Abstract PS5-03-21: Rewiring fibroblast function in breast cancer

Abstract In the United States, breast cancer (BCa) is the second most common cause of cancer-related death for women and the most commonly diagnosed malignancy. Cancer-associated fibroblasts (CAFs) are a crucial component of the tumor microenvironment (TME) that influences breast cancer progression. By interacting with cancer cells and altering the extracellular matrix (ECM), CAFs actively promote tumor growth and metastasis. In contrast, normal fibroblasts restrict tumor progression. Despite the significant role of CAFs, little is known about the mechanisms underlying the regulation and conversion of normal fibroblasts into CAFs within the TME. Lysine Deficient Protein Kinase 1 (WNK1) is a serine/threonine kinase that is important for maintaining electrolyte balance and tissue homeostasis in the body. It is overexpressed in the BCa microenvironment, particularly in CAFs. This study investigated the role of WNK1 in regulating CAFs in BCa. Studies were conducted using BCa patient tissues, available public data, orthotopic mouse 4T1 tumor models, and in vitro assays to identify the effect of WNK1 on regulating fibroblast functions and BCa progression. WNK1 expression levels were determined in BCa tissues, orthotopic 4T1 BCa mouse embryonic fibroblasts (MEFs), and 4T1 BCa cells using Western blot analysis, RT-PCR, immunofluorescence, and confocal microscopy. Quantitative RT-PCR (qRT-PCR) and flow cytometry were used to identify the molecular changes associated with WNK1 manipulation in MEFs in vitro. Our results indicated that WNK1 is overexpressed in BCa and is associated with a poor prognosis and worse disease outcomes. WNK1 is expressed by both cancer cells and CAFs in tumors; however, WNK1 expression in CAFs was significantly higher. Our in vitro data revealed molecular changes in MEFs, accompanied by the upregulation of WNK1, when these cells were exposed to 4T1 cell-conditioned medium. Furthermore, significant upregulation in the expression of CAF-specific markers, α-SMA, FAP, and FSP1, along with ECM crosslinking genes and fibronectin, was observed in 4T1 medium-stimulated MEFs, which was significantly repressed in the presence of WNK1 inhibitors. Interestingly, upon overexpression of WNK1 in MEFs through expression plasmid vector, CAF markers and ECM crosslinking genes were found to be expressed significantly higher in WNK-overexpressed MEFs compared to normal MEFs, as indicated by qRT-PCR and flow cytometry analysis. The findings suggest a critical role for WNK1 in fibroblast-to-CAF transition. Analyzed publicly available datasets also indicated a strong correlation between WNK1 expression and the expression of ECM crosslinking genes in BCa. Taken together, our findings indicate that WNK1 contributes to BCa progression by regulating CAF conversion and the production of ECM proteins. Our results suggest that WNK1 may be a potential therapeutic target in BCa. Citation Format: P. Suman, S. Kakkat, B. Kola, E. T. Herrera, A. Richter, A. Mahadevan, S. Atterberry, K. Sawrie, R. Gupta, D. T. Tambe, C. Sarkar, D. Chakroborty. Rewiring fibroblast function in breast cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS5-03-21.