SFRP1 suppressed hepatoma cells growth through Wnt canonical signaling pathway

Authors

  • Yu-Lueng Shih,

    1. Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei, Taiwan
    2. Division of Gastroenterology, Department of Internal Medicine, National Defense Medical Center, Tri-Service General Hospital, Taipei, Taiwan
    3. Laboratory of Epigenetics, National Defense Medical Center, Taipei, Taiwan
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  • Chung-Bao Hsieh,

    1. Laboratory of Epigenetics, National Defense Medical Center, Taipei, Taiwan
    2. Division of General Surgery, Department of Surgery, National Defense Medical Center, Tri-Service General Hospital, Taipei, Taiwan
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  • Hung-Cheng Lai,

    1. Laboratory of Epigenetics, National Defense Medical Center, Taipei, Taiwan
    2. Department of Obstetrics and Gynecology, National Defense Medical Center, Tri-Service General Hospital, Taipei, Taiwan
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  • Ming-De Yan,

    1. Institute of Cancer Research, National Health Research Institutes, Taipei, Taiwan
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  • Tsai-Yuan Hsieh,

    1. Division of Gastroenterology, Department of Internal Medicine, National Defense Medical Center, Tri-Service General Hospital, Taipei, Taiwan
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  • You-Chen Chao,

    1. Division of Gastroenterology, Department of Internal Medicine, National Defense Medical Center, Tri-Service General Hospital, Taipei, Taiwan
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  • Ya-Wen Lin

    Corresponding author
    1. Laboratory of Epigenetics, National Defense Medical Center, Taipei, Taiwan
    2. Department of Microbiology and Immunology, National Defense Medical Center, Taipei, Taiwan
    • Department of Microbiology and Immunology, National Defense Medical Center, No.161, Section 6, Min-Chuan East Road, Taipei 114, Taiwan
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    • Fax: +886-2-87917654.


Abstract

Oncogenic activation of the Wnt/β-catenin signaling pathway is common in hepatocellular carcinoma (HCC). The secreted frizzled-related proteins (SFRPs) function as negative regulators of Wnt signaling and have important implications for carcinogenesis. Promoter hypermethylation of SFRP genes is common in human cancers. However, the role of SFRPs in HCC is not clear. Recently, we have shown that SFRP1 is frequently downregulated through promoter hypermethylation. To confirm and extend these findings, the methylation status of the other SFRP members, including SFRP2, SFRP4 and SFRP5, was examined by methylation-specific polymerase chain reaction (MS-PCR). Hypermethylation of SFRP genes, except for SFRP4, is frequent in HCCs and the levels found here were significantly higher than those seen in cirrhotic livers, chronic hepatitis livers and normal controls (p < 0.0001 for SFRP1 and SFRP2, p < 0.05 for SFRP5). To investigate the role of SFRP1 in HCCs, we used re-expression of SFRP1 in β-catenin-dependent HCC cell lines: Huh6 and HepG2. Restoration of SFRP1 attenuated Wnt signaling in those Huh6 hepatoma cells with a β-catenin gene point mutation, decreased abnormal accumulation of β-catenin in the nucleus and suppressed cell growth. Conversely, restoration of SFRP1 in HepG2 hepatoma cells with truncated β-catenin could not block the Wnt signaling pathway. Furthermore, knocking down SFRP1 by RNA interference in β-catenin-deficient cell lines (SK-Hep1) stimulated Wnt signaling and promoted cell growth. Our data suggested that SFRP1 suppressed liver cancer cells growth through Wnt canonical signaling. Moreover, β-catenin-independent noncanonical pathway might be involved in Wnt signaling activation through unknown molecules in HCC. © 2007 Wiley-Liss, Inc.

Hepatocellular carcinoma (HCC) is one of the most common malignant human tumors worldwide.1, 2, 3, 4 The major factors associated with HCC include chronic hepatitis B and C viral infections, chronic alcohol consumption, aflatoxin-B1-contaminated food and virtually all cirrhosis-inducing conditions.4 Other etiological factors have also been proposed to lead to HCC. In addition, gender can also influence the risk and behavior of HCC, with men accounting for more cases.5 The molecular mechanisms of hepatocarcinogenesis and the complex interactions of genetic factors remain to be elucidated.

Wnt glycoproteins comprise a family of extracellular signaling ligands that play essential roles in proliferation, patterning and fate determination during normal developmental processes.6, 7, 8, 9, 10, 11, 12 Although this signaling is critical for normal embryonic development, the aberrant of activation of the Wnt signal transduction pathway has been closely linked to tumorigenesis in adults.13, 14, 15 β-Catenin is a crucial downstream component of the Wnt signaling pathway. When Wnt signaling is engaged, the adenomatosis polyposis coli (APC) and Axin proteins no longer bind β-catenin, with consequent β-catenin stabilization and translocation to the nucleus, where it associates with the T-cell factor (TCF) family of transcription factors. This transcription factor complex transactivates a host of target genes governing cancer-relevant processes, including MYC and cyclin D1.16 β-Catenin mutations and increased nuclear expression have been detected in human HCCs.17, 18 In some reports, β-catenin overexpression and mutations have been related to early-stage HCCs19, 20 and in others to cancer progression.21 These data suggest the participation of a WNT/β-catenin pathway in HCC progression, but the involvement of specific components in this pathway remains unclear. A few reports have linked a β-catenin-independent pathway to Wnt- dependentgrowth and to the inhibition of apoptosis.22, 23

Abbreviations:

CpG, phosphodiester-linked cytosine-guanine pair; HCC, hepatocellular carcinoma; MS-PCR, methylation-specific polymerase chain reaction; RT-PCR, reverse transcription-polymerase chain reaction; SFRP1, secreted frizzled-related gene 1; shRNA, short hairpin RNA; TCF, T-cell factor; TSG, tumor suppressor gene.

Aberrant hypermethylation of phosphodiester-linked cytosine–guanine pair (CpG) islands, which are CpG dinucleotide-rich areas located mainly in the promoter regions of many genes, serves as an alternative mechanism for inactivation of the tumor suppressor gene (TSG) in cancers.24, 25, 26, 27, 28, 29 Such hypermethylation of gene promoters has been increasingly implicated as an early event in hepatocellular carcinogenesis.30, 31, 32, 33 Secreted frizzled-related proteins (SFRPs), a family of 5 secreted glycoproteins, are extracellular signaling molecules that antagonize the Wnt signaling pathway.34SFRP genes are inactivated by promoter methylation in different human cancers and serve as epigenetic tumor biomarkers.35, 36, 37, 38 We have demonstrated that SFRP1 is a candidate TSG that is silenced in hepatocarcinogenesis through promoter hypermethylation.39 To confirm and extend these findings with respect to SFRPs, the methylation status of other SFRP members, including SFRP2, SFRP4 and SFRP5, was also examined by MS-PCR in our study. To further understand the role of SFRP1 in hepatocarcinogenesis, we tested whether SFRP1 could attenuate Wnt signaling in β-catenin-dependent hepatoma cells by restoration of gene action. Furthermore, we knocked down SFRP1 expression in β-catenin-deficient cell lines to test for any effects on cell growth.

Material and methods

Specimens

Tissue samples were obtained from surgical specimens with the informed consent of patients at the Tri-Service General Hospital. Fifty-four primary HCC samples and adjacent non-tumor tissues were collected during surgery, and were frozen immediately in liquid nitrogen and stored at −80°C until DNA/RNA extraction. The diagnosis of HCC was confirmed by histology. Experienced pathologist classified the non-tumor tissues as normal controls (3 cases), chronic hepatitis livers (11 cases) and cirrhosis livers (40 cases). Additionally, we collected liver biopsies from patients without HCC as normal controls. Two patients with liver cirrhosis were HBV-positive, 10 cases were chronic hepatitis B carriers and 12 cases were normal livers. The clinicopathological characteristics of patients and tumors have been summarized previously.39

Cell lines

We cultured the human HCC cell lines (HepG2, Hep3B, Huh7, SK-Hep1, PLC/PRF/5, Mahlavu, J5, J7, HA22T and Huh6) and a transformed cell line (Chang liver) in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (w/v) fetal bovine serum, penicillin at 100 U/ml, streptomycin at 100 μg/ml and L-glutamine at 2 mmol/l (all from Invitrogen, Carlsbad, CA) at 37°C in an atmosphere of 5% (v/v) CO2 in air.

DNA extraction

Genomic DNA was extracted from cell lines and tissue samples, using QIAmp Tissue Kits (Qiagen, Hilden, Germany). DNA was isolated according to previous protocol.39

Bisulfite treatment and MS-PCR

Genomic DNA isolated from cells and tissue was subjected to bisulfite methylation analysis, using an EZ DNA methylation kit (Zymo Research, Orange, CA), according to the manufacturer's protocol. Briefly, 1 μg of genomic DNA was denatured by incubation with 0.2 M NaOH. Aliquots of 10 mM hydroquinone and 3 M sodium bisulfite (pH 5.0) were added, and the solution was incubated at 50°C for 16 h. Treated DNA was purified on a Zymo-Spin I column, desulfonated with 0.3 M NaOH, repurified on a Zymo-Spin I column and resuspended in 20 μl elution buffer. MS-PCR40 was carried out in a volume of 25 μl containing 1 μl of the sodium-bisulfite-treated DNA with Gold Taq DNA polymerase (PE Applied Biosystems, Foster City, CA). After heating at 92°C for 10 min, PCR was performed in a thermal cycler (GeneAmp 2400, PE Applied Biosystems) for 35 cycles, each of which consisted of denaturation at 92°C for 30 sec, annealing at 60°C for 30 sec and extension at 72°C for 30 sec, followed by a final 10 min extension at 72°C. Normal DNA from human peripheral blood was bisulphite modified to serve as a control for the unmethylated promoter sequence. Normal human DNA was treated in vitro with SssI methyltransferase (New England Biolabs, Beverly, MA) to generate a positive control for methylated alleles.41 The PCR products were analyzed by electrophoresis on 3% agarose gels. Each experiment was repeated 3 times. Primer sequences42 are given in the supplementary table.

RT-PCR analysis

We isolated total RNA from each samples, using Qiagen RNeasy kits (Qiagen, Valencia, CA). An additional DNase I digestion procedure was included in the isolation of RNA to remove contaminating DNA, following the manufacturer's protocol. One microgram of total RNA from each sample was subject to cDNA synthesis, using Superscript III reverse transcriptase and random hexamers (Invitrogen). Then, cDNA was amplified by PCR with primers specific for SFRP1 and the glyceraldehyde-3-phosphate dehydrogenase gene (GPADH), using PCR Master Mix reagent kits (Applied Biosystems). After heating at 92°C for 10 min, PCR was performed in a thermal cycler (GeneAmp 2400, PE Applied Biosystems) for 32 cycles, each of which consisted of denaturation at 92°C for 30 sec, annealing at 55°C for 30 sec and extension at 72°C for 30 sec, followed by a final 10 min extension at 72°C. The PCR products were analyzed by electrophoresis on 2% agarose gels. RT-PCR primers are available in Supplemental Table I.

Table I. Epigenetic Silencing of SFRP Genes and β-Catenin Status in Human HCC Cell Lines
Cell nameSFRP1SFRP2SFRP4SFRP5Cytosolic β-catenin expression3
Methylation1Expression2Methylation1Expression2Methylation1Expression2Methylation1Expression2
  • M, presence of a methylation-specific PCR band; U, presence of an unmethylation-specific PCR band; +, presence of RT–PCR band; −, absence of an RT-PCR band; ND, not determine.

  • 1

    Methylation status was determined by methylation-specific PCR.

  • 2

    mRNA expression levels for the SFRP genes were determined by RT-PCR.

  • 3

    β-catenin protein expression was determined by western blotting.

HepG2MMU+M/U+
Hep3BMMU+M/U+
Huh6MMM/U+M/U++
SK-Hep1U+MU+M/U
PLC/PRF/5U+U+U+M/UND
MahlavuMM/U+U+M/U+
J5MMM/UM+
J7MMM/U+M+
Chang liverMM/UM/U+M/U
HA22TMMU+U++
Huh7M/U+MU+M+

Quantitative RT-PCR

Quantitative RT-PCR analysis was performed on an ABI PRISM 7700 Sequence Detector (Applied Biosystems). Primers and TaqMan probes were obtained from Applied Biosystems. The hypoxanthine ribosyltransferase gene (HPRT) was used as an internal control. Cycling was carried out using TaqMan PCR Master Mix reagent kits as earlier. Relative gene expression was determined based on the threshold cycles (Ct) of the gene of interest and of the internal reference gene. Messenger RNA levels were expressed as the ratio of that for the gene of interest to HPRT mRNA for each sample. The level of each mRNA in each set of short hairpin (sh) RNA-transfected cells was compared to the level in the vector control. The fold change in expression of the target gene relative to HPRT of each analyzed SFRP1 shRNA transfected samples was calculated. RT-PCR primers are available in Supplemental Table I.

Western blotting

A standard protocol was used. Anti-SFRP1 polyclonal antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-β-catenin and anti-lamin A/C antibodies were purchased from BD Transduction laboratory (BD Biosciences, Canada). Anti-β-actin antibody was purchased from Abcam (Cambridge, MA). For detecting alteration of β-catenin levels, cytosolic and nuclear extracts were prepared and examined as described.22, 23

Transfections

Cells were seeded in 6-well plates and were allowed to reach 80% confluence. The cells were transfected with pcDNA-SFRP1 or empty vector controls (kindly provided by Dr. Hiromu Suzuki), using LipofectAMINE 2000 (Invitrogen). After 6 hr of transfection, the medium was replaced with fresh serum-containing medium. For stable transfectants, medium was replaced after 48 hr with G418-containing medium and transfectants were selected.

Colony formation assay

For selection and colony formation assay, we transfected cells with pcDNA-SFRP1 or empty vector controls as earlier, stripped and plated them on 100-mm tissue culture dishes 48 hr after transfection and selected them for 14–21 days with G418.

RNA interference

Short hairpin RNA (shRNA) sequences were cloned downstream of the human H1 promoter in the vector pSuper as described.43 The target site for these was 5′-GACGGAGAGT TATCCTGAT-3′. Cell lines (SK-Hep1) that expressed SFRP1 were transfected with different shRNA constructs to evaluate any effects on colony formation.

MTS proliferation assay

MTS assays were performed with the Cell Titer 96 AQ One Solution Cell Proliferation Assay (Promega, Madison, WI).44 MTS reagent (20 μl/well) was added to 100 μl of medium containing cells in each well of 96-well plates and left for 1 hr at 37°C under humidified 5% CO2 in air. For colorimetric analysis, the absorbance at 490 nm was recorded using a microplate reader. Each condition was repeated at least 2 times. Total cells were harvested at the designated times after treatment.

Reporter assay

We plated 1 × 104 cells per well on 96-well tissue culture plates 24 hr before transfection. We transfected cells in each well with 50 ng of TOPflash (Millipore/Upstate), a luciferase reporter plasmid that contains wild type TCF binding sites, or FOPflash (Millipore/Upstate), containing mutated TCF binding sites as a negative control; pcDNA-SFRP1 with or without an empty vector to a total amount of 20 ng; and 2 ng of phRL-TK (Promega) as internal control using FuGENE6 (Roche Molecular Biochemicals). After 48 hr, we measured luciferase activities in a luminometer (BD Bioscience) and normalized the data for background Renilla luciferase activity, using Dual-Luciferase Reporter Assay Systems (Promega) according to the manufacturer's instructions.

Statistical analysis

The SPSS program (version 13) for Windows (SPSS, Chicago, IL) was used for statistical analyses. The associations between methylation of SFRPs and the liver disease status (control, chronic hepatitis, cirrhosis liver and HCC) were analyzed by using chi-square test. For SFRP1 re-expression and shRNA knockdown analysis, data are shown as means ± SDs. Student's t-test was used for comparing activities of different constructs. The significance level was defined as p < 0.05.

Results

Epigenetic silencing of SFRP genes in HCC cell lines and primary HCCs

We demonstrated previously that the SFRP1 promoter is frequently methylated and has a significant association with SFRP1 downregulation in human HCC cell lines and HCC tissues. To confirm and extend these findings with respect to SFRPs, the methylation status of the other SFRP members, including SFRP2, SFRP4 and SFRP5, was examined by MS-PCR. The frequency of SFRP gene methylation in HCC cell lines with promoter hypermethylation was 82% (9/11), 91% (10/11), 27% (3/11) and 91% (10/11) for SFRP1, SFRP2, SFRP4 and SFRP5, respectively (Table I). Representative cases were confirmed by bisulfite sequencing as reported previously (data not shown). According to the RT-PCR results (Table I), we also found strong correlations between promoter hypermethylation and gene silencing. To confirm that the lack of expression of the SFRP gene family in the hepatoma cell lines was caused by promoter hypermethylation, we treated cells with 5-aza-2′-deoxycytidine, an inhibitor of DNA methylation. After treatment, the SFRP transcripts were re-expressed (Supplemental data S1). Taken together, our data suggest that SFRP genes, except for SFRP4, are frequently hypermethylated in HCC cell lines and that this hypermethylation might be responsible for the absence of transcription of SFRPs.

Next, we investigated the frequency of SFRP gene promoter methylation in 54 primary HCCs and control tissues, using MS-PCR. Hypermethylation of SFRP1, SFRP2, SFRP4 and SFRP5 was detected in 48% (26/54), 54% (29/54), 6% (3/54) and 39% (21/54) of 54 HCC samples; 21% (9/42), 21% (9/42), 0% (0/42) and 17% (7/42) of cirrhosis samples; 14% (3/21), 27% (4/15), 0%(0/21) and 14% (3/21) of chronic hepatitis samples and 0% (0/15), 7% (1/15), 0% (0/15) and 7% (1/15) of normal control samples, respectively (Fig. 1). Thus, hypermethylation of SFRP genes, except for SFRP4, is frequent in primary HCCs and the levels found here were significantly higher than those seen in cirrhotic livers, chronic hepatitis livers and normal controls (p < 0.0001 for SFRP1 and SFRP2, p < 0.05 for SFRP5).

Figure 1.

Frequency of SFRP gene promoter methylation in liver tumors and control tissues. Methylation levels in HCC and control samples were detected using MS-PCR amplification. Methylation frequency is the number of positive samples out of total number of samples (percentage). There are only 15 chronic hepatitis samples analyzed for SFRP2 methylation status because of limited amount of tissues.

Identification of human HCC cell lines with constitutive Wnt pathway activation

Because SFRP proteins suppress cell growth in human colorectal cancer36 and lung cancer45 through inhibition of the Wnt/β-catenin canonical pathway, we hypothesized that they might also inhibit Wnt transcriptional activity in HCC cells. To begin exploring the role of epigenetic silencing of SFRP genes, we first looked for the levels of β-catenin and Wnt proteins in HCC cells. Oncogenic Wnt signaling activation led to frequent β-catenin accumulation in the cytoplasm of HCC cells (Table I), consistent with previous reports.46 Among the 4 HCC cell lines tested, we found that SFRP1 was hypermethylated in HepG2, Hep3B and HuH6, but not in SK-HepI (Fig. 2a). The analysis of mRNA levels in hepatoma cell lines showed clear disparities in the expression levels of methylated (HepG2, Hep3B and HuH6) and unmethylated SFRP1 (SK-HepI) (Fig. 2b). There was a large deletion in the β-catenin coding sequence (amino acids 25–140) and a point mutation (G34V) was observed in the HepG2 and Huh6 hepatoma cell lines, respectively. These mutations led to β-catenin accumulation in the cytoplasm (Fig. 2c).46 In addition to mutations of components of the Wnt signaling pathway, we found Wnt ligands (Wnt1, Wnt3 or Wnt5a) expressed in HCC cell lines containing upregulated β-catenin (Supplemental data S2). These data suggest that autocrine Wnt signaling might be involved in human hepatocarcinogenesis, except for cells carrying mutations of Wnt signaling components.

Figure 2.

Epigenetic silencing of SFRP1 in hepatocellular carcinoma cell lines. (a) Methylation in HCC cell lines was detected using methylation-specific polymerase chain reaction (MS-PCR) amplification. Peripheral blood lymphocyte (PBL) extract was substituted for DNA as a negative control and PBL DNA treated with SssI (CpG) methylase (New England Biolabs, Beverly, MA) was used as a positive control. M, methylated; U, unmethylated. (b) Expression of the SFRP1 transcript was analyzed by reverse transcription-PCR. Expression of the glyceraldehyde-3-phosphate dehydrogenase gene was determined as a control for RNA integrity. (c) Western blots analysis. Cytosolic proteins were prepared and used in western blotting; β-actin served as loading control. Truncated and full-length β-catenin proteins were identified in HepG2 and Hep3B cells, respectively. The G34V β-catenin mutation identified in Huh6 cells led accumulation of this protein. No β-catenin protein was detected in SK-Hep1 cells.

Restoration of SFRP1 inhibited TCF-dependent transcriptional activity and suppressed tumor growth in β-catenin-dependent cells

Next, we tested whether SFRP1 could downregulate Wnt signaling in HCC cell lines Huh6 and HepG2, which harbor mutations and deletions in the β-catenin gene, respectively. To confirm the restoration of SFRP1, we used RT-PCR to check whether SFRP1 was expressed in HCC cells (Fig. 3a). Then, we performed luciferase reporter assays on Huh6 and HepG2 cells. When cells were transfected with SFRP1, the relative TCF activity was suppressed significantly when compared to empty vector in Huh6 (p < 0.05), but not in HepG2 (Fig. 3b). Western blot analysis showed that Huh6 cells displayed a significantly decreased level of nuclear β-catenin after SFRP1 transfection, but there was no change in HepG2 cells (Fig. 3c). We tested the consequences for HCC cells of Wnt-pathway suppression by SFRP1. MYC, cyclin D1 and Survivin are well-known target genes of canonical Wnt signaling. We found that stable overexpression of SFRP1 in Huh6 cells resulted in downregulation of MYC and cyclin D1 mRNA after 4 weeks of selection (Fig. 4a). In contrast, HepG2 cells exhibited no difference in MYC and cyclin D1 expression, which is concordant with the results of our reporter assay and nuclear β-catenin experiments (Figs. 3b, 3c and 4a). There was no difference between transfected and control cells in the expression of Survivin, an apoptosis-related gene. We next examined the effects of SFRP1 on the growth and apoptosis of HCC cells. There was decreased cell proliferation and colony formation in Huh6 cells stably overexpressing SFRP1, but not in HepG2 (Figs. 4b, 4c and Supplemental data S3). Although SFRP1 has tumor growth suppressive activity by induction of apoptosis in colorectal cancers, no clear difference in the numbers of apoptotic cells was observed between transfected and control cells, using propidium iodide staining (data not shown). Moreover, we also found that restoration of SFRP1 expression in Hep3B cells could inhibit TCF-dependent transcriptional activity, downregulate the expression of Wnt-target genes, including MYC and Cyclin D1, and suppress tumor growth (Supplemental data S4).

Figure 3.

Restoration of SFRP1 expression reduced β-catenin production and its TCF-dependent transcriptional activity. (a) Expression of SFRP1 mRNA in Huh6 and HepG2 cells after being transfected with empty vector or a SFRP1 construct. (b) Huh6 cells displayed a significantly decreased level of nuclear β-catenin after SFRP1 transfection, but there was no change in HepG2 cells. Lamin A/C was used as a loading control. (c) Reporter gene assays were performed on the Huh6 and HepG2 cells, using phRL-TK, TOP-FLASH (the wild-type TCF reporter), or FOP-FLASH (the mutant TCF reporter). Cells grown in 96-well tissue culture plates were transfected with 20 ng of the pcDNA-SFRP1 or vector only, 50 ng of TOP-FLASH or FOP-FLASH and the internal control plasmid phRL-TK 2 ng. At 48 hr after transfection, reporter gene activity was measured using a dual luciferase reporter assay system (Promega). The luciferase activity was normalized to Renilla luciferase activity. Results are presented as the mean ± SD. Experiments were performed in triplicate. Statistical comparisons were made with Student's t-tests and p < 0.05 was assumed as significant.

Figure 4.

Restoration of SFRP1 suppressed both the canonical Wnt pathway downstream target gene expressions and cell growth. (a) Semiquantitative RT-PCR was used to confirm changes in mRNA expression for canonical Wnt pathway downstream genes before and after exogenously expressing SFRP1. (b) Cells were transfected with the SFRP1 expression vector, pcDNA3.1-His-SFRP1. Cells were transfected with 1 μg/well of the expression vector or control vector, using FuGene-6 (Roche Molecular Biochemicals), and cultured in 700 μg/ml G418 (G7034; Sigma-Aldrich, St Louis, MO) for 4 weeks. Numbers of colonies were counted after staining with methylene blue. The bars in the graph show the means ± SDs. Experiments were performed in triplicate, and representative ones are shown.

Knockdown of SFRP1 by shRNA in β-catenin-independent cells promoted tumor growth

To further explore the function of SFRP1 in canonical and noncanonical Wnt signaling pathway during carcinogenesis, we used a shRNA approach to block SFRP1 expression in SK-HepI cells with undetectable β-catenin (Figs. 2b and 2c). Quantitative RT-PCR was used to confirm shRNA-directed down-regulation of the SFRP1 expression. The mRNA levels were reduced to 60% for SFRP1 (p < 0.05) in the shRNA-transfected cells compared to controls (Fig. 5a). However, β-catenin was still undetectable in SFRP1 shRNA-transfeced SK-HepI cells (Supplemental data S5). Interestingly, in SK-HepI cells transfected with SFRP1 shRNA, we found increased expression of Wnt target genes, including MYC and Cyclin D1 (Fig. 5b). Furthermore, after selection of G418-resistant colonies for 4 weeks, we found significantly more colony numbers of SFRP1 shRNA-transfected cells than empty vector-transfected cells (p < 0.05; Fig. 5c). Taken together, these data suggest that SFRP1 expression in SK-HepI cells might be critical for their growth and that β-catenin-independent mechanisms might be responsible for the growth suppression induced by blocking Wnt signaling.

Figure 5.

Inhibition of SFRP1 expression by RNA interference in SK-Hep1 cells. (a) Quantitative RT-PCR was used to confirm shRNA-directed downregulation of SFRP1 expression. (b) Semiquantitative RT-PCR analysis of mRNA expression levels in canonical Wnt pathway downstream genes before and after SFRP1 shRNA treatment. (c) Numbers of colonies in the cells transfected with the SFRP1 shRNA (white bars) increased significantly (p < 0.05) when compared to those in the vector controls (gray bars). The bars show the means ± SDs. Experiments were performed in triplicate, and representative ones are shown.

Discussion

The Wnt/β-catenin signal transduction pathway is important in carcinogenesis and embryogenesis.6, 7, 8, 9, 10, 11, 12, 13, 14, 34, 35, 39, 46, 47, 48, 49 Dysregulation of this pathway can be caused by mutations in any components (CTNNB1, AXIN or FZD7) in colon cancers, HCCs and other cancers.46, 49, 50 Suzuki et al.36, 42 demonstrated recently that epigenetic loss of SFRP gene family functions may contribute to the progression of colorectal cancer. Interestingly, they found that Wnt signal activation by mutant β-catenin or APC could be suppressed partially by overexpression of SFRPs. Accordingly, it may be possible to suppress the tumor phenotype in Wnt-activated cancer cells by inhibiting the Fzd receptor through competition with antagonists. In fact, Suzuki et al.36 demonstrated that colon cancer cells that overexpressed SFRPs had reduced colony formation and a higher rate of apoptosis.

Our previous studies have shown that SFRP1 is frequently hypermethylated and that its downregulation is associated with methylation-mediated gene silencing in HCC.39 In our study, we investigated the frequency of promoter methylation for SFRPs in 54 primary HCCs using MS-PCR. Hypermethylation of SFRP genes, except for SFRP4, was common in HCCs and was significantly higher than the levels seen in cirrhotic livers, chronic hepatitis livers and normal controls (p < 0.0001 for SFRP1 and SFRP2, p < 0.05 for SFRP5). In addition to hypermethylation of genes encoding Wnt antagonists (SFRPs) and mutations of components of the Wnt signaling pathway, we found Wnt ligands at high levels in HCC cell lines containing upregulated β-catenin and producing 2 or more Wnt ligands. These data suggest that activation of Wnt signaling might be involved in human hepatocarcinogenesis.

Here, we demonstrated that SFRP1 inhibits the canonical Wnt signaling in Huh6 cells with a β-catenin gene mutation (G34V), but not in HepG2 cells with a large β-catenin gene deletion. It is possible that the loss of amino acids 25–140 of β-catenin leads to failure of the protein to be phosphorylated by GSK3β, leading to accumulation in the nucleus and an unaltered HepG2 cell phenotype (Figs. 3 and 4). However, this mutation (G34V) led to the accumulation of β-catenin in both the cytoplasm and the nucleus in Huh6 cells. Therefore, SFRP1 may attenuate Wnt transcriptional activity by regulating GSK3β activity and thereby inhibit tumor cell growth (Figs. 3, 4 and S3). Previous studies have shown that SFRP proteins can sensitize cells to proapoptotic stimuli through β-catenin gene downregulation. However, we found no differences between transfected and control cells in the numbers of apoptotic cells (data not shown) or in the expression of Survivin (Fig. 4a) an apoptosis-related gene. This may suggest that SFRP1 has less effect on apoptosis in HCC cells than in colorectal cancer cells. Our data demonstrated that it might be possible to suppress the tumor phenotype in subgroups of HCC cells carrying some β-catenin gene mutation sites by inhibiting the Fzd receptor through competition with antagonists.

Involvement of β-catenin-independent mechanisms in Wnt-induced growth inhibition has been reported only rarely. Recently, He et al.23 showed that restoration of SFRP4 expression in β-catenin-dependent and β-catenin-deficient mesothelioma cell lines induced apoptosis, suppressed cell growth and downregulated Wnt signaling. They suggested that the SFRP4 protein might induce apoptosis both through the canonical pathway and through β-catenin-independent noncanonical pathways, such as those dependent on c-jun-NH2 kinase.23 Interestingly, we found that β-catenin-deficient SK-HepI cells treated with SFRP1 shRNA showed enhanced cell growth (Figs. 5b and 5c). This implies that SFRP1 might influence HCC cell growth through a β-catenin-independent noncanonical pathway. Therefore, our data also support the idea that SFRP family genes, such as SFRP1, might inhibit Wnt signaling through the canonical pathway as well as through β-catenin-independent noncanonical pathways in HCC. However, what kind of signaling molecules involved in β-catenin-independent noncanonical pathway needs further study. In summary, the function of SFRP1 expression as an antagonist of the Wnt pathway provides a potential mechanism to suppress the abnormal activation of this pathway.

Acknowledgements

We thank Dr. Hiromu Suzuki (First Department of Internal Medicine, Sapporo Medical University, Sapporo, Japan) for kindly providing the construct plasmid, and we thank Dr. Steven B. Baylin (Department of Medicine, The Johns Hopkins University School of Medicine) for kindly providing the primer sequences of MSP for the SFRP genes.

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