Breast cancer-associated fibroblasts (bCAFs) comprise two pro-tumor populations: inflammatory CAFs (iCAFs) producing pro-inflammatory cytokines and myofibroblastic CAFs (myCAFs) characterized by their highly contractile and invasive phenotype. We have previously shown that bCAFs promote therapy resistance by overexpressing the anti-apoptotic protein MCL-1 and inducing its expression in luminal breast cancer cells by paracrine effects. This defines MCL-1 as a target in stroma-influenced breast cancers, and advocates for a comprehensive investigation of the effects of its targeting in distinct cellular components, such as myofibroblastic CAFs, of these tumor ecosystems.

In this study, we explored the role of MCL-1 in the primary culture of CAFs obtained from treatment-naive patients with invasive carcinoma after surgical resection by inhibiting MCL-1 pharmacologically with S63845, a BH3 mimetic developed to specifically antagonize its anti-apoptotic function or genetically by silencing MCL-1 gene expression using Crispr-Cas9.

Single-cell RNA sequencing analysis of bCAFs reveals that MCL-1 knock down induces a phenotypic shift from wound-myCAF to IL-iCAF. Notably, MCL-1 depletion in bCAFs leads to a reduction in the expression of key myofibroblast activation markers, including α-SMA and Myosin IIb, accompanied by a significant loss of contractile, migratory, and pro-invasive properties in a 3D co-culture model with luminal breast cancer cells. Mechanistically, pharmacological inhibition of MCL-1, which stabilizes the protein, enhances its interaction with DRP1, triggering mitochondrial network fragmentation and cytoplasmic retention of the transcription factor YAP1. Concurrently, MCL-1 targeting in bCAFs stimulates VEGFA secretion, which in turn enhances endothelial cell tubulogenesis and drives extensive tumor vascularization in the chicken chorioallantoic membrane (CAM) model. Mechanistically, we identified NF-κB activation as responsible for the overexpression of VEGF-A and the cytokines/chemokines CXCL1, CXCL8 and IL-1β leading to the acquisition of an inflammatory phenotype by bCAFs in response to MCL-1 targeting. Importantly, we established that chemotherapeutic treatment promotes inflammatory and pro-angiogenic phenotype of bCAFs too.

These findings highlight a novel role for MCL-1 in regulating the phenotypic plasticity of bCAFs, and provide new insights into the characterization of distinct bCAF subpopulations, particularly post-chemotherapy.