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The Transmodulation of HER2 and EGFR by Substance P in Breast Cancer Cells Requires c-Src and Metalloproteinase Activation.

Garcia-Recio S, Pastor-Arroyo EM, Marín-Aguilera M, Almendro V, Gascón P - PLoS ONE (2015)

Bottom Line: Overexpression of NK-1R in MDA-MB-231 and its chemical inhibition in SK-BR-3, BT-474 and MDA-MB-468 BC cells significantly modulated c-Src activation, suggesting that this protein is a mediator of NK-1R signaling.Moreover, the use of these inhibitors demonstrated that this Src and MMP-dependent signaling is important to the cell viability and migration capacity of HER2+ and EGFR+ cell lines.Our results indicate that the transactivation of HER2 and EGFR by the pro-inflammatory cytokine/neuropeptide SP in BC cells is a c-Src and MMP-dependent process.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Oncology, Hospital Clínic, Barcelona, Spain; Institut d'Investigacions Biomediques August Pi i Sunyer, Barcelona, Spain.

ABSTRACT

Background: Substance P (SP) is a pleiotropic cytokine/neuropeptide that enhances breast cancer (BC) aggressiveness by transactivating tyrosine kinase receptors like EGFR and HER2. We previously showed that SP and its cognate receptor NK-1 (SP/NK1-R) signaling modulates the basal phosphorylation of HER2 and EGFR in BC, increasing aggressiveness and drug resistance. In order to elucidate the mechanisms responsible for NK-1R-mediated HER2 and EGFR transactivation, we investigated the involvement of c-Src (a ligand-independent mediator) and of metalloproteinases (ligand-dependent mediators) in HER2/EGFR activation.

Results and discussion: Overexpression of NK-1R in MDA-MB-231 and its chemical inhibition in SK-BR-3, BT-474 and MDA-MB-468 BC cells significantly modulated c-Src activation, suggesting that this protein is a mediator of NK-1R signaling. In addition, the c-Src inhibitor 4-(4'-phenoxyanilino)-6,7-dimethoxyquinazoline prevented SP-induced activation of HER2. On the other hand, SP-dependent phosphorylation of HER2 and EGFR decreased substantially in the presence of the MMP inhibitor 1-10, phenanthroline monohydrate, and the dual inhibition of both c-Src and MMP almost abolished the activation of HER2 and EGFR. Moreover, the use of these inhibitors demonstrated that this Src and MMP-dependent signaling is important to the cell viability and migration capacity of HER2+ and EGFR+ cell lines.

Conclusion: Our results indicate that the transactivation of HER2 and EGFR by the pro-inflammatory cytokine/neuropeptide SP in BC cells is a c-Src and MMP-dependent process.

No MeSH data available.


Related in: MedlinePlus

NK-1R contributes to c-Src activation in BC cell lines.(A) The contribution of NK-1R to the activation of c-Src Y416 phosphorilation protein in the MDA-MB-231 cell line transfected with pcDNA3.1(+)-TACR1 or empty vector and treated for 6 and 10 minutes with SP 100 nM; (B) the effects of single NK-1R inhibition during 24h with L-733,060 (20 μM (SKBR3 and BT-474), 30 μM (MDA-MB-453)) or (C) combined NK-1R, NK-2R and NK-R3 inhibition during 24h with L-733,060 (20 μM), MEN 10376 (30 μM) and SB218795 (20 μM), respectively on c-Src (Y416). The blot was standardized to c-Src levels. All quantitative data are generated from a minimum of 3 replicates and are presented as mean + S.D. and compared by t-test (two-tailed) as * P<0.05, ** P<0.01 and *** P<0.001.
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pone.0129661.g002: NK-1R contributes to c-Src activation in BC cell lines.(A) The contribution of NK-1R to the activation of c-Src Y416 phosphorilation protein in the MDA-MB-231 cell line transfected with pcDNA3.1(+)-TACR1 or empty vector and treated for 6 and 10 minutes with SP 100 nM; (B) the effects of single NK-1R inhibition during 24h with L-733,060 (20 μM (SKBR3 and BT-474), 30 μM (MDA-MB-453)) or (C) combined NK-1R, NK-2R and NK-R3 inhibition during 24h with L-733,060 (20 μM), MEN 10376 (30 μM) and SB218795 (20 μM), respectively on c-Src (Y416). The blot was standardized to c-Src levels. All quantitative data are generated from a minimum of 3 replicates and are presented as mean + S.D. and compared by t-test (two-tailed) as * P<0.05, ** P<0.01 and *** P<0.001.

Mentions: We previously reported that the stable transfection of NK-1R into the HER2-negative MDA-MB-231 cell line can be used as a tool to study the mechanism by which SP contributes to the persistent transmodulation of the ERBB receptors [17]. These previous results demonstrated that the overexpression of NK-1R enhanced SP-mediated HER2 activation even in a HER2- negative and NK-1R-low cell line, the main reason we selected that particular cell line [17]. To further confirm the involvement of NK-1R in c-Src activation, in the present study we investigated the effects of NK-1R overexpression on c-Src activation in the MDA-MB-231 cell line. The MDA-MB-231 cells were transfected with pcDNA3.1(+)-TACR1 or the empty vector pcDNA3.1(+) and treated with SP 100 nM at 6 and 10 minutes. We observed that the basal levels of p-Src Y416 (at point 0, red bar) were 2.5-fold higher in the MDA-MB-231 cells overexpressing NK-1R compared to the control cells (at point 0, open bar) (Fig 2A). Then, in pcDNA3.1(+) transfected cells (left) (representing a basal situation), the treatment with SP for 10 minutes further increased the phosphorylation of c-Src Tyr416 (3.26 fold increased, open bar) as we found in MDA-MB-231 cell line in Fig 1. On the other hand, in the MDA-MB-231 cells overexpressing NK-1R (pcDNA3.1(+)-TACR1), the treatment with SP for 6 or 10 minutes further increased the phosphorylation of c-Src Tyr416 (5.5- and 4.6-fold, respectively, red bar) and in all cases were expressed by ratio of phospho/total protein (Fig 2A).


The Transmodulation of HER2 and EGFR by Substance P in Breast Cancer Cells Requires c-Src and Metalloproteinase Activation.

Garcia-Recio S, Pastor-Arroyo EM, Marín-Aguilera M, Almendro V, Gascón P - PLoS ONE (2015)

NK-1R contributes to c-Src activation in BC cell lines.(A) The contribution of NK-1R to the activation of c-Src Y416 phosphorilation protein in the MDA-MB-231 cell line transfected with pcDNA3.1(+)-TACR1 or empty vector and treated for 6 and 10 minutes with SP 100 nM; (B) the effects of single NK-1R inhibition during 24h with L-733,060 (20 μM (SKBR3 and BT-474), 30 μM (MDA-MB-453)) or (C) combined NK-1R, NK-2R and NK-R3 inhibition during 24h with L-733,060 (20 μM), MEN 10376 (30 μM) and SB218795 (20 μM), respectively on c-Src (Y416). The blot was standardized to c-Src levels. All quantitative data are generated from a minimum of 3 replicates and are presented as mean + S.D. and compared by t-test (two-tailed) as * P<0.05, ** P<0.01 and *** P<0.001.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4482606&req=5

pone.0129661.g002: NK-1R contributes to c-Src activation in BC cell lines.(A) The contribution of NK-1R to the activation of c-Src Y416 phosphorilation protein in the MDA-MB-231 cell line transfected with pcDNA3.1(+)-TACR1 or empty vector and treated for 6 and 10 minutes with SP 100 nM; (B) the effects of single NK-1R inhibition during 24h with L-733,060 (20 μM (SKBR3 and BT-474), 30 μM (MDA-MB-453)) or (C) combined NK-1R, NK-2R and NK-R3 inhibition during 24h with L-733,060 (20 μM), MEN 10376 (30 μM) and SB218795 (20 μM), respectively on c-Src (Y416). The blot was standardized to c-Src levels. All quantitative data are generated from a minimum of 3 replicates and are presented as mean + S.D. and compared by t-test (two-tailed) as * P<0.05, ** P<0.01 and *** P<0.001.
Mentions: We previously reported that the stable transfection of NK-1R into the HER2-negative MDA-MB-231 cell line can be used as a tool to study the mechanism by which SP contributes to the persistent transmodulation of the ERBB receptors [17]. These previous results demonstrated that the overexpression of NK-1R enhanced SP-mediated HER2 activation even in a HER2- negative and NK-1R-low cell line, the main reason we selected that particular cell line [17]. To further confirm the involvement of NK-1R in c-Src activation, in the present study we investigated the effects of NK-1R overexpression on c-Src activation in the MDA-MB-231 cell line. The MDA-MB-231 cells were transfected with pcDNA3.1(+)-TACR1 or the empty vector pcDNA3.1(+) and treated with SP 100 nM at 6 and 10 minutes. We observed that the basal levels of p-Src Y416 (at point 0, red bar) were 2.5-fold higher in the MDA-MB-231 cells overexpressing NK-1R compared to the control cells (at point 0, open bar) (Fig 2A). Then, in pcDNA3.1(+) transfected cells (left) (representing a basal situation), the treatment with SP for 10 minutes further increased the phosphorylation of c-Src Tyr416 (3.26 fold increased, open bar) as we found in MDA-MB-231 cell line in Fig 1. On the other hand, in the MDA-MB-231 cells overexpressing NK-1R (pcDNA3.1(+)-TACR1), the treatment with SP for 6 or 10 minutes further increased the phosphorylation of c-Src Tyr416 (5.5- and 4.6-fold, respectively, red bar) and in all cases were expressed by ratio of phospho/total protein (Fig 2A).

Bottom Line: Overexpression of NK-1R in MDA-MB-231 and its chemical inhibition in SK-BR-3, BT-474 and MDA-MB-468 BC cells significantly modulated c-Src activation, suggesting that this protein is a mediator of NK-1R signaling.Moreover, the use of these inhibitors demonstrated that this Src and MMP-dependent signaling is important to the cell viability and migration capacity of HER2+ and EGFR+ cell lines.Our results indicate that the transactivation of HER2 and EGFR by the pro-inflammatory cytokine/neuropeptide SP in BC cells is a c-Src and MMP-dependent process.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Oncology, Hospital Clínic, Barcelona, Spain; Institut d'Investigacions Biomediques August Pi i Sunyer, Barcelona, Spain.

ABSTRACT

Background: Substance P (SP) is a pleiotropic cytokine/neuropeptide that enhances breast cancer (BC) aggressiveness by transactivating tyrosine kinase receptors like EGFR and HER2. We previously showed that SP and its cognate receptor NK-1 (SP/NK1-R) signaling modulates the basal phosphorylation of HER2 and EGFR in BC, increasing aggressiveness and drug resistance. In order to elucidate the mechanisms responsible for NK-1R-mediated HER2 and EGFR transactivation, we investigated the involvement of c-Src (a ligand-independent mediator) and of metalloproteinases (ligand-dependent mediators) in HER2/EGFR activation.

Results and discussion: Overexpression of NK-1R in MDA-MB-231 and its chemical inhibition in SK-BR-3, BT-474 and MDA-MB-468 BC cells significantly modulated c-Src activation, suggesting that this protein is a mediator of NK-1R signaling. In addition, the c-Src inhibitor 4-(4'-phenoxyanilino)-6,7-dimethoxyquinazoline prevented SP-induced activation of HER2. On the other hand, SP-dependent phosphorylation of HER2 and EGFR decreased substantially in the presence of the MMP inhibitor 1-10, phenanthroline monohydrate, and the dual inhibition of both c-Src and MMP almost abolished the activation of HER2 and EGFR. Moreover, the use of these inhibitors demonstrated that this Src and MMP-dependent signaling is important to the cell viability and migration capacity of HER2+ and EGFR+ cell lines.

Conclusion: Our results indicate that the transactivation of HER2 and EGFR by the pro-inflammatory cytokine/neuropeptide SP in BC cells is a c-Src and MMP-dependent process.

No MeSH data available.


Related in: MedlinePlus