Systematic functional profiling of long noncoding RNAs identifies therapeutic vulnerabilities in doxorubicin-resistant triple-negative breast cancer

Triple-negative breast cancer (TNBC) is a highly aggressive and clinically challenging subtype of breast cancer, defined by the absence of estrogen receptor (ER), progesterone receptor (PR), and HER2 amplification. This molecular profile limits the use of targeted endocrine or HER2-directed therapies, leaving patients reliant on conventional chemotherapeutics such as doxorubicin. However, the frequent development of chemoresistance underscores the urgent need for new therapeutic strategies. Long noncoding RNAs (lncRNAs), a class of regulatory transcripts with emerging roles in cancer biology, remain largely unexplored in the context of TNBC pathogenesis and drug resistance. This study aimed to systematically identify lncRNAs that drive tumor survival and chemoresistance to uncover novel therapeutic targets and enhance the efficacy of doxorubicin-based treatment. We employed a high-throughput, dual-excision CRISPR-Cas9 screening system targeting the transcription start sites (TSSs) of approximately 1,000 lncRNAs. Functional screens were performed in both 2D monolayer and 3D organoid culture systems, with and without doxorubicin exposure, to closely mimic the tumor microenvironment and treatment stress. Integrative analysis of the screen data identified 22 high-confidence oncogenic lncRNAs essential for TNBC cell survival and resistance to doxorubicin, particularly under 3D conditions that recapitulate in vivo extracellular matrix architecture. Two of these lncRNAs were significantly overexpressed in patient-derived TNBC datasets, highlighting their clinical relevance. Functional validation using antisense oligonucleotides (ASOs) prioritized two top candidates—designated Candidate 100 and Candidate 305—whose knockdown resulted in marked inhibition of TNBC cell growth and viability in both 2D and 3D models. These effects were supported by impaired colony formation, reduced organoid growth, and AO/EtBr live-dead staining assays. This study presents the first comprehensive lncRNA essentiality map in TNBC under 3D culture conditions and reveals the context-dependent roles of lncRNAs in chemoresistance. Our findings establish Candidates 100 and 305 as actionable therapeutic targets, emphasizing the potential of RNA-based precision medicine approaches to overcome doxorubicin resistance in TNBC.