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Human Monoclonal Antibody Targeting the Heparan Sulfate Chains of Glypican-3 Inhibits HGF-Mediated Migration and Motility of Hepatocellular Carcinoma Cells.

Gao W, Kim H, Ho M - PLoS ONE (2015)

Bottom Line: In addition, an antibody that recognizes the core protein of GPC3 did not change the rate of cell motility.HCC cell migration and motility did not respond to either canonical or non-canonical Wnt induction, but did increase under hepatocyte growth factor (HGF) treatment.Blocking the HS chains on GPC3 with HS20 reduced c-Met activation in HGF-treated HCC cells and 3D-cultured spheroids.

View Article: PubMed Central - PubMed

Affiliation: Antibody Therapy Section, Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, United States of America.

ABSTRACT
Heparan sulfate proteoglycans (HSPGs) participate in many processes related to tumor development, including tumorigenesis and metastasis. HSPGs contain one or more heparan sulfate (HS) chains that are covalently linked to a core protein. Glypican-3 (GPC3) is a cell surface-associated HSPG that is highly expressed in hepatocellular carcinoma (HCC). GPC3 is involved in Wnt3a-dependent HCC cell proliferation. Our previous study reported that HS20, a human monoclonal antibody targeting the HS chains on GPC3, inhibited Wnt3a/β-catenin activation. In the current study, we showed that the HS chains of GPC3 could mediate HCC cells' migration and motility. Knocking down GPC3 or targeting the HS chains by HS20 inhibited HCC cell migration and motility. However, HS20 had no effect on GPC3 knockdown cells or GPC3 negative cells. In addition, an antibody that recognizes the core protein of GPC3 did not change the rate of cell motility. HCC cell migration and motility did not respond to either canonical or non-canonical Wnt induction, but did increase under hepatocyte growth factor (HGF) treatment. HS20-treated HCC cells exhibited less ability for HGF-mediated migration and motility. Furthermore, HS20 inhibited in vitro HCC spheroid formation and liver tumor growth in mice. GPC3 interacted with HGF; however, a mutant GPC3 lacking the HS chain showed less interaction with HGF. Blocking the HS chains on GPC3 with HS20 reduced c-Met activation in HGF-treated HCC cells and 3D-cultured spheroids. Taken together, our study suggests that GPC3 is involved in HCC cell migration and motility through HS chain-mediated cooperation with the HGF/Met pathway, showing how HS targeting has potential therapeutic implications for liver cancer.

No MeSH data available.


Related in: MedlinePlus

HGF knockdown reduced the inhibitory effect of HS20 in HCC cells.(A)Western blots to examine the expression of HGF in Hep3B, Huh-7 and SK-hep1 cells. (B) Western blots to examine the knockdown efficiency of HGF. (C) Hep3B cells and Huh-7 cells with HGF knockdown were treated with 50 μg/mL human IgG or HS20. Cell migration ability was then measured with a wound healing assay. The open wound area at 0 hours was regarded as 100%. Scale bar indicates 400 μm. Values represent mean ± SD from three replicates. P**<0.01 compare to IgG group. (D) Hep3B cells and Huh-7 cells with HGF knockdown were treated with 50 μg/mL human IgG or HS20. A Trans-well assay was performed to examine cell motility. The OD590nm value of control siRNA group with IgG treatment was set up as 100%. Scale bar indicates 50 μm. Values are mean ± SD from three replicates. P*<0.1 and P***<0.01 compared to the IgG group.
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pone.0137664.g003: HGF knockdown reduced the inhibitory effect of HS20 in HCC cells.(A)Western blots to examine the expression of HGF in Hep3B, Huh-7 and SK-hep1 cells. (B) Western blots to examine the knockdown efficiency of HGF. (C) Hep3B cells and Huh-7 cells with HGF knockdown were treated with 50 μg/mL human IgG or HS20. Cell migration ability was then measured with a wound healing assay. The open wound area at 0 hours was regarded as 100%. Scale bar indicates 400 μm. Values represent mean ± SD from three replicates. P**<0.01 compare to IgG group. (D) Hep3B cells and Huh-7 cells with HGF knockdown were treated with 50 μg/mL human IgG or HS20. A Trans-well assay was performed to examine cell motility. The OD590nm value of control siRNA group with IgG treatment was set up as 100%. Scale bar indicates 50 μm. Values are mean ± SD from three replicates. P*<0.1 and P***<0.01 compared to the IgG group.

Mentions: To investigate the underlying mechanism of how HS20 regulates cell migration and motility in HCC cells, we initially detected the effect of canonical and non-canonical Wnt signaling on HCC cell motility. Hep3B cells were treated with Wnt3a- or Wnt5a-conditioned media, but none of them had a significant effect on cell migration (data not shown). This observation suggests that GPC3 may coordinate with other signaling to regulate cell movement. Several studies report that glypican-1 (GPC1) and glypican-4 (GPC4) are involved in HGF-dependent signaling [29,30]. Therefore, we detected whether HGF is expressed in HCC cells. As shown in Fig 3A, Hep3B, Huh-7, and SK-hep1 cells all expressed endogenous HGF. We knocked down HGF with siRNA; at least 70% of HGF expression was reduced after transfection (Fig 3B). The HGF knockdown cells had a slower cell migration rate. When treated with HS20, cell migration of HGF knockdown cells was not significantly inhibited (Fig 3C). Similarly, HGF knockdown cells exhibited reduced cell motility. HS20 slightly inhibited cell motility in HGF knockdown cells but the inhibition was significantly less than that of control cells (Fig 3D). All of these observations indicated that HGF regulated HCC cell migration and motility.


Human Monoclonal Antibody Targeting the Heparan Sulfate Chains of Glypican-3 Inhibits HGF-Mediated Migration and Motility of Hepatocellular Carcinoma Cells.

Gao W, Kim H, Ho M - PLoS ONE (2015)

HGF knockdown reduced the inhibitory effect of HS20 in HCC cells.(A)Western blots to examine the expression of HGF in Hep3B, Huh-7 and SK-hep1 cells. (B) Western blots to examine the knockdown efficiency of HGF. (C) Hep3B cells and Huh-7 cells with HGF knockdown were treated with 50 μg/mL human IgG or HS20. Cell migration ability was then measured with a wound healing assay. The open wound area at 0 hours was regarded as 100%. Scale bar indicates 400 μm. Values represent mean ± SD from three replicates. P**<0.01 compare to IgG group. (D) Hep3B cells and Huh-7 cells with HGF knockdown were treated with 50 μg/mL human IgG or HS20. A Trans-well assay was performed to examine cell motility. The OD590nm value of control siRNA group with IgG treatment was set up as 100%. Scale bar indicates 50 μm. Values are mean ± SD from three replicates. P*<0.1 and P***<0.01 compared to the IgG group.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4557904&req=5

pone.0137664.g003: HGF knockdown reduced the inhibitory effect of HS20 in HCC cells.(A)Western blots to examine the expression of HGF in Hep3B, Huh-7 and SK-hep1 cells. (B) Western blots to examine the knockdown efficiency of HGF. (C) Hep3B cells and Huh-7 cells with HGF knockdown were treated with 50 μg/mL human IgG or HS20. Cell migration ability was then measured with a wound healing assay. The open wound area at 0 hours was regarded as 100%. Scale bar indicates 400 μm. Values represent mean ± SD from three replicates. P**<0.01 compare to IgG group. (D) Hep3B cells and Huh-7 cells with HGF knockdown were treated with 50 μg/mL human IgG or HS20. A Trans-well assay was performed to examine cell motility. The OD590nm value of control siRNA group with IgG treatment was set up as 100%. Scale bar indicates 50 μm. Values are mean ± SD from three replicates. P*<0.1 and P***<0.01 compared to the IgG group.
Mentions: To investigate the underlying mechanism of how HS20 regulates cell migration and motility in HCC cells, we initially detected the effect of canonical and non-canonical Wnt signaling on HCC cell motility. Hep3B cells were treated with Wnt3a- or Wnt5a-conditioned media, but none of them had a significant effect on cell migration (data not shown). This observation suggests that GPC3 may coordinate with other signaling to regulate cell movement. Several studies report that glypican-1 (GPC1) and glypican-4 (GPC4) are involved in HGF-dependent signaling [29,30]. Therefore, we detected whether HGF is expressed in HCC cells. As shown in Fig 3A, Hep3B, Huh-7, and SK-hep1 cells all expressed endogenous HGF. We knocked down HGF with siRNA; at least 70% of HGF expression was reduced after transfection (Fig 3B). The HGF knockdown cells had a slower cell migration rate. When treated with HS20, cell migration of HGF knockdown cells was not significantly inhibited (Fig 3C). Similarly, HGF knockdown cells exhibited reduced cell motility. HS20 slightly inhibited cell motility in HGF knockdown cells but the inhibition was significantly less than that of control cells (Fig 3D). All of these observations indicated that HGF regulated HCC cell migration and motility.

Bottom Line: In addition, an antibody that recognizes the core protein of GPC3 did not change the rate of cell motility.HCC cell migration and motility did not respond to either canonical or non-canonical Wnt induction, but did increase under hepatocyte growth factor (HGF) treatment.Blocking the HS chains on GPC3 with HS20 reduced c-Met activation in HGF-treated HCC cells and 3D-cultured spheroids.

View Article: PubMed Central - PubMed

Affiliation: Antibody Therapy Section, Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, United States of America.

ABSTRACT
Heparan sulfate proteoglycans (HSPGs) participate in many processes related to tumor development, including tumorigenesis and metastasis. HSPGs contain one or more heparan sulfate (HS) chains that are covalently linked to a core protein. Glypican-3 (GPC3) is a cell surface-associated HSPG that is highly expressed in hepatocellular carcinoma (HCC). GPC3 is involved in Wnt3a-dependent HCC cell proliferation. Our previous study reported that HS20, a human monoclonal antibody targeting the HS chains on GPC3, inhibited Wnt3a/β-catenin activation. In the current study, we showed that the HS chains of GPC3 could mediate HCC cells' migration and motility. Knocking down GPC3 or targeting the HS chains by HS20 inhibited HCC cell migration and motility. However, HS20 had no effect on GPC3 knockdown cells or GPC3 negative cells. In addition, an antibody that recognizes the core protein of GPC3 did not change the rate of cell motility. HCC cell migration and motility did not respond to either canonical or non-canonical Wnt induction, but did increase under hepatocyte growth factor (HGF) treatment. HS20-treated HCC cells exhibited less ability for HGF-mediated migration and motility. Furthermore, HS20 inhibited in vitro HCC spheroid formation and liver tumor growth in mice. GPC3 interacted with HGF; however, a mutant GPC3 lacking the HS chain showed less interaction with HGF. Blocking the HS chains on GPC3 with HS20 reduced c-Met activation in HGF-treated HCC cells and 3D-cultured spheroids. Taken together, our study suggests that GPC3 is involved in HCC cell migration and motility through HS chain-mediated cooperation with the HGF/Met pathway, showing how HS targeting has potential therapeutic implications for liver cancer.

No MeSH data available.


Related in: MedlinePlus