Estetrol/GPER/SERPINB2 transduction signaling inhibits the motility of triple-negative breast cancer cells.

BACKGROUND: Estetrol (E4) is a natural estrogen produced by the fetal liver during pregnancy. Due to its favorable safety profile, E4 was recently approved as estrogenic component of a new combined oral contraceptive. E4 is a selective ligand of estrogen receptor (ER)α and ERß, but its binding to the G Protein-Coupled Estrogen Receptor (GPER) has not been described to date. Therefore, we aimed to explore E4 action in GPER-positive Triple-Negative Breast Cancer (TNBC) cells. METHODS: The potential interaction between E4 and GPER was investigated by molecular modeling and binding assays. The whole transcriptomic modulation triggered by E4 in TNBC cells via GPER was explored through high-throughput RNA sequencing analyses. Gene and protein expression evaluations as well as migration and invasion assays allowed us to explore the involvement of the GPER-mediated induction of the plasminogen activator inhibitor type 2 (SERPINB2) in the biological responses triggered by E4 in TNBC cells. Furthermore, bioinformatics analysis was aimed at recognizing the biological significance of SERPINB2 in ER-negative breast cancer patients. RESULTS: After the molecular characterization of the E4 binding capacity to GPER, RNA-seq analysis revealed that the plasminogen activator inhibitor type 2 (SERPINB2) is one of the most up-regulated genes by E4 in a GPER-dependent manner. Worthy, we demonstrated that the GPER-mediated increase of SERPINB2 is engaged in the anti-migratory and anti-invasive effects elicited by E4 in TNBC cells. In accordance with these findings, a correlation between SERPINB2 levels and a good clinical outcome was found in ER-negative breast cancer patients. CONCLUSIONS: Overall, our results provide new insights into the mechanisms through which E4 can halt migratory and invasive features of TNBC cells.

GPER deletion triggers inhibitory effects in triple negative breast cancer (TNBC) cells through the JNK/c-Jun/p53/Noxa transduction pathway.

The G protein-coupled estrogen receptor (GPER) mediates estrogen action in different pathophysiological conditions, including cancer. GPER expression and signaling have been found to join in the progression of triple-negative breast cancer (TNBC), even though controversial data have been reported. In present study, we aimed at providing new mechanistic and biological discoveries knocking out (KO) GPER expression by CRISPR/Cas9 technology in MDA-MB-231 TNBC cells. GPER KO whole transcriptome respect to wild type (WT) MDA-MB-231 cells was determined through total RNA sequencing (RNA-Seq) and gene ontology (GO) enrichment analysis. We ascertained that anti-proliferative and pro-apoptotic gene signatures characterize GPER KO MDA-MB-231 cells. Thereafter, we determined that these cells exhibit a reduced proliferative, clonogenic and self-renewal potential along with an increased mitochondria-dependent apoptosis phenotype. In addition, we recognized that decreased cAMP levels trigger the JNK/c-Jun/p53/Noxa axis, which in turn orchestrates the pro-apoptotic effects observed in GPER KO cells. In accordance with these data, survival analyses in TNBC patients of the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) dataset indicated that high Noxa expression correlates with improved outcomes in TNBC patients. Furthermore, we demonstrated that GPER KO in TNBC cells impairs the expression and secretion of the well-acknowledged GPER target gene named CTGF, thus resulting in the inhibition of migratory effects in cancer-associated fibroblasts (CAFs). Overall, the present study provides novel mechanistic and biological insights on GPER KO in TNBC cells suggesting that GPER may be considered as a valuable target in comprehensive therapeutic approaches halting TNBC progression.

The Ephrin tyrosine kinase a3 (EphA3) is a novel mediator of RAGE-prompted motility of breast cancer cells.

BACKGROUND: The receptor for advanced glycation-end products (RAGE) and its ligands have been implicated in obesity and associated inflammatory processes as well as in metabolic alterations like diabetes. In addition, RAGE-mediated signaling has been reported to contribute to the metastatic progression of breast cancer (BC), although mechanistic insights are still required. Here, we provide novel findings regarding the transcriptomic landscape and the molecular events through which RAGE may prompt aggressive features in estrogen receptor (ER)-positive BC. METHODS: MCF7 and T47D BC cells stably overexpressing human RAGE were used as a model system to evaluate important changes like cell protrusions, migration, invasion and colony formation both in vitro through scanning electron microscopy, clonogenic, migration and invasion assays and in vivo through zebrafish xenografts experiments. The whole transcriptome of RAGE-overexpressing BC cells was screened by high-throughput RNA sequencing. Thereafter, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses allowed the prediction of potential functions of differentially expressed genes (DEGs). Flow cytometry, real time-PCR, chromatin immunoprecipitation, immunofluorescence and western blot assays were performed to investigate the molecular network involved in the regulation of a novel RAGE target gene namely EphA3. The clinical significance of EphA3 was explored in the TCGA cohort of patients through the survivALL package, whereas the pro-migratory role of EphA3 signaling was ascertained in both BC cells and cancer-associated fibroblasts (CAFs). Statistical analysis was performed by t-tests. RESULTS: RNA-seq findings and GSEA analysis revealed that RAGE overexpression leads to a motility-related gene signature in ER-positive BC cells. Accordingly, we found that RAGE-overexpressing BC cells exhibit long filopodia-like membrane protrusions as well as an enhanced dissemination potential, as determined by the diverse experimental assays. Mechanistically, we established for the first time that EphA3 signaling may act as a physical mediator of BC cells and CAFs motility through both homotypic and heterotypic interactions. CONCLUSIONS: Our data demonstrate that RAGE up-regulation leads to migratory ability in ER-positive BC cells. Noteworthy, our findings suggest that EphA3 may be considered as a novel RAGE target gene facilitating BC invasion and scattering from the primary tumor mass. Overall, the current results may provide useful insights for more comprehensive therapeutic approaches in BC, particularly in obese and diabetic patients that are characterized by high RAGE levels.

The AGEs/RAGE Transduction Signaling Prompts IL-8/CXCR1/2-Mediated Interaction between Cancer-Associated Fibroblasts (CAFs) and Breast Cancer Cells.

Advanced glycation end products (AGEs) and the cognate receptor, named RAGE, are involved in metabolic disorders characterized by hyperglycemia, type 2 diabetes mellitus (T2DM) and obesity. Moreover, the AGEs/RAGE transduction pathway prompts a dysfunctional interaction between breast cancer cells and tumor stroma toward the acquisition of malignant features. However, the action of the AGEs/RAGE axis in the main players of the tumor microenvironment, named breast cancer-associated fibroblasts (CAFs), remains to be fully explored. In the present study, by chemokine array, we first assessed that interleukin-8 (IL-8) is the most up-regulated pro-inflammatory chemokine upon AGEs/RAGE activation in primary CAFs, obtained from breast tumors. Thereafter, we ascertained that the AGEs/RAGE signaling promotes a network cascade in CAFs, leading to the c-Fos-dependent regulation of IL-8. Next, using a conditioned medium from AGEs-exposed CAFs, we determined that IL-8/CXCR1/2 paracrine activation induces the acquisition of migratory and invasive features in MDA-MB-231 breast cancer cells. Altogether, our data provide new insights on the involvement of IL-8 in the AGEs/RAGE transduction pathway among the intricate connections linking breast cancer cells to the surrounding stroma. Hence, our findings may pave the way for further investigations to define the role of IL-8 as useful target for the better management of breast cancer patients exhibiting metabolic disorders.

Estrogen receptor variant ERα46 and insulin receptor drive in primary breast cancer cells growth effects and interleukin 11 induction prompting the motility of cancer-associated fibroblasts.

Among the prognostic and predictive biomarkers of breast cancer (BC), the role of estrogen receptor (ER)α wild-type has been acknowledged, although the action of certain ERα splice variants has not been elucidated. Insulin/insulin receptor (IR) axis has also been involved in the progression and metastasis of BC. For instance, hyperinsulinemia, which is often associated with obesity and type 2 diabetes, may be a risk factor for BC. Similarly, an aberrant expression of IR or its hyperactivation may correlate with aggressive BC phenotypes. In the present study, we have shown that a novel naturally immortalized BC cell line (named BCAHC-1) is characterized by a unique expression of 46 kDa ERα splice variant (ERα46) along with IR. Moreover, we have shown that a multifaceted crosstalk between ERα46 and IR occurs in BCAHC-1 cells upon estrogen and insulin exposure for growth and pulmonary metastasis. Through high-throughput RNA sequencing analysis, we have also found that the cytokine interleukin-11 (IL11) is the main factor linking BCAHC-1 cells to breast cancer-associated fibroblasts (CAFs). In particular, we have found that IL11 induced by estrogens and insulin in BCAHC-1 cells regulates pro-tumorigenic genes of the "extracellular matrix organization" signaling pathway, such as ICAM-1 and ITGA5, and promotes both migratory and invasive features in breast CAFs. Overall, our results may open a new scientific avenue to identify additional prognostic and therapeutic targets in BC.