Oxygen-mediated Regulation of Tumor Cell Invasiveness
2002; Elsevier BV; Volume: 277; Issue: 38 Linguagem: Inglês
10.1074/jbc.m204529200
ISSN1083-351X
AutoresLynne‐Marie Postovit, Michael Adams, Gendie E. Lash, Jeremy P.W. Heaton, Charles H. Graham,
Tópico(s)Hemoglobin structure and function
ResumoTumor 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. 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 (1Brown J.M. Cancer Res. 1999; 59: 5863-5870PubMed Google Scholar, 2Matthews N.E. Adams M.A. Maxwell L.R. Gofton T.E. Graham C.H. J. Natl. Cancer Inst. 2001; 93: 1879-1885Crossref PubMed Scopus (142) Google Scholar, 3Teicher B.A. Cancer Metastasis Rev. 1998; 13: 139-168Crossref Scopus (478) Google Scholar), increased ability to invade the extracellular matrix in vitro (4Graham C.H. Forsdike J. Fitzgerald C.F. MacDonald-Goodfellow S. Int. J. Cancer. 1999; 80: 617-623Crossref PubMed Scopus (190) Google Scholar, 5Cuvier C. Jang A. Hill R.P. Clin. Exp. Metastasis. 1997; 15: 19-25Crossref PubMed Scopus (143) Google Scholar), and greater in vivo metastatic potential (6Young S.D. Marshall R.S. Hill R.P. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 9533-9537Crossref PubMed Scopus (355) Google Scholar, 7Cairns R.A. Kalliomaki T. Hill R.P. Cancer Res. 2001; 61: 8903-8908PubMed Google Scholar). 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) 1The abbreviations used are: uPAR, urokinase plasminogen activator receptor; HIF-1, hypoxia-inducible factor-1; sGC, soluble guanylyl cyclase; PKG, protein kinase G; GTN, glyceryl trinitrate; SNP, sodium nitroprusside; l-NMMA, N-monomethyl-l-arginine; DFO, desferrioxamine mesylate; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; Br, bromo; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase. (4Graham C.H. Forsdike J. Fitzgerald C.F. MacDonald-Goodfellow S. Int. J. Cancer. 1999; 80: 617-623Crossref PubMed Scopus (190) Google Scholar). Furthermore, hypoxia has been shown to increase metastasis of human melanoma cells transplanted into nude mice by up-regulating uPAR expression (8Rofstad E.K. Rasmussen H. Galappathi K. Mathiesen B. Nilsen K. Graff B.A. Cancer Res. 2002; 62: 1847-1853PubMed Google Scholar). Recently, it was shown that a causal link between hypoxia and the acquisition of resistance to chemotherapeutic agents is a reduction in the production of endogenous nitric oxide (NO) by tumor cells (2Matthews N.E. Adams M.A. Maxwell L.R. Gofton T.E. Graham C.H. J. Natl. Cancer Inst. 2001; 93: 1879-1885Crossref PubMed Scopus (142) Google Scholar). 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 (9Griffith O.W. Stuehr D.J. Annu. Rev. Physiol. 1995; 57: 707-736Crossref PubMed Google Scholar), 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 (10Sciorati C. Nistico G. Meldolesi J. Clementi E. Br. J. Pharmacol. 1997; 122: 687-697Crossref PubMed Scopus (45) Google Scholar). 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) (11Sogawa K. Numayama-Tsuruta K. Ema M. Abe M. Abe H. Fujii-Kuriyama Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7368-7373Crossref PubMed Scopus (217) Google Scholar, 12Liu Y. Christou H. Morita T. Laughner E. Semenza G.L. Kourembanas S. J. Biol. Chem. 1998; 273: 15257-15262Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 13Huang L.E. Willmore W.G., Gu, J. Goldberg M.A. Bunn H.F. J. Biol. Chem. 1999; 274: 9038-9044Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar). 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 (14Denninger J.W. Marletta M.A. Biochim. Biophys. Acta. 1999; 1411: 334-350Crossref PubMed Scopus (881) Google Scholar). 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 (15Nunokawa Y. Tanaka S. Biochem. Biophys. Res. Commun. 1992; 188: 409-415Crossref PubMed Scopus (109) Google Scholar, 16Sogo N. Magid K.S. Shaw C.A. Webb D.J. Megson I.L. Biochem. Biophys. Res. Commun. 2000; 279: 412-419Crossref PubMed Scopus (101) Google Scholar, 17Forslund T. Nilsson H.M. Sundqvist T. Biochem. Biophys. Res. Commun. 2000; 274: 482-487Crossref PubMed Scopus (21) Google Scholar). Furthermore, there is also evidence that cGMP can prevent the hypoxic up-regulation of vascular endothelial growth factor expression (12Liu Y. Christou H. Morita T. Laughner E. Semenza G.L. Kourembanas S. J. Biol. Chem. 1998; 273: 15257-15262Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). 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 (18Lincoln T.M. Dey N. Sellak H. J. Appl. Physiol. 2001; 91: 1421-1430Crossref PubMed Scopus (417) Google Scholar). 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. Glyceryl trinitrate (GTN; Sabex, Boucherville, Quebec, Canada) and sodium nitroprusside (SNP; Sigma) were used as NO-mimetic drugs. N-Monomethyl-l-arginine (l-NMMA; Calbiochem-Novabiochem) was used to inhibit endogenous NO production. 3-Isobutyl-1-methylxanthine (Sigma) was used to inhibit phosphodiesterase activity. Desferrioxamine mesylate (DFO; Sigma) was used as an iron chelator and inhibitor of heme biosynthesis. 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; Sigma) was used to selectively inhibit sGC. 8-Bromo-cGMP (8-Br-cGMP; Sigma) was used to activate PKG, and KT5823 (Calbiochem) was used to selectively inhibit PKG. The MDA-MB-231 cell line was maintained in monolayer culture in RPMI 1640 medium (Invitrogen) supplemented with 5% fetal bovine serum (Invitrogen) in a standard Sanyo CO2 incubator (5% CO2 in air at 37 °C; Esbe Scientific, Markham, Ontario, Canada). This is a metastatic breast cancer cell line that was initially isolated in 1973 from a pleural effusion obtained from a 51-year-old patient (19Cailleau R. Young R. Olive M. Reeves Jr., W.J. J. Natl. Cancer Inst. 1974; 53: 661-674Crossref PubMed Scopus (837) Google Scholar). To establish hypoxic conditions, cells were placed in airtight chambers (BellCo Biotechnology, Vineland, NJ) that were flushed with a gas mixture of 5% CO2 and 95% N2. Oxygen concentrations within these chambers were maintained at 0.5% using Pro-Ox Model 110 O2 regulators (BioSpherix, Redfield, NY) as described previously (2Matthews N.E. Adams M.A. Maxwell L.R. Gofton T.E. Graham C.H. J. Natl. Cancer Inst. 2001; 93: 1879-1885Crossref PubMed Scopus (142) Google Scholar). To determine the effect of hypoxia and the NO signaling pathway on the invasiveness of MDA-MB-231 cells, we used a previously described assay (4Graham C.H. Forsdike J. Fitzgerald C.F. MacDonald-Goodfellow S. Int. J. Cancer. 1999; 80: 617-623Crossref PubMed Scopus (190) Google Scholar) that employs reconstituted basement membrane (Matrigel®, Collaborative Biomedical Products, Bedford, MA) as the substrate for invasion. Briefly, Costar Transwell® plastic inserts with a 6.5-mm diameter polycarbonate membrane (8-μm pore; Corning Costar Corp., Cambridge, MA) were coated with 100 μl of a 1 mg/ml solution of Matrigel diluted in serum-free culture medium, placed in the wells of a 24-well tissue culture plate, and allowed to air-dry for ∼12 h. After reconstituting the Matrigel with serum-free medium, 5.0 × 104 cells in serum-containing medium were added to the inserts. Following a 24-h incubation under either hypoxic or standard conditions, in the absence or presence of drugs, cells on the surface of the Matrigel-coated polycarbonate membrane (non-invading cells) were removed by scraping with a cotton swab. Cells that invaded the Matrigel and the pores of the underlying membrane were fixed in Carnoy's fixative (25% acetic acid and 75% methanol) and stained with 1% toluidine blue in 1% sodium borate. Following several rinses in tap water, the membranes were removed with a small scalpel blade, placed on a microscope slide, and coverslipped. The "invasion index" was determined by counting, under a microscope, the total number of stained cells on the underside of the polycarbonate membranes. In a pilot study, we determined that the rate of MDA-MB-231 cell proliferation is identical at 1% versus 20% O2 for at least 48 h, thereby indicating that differences in cell numbers on the membranes at the end of the invasion assay are reflective of altered invasive ability alone. Following incubation, total cellular RNA from cells was isolated using a Gentra Purescript® RNA isolation kit (Gentra Systems, Inc., Minneapolis, MN). The isolated RNA was subsequently separated by electrophoresis, transferred to a charged nylon membrane (Micron Separations, Westborough, MA), and fixed with ultraviolet radiation using a UV cross-linker (Bio-Rad). The membranes were prehybridized at 42 °C in a hybridization incubator for ∼1 h using prewarmed ULTRAhyb® hybridization buffer (Ambion Inc., Austin, TX). They were then hybridized overnight at 42 °C with a uPAR cDNA probe that was cloned in a Bluescript plasmid vector and labeled with [32P]dCTP using anAmersham Biosciences Oligolabelling kit. Following serial washes, the membranes were used to expose Kodak X-Omat Blue film. After 1–4 days, the film was developed and analyzed. The density of the rRNA bands was used to normalize the amount of total RNA loaded in each well. To determine sGC activity, cellular cGMP levels were measured using a commercially available enzyme-linked immunosorbent assay (STI-Signal Transduction Products, San Clemente, CA). Briefly, cells were cultured for 6 h in 20% or 0.5% O2 in the presence or absence of GTN (1 μm). 3-Isobutyl-1-methylxanthine (500 μm) was included in the culture medium to inhibit phosphodiesterase activity, thereby allowing for a measurable accumulation of cGMP. Cells were subsequently extracted over ice in 1 ml of 6% trichloroacetic acid (BDH Laboratory Supplies, Poole, England). The homogenate was then centrifuged at 13,000 ×g for 10 min. The supernatant fraction was removed and extracted five times with 2 ml of water-saturated diethyl ether (BDH Laboratory Supplies). The cGMP contained in this fraction was subsequently acetylated and measured using the enzyme-linked immunosorbent assay kit. Following incubation, cells were lysed in 40 mm HEPES (pH 7.2), 100 mm NaCl, 20% glycerol, 0.1 mm EDTA (pH 8.0), 0.2% Triton X-100, 1 mm dithiothreitol, and 2 mm phenylmethylsulfonyl fluoride. The lysates were homogenized, followed by DNA shearing (10 times with a 25 58-gauge needle), boiling (5 min), and centrifugation (14,000 × g for 15 min). The supernatant was collected and stored at −80 °C until used. Samples were subjected to SDS-PAGE, and the resolved proteins were transferred onto an Immobilon-P membrane (Millipore Corp., Bedford, MA) using a wet transfer apparatus (Bio-Rad). The membranes were blocked overnight at 4 °C in a solution containing 1% phosphate-buffered saline and 0.01% Tween 20 (PBS-T) and 5% dry milk powder. The blots were subsequently incubated for 1.5 h with a monoclonal anti-uPAR antibody (2 μg/ml; monoclonal antibody 3937, American Diagnostica Inc., Greenwich, CT) or polyclonal anti-sGC antiserum (0.5 μg/ml; Cayman Chemical Co., Inc., Ann Arbor, MI), followed by six 5-min washes with PBS-T. The membranes incubated with the anti-uPAR antibody were incubated for 1 h with a horseradish peroxidase-labeled goat anti-mouse IgG secondary antibody (1:7500 dilution; Bio-Rad), and the membranes incubated with the anti-sGC antibody were incubated for 1 h with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:15000 dilution; Vector Laboratories, Inc., Burlingame, CA). Following six additional 5-min washes with PBS-T, secondary antibodies were detected by enhanced chemiluminescence (Amersham Biosciences) and exposure on Kodak X-Omat Blue film. X-ray films of Northern and Western blot experiments were scanned and analyzed using a SigmaGel densitometry software package (SPSS Inc., Chicago, IL). Data are presented as means ± S.D. Statistical analyses were performed using the StatView statistical software package (Abacus Concepts, Inc., Berkley, CA). Statistical significance was determined using one-way analysis of variance, followed by Fisher's post hoc analysis. Student's t test was used when only two sets of data were compared. All statistical tests were two-sided, and differences were considered statistically significant atp < 0.05. As shown in Fig.1, although hypoxia increased thein vitro invasiveness of MDA-MB-231 breast carcinoma cells by >5-fold, this effect of hypoxia was prevented by concomitant treatment with low concentrations of two different NO-mimetic drugs, GTN (1 pm and 0.1 μm; p < 0.002 and 0.001, respectively; one-way analysis of variance, followed by Fisher's test) and SNP (0.1 nm; p < 0.001), administered at the beginning of the 24-h invasion assay. In our previous study (4Graham C.H. Forsdike J. Fitzgerald C.F. MacDonald-Goodfellow S. Int. J. Cancer. 1999; 80: 617-623Crossref PubMed Scopus (190) Google Scholar), we showed that the hypoxic up-regulation of invasiveness is accompanied by increased uPAR expression and that a blocking anti-uPAR antibody could prevent the hypoxic up-regulation of invasiveness, thereby establishing a causal link between uPAR expression and hypoxia-induced invasion. In the present study, culture of MDA-MB-231 cells in 0.5% O2 for 24 h also resulted in up to 3.5-fold increases (p< 0.0001) in uPAR protein levels (Figs.2 A and 5 B) and up to 5-fold increases (p < 0.0002) in uPAR mRNA levels (Figs. 2 C, 4 A, and 5 A). Moreover, administration of single doses of GTN (1 pm, 1 nm, and 0.1 μm) to cells incubated for 24 h in 0.5% O2 was sufficient to prevent the up-regulation of uPAR protein expression (p < 0.004, 0.005, and 0.001 for each concentration of GTN, respectively) (Fig.2 A). Similarly, the hypoxia-mediated increase in uPAR mRNA levels was also inhibited when a low concentration of GTN (1 pm) was used (p < 0.001) (Fig.2 C).Figure 5Effect of 8-Br-cGMP on hypoxia- and DFO-induced uPAR expression in MDA-MB-231 breast carcinoma cells. A, Northern blot analysis of uPAR expression in cells cultured for 24 h in 20 or 0.5% O2 in the absence or presence of various concentrations of 8-Br-cGMP (n = 3). B, Western blot analysis of uPAR protein in cells cultured for 24 h in 20 or 0.5% O2 in the absence or presence of various concentrations of 8-Br-cGMP (n = 9). C, Northern blot analysis of the uPAR transcript in cells cultured for 24 h in the absence or presence of DFO (100 μm) alone or with 8-Br-cGMP (1 μm) (n = 6). Bars in A–C represent mean relative densities ± S.D. *, significantly different from control (20% O2) values. All p values are indicated under "Results."View Large Image Figure ViewerDownload (PPT)Figure 4Effect of sGC disruption on the NO-mediated inhibition of uPAR expression in MDA-MB-231 breast carcinoma cells. A, Northern blot analysis of uPAR mRNA expression in cells cultured for 24 h in 20 or 0.5% O2 with or without GTN (1 μm) and the selective sGC inhibitor ODQ (0.5 μm) (n = 5). B, Northern blot analysis of uPAR expression in cells cultured for 24 h in the presence or absence of the heme disrupter DFO (100 μm) alone or in combination with GTN (1 μm) (n = 5). Bars in bothA and B represent mean relative densities ± S.D. *, significantly different. p values for each condition are indicated under "Results."View Large Image Figure ViewerDownload (PPT) As demonstrated in a previous study, MDA-MB-231 cells express all three isoforms of NO synthase (2Matthews N.E. Adams M.A. Maxwell L.R. Gofton T.E. Graham C.H. J. Natl. Cancer Inst. 2001; 93: 1879-1885Crossref PubMed Scopus (142) Google Scholar). To assess whether endogenous NO inhibits uPAR expression, NO synthesis in MDA-MB-231 cells was blocked by incubation with the NO synthase inhibitorl-NMMA (0.5 μm). In a manner characteristic of cells exposed to hypoxia, a 24-h incubation with a single dose ofl-NMMA resulted in an overall 50% increase (p < 0.004) in uPAR protein levels (Fig.2 B) and a 2.8-fold increase (p < 0.04) in uPAR mRNA levels (Fig. 2 D) in MDA-MB-231 cells even when cultured in 20% O2. In contrast, compared with uPAR expression in cells incubated in 20% O2 alone, no significant increase in uPAR protein (p = 0.89) or transcript (p = 0.35) levels was observed in cells incubated with a combination of l-NMMA (0.5 mm) and GTN (0.1 nm) in 20% O2 (Fig. 2,B and D). The results obtained using the enzyme-linked immunosorbent assay for cGMP revealed that, compared with cells incubated in 20% O2, MDA-MB-231 breast carcinoma cells incubated in 0.5% O2 for 6 h in the presence of 3-isobutyl-1-methylxanthine (500 μm) exhibited a 50% reduction in accumulated cGMP levels (p < 0.002) (Fig.3 A). This effect of hypoxia on cGMP levels was prevented by co-incubation with the NO-mimetic drug GTN (1 μm). Western blot analysis was conducted to determine the effects of hypoxia on sGC protein levels. sGC is a heterodimeric protein consisting mainly of an α1- and a β1-subunit. Culture under hypoxic conditions for 24 h resulted in a 44% decrease in the levels of the β1-subunit (p < 0.0001). In contrast, culture under hypoxic conditions resulted in a 2.3-fold increase (p < 0.02) in the levels of the α1-subunit (Fig. 3 B). Although the ratio of the α1- and β1-subunits was altered during hypoxia, the total amount of sGC was not significantly changed. Fig. 4 A shows that, compared with incubation of cells under control conditions (20% O2 alone), incubation of MDA-MB-231 cells for 24 h with the selective sGC blocker ODQ (0.5 μm) resulted in a 2.7-fold increase (p < 0.05) in the levels of uPAR mRNA (Fig. 4 A). Although the presence of GTN (1 μm) prevented the hypoxic up-regulation of uPAR mRNA expression, GTN was unable to block the effect of hypoxia when ODQ was also present in the medium (Fig. 4 A). sGC is a heme-containing enzyme that requires ferrous iron for its biosynthesis and activity. Therefore, to further assess the participation of this enzyme in the regulation of uPAR expression by the NO signaling pathway, we cultured MDA-MB-231 cells in the presence of DFO (100 μm), an iron chelator and inhibitor of heme biosynthesis. The results showed that, in a manner similar to culture under hypoxic conditions or after pharmacological inhibition of NO synthase, culture in the presence of DFO resulted in a 4-fold increase (p < 0.007) in the levels of uPAR mRNA (Figs.4 B and 5 C). In contrast to hypoxia, the effect of DFO on uPAR mRNA expression was not prevented by 1 μm GTN. Furthermore, the up-regulation of uPAR mRNA and protein expression by hypoxia was significantly reduced (p < 0.0001) in a dose-dependent manner (up to 100%) by the presence of 8-Br-cGMP (0.1–10 μm, 24 h), a non-hydrolyzable analog of cGMP (Fig. 5, A andB). Interestingly, the presence of 8-Br-cGMP (1 μm) also resulted in the complete inhibition (p < 0.01) of the DFO-induced up-regulation of uPAR mRNA expression (Fig. 5 C). These results indicate that a major component of the hypoxia- and DFO-mediated stimulation of uPAR expression is the inhibition of sGC. As indicated earlier, cGMP-mediated activation of PKG is an important component of the NO signaling pathway. To further elucidate the role of NO signaling in the regulation of uPAR expression, MDA-MB-231 cells were incubated for 6 h with the PKG inhibitor KT5823 (10 μm). Northern and Western blot analyses revealed that selective inhibition of PKG, even in 20% O2, resulted in a 1.8-fold increase (p < 0.05) in uPAR protein and mRNA expression (Fig.6, A and B). These results demonstrate that PKG activation by cGMP is necessary for the inhibition of uPAR expression by NO. Results from the in vitro invasion assay using Matrigel as a substrate for invasion revealed that, compared with the invasiveness of cells incubated in 20% O2, hypoxia stimulated the invasiveness of MDA-MB-231 cells by 3.9-fold (p < 0.0001) (Fig.7). This effect of hypoxia on invasiveness was completely inhibited by the presence of various concentrations of 8-Br-cGMP (0.1 μm to 1 mm) (Fig. 7). 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 (20Dweik R.A. Laskowski D. Abu-Soud H.M. Kaneko F. Hutte R. Stuehr D.J. Erzurum S.C. J. Clin. Invest. 1998; 101: 660-666Crossref PubMed Scopus (261) Google Scholar). Indeed, exposure of cells to low levels of O2 (1–3%) inhibits NO production by up to 90% (21Whorton A.R. Simonds D.B. Piantadosi C.A. Am. J. Physiol. 1997; 272: L1161-L1166PubMed Google Scholar). 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. (22Taylor C.T. Lisco S.J. Awtrey C.S. Colgan S.P. J. Pharmacol. Exp. Ther. 1998; 284: 568-575PubMed Google Scholar) 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 (13Huang L.E. Willmore W.G., Gu, J. Goldberg M.A. Bunn H.F. J. Biol. Chem. 1999; 274: 9038-9044Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar, 23Gleadle J.M. Ebert B.L. Firth J.D. Ratcliffe P.J. Am. J. Physiol. 1995; 268: C1362-C1368Crossref PubMed Google Scholar, 24Wang G.L. Semenza G.L. Blood. 1993; 82: 3610-3615Crossref PubMed Google Scholar, 25Goldberg M.A. Dunning S.P. Bunn H.F. Science. 1988; 242: 1412-1415Crossref PubMed Scopus (877) Google Scholar). This has led to the hypothesis that a hemeprotein or changes in the redox status of cells are responsible for the adaptive responses to hypoxia (26Ratcliffe P.J. O'Rourke J.F. Maxwell P.H. Pugh C.W. J. Exp. Biol. 1998; 201: 1153-1162PubMed Google Scholar). 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 (27Lo L.W. Cheng J.J. Chiu J.J. Wung B.S. Liu Y.C. Wang D.L. J. Cell. Physiol. 2001; 188: 304-312Crossref PubMed Scopus (91) Google Scholar), and studies have shown that NO can prevent the phosphorylation of ERK through a PKG-mediated interference of Ras/Raf (28Yu S.M. Hung L.M. Lin C.C. Circulation. 1997; 95: 1269-1277Crossref PubMed Scopus (179) Google Scholar). This concept is supported by the study of Mitani et al. (29Mitani Y. Zaidi S.H. Dufourcq P. Thompson K. Rabinovitch M. FASEB J. 2000; 14: 805-814Crossref PubMed Scopus (44) Google Scholar), 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 (30Wang Y. Med. Res. Rev. 2001; 21: 146-170Crossref PubMed Scopus (118) Google Scholar). 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 (31Graham C.H. Fitzpatrick T.E. McCrae K.R. Blood. 1998; 91: 3300-3307Crossref PubMed Google Scholar). 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 (12Liu Y. Christou H. Morita T. Laughner E. Semenza G.L. Kourembanas S. J. Biol. Chem. 1998; 273: 15257-15262Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 13Huang L.E. Willmore W.G., Gu, J. Goldberg M.A. Bunn H.F. J. Biol. Chem. 1999; 274: 9038-9044Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar). 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 (32Friedl P. Brocker E.B. Cell. Mol. Life Sci. 2000; 57: 41-64Crossref PubMed Scopus (536) Google Scholar). In vitro invasiveness has been strongly correlated with the levels of cell-surface uPAR expression (4Graham C.H. Forsdike J. Fitzgerald C.F. MacDonald-Goodfellow S. Int. J. Cancer. 1999; 80: 617-623Crossref PubMed Scopus (190) Google Scholar, 33Kariko K. Kuo A. Boyd D. Okada S.S. Cines D.B. Barnathan E.S. Cancer Res. 1993; 53: 3109-3117PubMed Google Scholar, 34Xing R.H. Rabbani S.A. Int. J. Cancer. 1996; 67: 423-429Crossref PubMed Scopus (90) Google Scholar, 35Graham C.H. Postovit L.-M. Park H. Canning M.T. Fitzpatrick T.E. Placenta. 2000; 21: 443-450Crossref PubMed Scopus (87) Google Scholar). For example, using an in vitro assay similar to the one in this study, Kariko et al. (33Kariko K. Kuo A. Boyd D. Okada S.S. Cines D.B. Barnathan E.S. Cancer Res. 1993; 53: 3109-3117PubMed Google Scholar) showed that human osteosarcoma cells transfected with a human uPAR cDNA are four times more invasive than control cells. Xing and Rabbani (34Xing R.H. Rabbani S.A. Int. J. Cancer. 1996; 67: 423-429Crossref PubMed Scopus (90) Google Scholar) 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 (4Graham C.H. Forsdike J. Fitzgerald C.F. MacDonald-Goodfellow S. Int. J. Cancer. 1999; 80: 617-623Crossref PubMed Scopus (190) Google Scholar, 35Graham C.H. Postovit L.-M. Park H. Canning M.T. Fitzpatrick T.E. Placenta. 2000; 21: 443-450Crossref PubMed Scopus (87) Google Scholar). There is also clinical and experimental evidence that increased uPAR expression is associated with metastasis of prostate, colon, and breast carcinomas (34Xing R.H. Rabbani S.A. Int. J. Cancer. 1996; 67: 423-429Crossref PubMed Scopus (90) Google Scholar, 36Crowley C.W. Cohen R.L. Lucas B.K. Liu G. Shuman M.A. Levinson A.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5021-5025Crossref PubMed Scopus (367) Google Scholar, 37Ganesh S. Sier C.F. Heerding M.M. Griffioen G. Lamers C.B. Verspaget H.W. Lancet. 1994; 344: 401-402Abstract PubMed Scopus (173) Google Scholar). Furthermore, using human melanoma cells transplanted into nude mice, Rofstadet al. (8Rofstad E.K. Rasmussen H. Galappathi K. Mathiesen B. Nilsen K. Graff B.A. Cancer Res. 2002; 62: 1847-1853PubMed Google Scholar) 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.
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