Extracellular ATP activates multiple signalling pathways and potentiates growth factor-induced c-fos gene expression in MCF-7 breast cancer cells

S.C. Wagstaff1,2, W.B. Bowler1, J.A. Gallagher1 and R.A. Hipskind3,3

1 Human Bone Cell Research Group, Department of Human Anatomy and Cell Biology, University of Liverpool, Liverpool L69 3GE,
2 Department of Human and Biological Sciences, Liverpool Hope University College, Hope Park, Liverpool, L16 9JD, UK and
3 Institut de Genetique Moleculaire de Montpellier, UMR 5535, CNRS, 1919 Route de Mende, 34293 Montpellier cedex 5, France


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the human breast cancer cell line MCF-7, the nucleotides ATP{gamma}S and UTP, acting extracellularly through the purinergic receptor P2Y2, lead to elevated intracellular calcium levels and increased proliferation. ATP{gamma}S and UTP treatment of MCF-7 cells activated transcription of the immediate early gene c-fos, an important component in the response to proliferative stimulation. c-fos induction was enhanced by co-treatment with ATP{gamma}S and a variety of proliferative agents including growth factors, tumour promoters and stress. Stimulation with ATP{gamma}S or epidermal growth factor (EGF) led to extracellular signal-regulated kinase (ERK) activation and phosphorylation of the transcription factors CREB and Elk-1. Co-stimulation synergistically activated fos expression and notably led to increased levels of ERK, CREB and EGF receptor phosphorylation, as well as hyperphosphorylation of ternary complex factor. Nevertheless, the ERK pathway does not fully account for this synergy, since fos induction was differentially sensitive to the MEK inhibitor U0126, indicating that these two agonists signal differently to this immediate early gene. Thus, extracellular nucleotides co-operate with growth factors to activate genes linked to the proliferative response in MCF-7 cells through activation of specific purinergic receptors, which thereby represent important potential targets for arresting the neoplastic progression of breast cancer cells.

Abbreviations: CREB, camp response element binding protein; EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; PTHrP, parathyroid hormone-related peptide; TCF, ternary complex factor; TGFß1, transforming growth factor ß1.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell proliferation is controlled by a complex intracellular signalling network whose disruption is strongly linked to neoplastic transformation. This is well characterized for the pathway that links growth-factor activated receptors to the Ras/Raf/extracellular signal-regulated kinase (ERK) signalling cascade, where a number of its components are found mutated in a wide variety of cancers. Accordingly, these mutant proteins can function as potent oncogenes when over-expressed in vivo and in vitro. However, focus formation and tumorigenesis is much more efficient upon the concomitant expression of activated Ras and Myc, and a number of oncogenic signals augment the efficiency of Ras or Myc transformation. To understand and hence, reverse this process, it is essential to identify the factors capable of potentiating the transition to cancerous growth through complementation of the activated Ras/Raf/ERK signalling cascade.

Extracellular nucleotides have recently emerged as a novel class of proliferative agents and possible effectors of neoplastic transformation. Two types of receptor mediate responses to nucleotides: (i) P2Y, seven-transmembrane-domain receptors that are coupled to heterotrimeric G-proteins and (ii) P2X, ligand-gated ion channels (1). Extracellular ATP, which activates P2Y1 and P2Y2 receptors, synergizes with growth factors to induce mitogenesis in non-transformed smooth muscle cells, endothelial cells and astrocytes (24). In the breast cancer cell line MCF-7, ATP and UTP elevate intracellular calcium and stimulate proliferation via activation of the P2Y2 receptor (5). A variety of different signalling pathways could mediate these effects, since extracellular nucleotides lead to tyrosine phosphorylation of growth factor receptors (6) and activate the ERK cascade (7), phosphatidyl inositol 3-kinase (8), protein kinase A (9) and other Ca2+-dependent pathways depending upon the cell type (10).

An important component in the response to proliferative signals is the rapid, transient transcriptional activation of immediate early genes, such as the c-fos proto-oncogene. c-fos expression is regulated at multiple levels by intracellular signalling events, which makes it a useful paradigm to identify and characterize factors that affect cancer cell growth. This is particularly relevant in breast cancer models, since the expression of c-fos antisense RNA inhibits growth of MCF-7 tumours in athymic mice (11).

Extensive studies have linked distinct elements in the c-fos promoter to different signal transduction modules through the phosphorylation of transcription factors that interact with regulatory sites. Less well characterized is c-fos induction resulting from synergy between different signals, as appears to be the case with extracellular nucleotides. We have recently shown that nucleotide-induced c-fos activation in human osteosarcoma cells is strongly enhanced by co-treatment with parathyroid hormone (10). This synergy represented a novel example of distinct intracellular signalling pathways activating different regulatory elements in the c-fos promoter. This led us to investigate the effects of extracellular nucleotides alone and in combination with growth factors on c-fos induction in the breast cancer cell line MCF-7. We find that ATP{gamma}S and epidermal growth factor (EGF) alone lead to moderate increases in c-fos mRNA levels, but together they synergistically activate c-fos. This involves the ERK cascade, since synergy is partially sensitive to the MEK inhibitors, U0126 and PD98059, and another pathway correlating with increased CREB and ternary complex factor (TCF) phosphorylation. Thus, in MCF-7 cells, P2-receptor activation induces intracellular signalling events that co-operate with EGF to potentiate mitogenesis-linked gene activation.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Dulbecco's modified Eagle's medium (DMEM) was obtained from Flow Laboratories (GIBCO, Paisley, UK). Falcon culture plasticware was obtained from Fred Baker Scientific (Runcorn, Cheshire, UK). Fetal calf serum (FCS) was purchased from Myclone. Zetabind hybridization membrane was purchased from Cuno Products (Meriden, CT). Nucleotides, Tri-reagent, bovine serum albumin (BSA), total calf liver RNA, Torula tRNA, anisomycin, calcium ionophore and TPA were obtained from Sigma Chemical Co. (Poole, UK). Phospho-specific (Thr202Tyr204) p44/p42 (ERK 1 and ERK2) antibody, phospho-specific CREB (Ser133), and phospho-specific Elk-1 (Ser383), as well as control antibodies, were obtained from New England Biolabs (Hitchen, UK). Phospho-Tyr1173-specific EGF receptor antibody was obtained from Upstate Biotechnology (Buckingham, UK). PVDF membrane was purchased from Millipore (Bedford, MA, USA). [{alpha}-32P]UTP (800 Ci/mMol) was obtained from NEN Life Science Products (Boston, MA). EGF and TGFß1 were obtained from R& Systems (Abingdon, UK). Parathyroid hormone was purchased from Peninsula (St Helens, UK), and PTHrP from Thistle (Glasgow, UK). U0126 was purchased from Promega (Southampton, UK) and PD98059 from New England Biolabs.

Cell culture
MCF-7 cells were cultured in DMEM supplemented with 10% FCS, 100 µg/ml streptomycin, 100 U/ml penicillin and 2 mM L-glutamine and incubated at 37°C in a humidified atmosphere of 95% air and 5% CO2. At 75% confluence, medium was replaced with DMEM containing 0.5% serum and cells were cultured overnight. Cells were subsequently serum starved for 4 h before treatment. To obtain total RNA, cells were challenged with agonist for 45 min before extraction in Tri-reagent according to the manufacturer's protocol.

Nuclear extract preparation and gel retardation assays
MCF-7 cells were challenged for 10 min with agonist before preparation of nuclear extracts as previously described (28). Gel retardation assays were performed according to established protocols (10). Core SRF90–244 was produced in HeLa cells using a recombinant vaccina virus (29). The probe corresponded to the c-fos SRE subcloned in front of a G-free cassette plasmid (30). After digestion with EcoRI and NarI, the ends were labelled by a Klenow fill-in reaction containing [{alpha}-32P]dATP and cold dCTP, dGTP and dTTP fragments. Fragments were isolated from polyacrylamide gels by electroelution.

Northern analysis
Ten micrograms of total RNA was electrophoresed through a 0.8% (w/v) agarose–formaldehyde gel and transferred to Zetabind hybridization membrane according to the manufacturer's protocol. Blots were prehybridized in 50% formamide, 5x SSC, 5x Denhardt's, 50 mM NaHPO4, pH 6.8, 1% SDS, 5 mg/ml total calf liver RNA, 5 mg/ml Torula tRNA for 30 min at 65°C and probed with [32P]UTP-labelled riboprobes specific for the antisense strand of human c-fos exons 3 and 4 and either rat gapdh or ief-4{alpha} (31) as the internal control. Membranes were washed for 30 min in 0.2x SSC/1% SDS at 65°C and mRNA was visualized using phosphor storage technology and autoradiography with Kodak XAR film and intensifying screens at –70°C. Phosphorimager scans were quantified using ImageQuant (Molecular Dynamics) and standardized to the internal control.

Western analysis
To obtain whole protein cell extracts for western analysis, cells were challenged with agonist for 10 min before extraction in Laemlli loading buffer. Fifteen micrograms of cell extract were separated by SDS–PAGE on a 9% (w/v) minigel and electrotransferred to PVDF using the Milliblot-SDE system (Millipore) at 0.6 mA/cm2 for 1 h. Membranes were blocked with 5% (w/v) non-fat dry milk in 10 mM Tris–HCl, pH 7.5, 100 mM NaCl, 0.1% Tween 20 (TBST) for 3 h at room temperature. Membranes were probed with a polyclonal phospho-specific antiserum against ERK1 and ERK2, CREB, EGFR or Elk-1 according to the manufacturer's protocol. Equal protein loading was confirmed with duplicate blots probed with phosphorylation-state independent control antisera against ERK1 and ERK2, Elk-1 or CREB. For western analysis of EGF receptor, RIPA lysates (50 mM Tris–HCl pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM PMSF, 1 µg/ml each aprotinin, leupeptin and pepstatin, 1 mM Na3VO4 and 1 mM NaF) were acetone precipitated. Fifty micrograms of total protein were resuspended in reducing LDS sample buffer and separated on 3–8% Tris-acetate Novex minigels (Invitrogen, BU, Groningen, Netherlands) according to the manufacturer's instructions, before electrotransfer to nitrocellulose and immunodetection.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
ATP strongly induces c-fos gene expression in MCF-7 breast cancer cells
Stimulation of MCF-7 cells with ATP{gamma}S led to a dose-dependent induction of c-fos mRNA (Figure 1AGo). Treatment with 10–6 M ATP{gamma}S gave a weak response that increased several-fold with 10–5 M and jumped substantially to a maximum at 10–4 M ATP{gamma}S. This induction showed classic immediate early gene kinetics, with a transient peak 30–60 min after induction that returned to basal levels by 90 min after induction (Figure 1BGo). ATP{gamma}S did not affect levels of gapdh mRNA, which was equal in all lanes.



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Fig. 1. ATP{gamma}S induces c-fos in MCF-7 cells. Quiescent MCF-7 cells were either untreated (vehicle) or treated as indicated above each lane. Northern blots containing 10 µg total RNA per lane were co-hybridized with c-fos and gapdh riboprobes labelled with [32P]UTP. The positions of the corresponding mRNAs are indicated to the right of each panel. (A) Dose-dependence for c-fos activation by ATP{gamma}S. Cells were untreated (vehicle) or treated with 10% FCS or ATP{gamma}S as indicated, for 45 min. (B) Kinetics of c-fos induction by ATP{gamma}S.

 
c-fos is specifically induced by P2Y2 receptor agonists
Our previous studies have shown that MCF-7 breast cancer cells express the P2Y2 receptor (5), which is activated by ATP and UTP (1). To confirm that induction of c-fos mRNA by ATP{gamma}S resulted from P2Y2 receptor activation, serum-starved MCF-7 cells were challenged with a range of P2 receptor agonists. Both ATP{gamma}S and UTP (10 µM) strongly elevated c-fos mRNA levels (Figure 2Go), where UTP was clearly more effective. In contrast, neither the P2Y1 receptor agonists ADP and 2MeSATP, nor the P2X receptor agonist {alpha}ß-MeATP significantly activated c-fos (Figure 2Go). Equal loading and hybridization were confirmed by ethidium bromide staining to reveal 18S and 28S ribosomal RNAs (Figure 2Go) and hybridization with the housekeeping gene ief-4{alpha} (data not shown).



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Fig. 2. Specific induction of c-fos mRNA by P2Y2 receptor agonists. Quiescent MCF-7 cells were either untreated (vehicle) or treated as indicated above each lane, for 45 min. The northern blot was hybridized as indicated in the legend to Figure 1Go, except that RNA integrity and loading were confirmed by ethidium bromide staining (lower panel) to identify the two major rRNAs, and by hybridization with the housekeeping gene ief4-{alpha} (data not shown).

 
ATP potentiates EGF-induced c-fos transcription
Nucleotides can potentiate the proliferative effects of growth factors in a number of cell types. In order to determine if ATP potentiates growth factor-induced c-fos expression in MCF-7 breast cancer cells, we tested EGF, transforming growth factor ß1 (TGFß1) and synthetic human parathyroid hormone-related peptide (PTHrP1–35) alone and in combination with a submaximal concentration of ATP{gamma}S (10–5 M) (Figure 3AGo). EGF induced an increase in c-fos mRNA similar to that seen with ATP{gamma}S alone, whereas in combination, these two agents strongly potentiated c-fos induction. In contrast to this striking effect, c-fos induction by the combination of TGFß1 and ATP{gamma}S was no better than that by ATP{gamma}S alone, whilst co-stimulation with PTHrP and ATP{gamma}S elevated mRNA levels additively.



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Fig. 3. ATP{gamma}S potentiates c-fos induction by multiple inducers. Quiescent MCF-7 cells were either untreated (vehicle) or treated for 45 min as indicated below each bar. mRNAs were visualized by northern blotting as described in the legend to Figure 1Go, and the hybridization signals were quantified using a Molecular Dynamics Phosphorimager. The c-fos signal in each lane was standardized to the gapdh or ief-4{alpha} internal control; the level of induction is relative to that observed in the control lane, which is taken as 1. (A) ATP{gamma}S potentiates c-fos induction by EGF but not TGF-ß1 or PTHrP. (B) ATP{gamma}S differentially potentiates c-fos induction by activators of distinct intracellular signalling systems. Quiescent MCF-7 cells were induced with TPA, anisomycin or Ca2+ ionophore either alone or together with ATP{gamma}S as indicated.

 
ATP strongly potentiates MAPK-driven transcription
In order to define the intermediates involved in ATP and EGF potentiation, we assessed the ability of a submaximal dose of ATP{gamma}S to potentiate c-fos induction in combination with activators of distinct intracellular signalling systems. The phorbol ester TPA activates protein kinase C and ERK in MCF-7 cells (12), calcium ionophore increases intracellular Ca2+ levels and anisomycin activates both the SAPK/JNK and p38MAPK stress signalling cascades (13), both of which can target the serum response element (SRE) via TCF phosphorylation (14). All three inducers strongly activated c-fos expression in MCF-7 cells (Figure 3BGo) and each was affected differently by co-stimulation with ATP{gamma}S. c-fos induction by anisomycin was clearly potentiated, while the effect was less strong with TPA. Nevertheless, it was more than additive, unlike the degree of stimulation seen with Ca2+ ionophore and ATP{gamma}S. The latter effect clearly contrasts with that observed between EGF and ATP{gamma}S (Figure 3AGo) and underlines the significance of that synergy.

ATP and EGF induce phosphorylation and activation of ERK1 and ERK2 and the transcription factor CREB: differential sensitivity to MEK inhibitor U0126
ATP-induced increases in intracellular calcium levels may be linked to several different downstream intracellular signalling systems converging on the c-fos promoter. Activation of protein kinase A (14) or activation of CaMKs can result in activation, by phosphorylation on Ser133, of the transcription factor CREB, and thereby targets the CaCRE (position –60 in the c-fos promoter). Alternatively, extracellular ATP may lead to activation of the ERK signalling cascade and therefore signal to the c-fos SRE (position –300 in the c-fos promoter). Consequently, we have used phosphospecific antisera to determine if ATP treatment of MCF-7 cells leads to CREB phosphorylation or ERK activation. Antisera have been developed that are highly specific for Ser133-phosphorylated CREB, as well as for p44 (ERK1) and p42 (ERK2) doubly phosphorylated on Thr202 and Tyr204, the key step in the activation of these two kinases. Extracts were prepared from stimulated MCF-7 cells, separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and electroblotted on to polyvinylidene difluoride (PVDF) membranes. Immobilized proteins were immunodetected with the phospho-specific antisera (Figure 4A and BGo), which revealed that ATP{gamma}S treatment strongly increased both CREB and ERK phosphorylation relative to the uninduced cell extracts. Likewise, EGF treatment also strongly increased both ERK and CREB phosphorylation. Notably, co-stimulation with ATP{gamma}S and EGF led to increased levels of phospho-ERK and phospho-CREB relative to those observed with either factor alone. To test the contribution of the ERK pathway to CREB phosphorylation in our system, we treated MCF-7 cells with U0126, an inhibitor specific for the ERK-activating kinase, MEK. Interestingly, ATP{gamma}S and EGF-induced ERK and CREB phosphorylation was differentially sensitive to the MEK inhibitor, U0126. U0126, as expected, blocked ERK phosphorylation by ATP{gamma}S and EGF. In addition, CREB phosphorylation by EGF was sensitive to U0126. In contrast, CREB phosphorylation by ATP{gamma}S was unaffected by the inhibitor. Immunodetection with control antisera confirmed that induction did not affect the levels of ERK or CREB.



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Fig. 4. ATP{gamma}S induces phosphorylation and activation of ERK and the transcription factor CREB. Quiescent MCF-7 cells were stimulated for 10 min with 10–5 M ATP{gamma}S, 50 ng/ml EGF or the two together either alone or in combination with the MEK inhibitor U0126 (10 µM) as indicated above each lane. Levels of activated (phosphorylated) ERK and total ERK (A) or CREB (B) were assessed in protein extracts by western blotting. (A) Upper panel: western blot probed with a polyclonal phospho-specific antibody against ERK1 and ERK2. Lower panel: duplicate western blot probed with the control (phosphorylation-state independent) ERK antibody. (B) Upper panel: western blot probed with a polyclonal phospho-specific antibody against CREB. Lower panel: duplicate western blot probed with the control (phosphorylation-state independent) CREB antibody.

 
c-fos activation by ATP{gamma}S is only partially sensitive to inhibition of the ERK cascade
ATP{gamma}S-induced CREB activation, unlike EGF, was not sensitive to inhibition by U0126, suggesting that ATP activates CREB and ultimately targets the c-fos CaCRE, via an ERK-independent mechanism. We therefore investigated the effect of U0126 on fos mRNA induction following treatment with ATP{gamma}S and EGF. ATP{gamma}S and EGF activated c-fos alone and synergistically when combined. EGF-induced c-fos induction was completely inhibited by U0126, while the inhibitor only partially affected c-fos induction by ATP{gamma}S (Figure 5AGo). Similar results were obtained using the MEK inhibitor PD98059 (Figure 5BGo).



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Fig. 5. Effect of the MEK inhibitors U0126 and PD98059 on ATP{gamma}S- and EGF-induced c-fos transcription. Quiescent MCF-7 cells were treated with DMSO or the MEK inhibitor U0126 (A) or PD98059 (B) and either left unstimulated (vehicle) or stimulated for 45 min as indicated below each bar. mRNAs were visualized by northern blotting as described in the legend to Figure 1Go. The positions of the corresponding mRNAs are indicated to the right of each panel.

 
ATP and EGF co-stimulation potentiates EGF receptor phosphorylation
G-protein-coupled receptors have been linked to MAPK pathways via activation of tyrosine kinases that phosphorylate growth factor receptors. A more recent report has suggested that growth factor receptor phosphorylation links P2Y2 receptor activation to ERK phosphorylation (6). Consequently, we investigated whether EGF receptor phosphorylation occurred in response to nucleotide treatment, and whether co-stimulation with EGF and P2Y2 receptor agonists resulted in elevated levels of EGF receptor phosphorylation. As expected, treatment with EGF alone led to increased levels of EGF receptor phosphorylation, whilst the nucleotides ATP or UTP did not (Figure 6Go). Interestingly, co-stimulation with either nucleotide and EGF resulted in much greater levels of receptor phosphorylation than those seen with EGF stimulation alone.



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Fig. 6. ATP induces phosphorylation of EGF receptor. Quiescent MCF-7 cells were stimulated for 10 min with 10–5 M ATP{gamma}S, 10–5 M UTP, 50 ng/ml EGF or nucleotides and EGF together as indicated above lanes. Level of EGF receptor phosphorylation in RIPA extracts was assessed by immunoblotting with antiserum directed against the EGFR receptor phosphorylated on Tyr1173. A non-specific complex served as loading control.

 
ATP and EGF co-stimulation results in hyperphosphorylation of TCF
TCF is a main nuclear target for activated MAPK pathways (ERK, SAPK/JNK and p38MAPK). Resulting phosphorylation strongly potentiates transcriptional activation by TCF (14). Consequently, we have investigated the phosphorylation status of endogenous TCF following ATP{gamma}S and EGF stimulation. TCF can be visualized in bandshift assays where it forms a ternary complex on a 32P-labelled SRE probe, together with a recombinant truncated version of SRF that spans the MADS box and neighbouring amino acids. Phosphorylation of TCF leads to slowed mobility of the ternary complexes, which can be seen in response to EGF, ATP{gamma}S and anisomycin but not PTHrP stimulation (Figure 7Go). Interestingly, co-stimulation with both ATP{gamma}S and EGF appeared to result in TCF hyperphosphorylation, as indicated by a further mobility shift, an event only partially mirrored by anisomycin and ATP{gamma}S co-stimulation.



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Fig. 7. ATP and EGF co-stimulation results in hyperphosphorylation of TCF. Quiescent MCF-7 cells were stimulated for 10 min with 50 ng/ml EGF, 100 ng/ml PTHrP or 50 ng/ml anisomycin alone or in combination with 10–5 M ATP{gamma}S as indicated above each lane. Nuclear extracts were prepared and 10 µg of each was incubated for 30 min at room temperature with 32P-labelled SRE and core SRF90–245. Protein–DNA complexes were resolved by electrophoresis in 5% polyacrylamide gels containing 0.5x TBE for 4 h at 1 mA/cm. Phosphorylation of TCF changes the mobility of its ternary complexes with core SRF and the SRE from the uninduced position (U) to the slowed, induced position (I). The exaggerated induced mobility shift detected in extracts from cells challenged with both ATP{gamma}S and EGF represents a hyperphosphorylated form of TCF.

 
Elk-1 is the major component of TCF in human cells and represents a major nuclear target of activated ERK (15). To confirm that the induced complexes reflected Elk-1 phosphorylation, we probed western blots with affinity-purified antisera directed against Elk-1 phosphorylated on Ser383 (Figure 8Go). Indeed, all treatments that led to the induced bandshift complex showed Elk-1 phosphorylation. Notably, co-stimulation caused Elk-1 to migrate more slowly (Figure 8Go, ATP+EGF lane), an observation that is consistent with hyperphosphorylation as suggested by the bandshift analysis.



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Fig. 8. ATP and EGF induce activation of Elk-1. MCF-7 cells were stimulated for 10 min with 50 ng/ml EGF or 50 ng/ml anisomycin alone and in combination with 10–5 M ATP{gamma}S as indicated above each lane. Levels of Elk-1 phosphorylation in nuclear extracts were assessed by immunoblotting with antisera directed against Elk-1 phosphorylated on Ser383 or the Ets domain of Elk-1 (15).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Our previous studies have demonstrated that MCF-7 cells express P2Y2 receptors and proliferate in response to both ATP and UTP (5). These studies extend our previous observations and address the mechanisms underlying the proliferative response to extracellular nucleotides in vitro. Extracellular ATP{gamma}S strongly induced c-fos gene expression in the breast cancer cell line MCF-7 in a dose-dependent manner and with kinetics classically associated with induction of this immediate early gene. Furthermore, this induction was mediated solely by the P2Y2 receptor, since the P2Y2 receptor agonists ATP{gamma}S and UTP, but not the P2Y1, P2Y4, P2Y6 or P2X receptor agonists, gave rise to robust c-fos gene expression. Activation of c-fos is particularly relevant in the MCF-7 breast cancer model, since expression of fos antisense RNA blocks MCF-7 tumour growth in nude mice (11). Our observation that ATP induces c-fos expression is consistent with this model and significant in the context of the strong mitogenic effect of nucleotides on MCF-7 cells in culture.

Our previous demonstration that ATP potentiates c-fos transcription induced by systemic hormones in transformed cells (10) prompted us to assess the ability of ATP{gamma}S to modulate growth-factor-induced c-fos expression in MCF-7 cells. The results from co-stimulation with ATP{gamma}S and activators of different signalling cascades offer insight into the key pathways involved in ATP mediated-potentiation of growth factors and other environmental stimuli. Firstly, the effects of ATP{gamma}S and Ca2+ ionophore on c-fos induction were additive, suggesting that different signalling roles exist for calcium mobilized from intracellular pools (ATP{gamma}S) compared with extracellular calcium influx (calcium ionophore). The additivity also suggests that one calcium pathway cannot synergize with another. In contrast, ATP{gamma}S was able to co-operate with the signalling pathways activated by EGF, anisomycin and TPA. All of these factors induce c-fos expression, at least in part, via MAPK cascade activation, therefore adding support to the hypothesis raised in the Introduction, namely that factors complementing the Ras/Raf/ERK pathway have the potential to be important effectors of proliferative responses in normal and transformed cells.

Ca2+ mobilization following G-protein-coupled receptor–ligand interaction results in the activation of several signalling pathways converging upon the c-fos promoter, one targeting the CaCRE via CREB phosphorylation on Ser133 and the other involving activation of the SRE principally through the ERK cascade. In neuronal cells, P2Y2 receptor-activated MAPK signalling results from calcium-dependent trans-activation of growth factor receptors (16), PYK2 activation (6) and subsequent ERK phosphorylation (6,16). We have clearly shown, using anti-active transcription factor-specific antibodies in western analysis, that P2Y2 activation in MCF-7 cells leads to ERK1 and ERK2 phosphorylation. However, ATP or UTP activation alone did not result in EGF receptor phosphorylation in these cells (Figure 6Go). The pathways that couple ATP and UTP stimulation to activated ERK in MCF-7 cells are therefore unclear. Whilst nucleotide stimulation alone did not lead to EGF receptor phosphorylation, co-stimulation of MCF-7 cells with nucleotides and EGF resulted in higher levels of growth factor receptor phosphorylation than stimulation with EGF alone. It therefore appears that lateral signalling through growth factor receptor phosphorylation plays an important role in the synergic activation of gene expression observed in these studies.

As a major nuclear target for activated ERK, TCF is similarly phosphorylated in response to ATP{gamma}S, an event implicating the SRE in transcriptional activation. Furthermore, consistent with phosphorylation of TCF by nucleotides, Elk-1, the major component of human TCF, was similarly phosphorylated in response to nucleotide treatment. ATP{gamma}S-induced c-fos expression was only partially attenuated by the MEK inhibitors U0126 or PD98059. Accordingly, ATP{gamma}S was effective at inducing CREB phosphorylation independently of MAPK activation. These data demonstrate that in MCF-7 cells, as in human osteoblasts, ATP can activate multiple c-fos promoter elements via the independent activation of several distinct intracellular signalling pathways.

EGF, as expected, induced phosphorylation of both ERK and TCF and, like ATP{gamma}S, induced activation of CREB. However, inhibition of EGF-induced signalling with U0126 completely abolished c-fos gene expression (Figure 5Go), indicating that EGF-induced CREB phosphorylation is dependent upon ERK activation, most likely via ERK-mediated activation of the CREB kinase, RSK2 (17). Co-stimulation with ATP{gamma}S and EGF gave rise to increased levels of both CREB and ERK phosphorylation relative to those seen with either agonist alone and resulted in hyperphosphorylation of TCF. It is unclear at this stage to what extent individual components contribute toward the observed signalling synergy. However, on removal of the MAPK signalling component following MEK inhibition, levels of transcription in response to ATP{gamma}S and EGF were still significantly higher than ATP{gamma}S alone. These data suggest that pathway(s) other than growth factor receptor transactivation, MAPK and ATP-activated CREB contribute to the observed synergistic gene expression. One possible component would be calcium-activated CaMK targeting the SRE via direct SRF phosphorylation (18,19), especially since we have previously demonstrated that ATP can induce SRF phosphorylation in transformed cells (20), and since breast tumour cells express novel variants of CaMK (21). However, on investigation, inhibition of CaMK with the metabolic inhibitor KN92 did not attenuate ATP{gamma}S-induced signalling in MCF-7 cells (data not shown). Alternatively, ATP-activated TCF may lie downstream of MAPKs other than ERK, a hypothesis that requires further investigation.

The signalling synergy between nucleotides and growth factors clearly involves multiple components, a conclusion that concurs with the results from transgenic mice (22), and our in vitro experiments in cultured cells (10), both of which indicate that multiple elements of the c-fos promoter must be targeted in order to achieve high levels of activation. These data strongly suggest that ATP serves as a potentiator for a wide variety of signals known to have strong effects on cell proliferation, including growth factors (EGF), tumour promoters (TPA) and stress (anisomycin). This function extends to activation of genes regulating the cell cycle at later stages, since ATP induces the late G1 gene cyclin A and thereby promotes anchorage-independent cell growth (23). Since anchorage-independence is a characteristic of transformed cells and a prerequisite for metastasis, ATP may also potentiate tumour progression.

For ATP to influence neoplastic progression, it must exist in sufficient concentrations in the vicinity of the tumour to activate cell surface P2Y2 receptors. Transient high local ATP concentrations can exist in the extracellular environment as a result of cell lysis during ischaemia, necrosis or as a result of inflammation or lysis by cytotoxic or NK cells. In addition, we and others have demonstrated that a non-lytic mechanism for ATP release exists in primary and transformed cells (2426). It is therefore possible that breast cancer cells release ATP in vivo. Non-lytic ATP release may be particularly significant in the breast cancer model since a high intracellular nucleotide concentration in transformed cells has been reported (27). The P2Y2 receptor itself may play an important regulatory role, since non-lytic ATP release appears to be regulated, with UTP positively stimulating ATP release in a number of cell types (24).

A frequent complication in breast cancer is cancer cell invasion into bone. The high incidence of metastasis may be a reflection of a bone microenvironment suited to the further growth of these tumour cells. A variety of growth factors and other extracellular agents that are present in bone are likely to create conditions supporting cancer cell growth. Of these, ATP may be significant since human osteoblasts can release ATP into the bone microenvironment, a process that may, in turn, be modulated by factors secreted by tumour cells. ATP may therefore play a key role in this metastatic process, since it has the ability to synergize with a diverse range of signalling pathways, driving cells to proliferate.

In conclusion, these studies demonstrate that extracellular nucleotides can potently modulate gene expression in breast cancer cells via the activation of multiple signalling pathways. In addition, nucleotides can potentiate the effects of growth factors known to modulate transformed cell growth. In combination, these observations clearly implicate extracellular nucleotides in the multistage carcinogenic process. Extracellular nucleotides and their receptors therefore represent important potential targets for combating the neoplastic progression of breast tumour cells.


    Notes
 
3 To whom correspondence should be addressed Back


    Acknowledgments
 
The authors would like to acknowledge support from the following bodies: the North West Cancer Research Fund (S.C.W.), the Arthritis Research Campaign (W.B.B.) and the French `Association pour la Recherche sur le Cancer' and `Fondation de la Recherche Medicale' (R.A.H.).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received September 29, 1999; revised July 7, 2000; accepted August 15, 2000.