# Dissecting Context-Specific Effects of ERK5 Signaling in Triple-Negative Breast Cancer

## TL;DR

This study explores how ERK5 signaling affects triple-negative breast cancer progression using different lab models to find better treatment targets.

## Contribution

The study introduces a novel analysis of ERK5's role in TNBC using advanced in vitro models to identify potential therapeutic targets.

## Key findings

- ERK5 deletion impairs TNBC cell migration across multiple in vitro models.
- ERK5 influences genes related to EMT and matrix remodeling, with collagen downregulation observed in 3D models.
- NFκB pathway upregulation is specific to 2D cultures, not 3D models, highlighting model-dependent signaling differences.

## Abstract

Drug discovery research is hindered by a lack of models that mimic physiological conditions in cancer patients. Triple-negative breast cancer (TNBC) is clinically aggressive with high rates of relapse and limited treatment options. Kinases are attractive therapeutic targets due to their roles in cell fate, and irregular kinase activity drives tumor progression. Here, we analyzed a mitogen-activated kinase pathway in traditional and complex in vitro models to determine context-specific effects mediated by kinase expression, with the aim of applying these findings in the clinical setting. Our goal is to identify viable treatment targets to improve both overall and regression-free survival of TNBC patients.

Background: Triple-negative breast cancer (TNBC) is an aggressive subtype of cancer with poor clinical outcomes. There is a critical need to identify novel, druggable targets for TNBC to improve therapy response and patient outcomes. Due to their roles in critical processes driving cancer progression, kinases have been a major focus of drug discovery efforts. The role of extracellular signal-regulated kinase 5 (ERK5) in mediating TNBC extracellular matrix (ECM) has previously been described in 2D culture and in vivo. Here, we characterized the impact of ERK5 on breast cancer biology in 2D culture, 3D spheroids, and our 3D breast adipose-macrophysiological system (BA-MaPS). Methods: We assessed migration changes in MDA-MB-231 parental and ERK5-knockout (ERK5-ko) cells cultured in the three in vitro models using transwell, scratch, and spheroid pseudo-migration assays. Differential gene expression among these cell lines in the three platforms was assessed by RNA sequencing and pathway analysis. Stromal remodeling of adipocytes and matrix was evaluated by H&E and Masson’s Trichrome. Results: Across the in vitro models, ERK5 deletion impaired TNBC cell migration. ERK5-mediated transcriptomic changes included genes associated with epithelial-to-mesenchymal transition (EMT) and migration, with further analysis showing significant alterations in core and associated matrisome. Histological staining corroborated the downregulation of collagen with ERK5 depletion in the BA-MaPS. The NFκB pathway was significantly upregulated only in the ERK5-ko 2D-cultured cells, not in 3D spheroids nor the BA-MaPS model. Conclusions: These results indicate a link between ERK5 and TNBC progression through regulation of TME remodeling, EMT, and cell motility. Differences in 2D culture, 3D spheroid, and BA-MaPS underscore the importance of using physiologically relevant models in breast cancer research.