Y by aldosterone treatment, similar to what found in transfected cells under normoxia, was also found in cells exposed to lower oxygen concentration (Fig. 3B), allowing us to test the effect of MR activation also in cells grown under hypoxic conditions. We also showed that the competitive antagonist spironolactone caused the disappearance of most of the aldosterone induced post-translational modifications while inducing by itself some other specific ones (Fig. 3A lower panel) and largely, though incompletely, abolished the increase of luciferase Calcitonin (salmon) activity induced by aldosterone (Fig. 3B). Strikingly, the nuclear translocation of MR induced byaldosterone could not be blocked but rather was induced by spironolactone alone (Fig. 
3C). Functional validation of our cell model allowed us to investigate the possible causal relationship between MR activity and expression changes of mRNA coding for different angiogenic factors both in normoxic and hypoxic environment. We demonstrated that, in pchMR-transfected HTC116 cells grown under normoxic conditions, MR activation by aldosterone induces a significant decrease in VEGF mRNA expression, while it does not affect the mRNA expression 
levels of other angiogenic factors, namely bFGF, PGF2 and EGF. The aldosterone-induced decrease of VEGFA expression was specifically, albeit partially, inhibited byMR Activity Attenuates VEGF/KDR Pathways in CRCFigure 2. Overall survival stratified by CRC expression of CD34 (left), and CRC expression of MR (right)(Kaplan-Meyer method). doi:10.1371/journal.pone.0059410.gthe competitive MR antagonist spironolactone (Fig. 4). On the whole, these data point out VEGFA as a pro-angiogenic gene potentially regulated by MR activity. It is well known that hypoxia, a constant characteristic of solid tumor microenvironment, strongly induces VEGFA expression. On these bases we analysed the effect of MR activation on VEGFA expression levels under low oxygen concentration. At this purpose, we first demonstrated that, in pchMR-transfected HCT116 cells, the levels of VEGFA mRNA increase after their exposure to hypoxia similarly to wild type HCT116 cells and that the CoCl2 treatment, which mimics hypoxia, strongly increases VEGFA mRNA expression in transfected cells (Fig. 5B and 5A, respectively). We then demonstrated that the decrease of VEGFA expression induced by aldosterone in pchMR-transfected HCT116 cells during normoxia (Fig. 4A) was also found under CoCl2 treatment or hypoxic culture conditions 15755315 (Fig. 6A). These results are particularly significant since we showed that both hypoxia and CoCl2 treatment strongly increases VEGFA mRNA expression (Fig. 4B). Moreover, we also show that MR activation by the agonists naturally present in whole FCS is able to decrease VEGFA mRNA expression both under normoxic and hypoxic conditions (Fig. 6B ). Finally, on the bases of recent reports by Calvani and collaborators on the role of KDR expressed in HCT116 cells in sustaining cell survival after exposure to prolonged hypoxia, we analyzed, in pchMR transfected HCT116 cells, a possible causal relationship between MR activity and KDR expression changes. Indeed, these authors demonstrated that KDR mediates a late VEGF-dependent induction of HIF-1a, leading to the autocrine production of VEGFA, since they could inhibit both ML-240 HIF-1ainduction and survival of hypoxic HCT116 cells, with either antiVEGFA or anti-KDR antibodies. [21] Thus, as a preliminary experiment, we demonstrated the presence of a func.Y by aldosterone treatment, similar to what found in transfected cells under normoxia, was also found in cells exposed to lower oxygen concentration (Fig. 3B), allowing us to test the effect of MR activation also in cells grown under hypoxic conditions. We also showed that the competitive antagonist spironolactone caused the disappearance of most of the aldosterone induced post-translational modifications while inducing by itself some other specific ones (Fig. 3A lower panel) and largely, though incompletely, abolished the increase of luciferase activity induced by aldosterone (Fig. 3B). Strikingly, the nuclear translocation of MR induced byaldosterone could not be blocked but rather was induced by spironolactone alone (Fig. 3C). Functional validation of our cell model allowed us to investigate the possible causal relationship between MR activity and expression changes of mRNA coding for different angiogenic factors both in normoxic and hypoxic environment. We demonstrated that, in pchMR-transfected HTC116 cells grown under normoxic conditions, MR activation by aldosterone induces a significant decrease in VEGF mRNA expression, while it does not affect the mRNA expression levels of other angiogenic factors, namely bFGF, PGF2 and EGF. The aldosterone-induced decrease of VEGFA expression was specifically, albeit partially, inhibited byMR Activity Attenuates VEGF/KDR Pathways in CRCFigure 2. Overall survival stratified by CRC expression of CD34 (left), and CRC expression of MR (right)(Kaplan-Meyer method). doi:10.1371/journal.pone.0059410.gthe competitive MR antagonist spironolactone (Fig. 4). On the whole, these data point out VEGFA as a pro-angiogenic gene potentially regulated by MR activity. It is well known that hypoxia, a constant characteristic of solid tumor microenvironment, strongly induces VEGFA expression. On these bases we analysed the effect of MR activation on VEGFA expression levels under low oxygen concentration. At this purpose, we first demonstrated that, in pchMR-transfected HCT116 cells, the levels of VEGFA mRNA increase after their exposure to hypoxia similarly to wild type HCT116 cells and that the CoCl2 treatment, which mimics hypoxia, strongly increases VEGFA mRNA expression in transfected cells (Fig. 5B and 5A, respectively). We then demonstrated that the decrease of VEGFA expression induced by aldosterone in pchMR-transfected HCT116 cells during normoxia (Fig. 4A) was also found under CoCl2 treatment or hypoxic culture conditions 15755315 (Fig. 6A). These results are particularly significant since we showed that both hypoxia and CoCl2 treatment strongly increases VEGFA mRNA expression (Fig. 4B). Moreover, we also show that MR activation by the agonists naturally present in whole FCS is able to decrease VEGFA mRNA expression both under normoxic and hypoxic conditions (Fig. 6B ). Finally, on the bases of recent reports by Calvani and collaborators on the role of KDR expressed in HCT116 cells in sustaining cell survival after exposure to prolonged hypoxia, we analyzed, in pchMR transfected HCT116 cells, a possible causal relationship between MR activity and KDR expression changes. Indeed, these authors demonstrated that KDR mediates a late VEGF-dependent induction of HIF-1a, leading to the autocrine production of VEGFA, since they could inhibit both HIF-1ainduction and survival of hypoxic HCT116 cells, with either antiVEGFA or anti-KDR antibodies. [21] Thus, as a preliminary experiment, we demonstrated the presence of a func.
