Development of a Colon Cancer GEMM-Derived Orthotopic Transplant Model for Drug Discovery and Validation

Purpose: Effective therapies for KRAS-mutant colorectal cancer (CRC) are a critical unmet clinical need. Previously, we described genetically engineered mouse models (GEMM) for sporadic Kras-mutant and non-mutant CRC suitable for preclinical evaluation of experimental therapeutics. To accelerate drug discovery and validation, we sought to derive low-passage cell lines from GEMM Kras-mutant and wild-type tumors for in vitro screening and transplantation into the native colonic environment of immunocompetent mice for in vivo validation. Experimental Design: Cell lines were derived from Kras-mutant and non-mutant GEMM tumors under defined media conditions. Growth kinetics, phosphoproteomes, transcriptomes, drug sensitivity, and metabolism were examined. Cell lines were implanted in mice and monitored for in vivo tumor analysis. Results: Kras-mutant cell lines displayed increased proliferation, mitogen-activated protein kinase signaling, and phosphoinositide-3 kinase signaling. Microarray analysis identified significant overlap with human CRC-related gene signatures, including KRAS-mutant and metastatic CRC. Further analyses revealed enrichment for numerous disease-relevant biologic pathways, including glucose metabolism. Functional assessment in vitro and in vivo validated this finding and highlighted the dependence of Kras-mutant CRC on oncogenic signaling and on aerobic glycolysis. Conclusions: We have successfully characterized a novel GEMM-derived orthotopic transplant model of human KRAS-mutant CRC. This approach combines in vitro screening capability using low-passage cell lines that recapitulate human CRC and potential for rapid in vivo validation using cell line-derived tumors that develop in the colonic microenvironment of immunocompetent animals. Taken together, this platform is a clear advancement in preclinical CRC models for comprehensive drug discovery and validation efforts. Clin Cancer Res; 19(11); 2929–40. ©2013 AACR.

[1]  Steffen Jung,et al.  Utilization of Murine Colonoscopy for Orthotopic Implantation of Colorectal Cancer , 2011, PloS one.

[2]  Heidi Ledford,et al.  Translational research: 4 ways to fix the clinical trial , 2011, Nature.

[3]  J. Neuzil,et al.  K-Ras and mitochondria: Dangerous liaisons , 2011, Cell Research.

[4]  Erin M. Coffee,et al.  The Dual PI3K/mTOR Inhibitor NVP-BEZ235 Induces Tumor Regression in a Genetically Engineered Mouse Model of PIK3CA Wild-Type Colorectal Cancer , 2011, PloS one.

[5]  J. McCarter,et al.  STK33 kinase activity is nonessential in KRAS-dependent cancer cells. , 2011, Cancer research.

[6]  Sabine Tejpar,et al.  KRAS, BRAF, PIK3CA, and PTEN mutations: implications for targeted therapies in metastatic colorectal cancer. , 2011, The Lancet. Oncology.

[7]  Hans Clevers,et al.  The intestinal stem cell signature identifies colorectal cancer stem cells and predicts disease relapse. , 2011, Cell stem cell.

[8]  A. Giaccia,et al.  Harnessing synthetic lethal interactions in anticancer drug discovery , 2011, Nature Reviews Drug Discovery.

[9]  J. Arrowsmith.  Trial watch: Phase II failures: 2008–2010 , 2011, Nature Reviews Drug Discovery.

[10]  P. Spellman,et al.  Subtypes of Pancreatic Ductal Adenocarcinoma and Their Differing Responses to Therapy , 2011, Nature Medicine.

[11]  D. Hanahan,et al.  Hallmarks of Cancer: The Next Generation , 2011, Cell.

[12]  W. Hahn,et al.  Wilms tumor 1 (WT1) regulates KRAS-driven oncogenesis and senescence in mouse and human models. , 2010, The Journal of clinical investigation.

[13]  M. Wilkerson,et al.  Integrative genomic and proteomic analyses identify targets for Lkb1-deficient metastatic lung tumors. , 2010, Cancer cell.

[14]  S. Tejpar,et al.  New Strategies for Treatment of KRAS Mutant Metastatic Colorectal Cancer , 2010, Clinical Cancer Research.

[15]  T. Yeatman,et al.  Experimentally derived metastasis gene expression profile predicts recurrence and death in patients with colon cancer. , 2010, Gastroenterology.

[16]  J. DiMasi,et al.  Trends in Risks Associated With New Drug Development: Success Rates for Investigational Drugs , 2010, Clinical pharmacology and therapeutics.

[17]  Umar Mahmood,et al.  Development of a mouse model for sporadic and metastatic colon tumors and its use in assessing drug treatment , 2010, Proceedings of the National Academy of Sciences.

[18]  W. Zong,et al.  Akt and c-Myc Differentially Activate Cellular Metabolic Programs and Prime Cells to Bioenergetic Inhibition* , 2009, The Journal of Biological Chemistry.

[19]  M. Bertagnolli,et al.  Molecular origins of cancer: Molecular basis of colorectal cancer. , 2009, The New England journal of medicine.

[20]  A. Bardelli,et al.  Biomarkers Predicting Clinical Outcome of Epidermal Growth Factor Receptor–Targeted Therapy in Metastatic Colorectal Cancer , 2009, Journal of the National Cancer Institute.

[21]  Ben S. Wittner,et al.  Systematic RNA interference reveals that oncogenic KRAS-driven cancers require TBK1 , 2009, Nature.

[22]  Michael J. Emanuele,et al.  A Genome-wide RNAi Screen Identifies Multiple Synthetic Lethal Interactions with the Ras Oncogene , 2009, Cell.

[23]  L. Cantley,et al.  Understanding the Warburg Effect: The Metabolic Requirements of Cell Proliferation , 2009, Science.

[24]  D. Threadgill,et al.  Murine models of colorectal cancer , 2009, Mammalian Genome.

[25]  M. Taketo,et al.  Mouse models of colon cancer. , 2009, Gastroenterology.

[26]  S. Hanash,et al.  Comprehensive Proteome Analysis of an Apc Mouse Model Uncovers Proteins Associated with Intestinal Tumorigenesis , 2009, Cancer Prevention Research.

[27]  Ralph Weissleder,et al.  Effective Use of PI3K and MEK Inhibitors to Treat Mutant K-Ras G12D and PIK3CA H1047R Murine Lung Cancers , 2008, Nature Medicine.

[28]  Daniel J. Freeman,et al.  Wild-type KRAS is required for panitumumab efficacy in patients with metastatic colorectal cancer. , 2008, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[29]  E. Engleman,et al.  Orthotopic mouse model of colorectal cancer. , 2007, Journal of visualized experiments : JoVE.

[30]  L. Chin,et al.  Common and distinct genomic events in sporadic colorectal cancer and diverse cancer types. , 2007, Cancer research.

[31]  A. Bardelli,et al.  Knock-in of oncogenic Kras does not transform mouse somatic cells but triggers a transcriptional response that classifies human cancers. , 2007, Cancer research.

[32]  David A. Tuveson,et al.  Maximizing mouse cancer models , 2007, Nature Reviews Cancer.

[33]  S. Hanash,et al.  Innovative proteomic approaches for cancer biomarker discovery. , 2007, BioTechniques.

[34]  L. Chin,et al.  Chromosomally unstable mouse tumours have genomic alterations similar to diverse human cancers , 2007, Nature.

[35]  Oksana Gavrilova,et al.  p53 Regulates Mitochondrial Respiration , 2006, Science.

[36]  A. Lièvre,et al.  KRAS mutation status is predictive of response to cetuximab therapy in colorectal cancer. , 2006, Cancer research.

[37]  Jeffrey T. Chang,et al.  Oncogenic pathway signatures in human cancers as a guide to targeted therapies , 2006, Nature.

[38]  David Beach,et al.  Glycolytic enzymes can modulate cellular life span. , 2005, Cancer research.

[39]  R. Gillies,et al.  Why do cancers have high aerobic glycolysis? , 2004, Nature Reviews Cancer.

[40]  R. Hoffman,et al.  Models of human metastatic colon cancer in nude mice orthotopically constructed by using histologically intact patient specimens. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[41]  H. Lodish,et al.  Elevated levels of glucose transport and transporter messenger RNA are induced by ras or src oncogenes. , 1987, Science.

[42]  J S Fowler,et al.  A FLUORINATED GLUCOSE ANALOG, 2‐FLUORO-2‐DEOXY-D‐GLUCOSE (F‐18): NONTOXIC TRACER FOR RAPID TUMOR DETECTION , 1980, Journal of nuclear medicine : official publication, Society of Nuclear Medicine.

[43]  O. Warburg.  [Origin of cancer cells]. , 1956, Oncologia.

[44]  G. Naik.  Scientists' Elusive Goal: Reproducing Study Results , 2011 .

[45]  J. Mesirov,et al.  An oncogenic KRAS2 expression signature identified by cross-species gene-expression analysis , 2005, Nature Genetics.

[46]  B. Vogelstein,et al.  Prevalence of ras gene mutations in human colorectal cancers , 1987, Nature.