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GPER signalling in both cancer-associated fibroblasts and breast cancer cells mediates a feedforward IL1β/IL1R1 response.

De Marco P, Lappano R, De Francesco EM, Cirillo F, Pupo M, Avino S, Vivacqua A, Abonante S, Picard D, Maggiolini M - Sci Rep (2016)

Bottom Line: Cancer-associated fibroblasts (CAFs) contribute to the malignant aggressiveness through secreted factors like IL1β, which may drive pro-tumorigenic inflammatory phenotypes mainly acting via the cognate receptor named IL1R1.Thereby, ligand-activation of GPER generates a feedforward loop coupling IL1β induction by CAFs to IL1R1 expression by cancer cells, promoting the up-regulation of IL1β/IL1R1 target genes such as PTGES, COX2, RAGE and ABCG2.This regulatory interaction between the two cell types induces migration and invasive features in breast cancer cells including fibroblastoid cytoarchitecture and F-actin reorganization.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacy and Health and Nutritional Sciences, University of Calabria, 87036 Rende, Italy.

ABSTRACT
Cancer-associated fibroblasts (CAFs) contribute to the malignant aggressiveness through secreted factors like IL1β, which may drive pro-tumorigenic inflammatory phenotypes mainly acting via the cognate receptor named IL1R1. Here, we demonstrate that signalling mediated by the G protein estrogen receptor (GPER) triggers IL1β and IL1R1 expression in CAFs and breast cancer cells, respectively. Thereby, ligand-activation of GPER generates a feedforward loop coupling IL1β induction by CAFs to IL1R1 expression by cancer cells, promoting the up-regulation of IL1β/IL1R1 target genes such as PTGES, COX2, RAGE and ABCG2. This regulatory interaction between the two cell types induces migration and invasive features in breast cancer cells including fibroblastoid cytoarchitecture and F-actin reorganization. A better understanding of the mechanisms involved in the regulation of pro-inflammatory cytokines by GPER-integrated estrogen signals may be useful to target these stroma-cancer interactions.

No MeSH data available.


Related in: MedlinePlus

PTGES protein expression in SkBr3 (A–C) and MCF-7 (D–F) cells treated with 10 ng/ml IL1β alone or treated for 8 h with 10 nM E2 or 100 nM G-1 and then exposed to 10 ng/ml IL1β, as indicated. Protein levels of PTGES in SkBr3 (G,H) and MCF-7 (I–J) cells treated for 8 h with 10 nM E2 or 100 nM G-1 and then switched for additional 8 h to medium without serum in the presence of 10 ng/ml IL1β or conditioned medium collected from CAFs (CM/CAFs) treated for 8 h with vehicle [CM/CAFs (+vehicle)], 10 nM E2 [CM/CAFs (+E2)] and 100 nM G-1 [CM/CAFs (+G-1)]. SkBr3 and MCF-7 cells treated for 8 h with 10 nM E2 or 100 nM G-1 were also exposed to [CM/CAFs (+E2)] and [CM/CAFs (+G-1)] alone or in combination with 1 μM IL1R1 antagonist namely IL1R1a. β-actin serves as a loading control. Results shown are representative of at least two independent experiments.
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f5: PTGES protein expression in SkBr3 (A–C) and MCF-7 (D–F) cells treated with 10 ng/ml IL1β alone or treated for 8 h with 10 nM E2 or 100 nM G-1 and then exposed to 10 ng/ml IL1β, as indicated. Protein levels of PTGES in SkBr3 (G,H) and MCF-7 (I–J) cells treated for 8 h with 10 nM E2 or 100 nM G-1 and then switched for additional 8 h to medium without serum in the presence of 10 ng/ml IL1β or conditioned medium collected from CAFs (CM/CAFs) treated for 8 h with vehicle [CM/CAFs (+vehicle)], 10 nM E2 [CM/CAFs (+E2)] and 100 nM G-1 [CM/CAFs (+G-1)]. SkBr3 and MCF-7 cells treated for 8 h with 10 nM E2 or 100 nM G-1 were also exposed to [CM/CAFs (+E2)] and [CM/CAFs (+G-1)] alone or in combination with 1 μM IL1R1 antagonist namely IL1R1a. β-actin serves as a loading control. Results shown are representative of at least two independent experiments.

Mentions: In order to evaluate the transcriptional responses mediated by GPER through the up-regulation of IL1R1 in SkBr3 and MCF-7 cells, we assessed the changes of certain IL1β target genes3435. For instance, the mRNA expression of ATP-binding cassette G2 (ABCG2), cyclooxygenase-2 (COX2), prostaglandin E synthase-1 (PTGES) and receptor for advanced glycation end products (RAGE) was stimulated only in SkBr3 and MCF-7 cells treated with E2 and G-1 before IL1β exposure (Fig. 4A,B). In accordance with these findings, we determined that the protein levels of PTGES are up-regulated by IL1β only upon E2 and G-1 exposure in SkBr3 and MCF-7 cells (Fig. 5A–F), suggesting that the increase of IL1R1 by agonist-activated GPER does contribute to the aforementioned responses. Considering that E2 and G-1 trigger the expression of IL1β in CAFs (shown in Fig. 1) and IL1R1 in breast cancer cells (shown in Fig. 2), we then assessed that conditioned medium from CAFs exposed to E2 and G-1 does induce PTGES protein expression in SkBr3 (Fig. 5G–H) and MCF-7 (Fig. 5I,J) cells exposed to E2 or G-1. Using the IL1R1 antagonist, namely IL1R1a, the up-regulation of PTGES observed in the aforementioned experimental conditions was no longer evident (Fig. 5G–J). Moreover, an increased expression of PTGES was observed treating with IL1β both SkBr3 and MCF-7 cells exposed to E2 and G-1 (Fig. 5G–J). The up-regulation of PTGES in SkBr3 and MCF-7 cells treated with E2 and G-1 and cultured with conditioned medium from CAFs exposed to these ligands was not altered by increasing concentrations of the ER antagonist ICI up to 10 μM (data not shown). Collectively, these findings suggest that estrogenic GPER signalling generates a feedforward loop that couples IL1β induction in CAFs to IL1R1 expression by cancer cells, hence contributing to the functional cross-talk between the tumor microenvironment and breast cancer cells.


GPER signalling in both cancer-associated fibroblasts and breast cancer cells mediates a feedforward IL1β/IL1R1 response.

De Marco P, Lappano R, De Francesco EM, Cirillo F, Pupo M, Avino S, Vivacqua A, Abonante S, Picard D, Maggiolini M - Sci Rep (2016)

PTGES protein expression in SkBr3 (A–C) and MCF-7 (D–F) cells treated with 10 ng/ml IL1β alone or treated for 8 h with 10 nM E2 or 100 nM G-1 and then exposed to 10 ng/ml IL1β, as indicated. Protein levels of PTGES in SkBr3 (G,H) and MCF-7 (I–J) cells treated for 8 h with 10 nM E2 or 100 nM G-1 and then switched for additional 8 h to medium without serum in the presence of 10 ng/ml IL1β or conditioned medium collected from CAFs (CM/CAFs) treated for 8 h with vehicle [CM/CAFs (+vehicle)], 10 nM E2 [CM/CAFs (+E2)] and 100 nM G-1 [CM/CAFs (+G-1)]. SkBr3 and MCF-7 cells treated for 8 h with 10 nM E2 or 100 nM G-1 were also exposed to [CM/CAFs (+E2)] and [CM/CAFs (+G-1)] alone or in combination with 1 μM IL1R1 antagonist namely IL1R1a. β-actin serves as a loading control. Results shown are representative of at least two independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4829876&req=5

f5: PTGES protein expression in SkBr3 (A–C) and MCF-7 (D–F) cells treated with 10 ng/ml IL1β alone or treated for 8 h with 10 nM E2 or 100 nM G-1 and then exposed to 10 ng/ml IL1β, as indicated. Protein levels of PTGES in SkBr3 (G,H) and MCF-7 (I–J) cells treated for 8 h with 10 nM E2 or 100 nM G-1 and then switched for additional 8 h to medium without serum in the presence of 10 ng/ml IL1β or conditioned medium collected from CAFs (CM/CAFs) treated for 8 h with vehicle [CM/CAFs (+vehicle)], 10 nM E2 [CM/CAFs (+E2)] and 100 nM G-1 [CM/CAFs (+G-1)]. SkBr3 and MCF-7 cells treated for 8 h with 10 nM E2 or 100 nM G-1 were also exposed to [CM/CAFs (+E2)] and [CM/CAFs (+G-1)] alone or in combination with 1 μM IL1R1 antagonist namely IL1R1a. β-actin serves as a loading control. Results shown are representative of at least two independent experiments.
Mentions: In order to evaluate the transcriptional responses mediated by GPER through the up-regulation of IL1R1 in SkBr3 and MCF-7 cells, we assessed the changes of certain IL1β target genes3435. For instance, the mRNA expression of ATP-binding cassette G2 (ABCG2), cyclooxygenase-2 (COX2), prostaglandin E synthase-1 (PTGES) and receptor for advanced glycation end products (RAGE) was stimulated only in SkBr3 and MCF-7 cells treated with E2 and G-1 before IL1β exposure (Fig. 4A,B). In accordance with these findings, we determined that the protein levels of PTGES are up-regulated by IL1β only upon E2 and G-1 exposure in SkBr3 and MCF-7 cells (Fig. 5A–F), suggesting that the increase of IL1R1 by agonist-activated GPER does contribute to the aforementioned responses. Considering that E2 and G-1 trigger the expression of IL1β in CAFs (shown in Fig. 1) and IL1R1 in breast cancer cells (shown in Fig. 2), we then assessed that conditioned medium from CAFs exposed to E2 and G-1 does induce PTGES protein expression in SkBr3 (Fig. 5G–H) and MCF-7 (Fig. 5I,J) cells exposed to E2 or G-1. Using the IL1R1 antagonist, namely IL1R1a, the up-regulation of PTGES observed in the aforementioned experimental conditions was no longer evident (Fig. 5G–J). Moreover, an increased expression of PTGES was observed treating with IL1β both SkBr3 and MCF-7 cells exposed to E2 and G-1 (Fig. 5G–J). The up-regulation of PTGES in SkBr3 and MCF-7 cells treated with E2 and G-1 and cultured with conditioned medium from CAFs exposed to these ligands was not altered by increasing concentrations of the ER antagonist ICI up to 10 μM (data not shown). Collectively, these findings suggest that estrogenic GPER signalling generates a feedforward loop that couples IL1β induction in CAFs to IL1R1 expression by cancer cells, hence contributing to the functional cross-talk between the tumor microenvironment and breast cancer cells.

Bottom Line: Cancer-associated fibroblasts (CAFs) contribute to the malignant aggressiveness through secreted factors like IL1β, which may drive pro-tumorigenic inflammatory phenotypes mainly acting via the cognate receptor named IL1R1.Thereby, ligand-activation of GPER generates a feedforward loop coupling IL1β induction by CAFs to IL1R1 expression by cancer cells, promoting the up-regulation of IL1β/IL1R1 target genes such as PTGES, COX2, RAGE and ABCG2.This regulatory interaction between the two cell types induces migration and invasive features in breast cancer cells including fibroblastoid cytoarchitecture and F-actin reorganization.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacy and Health and Nutritional Sciences, University of Calabria, 87036 Rende, Italy.

ABSTRACT
Cancer-associated fibroblasts (CAFs) contribute to the malignant aggressiveness through secreted factors like IL1β, which may drive pro-tumorigenic inflammatory phenotypes mainly acting via the cognate receptor named IL1R1. Here, we demonstrate that signalling mediated by the G protein estrogen receptor (GPER) triggers IL1β and IL1R1 expression in CAFs and breast cancer cells, respectively. Thereby, ligand-activation of GPER generates a feedforward loop coupling IL1β induction by CAFs to IL1R1 expression by cancer cells, promoting the up-regulation of IL1β/IL1R1 target genes such as PTGES, COX2, RAGE and ABCG2. This regulatory interaction between the two cell types induces migration and invasive features in breast cancer cells including fibroblastoid cytoarchitecture and F-actin reorganization. A better understanding of the mechanisms involved in the regulation of pro-inflammatory cytokines by GPER-integrated estrogen signals may be useful to target these stroma-cancer interactions.

No MeSH data available.


Related in: MedlinePlus