A LINE-1 retrotransposon promotes SEMA3C expression as a cis-regulatory enhancer to sustain breast cancer stem cell survival.

Transposable elements (TEs), which are under tight epigenetic control, have been co-opted as cis-regulatory elements to regulate gene expression during development and cancer. Among them, long interspersed element 1 (LINE-1) retrotransposons are the most abundant and exhibit high activity in embryonic stem cells. However, the precise role of LINE-1 in breast cancer stem cells (BCSCs) remains poorly understood. Here, using RNA sequencing, an enhancer dual-luciferase reporter assay, and a nuclease-dead Cas9 (dCas9)-based CRISPR activation (CRISPRa) assay, we show that the LINE-1 retrotransposon L1Md_T within Sema3c (Sema3c_L1Md_T) is derepressed and functions as a cis-regulatory enhancer that drives SEMA3C expression to sustain mouse BCSC survival. In mouse BCSCs, Sema3c_L1Md_T results in the formation of more phase-separated nuclear condensates with the transcriptional coactivator BRD4 under conditions of increased chromatin accessibility. BRD4 puncta coincide with regions marked by histone H3 lysine 27 acetylation (H3K27ac), enhancing the transcription of SEMA3C. Aberrant SEMA3C expression contributes to mouse BCSC survival and self-renewal via its receptor NRP1 and the coreceptors PlexinA2/PlexinD1. Importantly, we also demonstrate that this regulatory mechanism is conserved in human breast cancer, where SEMA3C is highly expressed in human BCSCs. A human LINE-1 element (SEMA3C_L1ME4a) exhibits enhancer activity and colocalizes with BRD4 condensates in human BCSCs. These findings confirm that the LINE1-BRD4-SEMA3C regulatory axis is present in both mouse and human BCSCs, underscoring its translational relevance. Notably, pharmacological degradation of BRD4 using the proteolysis-targeting chimaera (PROTAC) MZ1 reduces SEMA3C levels and decreases BCSC viability both in vitro and in vivo. Our study reveals an oncogenic role for a LINE-1-derived enhancer in regulating SEMA3C transcription and sustaining BCSC properties, highlighting BRD4 as a therapeutic vulnerability in BCSC-driven breast cancer progression.