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Volume 10 Number 7 July 2008 pp. 680–686 680www.neoplasia.comRAS Signaling in ColorectalTerje Ahlquist* ,† , Irene Bottillo ‡,§ ,Carcinomas throughAlterations of RAS, RAF,NF1, and/or RASSF1A 1Stine A. Danielsen* ,† , Gunn I. Meling ,# ,Torleiv O. Rognum # , Guro E. Lind* ,† ,Bruno Dallapiccola ‡,§ and Ragnhild A. Lothe* ,†*Department of Cancer Prevention, Institute for CancerResearch, Norwegian Radium Hospital, RikshospitaletUniversity Hospital, Oslo, Norway; † Centre for CancerBiomedicine, University of Oslo, Oslo, Norway;‡ IRCCS-CSS, San Giovanni Rotondo and CSS-MendelInstitute, Rome, Italy; § Department of ExperimentalMedicine and Pathology, “Sapienza, University ofRome,” Rome, Italy; Surgical Department, FacultyDivision Akershus University Hospital, University ofOslo, Oslo, Norway; # Institute of Forensic Medicine,Rikshospitalet-Radiumhospitalet Medical Centre,University of Oslo, Oslo, NorwayAbstractMore than half of all colorectal carcinomas are known to exhibit an activated mitogen-activated protein kinasepathway. The NF1 gene, a negative regulator of KRAS, has not previously been examined in a series of colorectalcancer. In the present study, primary colorectal carcinomas stratified according to microsatellite instability statuswere analyzed. The whole coding region of NF1 was analyzed for mutations using denaturing high-performanceliquid chromatography and sequencing, and the copy number alterations of NF1 were examined using multipleligation-dependent probe amplification and real-time polymerase chain reaction. The mutational hot spots in KRASand BRAF were sequenced, and promoter hypermethylation status of RASSF1A was assessed with a methylationspecificpolymerase chain reaction. One sample had two missense mutations in NF1, whereas nine additional tumorshad intronic mutations likely to affect exon splicing. Interestingly, 8 of these 10 tumors were microsatellite-unstable.Four other tumors showed a duplication of NF1. Mutations in KRAS and BRAF were mutually exclusive and werepresent at 40% and 22%, respectively. RASSF1A was hypermethylated in 31% of the samples. We show that theRAS signaling network is extensively dysregulated in colorectal carcinomas, because more than 70% of the tumorshad an alteration in one or more of the four examined components.Neoplasia (2008) 10, 680–686IntroductionColorectal cancer (CRC) is one of the leading causes of cancer-relateddeaths in the western world today, and at least 50% of CRCs arethought to have a dysregulation of the RAS-RAF-MEK-ERK pathway,also known as the mitogen-activated protein kinase (MAPK)pathway [1]. When activated, this pathway leads to increased proliferationand reduced apoptosis, two of six crucial abilities of a cancercell, as described by Hanahan and Weinberg [2]. There are severalcomponents in this pathway, which, theoretically, could be affectedin cancer, and some are known mutational targets in cancer such asKRAS and BRAF. KRAS has been widely established as an importantAbbreviations: CRC, colorectal cancer; MAPK, mitogen-activated protein kinase;MSI, microsatellite instability; MSS, microsatellite stable; dHPLC, denaturing highperformanceliquid chromatography; MSP, methylation-specific PCR; MLPA, multipleligation-dependent probe amplificationAddress all correspondence to: Ragnhild A. Lothe, Department of Cancer Prevention,Institute for Cancer Research, Norwegian Radium Hospital, Montebello, 0310 Oslo,Norway. E-mail: ragnhild.a.lothe@rr-research.no1 This article refers to supplementary materials, which are designated by Tables W1and W2 and are available online at www.neoplasia.com.Received 19 February 2008; Revised 12 April 2008; Accepted 18 April 2008Copyright © 2008 Neoplasia Press, Inc. All rights reserved 1522-8002/08/$25.00DOI 10.1593/neo.08312