Nerve Growth Factor Stimulates Proliferation and Survival of Human Breast Cancer Cells through Two Distinct Signaling Pathways

We show here that the neurotrophin nerve growth factor (NGF), which has been shown to be a mitogen for breast cancer cells, also stimulates cell survival through a distinct signaling pathway. Breast cancer cell lines (MCF-7, T47-D, BT-20, and MDA-MB-231) were found to express both types of NGF receptors: p140trkA and p75NTR. The two other tyrosine kinase receptors for neurotrophins, TrkB and TrkC, were not expressed. The mitogenic effect of NGF on breast cancer cells required the tyrosine kinase activity of p140trkA as well as the mitogen-activated protein kinase (MAPK) cascade, but was independent of p75NTR. In contrast, the anti-apoptotic effect of NGF (studied using the ceramide analogue C2) required p75NTR as well as the activation of the transcription factor NF-kB, but neither p140trkA nor MAPK was necessary. Other neurotrophins (BDNF, NT-3, NT-4/5) also induced cell survival, although not proliferation, emphasizing the importance of p75NTR in NGF-mediated survival. Both the pharmacological NF-κB inhibitor SN50, and cell transfection with IkBm, resulted in a diminution of NGF anti-apoptotic effect. These data show that two distinct signaling pathways are required for NGF activity and confirm the roles played by p75NTR and NF-κB in the activation of the survival pathway in breast cancer cells. We show here that the neurotrophin nerve growth factor (NGF), which has been shown to be a mitogen for breast cancer cells, also stimulates cell survival through a distinct signaling pathway. Breast cancer cell lines (MCF-7, T47-D, BT-20, and MDA-MB-231) were found to express both types of NGF receptors: p140trkA and p75NTR. The two other tyrosine kinase receptors for neurotrophins, TrkB and TrkC, were not expressed. The mitogenic effect of NGF on breast cancer cells required the tyrosine kinase activity of p140trkA as well as the mitogen-activated protein kinase (MAPK) cascade, but was independent of p75NTR. In contrast, the anti-apoptotic effect of NGF (studied using the ceramide analogue C2) required p75NTR as well as the activation of the transcription factor NF-kB, but neither p140trkA nor MAPK was necessary. Other neurotrophins (BDNF, NT-3, NT-4/5) also induced cell survival, although not proliferation, emphasizing the importance of p75NTR in NGF-mediated survival. Both the pharmacological NF-κB inhibitor SN50, and cell transfection with IkBm, resulted in a diminution of NGF anti-apoptotic effect. These data show that two distinct signaling pathways are required for NGF activity and confirm the roles played by p75NTR and NF-κB in the activation of the survival pathway in breast cancer cells. nerve growth factor polyacrylamide gel electrophoresis nuclear factor-κB brain-derived neurotrophic factor neurotrophin polyADP-ribose polymerase tumor necrosis factor TATA box binding protein reverse transcriptase-polymerase chain reaction fetal calf serum dithiothreitol phosphate-buffered saline extracellular signal-regulated kinase green fluorescence protein dominant-negative IκBα mutant base pair(s) Park Davis 98059 Nerve growth factor (NGF)1 is the archetypal member of the neurotrophin superfamily, which also includes brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), NT-4/5, and NT-6 (1Barbacid M. Curr. Opin. Cell Biol. 1995; 7: 148-155Crossref PubMed Scopus (541) Google Scholar). NGF interacts with two classes of membrane receptor: the TrkA proto-oncogene product p140trkA, which possesses intrinsic tyrosine kinase activity, and a secondary receptor, p75NTR, that belongs to the tumor necrosis factor (TNF) receptor family (2Friedman W.J. Greene L.A. Exp. Cell Res. 1999; 253: 131-142Crossref PubMed Scopus (315) Google Scholar). The stimulation of cell survival and cell differentiation by NGF and other neurotrophins have been described primarily in neuronal cell systems (3Lewin G.R. Barde Y.-A. Ann. Rev. Neurosci. 1996; 19: 289-317Crossref PubMed Scopus (1802) Google Scholar). Although the neurotrophic effect through p140trkA is known to involve the MAPK cascade, the role of p75NTR is still controversial; there is evidence that it can both positively and negatively regulate neuronal cell death and differentiation, depending on the cell type examined (4Barker P.A. Cell Death Differ. 1998; 5: 346-356Crossref PubMed Scopus (123) Google Scholar). In some cases, p75NTR is an inducer of apoptosis, even without NGF stimulation (5Rabizadeh S. Oh J. Zhong L.-T. Yang J. Bitler C.M. Butcher L.L. Bredesen D.E. Science. 1993; 261: 345-348Crossref PubMed Scopus (757) Google Scholar), whereas in other cases the activation of p75NTR by NGF results in a protection from cell death (6Gentry J.J. Casaccia-Bonnefil P. Carter B.D. J. Biol. Chem. 2000; 275: 7558-7565Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). In addition to its neurotrophic function, other activities of NGF have been described. For example, NGF can modulate gene expression in monocytes (7Ehrhard P.B. Ganter U. Stalder A. Bauer J. Otten U. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5423-5427Crossref PubMed Scopus (175) Google Scholar), it is chemotactic for melanocytes (8Yaar M. Grossman K. Eller M. Gilchrest B.A. J. Cell Biol. 1991; 115: 821-828Crossref PubMed Scopus (173) Google Scholar), and its inhibition on p75NTR can block the migration of Schwann cells (9Anton E.S. Weskamp G. Reichardt L.F. Matthew W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2795-2799Crossref PubMed Scopus (307) Google Scholar). NGF also stimulates the proliferation of chromaffin cells (10Lillien L.E. Claude P. Nature. 1985; 317: 632-634Crossref PubMed Scopus (166) Google Scholar), lymphocytes (11Otten U. Ehrhard P. Peck R. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 10059-10063Crossref PubMed Scopus (364) Google Scholar), and keratinocytes (12Di Marco E. Mathor M. Bondanza S. Cutuli N. Marchisio P.C. Cancedda R. De Luca M. J. Biol. Chem. 1993; 268: 22838-22846Abstract Full Text PDF PubMed Google Scholar). We have previously shown that NGF is mitogenic for cancerous but not normal human breast cells (13Descamps S. Lebourhis X. Delehedde M. Boilly B. Hondermarck H. J. Biol. Chem. 1998; 273: 16659-16662Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), and these data, as well as others showing a role for NGF in the stimulation of prostatic cancer cells (14Djakiew D. Delsite R. Pflug B. Wrathall J. Lynch J.H. Onoda M. Cancer Res. 1991; 51: 3304-3310PubMed Google Scholar, 15Djakiew D. Pflug B.R. Delsite R. Onoda M. Lynch J.H. Arand G. Thompson E.W. Cancer Res. 1993; 53: 1416-1420PubMed Google Scholar, 16Pflug B. Djakiew D. Mol. Carcinog. 1998; 23: 106-114Crossref PubMed Scopus (56) Google Scholar, 17Sortino M.A. Condorelli F. Vancheri C. Chiarenza A. Bernardini R. Consoli U. Canonico P.L. Mol. Endocrinol. 2000; 14: 124-136Crossref PubMed Scopus (36) Google Scholar), implicate NGF in non-neuronal carcinogenesis. Both cellular proliferation as well as tumor cell survival are crucial for malignant progression. The effect of NGF on the survival of cancer cells through the p75NTR receptor has been shown for neuroblastoma (18Lièvremont J.P. Sciorati C. Morandi E. Paolucci C. Bunone G. Della Valle G. Meldolesi J. Clementi E. J. Biol. Chem. 1999; 274: 15466-15472Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) and schwannoma (6Gentry J.J. Casaccia-Bonnefil P. Carter B.D. J. Biol. Chem. 2000; 275: 7558-7565Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). In prostate cancer, p75NTR has been shown to be a mediator of NGF's effects during critical phases of developmental cell death and carcinogenic progression (19Rabizadeh S. Rabizadeh S. Ye X. Wong J.J. Bredesen D.E. Cell Death Differ. 1999; 6: 1222-1227Crossref PubMed Scopus (28) Google Scholar). To date only the mitogenic effect of NGF for breast cancer cells has been described (13Descamps S. Lebourhis X. Delehedde M. Boilly B. Hondermarck H. J. Biol. Chem. 1998; 273: 16659-16662Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), with its roles in the control of breast cancer cell survival unknown. In this study, we have shown that, in addition to its mitogenic effect, NGF is also an anti-apoptotic factor for breast cancer cells. These cells express mRNA for both p140trkA and p75NTRreceptors. Our results indicate that the mitogenic effect of NGF requires p140trkA and the MAPK cascade, but not the p75NTR receptor, whereas the promotion of cell survival strictly requires p75NTR as well as NF-κB, but not p140trkA and MAPK. Thus the mitogenic and anti-apoptotic effects of NGF on breast cancer cells are mediated through two different signaling pathways. Cell culture reagents were purchased from BioWhittaker (France) except insulin, which was obtained from Organon (France). Recombinant human nerve growth factor, brain derived growth factor (BDNF), and neurotrophins 3 (NT-3) and 4 (NT-4) were from R & D Systems (UK). K-252a (inhibitor of trk-tyrosine kinase activity) and PD98059 (inhibitor of MAPK cascade) were from Calbiochem (France). The mouse monoclonal anti-NGF receptor (p75NTR) antibody was from Euromedex (France) and was previously described for its ability to block the interaction between p75NTR and NGF (20Ross, A. H., Grob, P., Bothwell, M., Elder, D. E., Ernst, C. S., Marano, N., Ghrist, B. F., Slemp, C. C., Herlyn, M., Atkinson, B., and Koprowski, H. Proc. Natl. Acad. Sci.U. S. A. 81, 6681–6685.Google Scholar). The anti-lamin B (C-20), goat polyclonal IgG, and the polyclonal anti-p140trkA (trk763) were from Santa Cruz Biotechnology. C2 ceramide analogue (N-acetyl-d-sphingosine), Hoechst 33258, and electrophoresis reagents were from Sigma Chemical Co. (France). The SN50 NF-κB inhibitor peptide, the rabbit polyclonal anti-NF-κB p65 antibody, was obtained from TEBU (France). Anti-PARP antibody was from Oncogene Research Products (UK). Primers and probes for TrkA and p75NTR, probe for TATA box binding protein (TBP) were from Eurogentec (Belgium). RT-PCR reagents were from Applied Biosystems (France). Lipofectin reagent and Opti-MEM were provided by Life Technologies, Inc. (France). The green fluorescence protein plasmid (EGFPC1) was purchased from CLONTECH, and the dominant-negative IκBα mutant (IκBm) expression vectors (in PCDNA3) containing a Ser to Ala substitution at residues 32 and 36 were obtained from Dr. Jean Feuillard (UPRES EA 1625, Bobigny, France). p65 (rel-A) and c-rel cDNA were cloned at EcoRI site in PSVK3 expression plasmid. All vectors were obtained from Dr. Pascale Crépieux (McGill University, Montreal). The SY5Y subclone of SK-N-SH neuroblastoma cell line was a kind gift of Dr. Luc Buée (INSERM, U422, Lille, France). NT-2 (Ntera/D1) human neural precursor cells (Stratagene) are derived from a clone of the NT-2 teratocarcinoma. Breast cancer cell lines (MCF-7, T47-D, BT-20, and MDA-MB-231) were obtained from the American Type Culture Collection and routinely grown as monolayer cultures. Cells were maintained in minimal essential medium (Earle's salts) supplemented with 20 mm Hepes, 2 g/liter sodium bicarbonate, 2 mml-glutamine, 10% fetal calf serum (FCS), 100 units/ml of and The reverse transcription reaction 2 of from breast cancer cell NT-2 cells, SY5Y reverse transcription reaction mm mm of and of reverse were to of reaction All the reaction were at for and at for chain reaction was on without the for The for and RT-PCR in breast cancer cell lines were as and and The for RT-PCR of in cells were as and and and of was in RT-PCR reaction to To were of mm of of mm of cDNA mRNA of mm of and to a of The were as for 3 for were at for for 2 and for 3 The were for a at for the of cDNA the and the were in an were as previously described (13Descamps S. Lebourhis X. Delehedde M. Boilly B. Hondermarck H. J. Biol. Chem. 1998; 273: 16659-16662Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). were with 2 in 2 of medium containing 10% cells were with the medium was with 2 of serum medium containing 100 NGF of other neurotrophins (BDNF, NT-3, To the effect of pharmacological were with NGF 2 of NGF cells were by and using an of breast cancer cells was induced by the ceramide analogue which has been described as a for human breast cancer cells C.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, G. C. C. Cell Death Differ. 1999; 6: PubMed Scopus (44) Google Scholar). was obtained by with 2 C2 for To the anti-apoptotic activity of of this factor were we found that the effect was obtained for 100 this was in with pharmacological For of cell cells were with for and with phosphate-buffered saline with Hoechst for at in the Cells were with and with using The cells and were an fluorescence in of cells was examined for and results were as a of the of cells The of the data from cell and was using of were by the to the cell were by in the was with and In the of the was with mm 10% and at for The were to a membrane by and with at 4 The were at for 3 with G. of with the reaction was using the with were in mm mm 100 sodium to was with protein 4 2 the was with monoclonal antibody 4 was for and by 2 The was with and for in and were with serum were with antibody at 4 and with a for 3 at were at 4 with Cell nuclear were as described by M. S. Exp. Cell Res. PubMed Scopus Google Scholar). Cells were and in minimal essential medium containing 10% with cells were and in of mm Hepes, mm 2 mm 3 mm and 3 mm on of 10% was and were by for The nuclear was in mm Hepes, mm mm 10% 3 and 3 mm on with the nuclear was in a for nuclear protein was from the and in a and for in the nuclear were to and with an anti-NF-κB p65 control was with anti-lamin B were using Lipofectin as described by the cells were for in of Opti-MEM transfection medium containing of Lipofectin of green fluorescence protein and of of In the of c-rel cells were with of and of PSVK3 Cells were grown for with 10% minimal essential medium and for 2 in medium in medium in the of 100 NGF 2 C2 for Cells were with for and the of cell in cells was as described The effects of 100 NGF on cell proliferation and were by cell and Hoechst The results show that NGF an in cell for breast cancer cell lines We have previously that NGF has a mitogenic effect on breast cancer cells by cells in and by the In NGF breast cancer cells the survival was at The of cells this anti-apoptotic was distinct The of by C2 was found to involve of polymerase this was by NGF effect of Hoechst of cell in C2 and cells. Cells were in essential medium and with NGF was at 100 cells were and were Hoechst of was by p75NTR activation mediated by cells were in essential medium for and were with 100 NGF in the of 2 antibody for were of cell from breast cancer cells, and with RT-PCR was to show the expression of mRNA for both and NGF receptors in T47-D, BT-20, and cells the for the TrkA and a for the p75NTR were on that both p140trkA and p75NTR were in the breast cancer cell lines 3 RT-PCR that there was in the of TrkA and p75NTR in the of C2 not and that the of mRNA for TrkA and p75NTR in breast cancer cells was between and the in SY5Y neuroblastoma cells not that NGF receptor expression in breast cancer cells is be that, although mRNA of NGF receptors between breast cancer cells and the protein it has been shown that the of a cellular protein be from mRNA B.R. R. Mol. Biol. 1999; 19: PubMed Scopus Google Scholar). that the of mRNA protein for NGF between breast cancer cells and neuroblastoma cells, to the between mRNA and protein We a of and pharmacological to the of p140trkA and p75NTR in the stimulation of proliferation and cell survival induced by The tyrosine kinase inhibitor and the inhibitor both the effect of NGF on cells, but effect on its anti-apoptotic effects neither the antibody nor the NF-κB inhibitor SN50 proliferation, although both the anti-apoptotic effects The tyrosine kinase activity of p140trkA was by K-252a but not by the PD98059 the other the activity of the was by K-252a and PD98059 but not by the be that the SN50 inhibitor of NF-κB, to the the anti-apoptotic effect of NGF but neither its effect nor its activation of p140trkA and The effect of other neurotrophins on cell growth and survival was also In to effect was provided by NT-3, neurotrophins a effect on cells that was not in the of the inhibitor K-252a These data that receptors are not in NGF survival the of and in these can be are not in these breast cancer cells and MAPK cells were with 100 NGF in the of antibody, p140trkA and MAPK activation were using polyclonal and monoclonal and were with the For of TrkA and MAPK the of the with the of different neurotrophins on cells growth and survival. cells were in essential and the neurotrophins NT-3, 100 NT-4/5) were cells were and In with neither NT-3, nor to cells were in essential medium and with 2 with without neurotrophins NT-3, 100 cells were and was Hoechst an fluorescence For of both cell and apoptosis, results are as the of was using the TrkB and mRNA expression in cells. gel electrophoresis of RT-PCR TrkA but TrkB expression in breast cancer cells. cells were as control for the expression of TrkB and control without control without The effect of SN50 on the NGF anti-apoptotic activity the of NF-κB in the signaling to the activity of this growth To this we the effect of NGF on the nuclear of NF-κB, as well as the of transfection by inhibitor of by c-rel and of on the NGF-mediated anti-apoptotic activity in cells. in the nuclear of NF-κB during induced by C2 In contrast, the addition of NGF on cells induced a of NF-κB from to a p65 to the of the not this NF-κB nuclear was by the of antibody SN50, but was not by K-252a and in the of NGF was not to the nuclear of NF-κB, that NF-κB activation requires cell C. D. C. P.A. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). of cells with IkBm, an inhibitor of NF-κB, the anti-apoptotic effect of NGF a we cells with an effect was In transfection with of the NF-κB c-rel resulted in an inhibition of of cells, even in of the of NF-κB family in human breast cancer cell of NGF anti-apoptotic effect by and cells were with and c-rel using the Lipofectin were with both and and both PSVK3 and c-rel and cells were in essential medium and C2 neurotrophins for Cells were and the of in cells the expression of as a transfection Hoechst are as the of was using the that, in addition to its mitogenic activity, NGF is anti-apoptotic for breast cancer cells, and that these two effects are mediated by the p75NTR The growth of breast cancer results from a between cell proliferation and apoptosis, both of which can be by For example, growth factor, growth and growth can the proliferation and survival of breast cancer cells J. Natl. Cancer 1995; PubMed Scopus Google Scholar). the other as growth tumor necrosis can growth and in these cells X. Boilly B. Hondermarck H. Breast Cancer Res. 2000; PubMed Scopus Google Scholar). we have shown that which was primarily described for its neurotrophic is a mitogen for cancerous but not for normal human breast cells, a crucial for this factor in the and progression of human breast (13Descamps S. Lebourhis X. Delehedde M. Boilly B. Hondermarck H. J. Biol. Chem. 1998; 273: 16659-16662Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). In the study, we have shown that the breast cancer cells express for both TrkA and p75NTR In contrast, expression of TrkB and was found in of the breast cancer cells in with the that NT-3, have mitogenic effect for these cells. The of NGF receptors has been previously in breast cancer cells J. 1993; PubMed Scopus Google Scholar), and of NGF receptor expression have been in other breast cancer cell lines E. F. C. M. G. C. S. J. 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Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar), to the of a and between p140trkA and for the of by Our results indicate a stimulation of p140trkA tyrosine kinase activity and of the MAPK by and the of the pharmacological K-252a and PD98059 the for these in cell The of MAPK activity required p140trkA but p75NTR not to be not have effect on MAPK activation and cell In contrast, an inhibition of survival, to the of these the mitogenic activity of NGF requires the p140trkA and MAPK of the p75NTR signaling pathway for the effect to be to that which is described for the neurotrophic activity of this For example, in cells, of p75NTR not in an inhibition of the NGF activity, which is mediated by the pathway P.A. 1994; Full Text PDF PubMed Scopus Google Scholar). it has also been shown in cells that NGF survival and differentiation through two distinct signaling the activation of the MAPK is required for the but not the activity of NGF L.F. 1999; PubMed Scopus Google Scholar). These data the between the mitogenic and neurotrophic signaling pathways of The of NGF as a survival factor has been described for in both in and in P. H. Cell Death Differ. 1998; 5: PubMed Scopus Google Scholar). the signaling in the anti-apoptotic activity of NGF in The is described as for neuronal cell although there has been a of a pathway mediated by in cells J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). the p140trkA the of the role of p75NTR has (4Barker P.A. Cell Death Differ. 1998; 5: 346-356Crossref PubMed Scopus (123) Google Scholar). The p75NTR receptor belongs to the types and of the receptor, the and 1998; PubMed Scopus Google Scholar). The cellular to activation of this family of receptors are the activation of gene transcription nuclear and the of cell In some cases was shown to NGF binding to p75NTR, although in other cases it to in the of and was by NGF B.D. G.R. Full Text Full Text PDF PubMed Scopus Google Scholar). The C2 reagent here is known to in breast cancer cells as C.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, G. C. C. Cell Death Differ. 1999; 6: PubMed Scopus (44) Google Scholar). Hoechst and the inhibition of that NGF breast cancer cells from cell K-252a and PD98059 not the anti-apoptotic activity of that kinase and MAPK activities are not for the effect. have that NGF is to its effects through p75NTR receptors and of p140trkA in A. Barde Nature. 1996; PubMed Scopus Google Scholar, M. S. J. Neurosci. Res. PubMed Scopus Google Scholar) and Schwann cells B.D. C. B. N. R. P.A. Barde Science. 1996; PubMed Scopus Google Scholar). In a role for p75NTR in the cell survival effect was by the that other neurotrophins with p75NTR and not with are also to cells from death on cell The crucial role of p75NTR was by the of which the effect of NGF from NT-3, and NT-4/5, of which can can also breast cancer cell survival. TrkB and are not in breast cancer cells, these data the role played by p75NTR in the anti-apoptotic effect of of p75NTR NF-κB independent of p140trkA in cell Schwann cells B.D. C. B. N. R. P.A. Barde Science. 1996; PubMed Scopus Google Scholar). To the of NF-κB in the NGF survival effect, we SN50, which the nuclear of this transcription factor J. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). We found that it the anti-apoptotic effect of NGF without the cellular The of NF-κB was by transfection with a of which NF-κB to the cells by were not from by the of NF-κB in the anti-apoptotic activity mediated by p75NTR. have been in cells in which the of activation of NF-κB resulted in an of X. A. R. Greene J. Neurosci. 2000; PubMed Google Scholar). In by c-rel which are of NF-κB, a effect on cells by C2 in of NGF c-rel and to the NF-κB family of transcription The protection from transfection with this factor the role played by NF-κB in the control of breast cancer cell survival. In results that NGF is an anti-apoptotic factor for human breast cancer cells and that the signaling pathway to this survival activity is distinct from the signaling which to mitogenic Although p140trkA and the the mitogenic activity of its anti-apoptotic activity required p75NTR and NF-κB NGF is in the A. R. R. J. Res. 1985; 19: PubMed Scopus Google Scholar, C. 1993; 91: PubMed Scopus Google Scholar) as well as its E. S. M. X. B. and H. and that NGF is a crucial of tumor The inhibition of breast cancer progression through the p140trkA and p75NTR be as a for the of this We and G. of for critical of this

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