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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

Migration assays performed by Boyden Chamber assay in SkBr3 and MCF-7 cells transfected for 24 h with shRNA or shGPER and then treated for 8 h with vehicle (−) and 10 nM E2 (A) or vehicle (−) and 100 nM G-1 (B) before to be exposed for additional 8 h to conditioned medium collected from CAFs treated for 8 h with vehicle, 10 nM E2 [CM/CAFs (+E2)] or 100 nM G-1 [CM/CAFs (+G-1)]. Each data point is the average ± SD of three independent experiments performed in triplicate. (◼) p < 0.05 for cells receiving treatments versus vehicle.
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f8: Migration assays performed by Boyden Chamber assay in SkBr3 and MCF-7 cells transfected for 24 h with shRNA or shGPER and then treated for 8 h with vehicle (−) and 10 nM E2 (A) or vehicle (−) and 100 nM G-1 (B) before to be exposed for additional 8 h to conditioned medium collected from CAFs treated for 8 h with vehicle, 10 nM E2 [CM/CAFs (+E2)] or 100 nM G-1 [CM/CAFs (+G-1)]. Each data point is the average ± SD of three independent experiments performed in triplicate. (◼) p < 0.05 for cells receiving treatments versus vehicle.

Mentions: Upon IL1β stimulation, breast cancer cells acquire certain features of an invasive phenotype as the loss of cell-cell contact, the acquisition of a fibroblastoid cytoarchitecture and cell scattering141536. Nicely recapitulating the abovementioned results, medium collected from E2 and G-1 treated CAFs induced a fibroblast-like phenotype (as evaluated by the polarity index) in SkBr3 cells transfected with a shRNA and exposed to E2 and G-1, but not in SkBr3 cells transfected with a shGPER (Fig. 6A–D). Findings similar to those obtained using medium collected from E2 and G-1 treated CAFs were elicited in SkBr3 cells exposed to E2 and G-1 before IL1β treatment (data not shown). Then, SkBr3 cells were fixed and stained with rhodamine-phalloidin to visualize the F-actin pattern. Conditioned medium from E2 and G-1 treated CAFs triggered the F-actin reorganization in SkBr3 cells transfected with a shRNA and exposed to E2 and G-1, but not in SkBr3 cells transfected with a shGPER (Fig. 7A–H). Results comparable to those obtained using medium collected from E2 and G-1 treated CAFs were elicited in SkBr3 cells exposed to E2 and G-1 before IL1β treatment (data not shown). The aforementioned findings were further supported by time-lapse video microscopy performed in MCF-7 cells treated with E2 and cultured with conditioned medium from CAFs exposed to E2 (videos 1–2). As previously shown22, E2 and G-1 stimulated the migration of SkBr3 and MCF-7 cells. This effect was further potentiated culturing cells with medium collected from E2 and G-1 treated CAFs, while the response was no longer observed in both cell types transfected with a shGPER (Fig. 8).


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)

Migration assays performed by Boyden Chamber assay in SkBr3 and MCF-7 cells transfected for 24 h with shRNA or shGPER and then treated for 8 h with vehicle (−) and 10 nM E2 (A) or vehicle (−) and 100 nM G-1 (B) before to be exposed for additional 8 h to conditioned medium collected from CAFs treated for 8 h with vehicle, 10 nM E2 [CM/CAFs (+E2)] or 100 nM G-1 [CM/CAFs (+G-1)]. Each data point is the average ± SD of three independent experiments performed in triplicate. (◼) p < 0.05 for cells receiving treatments versus vehicle.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f8: Migration assays performed by Boyden Chamber assay in SkBr3 and MCF-7 cells transfected for 24 h with shRNA or shGPER and then treated for 8 h with vehicle (−) and 10 nM E2 (A) or vehicle (−) and 100 nM G-1 (B) before to be exposed for additional 8 h to conditioned medium collected from CAFs treated for 8 h with vehicle, 10 nM E2 [CM/CAFs (+E2)] or 100 nM G-1 [CM/CAFs (+G-1)]. Each data point is the average ± SD of three independent experiments performed in triplicate. (◼) p < 0.05 for cells receiving treatments versus vehicle.
Mentions: Upon IL1β stimulation, breast cancer cells acquire certain features of an invasive phenotype as the loss of cell-cell contact, the acquisition of a fibroblastoid cytoarchitecture and cell scattering141536. Nicely recapitulating the abovementioned results, medium collected from E2 and G-1 treated CAFs induced a fibroblast-like phenotype (as evaluated by the polarity index) in SkBr3 cells transfected with a shRNA and exposed to E2 and G-1, but not in SkBr3 cells transfected with a shGPER (Fig. 6A–D). Findings similar to those obtained using medium collected from E2 and G-1 treated CAFs were elicited in SkBr3 cells exposed to E2 and G-1 before IL1β treatment (data not shown). Then, SkBr3 cells were fixed and stained with rhodamine-phalloidin to visualize the F-actin pattern. Conditioned medium from E2 and G-1 treated CAFs triggered the F-actin reorganization in SkBr3 cells transfected with a shRNA and exposed to E2 and G-1, but not in SkBr3 cells transfected with a shGPER (Fig. 7A–H). Results comparable to those obtained using medium collected from E2 and G-1 treated CAFs were elicited in SkBr3 cells exposed to E2 and G-1 before IL1β treatment (data not shown). The aforementioned findings were further supported by time-lapse video microscopy performed in MCF-7 cells treated with E2 and cultured with conditioned medium from CAFs exposed to E2 (videos 1–2). As previously shown22, E2 and G-1 stimulated the migration of SkBr3 and MCF-7 cells. This effect was further potentiated culturing cells with medium collected from E2 and G-1 treated CAFs, while the response was no longer observed in both cell types transfected with a shGPER (Fig. 8).

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