Hypoxia Activates the K-Ras Proto-Oncogene to Stimulate Angiogenesis and Inhibit Apoptosis in Colon Cancer Cells Min Zeng, Hirotoshi Kikuchi, Maria S. Pino, Daniel C. Chung* Gastrointestinal Unit, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, United States of America Abstract The KRAS proto-oncogene plays a key role in the development of many human tumors and is commonly activated by somatic mutation or signaling through specific growth factor receptors. However, the interaction between the micro- environment and K-ras activity has not been defined. Hypoxia invariably develops as tumors outgrow their supply of oxygen. A series of well-orchestrated cellular adaptations occur that stimulate angiogenesis and enhance survival of the tumor in hypoxic conditions. Our previous studies demonstrated that mutant KRAS alleles can interact with hypoxia to induce vascular endothelial growth factor (VEGF) in colon cancer. We sought to determine whether similar hypoxic responses are also present in tumors without a KRAS mutation. Hypoxia consistently increased the levels of activated, GTP- bound K-ras in colon cancer cell lines with a wild-type KRAS gene, and this depended upon the activation of c-Src. Inhibition of c-Src by PP2 treatment or siRNA knockdown blocked the hypoxic activation of K-ras. This activation of K-ras did not depend upon EGFR and resulted in the phosphorylation of Akt and induction of VEGF expression. In addition, activation of K-ras significantly blocked apoptosis in hypoxic conditions. These studies reveal a unique adaptive mechanism in hypoxia that activates K-ras signaling in the absence of a mutant KRAS oncogene. Citation: Zeng M, Kikuchi H, Pino MS, Chung DC (2010) Hypoxia Activates the K-Ras Proto-Oncogene to Stimulate Angiogenesis and Inhibit Apoptosis in Colon Cancer Cells. PLoS ONE 5(6): e10966. doi:10.1371/journal.pone.0010966 Editor: Syed A. Aziz, Health Canada, Canada Received April 27, 2010; Accepted May 12, 2010; Published June 4, 2010 Copyright: 2010 Zeng et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by National Institutes of Health grant CA92594; Kate J. and Dorothy L. Clapp Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: dchung@partners.org. Introduction The development of tissue hypoxia is characteristically observed as malignant tumors rapidly increase in size. Such hypoxic conditions exert selective pressure on cancer cells, and the ability of tumor cells to survive in a hypoxic microenvironment has been associated with a poor prognosis and resistance to therapy [1]. One of the most critical and best characterized responses to hypoxia is the induction of vascular endothelial growth factor (VEGF), and hypoxia-inducible factor-1 (HIF-1) is a well-established mediator of this process. However, previous studies have demonstrated that HIF-independent mechanisms can also induce VEGF in hypoxia, and oncogenic K-ras plays a key role in this process [2,3,4]. K-ras is a small GTPase that cycles between inactive guanosine diphosphate (GDP)-bound and active guanosine triphosphate (GTP)-bound conformations (Ras-GDP and Ras-GTP, respective- ly) [5]. It serves as a signal switch molecule that couples receptor activation by specific growth factors with downstream effector pathways including the Ras-MEK-ERK and phosphatidylinositol 3-kinase (PI3K)-Akt cascades that control multiple cellular responses. Oncogenic mutations in KRAS impair the ability of K-Ras to hydrolyze bound GTP in a growth factor-independent manner, and constitutive signaling through these effector pathways results. The tumor microenvironment can have a profound influence on cellular behavior, and hypoxia has been shown to interact with many oncogenic signaling pathways. In particular, hypoxic activation of the PI3K-Akt, MEK-ERK, NF-kB, and hypoxia-inducible factor (HIF) signaling pathways has been described [6,7]. Although K-ras is a central regulator in all these pathways, it is unknown whether K-ras itself is specifically activated by hypoxia. Previous studies have demonstrated a strong synergistic interaction between hypoxia and mutant K-ras in the regulation of multiple target genes including vascular endothelial growth factor (VEGF), IL-8, and osteopontin [2,8,9]. However, fewer than 50% of colon tumors harbor KRAS mutations, and the relationship between Ras signaling and hypoxia in tumors with wild-type KRAS remains undefined. We sought to determine the role of K-ras in the hypoxic micro- environment in colon cancer, a tumor type that frequently harbors KRAS mutations. Wild-type K-ras was strongly activated by hypoxia, and c-Src was necessary for this hypoxic activation of K-ras. This resulted in the phosphorylation of Akt and induction of VEGF expression. In addition, hypoxic activation of wild-type K-ras blocked apoptosis. Collectively, these findings highlight a new mechanism for the hypoxic activation of oncogenic survival pathways in the absence of an oncogenic mutation. Results Hypoxic activation of K-Ras in colon cancer cells To determine whether hypoxia activates Ras, we measured GTP-bound Ras in normoxic and hypoxic conditions in a panel of colon cancer cell lines. Levels of GTP-bound Ras were barely detectable in Caco2, HT29, Colo320DM and Colo205 cells in PLoS ONE | www.plosone.org 1 June 2010 | Volume 5 | Issue 6 | e10966