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Targeting the Active B-Catenin Pathway to Treat Cancer Cells

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Targeting the Active B-Catenin Pathway to Treat Cancer Cells 2861 Targeting the active B-catenin pathway to treat cancer cells Hadas Dvory-Sobol,1,2 Eyal Sagiv,1,2 PUMA, whereas that of cells with low levels of B-catenin Diana Kazanov,1 Avri Ben-Ze’ev,3 and signaling was not. Growth inhibition was associated with Nadir Arber1,2 induction of apoptosis. Chemotherapy synergistically enhanced the effect of AdTOP-PUMA. A combination of 1Integrated Cancer Prevention Center, Tel Aviv Sourasky the adenovirus system with standard therapy may 2 Medical Center; Sackler School of Medicine, Tel-Aviv University, improve the efficacy and reduce the toxicity of therapy Tel-Aviv, Israel; 3Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel in humans. [Mol Cancer Ther 2006;5(11):2861–71] Abstract Introduction The adenomatous polyposis coli or b-catenin genes are h-Catenin is a multifunctional protein serving as a major frequently mutated in colorectal cancer cells, resulting in structural component of cell-to-cell adherens junctions. In oncogenic activation of B-catenin signaling. We tried to addition, it also acts as an important signaling molecule in establish in vitro and in vivo models for selectively the Wnt pathway that plays a key role in embryogenesis killing human cancer cells with an activated B-catenin/ and tumorigenesis (1–3). T-cell factor (Tcf) pathway. We used a recombinant In the absence of Wnt signaling, the cytoplasmic level of adenovirus that carries a lethal gene [p53-up-regulated h-catenin is kept low through interaction with a protein modulator of apoptosis (PUMA)] under the control of a complex [containing GSK3h-glycogen synthase kinase 3h, B-catenin/Tcf–responsive promoter (AdTOP-PUMA) to axin and adenomatous polyposis coli (APC)] that can selectively target human colorectal cancer cells phosphorylate h-catenin and target it to ubiquitin-mediated (SW480, HCT116, DLD-1, and LS174T), hepatocellular proteasomal degradation (4). Activation of Wnt signaling carcinoma (HepG2), and gastric cancer cells (AGS) in leads to inactivation of GSK3h, resulting in cytoplasmic which the B-catenin/Tcf pathway is activated, and accumulation of h-catenin (5). The increase in h-catenin compared its efficiency in killing cancer cells in which level is followed by its translocation into the nucleus, this pathway is inactive or only weakly active. AdFOP- where in complex with members of the T-cell factor (Tcf)/ PUMA, carrying a mutant Tcf-binding site, was used as lymphocyte enhancer–binding factor family of transcrip- control virus. The combined effect of AdTOP-PUMA with tion factors it activates the expression of target genes (6). several chemotherapeutic agents (5-florouracil, doxorubi- The APC tumor suppressor is mutated in f80% of the cin, and paclitaxel) was also evaluated. The effect of familial adenomatous polyposis syndrome and sporadic AdTOP-PUMA on colorectal cancer cells was also colorectal cancer patients (7). Loss of APC is believed to be examined in nude mice: SW480 cells were infected with one of the earliest initiating events in multistage colorectal the AdTOP-PUMA and AdFOP-PUMA, and then inoculat- carcinogenesis (8). Mutant APC loses its ability to direct h- ed s.c. into nude mice. The TOP-PUMA adenovirus catenin to degradation, resulting in nuclear accumulation inhibited cell growth in a dose-dependent fashion, and inappropriate activation of h-catenin–mediated trans- depending on the signaling activity of B-catenin. The activation. Mutations in h-catenin in the GSK-3h phosphor- growth of cells displaying high levels of active B-catenin/ ylation sites have been identified in 50% of colorectal Tcf signaling was inhibited after infection with AdTOP- 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 h-catenin/Tcf with direct implications in tumorigenesis (15–19). Activating h-catenin Received 3/6/06; revised 8/19/06; accepted 9/11/06. mutations have also been identified in a variety of other Grant support: Israel Cancer Association (N. Arber), Israel Science Foundation (A. Ben-Ze’ev), and the German-Israel Foundation for Scientific tumors, including melanomas (20), hepatocellular carcino- Research and Development (A. Ben-Ze’ev). mas (21), skin (22), breast (23), and prostate cancers (24), The costs of publication of this article were defrayed in part by the whereas the h-catenin–Tcf pathway is not activated in payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to most normal tissues. Therefore, a therapeutic strategy that indicate this fact. targets this pathway could be applied to patients with Note: This work was part of the requirements of Hadas Dvory-Sobol for primary or metastatic colorectal cancer. her Ph.D. degree at the Sackler School of Medicine at Tel Aviv University. p53-up-regulated modulator of apoptosis (PUMA) is a Requests for reprints: Nadir Arber, Director-Integrated Cancer Prevention potent mediator of the p53 apoptotic response (25, 26). Center, Tel-Aviv Medical Center, 6 Weizmann Street, Tel-Aviv 64239, Israel. Phone: 972-3-6974968; Fax: 972-3-6950339. It belongs to the group of BH3-only proteins that have E-mail: [email protected] or [email protected] been shown to function by dimerization with other BH3 Copyright C 2006 American Association for Cancer Research. domain–containing proteins, including Bcl-2 and Bcl-XL, doi:10.1158/1535-7163.MCT-06-0122 that results in the release of cytochrome c from Mol Cancer Ther 2006;5(11). November 2006 Downloaded from mct.aacrjournals.org on September 23, 2021. © 2006 American Association for Cancer Research. 2862 Targeting activated b-Catenin in Human Cancer Cells mitochondria and induction of apoptosis by activation of the human PUMA cDNA fused to a double hemagglutinin- caspase-3 and caspase-9 (27). epitope tag) from pCEP4-PUMA (a generous gift from Bert A substantial limitation of conventional cancer chemo- Vogelstein, Johns Hopkins Oncology Center, Baltimore, therapy and radiotherapy is the toxicity of these agents to MD) was cloned downstream to the TOP/FOP elements normal tissue. The toxicity of currently available gene in the pAd-Track vector. The resultant plasmids were delivery systems to the normal cell population results from designated pAdTrack-TOP/FOP-PUMA. These shuttle their toxicity to the normal cell population. Here, we propose vectors were linearized with PmeI and cotransformed with a novel gene therapy approach that selectively expresses a E1-deleted adenoviral backbone AdEasy-1 into the compe- lethal gene by targeting the active h-catenin–Tcf pathway in tent bacterial strain BJ5183, which enables efficient recom- human colorectal, gastric, and hepatic cancer cells. More- bination. A panel of Ad-TOP-PUMA and Ad-FOP-PUMA over, we show that combining this strategy with standard recombinant adenoviruses were generated. chemotherapy results in a synergistic growth inhibition of Adenovirus Production and Titering colorectal cancer cells that overcomes cancer cell resistance To produce viruses, 4 Ag PacI-linearized adenoviral to therapy. This approach may pave the way to a novel DNA was transfected into 50% to 70% confluent 293 cells treatment of primary and metastatic colorectal cancer. in 10-cm dishes using LipofectAMINE and Plus Reagents (Invitrogen Life Technologies, Carlsbad, CA). Between 5 and 7 days posttransfection, colonies expressing GFP were Materials and Methods observed under a fluorescent microscope, the cells were Cell Culture harvested and lysed in PBS by four cycles of freeze/thaw/ Human colorectal cancer (SW480, DLD-1, HT-29, vortex (Fig. 1C). The supernatant was collected and half of HCT116, LS174T), gastric (AGS), hepatic (HepG2, it was used to reinfect 50% to 70% confluent 293 cells. SK-Hep-1), pancreatic (Colo357, Panc-1), and embryonic Viruses were collected 2 to 3 days postinfection when a kidney (293) cell lines were obtained from the American cytopathic effect became evident. Further amplification Type Culture Collection (Manassas, VA). They were and concentration of the virus stocks was achieved cultured in DMEM (Sigma, Rehovot, Israel) containing through several rounds of infection. To titer the viruses, 5% to 10% fetal bovine serum (Biological Industries, Beit 50% to 70% confluent 293 cells in 96-well dishes were Haemek, Israel), 1% penicillin, and 1% streptomycin, at infected with serial dilutions of the virus stocks. GFP- j 37 C, in an atmosphere of 95% oxygen and 5% CO2 positive colonies were counted 5 days postinfections. The (complete medium). control Ad-CMV-GFP adenovirus containing the GFP gene Construction of Plasmids and Adenoviral Vectors under the control of a full-length CMV promoter was a Two sets of h-catenin/Tcf–responsive promoters were generated: one contains wild-type Tcf/lymphocyte enhanc- er–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). 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 Figure 1.
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