Novel mechanisms involved by G-protein estrogen receptor (GPER) in the progression of Supervisore estrogen receptor-negative breast cancer

Abstract

The G protein-coupled estrogen receptor (GPER) is a seven transmembrane receptor that mediates estrogen signals in both normal and malignant cells, including breast cancer. In particular, GPER activation triggers diverse transduction pathways prompting transcriptional and biological pro-tumorigenic responses. In this context, we aimed to perform in-silico analysis that show a correlation of GPER expression levels with worse clinical-pathological features of breast cancer. By gene expression correlation, gene set enrichment analysis (GSEA) and KEGG pathway enrichment analysis on the transcriptomics data of ER-negative breast tumors provided by TCGA and METABRIC datasets, we also ascertained that the levels of GPER are associated with pro-migratory and metastatic genes. In particular, a strong association was found between the expression of GPER and that of genes belonging to cell adhesion molecules (CAMs), extracellular matrix (ECM)-receptor interaction and focal adhesion (FA) signaling pathways. Accordingly, we found that high GPER levels are predictive of a shorter disease-free interval (DFI) in ER-negative and HER2-negative breast cancer patients. Overall, our results may pave the way to further dissect the network triggered by GPER in breast malignancies lacking ER. Next, starting from a further bioinformatics analysis on TCGA and METABRIC triple negative breast cancer (TNBC) cohorts of patients, we determined that the expression of the pro-inflammatory cytokine interleukin-1β (IL-β) correlates with the levels of the hypoxia inducible factor-1α (HIF-1α) as well as with a hypoxia related gene signature. Then, through gene and protein expression studies, immunofluorescence analysis, co-immunoprecipitation, ChiP and ELISA assays we demonstrated that hypoxia triggers a functional liaison among HIF-1α, GPER and the IL-1β/IL1R1 signaling toward a metastatic gene signature and a feed-forward loop of IL-1β. Cell spreading, invasion and spheroid formation assays showed that IL-1β/IL1R1 axis leads to proliferative and invasive responses in TNBC cells. Furthermore, we found that the IL-1β released in the conditioned medium of TNBC cells exposed to hypoxic conditions promotes an invasive phenotype of breast cancer-associated fibroblasts (CAFs). Together, these findings may contribute to unveil the mechanisms involved in the hypoxic activation of the IL-1β/IL1R1 signaling toward aggressive features of both TNBC cells and CAFs, suggesting novel targets needed for innovative and more comprehensive therapeutic strategies in TNBC patients.