IL-3/STAT5/miR-155-5p axis supports stem-related pathway reprogramming in TNBC

Triple negative breast cancer (TNBC) remains one of the most aggressive subtypes of cancer with a poor prognosis and limited treatment options. Building on our previous findings of elevated Interleukin-3-Receptor-α (IL-3Rα) expression in TNBC, this study investigates the mechanisms underpinning IL-3-mediated actions in TNBC. GEO database (GSE25066) was interrogated to evaluate the expression of IL-3. RNAseq data were acquired from the TCGA-BRCA (Breast Carcinoma) project. Seven TNBC cell lines were used to validate the expression of IL-3 by ELISA assay. Chromatin immunoprecipitation assay was performed to evaluate the binding of STAT5A to the miR-155-5p promoter in TNBC cells. FACS analysis and ALDH activity were performed to evaluate the expansion of ALDH-1A1 + and CD44high/CD24low subpopulations. Mammosphere formation efficiency (MFE) was evaluated using the standard assay, while chemoresistance by applying the incucyte cell viability assay. miR155-5p silencing served to validate the expression of all target proteins both in vitro and in vivo. Bioinformatic analysis of breast cancer patient gene datasets revealed significant upregulation of the IL-3 gene in TNBC patient samples compared to the non-TNBC group (GEO: p = 0.004: TCGA p = 2.7e−30 respectively). We also found that TNBC cells secrete IL-3, which activates STAT5A promoting miR-155-5p expression by binding to its promoter in TNBC cells. Correlation analysis based on TCGA-BRCA confirmed elevated miR-155-5p levels in TNBC compared to non-tumoral tissues (p = 2.1e−33) and non-TNBC (p = 6.5e−30), with positive correlations between the IL-3 and miR-155-5p (r = 0.157, p < 0.001), as well as between miR-155-5p and miR-155-3p and STAT5A (r = 0.250, p = 0.002; r = 0.245, p < 0.005 respectively). Functional studies demonstrated that miR-155-5p downregulates programmed cell death 4, APC, and GSK-3β, enhancing β-catenin nuclear translocation and c-myc expression. Silencing miR-155-5p reversed all these effects. IL-3, via miR-155-5p, also drives ALDH-1A1 + and CD44high/CD24low subpopulation expansion and ALDH activity, enhances MFE and chemoresistance. Notably, blocking IL-3 impaired MFE, suggesting an autocrine loop sustaining IL-3 action in TNBC. In vivo, IL-3 promoted tumour growth, β-catenin activity, and metastasis, while miR-155-5p silencing mitigated these effects. Overall, our results underscore the crucial role of IL-3 in tumour progression, thereby advocating IL-3/IL-3Rα axis targeting as a promising therapeutic approach for TNBC.