CONSTRUCTING GENE REGULATORY NETWORKS FOR BREAST CANCER STEM CELLS USING SINGLE-CELL MULTI-OMICS

Abstract

Breast cancer mortality is primarily driven by metastasis and therapeutic relapse; processes strongly linked to breast cancer stem cells (BCSCs). These cells are believed to orchestrate tumor initiation, resistance, and recurrence through complex gene regulatory networks (GRNs) that remain poorly characterized. This study aimed to construct and compare GRNs of BCSCs and normal mammary stem cells (MaSCs) using a single-cell multi-omics framework. Datasets that were publicly available, containing single-cell RNA sequencing (scRNA-seq) and single-cell chromatin accessibility (scATAC-seq) profiles, were sourced from normal breast tissue, primary tumors, recurrent tumors, and BCSC-enriched mammospheres. The datasets were subjected to strict preprocessing that involved filtering, normalization, and quality control. The scGLUE model, which uses a graph neural network to integrate multiple omics including transcriptomic and epigenomic data into a single latent space while conserving biological identity, was used to integrate the datasets. pySCENIC pipeline was then used, which combines co-expression analysis, cis-regulatory motif enrichment, and pruning to reconstruct gene regulatory networks, and high-confidence regulons were generated for BCSCs and MaSCs. Comparative network analysis revealed extensive “regulatory rewiring” in BCSCs, with transcription factors such as JUNB, FOSB, LEF1, SOX4, and MAFB emerging as master regulators absent or significantly altered in normal stem cells. Functional enrichment of BCSC-exclusive targets highlighted pathways central to metastasis and recurrence, including extracellular matrix remodeling, adhesion, migration, and growth factor signaling. Disease ontology mapping further confirmed strong associations with invasive breast carcinoma and therapy resistance. Collectively, this study provides one of the first integrated single-cell GRN maps contrasting BCSCs with their normal counterparts, establishing mechanistic links between regulatory rewiring and cancer hallmarks. The identification of BCSC-specific master regulators offers promising therapeutic entry points for interventions aimed at eradicating the root drivers of breast cancer relapse and metastasis.