contributed to the activation of Ras by hypoxia, we pretreated Caco2 cells with PP2, a potent and selective Src tyrosine kinase inhibitor. A 50% reduction in the hypoxic induction of Ras was detected in Caco2 cells, while there was no effect on the levels of total Ras (Fig. 2B, left panel). This effect was dose-dependent, with a maximal effect seen at 20 mM (data not shown). To more directly assess the role of c-Src, we knocked down endogenous c-Src in Caco2 cells via RNA interference. Knock- down of c-Src led to a 33% reduction in the hypoxic activation of GTP-bound Ras (Fig. 2B, right panel). Furthermore, Caco2 cells expressing a constitutively active c-Src vector (SrcY527F) exhib- ited a 70% increase in the level of GTP-bound Ras (Fig. 2C) when compared with empty vector (pEVX). Western blotting verified the K-ras isoform was activated by expression of Src (Fig. S2). Reactive oxygen species (ROS) generated by hypoxia have previously been shown to activate Src. Exogenous administration of H2O2 (5 mM) potently induced phosphorylation of Src at Tyr416 in Caco2 cells (Fig. 2D, left panel). This effect was significantly reduced by NAC (20 mM), an antioxidant that inhibits ROS. We then sought to determine whether NAC could block the hypoxic activation of endogenous Src and Ras. NAC treatment reduced the induction of p-Src416 in hypoxia by approximately 30% with a concomitant 17% reduction of GTP- bound Ras (Fig. 2D). These findings suggest that generation of ROS may partially contribute to the hypoxic activation of c-Src in colon cancer cells. Recent reports have suggested that hypoxia can increase levels of EGFR and that phosphorylation of EGFR may be an intermediate step in the signaling from Src to Ras [11,12]. To determine whether EGFR may play such a role, we measured total EGFR and phospho-EGFR levels in Caco2 cells incubated in hypoxia. There was a small increase in total EGFR in hypoxia. However, there was no increase in EGFR phosphorylation at Tyr1068 (Fig. 2E). Overall, the data suggest that up-regulation of c- Src activity by hypoxia is mediated in part through ROS but that activation of K-Ras by c-Src in hypoxia does not depend upon EGFR. Activation of Akt by hypoxia is downstream of c-Src and K-ras We next sought to determine which downstream effector pathways were activated by c-Src and K-ras in hypoxia. Exposure to hypoxic conditions induced phosphorylation of Akt at Ser473 in both Caco2 and HT29 cells, while no activation of ERK was detected under the same conditions (Fig. 3A). Akt was activated in a time-dependent manner and reached a maximum level at 12 hours. To determine whether Src was involved in the hypoxic activation of Akt, we pretreated Caco2 cells with the specific Src inhibitor PP2 for 12 hours before exposure to hypoxia. As shown in Figure 3B, the hypoxic activation of Akt was reduced by PP2 treatment in a dose- dependent manner. Consistent with these findings, knock down of c- Src also completely blocked the hypoxic activation of Akt when compared to cells transfected with a control siRNA (Fig. 3C). Because Akt is known to be downstream of K-ras, we tested whether Akt is also a specific target of K-ras under hypoxic conditions by utilizing K-ras siRNA oligos. Silencing of K-ras reduced the levels of Akt in hypoxia when compared to cells transfected with control siRNA (Fig. 3C). These data indicate that hypoxic activation of Akt is mediated through both Src and K-ras. No changes in phospho-Src416 and total Src protein levels were observed when K-ras was silenced (Fig. 3C), indicating that Src is indeed upstream of K-ras in this hypoxic signaling pathway. To independently verify that K-ras was downstream of Src in this hypoxia-induced phosphorylation of Akt, we performed Hypoxic Activation of K-Ras experiments in a Caco2 cell line stably expressing the mutant oncogene KRASV12 (Caco2/pCSGWK-rasV12). The hypoxic induction of p-Akt was not blocked by silencing of c-Src with siRNA (Fig. 3D). These findings indicate that c-Src functions upstream of K-ras in the hypoxic activation of Akt. K-ras and Src mediate resistance to apoptosis in hypoxia Akt plays a pivotal role in regulating apoptosis and cell cycle progression. Since Akt is a downstream target of K-ras in this hypoxia-triggered intracellular signaling pathway, we sought to determine whether silencing of K-ras would affect cell survival. Knock-down of K-ras decreased cell viability even in normoxic conditions by 20% in HCT116 cells, but there were no significant changes in DLD1 or Caco2 cells. In contrast, K-ras knockdown in hypoxic conditions reduced cell counts to a much greater extent: a 40% reduction in HCT116 (P,0.05) and 50% reductions in DLD1 and Caco2 cells (P,0.01) were observed (Fig. 4A). Silencing of K-ras in Caco2 cells resulted in a survival rate in hypoxia comparable to DLD1 and HCT116 cells that harbor a mutant KRAS gene, suggesting that the wild-type K-ras protein also plays an important role in the adaptive mechanism in hypoxia. We then labeled cells with FITC-conjugated annexin V and propidium iodide (PI) to measure rates of apoptosis. Another wild- type KRAS cell line, Colo320DM, was studied because its growth pattern as single cells permits visualization of apoptotic membrane changes more clearly. Silencing of K-ras resulted in alterations in cell morphology, including blebbing, shrinkage, and nuclear fragmentation, as well as reduced attachment to the tissue culture dish and increased floating. Silencing of K-ras also increased the number of Annexin V positive cells compared with control siRNA (Fig. 4B). To quantify the anti-apoptotic effects of K-ras in hypoxia, we analyzed apoptotic cell populations by flow cytometry (FACS). Caco2 cells transfected with either a non targeting control or K-ras siRNA oligos were incubated in normoxia or hypoxia for 48 hours. Cells without any treatment served as a negative control, and cells exposed to UV light for 10 minutes and cultured with TNF-a and Cycloheximide (CHX) served as a positive control (Fig. 4C, left panel). A histogram of Annexin V-FITC positive cells revealed that knock-down of K-ras in normoxia did not significantly alter rates of apoptosis. However, under hypoxic conditions, loss of K-ras significantly increased rates of cell death from 22% to 57% when compared with control siRNA treatment (Fig. 4C, middle and right panel). We further analyzed the apoptotic cells as two subpopulations: early apoptotic cells (Annexin V+, PI-) and late apoptotic/necrotic cells (Annexin V+, PI+). Only when K-ras was knocked-down under hypoxic conditions was a significant induction of apoptosis observed by FACS analysis; there was a 1.3-fold increase in the number of early apoptotic cells (P,0.05) and 2.0-fold increase in the number of late apoptotic cells (P,0.01) when compared to control siRNA in hypoxia (Fig. 4D). These studies indicate that K-ras can inhibit apoptosis in hypoxia. Our data suggested that Src may also regulate survival pathways that block apoptosis in hypoxia. We defined the relationship between Src and cell survival directly by counting viable cells that excluded trypan blue. Knock-down of Src in DLD1 and HCT116 cells did not alter cell survival in normoxia (1% and 9% reductions in cell counts, respectively), whereas in hypoxia, cell counts decreased by 36% and 45%, respectively (Fig. 4E). Similarly, knockdown of c-Src in Caco2 cells reduced cell counts by 20% in normoxia compared to a more significant 58% reduction in hypoxia. Therefore, the activation of K-ras or Src under hypoxic conditions enhances the survival of colon cancer cells. PLoS ONE | www.plosone.org 4 June 2010 | Volume 5 | Issue 6 | e10966