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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

Blockade of c-Src, MMPs or NK-1R inhibits tumor cell viability and migration in SK-BR-3 and MDA-MB-468 cells.(A) Cell viability quantification of SK-BR-3 cells and MDA-MB-468 cells treated for 24h with IC50 values of MMP inhibitor, c-Src inhibitor, L-733,060 antagonist or with the combinations of drugs: MMP inhibitor + Src inhibitor, MMP inhibitor + L-733,060), Src inhibitor + L-733,060 and MMP inhibitor + Src inhibitor + L-733,060 in serum free medium. After 24h, the cells were treated with calcein (2 μM) for 45 min and calcein AM fluorescence was measured to determine cell viability. (B) Migration rate quantification of SK-BR-3 cells and MDA-MB-468 cells treated for 24h with IC50 values of MMP inhibitor, c-Src inhibitor, L-733,060 antagonist or with the combination of MMP inhibitor + Src inhibitor in serum-free medium. After 24h, detection of cell migration was quantified using calcein AM. Results are represented as mean of % viability or % migration ± SD. All the quantitative data are for a minimum of 6 replicates. Significant differences by ANOVA with Tukey Multiple Comparison post-hoc test are indicated as * P<0.05, ** P<0.01 and *** P<0.001.
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pone.0129661.g005: Blockade of c-Src, MMPs or NK-1R inhibits tumor cell viability and migration in SK-BR-3 and MDA-MB-468 cells.(A) Cell viability quantification of SK-BR-3 cells and MDA-MB-468 cells treated for 24h with IC50 values of MMP inhibitor, c-Src inhibitor, L-733,060 antagonist or with the combinations of drugs: MMP inhibitor + Src inhibitor, MMP inhibitor + L-733,060), Src inhibitor + L-733,060 and MMP inhibitor + Src inhibitor + L-733,060 in serum free medium. After 24h, the cells were treated with calcein (2 μM) for 45 min and calcein AM fluorescence was measured to determine cell viability. (B) Migration rate quantification of SK-BR-3 cells and MDA-MB-468 cells treated for 24h with IC50 values of MMP inhibitor, c-Src inhibitor, L-733,060 antagonist or with the combination of MMP inhibitor + Src inhibitor in serum-free medium. After 24h, detection of cell migration was quantified using calcein AM. Results are represented as mean of % viability or % migration ± SD. All the quantitative data are for a minimum of 6 replicates. Significant differences by ANOVA with Tukey Multiple Comparison post-hoc test are indicated as * P<0.05, ** P<0.01 and *** P<0.001.

Mentions: To study the role of c-Src, MMPs, and NK-1R in cell viability and migration capacities, we treated the HER2+ SK-BR-3 and EGFR+ MDA-MB-468 cell lines with NK-1R antagonist L-733,060, c-Src inhibitor 4-(4′-phenoxyanilino)-6,7-dimethoxyquinazoline and MMP inhibitor 1–10, phenanthroline monohydrate. Cell viability was significantly decreased, above all under NK-1R antagonist in both cell lines (Fig 5A). Inhibition of c-Src and MMPs activity also significantly decreased cell viability and was almost completely abolished after the combination of each drug with NK-1R antagonist and after triple inhibition.


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)

Blockade of c-Src, MMPs or NK-1R inhibits tumor cell viability and migration in SK-BR-3 and MDA-MB-468 cells.(A) Cell viability quantification of SK-BR-3 cells and MDA-MB-468 cells treated for 24h with IC50 values of MMP inhibitor, c-Src inhibitor, L-733,060 antagonist or with the combinations of drugs: MMP inhibitor + Src inhibitor, MMP inhibitor + L-733,060), Src inhibitor + L-733,060 and MMP inhibitor + Src inhibitor + L-733,060 in serum free medium. After 24h, the cells were treated with calcein (2 μM) for 45 min and calcein AM fluorescence was measured to determine cell viability. (B) Migration rate quantification of SK-BR-3 cells and MDA-MB-468 cells treated for 24h with IC50 values of MMP inhibitor, c-Src inhibitor, L-733,060 antagonist or with the combination of MMP inhibitor + Src inhibitor in serum-free medium. After 24h, detection of cell migration was quantified using calcein AM. Results are represented as mean of % viability or % migration ± SD. All the quantitative data are for a minimum of 6 replicates. Significant differences by ANOVA with Tukey Multiple Comparison post-hoc test are indicated as * P<0.05, ** P<0.01 and *** P<0.001.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0129661.g005: Blockade of c-Src, MMPs or NK-1R inhibits tumor cell viability and migration in SK-BR-3 and MDA-MB-468 cells.(A) Cell viability quantification of SK-BR-3 cells and MDA-MB-468 cells treated for 24h with IC50 values of MMP inhibitor, c-Src inhibitor, L-733,060 antagonist or with the combinations of drugs: MMP inhibitor + Src inhibitor, MMP inhibitor + L-733,060), Src inhibitor + L-733,060 and MMP inhibitor + Src inhibitor + L-733,060 in serum free medium. After 24h, the cells were treated with calcein (2 μM) for 45 min and calcein AM fluorescence was measured to determine cell viability. (B) Migration rate quantification of SK-BR-3 cells and MDA-MB-468 cells treated for 24h with IC50 values of MMP inhibitor, c-Src inhibitor, L-733,060 antagonist or with the combination of MMP inhibitor + Src inhibitor in serum-free medium. After 24h, detection of cell migration was quantified using calcein AM. Results are represented as mean of % viability or % migration ± SD. All the quantitative data are for a minimum of 6 replicates. Significant differences by ANOVA with Tukey Multiple Comparison post-hoc test are indicated as * P<0.05, ** P<0.01 and *** P<0.001.
Mentions: To study the role of c-Src, MMPs, and NK-1R in cell viability and migration capacities, we treated the HER2+ SK-BR-3 and EGFR+ MDA-MB-468 cell lines with NK-1R antagonist L-733,060, c-Src inhibitor 4-(4′-phenoxyanilino)-6,7-dimethoxyquinazoline and MMP inhibitor 1–10, phenanthroline monohydrate. Cell viability was significantly decreased, above all under NK-1R antagonist in both cell lines (Fig 5A). Inhibition of c-Src and MMPs activity also significantly decreased cell viability and was almost completely abolished after the combination of each drug with NK-1R antagonist and after triple inhibition.

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