CRISPR screens in 3D tumourspheres identified miR-4787-3p as a transcriptional start site miRNA essential for breast tumour-initiating cell growth

Abstract

Breast cancer (BC) is a heterogeneous malignancy with distinct molecular subtypes and clinical characteristics. Tumour-initiating cells (TICs) are a small subset of cancer cells that are responsible for tumour initiation and progression. Our study employed pooled CRISPR screens, integrating 2D and 3D culture models, to identify miRNAs critical in BC tumorsphere formation. These screens combined with RNA-seq experiments allowed us to identify the miRNA signature and their targets that are essential for tumoursphere growth. Amongst them, miR-4787-3p exhibited significant up-regulation in BC, particularly in basal-like BCs, suggesting its association with aggressive disease phenotypes. Surprisingly, despite its location within the 5UTR of a protein coding gene, which define DROSHA-independent transcription start site (TSS)-miRNAs, our findings revealed its dependence on both DROSHA and DICER1 for maturation. Inhibition of miR-4787-3p hindered tumorsphere formation, highlighting its potential as a therapeutic target in BC. Moreover, our study proposes elevated miR-4787-3p expression as a potential prognostic biomarker for adverse outcomes in BC patients. We found that protein-coding genes positively selected in the CRISPR screens were enriched of miR-4787-3p putative targets. Amongst these identified key targets, we selected ARHGAP17, FOXO3A, and PDCD4 because are known tumour suppressors in cancer and experimentally validated the interaction of miR-4787-3p with their 3UTRs. Our work illuminates the molecular mechanisms underpinning miR-4787-3ps oncogenic role in BC. These findings advocate for further clinical investigations targeting miR-4787-3p and underscore its prognostic significance, offering promising avenues for tailored therapeutic interventions and prognostic assessments in BC.