Limits...
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

SP transmodulates EGFR by c-Src and MMP-dependent mechanisms in the MDA-MB-468 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 EGFR 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-) EGFR (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
getmorefigures.php?uid=PMC4482606&req=5

pone.0129661.g004: SP transmodulates EGFR by c-Src and MMP-dependent mechanisms in the MDA-MB-468 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 EGFR 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-) EGFR (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 determine whether the transmodulation of EGFR by SP was also dependent on c-Src and MMPs in BC cells, we performed similar experiments in the EGFR positive cell line MDA-MB-468. In the control situation, addition of SP increased EGFR phosphorylation at 6 min (1.17-fold), 10 min (1.45-fold) and particularly at 15 min (2.61-fold). No increase occurred in the presence of the inhibitors (alone or in combination) under SP treatment, as we observed in the western blot and densitrometic quantification of phospho EGFR/EGFR ratio (Fig 4A and 4B). In particular, c-Src inhibition significantly decreased the capability of SP to induce EGFR phosphorylation. Similarly, MMP inhibition affected the phosphorylation of EGFR induced by SP, as did the concomitant inhibition of both c-Src and MMPs, especially with both c-Src and MMPs inhibitor treatment at 15 min point (0.46 fold decrease) compared with point 0 (Fig 4B, right diagram).We also observed that c-Src inhibition significantly decreased the capability of SP to induce EGFR phosphorylation. Similarly, MMP inhibition affected the phosphorylation of EGFR induced by SP, as did the concomitant inhibition of both c-Src and MMPs, especially with both c-Src and MMPs inhibitor treatment at 15 min point (0.46 fold decrease) compared with the point 0, right diagram (Fig 4B). As before, the inhibition of c-Src and MMPs in this cell line did not block MAPK signaling due to its modulation by both NK-1R and RTKs (Fig 4A and 4C).


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 EGFR by c-Src and MMP-dependent mechanisms in the MDA-MB-468 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 EGFR 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-) EGFR (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.g004: SP transmodulates EGFR by c-Src and MMP-dependent mechanisms in the MDA-MB-468 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 EGFR 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-) EGFR (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 determine whether the transmodulation of EGFR by SP was also dependent on c-Src and MMPs in BC cells, we performed similar experiments in the EGFR positive cell line MDA-MB-468. In the control situation, addition of SP increased EGFR phosphorylation at 6 min (1.17-fold), 10 min (1.45-fold) and particularly at 15 min (2.61-fold). No increase occurred in the presence of the inhibitors (alone or in combination) under SP treatment, as we observed in the western blot and densitrometic quantification of phospho EGFR/EGFR ratio (Fig 4A and 4B). In particular, c-Src inhibition significantly decreased the capability of SP to induce EGFR phosphorylation. Similarly, MMP inhibition affected the phosphorylation of EGFR induced by SP, as did the concomitant inhibition of both c-Src and MMPs, especially with both c-Src and MMPs inhibitor treatment at 15 min point (0.46 fold decrease) compared with point 0 (Fig 4B, right diagram).We also observed that c-Src inhibition significantly decreased the capability of SP to induce EGFR phosphorylation. Similarly, MMP inhibition affected the phosphorylation of EGFR induced by SP, as did the concomitant inhibition of both c-Src and MMPs, especially with both c-Src and MMPs inhibitor treatment at 15 min point (0.46 fold decrease) compared with the point 0, right diagram (Fig 4B). As before, the inhibition of c-Src and MMPs in this cell line did not block MAPK signaling due to its modulation by both NK-1R and RTKs (Fig 4A and 4C).

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