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. 2008 Jul;10(7):697–705. doi: 10.1593/neo.08320

Frizzled-7 as a Potential Therapeutic Target in Colorectal Cancer1

Koji Ueno *, Mikako Hiura *, Yutaka Suehiro *, Shoichi Hazama †, Hiroshi Hirata ‡, Masaaki Oka †, Kohzoh Imai §, Rajvir Dahiya ‡, Yuji Hinoda *
PMCID: PMC2435005  PMID: 18592008

Abstract

We investigated whether one of the Wnt receptors, frizzled-7 (FZD7), functions in the canonical Wnt signaling pathway of colorectal cancer (CRC) cells harboring an APC or CTNNB1 mutation and may be a potential therapeutic target for sporadic CRCs. The expression level of FZD gene family members in colon cancer cells and primary CRC tissues were determined by real-time PCR. Activation of the Wnt signaling pathway was evaluated by TOPflash assay. The expression level of Wnt target genes was determined by real-time polymerase chain reaction and/or Western blot analysis. Cell growth and cell invasion were assessed by MTS and matrigel assays, respectively. Among 10 FZD gene family members, FZD7 mRNA was predominantly expressed in six colon cancer cell lines with APC or CTNNB1 mutation. These six cell lines were transfected with FZD7 cDNA together with a TOPflash reporter plasmid, resulting in a 1.5- to 24.3-fold increase of Tcf transcriptional activity. The mRNA expression levels of seven known Wnt target genes were also increased by 1.5- to 3.4-fold after transfection of FZD7 cDNA into HCT-116 cells. The six cell lines were then cotransfected with FZD7-siRNA and a TOPflash reporter plasmid, which reduced Tcf transcriptional activity to 20% to 80%. FZD7-siRNA was shown to significantly decrease cell viability and in vitro invasion activity after transfection into HCT-116 cells. Our present data demonstrated that FZD7 activates the canonical Wnt pathway in colon cancer cells despite the presence of APC or CTNNB1 mutation and that FZD7-siRNA may be used as a therapeutic reagent for CRCs.

Introduction

The canonical Wnt signaling pathway that controls cell fate and proliferation is initiated by binding of Wnt ligands to the transmembrane receptors, frizzleds and low-density lipoprotein receptor-related proteins. The resultant signals prevent β-catenin phosphorylation by a multiprotein complex composed of adenomatous polyposis coli (APC), glycogen synthase kinase 3β, casin kinase 1, and axins, and its subsequent proteosomal degradation. β-Catenin associates with T-cell factor (TCF)/lymphocyte enhancer transcription factors to activate target genes that are involved in cell survival, proliferation, or invasion [1,2]. This signaling pathway is activated in most sporadic colorectal cancers (CRCs; up to 80%), which is mainly caused by mutations of APC [3,4]. Mutations of the β-catenin gene, CTNNB1, most of which preclude its phosphorylation, are also detected but only in a small proportion of cases (∼10%) [4]. APC mutations mostly bring about the deletion of the C-terminal half of the protein, leading to the failure to assemble a functional destruction complex, which ultimately results in the constitutive stabilization of β-catenin [3,4]. Until recently, little attention was paid to the role of Wnt ligands or receptors that function upstream of APC in CRCs.

We have demonstrated that secreted frizzled-related proteins, the Wnt antagonists that were frequently down-regulated (>90%) by promoter hypermethylation in CRCs, decreased colony formation and induced apoptosis when overexpressed in colon cancer HCT-116 or SW-480 cells with CTNNB1 or APC mutations, respectively, suggesting that Wnt ligands could affect the proliferation and survival of the CRC cells with these mutations [5]. Another Wnt antagonist, Wnt inhibitory factor 1 (WIF-1), was thereafter revealed to be epigenetically inactivated in CRCs, and its overexpression in SW-480 cells induced apoptosis [6]. In that report, cell death was also induced by the suppression of Wnt-1 with small interfering RNA (siRNA) or a monoclonal antibody in cell lines (SW-480 and HCT-116) or in primary cultured colon cancer cells. These findings suggest that Wnt ligands and receptors may be promising therapeutic targets for sporadic CRCs.

Frizzled-7 (FZD7) is 1 of 10 members of the frizzled gene family. Its expression is limited in normal tissues. The mRNA has been detected in fetal kidney and lung and in adult skeletal muscle, heart, brain, and placenta [7]. In contrast, FZD7 mRNA has been found in a wide variety of cancer cells including melanoma, lung cancer [7], esophageal cancer [8], gastric cancer [9], colon cancer [7,10,11], hepatocellular carcinoma [12], and lymphoblastic leukemia [13]. A recent study using in situ hybridization showed that FZD7 mRNA expression was confined to the epithelium of the crypt bottom of the colon [14], suggesting that it is expressed by immature colonocytes. Activation of the canonical Wnt pathway with FZD7 in cancer cells was suggested by previous studies using esophageal cancer KYSE150 cells [8], several kinds of hepatocellular carcinoma cells [12], and colon cancer LIM1863 cells [11]. However, to the best of our knowledge, there is no information about FZD7 in colon cancer cells harboring the APC or CTNNB1 mutation. In this study, we investigated whether FZD7 functions as a receptor for the canonical Wnt signaling pathway in the colon cancer cells and may be a potential therapeutic target for sporadic CRCs.

Materials and Methods

Cell Cultures

Human colon cancer cell lines SW-480, HCT-116, DLD-1, LoVo, and HT-29 were purchased from American Type Culture Collection (ATCC, Manassas, VA). Human colon cancer Caco-2 and human embryonic kidney 293T cells were purchased from RIKEN BRC (Tsukuba, Japan). 293T cells were cultured in DMEM (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 10% heat-inactivated fetal bovine serum (FBS; ATCC), 100 IU/ml penicillin, and 100 µg/ml streptomycin (Sigma, St. Louis, MO). SW-480 cells were cultured in Leibovitz's L-15 medium (Gibco/Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated FBS, 100 IU/ml penicillin, and 100 µg/ml streptomycin. HCT-116 and HT-29 cells were cultured in McCoy's 5A medium (Gibco) supplemented with 10% heat-inactivated FBS, 100 IU/ml penicillin, and 100 µg/ml streptomycin. DLD-1 cells were cultured in RPMI 1640 medium (Gibco) supplemented with 10% heat-inactivated FBS, 100 IU/ml penicillin, and 100 µg/ml streptomycin. Caco-2 cells were cultured in minimum essential medium (Eagle; Sigma) supplemented with 20% heat-inactivated FBS, 100 IU/ml penicillin, and 100 µg/ml streptomycin. LoVo cells were cultured in Ham's F-12K medium (SIGMA) supplemented with 10% heat-inactivated FBS, 100 IU/ml penicillin, and 100 µg/ml streptomycin.

Primary CRC Tissues

Primary CRC tissues (n = 13) were obtained from the patients who underwent surgical operation in our institution. Mean age was 68.2 years (range, 48–83 years), and male/female ratio was 3:10. According to pTNM classification, three patients were in stage II, four in stage III, and six in stage IV Written informed consent was obtained from those patients, and the experimental protocol was approved by the institutional ethics committee of the Yamaguchi University Graduate School of Medicine.

Plasmid Construction

Human frizzled-1 (FZD1), frizzled-6 (FZD6), and frizzled-7 (FZD7) were amplified from human testis total RNA (Clontech, Palo Alto, CA) by transcription-polymerase chain reaction (RT-PCR). The following primers were used: FZD1 forward primer, 5′-gccgagaaagtatggctgcgg-3′; FZD1 reverse primer, 5′-agcctgcgaaagagagttgtc-3′; FZD6 forward primer, 5′-gaattctaattgacccaggactcattttcag-3′; FZD6 reverse primer, 5′-ctcgagccagtgtaacacaaatttgcttctg-3′; FZD7 forward primer, 5′-gcctcgtcgcactcctcag-3′; FZD7 reverse primer, 5′-ggggctcataccgcagtctc-3′. Polymerase chain reaction products were cloned into pTargeT-Mammalian Expression Vector System (Promega, Madison, WI). pcDNA3.1-FZD1 and pcDNA3.1-FZD7 were obtained by subcloning a NheI-EcoRI fragment from pTargeT-FZD1 and pTargeT-FZ7 into the NheI-EcoRI site of pcDNA3.1 (Invitrogen). pcDNA3.1-FZD6 were obtained by subcloning a EcoRI- XhoI fragment from pTargeT-FZD6 into the EcoRI- XhoI site of pcDNA3.1. EcoRI- EcoRI fragments of pTargeT-FZD7 were subcloned into the EcoRI site of pEF4-V5 (Invitrogen). The following primers were used: FZD7 forward primer, 5′-gccatgggccaggtagac-3′; FZD7 reverse primer, 5′-ttcgaataccgcagtctcccccttgc-3′. The PCR products were cloned into pGEM-T Easy Vector (Promega). pEF-FZD7-V5 was obtained by subcloning a BlpI- BstBI fragment from pGEM-T-FZD7 into the BlpI- BstBI site of pEF4-FZD7.

Transfection

Cells growing in culture dishes were transfected at 50% to 70% confluence by FuGENE HD (Roche Diagnostics, Basel, Switzerland) according to the manufacturer's instructions.

Quantitative PCR

Total RNA was isolated from colon cancer cell lines using the RNeasy Mini Kit (Qiagen, Hilden, Germany). The total RNA from primary CRC tissues was isolated using TRIzol (Invitrogen). The extracted total RNA was reverse-transcribed into single-stranded cDNA using High Capacity cDNA Archive Kit (Applied Bio-systems, Warrington, UK). Real-time PCR was performed using first-strand cDNA with TaqMan Universal PCR Master Mix (Applied Biosystems). The assay numbers for the endogenous control (GAPDH) and target genes were as follows: 4326317 (GAPDH); Hs00268943_s1 (frizzled-1); Hs00361432_s1 (frizzled-2); Hs00184043_m1 (frizzled-3); Hs00201853_m1 (frizzled-4); Hs00361869_g1 (frizzled-5); Hs00171574_m1 (frizzled-6); Hs00275833_s1 (frizzled-7); Hs00259040_s1 (frizzled-8); Hs00268954_s1 (frizzled-9); and Hs00273077_s1 (frizzled-10). Quantitative PCR was performed on an ABI Prism 7900HT Sequence Detection System (Applied Biosystems). Quantitative PCR parameters for cycling were as follows: 50°C for a 2-minute hold, 95°C for 10 minutes of 40 cycles of PCR at 95°C for 15 seconds, and 60°C for 1 minute. All reactions were done in a 20-µl reaction volume in triplicate. The mRNA expression level was determined using the 2-Δ CT method.

For target gene assays and interferon assay, total RNA was isolated from colon cancer cell lines using the RNeasy Mini Kit (Qiagen) 48 hours after transfection. The extracted total RNA was reverse-transcribed into single-stranded cDNA using High Capacity cDNA Archive Kit (Applied Biosystems). Real-time PCR was performed using first-strand cDNA with Power SYBR Green PCR Master Mix (Applied Biosystems). The primers used were as follows: Myc forward primer, 5′-tcaagaggtgccacgtctcc-3′; Myc reverse primer, 5′-tcttggcagcaggatagtcctt-3′; Cyclin D1 forward primer, 5′-ccgtccatgcggaagatc-3′; Cyclin D1 reverse primer, 5′-atggccagcgggaagac-3′; CD44 forward primer, 5′-caggtttggtggaagatttgg-3′; CD44 reverse primer, 5′-tgtcagagtagaagttgttggatgg-3′; VEGF forward primer, 5′-gccttgccttgctgctctac-3′; VEGF reverse primer, 5′-atgattctgccctcctccttc-3′; Survivin forward primer, 5′-cggttgcgctttcctttc-3′; Survivin reverse primer, 5′-tgttcttggctctttctctgtcc-3′; Id-2 forward primer, 5′-gacccgatgagcctgctataca-3′; Id-2 reverse primer, 5′-ggtgctgcaggatttccatct-3′; Jun forward primer, 5′-ggaaacgaccttctatgacgatg-3′; Jun reverse primer, 5′-agggtcatgctctgtttcagg-3′; OAS1 forward primer, 5′-gtcttcctcagtcctctcaccac-3′; OAS1 reverse primer, 5′-gagcctggacctcaaacttcac-3′; GAPDH forward primer, 5′-ccatgttcgtcatgggtgtg-3′; GAPDH reverse primer, 5′-ggtgctaagcagttggtggtg-3′. Quantitative PCR was performed on an ABI Prism 7900HT Sequence Detection System (Applied Biosystems). Quantitative PCR parameters for cycling were as follows: 95°C for 10 minutes of 40 cycles of PCR at 95°C for 15 seconds, and 60°C for 1 minute. All reactions were done in a 20-µl reaction volume in triplicate. The mRNA expression level was determined using the 2 -Δ CT method.

Luciferase Reporter Assay

For Tcf luciferase assays, cells were transfected with 0.1 µg of TOPflash (Upstate, Lake Placid, NY), 0.1 µg of pcDNA3.1, pcDNA3.1-FZD1, pcDNA3.1-FZD6, pcDNA3.1-FZD7, piGENE or piGENE-FZD7, and 0.005 µg of pRL-TK Vector (Promega) according to the manufacturer's instructions. The total amount of DNA was adjusted to equal amounts with empty vector. At 48 hours after transfection, the luciferase levels were measured by using the Dual-Luciferase Reporter Assay System (Promega). Data are presented as mean values and SD for three independent experiments compared with the level of luciferase activity obtained in the presence of empty vector that is presented as 1.

Western Blot Analysis

After plasmid transfection, cells were washed in ice-cold phosphate-buffered saline and resuspended in cold buffer containing 10 mM HEPES, pH 7.9, 10 mM KCl, 1 mM dithiothreitol, 0.1 mM EGTA, 0.1 mM EDTA, and 0.5 mM phenylmethylsulfonyl fluoride. Resuspended cells were lysed in 0.5% Nonidet P-40 for 15 minutes followed by centrifugation at 1000 g for 5 minutes at 4°C. A total of 10 µg of cytosolic fraction was analyzed by Western blot analysis using mouse monoclonal antibody or rabbit polyclonal antibody, and antimouse and -rabbit IgG HRP-conjugated secondary antibodies (Dako, Glostrup, Denmark) were visualized with a LumiGLO Reagent and Peroxidet (Cell Signaling Technology, Beverly, MA).

Primary antibodies used were mouse anti-β-catenin (BD Biosciences, San Jose, CA), anti-Myc (Novus Biologicals, Littleton, CO), anti-Jun (BD Biosciences), anti-V5 (Invitrogen), anti-caspase-3 (BD Biosciences), anti-cytochrome c (BD Biosciences), anti-β-actin (Abcam, Cambridge, UK) monoclonal antibodies, rabbit anti-FZD7 (Aviva Systems Biology, San Diego, CA) and anti-survivin polyclonal antibody (Abcam).

RNA Interference

Small interfering RNA were constructed in the piGENE hU6 Vector (Clontech). The 21-nucleotide target sequence for FZD7 was 5′-tcacctacctggtggacatgc-3′. pcDNA3.1-U6 and pcDNA3.1-U6FZD7-siRNA were obtained by subcloning a EcoRI- HindIII fragment from piGENE and piGENE-FZD7 into the EcoRI- HindIII site of pcDNA3.1.

Interferon Assay

HCT-116 and Caco-2 cells were transfected with piGENE or pi-GENE-FZD7 or treated with 1 µg/ml poly(I:C) (InvivoGen, San Diego, CA). At 24 or 48 hours after transfection, total RNA was isolated from cells and analyzed by real-time PCR for 2′,5′-oligoadenylate synthetase (OAS1).

Cell Viability Assay

HCT-116 and HT-29 cells were transfected with pcDNA3.1-U6 or pcDNA3.1-U6FZD7. At 24 or 48 hours after transfection, cells were maintained in medium supplemented with 500 µg/ml G418. During 6 or 17 days after transfection, cell viability was measured by the MTS (3-(4,5-dimethylthiazol-2-yl-5-(3-carboxymethoxyphenyl-2-(4-sulfonphenyl)-2H-tetrazolium, inner salt) assay. The CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega) was used for the MTS assay, and instructions from the manufacturer were followed.

Cell Invasion Assay

Matrigel (1:5; BD Biosciences) was added to the Transwell membrane filter inserts with 8.0-µm pore size (Costar, Cambridge, MA) and was incubated for 5 hours at 37°C in a 5% C02 tissue culture incubator. HCT-116 and HT-29 cells were transfected with pcDNA3.1-U6 or pcDNA3.1-U6FZD7. At 24 or 48 hours after transfection, cells were harvested and resuspended in serum-free medium supplemented with 500 µg/ml G418. An aliquot (105 cells) of the prepared cell suspension was added into the upper chamber and cultured into the lower chamber filled with 600 µl of culture medium containing 5 µg/ml fibronectin (Sigma), as an adhesive substrate for 48 hours at 37°C in a 5% CO2 tissue culture incubator. Invasive cells were stained with Diff-Quick solution (Fisher Scientific, Pittsburgh, PA). Cells were counted under a microscope. The average number of cells in five fields per membrane was counted in triplicate assay.

Results

The mRNA Expression level of Frizzled Family Members in Colon Cancer Cell Lines

To reveal the expression of FZD gene family members in colon cancer cells harboring the APC or CTNNB1 mutation, the mRNA levels of 10 FZD genes in SW-480, HCT-116, Caco-2, DLD-1, LoVo, and HT-29 cells were examined by real-time PCR (Figure 1A). All the cell lines tested, except for HCT-116, possess the APC mutation [15,16]. HCT-116 exhibits the CTNNB1 mutation [16]. Among the 10 FZD genes, FZD6 and FZD7 were predominantly expressed in all cell lines. The protein levels of FZD7 in six colon cancer cells was indicated by Western blot analysis. 293T cells was transfected with FZD7 as a positive control (Figure 1B).

Figure 1.

Figure 1

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The mRNA and protein expression level of frizzled family members in colon cancer cell lines. (A) The extracted total RNA was reverse-transcribed into single-stranded cDNA. Real-time PCR was performed using first-strand cDNA with TaqMan. GAPDH was used as an endogenous control. Data are presented as mean values and SD for three independent experiments compared with the level of FZD1 that is presented as 1. (B) Western blot analysis of protein expression of FZD7 in six colon cancer cells. 293T cells was transfected with FZD7 as a positive control.

Effect of FZD7 Overexpression on Tcf-reporter Transcriptional Activity

Because FZD6 has been shown to be involved in the noncanonical Wnt pathway [17], we examined whether FZD7 functions as a receptor transducing the canonical Wnt signaling in colon cancer cells. Six colon cancer cells were transfected with FZD1, FZD6, or FZD7 cDNA together with a TOPflash reporter plasmid. FZD1 was used as a positive control because FZD1 is known to activate the canonical pathway [18]. FZD6 was used as a negative control because it is known to activate the noncanonical Wnt pathway. As shown in Figure 2A, Tcf transcriptional activity was increased by 1.5- to 24.3-fold when FZD7 was transfected, suggesting that FZD7 could activate the canonical pathway in colon cancer cells.

Figure 2.

Figure 2

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Effects of FZD7 overexpression on canonical Wnt signaling. (A) Effects of FZD7 overexpression on Tcf-reporter transcriptional activiry. Colon cancer cell lines were transiently cotransfected with either mock, FZD1, FZD6, or FZD7, TOPflash reporter plasmid, and pRL-TK plasmid encoding Renilla luciferase as an internal control for transfection efficiency. At 48 hours after transfection, cell lysates were measured for relative luciferase activities. Data are presented as mean values and SD for three independent experiments compared with the level of luciferase activity obtained in the presence of mock that is presented as 1. (B) Real-time PCR analysis of expression of Wnt target genes in HCT-116 cells transfected with mock, FZD1, or FZD7. HCT-116 cells were transiently transfected with mock, FZD1, or FZD7. At 48 hours after transfection, total RNA were reverse-transcribed and amplified with primers specific for each indicated Wnt target as described in the Materials and Methods section. (C) Western blot analysis of expression of Wnt target genes in HCT-116 cells transfected with mock, FZD1, or FZD7. HCT-116 cells were transiently transfected with mock, FZD1, or FZD7. At 48 hours after transfection, each cytosolic protein was prepared, and 10 µg of each was subjected to electrophoresis. Expression of β-catenin, Myc, Survivin, and Jun were assessed. β-Actin served as a loading control.

Real-Time PCR and Western Blot Analyses of Expression of Wnt Target Genes in FZD7-Transfected HCT-116 Cells

To assess whether FZD7 indeed affected the expression of target genes of the canonical pathway, we transfected FZD1 or FZD7 cDNA into HCT-116 cells and measured the levels of mRNA expression of seven known target genes including Myc, Cyclin D1, CD44, VEGF, Survivin, Id-2, and Jun using real-time PCR. The transcriptional levels of these genes increased 1.5- to 3.4-fold with FZD7 transfection compared to mock transfection (Figure 2B).

We then performed Western blot analysis of FZD7-transfected HCT-116 cells for cytosolic β-catenin, Myc, Survivin and Jun proteins. β-Catenin was up-regulated in HCT-116 after FZD7 transfection (Figure 2C). The expression levels of Myc, Survivin, and Jun were also increased, which was consistent with the results of real-time PCR.

FZD7-siRNA Specifically Inhibits the Expression of FZD7

We constructed an expression vector harboring siRNA against FZD7 (FZD7-siRNA) to investigate the effect of decreased FZD7 expression. Initially, FZD7-V5 and FZD7-siRNA were cotransfected into 293T cells, and the whole proteins were subjected to immunoblot analysis with an anti-V5 antibody. As shown in Figure 3A, FZD7-siRNA abolished the expression of FZD7-V5 protein, confirming the inhibitory action of the siRNA.

Figure 3.

Figure 3

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Effects of FZD7-siRNA on canonical Wnt signaling. (A) FZD7-siRNA specifically inhibits the expression of FZD7. 293T cells were transiently transfected with various combinations of pEF4, pEF4-FZD-V5, piGENE, and FZD7-siRNA. A total of 30 µg of cell lysates was analyzed by Western blot analysis using anti-V5 or anti-β-actin antibodies. (B) Effects of FZD7-siRNA on Tcf-reporter transcriptional activity. Colon cancer cell lines were transiently cotransfected with either piGENE or FZD7-siRNA, TOPflash reporter vector, and pRL-TK plasmid encoding Renilla luciferase as an internal control for transfection efficiency. At 48 hours after transfection, cell lysates were measured for relative luciferase activities. Data are presented as mean values and SD for three independent experiments compared with the level of luciferase activity obtained in the presence of piGENE that is presented as 1. (C) Western blot analysis of the expression of Wnt target genes in HCT-116 cells transfected with piGENE or FZD7-siRNA. HCT-116 cells were transiently transfected with piGENE or FZD7-siRNA. At 48 hours after transfection, 10 µg of cytosolic protein was prepared. Expression of β-catenin, Myc, Survivin and Jun were assessed. β-Actin served as a loading control.

Effects of FZD7-siRNA on Tcf-Reporter Transcriptional Activity and Expression Level of Wnt Target Genes

The effect of FZD7-siRNA on the canonical Wnt signal-transducing activity of FZD7 was then observed in six colon cancer cell lines. The cells were transfected with piGENE or FZD7-siRNA together with a TOPflash reporter plasmid, and Tcf transcriptional activity was measured in the same manner as in Figure 2A. We found that Tcf activity was decreased by 20% to 80% with FZD7-siRNA (Figure 3B).

The effect of FZD7-siRNA on the expression level of target proteins of the canonical pathway was finally examined in HCT-116 cells transfected with piGENE or FZD7-siRNA. As shown in Figure 3C, the band intensities of B-catenin, Myc, Survivin, and Jun were decreased by FZD7-siRNA transfection. These data suggested that FZD7 is involved in the activation of the canonical Wnt pathway in colon cancer cells despite the presence of the APC or CTNNB1 mutation.

Effects of FZD7-siRNA on Cell Viability and Cell Invasion

We tested whether FZD7-siRNA affects cell viability and migration to evaluate its potential therapeutic effects on colon cancer cells. HCT-116 and HT-29 cells were transfected with pcDNA3.1-U6 or pcDNA3.1-U6FZD7-siRNA, and the number of viable cells selected by G418 was measured by MTS assay 6 or 17 days after transfection (Figure 4A). As the growth of HT-29 was much slower than HCT-116, the longer incubation time was required for the evaluation of HT-29 viability. The viable cell number in HCT-116 and HT-29 was decreased to 20% or 80%, respectively. A significant impairment of cell viability activity was observed in HCT-116 (P = .0042) and HT-29 cells (P = .0256) FZD7-siRNA transfer compared to vector control alone. G418 selection was used for excluding nontransfected cells.

Figure 4.

Figure 4

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Functional effects of FZD7-siRNA in HCT-116 and HT-29 cells. (A) Effect of FZD7-siRNA on cell viability. HCT-116 and HT-29 cells were transiently transfected with pcDNA3.1-U6 or pcDNA3.1-U6FZD7-siRNA. At 24 or 48 hours after transfection, HCT-116 and HT-29 cells were maintained in medium supplemented with G418. During 6 or 17 days after transfection, cell viability was tested by the MTS assay. (B) Effect of FZD7-siRNA on cell invasion. HCT-116 and HT-29 cells were transiently transfected with pcDNA3.1-U6 or pcDNA3.1-U6FZD7-siRNA. At 24 or 48 hours after transfection, an aliquot (105 cells) of the prepared cell suspension was added into the upper chamber and cultured into the lower chamber filled with the culture medium containing fibronectin for 48 hours. Invasive cells were stained, and the average number of cells in five fields per membrane was counted in triplicate assay. (C) Effect of FZD7-siRNA on interferon response gene, OAS1. HCT-116 and Caco-2 cells were transiently transfected with piGENE or FZD7-siRNA. At 48 hours after transfection, total RNA were reverse-transcribed and amplified with primers specific for OAS1 as described in the Materials and Methods section. Poly(I:C) served as a positive control. OAS1 expression was normalized to GAPDH mRNA. Caco-2 served as a negative control.

FZD7-siRNA also affected the in vitro invasion activity of HCT-116 and HT-29 cells. The cell invasion number in HCT-116 and HT-29 was decreased to 40% or 60%, respectively. A significant impairment of cell invasion activity was observed in HCT-116 (P = .0002) and HT-29 cells (P = .0006; Figure 4B).

FZD7-siRNA Does Not Induce Interferon Response Gene, OAS1

To exclude the possibility that FZD7-siRNA triggers an interferon response in colon cancer cells, HCT-116 and Caco-2 cells were transfected with piGENE or FZD7-siRNA, and the interferon response was observed using real-time PCR for OAS1. As shown in Figure 4C, OAS1 expression was induced by poly(I:C), used as a positive control, whereas it was not found at all after piGENE or FZD7-siRNA transfection.

FZD7-siRNA Does Not Induce Apoptosis

The effect of FZD7-siRNA on the expression level of apoptotic proteins was examined in HCT-116 cells transfected with piGENE or FZD7-siRNA. As shown in Figure 5A, neither cleavage of caspase-3 nor increase of cytochrome c release was detected by FZD7-siRNA transfection.

Figure 5.

Figure 5

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Effect of FZD7-siRNA on apoptosis (A) and the mRNA expression level of FZD7 in primary CRC tissues (B). (A) Western blot analysis of apoptotic proteins in HCT-116 cells transfected with piGENE or FZD7-siRNA. HCT-116 cells were transiently transfected with piGENE or FZD7-siRNA. At 48 hours after transfection, 10 µg of cytosolic protein was prepared. Cleavage of caspase-3 and cytochrome c release were assessed. β-Actin served as a loading control. (B) The mRNA levels of FZD7 in primary CRC tissues and colon cancer cell lines were examined by real-time PCR. Data are presented as mean values and SD for three independent experiments compared with the level of GAPDH that is presented as 1.

The mRNA Expression level of FZD7 in Primary CRC

To reveal the expression of FZD7 in primary CRC, the mRNA levels of FZD7 in surgically resected cancer tissues were examined by real-time PCR (Figure 5B). The FZD7 expression level of primary cancer tissues was various, but it was equivalent to that of cell lines in about half of the cases.

Discussion

Our present data demonstrate that FZD7 is the predominantly expressed member of the frizzled gene family and activates the canonical Wnt signaling pathway in colon cancer cells possessing the APC or CTNNB1 mutation. Overexpression of FZD7 in these cell lines caused an increase of Tcf transcriptional activity and expression of target genes of the canonical Wnt pathway (Figure 2). More importantly, FZD7-siRNA decreased Tcf activity and Wnt target gene expression (Figure 3, B and C), suggesting that FZD7 transmits the canonical Wnt signals to downstream molecules despite the presence of APC or CTNNB1 mutation. The findings seem to be compatible with the “just right” hypothesis for APC mutations in CRCs [3]. Accumulating evidence suggests that a pertinent combination of the first hit and second hit provides a superior growth advantage for tumor cells [3]. Resultant truncated APC proteins are almost invariably retained in CRC cells, and many of them preserve some ability to bind and, perhaps, degrade β-catenin [3,4]. With regard to the CTNNB1 mutation, WNT signaling controls the phosphorylation status through several different crucial serine and threonine sites, and hence it may be surmised that even if mutation occurs in one site, other sites will be phosphorylated to adjust the level suitable for tumor cell survival and proliferation [5]. Indeed, individual CRCs with CTNNB1 mutations contain only one mutant site at a time [16,19,20].

We then investigated the effect of FZD7-siRNA on the viability of HCT-116 and HT-29 cells under G418 selection and found that it exhibited a significant suppressive activity, especially in HCT-116 cells (Figure 4A). However, this siRNA did not induce apoptosis of HCT-116 cells as shown in Figure 5A. The effect of the siRNA on cell cycle is now under investigation. Furthermore, the siRNA decreased the invasion activity of HCT-116 and HT-29 cells to 40% and 60%, respectively (Figure 4B). When considering that our lipofection efficiency in HCT-116 cells was ∼60%, as assessed by EGFP expression, the invasion suppression activity of the siRNA in these cells may be very strong. The molecular mechanism for the activation of cell invasion with FZD7 is unknown now. One possible explanation for our result is the decreased expression of Wnt target genes affecting cell invasion activity, which may include CD44 [21], matrix metalloproteinase 7 (MMP-7) [22], MT1-MMP/MMP-14 [23], urokinase plasminogen activator [24], S100A4 [25], etc. Although some developmental studies have suggested that FZD7 is involved in the noncanonical Wnt signaling pathway and regulates tissue movement or cell migration [26,27], its role in human tissues and cancer cells remains to be investigated.

We finally demonstrated the FZD7 expression in primary CRC tissues (Figure 5B). Although the expression level was various in each tissue, it was equivalent to that of cell lines in around half of the cases. Of note, samples #346, 347, and 404 with the lower expression level were all in stage II, which might suggest the FZD7 expression in advanced stages.

There has been little knowledge about Wnts that interact with FZD7 in cancer cells. Very recently, Kim et al. [28] reported that Wnt3 directly interacts with FZD7 in hepatoma cells. It was previously shown that Wnt2, Wnt3, and Wnt3a were detected in HCT-116 cells by RT-PCR [29], Wnt3 may also interact with FZD7 in CRC cells.

In conclusion, we demonstrated for the first time that FZD7 activates the canonical Wnt pathway in colon cancer cells despite the presence of APC or CTNNB1 mutation and that FZD7-siRNA could be used as a therapeutic reagent for CRCs.

Footnotes

1

This study was supported by a Scientific Research on Priority Areas (No. 17016049) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

References

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