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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

c-Src phosphorylation levels at baseline or under SP treatment.Representative images of Western blots corresponding to experiments showing (A) the basal levels of c-Src Y416 phosphorylation in BC cell lines and (B) c-Src (Y416) phosphorylation at 0, 1, 2, 4, 6, 8, 10, 15 and 30 minutes after SP 100 nM stimulation in different BC cell lines. The blot was standardized to c-Src levels. The plots accompanying each panel show the densitometric quantification of Western blots (the ratio of intensities of the bands corresponding to phospho-Y416 and total c-Src) relative to the expression of tubulin or actin, which was used to ensure equal protein loading.
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pone.0129661.g001: c-Src phosphorylation levels at baseline or under SP treatment.Representative images of Western blots corresponding to experiments showing (A) the basal levels of c-Src Y416 phosphorylation in BC cell lines and (B) c-Src (Y416) phosphorylation at 0, 1, 2, 4, 6, 8, 10, 15 and 30 minutes after SP 100 nM stimulation in different BC cell lines. The blot was standardized to c-Src levels. The plots accompanying each panel show the densitometric quantification of Western blots (the ratio of intensities of the bands corresponding to phospho-Y416 and total c-Src) relative to the expression of tubulin or actin, which was used to ensure equal protein loading.

Mentions: We first investigated whether SP used the c-Src protein as a cell-signaling mediator in BC cells, as previously shown in other cell types [36, 37]. First, we checked a panel of BC cell lines under basal conditions without stimulation (Fig 1A) and we detected different levels of phosphorylated c-Src protein relative to total levels. Second, using time-course studies, we observed that SP treatment induced the phosphorylation of c-Src Tyr416 (indicative of Src activation [38, 39]) at the indicated time points (Fig 1B) in all the cell lines, including the HER2 negative cell lines MDA-MB-231 or MCF7. Some lines have more pronounced phosphorilation than others, and this activation is not consistent in all time points used because NK-1R activation by SP is a cyclic activation as we previously described [16]. For these reason, the activation of c-Src Tyr416 within this time frame was consistently observed in all the replicates conducted, although the exact time point and intensity of maximum activation varied. Lower activation of c-Src was found after SP treatment in the MDA-MB-453 cell line and was slightly pronounced in MDA-MB-468 line, probably due to the very low or very high basal levels of c-Src (phosphorylated and total protein) [30], respectively.


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)

c-Src phosphorylation levels at baseline or under SP treatment.Representative images of Western blots corresponding to experiments showing (A) the basal levels of c-Src Y416 phosphorylation in BC cell lines and (B) c-Src (Y416) phosphorylation at 0, 1, 2, 4, 6, 8, 10, 15 and 30 minutes after SP 100 nM stimulation in different BC cell lines. The blot was standardized to c-Src levels. The plots accompanying each panel show the densitometric quantification of Western blots (the ratio of intensities of the bands corresponding to phospho-Y416 and total c-Src) relative to the expression of tubulin or actin, which was used to ensure equal protein loading.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0129661.g001: c-Src phosphorylation levels at baseline or under SP treatment.Representative images of Western blots corresponding to experiments showing (A) the basal levels of c-Src Y416 phosphorylation in BC cell lines and (B) c-Src (Y416) phosphorylation at 0, 1, 2, 4, 6, 8, 10, 15 and 30 minutes after SP 100 nM stimulation in different BC cell lines. The blot was standardized to c-Src levels. The plots accompanying each panel show the densitometric quantification of Western blots (the ratio of intensities of the bands corresponding to phospho-Y416 and total c-Src) relative to the expression of tubulin or actin, which was used to ensure equal protein loading.
Mentions: We first investigated whether SP used the c-Src protein as a cell-signaling mediator in BC cells, as previously shown in other cell types [36, 37]. First, we checked a panel of BC cell lines under basal conditions without stimulation (Fig 1A) and we detected different levels of phosphorylated c-Src protein relative to total levels. Second, using time-course studies, we observed that SP treatment induced the phosphorylation of c-Src Tyr416 (indicative of Src activation [38, 39]) at the indicated time points (Fig 1B) in all the cell lines, including the HER2 negative cell lines MDA-MB-231 or MCF7. Some lines have more pronounced phosphorilation than others, and this activation is not consistent in all time points used because NK-1R activation by SP is a cyclic activation as we previously described [16]. For these reason, the activation of c-Src Tyr416 within this time frame was consistently observed in all the replicates conducted, although the exact time point and intensity of maximum activation varied. Lower activation of c-Src was found after SP treatment in the MDA-MB-453 cell line and was slightly pronounced in MDA-MB-468 line, probably due to the very low or very high basal levels of c-Src (phosphorylated and total protein) [30], respectively.

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