Functional CRISPR screens reveal TPL1 lncRNA as a regulator of triple-negative breast cancer hallmarks

Despite the rapidly expanding catalog of long noncoding RNAs (lncRNAs), the functional roles of most remain poorly characterized in cancer. In triple-negative breast cancer (TNBC), an aggressive subtype with limited targeted treatment options, defining lncRNA-associated molecular mechanisms could uncover novel therapeutic vulnerabilities.

This study aimed to systematically identify TNBC-enriched lncRNAs associated with cellular fitness and drug response, and to functionally characterize prioritized lncRNA candidates with potential therapeutic relevance.

We generated comprehensive lncRNA annotation by integrating GENCODE, BIGTranscriptome, and MiTranscriptome databases. A CRISPR-Cas9 deletion screen targeting 1,029 TNBC-enriched lncRNAs was conducted. Functional validation included proliferation assays in two-dimensional (2D) and three-dimensional (3D) cultures, invasion assays using an organ-on-chip model, and transcriptomic and proteomic profiling following lncRNA knockdown. Candidate lncRNA-protein associations were assessed using proteomic array analysis. miRNA expression profiling, miRanda-based interaction prediction, and Ingenuity Pathway Analysis (IPA) were used to construct candidate competing endogenous RNA (ceRNA)-like regulatory networks and downstream signaling pathways.

Our CRISPR screen identified fourteen recurrent candidate lncRNA dependencies across both TNBC models, with TNBC Promoting LncRNA 1 (TPL1) emerging among the top candidates. ASO-mediated TPL1 suppression significantly reduced TNBC cell proliferation, clonogenic growth, 3D growth, and invasive capacity. TPL1 was significantly overexpressed in TNBC tissues, particularly within the basal-like immune-suppressed (BLIS) subtype. RNA-FISH analysis showed nuclear/perinuclear and cytoplasmic TPL1 localization. Transcriptomic and proteomic analyses revealed suppression of pathways related to extracellular matrix-receptor interaction, focal adhesion, cell migration, and PI3K-Akt signaling following TPL1 knockdown. Proteomic array, RBPsuite, miRNA profiling, and transcriptomic integration supported candidate protein-associated and ceRNA-like regulatory mechanisms involving TPL1.

This study identifies TPL1 as a functionally relevant lncRNA associated with TNBC growth, invasion, and molecular regulatory programs. These findings support TPL1 as a candidate RNA-targetable vulnerability in TNBC and provide a framework for further mechanistic and translational investigation.