Wnt signaling promotes inflammation and EMT-associated gene expression in mesenchymal TNBC

Aberrant activation of the Wnt signaling pathway in triple-negative breast cancer (TNBC) is linked to treatment resistance and recurrence, yet its role in tumoral heterogeneity remains unclear. We developed Wnt reporter cell lines from two mesenchymal TNBC models (MDA-MB-231 and MDA-MB-436) using a Tcf/Lef-eGFP vector and observed intra- and inter-tumoral variation in Wnt activity. Paired Wnt-positive and Wnt-negative TNBC cell lines were established and profiled by RNA-Seq. Integrative analyses revealed that Wnt-positive cells consistently upregulate genes involved in epithelial-to-mesenchymal transition, inflammation (e.g., IL6/JAK/STAT3, TNFα via NF-κB), and extracellular matrix remodeling. A 55-gene Wnt signature common to both nutrient conditions captured these features. Wnt-related gene sets were also enriched in the Mesenchymal-like Immune-Altered subtype of 699 primary TNBC tumors. These findings highlight the role of basal Wnt activity in driving pro-tumorigenic transcriptional programs in TNBC and provide new insight into its contribution to subtype-specific disease features. Two mesenchymal triple-negative breast cancer (TNBC) cell lines, MDA-MB-231 and MDA-MB-436, were stably transfected with the 7TGP Wnt reporter vector, which drives eGFP expression under the control of TCF/LEF-responsive elements. Fluorescence-activated cell sorting (FACS) was used to isolate and expand paired Wnt-positive and Wnt-negative sublines from each model. These reporter-defined cell lines were cultured under two serum conditions: low serum (0.5% FBS) for 3 days and standard serum (10% FBS) for 6 days. Total RNA was extracted from two biological replicates per condition for each cell line for subsequent bulk RNA sequencing.