APC and Oncogenic KRAS Are Synergistic in Enhancing Wnt Signaling in Intestinal Tumor Formation and Progression
2006; Elsevier BV; Volume: 131; Issue: 4 Linguagem: Inglês
10.1053/j.gastro.2006.08.011
ISSN1528-0012
AutoresKlaus‐Peter Janssen, Paola Alberici, Hafida Fsihi, Cláudia Gaspar, Cor Breukel, Patrick Franken, Christophe Rosty, Miguel Abal, Fatima El Marjou, Ron Smits, Daniel Louvard, Riccardo Fodde, Sylvie Robine,
Tópico(s)Digestive system and related health
ResumoBackground & Aims: Synchronous activation of the Wnt signaling pathway, mostly because of loss of function of the APC tumor suppressor, and of the oncogenic KRAS-signaling pathway is very frequent in colorectal cancer and is associated with poor prognosis. Methods: We have generated a compound transgenic mouse model, KRASV12G/Apc+/1638N, to recapitulate the human disease and compared it with single transgenic littermates. Results: Compound mutant mice are characterized by a 10-fold increase in tumor multiplicity and by accelerated tumor progression, resulting in strongly enhanced morbidity and mortality. Tumors from compound mutant mice proliferate faster and show decreased levels of apoptosis. Several lines of evidence indicate that the observed increase in tumor multiplicity and malignant transformation is caused by the synergistic activation of Wnt signaling in cells with oncogenic KRAS and loss-of-function Apc mutations. Activated KRAS is known to induce tyrosine phosphorylation of β-catenin, leading to its release from E-cadherin at the adherens junction. This results in an increased β-catenin pool in the cytoplasma, its subsequent translocation to the nucleus, and the transcriptional activation of Wnt downstream target genes. Accordingly, intestinal tumors from KRASV12G/Apc+/1638N mice show a significant increase in cells with nuclear accumulation of β-catenin when compared with Apc+/1638N animals. Moreover, Apc/KRAS-mutant embryonic stem cells show a significantly enhanced β-catenin/T-cell factor–mediated transcriptional activation, accompanied by increased β-catenin nuclear localization. Conclusions: This KRAS-induced increase in Wnt/β-catenin signaling may enhance the plasticity and self-renewal capacity of the tumor, thus resulting in the drastically augmented tumor multiplicity and malignant behavior in compound mutant animals. Background & Aims: Synchronous activation of the Wnt signaling pathway, mostly because of loss of function of the APC tumor suppressor, and of the oncogenic KRAS-signaling pathway is very frequent in colorectal cancer and is associated with poor prognosis. Methods: We have generated a compound transgenic mouse model, KRASV12G/Apc+/1638N, to recapitulate the human disease and compared it with single transgenic littermates. Results: Compound mutant mice are characterized by a 10-fold increase in tumor multiplicity and by accelerated tumor progression, resulting in strongly enhanced morbidity and mortality. Tumors from compound mutant mice proliferate faster and show decreased levels of apoptosis. Several lines of evidence indicate that the observed increase in tumor multiplicity and malignant transformation is caused by the synergistic activation of Wnt signaling in cells with oncogenic KRAS and loss-of-function Apc mutations. Activated KRAS is known to induce tyrosine phosphorylation of β-catenin, leading to its release from E-cadherin at the adherens junction. This results in an increased β-catenin pool in the cytoplasma, its subsequent translocation to the nucleus, and the transcriptional activation of Wnt downstream target genes. Accordingly, intestinal tumors from KRASV12G/Apc+/1638N mice show a significant increase in cells with nuclear accumulation of β-catenin when compared with Apc+/1638N animals. Moreover, Apc/KRAS-mutant embryonic stem cells show a significantly enhanced β-catenin/T-cell factor–mediated transcriptional activation, accompanied by increased β-catenin nuclear localization. Conclusions: This KRAS-induced increase in Wnt/β-catenin signaling may enhance the plasticity and self-renewal capacity of the tumor, thus resulting in the drastically augmented tumor multiplicity and malignant behavior in compound mutant animals. 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Khan P.M. et al.A targeted chain-termination mutation in the mouse Apc gene results in multiple intestinal tumors.Proc Natl Acad Sci U S A. 1994; 91: 8969-8973Crossref PubMed Scopus (467) Google Scholar was bred with the transgenic model22Janssen K.P. el-Marjou F. Pinto D. Sastre X. Rouillard D. Fouquet C. Soussi T. Louvard D. Robine S. Targeted expression of oncogenic K-ras in intestinal epithelium causes spontaneous tumorigenesis in mice.Gastroenterology. 2002; 123: 492-504Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar pVillin-KRASV12G in the genetic background B6D2 (C57Bl/6J × DBA/2). To control for genetic background effects, littermates were always used as controls. Mice were maintained under a 12-hour light-dark cycle and fed with standard diet and water ad lib. Genotyping was performed on DNA extracted from mouse tails as previously described.22Janssen K.P. el-Marjou F. Pinto D. Sastre X. Rouillard D. Fouquet C. Soussi T. Louvard D. Robine S. Targeted expression of oncogenic K-ras in intestinal epithelium causes spontaneous tumorigenesis in mice.Gastroenterology. 2002; 123: 492-504Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 23Smits R. van der Houven van Oordt W. Luz A. Zurcher C. Jagmohan-Changur S. Breukel C. Khan P.M. Fodde R. Apc1638N: a mouse model for familial adenomatous polyposis-associated desmoid tumors and cutaneous cysts.Gastroenterology. 1998; 114: 275-283Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar The median age of the analyzed animals was 5.5 months (KRASV12G/Apc+/1638N), 7 months (Apc+/1638N), and 9 months (KRASV12G). Animals were killed at the ages indicated or at the appearance of signs of distress, and the gross study of the tissues was carried out as described.22Janssen K.P. el-Marjou F. Pinto D. Sastre X. Rouillard D. Fouquet C. Soussi T. Louvard D. Robine S. Targeted expression of oncogenic K-ras in intestinal epithelium causes spontaneous tumorigenesis in mice.Gastroenterology. 2002; 123: 492-504Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar Macroscopically visible tumors were resected and embedded in paraffin according to standard procedures. Tumors were classified according to standard World Health Organization (WHO) histopathologic criteria by an experienced pathologist. In addition to the processing for histopathologic analysis, a subset of freshly isolated tumors was also snap frozen in liquid nitrogen and stored at −80°C. Frozen tumors were either used for DNA/RNA extraction (Qiagen, Hilden, Germany) or embedded in Tissue-Tek (Sakura B.V., Zoeterwoude, The Netherlands) and processed for cryosections. Kidneys, liver, and lungs of all animals were also investigated for the presence of metastases by macroscopic and, in a selected number of cases, microscopic analysis of serial sections. For protein analysis, snap-frozen mouse tissue or scrapings of intestinal mucosa were lysed in ice-cold lysis buffer (50 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 1 mmol/L benzamidine, 1 mmol/L PMSF, 1 mmol/L DTT, 2 mmol/L EGTA, 1% Triton X-100, 1% NP-40, Mammalian Protease Inhibitor Cocktail; Sigma Chemical Co, St. Louis, MO) using a 1-mL Dounce Homogenizer. After centrifugation (15,000g, 15 minutes, 4°C), supernatants were collected, and protein concentration was determined (Bio-Rad assay, Richmond, CA). LOH of the Apc and Tp53 genes was determined by PCR amplification of dinucleotide repeat markers.22Janssen K.P. el-Marjou F. Pinto D. Sastre X. Rouillard D. Fouquet C. Soussi T. Louvard D. Robine S. Targeted expression of oncogenic K-ras in intestinal epithelium causes spontaneous tumorigenesis in mice.Gastroenterology. 2002; 123: 492-504Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 24Smits R. Kartheuser A. Jagmohan-Changur S. Leblanc V. 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Analysis of transcript mutations due to transcriptional slippage in rat p53 tumor suppressor gene with the use of yeast functional assay.Hokkaido Igaku Zasshi. 1999; 74: 173-188PubMed Google Scholar C-myc and cyclin D1 messenger RNA (mRNA) expression levels were analyzed in normal mucosa and tumors (n = 5 mice/genotype). RNA was harvested from snap-frozen tissues using the RNeasy extraction kit (Qiagen). Up to 2 μg of total RNA was then subjected to reverse transcription using Superscript II Reverse Transcriptase (Invitrogen Life Technologies, Carlsbad, CA) and oligo dT primers (pd(N)6, Roche, Mannheim, Germany). Reactions were carried out in SybrGreen PCR Master mix (Applied Biosystems, Courtaboeuf Cedex, France) under recommended conditions, run on ABI PRISM 7900, and analyzed with Sequence Detector Software (Applied Biosystems). Relative quantities were calculated using the ddCT formula and normalized to the transcript levels of the housekeeping gene TATA binding protein (TBP). Assays were performed in triplicate. Primer sequences used were as follows: TBP: forward, CCACGGACAACTGCGTTGAT; reverse, GGCTCATAGCTACTGAACTG. c-myc: forward, TAGTGCTGCATGAGGAGACA; reverse, GGTTTGCCTCTTCTCCACAG. cyclinD1: forward, CACAACGCACTTTCTTTCCAG; reverse, CGCAGGCTTGACTCCAGAAG. Mouse livers were dissected under sterile conditions to avoid contamination. Liver RNA was extracted from different tissues using the RNA Now Kit (Ozyme, St Quentin, France). RT-PCR reactions were performed as previously described.22Janssen K.P. el-Marjou F. Pinto D. Sastre X. Rouillard D. Fouquet C. Soussi T. Louvard D. Robine S. Targeted expression of oncogenic K-ras in intestinal epithelium causes spontaneous tumorigenesis in mice.Gastroenterology. 2002; 123: 492-504Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 28Araki Y. Okamura S. Hussain S.P. Nagashima M. He P. Shiseki M. Miura K. Harris C.C. Regulation of cyclooxygenase-2 expression by the Wnt and ras pathways.Cancer Res. 2003; 63: 728-734PubMed Google Scholar Primers were used that amplify specifically the transgenic KRASV12G gene under control of the villin promoter. The sense primer is specific to the villin promoter, CAAGCCTGGCTCGACGGCC, and the antisense primer recognizes the coding sequence of the human KRASV12G gene, ATTTGCGGCCGCTTTACATAATTACACACT, yielding a fragment of 400 base pair. PCR reactions were repeated twice for each sample, and RNA was extracted twice from each tissue to confirm the result. Direct sequencing of the fragment confirmed the identity of the transgenic KRASV12G. Equal amounts (40 μg) of protein lysate were separated on 13% polyacrylamide gels and further subjected to immunoblotting according to standard procedures. Primary antibodies used were as follows: anti-pan-Ras (Transduction Laboratories, Lexington, KY), anti-β-Actin (Sigma), anti-β-catenin (Transduction Lab.), anti-E-cadherin, anti-phospho-(Thr202, Tyr204)-p44/42 MAPK, anti-p44/42 MAPK, anti-phospho-(Thr308)-Akt, anti-phospho-(Ser473)-Akt, anti-Akt (all Cell Signaling, New England Biolabs, Beverly, MA). Peroxidase-conjugated secondary antibodies (Jackson Immunoresearch, West Grove, PA) were visualized with an ECL kit (Pierce, Rockford, IL).22Janssen K.P. el-Marjou F. Pinto D. Sastre X. Rouillard D. Fouquet C. Soussi T. Louvard D. Robine S. Targeted expression of oncogenic K-ras in intestinal epithelium causes spontaneous tumorigenesis in mice.Gastroenterology. 2002; 123: 492-504Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar GTP-Ras pull-down assays were performed on mouse tissue lysates as previously described.22Janssen K.P. el-Marjou F. Pinto D. Sastre X. Rouillard D. Fouquet C. Soussi T. Louvard D. Robine S. Targeted expression of oncogenic K-ras in intestinal epithelium causes spontaneous tumorigenesis in mice.Gastroenterology. 2002; 123: 492-504Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar GTP-Ras pull-down assays on embryonic stem (ES) cells followed essentially the same protocol; the cells were first washed with ice-cold phosphate-buffered saline (PBS) and then incubated in lysis buffer.22Janssen K.P. el-Marjou F. Pinto D. Sastre X. Rouillard D. Fouquet C. Soussi T. Louvard D. Robine S. Targeted expression of oncogenic K-ras in intestinal epithelium causes spontaneous tumorigenesis in mice.Gastroenterology. 2002; 123: 492-504Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 28Araki Y. Okamura S. Hussain S.P. Nagashima M. He P. Shiseki M. Miura K. Harris C.C. Regulation of cyclooxygenase-2 expression by the Wnt and ras pathways.Cancer Res. 2003; 63: 728-734PubMed Google Scholar Samples were run on 13% SDS-PAGE gels and transferred to membranes. Immunodetection was performed with anti-Ras antibody (Cell Signaling). The amount of GTP-bound GTPases was normalized to the total amount of GTPases present in whole cell lysates. Flow cytometry from mouse tissue was carried out essentially as described.22Janssen K.P. el-Marjou F. Pinto D. Sastre X. Rouillard D. Fouquet C. Soussi T. Louvard D. Robine S. Targeted expression of oncogenic K-ras in intestinal epithelium causes spontaneous tumorigenesis in mice.Gastroenterology. 2002; 123: 492-504Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar For each genotype, 2 mice were injected intraperitoneally with 0.01 mL/g body weight of a 6 mg/mL solution of 5-bromo-2′-deoxyuridine (BrdU; Sigma). Animals were killed 2 hours after bromodeoxyuridine (BrdU) injection and dissected. Bivariate distributions of BrdU content (FITC) vs DNA content (propidium iodide) were measured using a FACScan flow cytometer (Becton Dickinson, San Jose, CA). Doublets and clumps were excluded from the analysis by gating on a bivariate distribution of the propidium iodide area vs signal width. Cryosections of Tissue-tek OCT (Sakura) embedded mouse tissues were cut at 5-μm thickness, air-dried, and fixed with 3% paraformaldehyde at room temperature for 20 minutes. The paraformaldehyde-fixed sections were treated with 50 mmol/L NH4Cl in PBS for 20 minutes, and solubilized with 0.1% Triton X-100 for 5 minutes. Antibodies and reagents used were as follows: anti-β-catenin (dilution 1:200; Transduction Laboratories Clone 14); pAb anti-Ki67 (Novocastra, Newcastle, UK); cleaved caspase-3 (Cell Signaling); Peroxidase-, Cy3-, or Alexa488-conjugated secondary antibodies (Jackson Immunoresearch); and TRITC-phalloidin and Hoechst 33258 (Sigma). Cells and tissue sections were viewed using a fluorescence microscope (Zeiss, Göettingen, Germany) or a confocal microscope (LSM510, Zeiss, Göettingen, Germany). Images were processed using Adobe Photoshop Software (San Jose, CA). For evaluation of β-catenin staining, antigen retrieval treatment was performed (10 minutes in boiling 10 mmol/L Tris/Cl, 1 mmol/L EDTA, pH 8.0), followed by incubation with the specific antibody overnight at 4°C. Localization of peroxidase activity was detected with the SIGMA FAST DAB system (Sigma), after brief hematoxylin counterstaining. For the comparative evaluation of nuclear β-catenin accumulation between tumors derived from Apc+/1638N and KRASV12G/Apc+/1638N mice, a previously established protocol was used29Jung A. Schrauder M. Oswald U. Knoll C. Sellberg P. Palmqvist R. Niedobitek G. Brabletz T. Kirchner T. The invasion front of human colorectal adenocarcinomas shows co-localization of nuclear β-catenin, cyclin D1, and p16INK4A and is a region of low proliferation.Am J Pathol. 2001; 159: 1613-1617Abstract Full Text Full Text PDF PubMed Google Scholar based on 2 independent blinded observers. In brief, tumor cells with nuclear β-catenin were counted from immunohistochemistry (IHC) sections and scored according to staining intensity when compared with the few normal crypt cells within the same section that, as previously reported, also encompass nuclear β-catenin. The ratio between the tumor area, measured in millimeters squared, using the PALM MicroBeam microscope system (P.A.L.M. Microlaser Technologies AG- Bernried, Germany), and the absolute numbers of positive cells were calculated for each tumor sample based on at least 2 serial sections representative of the whole tumor. Statistical analysis was performed using R software (version 1.9.1., Free Software Foundation, Boston, MA). ES cell lines containing both Apc and KRAS mutations were generated by stable cotransfection by electroporation of wild-type and Apc1638N/1638N ES cell lines (E14; 129 Ola) with a pPGK expression vector containing either the human wild-type KRAS or the human oncogenic KRASV12G together with a pPGK-puromycin selection vector. To select for stable clones, the ES cells were cultured in the presence of puromycin (Sigma) at a final concentration of 2 μg/mL for 2 weeks. Twenty hours before transfection, 105 ES cells per well were plated on tissue culture plates coated by primary, mitomycin C inactivated, murine embryonic fibroblasts. ES cells were transfected in each well with 500 ng of pTOPFLASH or pFOPFLASH vector (kindly provided by Dr H. Clevers) and 5 ng luciferase from Renilla reniformis using Lipofectamine 2000 (Life Technologies) as recommended by the manufacturer. After 24 hours, luciferase activities were measured in a luminometer (Lumat LB 9507, Berthold, Bad Wildbad, Germany) and normalized for transfection efficiency by the Dual Luciferase Reporter Assay system (Promega, Madison, WI). Luciferase activities were evaluated as ratio of pTOPFLASH vs pFOPFLASH levels for 3 different experiments, each carried out in duplicate. ES cells were prepared following the Vibrant Apoptosis Assay kit No. 2 protocol (Molecular Probes). Five thousand events were analyzed per test in list mode using a FACScan flow cytometer (Becton Dickinson, San Jose, CA). To quantify changes associated with cell differentiations, 2 regions were created on the dot-plot graphs using the Forward Scatter/Side Scatter. Bivariate distributions of Annexin-V content (Alexa 488) vs DNA content (PI) were measured. Mean ± SD of 3 experiments is shown. Tumor-specific expression profiles were analyzed in 4 Apc+/1638N and 4 KRASV12G/Apc+/1638N tumors collected from a total of 7 mice of 6–8 months of age and same genetic background. All tumors were highly dysplastic and localized in the upper duodenum. Samples were laser-capture microdissected (LCM) from cryosections of Tissue-tek OCT (Sakura) embedded snap-frozen tumors. Ten-micrometer sections were briefly stained with H&E, and consecutive sections were carefully microdissected using a PALM MicroBeam microscope system (P.A.L.M. Microlaser Technologies AG- Bernried, Germany). On average, 2000 ce
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