JOURNAL OF CELLULAR PHYSIOLOGY 210:740–749 (2007) Oncogenic KRAS Provides a Uniquely Powerful and Variable Oncogenic Contribution Among RAS Family Members in the Colonic Epithelium JEFFREY W. KELLER, 1 JEFFREY L. FRANKLIN, 1 RAMONA GRAVES-DEAL, 1 DAVID B. FRIEDMAN, 2,3 CORBIN W. WHITWELL, 2,3 AND ROBERT J. COFFEY 1,4 * 1 Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, Tennessee 2 Mass Spectrometry Research Center, Vanderbilt University Medical Center, Nashville, Tennessee 3 Department of Biochemistry, Vanderbilt University Medical Center, Nashville, Tennessee 4 Department of Veterans Affairs Medical Center, Nashville, Tennessee Activating mutations of the RAS family of small GTPases are among the most common genetic events in human tumorigenesis. Constitutive activation of the three canonical family members, KRAS, NRAS, and HRAS segregate strongly by tissue type. Of these, KRAS mutations predominate in human tumors, including those arising from the colon and lung. We sought to compare the oncogenic contributions of different RAS isoforms in a comparable genetic setting and to explore downstream molecular changes that may explain the apparent differential oncogenic effects of the various RAS family members. We utilized colorectal cancer cell lines characterized by oncogenic KRAS in parallel with isogenically derived lines in which the mutant allele has been disrupted. We additionally attempted to reconstitute the isogenic derivatives with oncogenic forms of other RAS family members and analyze them in parallel. Pairwise analysis of HCT 116 and DLD-1 cell lines as well as their isogenic derivatives reveals distinct K-RAS G13D signatures despite the genetic similarities of these cell lines. In DLD-1, for example, oncogenic K-RAS enhances the motility of these cells by downregulation of Rap1 activity, yet is not associated with increased ERK1/2 phosphorylation. In HCT 116, however, ERK1/ 2 phosphorylation is elevated relative to the isogenic derivative, but Rap1 activity is unchanged. K-RAS is uniquely oncogenic in the colonic epithelium, though the molecular aspects of its oncogenic contribution are not necessarily conserved across cell lines. We therefore conclude that the oncogenic contribution of K-RAS is a function of its multifaceted functionality and is highly context- dependent. J. Cell. Physiol. 210: 740 – 749, 2007. ß 2006 Wiley-Liss, Inc. Activating mutations of the members of the RAS family of small GTPases are among the most common genetic events in human tumorigenesis and character- ize approximately 30% of all human cancers (Bos, 1989). Constitutive RAS signaling contributes to the trans- formed phenotype by promoting proliferation, enhanced motility, survival and loss of anchorage dependence. Activating mutations of RAS family members do not occur with equal frequency across tumor types, but rather segregate strongly by tissue of origin, with one isoform predominating in any particular RAS-driven tumor. For example, mutations to KRAS are the most frequent RAS family mutations and occur in 30% of nonsmall cell lung cancers, 50% of colorectal adenocar- cinomas and 90% of pancreatic adenocarcinomas. Fifteen percent of melanomas and 30% of acute myelogenous leukemias display activating NRAS muta- tions. Activating mutations of HRAS are far less common but contribute to squamous cell carcinomas, bladder carcinomas, and to renal cancers (Downward, 2003). Isoform-specific functions may explain the observed segregation by tumor type. Numerous recent studies have refuted the long-held belief that RAS isoforms are functionally interchange- able in vivo (Yan et al., 1997a,b, 1998; Rodriguez- Viciana et al., 2004). Despite their extensive sequence identity, differences in primary sequence and posttran- slational modifications of the C-terminal 24 amino acids provide the cell biological basis for functional differences in the members of this protein family. Because of the multiplicity of RAS function, discerning specific path- ways and isolating their contributions to tumorigenesis has proved daunting. This complexity is further magni- fied by the differential requirements for RAS signaling at different stages of tumor development (Chin et al., 1999; Lim and Counter, 2005). The frequency of KRAS mutations across a broad range of human tumors suggests the potency of the oncogenic contribution of the constitutively active form of this protein. In an attempt to better understand the basis for the prevalence of the KRAS oncogene in colorectal cancer, we employed isogenic pairs of colo- rectal cancer cell lines that owe much of their trans- formed phenotype to the presence of an activating mutation (G13D) of KRAS (Shirasawa et al., 1993). Using homologous recombination, the mutant allele has ß 2006 WILEY-LISS, INC. This article includes Supplementary Material available from the authors upon request or via the Internet at http://www.inters- cience.wiley.com/jpages/0021-9541/suppmat. Contract grant sponsor: NCI SPORE; Contract grant number: 95103; Contract grant sponsor: NIH; Contract grant numbers: CA46413 and CA95103. *Correspondence to: Robert J. Coffey, Suite 4140 MRBIII, Vanderbilt University, 465 21st Avenue South, Nashville, Tennessee 37232. E-mail: robert.coffey@vanderbilt.edu Received 20 May 2006; Accepted 28 August 2006 DOI: 10.1002/jcp.20898