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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

Proposed model of EGFR and HER2 transactivation by SP.NK-1R induces signal transduction through the activation of G proteins. Heterotrimeric G proteins consist of three different subunits: the Gα subunit that binds GDP/GTP, and the Gβ and Gγ subunits that form the Gβγ complex. The binding of an agonist (in this case Substance P) to NK-1R induces the activation of G proteins which in turn induce their own signaling cascade, such as the activation of the MAPK pathway or the phosphorylation of c-Src. The activation of the MAPK pathway in turn contributes to raising MMP secretion, which increases the cleavage of membrane-anchored ligands that in turn will bind the EGFR receptor. On the other hand, c-Src directly phosphorylates the cytoplasmic tails of both EGFR and HER2, allowing the binding of scaffold proteins that will further activate signal transduction. Based on our results, we propose that this direct phosphorylation of the cytoplasmic tails of EGFR and HER2 by c-Src may overcome the effects of tyrosine kinase inhibitors or antibodies or molecules against extracellular domains [17], since c-Src may act independently of EGFR and HER2 tyrosine kinase activity. This figure has been made using Servier Medical Art collection (http://creativecommons.org/licenses/by/3.0).
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pone.0129661.g006: Proposed model of EGFR and HER2 transactivation by SP.NK-1R induces signal transduction through the activation of G proteins. Heterotrimeric G proteins consist of three different subunits: the Gα subunit that binds GDP/GTP, and the Gβ and Gγ subunits that form the Gβγ complex. The binding of an agonist (in this case Substance P) to NK-1R induces the activation of G proteins which in turn induce their own signaling cascade, such as the activation of the MAPK pathway or the phosphorylation of c-Src. The activation of the MAPK pathway in turn contributes to raising MMP secretion, which increases the cleavage of membrane-anchored ligands that in turn will bind the EGFR receptor. On the other hand, c-Src directly phosphorylates the cytoplasmic tails of both EGFR and HER2, allowing the binding of scaffold proteins that will further activate signal transduction. Based on our results, we propose that this direct phosphorylation of the cytoplasmic tails of EGFR and HER2 by c-Src may overcome the effects of tyrosine kinase inhibitors or antibodies or molecules against extracellular domains [17], since c-Src may act independently of EGFR and HER2 tyrosine kinase activity. This figure has been made using Servier Medical Art collection (http://creativecommons.org/licenses/by/3.0).

Mentions: The modulation of the steady state of RTKs like HER2 and EGFR by neuropeptides such as SP can influence the clinical response of a tumor [17]. Although the oncogenic addiction to RTKs is therapeutically exploited for BC treatment, the transmodulation of RTKs by SP and other neuropeptides and pro-inflammatory mediators [42, 43] can influence the cancer cell response to RTK inhibitors since it serves as a mechanism for RTK activation in a ligand-independent way [14]. The protein tyrosine kinase c-Src can directly phosphorylate Tyr residues in the kinase domain HER2 [30, 32] and the cytoplasmic tail of EGFR [31], allowing the formation of stable homo- or heterocomplexes with other receptors or the binding of scaffold proteins and the activation of signal transduction. In addition, activated RTKs will reciprocally activate c-Src, thereby creating a positive regulatory loop. This overactivation may contribute to the permanent signaling through the RTKs and the maintenance of multiple signaling pathways downstream of the receptor [44]. Then, the transactivation of these receptors by c-Src-dependent mechanisms may contribute to the persistence of RTK-related signaling pathways even in the presence of tyrosine kinase inhibitors or antibodies against extracellular domains of these receptors (Fig 6).


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)

Proposed model of EGFR and HER2 transactivation by SP.NK-1R induces signal transduction through the activation of G proteins. Heterotrimeric G proteins consist of three different subunits: the Gα subunit that binds GDP/GTP, and the Gβ and Gγ subunits that form the Gβγ complex. The binding of an agonist (in this case Substance P) to NK-1R induces the activation of G proteins which in turn induce their own signaling cascade, such as the activation of the MAPK pathway or the phosphorylation of c-Src. The activation of the MAPK pathway in turn contributes to raising MMP secretion, which increases the cleavage of membrane-anchored ligands that in turn will bind the EGFR receptor. On the other hand, c-Src directly phosphorylates the cytoplasmic tails of both EGFR and HER2, allowing the binding of scaffold proteins that will further activate signal transduction. Based on our results, we propose that this direct phosphorylation of the cytoplasmic tails of EGFR and HER2 by c-Src may overcome the effects of tyrosine kinase inhibitors or antibodies or molecules against extracellular domains [17], since c-Src may act independently of EGFR and HER2 tyrosine kinase activity. This figure has been made using Servier Medical Art collection (http://creativecommons.org/licenses/by/3.0).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0129661.g006: Proposed model of EGFR and HER2 transactivation by SP.NK-1R induces signal transduction through the activation of G proteins. Heterotrimeric G proteins consist of three different subunits: the Gα subunit that binds GDP/GTP, and the Gβ and Gγ subunits that form the Gβγ complex. The binding of an agonist (in this case Substance P) to NK-1R induces the activation of G proteins which in turn induce their own signaling cascade, such as the activation of the MAPK pathway or the phosphorylation of c-Src. The activation of the MAPK pathway in turn contributes to raising MMP secretion, which increases the cleavage of membrane-anchored ligands that in turn will bind the EGFR receptor. On the other hand, c-Src directly phosphorylates the cytoplasmic tails of both EGFR and HER2, allowing the binding of scaffold proteins that will further activate signal transduction. Based on our results, we propose that this direct phosphorylation of the cytoplasmic tails of EGFR and HER2 by c-Src may overcome the effects of tyrosine kinase inhibitors or antibodies or molecules against extracellular domains [17], since c-Src may act independently of EGFR and HER2 tyrosine kinase activity. This figure has been made using Servier Medical Art collection (http://creativecommons.org/licenses/by/3.0).
Mentions: The modulation of the steady state of RTKs like HER2 and EGFR by neuropeptides such as SP can influence the clinical response of a tumor [17]. Although the oncogenic addiction to RTKs is therapeutically exploited for BC treatment, the transmodulation of RTKs by SP and other neuropeptides and pro-inflammatory mediators [42, 43] can influence the cancer cell response to RTK inhibitors since it serves as a mechanism for RTK activation in a ligand-independent way [14]. The protein tyrosine kinase c-Src can directly phosphorylate Tyr residues in the kinase domain HER2 [30, 32] and the cytoplasmic tail of EGFR [31], allowing the formation of stable homo- or heterocomplexes with other receptors or the binding of scaffold proteins and the activation of signal transduction. In addition, activated RTKs will reciprocally activate c-Src, thereby creating a positive regulatory loop. This overactivation may contribute to the permanent signaling through the RTKs and the maintenance of multiple signaling pathways downstream of the receptor [44]. Then, the transactivation of these receptors by c-Src-dependent mechanisms may contribute to the persistence of RTK-related signaling pathways even in the presence of tyrosine kinase inhibitors or antibodies against extracellular domains of these receptors (Fig 6).

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