Hypoxic Activation of K-Ras with siRNA control oligos (shown in top rows) or siRNA oligos to K-ras (shown in bottom rows) for 24 hours, followed by incubation in hypoxia for 48 hours. Early apoptotic (FITC+PI-) and late apoptotic/necrotic cells (FITC+PI+) were detected. Left panels: 20x phase contrast and 20x fluorescence green and red channel merged images. Right panels: 20x and 40x fluorescence green and red channel merged images. C, Caco2 cells were transfected with K-ras siRNA or control siRNA, and then incubated in normoxia or hypoxia for 48 hours. Cell death was determined by FACS as described in Materials and Methods. D, Early apoptotic (Annexin V+PI-) and late apoptotic/necrotic (Annexin V+PI+) cells were determined by FACS analysis. Mean 6 SD of three independent experiments is shown. *, P,0.05. **, P,0.01. E, DLD1, HCT116 and Caco2 cells were transfected with control siRNA or c-Src siRNA oligos (20 nM) for 24 hours and then exposed to hypoxia for 48 hours. Cells excluding trypan blue were counted and results are expressed as percentage of viable cells compared with siRNA control transfected cells in normoxia. Mean 6 SD of three independent experiments is shown. *, P,0.05. **, P,0.01. doi:10.1371/journal.pone.0010966.g004 siRNA oligos to define the relationship between K-ras activation and VEGF production. In Caco2 cells transfected with a control siRNA, hypoxia increased VEGF mRNA levels 4.9-fold. Knock- down of K-ras resulted in a decrease of VEGF mRNA expression by 60% and 76% in normoxic and hypoxic conditions, respectively, when compared to cells transfected with a control siRNA (Fig. 5A). We next used a VEGF promoter reporter construct (Fig. 5B). Hypoxic conditions induced VEGF promoter activity by 2.2 fold. Consistent with our qRT-PCR results, silencing of K-ras dramatically reduced the hypoxic induction of VEGF promoter activity by 72%. In normoxia, there was a 38% reduction in VEGF promoter activity after K-ras silencing. This decrease in VEGF promoter activity correlated with the changes seen in K-ras activation. Finally, an ELISA assay demonstrated that secreted VEGF protein levels were up-regulated 2.6-fold by hypoxia. Knockdown of wild-type KRAS in Caco2 cells decreased VEGF protein levels by only 8% in normoxia but reduced VEGF protein levels more significantly in hypoxia by 51% (Fig. 5C). These results suggest that a wild-type KRAS gene is also a critical regulator of VEGF in hypoxic conditions. Hypoxic induction of VEGF is also Src-dependent Although previous evidence has indicated that c-Src signal transduction pathways can regulate VEGF expression, the relationship between activation of Src by hypoxia and production of VEGF in colon tumor cells has not yet been clarified. Rather than overexpressing v-Src, we blocked endogenous c-Src with PP2. PP2 treatment for 24 hours in Caco2 cells suppressed VEGF mRNA levels by 87% in hypoxia compared to a 60% decrease in normoxia (Fig. 6A). PP2 also suppressed VEGF promoter activity by 85% in hypoxia compared to a 50% reduction in normoxia (Fig. 6B). To verify the specific role of c-Src in the hypoxic induction of VEGF, we utilized Src siRNA oligos. With this approach, mRNA levels of VEGF were unchanged in normoxic conditions but reduced by 39% in hypoxia (Fig. 6C), and VEGF promoter activity decreased 56% compared with a 33% decrease in normoxia (Fig. 6D). To confirm this effect was not unique to Caco2 cells, HT29 cells were also analyzed. Treatment with PP2 or Src siRNA oligos in HT29 cells under normoxic conditions had negligible effects on VEGF mRNA or promoter activity. In contrast, under hypoxic conditions, PP2 suppressed VEGF mRNA levels and promoter activity by 67% and 53%, respectively (Fig. 6A and 6B, right panels); similarly, c-Src siRNA oligos reduced VEGF mRNA levels and promoter activity by 37% and 76% in hypoxia, respectively (Fig. 6C and 6D, right panels). Collectively, these results also implicate c-Src as a critical regulator of VEGF in hypoxia. Discussion Hypoxia is an unavoidable consequence of rapid tumor growth that outstrips the existing blood supply. A carefully orchestrated set of adaptive responses ensures survival of the tumor cell in these hypoxic conditions. The HIF-1 transcription factor is known to play a role in this hypoxic response [13,14,15]. We sought to determine whether additional oncogenic pathways may enhance these adaptive responses. In colon cancer, previous studies have demonstrated a synergistic interaction between KRAS mutations and hypoxia in the regulation of multiple genes including VEGF [2,8,9]. However, KRAS mutations are identified in less than half of all colon cancers, and the role of wild-type KRAS in the hypoxic response is less certain. To our knowledge, this is the first demonstration of the hypoxic activation of K-ras in colon cancer. Our data demonstrate that hypoxia is a potent activator of wild- type K-Ras in colon cancer cells. Activation of K-ras resulted in the downstream activation of Akt, induction of VEGF, and inhibition of apoptosis. These effects on angiogenesis as well as apoptosis are both critical for tumor survival in conditions of sustained hypoxia. The recruitment of the Akt pathway is consistent with previous reports of hypoxic signaling by mutant K-ras in the regulation of OPN gene expression [9]. K-ras can activate hypoxia-inducible factor-1 (HIF-1) through protein phosphorylation, and some of the observed effects on the induction of VEGF may potentially be mediated through HIF-1 [16]. However, it is unlikely that HIF-1 is the only mediator of this process, as K-ras can also regulate VEGF through HIF-1 independent pathways in hypoxia [17]. The activation of K-ras in hypoxia depended upon the upstream activation of c-Src. There is a precedent for the hypoxic activation of Src, which has been demonstrated both in vivo and in vitro in other cell types [18]. There are a number of specific mediators that link Src to K-ras, and a role for the activation of EGFR is well-described [11,19,20]. Despite its established link to colonic tumorigenesis, EGFR does not play a role in the hypoxic activation of K-ras. Cellular responses to hypoxia seek to preserve survival in a hostile microenvironment. The activation of K-ras in hypoxic conditions implies a key role in the hypoxic response, and our studies highlight two important functions: inhibition of apoptosis and stimulation of angiogenesis. The role of K-ras in the regulation of apoptosis is highly dependent upon context and cell type. In some cases, K-ras can be pro-apoptotic through the activation of RASSF1 or Nore1, but in other scenarios it may serve anti-apoptotic functions through PI3K or Tiam1 [21]. Our studies in colon cancer cells demonstrate a key role for activation of the Akt pathway in hypoxia, which has been previously shown to block apoptosis in other cellular systems [22,23,24]. Interestingly, ERK was not activated by hypoxia in our system. The role of ERK activation in colon tumors is not straightforward. Although expression of oncogenic K-ras in normal colonic epithelial cells can strongly activate ERK in vivo, only limited ERK signaling was observed in colon tumors that developed in these animals [10]. In addition, colon cancer cells carrying a K-ras mutation failed to demonstrate significant activation of ERK, and no reliable correlation between KRAS mutation status and ERK activation has been observed in human colon cancer samples [10,25]. The specific mechanisms that determine which effector pathways are PLoS ONE | www.plosone.org 7 June 2010 | Volume 5 | Issue 6 | e10966