ΕGFR/ERβ-Mediated Cell Morphology and Invasion Capacity Are Associated with Matrix Culture Substrates in Breast Cancer

Abstract

Breast cancer accounts for almost one in four cancer diagnoses in women. Studies in breast cancer patients have identified several molecular markers, indicators of aggressiveness, which help toward more individual therapeutic approaches. In triple-negative breast cancer (TNBC), epidermal growth factor receptor (EGFR) overexpression is associated with increased metastatic potential and worst survival rates. Specifically, abnormal EGFR activation leads to altered matrix metalloproteinases’ (MMPs) expression and, hence, extracellular matrix (ECM) degradation, resulting in induced migration and invasion. The use of matrix substrates for cell culture gives the opportunity to mimic the natural growth conditions of the cells and their microenvironment, as well as cell–cell and cell–matrix interactions. The aim of this study was to evaluate the impact of EGFR inhibition, estrogen receptor beta (ERβ) and different matrix substrates [type I collagen and fibronectin (FN)] on the functional properties, expression of MMPs and cell morphology of ERβ-positive TNBC cells and shERβ ones. Our results highlight EGFR as a crucial regulator of the expression and activity levels of MMPs, while ERβ emerges as a mediator of MMP7 and MT1-MMP expression. In addition, the EGFR/ERβ axis impacts the adhesion and invasion potential of breast cancer cells on collagen type I. Images obtained by scanning electron microscope (SEM) from cultures on the different matrix substrates revealed novel observations regarding various structures of breast cancer cells (filopodia, extravesicles, tunneling nanotubes, etc.). Moreover, the significant contribution of EGFR and ERβ in the morphological characteristics of these cells is also demonstrated, hence highlighting the possibility of dual pharmacological targeting.

EGFR is a pivotal player of the E2/ERβ – mediated functional properties, aggressiveness, and stemness in triple‐negative breast cancer cells

“…Breast cancer cells' morphological characteristics are useful indicators of the interactions that take place between cells and tumor microenvironment. We have recently showed the importance of the EGFR/ERβ axis in the adhesion and invasion potential of breast cancer cells as well as the development of specific morphological structures (filopodia and extracellular vesicles) in artificial 3D cultures using various ECM substrates [37]. Herein, we assessed the impact of E2 and EGFR inhibition on cell morphology and cytoskeletal organization in standard 2D cultures by scanning electron microscopy (SEM), IF microscopy and AFM.…”

“…Furthermore, EGFR is significantly overexpressed in TNBC, and despite the fact that various clinical trials have targeted the receptor's activation, the results until now have not been promising [52,53]. EGFR and ERb are known to interact and together modulate ECM remodeling of TNBC cells [37]. To that end, in the current study we chose the triple-negative, ERb+ MDA-MB-231 cells, as well as the stably transfected, shERb MDA-MB-231 cell line generated in our laboratories, to showcase the synergistic effects of EGFR and ERb in the aggressiveness and CSC-like capabilities of TNBC cells.…”

“…Our group has shown that ERb fine tunes the mRNA and protein expression of several matrix partners and EMT markers, to alter ECM composition, guide cell behavior, and cause changes in the morphological characteristics of the highly aggressive MDA-MB-231 cells [33,36]. In addition, the EGFR/ ERb axis determines the morphological characteristics while mediating the adhesion and invasion potential of TNBC cells [37]. The significant role of ERb in TNBC cells together with its established signature in ECM remodeling and interplay with EGFR, prompted us to take a step forward and assess the role of EGFR in the functional properties (i.e., migration, proliferation, angiogenesis) of TNBC cells with different ERb status (ERb+ and shERb MDA-MB-231 cells) in the presence and absence of E2.…”

ESR2 Drives Mesenchymal-to-Epithelial Transition in Triple-Negative Breast Cancer and Tumorigenesis In Vivo

“…In this study, we reported that high ESR2 expression rates have been correlated to decreased overall survival rates in breast cancer patients diagnosed with invasive breast carcinoma. Recent reports indicate that ERb target genes mostly regulate cell survival, movement, and growth (50), and that ERb signaling pathway intersects with EGFR cascade to mediate TNBC cell morphology and stemness (13,15). These data have been corroborated by the strong implication of ERb in EMT process since it acts as EMT promoter by activating the TGFb/Smad3 pathway to promote tumor growth and invasion in metastatic renal cell carcinoma (51).…”

“…The discovery of ERb in 1996 as the second nuclear receptor for steroid/thyroid hormones (i.e., 17b-estradiol, E2), after ERa, reserved a new era in the diagnosis, survival estimation and therapeutic targeting of breast cancer (12). While less well established, ERb dynamically communicates with major matrix components, including PGs and epidermal growth factor receptor (EGFR) to stimulate cancer cell behavior, epithelialto-mesenchymal transition (EMT) and stem-like characteristics (13)(14)(15).…”