Autophagy-associated exosomal microRNAs in triple-negative breast cancer: a discovery-phase study

Abstract

Triple-negative breast cancer (TNBC) is characterised by aggressive pathobiology and frequent chemoresistance, yet the mechanisms underpinning treatment failure remain incompletely understood. Chemotherapy-induced autophagy has been implicated in small extracellular vesicle (sEV) biogenesis and cargo remodelling; whether the exosomal microRNA (exomiR) landscape of TNBC is altered under autophagy-inducing chemotherapy has not been established. sEVs were isolated from MDA-MB-231 and MDA-MB-468 cells under doxorubicin-induced autophagy-inducing conditions (10 nM, 48 h) and from matched vehicle controls, and were confirmed by CD81 and CD63 immunoblotting with Calnexin as a negative purity control. Autophagy induction was confirmed by Beclin-1 upregulation, p62/SQSTM1 turnover and LC3 puncta formation. Small RNA sequencing (12 libraries; n = 3 biological replicates per condition; DESeq2, nominal p < 0.01 with |log₂FC| > 1) identified 10 differentially expressed exomiRs in MDA-MB-231 and 7 in MDA-MB-468, of which three per cell line additionally satisfied a Benjamini-Hochberg false discovery rate threshold (adj. p < 0.05). In-silico target analysis of these 17 exomiRs identified eight convergent autophagy and apoptosis pathway nodes, most prominently BECN1 (targeted by 6 of the 17). Exploratory qRT-PCR confirmed increased hsa-miR-1468-5p in MDA-MB-231 (23.97-fold; adjusted p = 0.007) but did not confirm the remaining five candidates, one of which changed significantly in the opposite direction. Doxorubicin treatment under autophagy-inducing conditions is associated with cell-line-specific remodelling of the sEV miRNA cargo of TNBC cells. The present data do not provide direct evidence that autophagy causes these changes; establishing causality will require pharmacological or genetic disruption of the autophagy pathway. These discovery-phase findings identify hsa-miR-1468-5p as the priority candidate for prospective functional and clinical validation, and indicate that the remaining candidates require methodological refinement before their biological relevance can be assessed. Triple-negative breast cancer (TNBC) cells, defined by the absence of oestrogen receptor (ER), progesterone receptor (PR), and HER2/neu expression, undergo autophagy induction in response to cytotoxic chemotherapy. Both TNBC cells and autophagy-induced TNBC cells release small extracellular vesicles carrying microRNA cargo (exomiRs), which were isolated and profiled by small RNA sequencing (RNA-seq) in this study. Differential exomiR expression analysis identified candidate species potentially linked to autophagy regulation in cancer cells and to tumour survival and disease progression. Question marks denote the mechanistic and translational relationships interrogated by the present work and identified as priorities for future functional and clinical validation. Schematic created in BioRender.com. Triple-negative breast cancer (TNBC) cells, defined by the absence of oestrogen receptor (ER), progesterone receptor (PR), and HER2/neu expression, undergo autophagy induction in response to cytotoxic chemotherapy. Both TNBC cells and autophagy-induced TNBC cells release small extracellular vesicles carrying microRNA cargo (exomiRs), which were isolated and profiled by small RNA sequencing (RNA-seq) in this study. Differential exomiR expression analysis identified candidate species potentially linked to autophagy regulation in cancer cells and to tumour survival and disease progression. Question marks denote the mechanistic and translational relationships interrogated by the present work and identified as priorities for future functional and clinical validation. Schematic created in BioRender.com.