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Research Scholar from the Heart and Stroke Foundation of Canada. To whom correspondence and reprint requests should be addressed: Dept. of Anatomy and Cell Biology, Botterell Hall, Rm. 859, Queen's University, Kingston, Ontario K7L 3N6, Canada. Tel.: 613-533-2852; Fax: 613-533-2566;
* This work was supported in part by a grant from the Canadian Institutes of Health Research (to C. H. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § Recipient of a joint studentship from the Canadian Institutes of Health Research and the Heart and Stroke Foundation of Canada. ‖ Recipient of a postdoctoral fellowship from the Canadian Hypertension Society.
Tumor hypoxia is associated with a poor prognosis for patients with various cancers, often resulting in an increase in metastasis. Moreover, exposure to hypoxia increases the ability of breast carcinoma cells to invade the extracellular matrix, an important aspect of metastasis. Here, we demonstrate that the hypoxic up-regulation of invasiveness is linked to reduced nitric oxide signaling. Incubation of human breast carcinoma cells in 0.5% versus 20% oxygen increased their in vitro invasiveness and their expression of the urokinase receptor, an invasion-associated molecule. These effects of hypoxia were inhibited by nitric oxide-mimetic drugs; and in a manner similar to hypoxia, pharmacological inhibition of nitric oxide synthesis increased urokinase receptor expression. The nitric oxide signaling pathway involves activation of soluble guanylyl cyclase (sGC) and the subsequent activation of protein kinase G (PKG). Culture of tumor cells under hypoxic conditions (0.5% versus 20% oxygen) resulted in lower cGMP levels, an effect that could be prevented by incubation with glyceryl trinitrate. Inhibition of sGC activity with a selective blocker or with the heme biosynthesis inhibitor desferrioxamine increased urokinase receptor expression. These compounds also prevented the glyceryl trinitrate-mediated suppression of urokinase receptor expression in cells incubated under hypoxic conditions. In contrast, direct activation of PKG using 8-bromo-cGMP prevented the hypoxia- and desferrioxamine-induced increases in urokinase receptor expression as well as the hypoxia-mediated enhanced invasiveness. Further involvement of PKG in the regulation of invasion-associated phenotypes was established using a selective PKG inhibitor, which alone increased urokinase receptor expression. These findings reveal that an important mechanism by which hypoxia increases tumor cell invasiveness (and possibly metastasis) requires inhibition of the nitric oxide signaling pathway involving sGC and PKG activation.
Hypoxia in cancers is associated with resistance to therapy and with increased tumor growth and metastatic potential. Several studies have demonstrated that tumor cells exposed to hypoxia exhibit reduced sensitivity to radiation and drug therapy (
). Exposure of human MDA-MB-231 breast carcinoma cells to hypoxia enhances their ability to invade the extracellular matrix (Matrigel), and this effect of hypoxia is linked to increased expression of the cell-surface urokinase plasminogen activator receptor (uPAR)
). In that study, the increase in drug resistance caused by hypoxia was prevented by low concentrations of NO-mimetic drugs; and in a manner similar to hypoxia, pharmacological inhibition of endogenous NO production with an NO synthase inhibitor led to a drug resistance phenotype. Those findings suggested that NO may play a function in the regulation of tumor cell adaptive responses to alterations in local oxygenation levels.
Nitric oxide is produced endogenously by the enzyme NO synthase (
), and has been implicated in several biological processes such as gene regulation. For example, NO has been shown to activate AP-1 (activatorprotein-1)-regulated genes via a pathway dependent on cGMP production (
). Nitric oxide also modulates hypoxic gene expression. Studies have revealed that NO inhibits the hypoxic induction of erythropoietin, vascular endothelial growth factor, and hypoxia-inducible factor-1 (HIF-1) (
Soluble guanylyl cyclase (sGC) is a well characterized receptor for NO. This heterodimeric protein catalyzes the conversion of GTP to cGMP. Nitric oxide binds to the heme moiety of sGC, thereby inducing conformational changes that result in sGC activation. cGMP is, in turn, a second messenger that amplifies NO signals to downstream effectors (
). Elevated levels of cGMP have been negatively correlated with vascular smooth muscle growth and have been shown to prevent platelet aggregation as well as the adherence of neutrophils to endothelial cells (
Several proteins interact with cGMP and potentially regulate gene expression and cell phenotype at various levels of the signaling cascade. These include protein kinase G (PKG), cGMP-activated phosphodiesterases, and cGMP-gated ion channels. Of these, it is thought that PKG is responsible for the majority of the cellular effects of cGMP. PKG is a serine/threonine kinase that is activated following cGMP binding (
). Upon activation, PKG phosphorylates many intracellular targets, often resulting in alterations in gene expression. Based on the previous knowledge that NO plays a role in the regulation of cellular adaptive responses to hypoxia and given the importance of cGMP-dependent signaling in the actions of NO, we sought to investigate the role of sGC, cGMP, and PKG in the NO-mediated regulation of tumor cell invasion.
The major finding of this study is that NO signaling plays an important role in the regulation of hypoxia-induced invasiveness of human MDA-MB-231 breast carcinoma cells. Furthermore, our results strongly suggest that the mechanisms by which cells adapt to hypoxia and reduced NO activity involve convergent processes.
This study also presents a novel role for cGMP-dependent signaling in the regulation of cellular invasiveness. Specifically, it was shown that the NO-mediated inhibition of uPAR expression is dependent on the sequential activation of sGC and PKG. Furthermore, it was determined that the hypoxic up-regulation of uPAR expression and the concomitant enhancement of thein vitro invasiveness are associated with reduced levels of sGC and PKG signaling. Because invasion of the extracellular matrix is an essential component of the metastatic process, these results suggest that perturbations in the cGMP-dependent signaling pathway could lead to increases in metastatic potential.
Our results show that the effects of hypoxia on sGC activity and uPAR expression can be prevented by low concentrations of GTN. These findings point to a mechanism of oxygen sensing and gene regulation whereby phenotypes are modified in response to a decrease in NO-mediated signaling. As shown in Fig.8, we propose that this phenomenon is due to a reduction in endogenous NO synthesis. Molecular oxygen is obligatory for the conversion of l-arginine into NO andl-citrulline by the enzyme NO synthase (
). Due to the reduced NO levels associated with hypoxia, there is a decrease in guanylyl cyclase activity and a consequential reduction in cGMP levels (Fig. 8). Supporting this concept, Taylor et al. (
) showed that culturing intestinal epithelial cells under hypoxic conditions (1% O2) results in a significant decrease in basal and stimulated cGMP levels. Here, we have similarly shown that low O2 levels decrease cGMP generation. Furthermore, we have demonstrated that the decrease in cGMP signaling is correlated with an enhancement of uPAR expression as well as with increased invasiveness. To strengthen the concept that uPAR expression is regulated through sGC activation, this study showed that GTN was unable to inhibit uPAR expression when sGC activity was directly blocked with either ODQ or DFO (Fig. 4).
Previous studies have shown that treatment with DFO results in cellular adaptive responses similar to those induced by hypoxia (
). In the present study, the DFO-mediated up-regulation of uPAR expression was inhibited by 8-Br-cGMP (Fig. 5). This indicates that a major component of the DFO-mediated stimulation of uPAR expression is inhibition of guanylyl cyclase activity and that DFO does not interfere with signals acting downstream of guanylyl cyclase.
There are many potential targets that could be phosphorylated by PKG and therefore serve as downstream effectors in the NO signaling pathway. One possible mechanism by which PKG regulates cellular adaptations to changes in oxygenation involves a perturbation of the MAPK pathway. This pathway is activated by hypoxia (
), who showed that NO donors and cGMP-mimetic drugs reduce elastase expression by suppressing ERK phosphorylation. This leads to a subsequent reduction in the activation and DNA-binding capacity of AML1B (the transcription factor for elastase). It is possible that cGMP- dependent NO signaling similarly inhibits hypoxia-induced ERK phosphorylation, thereby decreasing the activation of the transcription factors responsible for the up-regulation of uPAR expression.
The promoter region of uPAR contains binding sites for transcription factors such as AP-1, Sp1/3, and nuclear factor-κB (
). HIF-1 levels may also contribute to the transcriptional activation of the uPAR gene, as previous examination of the sequences upstream of the uPAR initiation codon revealed the presence of potential HIF-1-binding sites (
). It has also been shown that HIF-1 accumulation and transcriptional activity can be reduced by relatively high concentrations (2.5–500 μm) of NO-mimetic drugs such as SNP, S-nitroso-l-glutathione, and 3-morpholinosydnonimine (
). Preliminary studies in our laboratory have confirmed that high concentrations (0.1–1 mm) of GTN and SNP inhibit HIF-1 accumulation and transactivating activity.
The invasive potential of a cell is dependent upon its ability to break down components of the extracellular matrix and basement membranes. This process requires the participation of several proteolytic enzyme systems, among which the urokinase plasminogen activator system figures prominently (
) obtained similar results using a rat mammary carcinoma cell line. We have previously demonstrated that uPAR availability for urokinase plasminogen activator binding is essential for the hypoxic stimulation of invasiveness (
) recently demonstrated that hypoxia-induced metastasis is dependent on uPAR up-regulation. Thus, we postulate that the cGMP-dependent inhibition of invasiveness observed in the present study was partially due to down-regulation of uPAR expression. Although the role of uPAR in invasion and tumor progression has been studied extensively, the mechanisms governing its expression are not fully understood. In characterizing the contribution of the plasminogen activator system to the regulation of invasiveness and metastasis, the present study confirmed that uPAR message and protein are up-regulated during hypoxia. In addition, we demonstrated that the NO signaling pathway involving sGC and PKG activation is an integral component of the mechanism that regulates uPAR expression as well as invasiveness.
In summary, this study specifically links NO-mediated activation of sGC and PKG to the regulation of tumor cell invasiveness. Furthermore, our results suggest that sGC and PKG may be useful pharmacological targets for the prevention of cancer invasion and metastasis.
We thank Shannyn MacDonald-Goodfellow, Lori Maxwell, and Judy Pang for technical assistance. We also thank Drs. S. Pang and J. Elce for helpful suggestions.