 ng/mL) for 24 hours, washed, and lysed, and western immunoblotting was performed with antibodies against Wnt3a and actin (the loading control). SULF2 increased basal and Wnt3a-induced Wnt3a expression. (B,C) The TOPFLASH luciferase assay showed 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 an SULF2-expressing construct or an empty vector. After serum starvation, the cells were treated with 5 ng/mL Wnt3a, and the TOPFLASH luciferase activity was measured after (B) 6 or (C) 24 hours. SULF2 enhanced Wnt3a-induced luciferase activity as early as 6 hours, and the effect was sustained over 24 hours (P < 0.0002).

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Next, we determined whether Wnt3a binding to HCC cells is HS-dependent. Wnt3a binding was inhibited by HS in a dose-dependent manner (Fig. 2A-C). Because GPC3 is the most highly up-regulated HSPG in HCC and has been shown to bind Wnt3a and activate the Wnt/β-catenin pathway,5, 10 we hypothesized that knockdown of GPC3 would abrogate the binding of Wnt3a at the cell surface. To test this hypothesis, we transiently transfected green fluorescent protein (GFP) plasmid constructs coexpressing shRNA targeting the GPC3 messenger RNA (mRNA) or control scrambled shRNA into Hep3B SULF2-H cells. GPC3 knockdown significantly decreased Wnt3a binding to Hep3B cells. Wnt3a binding was also further decreased by HS (Fig. 2D).

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Figure 2. Wnt3a binding to HCC cells is HS-dependent and is mediated by GPC3. (A-C) 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 of HS. After streptavidin staining, 20,000 cells were counted by flow cytometry. There was dose-dependent inhibition of Wnt3a binding by HS 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. The addition of HS decreased binding further.

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SULF2, GPC3, and Wnt3a Associate in a Possible Ternary Complex

To determine whether SULF2, GPC3, and Wnt3a associate in HCC cells, we treated Hep3B vector and Hep3B SULF2-H cells with the Wnt3a ligand (10 ng/mL) and performed immunoprecipitation with antibodies against SULF2 and GPC3. The SULF2 antibody pulled down GPC3 and Wnt3a (Fig. 3A), and the GPC3 antibody pulled down SULF2 and Wnt3a (Fig. 3B); this suggests that all three molecules associate in a molecular complex. Because GPC3 and SULF2 are primarily located at the cell surface, we confirmed the cell surface colocalization of SULF2 and GPC3 by immunocytochemistry (Fig. 3C).

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Figure 3. SULF2, GPC3, and Wnt3a associate at the cell surface in a possible ternary complex. (A,B) Lysates from Hep3B vector and Hep3B SULF2 cells were used for immunoprecipitation (IP) with anti-SULF2 and anti-GPC3 antibodies. Western immunoblotting was performed with antibodies 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 an antibody against Wnt3a (see the lower panels). (C) Immunocytochemistry showed colocalization of SULF2 and GPC3 in Hep3B cells transiently transfected with SULF2. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI).

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SULF2 Up-Regulates Wnt3a and GPC3 and Activates the Wnt/β-Catenin Pathway

GPC3-dependent Wnt/β-catenin pathway activation and consequent HCC cell proliferation have been demonstrated with exogenous Wnt3a.5, 10 Because SULF2-expressing Hep3B cells have higher Wnt3a expression and may activate the Wnt/β-catenin pathway in an autocrine fashion (Fig. 1A-C), we investigated the relationship between SULF2, GPC3, and Wnt signaling in the absence of exogenous Wnt3a. We have previously shown by western immunoblotting that SULF2 induces up-regulation of the GPC3 protein.11 SULF2-induced changes in the expression of Wnt3a and the Wnt/β-catenin molecules phospho-GSK3β and β-catenin were assessed by western immunoblotting. Forced expression of SULF2 increased Wnt3a, increased phospho-GSK3β, and increased total β-catenin, and this was consistent with canonical Wnt/β-catenin activation (Fig. 4A). Total GSK3β was unchanged, and inactive phospho-β-catenin was decreased (Supporting Fig. 2). Immunocytochemistry showed increased cell surface localization of SULF2, GPC3, and Wnt3a and membrane, cytoplasmic, and nuclear accumulation of β-catenin in Hep3B SULF2-H cells (Fig. 4B and Supporting Fig. 3).

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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 with 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 the 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, down-regulation of SULF2 decreased cyclin D1 levels.

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To determine the functional effects of SULF2 downstream of β-catenin, we measured β-catenin–dependent Tcf/lymphoid enhancer-binding factor (Lef) transcriptional activity with the TOPFLASH reporter plasmid. Forced expression of SULF2 significantly increased Tcf/Lef transcription in Hep3B SULF2-H cells (P < 0.05; Fig. 4C) and also increased expression of the target gene cyclin D1 (Fig. 4D). Furthermore, the increase in cyclin D1 was reversed by knockdown of SULF2 in Hep3B SULF2-H cells (Fig. 4D).

Knockdown of SULF2 Down-Regulates Wnt3a and Inhibits Wnt/β-Catenin Signaling

Because most HCC cell lines overexpress SULF2, we examined the effects of down-regulation of SULF2 on Wnt/β-catenin signaling in SULF2-positive Huh7 cells. We have previously shown that knockdown of SULF2 down-regulates GPC3 in Huh7 cells.11 Knockdown of SULF2 decreased the levels of Wnt3a, phospho-GSK3β, and β-catenin by western immunoblotting (Fig. 5A) and the level of β-catenin by immunocytochemistry (Fig. 5B). Knockdown of SULF2 also significantly decreased Tcf/Lef transcriptional activity in Huh7 cells (P < 0.05; Fig. 5C), and there was an associated decrease in the expression of cyclin D1 (Fig. 5D).

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Figure 5. Knockdown of SULF2 down-regulates Wnt3a 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 with antibodies against Wnt3a, phospho-GSK3β, β-catenin, and actin (the loading control). Down-regulation 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 an 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 versus the untransfected cells without green fluorescence. (C) Huh7 vector and Huh7 SULF2 shRNA stable clones were transiently transfected with TOPFLASH and FOPFLASH plasmids. Down-regulation 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).

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SULF2 Up-Regulates GPC3 and Wnt3a and Increases Cell Proliferation in Nude Mouse Xenografts In Vivo

We have previously shown that SULF2 increases the proliferation and viability of HCC cell lines in vitro. To confirm this observation in vivo, we inoculated stably transfected HCC cells subcutaneously in nude mice. SULF2 significantly increased tumor growth and reduced the median time to a tumor size of 1000 mm3 by 28 days.11 To confirm the SULF2-induced changes in GPC3 and Wnt signaling in vivo, we performed immunohistochemistry with antibodies against SULF2, GPC3, Wnt3a, and β-catenin in consecutive sections of xenografts derived from Hep3B vector and Hep3B SULF2-H cells. SULF2 induced up-regulation of GPC3, Wnt3a, and β-catenin in vivo (Fig. 6A). Furthermore, the dominant effect of SULF2 on HCC cell growth occurred through increased proliferation (Ki-67 assay; Fig. 6B,C) rather than decreased apoptosis (cleaved caspase-3 and TUNEL assays; Fig. 7).

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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 10 nude mice. SULF2 significantly enhanced tumor growth in vivo.11 (A) Successive sections from paraffin-embedded xenografts from Hep3B vector (top) and Hep3B SULF2-H cells (bottom) were immunostained with antibodies against SULF2, GPC3, and Wnt3a; 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 versus Hep3B vector xenografts. Xenografts from Hep3 SULF2 cells showed smaller but more numerous brown-staining proliferative cells (arrows; magnification, ×200). (C) Graph quantifying increased cell proliferation in Hep3B SULF2–derived xenografts versus Hep3B vector xenografts (averages of six HPFs).

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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) The caspase-3 assay showed few apoptotic cells in Hep3B vector–derived and Hep3B SULF2–derived xenograft sections (magnification, ×200). (B) There was no significant difference in active caspase-3 between the two groups. (C) TUNEL staining also showed no difference between the groups. The values are means of TUNEL-positive cells in six HPFs.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The mechanisms regulating Wnt/β-catenin pathway activation in HCC have not been completely elucidated.2 Many cell growth signaling pathways have ligands for which cell surface and extracellular matrix proteoglycans serve as coreceptors or storage sites. GPC3 is a cell surface HSPG that is highly overexpressed in HCC and can sequester growth factor ligands and cytokines via its sulfated HSGAG side chains. GPC3 has been shown to mediate activation of the canonical Wnt/β-catenin pathway, and anchorage of GPC3 to the cell membrane has been shown to be critical for Wnt/β-catenin activation and growth of HCC cells.5, 16, 17 The HS-degrading endosulfatase SULF2 may release sequestered factors from HSGAGs, allow binding to their receptors, and thus enhance growth signaling.18 We therefore hypothesized that GPC3-mediated activation of the Wnt/β-catenin pathway in human HCC would be enhanced by SULF2. In this article, we explore the contribution of SULF2 expression to GPC3-mediated Wnt pathway activation in HCC.

The principal findings of this study are as follows:

  • 1
    SULF2 increases endogenous Wnt3a expression and stimulates basal and Wnt3a-induced Tcf/Lef transcriptional activation in SULF2-negative Hep3B HCC cells.
  • 2
    The binding of exogenous Wnt3a to the cell surface is dependent on the HSPG GPC3.
  • 3
    SULF2, GPC3, and Wnt3a associate in a possible ternary complex.
  • 4
    The canonical Wnt pathway is activated by SULF2, which increases GPC3 and Wnt3a expression, GSK3β phosphorylation, membrane, cytosolic, and nuclear β-catenin, and downstream cyclin D1 expression.

Down-regulation of SULF2 in the SULF2-positive Huh7 cell line leads to opposite effects on Wnt/β-catenin signaling. The SULF2-induced increase in GPC3, Wnt3a, and β-catenin occurs in HCC xenografts in vivo and is primarily associated with activation of cell proliferation.

Previous work on GPC3-mediated Wnt/β-catenin signaling has used exogenous Wnt3a. However, because we found that SULF2 up-regulates endogenous Wnt3a, we assessed changes in Wnt/β-catenin pathway activity both in HCC cell lines treated with exogenous Wnt3a (as would occur in paracrine signaling) and in HCC cells transfected with SULF2 (mimicking autocrine signaling). Using SULF2-transfected and GPC3-knockout cell models, we demonstrated that the effect of HS on Wnt3a binding at the cell surface is dose-dependent and that GPC3 is a mediator of Wnt3a binding. In addition, by immunoprecipitation and immunocytochemistry with antibodies against SULF2 and GPC3, we provide evidence for the cellular interaction of SULF2, GPC3, and Wnt3a.

To determine the functional consequences of the cell surface association of GPC3, SULF2, and Wnt3a, we examined the effect of forced expression of SULF2 in the SULF2-negative Hep3B HCC cell line and also studied the impact of SULF2 knockdown in Huh7 HCC cells, which endogenously express SULF2.11 In Hep3B cells, SULF2 expression increased GPC3 and Wnt3a expression and activated the Wnt/β-catenin pathway, as evidenced by increased phosphorylation of GSK3β and consequent accumulation of β-catenin. Conversely, knockdown of SULF2 in Huh7 cells decreased GPC3, Wnt3a, phosphorylated GSK3β, and β-catenin. The functional significance of these changes in β-catenin expression was confirmed by the measurement of the β-catenin–dependent Tcf/Lef transcriptional activity with the TOPFLASH/FOPFLASH luciferase reporter assay and the corresponding expression of the target gene cyclin D1. These findings demonstrate that Wnt/β-catenin pathway activation is mediated by both SULF2 and the HSPG GPC3 in a complex involving Wnt3a.

Finally, we have provided in vivo evidence of SULF2-induced up-regulation of GPC3, Wnt3a, and β-catenin expression in HCC xenografts from nude mice. Together, our results support a working model showing that SULF2-mediated desulfation of GPC3 HSGAGs at the cell surface releases Wnt from storage-type HSGAGs to enable Wnt activation of its Frizzled receptors and downstream Wnt/β-catenin signaling (Fig. 8). Because the primary action of the sulfatases is on the HSGAG chains attached to core proteins, this model suggests that the HSGAG chains of GPC3 play a role in GPC3-mediated activation of the Wnt pathway in HCC. This supports earlier work that described HSGAG chains as essential to GPC3-mediated Wnt signaling in both canonical and noncanonical Wnt pathway activation.19 Although it has been suggested that the HSGAG chains of GPC3 are not absolutely required for canonical Wnt signaling in HCC,5 our findings strongly suggest that SULF2-induced changes in the sulfation state of GPC3 HSGAGs modulate GPC3-mediated Wnt/β-catenin signaling in HCC cells both in vitro and in vivo.

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Figure 8. Working model of the effect of SULF2 on Wnt/β-catenin signaling in HCC cells. The expression of SULF2 in HCC cells up-regulates cell surface GPC3 and Wnt3a. SULF2 desulfates GPC3 HSGAGs and leads to the release of Wnt, the binding and activation of the Wnt receptor Frizzled, and the phosphorylation of GSK3β. Phosphorylation of GSK3β results in the dissolution of the complex responsible for the degradation of β-catenin and thus allows 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 the resultant promotion of cell proliferation in vitro and tumor growth in vivo.

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In summary, this work supports the hypothesis that SULF2 acts as an oncogenic protein in HCCs at least in part by increasing Wnt3a and GPC3 expression, activating the Wnt/β-catenin pathway, and thus promoting growth of HCC cell lines and xenografts. We have previously shown that SULF2 also enhances signaling by receptor tyrosine kinases such as FGF2.11 This ability of SULF2 to modulate FGF2 and Wnt binding at the cell surface and hence activate the mitogen-activated protein kinase, protein kinase B, and Wnt pathways likely accounts for the high tumor recurrence rate and poor survival of HCC patients whose tumors express high levels of SULF2.11 Our findings also confirm the concept that, in contrast to its tumor suppressor-like homolog sulfatase 1, SULF2 has an oncogenic effect in human HCCs. Agents that inhibit SULF2 may therefore be effective for the prevention and/or treatment of HCCs.12, 20, 21 A recent study has also shown an analogous oncogenic effect of SULF2 in lung cancer.22

We are currently pursuing studies to determine the exact mechanism by which desulfation of HS regulates GPC3 function and to determine how this modulates Wnt3a–Frizzled 7 binding and Wnt pathway activation at the cell surface. In particular, we propose that 6-O desulfation of the HS chains of GPC3 by SULF2 will release more Wnt proteins from their storage sites and make them available to bind to and stimulate their cognate Frizzled receptors.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The authors thank Dr. Shin-Ichiro Kojima for the pG-SUPER vector, Dr. Daniel D. Billadeau for the pSSH1p and TOPFLASH vectors, Dr. Wanguo Liu for the TOPFLASH and FOPFLASH vectors, Patrick L. Splinter and Linda M. Murphy for technical assistance, Victoria Campion for secretarial assistance, and Dr. Gregory J. Gores and Dr. Rosebud O. Roberts for critical reviews of the manuscript.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
HEP_23848_sm_SuppFig-1.tif818KSupporting Figure 1: SULF2 increases Wnt/β-catenin signaling in PLC/PRF/5 cells PLC/PRF/5 cells were transfected with TOPFLASH reporter construct and either a SULF2-expressing construct or empty vector. After serum-starvation, cells were treated with 5 ng/ml Wnt3a ligand and TOPFLASH luciferase activity was measured after 6 hours (A) or 24 hours (B). SULF2 enhanced Wnt3a-induced luciferase activity as early as 6 hours and the effect was sustained over 24 hours (p<0.0271).
HEP_23848_sm_SuppFig-2.tif122KSupporting Figure 2: Expression of SULF2 up-regulates the canonical Wnt signaling pathway Western immunoblotting was performed on whole cell lysates from Hep3B Vector (control), Hep3B SULF2-L (low SULF2 expression) and Hep3B SULF2-H (high SULF2 expression) cells using antibodies against phospho-GSK3β, total GSK3β, β-catenin, phospho-β-catenin and actin (loading control). Increased SULF2 expression resulted in increased phosphorylation of GSK3β and a concomitant decrease in phospho-β-catenin, which is then ubiquitinated and degraded.
HEP_23848_sm_SuppFig-3.tif128KSupporting Figure 3: SULF2 increases membrane and cytoplasmic β-catenin in HCC cells Hep3B cells were transiently transfected with either SULF2-expressing plasmid construct or an empty vector. SULF2 was associated with both increased membrane and cytoplasmic β-catenin as seen by immunofluorescence.

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