Interleukin-3 boosts the stemness phenotype of triple negative breast cancer cells through miR-155-5p

Triple negative breast cancer (TNBC) persists as one of the most aggressive subtypes, carrying a grim prognosis lacking effective treatment strategies. Building upon our prior research indicating elevated expression of Interleukin-3-Receptor-α (IL-3Rα) in TNBC, the current study endeavours to unravel the mechanisms underpinning IL-3 -mediated actions. Initially, a bioinformatic analysis on two breast cancer patient gene datasets (TCGA-BRCA and GSE25066 dataset) found the IL-3 gene significantly upregulated in TNBC patient samples compared to the non-TNBC group, and adjacent normal tissues. In addition, we established that TNBC cells secrete IL-3 cytokine, which activates STAT5A that by binding to miR-155-5p promoter increases its expression in MDA-MB-436 and Hs-578T cell lines. Interestingly, a correlation analysis based on TCGA-BRCA demonstrated a positive association between miR-155-5p in TNBC compared to non-tumoral tissues and non-TNBC. Furthermore, a positive correlation was also found between the expressions of miR-155-5p with IL-3 and STAT5A. Functional studies demonstrated that miR-155-5p downregulates APC and GSK-3β, affecting β-catenin nuclear translocation and c-myc expression. Therefore, IL-3 fosters the expansion of ALDH1A1+ and CD44high/CD24low subpopulations via miR-155-5p targeting, as well as enhances mammosphere formation efficiency (MFE) and drug resistance. Notably, blockade of IL-3 or its receptor IL-3Rα impedes MFE, suggesting an autocrine loop sustaining IL-3 action in TNBC. Consistent with these findings, mice bearing tumours experiencing IL-3 display intratumorally active β-catenin over-expression, tumour outgrowth and metastatic dissemination, blunted by miR-155-5p silencing. Noteworthy, tumorspheres showed an increased IL-3 release compared to adherent bulk cells, with miR-155-5p acting in a feed-forward mechanism. Finally, also the tumour suppressor PDCD4 was targeted and suppressed by the IL-3 -mediated miR-155-5p expression. Overall, our results underscore the crucial role of IL-3 in maintaining stemness and advocate the targeting of IL-3/IL-3Rα axis as a promising therapeutic approach for TNBC.