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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.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.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

SP transmodulates HER2 by c-Src and MMP-dependent mechanisms in SKBR3 cell line.(A) Representative images of Western blots evaluating the effects of the single or combined inhibition of c-Src (Y416) with 4-(4′-phenoxyanilino)-6,7-dimethoxyquinazoline (1μM) and MMPs with 1–10, phenanthroline monohydrate (7μM) on the activation of HER2 and p42/44 MAPK triggered by SP 100 nM for 6, 10 and 15 minutes. The plots show the densitometric quantification of the Western blots on phosphorylated (denoted by p-) HER2 (B) and p42/44 MAPK (C) relative to the expression of tubulin, which was used to ensure equal protein loading. Western blots are representative of at least two independent experiments.
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pone.0129661.g003: SP transmodulates HER2 by c-Src and MMP-dependent mechanisms in SKBR3 cell line.(A) Representative images of Western blots evaluating the effects of the single or combined inhibition of c-Src (Y416) with 4-(4′-phenoxyanilino)-6,7-dimethoxyquinazoline (1μM) and MMPs with 1–10, phenanthroline monohydrate (7μM) on the activation of HER2 and p42/44 MAPK triggered by SP 100 nM for 6, 10 and 15 minutes. The plots show the densitometric quantification of the Western blots on phosphorylated (denoted by p-) HER2 (B) and p42/44 MAPK (C) relative to the expression of tubulin, which was used to ensure equal protein loading. Western blots are representative of at least two independent experiments.

Mentions: To investigate the role of c-Src in SP-mediated HER2 and EGFR activation [30] we next performed time-course studies with SP in the presence of the c-Src inhibitor 4-(4′-phenoxyanilino)-6,7-dimethoxyquinazoline [40]. Inhibition of c-Src activity in the HER2 positive SK-BR-3 cell line significantly blocked SP-induced phosphorylation of HER2 Tyr1248 compared to control cells (Fig 3A and 3B). HER2 transactivation by SP was also substantially inhibited in the presence of the MMP inhibitor 1–10, phenanthroline monohydrate, and almost completely abolished after the inhibition of both pathways, suggesting that the transactivation of HER2 by SP in BC cells is a c-Src and MMP-dependent process (Fig 3A and 3B). SP signaling activates the mitogen-activated protein kinase (MAPK) pathway [10, 26, 41]; therefore, the phosphorylation of p42/44 MAPK was used to control of NK-1R downstream activation. For this reason, the phosphorylation of p42/44 MAPK was not always reduced in the presence of c-Src and MMP inhibitors (Fig 3A and 3C), since the activation of the MAPK pathway can be triggered by both NK-1R and ERBB signaling [14, 37].


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)

SP transmodulates HER2 by c-Src and MMP-dependent mechanisms in SKBR3 cell line.(A) Representative images of Western blots evaluating the effects of the single or combined inhibition of c-Src (Y416) with 4-(4′-phenoxyanilino)-6,7-dimethoxyquinazoline (1μM) and MMPs with 1–10, phenanthroline monohydrate (7μM) on the activation of HER2 and p42/44 MAPK triggered by SP 100 nM for 6, 10 and 15 minutes. The plots show the densitometric quantification of the Western blots on phosphorylated (denoted by p-) HER2 (B) and p42/44 MAPK (C) relative to the expression of tubulin, which was used to ensure equal protein loading. Western blots are representative of at least two independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0129661.g003: SP transmodulates HER2 by c-Src and MMP-dependent mechanisms in SKBR3 cell line.(A) Representative images of Western blots evaluating the effects of the single or combined inhibition of c-Src (Y416) with 4-(4′-phenoxyanilino)-6,7-dimethoxyquinazoline (1μM) and MMPs with 1–10, phenanthroline monohydrate (7μM) on the activation of HER2 and p42/44 MAPK triggered by SP 100 nM for 6, 10 and 15 minutes. The plots show the densitometric quantification of the Western blots on phosphorylated (denoted by p-) HER2 (B) and p42/44 MAPK (C) relative to the expression of tubulin, which was used to ensure equal protein loading. Western blots are representative of at least two independent experiments.
Mentions: To investigate the role of c-Src in SP-mediated HER2 and EGFR activation [30] we next performed time-course studies with SP in the presence of the c-Src inhibitor 4-(4′-phenoxyanilino)-6,7-dimethoxyquinazoline [40]. Inhibition of c-Src activity in the HER2 positive SK-BR-3 cell line significantly blocked SP-induced phosphorylation of HER2 Tyr1248 compared to control cells (Fig 3A and 3B). HER2 transactivation by SP was also substantially inhibited in the presence of the MMP inhibitor 1–10, phenanthroline monohydrate, and almost completely abolished after the inhibition of both pathways, suggesting that the transactivation of HER2 by SP in BC cells is a c-Src and MMP-dependent process (Fig 3A and 3B). SP signaling activates the mitogen-activated protein kinase (MAPK) pathway [10, 26, 41]; therefore, the phosphorylation of p42/44 MAPK was used to control of NK-1R downstream activation. For this reason, the phosphorylation of p42/44 MAPK was not always reduced in the presence of c-Src and MMP inhibitors (Fig 3A and 3C), since the activation of the MAPK pathway can be triggered by both NK-1R and ERBB signaling [14, 37].

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.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.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