PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of neoplasiaLink to Publisher's site
 
Neoplasia. Aug 2011; 13(8): 735–747.
PMCID: PMC3156664
Suppression of Glypican 3 Inhibits Growth of Hepatocellular Carcinoma Cells through Up-Regulation of TGF-β21,2
Chris K Sun, Mei-Sze Chua, Jing He, and Samuel K So
Asian Liver Center and Department of Surgery, Stanford University School of Medicine, Stanford University, Stanford, CA, USA
Address all correspondence to: Dr. Mei-Sze Chua, PhD, Department of Surgery, Stanford University School of Medicine, Stanford University, 1201 Welch Rd, MSLS Bldg, P228, Stanford, CA 94305-5655. E-mail: mchua/at/stanford.edu
Received May 11, 2011; Revised June 6, 2011; Accepted June 7, 2011.
Glypican 3 (GPC3) is a valuable diagnostic marker and a potential therapeutic target in hepatocellular carcinoma (HCC). To evaluate the efficacy of targeting GPC3 at the translational level, we used RNA interference to examine the biologic and molecular effects of GPC3 suppression in HCC cells in vitro and in vivo. Transfection of Huh7 and HepG2 cells with GPC3-specific small interfering RNA (siRNA) inhibited cell proliferation (P < .001) together with cell cycle arrest at the G1 phase, down-regulation of antiapoptotic protein (Bcl-2, Bcl-xL, and Mcl-1), and replicative senescence. Gene expression analysis revealed that GPC3 suppression significantly correlated with transforming growth factor beta receptor (TGFBR) pathway (P = 4.57e-5) and upregulated TGF-β2 at both RNA and protein levels. The effects of GPC3 suppression by siRNA can be recapitulated by addition of human recombinant TGF-β2 to HCC cells in culture, suggesting the possible involvement of TGF-β2 in growth inhibition of HCC cells. Cotransfection of siRNA-GPC3 with siRNA-TGF-β2 partially attenuated the effects of GPC3 suppression on cell proliferation, cell cycle progression, apoptosis, and replicative senescence, confirming the involvement of TGF-β2 in siRNA-GPC3-mediated growth suppression. In vivo, GPC3 suppression significantly inhibited the growth of orthotopic xenografts of Huh7 and HepG2 cells (P < .05), accompanied by increased TGF-β2 expression, reduced cell proliferation (observed by proliferating cell nuclear antigen staining), and enhanced apoptosis (by TUNEL staining). In conclusion, molecular targeting of GPC3 at the translational level offers an effective option for the clinical management of GPC3-positive HCC patients.
Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and the third most common cause of cancer mortality [1]. It is typically aggressive and intrinsically resistant to standard chemotherapeutic agents, underscoring the need for developing more effective therapies for HCC patients [2,3]. Recent studies have implicated glypican 3 (GPC3) as an important protein in HCC. As a histologic marker, GPC3 staining could distinguish malignant HCC from preneoplastic, cirrhotic, or benign liver lesions [4–9]. As a serum marker, the cleaved and secreted form of GPC3 was found to be elevated in the serum of HCC patients, making it a valuable, noninvasive diagnostic marker of HCC [10–12]. Functionally, GPC3 promotes growth of HCC cells through stimulation of the canonical Wnt signaling pathway [13], which regulates the expression of many downstream oncogenic proteins such as c-MYC [14]. Given the importance of Wnt signaling in HCC [15], GPC3 may also have therapeutic potential in the clinical management of HCC.
GPC3 was first identified in patients with Simpson-Golabi-Behmel syndrome, an X-linked disease characterized by prenatal and postnatal overgrowth caused by mutation of the GPC3 gene [16]. It is one of six members of the glypican family, which are heparan sulfate proteoglycans that are linked to the exocytoplasmic surface of the plasma membrane by a glycosyl-phosphatidylinositol (GPI) anchor [17]. Glypicans play a critical role in the regulation of cell proliferation and survival, particularly during development and malignant transformation [18–21]. The main function of membrane-attached glypicans is to regulate the signaling of Wnts, Hedgehogs (Hhs), fibroblast growth factors, and bone morphogenic proteins (BMPs) [20,22–24]. Depending on the cellular context, glypicans may have a stimulatory or inhibitory role in these signaling pathways: in tissues where cell proliferation is predominantly controlled by Hh signaling, GPC3 overexpression inhibits proliferation, whereas in tissues where canonical Wnt signaling predominates, GPC3 overexpression stimulates cell proliferation [25].
In HCC, GPC3 interacts with Wnt ligands acting in the canonical pathway to stimulate cell proliferation [15]. Cellular proliferation induced by Wnt3a has recently been attributed to activation of both the extracellular signal-regulated kinase (ERK) and Wnt pathways [26], both of which are implicated in hepatocarcinogenesis associated with hepatitis B or C virus infections, the major risk factors of HCC [27]. In addition, GPC3 has been reported to confer hepatocarcinogenesis through the interaction between insulin-like growth factor II and its receptor, with subsequent activation of the insulin growth factor signaling pathway [28]. The interaction between GPC3 and fibroblast growth factor basic has also been reported to modulate proliferation of HCC cells [23] and could, in part, mediate the oncogenic effect of sulfatase 2 in HCC [29]. Indeed, the overexpression of GPC3 in HCC patients has been positively correlated with fibroblast growth factor receptor 1, insulin-like growth factor 1 receptor, and nuclear localization of β-catenin [30].
The elevated expression of GPC3 in HCC and its potential involvement in multiple signaling pathways contributing to hepatocarcinogenesis together suggest that inhibition of GPC3 may offer a potent and molecularly targeted strategy for intervening with HCC progression. In this study, we confirmed that suppression of GPC3 in HCC cells by RNA interference led to inhibitory effects on cell growth and cell cycle progression. We additionally provide further mechanistic insights into another signaling pathway, the transforming growth factor β (TGF-β) pathway, which might be involved in GPC3-mediated functions in HCC. Taken together, our data provide support that molecular targeting of GPC3 at the translational level is an effective treatment strategy for HCC, which has heterogeneous pathology.
Cell Culture
The human HCC cell lines Huh7 and HepG2 were cultured in Dulbecco modified Eagle medium and minimum essential medium, respectively (Gibco/Invitrogen, Carlsbad, CA). Media were supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 µg/ml streptomycin (Invitrogen). Cells were cultured at 37°C in a humidified atmosphere of 5% CO2.
Small Interfering RNA Transfection In Vitro
GPC3-specific small interfering RNA (siRNA-GPC3), TGF-β2-specific siRNA (siRNA-TGFβ2), and silencer-negative control siRNA (siRNA-N) were purchased from Ambion, Inc (Austin, TX). Sequences for GPC3 and TGF-β2-specific siRNAs used for the experiments were as follows:
  • siRNA-GPC3: (GGCUCUGAAUCUUGGAAUUtt), s14750
  • siRNA-TGF-β2: (CACUCGAUAUGGACCAGUUtt), s14059
The transfection reagent Lipofectamine RNAiMAX (Invitrogen) was used to transfect siRNA oligos into Huh7 and HepG2 cells, using the reverse transfection method according to the manufacturer's instructions.
Cell Proliferation Assay
Huh7 and HepG2 cells reverse transfected with siRNA-GPC3 (25 nM) or siRNA-N (25 nM) were seeded onto 96-well plates in triplicate wells (4000 cells/well) in culture medium without antibiotics. At the indicated time points, cell viability was determined by the CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI) according to the manufacturer's protocol. Optical density (OD) was read at 490 nm using a microplate reader (BioTek Instruments, Inc, Winooski, VT). Once cells have attached (approximately 4 hours after transfection), an OD value was obtained as the first time point. The background values (OD values from wells with only the AQueous One Solution) were subtracted from all data. Three independent experiments were done. For experiments with recombinant human (rh) TGF-β2 (R&D Systems, Minneapolis, MN), 1 or 5 ng/ml of TGF-β2 was added to the culture medium, and the effects on proliferation of Huh7 and HepG2 cells were determined as described previously.
Cell Cycle Analysis by Flow Cytometry
Huh7 and HepG2 cells were synchronized at the G0/G1 phase by culturing in serum-free medium for 24 hours, then transfected with siRNA-N, siRNA-GPC3 alone, or siRNA-GPC3 with siRNA-TGF-β2 for 48 hours. Cells were then stimulated with 20%FBS for 24 hours and trypsinized, washed, and fixed in 70% ethanol for 30 minutes at 4°C. Fixed cells were stained with propidium iodide with RNaseA in 1x PBS for data acquisition using BD LSRII flow cytometer (BD BioSciences, Franklin Lakes, NJ). Data were analyzed by using FlowJo version 7.5.5 (Tree Star, Ashland, OR). Three independent experiments were done for each experiment set.
Protein Extraction and Western Blot Analysis
Transfected cells were harvested and lysed in T-PER Tissue Protein Extraction Reagent (Thermo Scientific, Inc, Waltham, MA) for isolation of total cell lysates. For the isolation of nuclear and cytoplasmic fractions, NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific) were used according to the manufacturer's instructions. Western blot analysis was performed using standard protocols with specific antibodies against the antigens according to the manufacturer's recommendations. Monoclonal antibody against human GPC3 (clone 1G12) was from BioMosaics, Inc (Burlington, VT);monoclonal antibody against TGF-β2 and Histone H3 were from AbCam (Cambridge, MA); polyclonal antibodies against PCNA and β-actin were from Santa Cruz Biotechnology (Santa Cruz, CA); and monoclonal antibody against cyclin D1 was from Thermo Scientific. All other antibodies were purchased from Cell Signaling Technology, Inc (Danvers, MA).
Oligonucleotide Microarray Analysis
Genome-wide expression analysis was done using the Agilent 60-mer Oligonucleotide Two-ColorMicroarray System (Agilent Technologies, Inc, Santa Clara, CA). Total RNA was extracted from siRNA-N- or siRNA-GPC3-treated cells by using RNeasy mini kit (Qiagen, Valencia, CA). The integrity of the RNA was confirmed by the Agilent 2100 Bioanalyzer (Agilent Technologies). RNA samples with RIN number 8 or higher were used for further experiments. The RNA samples were amplified and labeled using the Agilent Quick Amp labeling kit according to the manufacturer's instructions. After fragmentation, 1650 ng of complementary RNA was hybridized onto a 4 x 44K oligonucleotide array at 65°C for 17 hours. The array was then washed and scanned using GenePix 4000A scanner (Agilent Technologies). Data were extracted by using Feature Extraction software and uploaded onto the Stanford Microarray Database (SMD; http://smd.stanford.edu). Supervised analysis using Significance Analysis of Microarrays was used to identify genes that are significantly differentially expressed between siRNA-GPC3-treated cells and siRNA-N-treated cells (a two-fold change cutoff was used). Significantly, differentially regulated pathways were identified using GeneSpring GX11 (Agilent Technologies).
Semiquantitative Real-time Polymerase Chain Reaction
Total RNA was extracted using RNeasy Mini kit (Qiagen) and reverse transcribed using TaqMan Reverse Transcription Reagent (Applied Biosystems, Foster City, CA). Semiquantitative real-time polymerase chain reaction (PCR) was carried out, and data were analyzed using a MX3000P Real-time PCR machine (Stratagene, La Jolla, CA). Primers and probe reagents for human GPC3 (assay no. Hs00170471_m1), TGF-β1 (assay no. Hs00998129_m1), TGF-β2 (assay no. Hs00234244_m1), TGF-β3 (assay no. Hs01086000_m1), and 18S rRNA (normalization control; assay no. 4333760F) were purchased as Pre-Developed TaqMan Gene Expression Assay reagents from Applied Biosystems. Transcript quantification was performed in at least duplicate for every sample. The amount of each target gene was normalized with 18S rRNA to control for RNA amount variation.
Senescence-Associated β-Gal Staining
Senescence β-galactosidase (SA-β-Gal) staining was performed by using the staining kit according to the manufacturer's instructions (Cell Signaling Technology, Inc, Danvers, MA). Briefly, cells were transfected with siRNA-GPC3 or incubated with rhTGF-β2 (1 or 5 ng/ml) for 48 hours before fixing and staining. After SA-β-Gal staining, cells were counterstained with Nuclear Fast Red Solution (Electron Microscopy Sciences, Hatfield, PA), and images were acquired using a fluorescence microscope (Nikon, Melville, NY).
Lentiviral Labeling of Cell Lines and Cell Sorting
Huh7 and HepG2 cells were transduced with self-inactivating lentivirus carrying an ubiquitin promoter driving a trifusion reporter gene, which harbors a bioluminescence (firefly luciferase [Fluc]), a fluorescence (mrfp or egfp), and a positron emission tomography reporter gene (ttk) at a multiplicity of infection of 5 as reported previously [31]. Stable expressors were isolated by sorting at 45 pound-force per square inch (BD FACS Aria II; BD BioSciences).
Animal Studies
Animal experiments were approved by the Administrative Panel on Laboratory Animal Care of Stanford University. Orthotopic liver tumor models of Huh7 and HepG2 cells (stably expressing the trifusion reporter gene) were established in nude mice as reported previously with some modifications [32]. Briefly, ~1 x 107 cells in 200 µl of culture medium were injected subcutaneously into the right flank of nu/nu nude mice (4 weeks old; Charles River Laboratories, Wilmington, MA). Tumor development was monitored daily. Once the subcutaneous tumor reached 1 cm in diameter, it was removed and cut into ~1- to 2-mm3 cubes, which were then surgically implanted into the left lobe of liver in another group of nude mice (6 weeks old). The mice were given intraperitoneal injections of 25 nM siRNA-GPC3 in 100 µl of PBS or siRNA-N as control (five mice each) every 3 days. Tumor growth was monitored once a week for 7 weeks using the Xenogen IVIS in vivo imaging system (Caliper Life Sciences, Hopkinton, CA). Growth curves were plotted using average bioluminescence within each group.
Immunohistochemistry for Cell Proliferation and Apoptosis
Immunostaining was performed as reported previously [32]. Briefly, paraffin slides were stained with anti-proliferating cell nuclear antigen (PCNA; Santa Cruz Biotechnology) or anti-TGF-β2 monoclonal antibody (AbCam) at 4°C overnight followed by the avidin-biotinperoxidase protocol. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assay was done according to the manufacturer's protocol (Roche Applied Science, Indianapolis, IN).
Statistical Analysis
The statistical analyses were done using the SPSS version 15.0 software package (SPSS, Inc, Chicago, IL). Statistical significance was determined by independent-samples t test. P < .05 and P < .01 were considered statistically significant and highly significant, respectively.
Suppression of GPC3 Inhibits In Vitro Proliferation and Cell Cycle Progression in Huh7 and HepG2 Cells
We transfected two tumorigenic hepatoma cell lines (Huh7 and HepG2) that overexpress GPC3 protein (based on Western blot results) with 25 nM of a GPC3-specific siRNA (siRNA-GPC3) and observed greatly reduced expression of glycanated and core GPC3 protein in both cell lines compared with cells transfected with vehicle control or with silencer-negative siRNA (siRNA-N; Figure 1A). Transfection with siRNA-GPC3 also reduced proliferation of Huh7 cells (P < .001 from 72 hours) and HepG2 cells (P < .001 from 48 hours) when assessed daily during a 5-day period (Figure 1B).
Figure 1
Figure 1
Suppression of GPC3 by siRNA-GPC3 inhibits in vitro cell proliferation and cell cycle progression of Huh7 and HepG2 cells. (A) Transfection of GPC3-specific siRNA (siRNA-GPC3) efficiently suppressed expression of core and glycanated forms of GPC3 protein (more ...)
To determine whether the growth-inhibitory effects of GPC3 suppression could result from changes in the cell cycle, we measured cellular DNA content by flow cytometry after cell synchronization by serum starvation, followed by transfection with siRNA-GPC3 for 48 hours and restimulation with 10% FBS for 24 hours. Suppression of GPC3 caused a significant increase in G1 peak in HepG2 cells (P = .04 compared with siRNA-N), together with a decrease in S-phase cells (P = .039; Figure 1C). The expression of cell cycle regulators, cyclin A and cyclin D1, were also downregulated by siRNA-GPC3 in HepG2 cells (Figure 1C). These changes in the cell cycle were less obvious in the Huh7 cells, which showed a small increase in the G0/G1 peak (P = .31 compared with siRNA-N) but an insignificant change in the S peaks (P = .90; Figure 1D). In Huh7 cells, the levels of cyclin A and cyclin D1 were also reduced to a lesser extent than in HepG2 cells (Figure 1D). Thus, the effect of GPC3 suppression on cell growth and cell cycle progression is more marked in HepG2 cells, which have higher levels of GPC3 than Huh7 cells.
Identification of TGF-β2 as an Important Modulator of GPC3-Mediated Signaling
To characterize the signaling pathways and molecular changes regulated by GPC3 in HCC cells, we studied the gene expression changes induced by siRNA-GPC3 compared with their siRNA-N-transfected controls. Pathway analysis using GeneSpring GX 11 identified five signaling pathways that are significantly regulated by GPC3 suppression: nuclear factor κB (P = 3.73e-6), TGFBR (P = 4.57e-5), EGFR1 (P = 1.43e-4), IL-6 (P = 6.43e-4), and Wnt (P = .008). Using Significance Analysis of Microarrays, biologically relevant genes that are commonly, significantly upregulated or downregulated in both HepG2 and Huh7 cell lines were identified using a two-fold change cutoff and P < .0001 (Table 1 and Figure 2A). Biologic functions of encoded proteins were compiled from the SOURCE database available at the SMD. A member of theTGFBR pathway, TGF-β2 (but not TGF-β1 or TGF-β3), was significantly upregulated by GPC3 suppression in both Huh7 and HepG2 cells (P < .05, siRNA-N vs siRNA-GPC3; Figure 2B). The up-regulation of TGF-β2 by siRNA-GPC3 was independently validated using semiquantitative RT-PCR (Figure 2B) and Western blot analysis (Figure 2C) to confirm the negative correlation at both transcript and protein levels, respectively. Two additional GPC3-specific siRNAs (GGGAACCACUUUCUUAUUUtt; GACGUGACCUGAAAGUAUUtt) were used to verify this observation (data not shown). Further analysis of GPC3 and TGF-β2 protein expression in a panel of nine HCC cell lines confirmed this negative correlation: cell lines expressing GPC3 have lower levels of TGF-β2, whereas cell lines with undetectable GPC3 have higher levels of TGF-β2 (Figure 2D). Our results suggest a close association between GPC3 and TGF-β2 in the TGFBR signaling pathway.
Table 1
Table 1
Genes Commonly Significantly Upregulated or Downregulated by GPC3 Suppression in HepG2 and Huh7 Cells.
Figure 2
Figure 2
Identification of TGF-β2 as an important mediator in GPC3 signaling by oligonucleotide microarray analysis. (A) Heat map showing nine significantly upregulated or downregulated genes associated with GPC3 suppression in both Huh7 and HepG2 cells. (more ...)
TGF-β2 Suppresses Cell Proliferation by Activating R-SMADs in Huh7 and HepG2 Cells
To determine whether the growth-inhibitory effects of GPC3 suppression might be partially mediated by TGF-β2, we next looked at the effects of rhTGF-β2 on HCC cell proliferation. Cells were cultured with or without rhTGF-β2 and cell growth was assessed daily for 5 days. We found that rhTGF-β2 significantly inhibited cell proliferation in both Huh7 and HepG2 cells (P < .01 for both cell lines; Figure 3A). Important components of the TGF-β pathway, SMAD2 and SMAD3, were hyperphosphorylated on addition of rhTGF-β2 together with nuclear translocation of SMAD 6 (Figure 3B). Our results imply that TGF-β2 negatively regulates HCC cell proliferation through the activation of R-SMADs, allowing transduction of extracellular TGF-β2 superfamily ligand signaling from cell membrane-bound TGF-β receptors into the nucleus where they activate the transcription of TGF-β target genes.
Figure 3
Figure 3
Human recombinant TGF-β2 inhibits cell proliferation and activates R-SMAD in HepG2 and Huh7 cells. (A) TGF-β2 suppressed proliferation of HCC cells as shown by cell proliferation assay. Cells were cultured in media with different concentrations (more ...)
TGF-β2 Suppresses Cell Cycle Progression and Induces Replicative Senescence in Huh7 and HepG2 Cells
To determine whether the cell cycle effects of GPC3 suppression might be partially mediated by TGF-β2, we looked at the effect of rhTGF-β2 on cell cycle progression of HCC cells. Huh7 and HepG2 cells were grown in culture medium supplemented with rhTGF-β2 (1 or 5 ng/ml) for a period of 48 hours before flow cytometry analyses. Similar to the effects of GPC3 suppression (Figure 1C), significant increases in the G1 peak (P = .016 for Huh7 cells and P < .001 for HepG2 cells), together with a decrease in S- and G2-phase cells were observed in both cell lines compared with control cells not treated with rhTGF-β2 (P < .001 and P = .034 for S and G2 phases, respectively, for HepG2 cells; P = .017 and P = .004 for S and G2 phases, respectively, for Huh7 cells) (Figure 4A). Western blot analysis further showed that rhTGF-β2 decreased the expression of cell cycle regulators cyclin A and cyclin D1 (Figure 4B), as observed with GPC3 suppression (Figure 1C). In addition, phosphorylation of retinoblastoma (Rb) protein was decreased, whereas the expression of p15Ink4b and p21Cip1 were increased in both Huh7 and HepG2 cells, indicating a G1 cell cycle arrest (Figure 4B). The addition of TGF-β2 also increased the apoptotic response, demonstrated by the reduced expression of antiapoptotic proteins (Bcl-xL and Mcl-1) in both cell lines, and the reduced expression of Bcl-2 in HepG2 cells only (Huh7 cells do not express Bcl-2) (Figure 4B).
Figure 4
Figure 4
Human recombinant TGF-β2 inhibits cell cycle progression and induces replicative senescence in HCC cells. (A) Addition of rhTGF-β2 to the culture media inhibited cell cycle progression through G1 arrest in both HepG2 and Huh7 cells. Only (more ...)
Consistent with a recent report that TGF-β1 induces p15Ink4b- and p21Cip1-dependent senescence in HCC cells [33], we also observed that addition of rhTGF-β2 induced cellular senescence in both Huh7 and HepG2 cells as shown by enhanced accumulation of SA-β-Gal (Figure 4C). Taken together, these results suggest that TGF-β2 negatively mediates cell cycle progression through modulation of various cell cycle regulators, thereby contributing to G1 arrest, apoptosis, and senescence.
TGF-β2 siRNA Partially Reverses Growth Inhibition, Cell Cycle Arrest, and Replicative Senescence Caused by siRNA-GPC3
We next cotransfected siRNA-TGF-β2 with siRNA-GPC3 in HCC cells to interfere with the rise in TGF-β2 levels caused by siRNA-GPC3 alone and to observe the resulting effects on cell proliferation and cell cycle progression. The cotransfection of siRNA-TGF-β2 with siRNA-GPC3 partially reversed the growth-inhibitory effects of GPC3 suppression in HepG2 cells but had negligible effect in Huh7 cells (Figure 5A). When the increase in TGF-β2 levels was reduced by siRNA-TGF-β2, cells proliferated faster than when treated with siRNA-GPC3 alone. These effects of cell proliferation were also reflected by the levels of SMAD2 and SMAD3 phosphorylation: siRNA-GPC3 alone caused an increase in SMAD2 and SMAD3 phosphorylation in both Huh7 and HepG2 cells; this increase was partially attenuated when cells were cotransfected with siRNA-TGF-β2 (Figure 5B). Thus, TGF-β2 may partially mediate the effect of GPC3 on HCC cell proliferation.
Figure 5
Figure 5
Transfection of siRNA-TGF-β2 partially reverses the growth inhibition caused by GPC3 suppression. (A) Cotransfection of siRNA-TGF-β2 with siRNA-GPC3 partially reversed the growth inhibition caused by siRNA-GPC3 alone in HepG2 cells, with (more ...)
Cell cycle arrest at the G1 phase caused by siRNA-GPC3 was partially reversed when cells were cotransfected with siRNA-TGF-β2. In HepG2 cells, cotransfection with siRNA-TGF-β2 decreased the G1 peak when compared with siRNA-GPC3-treated controls; a similar but less pronounced effect was observed in Huh7 cells (Figure 6A). Cotransfection with siRNA-TGF-β2 also negated the decrease in cyclin A and cyclin D1 levels caused by GPC3 siRNA in HepG2 cells (Figure 6B). In addition, cotransfection with TGF-β2 siRNA decreased the levels of p15Ink4b and p21Cip1 while increasing the levels of antiapoptotic proteins Bcl-2, Bcl-xL, and Mcl-1 that were regulated by siRNA-GPC3 in HepG2 cells (Figure 6B). In Huh7 cells, cotransfection with siRNA-TGF-β2 caused little effect on the levels of cyclin A, cyclinD1, p15Ink4b, or p21Cip1 but instead increased the phosphorylation of Rb and the levels of Bcl-xL and Mcl-1 that were reduced by siRNA-GPC3 (Figure 6B). Transfection with siRNA-GPC3 alone caused marked accumulations of SA-β-Gal in both cell lines, which were significantly reduced when both cell lines were cotransfected with siRNA-TGF-β2 (Figure 6C). These results showed that cotransfection with siRNA-TGF-β2 can partially reverse the cell cycle arrest and replicative senescence induced by GPC3 suppression, implying that TGF-β2 may partially mediate the effects of GPC3 on regulation of cell cycle and senescence.
Figure 6
Figure 6
Transfection of siRNA-TGF-β2 partially reverses the cell cycle arrest caused by GPC3 suppression. (A) Cell cycle analysis by flow cytometry indicated that siRNA-TGF-β2 partially reversed the G1 arrest induced by GPC3 suppression in HepG2 (more ...)
Suppression of GPC3 Delays HCC Xenograft Growth and Upregulates Cytoplasmic TGF-β2 In Vivo
Finally, we generated orthotopic tumors of Huh7 and HepG2 cells in nude mice to confirm the growth-inhibitory effects of siRNA-GPC3 in vivo. Tumor-bearing mice were given intraperitoneal injections of 25 nM siRNA-GPC3 (n = 5) or siRNA-N (n = 5) in 100 µl of PBS every 3 days until the tumors reached the criteria for euthanasia. Tumor growth was monitored by noninvasive in vivo luciferase imaging. In the HepG2 group, all mice treated with siRNA-GPC3 had a significantly smaller tumor size compared with that observed in mice treated with siRNA-N (P < .05 from week 3 onward) (Figure 7A). In the Huh7 group, mice treated with siRNA-GPC3 had smaller tumor volume when compared with mice treated with siRNA-N, although statistical significance was not reached (P > .05; Figure 7B). Concomitantly, tumors treated with siRNA-GPC3 had markedly lower expression of GPC3 but enhanced expression of cytoplasmic TGF-β2 in the tumor cells (Figure 7C). In addition, we determined the effects of GPC3 suppression on cell proliferation by PCNA immunohistochemistry and on apoptosis by the TUNEL assay (Figure 7C). The siRNA-GPC3-treated tumors expressed reduced levels of PCNA and contained more apoptotic nuclei in both Huh7 and HepG2 xenografts, suggesting that GPC3 suppression inhibited xenograft growth by reducing cell proliferation while inducing apoptosis in vivo. These effects positively correlated with up-regulation of TGF-β2.
Figure 7
Figure 7
Suppression of GPC3 in vivo delays tumor growth and upregulates cytoplasmic TGF-β2. (A, B) Orthotopic liver tumor models of Huh7 and HepG2 cells stably expressing a trifusion reporter gene were given intraperitoneal injections of siRNA-GPC3 (25 (more ...)
Our previous gene expression study of human HCC identified GPC3 as the most highly overexpressed membrane-bound protein in HCC compared with nontumor liver and the second most highly overexpressed secretory protein (after AFP) [34]. Until lately, much of the focus on GPC3 has been on its diagnostic potential. In this study, we validated the therapeutic potential of GPC3 in HCC and reported the involvement of TGF-β2 in GPC3-mediated signaling in HCC cells. Specifically, we showed that suppression of GPC3 in HCC cells enhanced TGF-β2 expression and signaling, which inhibited cell proliferation and cell cycle progression, and induced replicative senescence.
GPC3 is a rational target for the treatment of HCC because it is predominantly expressed in HCC tumors compared with its adjacent nontumor or cirrhotic tissues [5], implying specificity. In addition, it potentially regulates multiple pathways involved in hepatocarginogensis [20,22–24], implying a broad spectrum of activity. Several recent studies have investigated different approaches of targeting GPC3, such as the use of anti-GPC3 monoclonal antibody [35] and HLA-A2- and -A24-restricted GPC3-derived peptide for the immunotherapy for HCC [36]. These approaches are based on the induction of antibody-dependent cellular cytotoxicity and/or complement-dependent cytotoxicity and on the peptide induction of cytotoxic T lymphocytes, respectively, which, in turn, reduced HCC tumor mass. A mutated, soluble form of GPC3 was also reported to inhibit Wnt signaling in HCC cells, leading to antitumor effects [37]. Our study demonstrates for the first time that targeting GPC3 at the translational level in GPC3-positive HCC cells activates TGF-β signaling, which, in turn, partially mediates the antitumor effects of GPC3 suppression in HCC cells in vitro and in vivo.
The TGF superfamily, including TGF-β, activin, and BMPs, modulates many cellular responses, such as cell division, differentiation, and cell fate decision [38]. Three different isoforms of TGF-β, TGF-β1, TGF-β2, and TGF-β3, have similar but not identical biologic activities [39]. TGF-β signaling is mediated through the binding of TGF-β with TGF-β receptors (TGFBR), which, in turn, recruit and phosphorylate downstream receptor-regulated SMADs (R-SMADs), SMAD2 or SMAD3. Activation of R-SMADs may form signaling complexes with SMAD4 and translocate to the nucleus eliciting tumor suppressive or oncogenic effects [40]. Whereas TGF-β/SMAD signaling can be both promoting and suppressing, its role in HCC progression is not completely understood [41]. Recently, TGF-β1 was reported to induce cellular senescence and inhibit tumor growth in HCC cell lines by a p53-independent and p21Cip1-dependent pathway [33].
Our results indicate that the antiproliferative effects of GPC3 in HCC cells are partially mediated by TGF-β signaling. Suppression of GPC3 in HCC cells overexpressing GPC3 inhibited cell proliferation associated with an increase in phosphorylation of SMAD2/3 and also arrested cell cycle progression at the G1 phase, associated with down-regulation of cyclin A and cyclin D1, accumulation of p15Ink4b and p21Cip1, and down-regulation of Rb phosphorylation. GPC3 suppression also caused an accumulation of SA-β-Gal, an indicator of replicative senescence. These cellular changes were recapitulated by the addition of rhTGF-β2 to the HCC cells in culture, confirming the involvement of TGF-β2 in cell cycle arrest and replicative senescence. Moreover, the cotransfection of siRNAs against GPC3 and TGF-β2 partially reversed these effects on cell proliferation, cell cycle progression, replicative senescence, and the associated molecular changes. We further observed an inverse correlation between the expression of GPC3 and TGF-β2, as GPC3 suppression upregulated TGF-β2 at both transcriptional and translational levels. Thus, GPC3 may inversely regulate TGF-β2, which, in turn, partially mediates the subsequent cellular and molecular changes observed in HCC cells on GPC3 suppression.
The two HCC cell lines studied, HepG2 and Huh7, responded to different extents toward GPC3 suppression, probably because of the different genetic contexts between the two cell lines. The poorer response of Huh7 cells may be explained by the following reasons. First, Huh7 cells have shorter cell doubling time (24 hours) than HepG2 cells (33 h) and have a lower percentage of cells in the G1 phase (56.39% ± 2.47%) than HepG2 cells (64.57% ± 2.84%) when cultured in Dulbecco modified Eagle medium supplemented with 10% FBS (P = .01). The shorter doubling time of Huh7 cells may allow the cells to escape faster from the effect of transient siRNA knockdown. Second, we observed that Wnt signaling was inactivated in HepG2 cells but not in Huh7 cells after GPC3 suppression (Figure W1, A–C). Cooperatively, the regulation of Wnt and TGF-β signaling pathways by GPC3 in HepG2 cells may lead to greater inhibitory effects on cell growth and cell cycle progression in this cell line. Third, in Huh7 cells, the ERK pathway might be the predominant pathway regulating proliferation that is affected by GPC3 suppression (Figure W1D), and therefore, we observed a negligible effect of TGF-β2 siRNA on reversing cell growth inhibition caused by GPC3 suppression. Fourth, Huh7 cells have endogenous TGF-β2 expression but HepG2 cells do not. The endogenous expression of TGF-β2 may mask the effect contributed by the low and transient increase in TGF-β2 levels caused by GPC3 suppression.
In summary, we have shown that suppression of GPC3 activates the TGF-β signaling pathway in HCC cells, eventually inhibiting cell proliferation, arresting cell cycle progression at the G1 phase, and inducing replicative senescence. The extent and biologic effects of GPC3 suppression vary, depending largely on the genetic context of the cells, which reflects the heterogeneity of HCC tumors. Combined with previous reports that interference with GPC3 expression additionally regulates at least two major signaling pathways (Wnt/β-catenin and ERK/MAPK) in HCC, we suggest that interfering with GPC3 expression and function exert broad-spectrum biologic effects that might be beneficial for the treatment of the heterogeneous subtypes of HCC. Targeting GPC3 in HCC is widely applicable because it is overexpressed in a large percentage (>60%) of HCC patients [4]. Further studies of the mechanisms by which GPC3 regulates multiple signaling pathways may reveal other points of intervention for this important target.
Supplementary Material
Supplementary Figures and Tables
Acknowledgments
The authors thank Xinrui Yan, Department of Radiology, Stanford University, for labeling the Huh7 and HepG2 cells with trifusion reporter lentivirus.
Abbreviations
BMPbone morphogenic protein
GPC3glypican 3
GPIglycosylphosphatidylinositol
HCChepatocellular carcinoma
SA-β-Galsenescence-associated-β-galactosidase
SMDStanford Microarray Database
TGF-β2transforming growth factor, beta 2

Footnotes
1This work is supported by grants to the Asian Liver Center at Stanford University from the H. M. Lui Foundation, the C. J. Huang Foundation, and the T. S. Kwok Liver Research Foundation. The authors declare no conflict of interest.
2This article refers to a supplementary material, which is designated by Figure W1 and is available online at www.neoplasia.com.
1. Parkin DM. Global cancer statistics in the year 2000. Lancet Oncol. 2001;2:533–543. [PubMed]
2. Hayashi PH, Di Bisceglie AM. The progression of hepatitis B- and C-infections to chronic liver disease and hepatocellular carcinoma: presentation, diagnosis, screening, prevention, and treatment of hepatocellular carcinoma. Infect Dis Clin North Am. 2006;20:1–25. [PubMed]
3. Poon RT, Fan ST, Lo CM, Ng IO, Liu CL, Lam CM, Wong J. Improving survival results after resection of hepatocellular carcinoma: a prospective study of 377 patients over 10 years. Ann Surg. 2001;234:63–70. [PubMed]
4. Baumhoer D, Tornillo L, Stadlmann S, Roncalli M, Diamantis EK, Terracciano LM. Glypican 3 expression in human nonneoplastic, preneoplastic, and neoplastic tissues: a tissue microarray analysis of 4,387 tissue samples. Am J Clin Pathol. 2008;129:899–906. [PubMed]
5. Libbrecht L, Severi T, Cassiman D, Vander Borght S, Pirenne J, Nevens F, Verslype C, van Pelt J, Roskams T. Glypican-3 expression distinguishes small hepatocellular carcinomas from cirrhosis, dysplastic nodules, and focal nodular hyperplasia-like nodules. Am J Surg Pathol. 2006;30:1405–1411. [PubMed]
6. Ligato S, Mandich D, Cartun RW. Utility of glypican-3 in differentiating hepatocellular carcinoma from other primary and metastatic lesions in FNA of the liver: an immunocytochemical study. Mod Pathol. 2008;21:626–631. [PubMed]
7. Man XB, Tang L, Zhang BH, Li SJ, Qiu XH, Wu MC, Wang HY. Upregulation of glypican-3 expression in hepatocellular carcinoma but downregulation in cholangiocarcinoma indicates its differential diagnosis value in primary liver cancers. Liver Int. 2005;25:962–966. [PubMed]
8. Wang XY, Degos F, Dubois S, Tessiore S, Allegretta M, Guttmann RD, Jothy S, Belghiti J, Bedossa P, Paradis V. Glypican-3 expression in hepatocellular tumors: diagnostic value for preneoplastic lesions and hepatocellular carcinomas. Hum Pathol. 2006;37:1435–1441. [PubMed]
9. Zhu ZW, Friess H, Wang L, Abou-Shady M, Zimmermann A, Lander AD, Korc M, Kleeff J, Buchler MW. Enhanced glypican-3 expression differentiates the majority of hepatocellular carcinomas from benign hepatic disorders. Gut. 2001;48:558–564. [PMC free article] [PubMed]
10. Capurro M, Wanless IR, Sherman M, Deboer G, Shi W, Miyoshi E, Filmus J. Glypican-3: a novel serum and histochemical marker for hepatocellular carcinoma. Gastroenterology. 2003;125:89–97. [PubMed]
11. Filmus J, Capurro M. Glypican-3 and alphafetoprotein as diagnostic tests for hepatocellular carcinoma. Mol Diagn. 2004;8:207–212. [PubMed]
12. Hippo Y, Watanabe K, Watanabe A, Midorikawa Y, Yamamoto S, Ihara S, Tokita S, Iwanari H, Ito Y, Nakano K, et al. Identification of soluble NH2-terminal fragment of glypican-3 as a serological marker for early-stage hepatocellular carcinoma. Cancer Res. 2004;64:2418–2423. [PubMed]
13. Capurro MI, Xiang YY, Lobe C, Filmus J. Glypican-3 promotes the growth of hepatocellular carcinoma by stimulating canonical wnt signaling. Cancer Res. 2005;65:6245–6254. [PubMed]
14. Paul S, Dey A. Wnt signaling and cancer development: therapeutic implication. Neoplasma. 2008;55:165–176. [PubMed]
15. Takigawa Y, Brown AM. Wnt signaling in liver cancer. Curr Drug Targets. 2008;9:1013–1024. [PubMed]
16. Pilia G, Hughes-Benzie RM, MacKenzie A, Baybayan P, Chen EY, Huber R, Neri G, Cao A, Forabosco A, Schlessinger D. Mutations in GPC3, a glypican gene, cause the Simpson-Golabi-Behmel overgrowth syndrome. Nat Genet. 1996;12:241–247. [PubMed]
17. Filmus J, Shi W, Wong ZM, Wong MJ. Identification of a new membrane-bound heparan sulphate proteoglycan. Biochem J. 1995;311(pt 2):561–565. [PubMed]
18. Cano-Gauci DF, Song HH, Yang H, McKerlie C, Choo B, Shi W, Pullano R, Piscione TD, Grisaru S, Soon S, et al. Glypican-3-deficient mice exhibit developmental overgrowth and some of the abnormalities typical of Simpson-Golabi-Behmel syndrome. J Cell Biol. 1999;146:255–264. [PMC free article] [PubMed]
19. Filmus J. Glypicans in growth control and cancer. Glycobiology. 2001;11:19R–23R. [PubMed]
20. Filmus J, Capurro M. The role of glypican-3 in the regulation of body size and cancer. Cell Cycle. 2008;7:2787–2790. [PubMed]
21. Gonzalez AD, Kaya M, Shi W, Song H, Testa JR, Penn LZ, Filmus J. OCI-5/GPC3, a glypican encoded by a gene that is mutated in the Simpson-Golabi-Behmel overgrowth syndrome, induces apoptosis in a cell line-specific manner. J Cell Biol. 1998;141:1407–1414. [PMC free article] [PubMed]
22. Capurro MI, Xu P, Shi W, Li F, Jia A, Filmus J. Glypican-3 inhibits hedgehog signaling during development by competing with patched for hedgehog binding. Dev Cell. 2008;14:700–711. [PubMed]
23. Midorikawa Y, Ishikawa S, Iwanari H, Imamura T, Sakamoto H, Miyazono K, Kodama T, Makuuchi M, Aburatani H. Glypican-3, overexpressed in hepatocellular carcinoma, modulates FGF2 and BMP-7 signaling. Int J Cancer. 2003;103:455–465. [PubMed]
24. Song HH, Shi W, Xiang YY, Filmus J. The loss of glypican-3 induces alterations in wnt signaling. J Biol Chem. 2005;280:2116–2125. [PubMed]
25. Filmus J, Capurro M, Rast J. Glypicans. Genome Biol. 2008;9:224. [PMC free article] [PubMed]
26. Yun MS, Kim SE, Jeon SH, Lee JS, Choi KY. Both ERK and Wnt/β-catenin pathways are involved in Wnt3a-induced proliferation. J Cell Sci. 2005;118:313–322. [PubMed]
27. Aravalli RN, Steer CJ, Cressman EN. Molecular mechanisms of hepatocellular carcinoma. Hepatology. 2008;48:2047–2063. [PubMed]
28. Cheng W, Tseng CJ, Lin TT, Cheng I, Pan HW, Hsu HC, Lee YM. Glypican-3-mediated oncogenesis involves the insulin-like growth factor-signaling pathway. Carcinogenesis. 2008;29:1319–1326. [PubMed]
29. Lai JP, Sandhu DS, Yu C, Han T, Moser CD, Jackson KK, Guerrero RB, Aderca I, Isomoto H, Garrity-Park MM, et al. Sulfatase 2 up-regulates glypican 3, promotes fibroblast growth factor signaling, and decreases survival in hepatocellular carcinoma. Hepatology. 2008;47:1211–1222. [PMC free article] [PubMed]
30. Akutsu N, Yamamoto H, Sasaki S, Taniguchi H, Arimura Y, Imai K, Shinomura Y. Association of glypican-3 expression with growth signaling molecules in hepatocellular carcinoma. World J Gastroenterol. 2010;16:3521–3528. [PMC free article] [PubMed]
31. Ray P, De A, Min JJ, Tsien RY, Gambhir SS. Imaging tri-fusion multimodality reporter gene expression in living subjects. Cancer Res. 2004;64:1323–1330. [PubMed]
32. Sun CK, Man K, Ng KT, Ho JW, Lim ZX, Cheng Q, Lo CM, Poon RT, Fan ST. Proline-rich tyrosine kinase 2 (Pyk2) promotes proliferation and invasiveness of hepatocellular carcinoma cells through c-Src/ERK activation. Carcinogenesis. 2008;29:2096–2105. [PubMed]
33. Senturk S, Mumcuoglu M, Gursoy-Yuzugullu O, Cingoz B, Akcali KC, Ozturk M. Transforming growth factor-β induces senescence in hepatocellular carcinoma cells and inhibits tumor growth. Hepatology. 2010;52:966–974. [PubMed]
34. Patil MA, Chua MS, Pan KH, Lin R, Lih CJ, Cheung ST, Ho C, Li R, Fan ST, Cohen SN, et al. An integrated data analysis approach to characterize genes highly expressed in hepatocellular carcinoma. Oncogene. 2005;24:3737–3747. [PubMed]
35. Ishiguro T, Sugimoto M, Kinoshita Y, Miyazaki Y, Nakano K, Tsunoda H, Sugo I, Ohizumi I, Aburatani H, Hamakubo T, et al. Anti-glypican 3 antibody as a potential antitumor agent for human liver cancer. Cancer Res. 2008;68:9832–9838. [PubMed]
36. Komori H, Nakatsura T, Senju S, Yoshitake Y, Motomura Y, Ikuta Y, Fukuma D, Yokomine K, Harao M, Beppu T, et al. Identification of HLA-A2- or HLA-A24-restricted CTL epitopes possibly useful for glypican-3-specific immunotherapy of hepatocellular carcinoma. Clin Cancer Res. 2006;12:2689–2697. [PubMed]
37. Zittermann SI, Capurro MI, Shi W, Filmus J. Soluble glypican 3 inhibits the growth of hepatocellular carcinoma in vitro and in vivo. Int J Cancer. 2010;126:1291–1301. [PubMed]
38. Ikushima H, Miyazono K. TGFβ signalling: a complex web in cancer progression. Nat Rev Cancer. 2010;10:415–424. [PubMed]
39. Matsuzaki K, Date M, Furukawa F, Tahashi Y, Matsushita M, Sugano Y, Yamashiki N, Nakagawa T, Seki T, Nishizawa M, et al. Regulatory mechanisms for transforming growth factor beta as an autocrine inhibitor in human hepatocellular carcinoma: implications for roles of SMADs in its growth. Hepatology. 2000;32:218–227. [PubMed]
40. Feng XH, Derynck R. Specificity and versatility in TGF-β signaling through SMADs. Annu Rev Cell Dev Biol. 2005;21:659–693. [PubMed]
41. Giannelli G, Mazzocca A, Fransvea E, Lahn M, Antonaci S. Inhibiting TGF-β signaling in hepatocellular carcinoma. Biochim Biophys Acta. 2011;1815:214–223. [PubMed]
Articles from Neoplasia (New York, N.Y.) are provided here courtesy of
Neoplasia Press