GPER Mediates a Feedforward FGF2/FGFR1 Paracrine Activation Coupling CAFs to Cancer Cells Toward Breast Tumor Progression

Maria Francesca Santolla,Francesca Cirillo,Anna Maria Miglietta,Marcello Maggiolini,Marianna Talia,Giulia Raffaella Galli,Giuseppe Brunetti,Adele Vivacqua,Antonino Belfiore,Rosamaria Lappano,Damiano Cosimo Rigiracciolo

doi:10.3390/cells8030223

Abstract

The FGF2/FGFR1 paracrine loop is involved in the cross-talk between breast cancer cells and components of the tumor stroma as cancer-associated fibroblasts (CAFs). By quantitative PCR (qPCR), western blot, immunofluorescence analysis, ELISA and ChIP assays, we demonstrated that 17β-estradiol (E2) and the G protein estrogen receptor (GPER) agonist G-1 induce the up-regulation and secretion of FGF2 via GPER together with the EGFR/ERK/c-fos/AP-1 signaling cascade in (ER)-negative primary CAFs. Evaluating the genetic alterations from METABRIC and TCGA datasets, we then assessed that FGFR1 is the most frequently amplified FGFRs family member and its amplification/expression associates with shorter survival rates in breast cancer patients. Therefore, in order to assess the functional FGF2/FGFR1 interplay between CAFs and breast cancer cells, we generated the FGFR1-knockout MDA-MB-231 cells using CRISPR/Cas9 genome editing strategy. Using conditioned medium from estrogen-stimulated CAFs, we established that the activation of FGF2/FGFR1 paracrine signaling triggers the expression of the connective tissue growth factor (CTGF), leading to the migration and invasion of MDA-MB-231 cells. Our findings shed new light on the role elicited by estrogens through GPER in the activation of the FGF2/FGFR1 signaling. Moreover, our findings may identify further biological targets that could be considered in innovative combination strategies halting breast cancer progression.

Highlights

Cross-talk between stromal and epithelial cells plays an important role in diverse pathophysiological conditions, including malignant diseases [1,2,3]
In order to provide novel insights into the FGF2 regulation by estrogens within the tumor microenvironment, we sought to address whether estrogens may regulate FGF2 levels in extracellular signal-regulated kinase (ERK)/c-fos/AP-1 signaling cascade in (ER)-negative/ G protein estrogen receptor (GPER)-positive cancer-associated fibroblasts (CAFs) isolated from breast tumor patients
As GPER activation induces the stimulation of diverse transduction pathways [23], we found that FGF2 up-regulation prompted by E2 and G-1 was prevented either by the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor AG1478 (AG) or the MEK inhibitor PD98059 (PD), but not by the phosphatidylinositol 3-kinase (PI3K) inhibitor Wortmannin (WM) (Supplementary Figure S3a,b)

Summary

Introduction

Cross-talk between stromal and epithelial cells plays an important role in diverse pathophysiological conditions, including malignant diseases [1,2,3]. In this regard, it has been largely reported that the acquisition of an aggressive phenotype does not depend exclusively on the intrinsic cancer cell properties, and on stromal features [4]. Cells 2019, 8, 223 mediators secreted by CAFs, such as cytokines and growth factors, exert an important role in the acquisition of malignant features [6,7]. The fibroblast growth factor (FGF)-FGF receptor (FGFR) axis is one of the major signal transduction pathways mediating the interaction between tumor stroma and cancer cells [8,9].

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Conclusion