β-Catenin/Tcf-4 Inhibition After Progastrin Targeting Reduces Growth and Drives Differentiation of Intestinal Tumors
2007; Elsevier BV; Volume: 133; Issue: 5 Linguagem: Inglês
10.1053/j.gastro.2007.08.023
ISSN1528-0012
AutoresJulie Pannequin, Nathalie Delaunay, Michael Büchert, Fanny Surrel, Jean–François Bourgaux, Joanne Ryan, Stéphanie Boireau, Jessica Coelho, André Pèlegrin, Pomila Singh, Arthur Shulkes, Mildred Yim, Graham S. Baldwin, C Pignodel, Gérard Lambeau, Philippe Jay, Dominique Joubert, Frédéric Hollande,
Tópico(s)Genetic factors in colorectal cancer
ResumoBackground & Aims: Aberrant activation of the β-catenin/Tcf-4 transcriptional complex represents an initiating event for colorectal carcinogenesis, shifting the balance from differentiation toward proliferation in colonic crypts. Here, we assessed whether endogenous progastrin, encoded by a target gene of this complex, was in turn able to regulate β-catenin/Tcf-4 activity in adenomatous polyposis coli (APC)-mutated cells, and we analyzed the impact of topical progastrin depletion on intestinal tumor growth in vivo. Methods: Stable or transient RNA silencing of the GAST gene was induced in human tumor cells and in mice carrying a heterozygous Apc mutation (APCΔ14), which overexpress progastrin but not amidated or glycine-extended gastrin. Results: Depletion of endogenous progastrin production strongly decreased intestinal tumor growth in vivo through a marked inhibition of constitutive β-catenin/Tcf-4 activity in tumor cells. This effect was mediated by the de novo expression of the inhibitor of β-catenin and Tcf-4 (ICAT), resulting from a down-regulation of integrin-linked kinase in progastrin-depleted cells. Accordingly, ICAT down-regulation was correlated with progastrin overexpression and Tcf-4 target gene activation in human colorectal tumors, and ICAT repression was detected in the colon epithelium of tumor-prone, progastrin-overexpressing mice. In APCΔ14 mice, small interfering RNA–mediated progastrin depletion not only reduced intestinal tumor size and numbers, but also increased goblet cell lineage differentiation and cell apoptosis in the remaining adenomas. Conclusions: Thus, depletion of endogenous progastrin inhibits the tumorigenicity of APC-mutated colorectal cancer cells in vivo by promoting ICAT expression, thereby counteracting Tcf-4 activity. Progastrin targeting strategies should provide an exciting prospect for the differentiation therapy of colorectal cancer. Background & Aims: Aberrant activation of the β-catenin/Tcf-4 transcriptional complex represents an initiating event for colorectal carcinogenesis, shifting the balance from differentiation toward proliferation in colonic crypts. Here, we assessed whether endogenous progastrin, encoded by a target gene of this complex, was in turn able to regulate β-catenin/Tcf-4 activity in adenomatous polyposis coli (APC)-mutated cells, and we analyzed the impact of topical progastrin depletion on intestinal tumor growth in vivo. Methods: Stable or transient RNA silencing of the GAST gene was induced in human tumor cells and in mice carrying a heterozygous Apc mutation (APCΔ14), which overexpress progastrin but not amidated or glycine-extended gastrin. Results: Depletion of endogenous progastrin production strongly decreased intestinal tumor growth in vivo through a marked inhibition of constitutive β-catenin/Tcf-4 activity in tumor cells. This effect was mediated by the de novo expression of the inhibitor of β-catenin and Tcf-4 (ICAT), resulting from a down-regulation of integrin-linked kinase in progastrin-depleted cells. Accordingly, ICAT down-regulation was correlated with progastrin overexpression and Tcf-4 target gene activation in human colorectal tumors, and ICAT repression was detected in the colon epithelium of tumor-prone, progastrin-overexpressing mice. In APCΔ14 mice, small interfering RNA–mediated progastrin depletion not only reduced intestinal tumor size and numbers, but also increased goblet cell lineage differentiation and cell apoptosis in the remaining adenomas. Conclusions: Thus, depletion of endogenous progastrin inhibits the tumorigenicity of APC-mutated colorectal cancer cells in vivo by promoting ICAT expression, thereby counteracting Tcf-4 activity. Progastrin targeting strategies should provide an exciting prospect for the differentiation therapy of colorectal cancer. Various strategies that aim to reduce tumor development through the induction of tumor cell differentiation have been developed in recent years, providing very promising results in animal models and in human patients. In particular, the use of all-trans–retinoic acid has proven extremely useful in the treatment of acute promyelocytic leukemia,1Lallemand-Breitenbach V. Zhu J. Kogan S. et al.Opinion: how patients have benefited from mouse models of acute promyelocytic leukaemia.Nat Rev Cancer. 2005; 5: 821-827Crossref PubMed Scopus (35) Google Scholar, 2Wang Z.Y. Chen Z. Differentiation and apoptosis induction therapy in acute promyelocytic leukaemia.Lancet Oncol. 2000; 1: 101-106Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar and is considered a promising approach to force tumor differentiation in advanced thyroid cancer.3Coelho S.M. Vaisman M. Carvalho D.P. Tumour re-differentiation effect of retinoic acid: a novel therapeutic approach for advanced thyroid cancer.Curr Pharm Des. 2005; 11: 2525-2531Crossref PubMed Scopus (47) Google Scholar In the intestine, great hopes have been placed on agents modulating 2 major pathways involved in the control of cell proliferation and differentiation, namely the Notch cascade and the Wnt/β-catenin/Tcf-4 pathway.4Radtke F. Clevers H. Self-renewal and cancer of the gut: two sides of a coin.Science. 2005; 307: 1904-1909Crossref PubMed Scopus (594) Google Scholar, 5van Es J.H. Clevers H. Notch and Wnt inhibitors as potential new drugs for intestinal neoplastic disease.Trends Mol Med. 2005; 11: 496-502Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar Notch activation is known to inhibit cell differentiation while amplifying the intestinal progenitor pool,6Fre S. Huyghe M. Mourikis P. et al.Notch signals control the fate of immature progenitor cells in the intestine.Nature. 2005; 435: 964-968Crossref PubMed Scopus (742) Google Scholar and inactivation of Notch signaling with γ-secretase inhibitors was found to redirect proliferative intestinal tumor cells toward a goblet cell phenotype in intestinal crypts and adenomas.7van Es J.H. van Gijn M.E. Riccio O. et al.Notch/gamma-secretase inhibition turns proliferative cells in intestinal crypts and adenomas into goblet cells.Nature. 2005; 435: 959-963Crossref PubMed Scopus (1302) Google Scholar In addition, conditional inactivation of the Tcf-4 target oncogene MYC was shown to push sarcoma cells toward differentiation,8Jain M. Arvanitis C. Chu K. et al.Sustained loss of a neoplastic phenotype by brief inactivation of MYC.Science. 2002; 297: 102-104Crossref PubMed Scopus (554) Google Scholar and modulation of the β-catenin/Tcf-4 pathway in transgenic mice regulates several genes associated with cell differentiation in the intestine.9van de Wetering M. Sancho E. Verweij C. et al.The beta-catenin/TCF-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells.Cell. 2002; 111: 241-250Abstract Full Text Full Text PDF PubMed Scopus (1773) Google Scholar Several new compounds inhibiting the association between β-catenin and Tcf-4 were identified recently through large-scale library screening,10Lepourcelet M. Chen Y.-N.P. France D.S. et al.Small-molecule antagonists of the oncogenic Tcf/[beta]-catenin protein complex.Cancer Cell. 2004; 5: 91-102Abstract Full Text Full Text PDF PubMed Scopus (607) Google Scholar and could offer promising avenues for colorectal cancer (CRC) treatment by inducing the differentiation of tumor cells. Yet, these pathways are essential for the homeostasis of intestinal crypts, and approaches directly targeting them in colon tumors are likely to have adverse effects on overall intestinal physiology. Ideally, such strategies would greatly benefit from the capacity to identify targets that are activated selectively, or stimulated strongly in tumor cells, while being absent or inactivated in the surrounding tissues. Among the targets specifically activated in intestinal tumor cells, partially processed gastrin gene (GAST) products, such as glycine-extended gastrin (Ggly) and progastrin, offer an interesting prospect for the selective targeting of tumor growth. Indeed, the GAST gene itself is a target of both Tcf-4 and K-Ras,11Chakladar A. Dubeykovskiy A. Wojtukiewicz L.J. et al.Synergistic activation of the murine gastrin promoter by oncogenic Ras and beta-catenin involves SMAD recruitment.Biochem Biophys Res Commun. 2005; 336: 190-196Crossref PubMed Scopus (35) Google Scholar, 12Koh T.J. Bulitta C.J. Fleming J.V. et al.Gastrin is a target of the beta-catenin/TCF-4 growth-signaling pathway in a model of intestinal polyposis.J Clin Invest. 2000; 106: 533-539Crossref PubMed Scopus (175) Google Scholar two pathways that frequently are activated and act synergistically in colorectal cancers,13Janssen K.P. Alberici P. Fsihi H. et al.APC and oncogenic KRAS are synergistic in enhancing Wnt signaling in intestinal tumor formation and progression.Gastroenterology. 2006; 131: 1096-1109Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar and these GAST-derived peptides seem to stimulate proliferation via an autocrine/paracrine loop on tumor cells.14Hollande F. Imdahl A. Mantamadiotis T. et al.Glycine-extended gastrin acts as an autocrine growth factor in a nontransformed colon cell line.Gastroenterology. 1997; 113: 1576-1588Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 15Hollande F. Lee D.J. Choquet A. et al.Adherens junctions and tight junctions are regulated via different pathways by progastrin in epithelial cells.J Cell Sci. 2003; 116: 1187-1197Crossref PubMed Scopus (70) Google Scholar The proliferative effect of Ggly was discovered during the past few years,16Seva C. Dickinson C.J. Yamada T. Growth-promoting effects of glycine-extended progastrin.Science. 1994; 265: 410-412Crossref PubMed Scopus (318) Google Scholar and, more recently, progastrin was shown to stimulate proliferation17Ottewell P.D. Varro A. Dockray G.J. et al.COOH-terminal 26-amino acid residues of progastrin are sufficient for stimulation of mitosis in murine colonic epithelium in vivo.Am J Physiol. 2005; 288: G541-G549Crossref PubMed Scopus (36) Google Scholar, 18Singh P. Velasco M. Given R. et al.Mice overexpressing progastrin are predisposed for developing aberrant colonic crypt foci in response to AOM.Am J Physiol. 2000; 278: G390-G399PubMed Google Scholar and to modulate epithelial cell/cell adhesion and migration.15Hollande F. Lee D.J. Choquet A. et al.Adherens junctions and tight junctions are regulated via different pathways by progastrin in epithelial cells.J Cell Sci. 2003; 116: 1187-1197Crossref PubMed Scopus (70) Google Scholar, 19Hollande F. Shulkes A. Baldwin G.S. Signaling the junctions in gut epithelium.Sci STKE. 2005; 2005: pe13PubMed Google Scholar In addition, experiments using animals with a targeted deletion of the GAST gene argued in favor of a tumor-promoting role for products of this gene.12Koh T.J. Bulitta C.J. Fleming J.V. et al.Gastrin is a target of the beta-catenin/TCF-4 growth-signaling pathway in a model of intestinal polyposis.J Clin Invest. 2000; 106: 533-539Crossref PubMed Scopus (175) Google Scholar Yet, they provided little information concerning the potential for treatments impairing the function of these peptides to slow down or reverse the growth of pre-existing tumors. Critically, progastrin is produced and secreted in significant quantities by almost 80% of human colorectal tumors and tumor cell lines, whereas its secretion mostly decreases to levels less than what can be detected, under physiologic conditions.20Ciccotosto G.D. McLeish A. Hardy K.J. et al.Expression, processing, and secretion of gastrin in patients with colorectal carcinoma.Gastroenterology. 1995; 109: 1142-1153Abstract Full Text PDF PubMed Scopus (183) Google Scholar, 21Konturek P.C. Bielanski W. Konturek S.J. et al.Progastrin and cyclooxygenase-2 in colorectal cancer.Dig Dis Sci. 2002; 47: 1984-1991Crossref PubMed Scopus (34) Google Scholar, 22Siddheshwar R.K. Gray J.C. Kelly S.B. Plasma levels of progastrin but not amidated gastrin or glycine extended gastrin are elevated in patients with colorectal carcinoma.Gut. 2001; 48: 47-52Crossref PubMed Scopus (72) Google Scholar, 23Van Solinge W.W. Nielsen F.C. Friis-Hansen L. et al.Expression but incomplete maturation of progastrin in colorectal carcinomas.Gastroenterology. 1993; 104: 1099-1107Abstract PubMed Scopus (0) Google Scholar Therefore, because the constitutive activation of the Wnt signaling pathway represents a hallmark of sporadic CRC, as well as of the hereditary condition of adenomatous polyposis,24Fodde R. Smits R. Clevers H. APC, signal transduction and genetic instability in colorectal cancer.Nat Rev Cancer. 2001; 1: 55-67Crossref PubMed Scopus (785) Google Scholar and because the GAST gene is itself a target of Tcf-4, we investigated whether progastrin was able, in turn, to modulate adenomatous polyposis coli (APC) mutation–driven tumorigenesis, and whether targeting progastrin could represent a relevant strategy to reduce the growth of intestinal tumors. We show that RNA interference-mediated progastrin depletion was capable of inhibiting tumor growth induced by a mutation of the APC gene in vivo. We then show that this inhibition reflected the capacity of progastrin to modulate the level of β-catenin/Tcf-4 transcriptional activity in tumor cells, despite its constitutive activation triggered by the APC mutation. De novo expression of the inhibitor of β-catenin and Tcf-4 binding (the inhibitor of β-catenin and Tcf-4 [ICAT]) was instrumental in inhibiting the β-catenin/Tcf-4 transcriptional pathway and in decreasing tumorigenicity in progastrin-depleted tumor cells, whereas repression of ICAT was detected in progastrin-overexpressing mouse colonic mucosa, and in human and mouse intestinal tumors. Finally, treatment with GAST-specific small interfering RNA (siRNA) induced a terminal differentiation toward the goblet cell lineage, not only of human CRC cells in vitro, but also of the progastrin-secreting intestinal adenomas of mice carrying a heterozygous mutation of the Apc gene. Primary antibodies were as follows: monoclonal antidephosphorylated β-catenin (AG Scientific, Paris, France); anti–β-catenin and anti–E-cadherin (Transduction Laboratories, InVitrogen, Lexington, KY); anti–Muc-2 and anti–CD 44 (Neomarkers, Lab Vision, Astmoor, UK); rabbit anti-ICAT,25Gottardi C.J. Gumbiner B.M. Role for ICAT in beta-catenin-dependent nuclear signaling and cadherin functions.Am J Physiol. 2004; 286: C747-C756Crossref PubMed Scopus (66) Google Scholar anti-phosphatase and tensin homologue deleted from chromosome 10 (Upstate Cell Signaling, Lake Placid, NY); antiphosphoserin and anti–claudin-1 (Zymed, San Francisco, CA); anti–activated caspase-3 (Upstate Cell Signaling); anti–cyclin D1 (Neomarkers); and anti–Sox-9,26Blache P. van de Wetering M. Duluc I. et al.SOX9 is an intestine crypt transcription factor, is regulated by the Wnt pathway, and represses the CDX2 and MUC2 genes.J Cell Biol. 2004; 166: 37-47Crossref PubMed Scopus (396) Google Scholar goat anti–Tcf-4 (Santa Cruz Biotechnology; Tebu-Bio SA, Le Perray en Yvelines, France). The Src family kinase inhibitor PP2 (4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) and the MEK-1 inhibitor PD98059 (2′-amino-3′-methoxyflavone) were from Calbiochem (Merck Chemicals Ltd, Nottingham, UK).26Blache P. van de Wetering M. Duluc I. et al.SOX9 is an intestine crypt transcription factor, is regulated by the Wnt pathway, and represses the CDX2 and MUC2 genes.J Cell Biol. 2004; 166: 37-47Crossref PubMed Scopus (396) Google Scholar Specimens of colon tumors and histologically normal epithelium (taken at a significant distance from the tumor from 23 patients) were obtained from the pathologist after resection according to French government regulations and with approval of the ethical committee (Nîmes Hospital). Informed consent was obtained from all patients. Tissue samples were stored in liquid nitrogen until further use. Short hairpin RNA (shRNA) oligonucleotides for ICAT (supplementary Figure 1; see supplementary material online at www.gastrojournal.org) were cloned into pSIREN-retroQ (Clontech, Ozyme, Saint Quentin Yvelines, France). The amphotropic packaging cell line (ΦNX; Dr Garry Nolan, Stanford University, Stanford, CA) was transfected as described in Korinek et al.27Pear W.S. Nolan G.P. Scott M.L. et al.Production of high-titer helper-free retroviruses by transient transfection.Proc Natl Acad Sci U S A. 1993; 90: 8392-8396Crossref PubMed Scopus (2335) Google Scholar The medium containing the virus was removed 48 hours later and centrifuged (1500 rpm, 5 min) to pellet cell debris. Target cells (2 × 105) were infected in the presence of polybrene (8 μg/mL) with 2 mL of virus-containing medium. Positive clones were selected using puromycin (5 μg/mL). Tissue sections were prepared from liquid nitrogen–frozen tumor samples. Laser capture microdissection was performed using a PixCell IIe microdissector (Arcturus/Alphelys, Plaisir, France), using the following settings: 265 mV, 45 mWh, 15 μm diameter, 1.8 ms. RNA was prepared from microdissected tissues using the RNAeasy Microkit (Qiagen, Courtaboeuf, France). The quality and amount of RNA recovered were assessed using RNA pico Labchips (Agilent Technologies, Palo Alto, CA). In vivo experiments were performed following the French guidelines for experimental animal studies (Direction des Services Vétérinaires, Ministère de l'Agriculture, Agreement no. B34-172-27) and fulfilled the United Kingdom Coordinating Committee on Cancer Research guidelines for the welfare of animals in experimental neoplasia. Cells (2 × 106) were injected subcutaneously into 6-week-old athymic BALB/c-nu/nu (nude) mice. Tumor growth was followed up regularly thereafter (estimated tumor volume = [length × width × thickness]/2). APCΔ14 mice were housed in conventional conditions, and randomized into 2 groups. The first group (n = 9) was treated once daily by intraperitoneal administration of 250 μg/kg siRNA against the murine GAST messenger RNA (mRNA). Control mice (n = 9) were treated with a similar dose of siRNA against luciferase (siRNA sequence in Supplementary Figure 1; see supplementary material online at www.gastrojournal.org). SiRNA design incorporated 3' overhangs and did not contain a 5'UGUGU3' sequence, to minimize proinflammatory response and degradation in vivo.28Judge A.D. Sood V. Shaw J.R. et al.Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA.Nat Biotechnol. 2005; 23: 457-462Crossref PubMed Scopus (1033) Google Scholar, 29Marques J.T. Devosse T. Wang D. et al.A structural basis for discriminating between self and nonself double-stranded RNAs in mammalian cells.Nat Biotechnol. 2006; 24: 559-565Crossref PubMed Scopus (329) Google Scholar After a 10-day treatment, mice were culled, their blood collected, and their intestine dissected out. Adenoma scoring in the intestine and colon has been described by Sekiya et al.30Colnot S. Niwa-Kawakita M. Hamard G. et al.Colorectal cancers in a new mouse model of familial adenomatous polyposis: influence of genetic and environmental modifiers.Lab Invest. 2004; 84: 1619-1630Crossref PubMed Scopus (146) Google Scholar Samples of healthy intestinal mucosa, as well as intestinal and colon adenomas, were fixed in 4% paraformaldehyde (PFA) and paraffin-embedded, or frozen in liquid nitrogen, for Western blotting, immunohistochemistry, or RNA extraction. Plasma was prepared and assayed for interleukin-6 and tumor necrosis factor α using the FACSarray system (Becton Dickinson; Pont de Claix, France). Statistical analyses were performed using SAS 9.1 for Windows (Cary, NC). The Student t test was used to assess the significance of differences in in vitro studies. The Spearman rank test was used to determine the correlation between the non-normally distributed GAST and ICAT gene expressions in human tumors. The chi-square test was used to assess differences in tumor size between APCΔ14 animals treated with GAST or luciferase siRNA. The Fisher exact test was used to perform similar analyses on colon tumors, in view of the smaller sample size. The Mann–Whitney rank sum test was used to analyze differences between goblet cell counts and Muc-2 mRNA levels in APCΔ14 mice. To assess whether a strategy aimed at selectively targeting endogenous progastrin in pre-existing tumors would be able to antagonize Tcf-4–promoted tumor growth in vivo, we used Balbc/nude mice xenografts from the colorectal cell line SW480,31Morin P.J. Sparks A.B. Korinek V. et al.Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC.Science. 1997; 275: 1787-1790Crossref PubMed Scopus (3565) Google Scholar as well as a mouse model of spontaneous intestinal tumorigenesis, APCΔ14.30Colnot S. Niwa-Kawakita M. Hamard G. et al.Colorectal cancers in a new mouse model of familial adenomatous polyposis: influence of genetic and environmental modifiers.Lab Invest. 2004; 84: 1619-1630Crossref PubMed Scopus (146) Google Scholar These 2 models bear mutations of APC and consequently display a constitutive activation of the β-catenin/Tcf-4 pathway, similar to that found in human familial adenomatous polyposis and sporadic CRC.32Korinek V. Barker N. Morin P.J. et al.Constitutive transcriptional activation by a beta-catenin-Tcf complex in APC-/- colon carcinoma.Science. 1997; 275: 1784-1787Crossref PubMed Scopus (2983) Google Scholar Control SW480/βgal(-) cells were shown to express high progastrin levels but little, if any, of the amidated and glycine-extended forms of gastrin (Figure 1A). Within 7 weeks of subcutaneous injection in Balbc/nude mice, these cells produced large tumors (Figure 1B). In contrast, the ability to form tumors within the same time span was greatly reduced when progastrin secretion was down-regulated by stable expression of shRNA, directed against the GAST gene (SW480/GAST[-] cells) (Figure 1A and B). Similar results were obtained after injection of previously characterized15Hollande F. Lee D.J. Choquet A. et al.Adherens junctions and tight junctions are regulated via different pathways by progastrin in epithelial cells.J Cell Sci. 2003; 116: 1187-1197Crossref PubMed Scopus (70) Google Scholar DLD-1 CRC cells expressing antisense GAST gene complementary DNA (cDNA) (Figure 1B). Next, we showed that progastrin inhibition decreased the growth of pre-existing intestinal tumors in a more pathologically relevant context, using APCΔ14 mice. These animals develop spontaneous tumors in the ileum but also more colon tumors than the APCmin+/- model,26Blache P. van de Wetering M. Duluc I. et al.SOX9 is an intestine crypt transcription factor, is regulated by the Wnt pathway, and represses the CDX2 and MUC2 genes.J Cell Biol. 2004; 166: 37-47Crossref PubMed Scopus (396) Google Scholar thus partially recapitulating the intestinal epithelium phenotype found in familial adenomatous polyposis and CRC patients. APCΔ14 mice always display multiple adenomas in the ileum and colon from the age of 3 months (Colnot et al30Colnot S. Niwa-Kawakita M. Hamard G. et al.Colorectal cancers in a new mouse model of familial adenomatous polyposis: influence of genetic and environmental modifiers.Lab Invest. 2004; 84: 1619-1630Crossref PubMed Scopus (146) Google Scholar and data not shown), and progastrin levels, but not those of amidated or glycine-extended gastrin, were found to be increased in these adenomas (supplementary Figure 2; see supplementary material online at www.gastrojournal.org). These animals therefore provided us with an ideal in vivo model to specifically study the role of progastrin modulation on Tcf-4–promoted tumor growth. Daily treatment for 2 weeks with siRNA directed against the murine GAST gene (APCΔ14/GAST[-]), previously shown to be effective in mouse cells (supplementary Figure 2; see supplementary material online at www.gastrojournal.org), induced a reduction in GAST gene and progastrin expression in intestinal adenomas, compared with that found after treatment with a luciferase-specific siRNA (APCΔ14/Luc[-]) (Figure 1C and D, and supplementary Figure 2; see supplementary material online at www.gastrojournal.org). In correlation with the reduced GAST expression, a significant reduction in tumor size was detected in the intestine of APCΔ14/GAST(-) animals, compared with controls (χ2, P < .0001, n = 9 per group). This reduction was particularly marked in the ileum (Figure 1C), but a similar trend was visible in the colon, although not reaching significance because of the smaller number of tumors located in this area (Fisher exact test, P = .228) (Figure 1D). In addition, a 20% decrease in the total number of intestinal tumors was detected overall in APCΔ14/GAST(-) mice compared with control animals. In particular, this reduction was drastic in 2 animals of the APCΔ14/GAST(-) group, in which the ileum or colon were found to be tumor-free. In contrast, the GAST-specific siRNA treatment failed to down-regulate GAST gene levels in one mouse from the same group, and this animal displayed a similar number and size of tumors as those found in controls (Figure 1C). Therefore, although the efficiency of the siRNA-mediated down-regulation of progastrin production varied among APCΔ14/GAST(-) animals, tumor sizes and numbers were decreased consistently in mice displaying reduced progastrin levels. Taken together, these results show that progastrin secretion is essential for the tumorigenicity of APC-mutated human CRC cells, and that a treatment inhibiting progastrin production in vivo strongly decreased tumor growth in a mouse model recapitulating the early stages of adenomatous polyposis and human colorectal tumorigenesis. Because we showed that targeting progastrin inhibited tumor growth in 2 different APC-mutated intestinal tumor models, we postulated that this effect was owing to the capacity of progastrin to modulate the activity levels of the β-catenin/Tcf-4 transcriptional complex, known to be activated constitutively by the APC mutation. This activity, quantified by transcription of a luciferase reporter gene (TOP/FOP),32Korinek V. Barker N. Morin P.J. et al.Constitutive transcriptional activation by a beta-catenin-Tcf complex in APC-/- colon carcinoma.Science. 1997; 275: 1784-1787Crossref PubMed Scopus (2983) Google Scholar indeed was increased in control cells (SW480/βgal[-]), but was inhibited significantly in progastrin-depleted clones (Figure 2A), without detectable modulation of β-catenin or Tcf-4 levels (supplementary Figure 3; see supplementary material online at www.gastrojournal.org). A similar result was obtained in previously established15Hollande F. Lee D.J. Choquet A. et al.Adherens junctions and tight junctions are regulated via different pathways by progastrin in epithelial cells.J Cell Sci. 2003; 116: 1187-1197Crossref PubMed Scopus (70) Google Scholar DLD-1 cells expressing an antisense GAST cDNA (supplementary Figure 3; see supplementary material online at www.gastrojournal.org). Treatment with 5 nmol/L recombinant progastrin, but also endogenous progastrin produced by expression of a codon-optimized, shRNA-insensitive, preprogastrin construct in SW480/GAST(-) clones, were found to restore a strong level of transcription of the luciferase reporter gene, confirming that progastrin is able to stimulate this transcriptional pathway (Figure 2A). In addition, immunofluorescent staining indicated that the reduction of β-catenin/Tcf-4 activity correlated with a marked reduction in the nuclear amounts of Tcf-4 and dephosphorylated β-catenin in SW480/GAST(-) cells, and treatment of these cells with 5 nmol/L recombinant progastrin partially restored the nuclear compartmentation of β-catenin and Tcf-4 (Figure 2B). Similarly, the expression of several Tcf-4 target genes (c-myc, cyclin D1, Sox-9, and claudin-1)9van de Wetering M. Sancho E. Verweij C. et al.The beta-catenin/TCF-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells.Cell. 2002; 111: 241-250Abstract Full Text Full Text PDF PubMed Scopus (1773) Google Scholar, 26Blache P. van de Wetering M. Duluc I. et al.SOX9 is an intestine crypt transcription factor, is regulated by the Wnt pathway, and represses the CDX2 and MUC2 genes.J Cell Biol. 2004; 166: 37-47Crossref PubMed Scopus (396) Google Scholar was down-regulated strongly after progastrin depletion, and was stimulated significantly by re-expression of progastrin or treatment with the recombinant peptide (Figure 2C and supplementary Figure 4; see supplementary material online at www.gastrojournal.org). In contrast, treatment with amidated or glycine-extended gastrin17, or with the amidated gastrin (Cholecystokinin-type B [CCK-B]) receptor antagonist L365, 260, did not affect β-catenin/Tcf-4 activity in these cells (supplementary Figure 3; see supplementary material online at www.gastrojournal.org), indicating that short processed forms of gastrin are unable to mimic the regulation of β-catenin/Tcf-4 activity by progastrin in these cells. The earlier-described data clearly show that progastrin produced by SW480 CRC cells stimulates the activity of the β-catenin/Tcf-4 complex. It also shows that blocking progastrin production results in a strong inhibition of this transcriptional pathway, despite its constitutive activation in APC-mutated cells. In view of the mutated status of the APC gene in the CRC cells used, and because the nuclear compartmentation of Tcf-4 and β-catenin was altered significantly in progastrin-depleted cells (Figure 2A), we hypothesized that the decreased β-catenin/Tcf-4 activity could result from an increased interaction of β-catenin with another of its partners. One of these partners, ICAT, was identified recently as a direct inhibitor of the association between these 2 proteins,33Tago K. Nakamura T. Nishita M. et al.Inhibition of Wnt signaling by ICAT, a novel beta-catenin-interacting protein.Genes Dev. 2000; 14: 1741-1749PubMed Google
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