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Mol Cancer Ther. 2006;5:2861-2871
© 2006 American Association for Cancer Research

Research Articles: Therapeutics, Targets, and Development

Targeting the active ß-catenin pathway to treat cancer cells

Hadas Dvory-Sobol1,2, Eyal Sagiv1,2, Diana Kazanov1, Avri Ben-Ze'ev3 and Nadir Arber1,2

1 Integrated Cancer Prevention Center, Tel Aviv Sourasky Medical Center; 2 Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel; 3 Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel

Requests for reprints: Nadir Arber, Director-Integrated Cancer Prevention Center, Tel-Aviv Medical Center, 6 Weizmann Street, Tel-Aviv 64239, Israel. Phone: 972-3-6974968; Fax: 972-3-6950339. E-mail: narber{at}post.tau.ac.il or nadir{at}tasmc.health.gov.il

Abstract

The adenomatous polyposis coli or ß-catenin genes are frequently mutated in colorectal cancer cells, resulting in oncogenic activation of ß-catenin signaling. We tried to establish in vitro and in vivo models for selectively killing human cancer cells with an activated ß-catenin/T-cell factor (Tcf) pathway. We used a recombinant adenovirus that carries a lethal gene [p53-up-regulated modulator of apoptosis (PUMA)] under the control of a ß-catenin/Tcf–responsive promoter (AdTOP-PUMA) to selectively target human colorectal cancer cells (SW480, HCT116, DLD-1, and LS174T), hepatocellular carcinoma (HepG2), and gastric cancer cells (AGS) in which the ß-catenin/Tcf pathway is activated, and compared its efficiency in killing cancer cells in which this pathway is inactive or only weakly active. AdFOP-PUMA, carrying a mutant Tcf-binding site, was used as control virus. The combined effect of AdTOP-PUMA with several chemotherapeutic agents (5-florouracil, doxorubicin, and paclitaxel) was also evaluated. The effect of AdTOP-PUMA on colorectal cancer cells was also examined in nude mice: SW480 cells were infected with the AdTOP-PUMA and AdFOP-PUMA, and then inoculated s.c. into nude mice. The TOP-PUMA adenovirus inhibited cell growth in a dose-dependent fashion, depending on the signaling activity of ß-catenin. The growth of cells displaying high levels of active ß-catenin/Tcf signaling was inhibited after infection with AdTOP-PUMA, whereas that of cells with low levels of ß-catenin signaling was not. Growth inhibition was associated with induction of apoptosis. Chemotherapy synergistically enhanced the effect of AdTOP-PUMA. A combination of the adenovirus system with standard therapy may improve the efficacy and reduce the toxicity of therapy in humans. [Mol Cancer Ther 2006;5(11):2861–71]

Introduction

ß-Catenin is a multifunctional protein serving as a major structural component of cell-to-cell adherens junctions. In addition, it also acts as an important signaling molecule in the Wnt pathway that plays a key role in embryogenesis and tumorigenesis (1–3).

In the absence of Wnt signaling, the cytoplasmic level of ß-catenin is kept low through interaction with a protein complex [containing GSK3ß-glycogen synthase kinase 3ß, axin and adenomatous polyposis coli (APC)] that can phosphorylate ß-catenin and target it to ubiquitin-mediated proteasomal degradation (4). Activation of Wnt signaling leads to inactivation of GSK3ß, resulting in cytoplasmic accumulation of ß-catenin (5). The increase in ß-catenin level is followed by its translocation into the nucleus, where in complex with members of the T-cell factor (Tcf)/lymphocyte enhancer–binding factor family of transcription factors it activates the expression of target genes (6).

The APC tumor suppressor is mutated in ~80% of the familial adenomatous polyposis syndrome and sporadic colorectal cancer patients (7). Loss of APC is believed to be one of the earliest initiating events in multistage colorectal carcinogenesis (8). Mutant APC loses its ability to direct ß-catenin to degradation, resulting in nuclear accumulation and inappropriate activation of ß-catenin–mediated transactivation. Mutations in ß-catenin in the GSK-3ß phosphorylation sites have been identified in 50% of colorectal cancer cases that retain wild-type APC (9–11). c-MYC (12) and cyclin D1 (13, 14), which positively regulate cell proliferation, are target genes of ß-catenin/Tcf with direct implications in tumorigenesis (15–19). Activating ß-catenin mutations have also been identified in a variety of other tumors, including melanomas (20), hepatocellular carcinomas (21), skin (22), breast (23), and prostate cancers (24), whereas the ß-catenin–Tcf pathway is not activated in most normal tissues. Therefore, a therapeutic strategy that targets this pathway could be applied to patients with primary or metastatic colorectal cancer.

p53-up-regulated modulator of apoptosis (PUMA) is a potent mediator of the p53 apoptotic response (25, 26). It belongs to the group of BH3-only proteins that have been shown to function by dimerization with other BH3 domain–containing proteins, including Bcl-2 and Bcl-XL, that results in the release of cytochrome c from mitochondria and induction of apoptosis by activation of caspase-3 and caspase-9 (27).

A substantial limitation of conventional cancer chemotherapy and radiotherapy is the toxicity of these agents to normal tissue. The toxicity of currently available gene delivery systems to the normal cell population results from their toxicity to the normal cell population. Here, we propose a novel gene therapy approach that selectively expresses a lethal gene by targeting the active ß-catenin–Tcf pathway in human colorectal, gastric, and hepatic cancer cells. Moreover, we show that combining this strategy with standard chemotherapy results in a synergistic growth inhibition of colorectal cancer cells that overcomes cancer cell resistance to therapy. This approach may pave the way to a novel treatment of primary and metastatic colorectal cancer.

Materials and Methods

Cell Culture
Human colorectal cancer (SW480, DLD-1, HT-29, HCT116, LS174T), gastric (AGS), hepatic (HepG2, SK-Hep-1), pancreatic (Colo357, Panc-1), and embryonic kidney (293) cell lines were obtained from the American Type Culture Collection (Manassas, VA). They were cultured in DMEM (Sigma, Rehovot, Israel) containing 5% to 10% fetal bovine serum (Biological Industries, Beit Haemek, Israel), 1% penicillin, and 1% streptomycin, at 37°C, in an atmosphere of 95% oxygen and 5% CO2 (complete medium).

Construction of Plasmids and Adenoviral Vectors
Two sets of ß-catenin/Tcf–responsive promoters were generated: one contains wild-type Tcf/lymphocyte enhancer–binding factor binding sites fused with cFos (TOP-cFos-Luc-TOPFLASH) and the other, the SV40 (TOP-SV40-Luc) minimal promoter upstream to a luciferase (Luc) reporter gene. The corresponding control plasmids were constructed for each promoter by replacing the TOP oligomers with mutant Tcf-binding oligomers (FOP), e.g., FOP-cFos-Luc (FOPFLASH) and FOP-SV40-Luc (see the TOP and FOP sequences in Fig. 1A ). To construct the TOP/FOP-cFos-Luc plasmids, an XbaI fragment containing the TOP-cFos, and the FOP-cFos from TOPFLASH and FOPFLASH plasmids (generous gifts from Hans Clevers, Utrecht University, Utrecht, the Netherlands), was cloned into the NheI site upstream to the Luc gene in the pGL3-basic plasmid (Promega, Rehovot, Israel). To construct the TOP/FOP-SV40-Luc plasmids, the BglII-NheI TOP and FOP fragments were cloned into the NheI and BglII sites upstream of the SV40 minimal promoter in the pGL3-promoter plasmid (Promega).


Figure 1
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  		Figure 1. Construction of adenoviruses. A, ß-catenin/Tcf activatable promoters containing TOP or FOP sequences. B, schematic representation of different adenovirus constructs. C, the PacI-digested recombinant adenoviral vector pAdTOP-PUMA was transfected into 293 cells and GFP expression was visualized by fluorescence microscopy at the indicated times. Comet-like adenovirus–producing foci became apparent after 5 to 7 d.

 
The AdEasy system (28) was used to generate the AdTOP-PUMA and AdFOP-PUMA (AdTOP/FOP-PUMA) adenoviruses. The TOP and FOP sequences were obtained from the TOP-cFos-Luc and FOP-cFos-Luc plasmids and cloned into the shuttle vector-pAd-Track. The pAdTrack also contains a green fluorescent protein (GFP) gene under the control of the cytomegalovirus (CMV) promoter (Fig. 1B). The blunted HA-PUMA fragment (containing the human PUMA cDNA fused to a double hemagglutinin-epitope tag) from pCEP4-PUMA (a generous gift from Bert Vogelstein, Johns Hopkins Oncology Center, Baltimore, MD) was cloned downstream to the TOP/FOP elements in the pAd-Track vector. The resultant plasmids were designated pAdTrack-TOP/FOP-PUMA. These shuttle vectors were linearized with PmeI and cotransformed with E1-deleted adenoviral backbone AdEasy-1 into the competent bacterial strain BJ5183, which enables efficient recombination. A panel of Ad-TOP-PUMA and Ad-FOP-PUMA recombinant adenoviruses were generated.

Adenovirus Production and Titering
To produce viruses, 4 µg PacI-linearized adenoviral DNA was transfected into 50% to 70% confluent 293 cells in 10-cm dishes using LipofectAMINE and Plus Reagents (Invitrogen Life Technologies, Carlsbad, CA). Between 5 and 7 days posttransfection, colonies expressing GFP were observed under a fluorescent microscope, the cells were harvested and lysed in PBS by four cycles of freeze/thaw/vortex (Fig. 1C). The supernatant was collected and half of it was used to reinfect 50% to 70% confluent 293 cells. Viruses were collected 2 to 3 days postinfection when a cytopathic effect became evident. Further amplification and concentration of the virus stocks was achieved through several rounds of infection. To titer the viruses, 50% to 70% confluent 293 cells in 96-well dishes were infected with serial dilutions of the virus stocks. GFP-positive colonies were counted 5 days postinfections. The control Ad-CMV-GFP adenovirus containing the GFP gene under the control of a full-length CMV promoter was a kind gift of Hila Giladi (Hadassah School of Medicine, Jerusalem, Israel) and was amplified in 293 cells.

Human cDNA of caspase-8, Bak, and Bax were a kind gift from Atan Gross (Weizmann Institute of Science, Rehovot, Israel). The expression constructs of PUMA and PUMA-{Delta}BH3, a mutant PUMA without activity, were generous gifts from Bert Vogelstein. PKGIß that encodes a mutant PKG sequence with an NH2-terminal truncation (29), was a gift from I. Bernard Weinstein (Columbia University, New York, NY). This deletion renders PKG independent of cyclic guanosine 3',5'-monophosphate, and it is constitutively active.

Luciferase Assays
Transfections were done using LipofectAMINE and Plus Reagents (Invitrogen) according to the instructions from the manufacturer. A total of 5 x 105 cells were seeded in six-well plates. The next day, 50% confluent dishes were cotransfected with 1 µg vectors plus 0.1 µg pRL-TK (Promega). Luc assay was done 24 hours posttransfection. Briefly, cells were washed once with PBS and lysed in 400 µL lysis buffer for 15 minutes at room temperature. The lysates were centrifuged at 14,000 rpm for 5 minutes, and 20 µL of each lysate were used to measure Luc reporter gene expression. Luc activity was normalized to Renilla Luc activity from a parallel cotransfection of pRL-TK (Dual Luc system, Promega). All experiments were done in triplicate at least thrice and gave similar results.

The Potency of PUMA in Cell Killing
SW480 cells were transfected with constructs encoding for PUMA or a mutant PUMA-{Delta}BH3, without activity. Cells were harvested 24 hours after transfection, and an equal number of cells was diluted in duplicates (1:10 and 1:25) into 10-cm dishes and grown under hygromycin B selection for 3 weeks, after which the cells were fixed and stained with 0.2% Coomassie blue, 50% methanol, 10% acetic acid, and 40% H2O.

Cell Viability Assays
Between 2 x 104 and 5 x 104 cells in 100 µL complete medium were plated in 96-well dishes. The next day, six wells were infected with each adenovirus at a different multiplicity of infection (0.1–50 MOI). Cell viability was assessed by methylene blue staining after 48 hours. The cells were washed once with PBS and fixed in 150 µL formaldehyde (4%) for 2 hours at room temperature, washed with 0.1 mol/L sodium borate (pH 8.5), and stained with 0.5% methylene blue for 10 minutes, then washed with tap water and 150 µL of 0.1 mol/L HCl to dilute the cell-bound dye. Absorbance was measured at 590 nm. Cell viability is expressed as percentage absorbance relative to mock-infected cells. The average of at least two independent experiments with six replicates was recorded.

Chemicals
Paclitaxel, doxorubicin, and 5-florouracil were obtained from Sigma. Cells were infected with AdTOP-PUMA, AdFOP-PUMA, or Ad-CMV-GFP (5 MOI); after 5 hours were treated with 0.05 µmol/L paclitaxel, 1 µmol/L doxorubicin, or 0.05 µmol/L 5-florouracil; and were then cultured for 48 hours.

Western Blot Analysis
Infected cells were harvested and protein concentrations were determined using the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA). An equal amount of protein from each lysate was analyzed by SDS-PAGE and the proteins were transferred to hybond-C extra nitrocellulose membranes (Amersham Life Science, Buckinghamshire, United Kingdom). Membranes were blocked with buffer containing 5% low-fat milk and 0.05% Tween 20 in PBS for 1 hour, incubated with primary antibodies for 1 hour with peroxidase-conjugated secondary antibodies, and developed with a Supersignal West Pico chemiluminescent substrate (Pierce, Rockford, IL). Antibodies against hemagglutinin, actin, ß-catenin, and caspase-3 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Apoptosis Analysis
Flow Cytometry. Cells were plated at 5 x 106/10-cm dish 24 hours before infection, and were infected with recombinant adenoviral vectors at 5 MOI. Twenty-four hours later, both adherent and floating cells were harvested, washed with PBS, and fixed in 80% ethanol for 1 hour and stained with propidium iodide for analysis of DNA content. The number of subdiploid cells, representing apoptotic cells, was quantified by FACScan using the CellQuest software (Becton Dickinson Immunocytometry Systems, San Jose, CA). Necrotic cells were excluded by staining with trypan blue. The average of at least three independent experiments with two replicates was recorded.

ssDNA. For the ssDNA assay, 104 cells were seeded in 96-well microplates, and after 24 hours infected with recombinant adenoviral vectors at 5 MOI. The following day, the ssDNA Apoptosis ELISA kit was used (Chemicon International, Inc., Temecula, CA). Based on the selective, formamide-induced denaturation of DNA, this method identifies apoptotic cells (30), by staining ssDNA using a mixture of anti-ssDNA monoclonal antibody and peroxidase-conjugated anti-mouse IgM. The average of at least two independent experiments with two replicates was recorded.

Fluorogenic assay for caspase-3 activity.
Cells were plated at 1 x 106 per well onto a six-well plate 24 hours before infection, and were infected with recombinant adenoviral vectors at 5 MOI. Cells were harvested with a rubber policeman; washed; resuspended in 50 mmol/L Tris-HCl buffer (pH 7.4), 1 mmol/L EDTA, and 10 mmol/L EGTA; and lysed by three successive freeze-thaw cycles on dry ice. Cell lysates were centrifuged at 20,000 x g for 5 minutes, and the supernatants were stored at –70°C. The protein concentration of each sample was determined using the Bradford Bio-Rad protein assay. For caspase-3 activity, a total of 50 µg protein was incubated with 50 mmol/L ac-DEVD-AMC (from BIOMOL Research Laboratories, Plymouth Meeting, PA) at 37°C, for 30 minutes in the dark. The release of 7-amino-4-methylcoumarine was monitored by a spectrofluorometer using an excitation wavelength of 360 nm and an emission wavelength of 460 nm.

Tumorigenic Assays
CD1 nude mice housed in sterile cages were handled under aseptic conditions. The animals were maintained in facilities approved by the Israeli Association for Accreditation of Laboratory Animal Care and in accordance with current regulations and the standard of care of the Israeli Ministry of Health. SW480 colorectal cancer cells were infected with 5 MOI of adenoviral vectors in serum-free medium. Five hours after infection, the medium was changed to 10% fetal bovine serum medium and the infected cells were incubated at 37°C overnight. Twenty-four hours after adding the virus, the cells were trypsinized and inoculated s.c. on the backs of nude mice (3 x 106 per animal). The number of mice with tumors (incidence) and their volume (tumor burden) were determined after different times.

Statistical Analysis
Statistical analysis was done by using InStat software version 3.01 (GraphPad Software, Inc., San Diego, CA). In the tissue culture experiments, the comparison between two samples was done using Student's t test and between more than two samples using one-way ordinary parametric ANOVA followed by Tukey-Kramer multiple comparison test. For all statistical tests, preliminary evaluation of the homoscadacity and normality of the compared samples was done using Bartlett and Kolmogorov-Smirnov tests, respectively.

Results

ß-Catenin/Tcf–Mediated Luc Activity in Different Colorectal Cancer Cells
ß-Catenin/Tcf–dependent activity was determined in human cell lines displaying different levels of ß-catenin (Fig. 2A ). These cell lines harbored mutant APC proteins, except for HCT116 that have a deletion at residue S45 of the ß-catenin protein (11). To evaluate which ß-catenin/Tcf reporter construct is more readily detectable in these cells, the ß-catenin/Tcf–responsive promoters fused to SV40 and cFos minimal promoters were used using the luciferase (Luc) assay. ß-Catenin–activated promoters containing four copies of the Tcf-binding site (TOP) fused to either the cFos or the minimal SV40 promoter were analyzed. The TOP-cFos (TOPFLASH) construct exhibited higher activity than TOP-SV40 and was therefore used in the following experiments. The relative activity of TOPFLASH is shown in Table 1 . Luc activity, determined as fold induction of TOPFLASH, was 4.1- to 25.3-fold higher than that of the control FOPFLASH in colorectal cancer cells (SW480, DLD-1, HCT116, and LS174T), whereas HT-29 cells did not show significant transcriptional activation of this reporter construct (Table 1).


Figure 2
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  		Figure 2. PUMA suppresses the growth of colon cancer cells. A, comparison of ß-catenin levels in different cell types. Cell extracts were prepared from colorectal cancer lines (SW480, HCT116, DLD-1, and HT-29). Twenty micrograms of protein from each sample were subjected to Western blot analysis using anti-ß-catenin and anti-ß-actin antibodies. B, 293 cells were transiently transfected with human cDNA of caspase-8, Bid, Bak, Bax, PUMA, PKGIß, and pcDNA3. Forty-eight hours after transfection, the percentage of apoptotic (sub-G1) cells was determined by FACS analysis. *, P < 0.05, significantly different from control. **, P < 0.01, significantly different from control. C, SW480 cell lines were transfected with constructs encoding PUMA and PUMA-{Delta}BH3. Cells were harvested 24 h after transfection, and equal cell numbers were diluted in duplicates in 10-cm dishes and grown under selection in hygromycin B for 3 wks, then fixed and stained with Coomassie blue. Colony formation in representative dishes (1:10 and 1:25 dilution) of transfected SW480 cells is shown. D, the number of colonies formed with cells infected with PUMA and PUMA-{Delta}BH3 is shown. *, P < 0.05, significantly different from PUMA-{Delta}BH3.

 

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  		Table 1. ß-catenin/Tcf–regulated transcription activity

 
PUMA Induces Cell Death in SW480 Cells
To identify the most potent proapoptotic gene in colorectal cancer cells, we tested several full-length cDNAs, including those encoding for Bak, caspase-8, PUMA, PKGIß, Bax, and Bid that we transfected into 293 cells. Forty-eight hours after transfection, the number of subdiploid DNA-containing cells, representing apoptotic cells, was quantified by FACScan (Fig. 2B). PUMA and Bax induced the highest apoptotic activity. We have chosen PUMA because it was more effective in SW480 cells, which we used in the following studies (Fig. 2C).

To determine the effect of PUMA expression on colon cancer cell growth, an expression vector containing PUMA under the control of the CMV promoter and a hygromycin B–resistant gene (pCEP4-PUMA) was transfected into SW480 cells. PUMA-{Delta}BH3 encoding for a nonfunctional PUMA (without its BH3 domain) was used as control. The results shown in Fig. 2C and D show a drastic reduction in colony formation by cells after transfection with PUMA compared with the mutant PUMA vector.

Next, we used an adenoviral vector selected for gene delivery with PUMA placed downstream to the cFos minimal promoter. The promoter contained either the wild-type (AdTOP-PUMA) or the mutant (AdFOP-PUMA) Tcf/lymphocyte enhancer–binding factor binding sites. The Ad-CMV-GFP vector was used as control for viral toxicity. The ability of AdTOP-PUMA and AdFOP-PUMA adenoviral vectors to kill cells with different levels of ß-catenin signaling was evaluated by cell viability assays 48 hours after infection with adenoviruses at varying doses (Fig. 3A ). Cells displaying elevated ß-catenin transactivation (Table 1), such as SW480, HCT116, and DLD-1, were killed efficiently by infection with AdTOP-PUMA, in a dose-dependent manner. Although the infection efficiency of the adenoviral vectors in the human colorectal cancer cell line HT-29 was high (shown by the number of GFP positive cells in Fig. 3D), neither AdTOP-PUMA nor AdFOP-PUMA caused cell death in this cell line (Fig. 3A), most probably because the level of ß-catenin/Tcf transactivation in these cells is low (Table 1). The number of viable cells after infection with AdTOP-PUMA was proportional to the methylene blue color intensity as shown for SW480 cells infected with adenoviral constructs (Fig. 3B). Representative pictures of SW480, DLD-1, and HT-29 cells, 48 hours after infection with either AdTOP-PUMA or AdFOP-PUMA, are shown in Fig. 3C.


Figure 3
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  		Figure 3. AdTOP-PUMA suppresses the survival of colon cancer cells. A, SW480, DLD-1, HCT116, and HT-29 cells were infected with AdTOP-PUMA, AdFOP-PUMA, and Ad-CMV-GFP adenoviruses in 96-well culture plates. Cell viability expressed as percentage absorbance relative to mock-infected cells was measured by methylene blue staining 48 h after adenoviral infection. Average of at least two independent experiments with six replicates. Statistical difference was observed between AdTOP-PUMA and the control groups (AdFOP-PUMA and Ad-CMV-GFP; ***P < 0.001; **P < 0.01). B, the number of viable cells is proportional to the intensity of methylene blue staining shown here for SW480 cells 48 h after adenoviral infection. C, SW480 cells 48 h following infection with either AdFOP-PUMA (left) or AdTOP-PUMA (right). AdTOP-PUMA and AdFOP-PUMA also contain a GFP gene under the control of a CMV promoter. GFP expression was visualized by fluorescence microscopy. D, HT-29 and DLD-1 cells were infected (at 5 MOI) with either AdFOP-PUMA (left) or AdTOP-PUMA (right) and GFP expression was visualized by fluorescence microscopy after 48 h.

 
Next, the number of cells in the sub-G1 population was determined by fluorescence-activated cell sorting (FACS) analysis in the different colorectal cancer cell lines infected with the various adenovirus constructs. By this method, SW480 and HCT116 cells displayed only a very low number of cells in the sub-G1 population (representing apoptotic cells) 24 hours after treatment with AdFOP-PUMA or Ad-CMV-GFP (Fig. 4A ). Apoptosis induced by AdTOP-PUMA was detected in SW480, DLD-1, and HCT116 cells 48 hours after infection, by the ssDNA assay, but not in HT-29 cells (Fig. 4B). The number of apoptotic cells was proportional to the increase in methylene blue color intensity as shown for SW480 cells infected with adenoviral constructs and stained with this dye after 48 hours (Fig. 4C). Taken together, the results of these different approaches suggest that AdTOP-PUMA is capable of inducing the death of cells that have elevated ß-catenin/Tcf transcriptional activity.


Figure 4
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  		Figure 4. Cell killing by AdTOP-PUMA adenovirus. A, SW480 and HCT116 cells were infected with AdTOP-PUMA, AdFOP-PUMA, or Ad-CMV-GFP adenovirus constructs at 5 MOI. Percentage apoptotic (sub-G1) cells was determined by FACS analysis 24 h after treatment. Significantly different from the control groups (no virus, AdFOP-PUMA, Ad-CMV-GFP; *P < 0.05; **P < 0.01). B, induction of apoptosis determined by the apoptosis ELISA kit described in Materials and Methods in cultures grown in 96-well plates and infected with adenoviruses for 48 h. *, significantly different from the control groups (no virus, P < 0.01; AdFOP-PUMA, P < 0.05; Ad-CMV-GFP, P < 0.01). #, significantly different from the control groups (no virus, P < 0.01; AdFOP-PUMA, P < 0.05; Ad-CMV-GFP, P < 0.05). &, significantly different from the control groups (no virus, P < 0.001; AdFOP-PUMA, P < 0.001; Ad-CMV-GFP, P < 0.001). C, color intensity is proportional the number of apoptotic SW480 cells 48 h after adenoviral infection.

 
Activated ß-Catenin/Tcf Signaling Induces Apoptosis by Up-regulating PUMA Expression
In SW480, HCT116, and DLD-1 cells, high levels of PUMA protein were detected after infection with AdTOP-PUMA, but not after AdFOP-PUMA or Ad-CMV-GFP infection (Fig. 5A ). HT29 colorectal cancer cells that express lower levels of ß-catenin and low levels of ß-catenin–Tcf transactivation did not express the PUMA protein after infection with AdTOP-PUMA (Fig. 5A). The infection of SW480 cells with AdTOP-PUMA induced PUMA expression as early as 12 hours after infection, as shown in Fig. 5B. Induction of apoptosis by AdTOP-PUMA was apparently mediated by the activation of caspases. Western blot analysis of SW480 cells after infection with AdTOP-PUMA, but not with AdFOP-PUMA or Ad-CMV-PUMA, detected a cleaved form of caspases-3 (p17; Fig. 5C). Also, caspase-3 activation occurred 48 hours following infection with AdTOP-PUMA, but not after infection with control viruses (AdFOP-PUMA or Ad-CMV-PUMA; Table 2 ).


Figure 5
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  		Figure 5. Up-regulation of PUMA after infection of cells with AdTOP-PUMA adenovirus. A, SW480, HCT116, DLD-1, and HT-29 cells were infected with AdTOP-PUMA, AdFOP-PUMA, or Ad-CMV-GFP (Ad-GFP) adenoviruses. Cells were harvested after 48 h and the lysates, normalized for protein concentration, were analyzed by Western blotting using antihemagglutinin (for PUMA detection) and anti-ß-actin antibodies. Cells were treated with PBS (left lane in each blot). B, immunoblot analysis of PUMA protein in SW480 cells 12, 24, 36, and 48 h postinfection with AdTOP-PUMA (5 MOI). C, Western blot analysis of caspase-3 activation in SW480 cells 24 h after infection. The antibody detected both the procaspase-3 (32 kDa) and the cleaved fragment of caspase-3 (17 kDa).

 

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  		Table 2. Caspase-3 is activated by the AdTOP-PUMA construct

 
AdTOP-PUMA Inhibits Tumor Growth In vivo
Next, we evaluated whether AdTOP-PUMA infection can induce a cell killing effect in s.c. tumors established in nude mice with colorectal cancer cells. SW480 cells were infected with adenoviruses in vitro, harvested after 24 hours, and then inoculated s.c. into nude mice. As shown in Table 3 , mice that were injected with SW480 cells that were infected with AdTOP-PUMA virus failed to develop tumors. In contrast, AdFOP-PUMA and Ad-CMV-GFP did not suppress the growth of the implanted SW480 cells.


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  		Table 3. AdTOP-PUMA suppresses the growth of tumors formed by SW480 cells displaying hyperactive ß-catenin

 
ß-Catenin Activates Expression of PUMA from Wild-Type Tcf/Lymphocyte Enhancer–Binding Factor–Responsive Constructs in Other Tumor Cells
The effect of AdTOP-PUMA was determined in other tumor cell lines with hyperactive ß-catenin/Tcf signaling. Luc activity was analyzed in AGS (gastric cancer), HepG2 (hepatocellular carcinoma), and LS174T (a colorectal cancer) cell lines. The results (Table 1) show that these cell lines displayed significant levels of ß-catenin/Tcf activity. In contrast, pancreatic cells (Colo357 and Panc-1) and SK-Hep-1 (hepatocellular carcinoma) showed only basal levels of ß-catenin/Tcf activity. Next, we examined the effect of Ad-PUMA constructs on these cells. As shown in Fig. 6A , AGS, HepG2, and LS174T were efficiently killed by AdTOP-PUMA infection, whereas Colo357 and Panc-1 cells were resistant, despite the high adenovirus infection efficiency in these cells (Fig. 6A and B).


Figure 6
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  		Figure 6. The effect of AdTOP-PUMA in different tumor cell lines. A, AGS (gastric cancer), HepG2 (hepatocellular carcinoma), LS174T (colon cancer), and Colo357 and Panc-1 (pancreatic cancer) cells were infected with AdTOP-PUMA, AdFOP-PUMA, and Ad-CMV-GFP viruses. Cell viability was measured 48 h after adenoviral infection by methylene blue staining. Cell viability is expressed as percentage absorbance relative to mock-infected cells. Average of at least two independent experiments with six replicates. Statistical difference was observed between AdTOP-PUMA and the control groups (AdFOP-PUMA and Ad-CMV-GFP) at 5, 10, and 25 MOI (***P < 0.001, **P < 0.01, *P < 0.05). B, Colo357 and Panc-1 cells were infected (at 5 MOI) with either AdFOP-PUMA (left) or AdTOP-PUMA (right), and GFP expression was visualized by fluorescence microscopy after 48 h.

 
AdTOP-PUMA and Chemotherapy Synergistically Induce Tumor Cell Death
We investigated whether the combination of AdTOP-PUMA adenovirus and chemotherapeutic agents more efficiently induce apoptosis in colorectal cancer cells than each treatment alone. HCT116, SW480, and HT29 cells were infected with either AdTOP-PUMA, AdFOP-PUMA, or Ad-CMV-GFP adenoviruses (at 5 MOI) and cultured for 48 hours in the presence or absence of paclitaxel (0.05 µmol/L), doxorubicin (1 µmol/L), or 5-florouracil (0.05 µmol/L). AdTOP-PUMA adenovirus dramatically enhanced the killing effect by all three chemotherapeutic agents in both SW480 and HCT116 cells, but not in HT29 cells (Fig. 7 ). The efficacy of the chemotherapeutic agents was not augmented by exposure of the different cells to either Ad-CMV-GFP or AdFOP-PUMA adenovirus constructs (Fig. 7).


Figure 7
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  		Figure 7. Combination effect of AdTOP-PUMA and chemotherapeutic agents in colon cancer cells. Cells were infected with the different viruses (5 MOI) and treated with various anticancer agents [paclitaxel, doxorubicin, and 5-florouracil (5-FU)]. One hundred percent indicates the viability of cells treated with PBS, without any agent or viral vector. Cell viability was determined 48 h after treatment with the various agents. Columns, mean of two quadruplicate assays; bars, SD. **, P < 0.01, significantly different from the AdTOP-PUMA (virus alone).

 
Discussion

Although gene therapy–based clinical trials showed significant success (31–33) in a variety of human tumors, thus far they failed to show a significant therapeutic effect in colorectal cancer. By contrast, in cell lines and in experimental animals, remarkable results, including complete regression of the tumor, have been shown by this approach (34, 35). The lack of efficacy of this approach in humans might result from the poor targeting selectivity of the vectors that could lead to low levels of expression of the transferred gene in tumor cells and to high toxicity of the gene product in normal cells.

Adenoviral systems are easy to produce and they have a highly effective nuclear entry mechanism and display very low pathogenicity in humans (36). The adenoviral vectors can transduce cells in vivo and they do not integrate into the host cell genome (36). Mulvihill et al. (37) conducted a phase I clinical trial of adenoviral administration using ONYX-015 that consists of an adenovirus expressing a deletion mutant of E1B-55 that selectively replicates in and lyses tumor cells displaying a mutant p53. The virus was injected through hepatic arterial catheters to patients with colorectal cancer liver metastases, and was well tolerated at doses up to 1011 plaque-forming units. A phase I clinical trial using the Escherichia coli cytosine deaminase for the treatment of metastatic colon cancer is currently being conducted (38).

In the present study, we show that targeting ß-catenin/Tcf responsive transcription can selectively and efficiently kill tumor cells displaying high levels of ß-catenin/Tcf transactivation, with minimal toxicity to cells displaying low levels of ß-catenin–Tcf signaling. Similar strategies were used and recently reported by others (39, 40). In Chen and McCormick's work (39), an adenoviral vector, AdWt-Fd, containing the thymidine kinase promoter carrying the proapoptotic gene Fadd, selectively killed colorectal cancer cells in vitro. Kwong et al. (40) used an in vitro-in vivo animal model similar to the one used in the present study. They showed selective killing of DLD-1 colorectal cancer cells in an ex vivo animal model, by the adenoviral vector AdTOP-CMV-TK that contains a herpes simplex virus thymidine kinase gene under the control of a ß-catenin/Tcf–responsive promoter linked to a minimal CMV promoter. Lipinski et al. (41) optimized the activity and specificity profile of a synthetic catenin-dependent promoter by varying its basal promoter, the number of Tcf-binding sites, and the distance between them and the basal promoter. The optimal promoter showed virtually undetectable expression in cells with normal ß-catenin regulation, but displayed high activity in cells expressing deregulated ß-catenin. Malerba et al. (42) inserted Tcf-binding sites into the viral P4 promoter and showed that reduction of the number of Tcf sites from four to two leads to an increase in the efficiency of replication and toxicity of the virus in Co115 colon cancer cells.

The current study took this strategy further in several aspects: (1) Several genes known to be associated with apoptosis induction (Bax, Bak, Bid, and caspase-8) were evaluated for their apoptotic effect. Among these, we found that PUMA, a critical mediator of p53-dependent apoptosis following DNA damage (25, 26), was the most effective one. There are only two more recent reports demonstrating the therapeutic value of PUMA: one in malignant glioma (43) and another in esophageal carcinoma cells (44). (2) Many cancers harbor mutations in the p53 gene. Adenovirus-mediated p53 gene transfer has been extensively studied as a plausible novel gene therapy strategy in various cancers. However, a variety of cancer cells are resistant to this novel therapy (45, 46). We hypothesized that the PUMA gene located downstream to p53 pathways may serve as a good alternative strategy to p53 gene therapy as it can kill cancer cells directly. (3) In this study, PUMA expression was induced in four human colorectal cancer cell lines (SW480, HCT116, DLD-1, and LS174T) that display hyperactive ß-catenin/Tcf signaling. The induction of PUMA expression in these cells using the TOP, but not the FOP, construct resulted in significant cell killing. PUMA expression was not induced in cells with low or undetectable ß-catenin signaling (HT-29). (4) Hyperactivity of ß-catenin signaling is not unique to colorectal cancer cells. Hence, the effect of these viruses was also evaluated in hepatocellular carcinoma (HepG2) and gastric cancer cells (AGS that harbor activating ß-catenin mutations; refs. 21, 47). We found that both cell lines were very sensitive to treatment with the AdTOP-PUMA adenovirus. In particular, HepG2 cells were 10 times more sensitive than the other cell lines, including SW480 cells that contain a very potent transactivating ß-catenin/Tcf complex (Fig. 6). In contrast, pancreatic cancer cell lines (Colo357 and Panc-1) that do not possess an active ß-catenin signaling pathway (48–51) were not affected by this therapy. Of special note is that although SW480 cells have the highest ß-catenin/Tcf activity (Table 1), the number of apoptotic SW480 cells is lower after 24 hours (Fig. 4A) than in HCT116 cells that have weaker ß-catenin/Tcf transactivation. This suggests that the effect of AdTOP-PUMA virus most probably depends on additional regulatory mechanism(s) in the cell besides transactivation by ß-catenin/Tcf, and different cancer cells display varying sensitivities to different killing agents as was shown in Fig. 7.

A proof of concept of this gene therapy strategy was shown in the in vitro-in vivo experiments. Indeed, s.c. injection of SW480 cells infected with AdTOP-PUMA did not produce tumors in any of the animals, whereas injection of SW480 cells infected with AdFOP-PUMA or Ad-CMV-GFP induced tumors in the majority of nude mice. This confirms that this strategy could be useful in targeting human cancer cells.

We have also shown that a combination of the gene therapy approach with chemotherapeutic agents that have a distinct mechanism of action might be an effective route in achieving a better antitumor response. Thus, the combination of AdTOP-PUMA adenovirus and paclitaxel, doxorubicin, and 5-florouracil dramatically enhanced the cell killing effect. This strategy could be particularly useful in the treatment of chemotherapy-resistant colorectal cancer (52) because it might minimize the toxicity of this regimen. Adenoviruses practically infect all types of cells in the body and, therefore, PUMA will probably be activated only in cells with hyperactive ß-catenin signaling. Hence, in the clinical setting, combination therapy using adenovirus and chemotherapeutic agents would be suitable not only for the metastatic disease, but also at stages II and III of the disease, aiming to eradicate microscopic residual tumor cells.

Recently, we showed that a Ras-responsive element selectively kills tumor cells without affecting the growth and survival of normal cells (53). Introduction of cell death genes (bax, caspase-8, and mutant PKG) under the control of a promoter containing wild-type Ets and activator protein-1 binding sites resulted in preferential killing of cells displaying a mutant, but not a normal, Ras pathway.

In conclusion, this strategy involving an active ß-catenin/Tcf pathway is not a cell- or organ-specific approach, but could be applied to a much wider range of cases because hyperactive ß-catenin/Tcf signaling is found in a significant percentage of almost all types of cancer (2, 54). The results of the current study may pave the way to new combined approaches with chemotherapy in the setting of all stages of colorectal cancer with activated ß-catenin signaling.

Acknowledgments

We thank Moshe Oren (Weizmann Institute of Science, Israel) for helpful advice and thorough discussions, Hila Giladi (Hebrew University, Jerusalem, Israel) for assistance with adenovirus production, and Naham Kariv (Tel Aviv University, Israel) for assistance with the animal studies.

Footnotes

Grant support: Israel Cancer Association (N. Arber), Israel Science Foundation (A. Ben-Ze'ev), and the German-Israel Foundation for Scientific Research and Development (A. Ben-Ze'ev).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: This work was part of the requirements of Hadas Dvory-Sobol for her Ph.D. degree at the Sackler School of Medicine at Tel Aviv University.

Received 3/ 6/06; revised 8/19/06; accepted 9/11/06.

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