Triple-negative breast cancer (TNBC) is a highly aggressive form of breast cancer, marked by frequent metastasis and limited targeted therapies. The mechanisms by which epigenomic regulation influences matrix metalloprotease (MMP) activity—and thereby tumor invasiveness—remain incompletely understood. We integrated transcriptomic and chromatin conformation datasets to identify candidate insulator elements that might coordinate MMP gene expression and invasive potential. Using CRISPR/Cas9, we specifically disrupted a CTCF binding site within an insulator element downstream of MMP8 (IE8) in two TNBC cell models. The resulting cells were profiled through Hi-C, ATAC-seq, and RNA-seq, alongside functional assays to assess invasion. We also evaluated the prognostic relevance of these regulatory elements in clinical ductal carcinoma in situ (DCIS) samples. We characterized a functional insulator at the Chr11q22.2 locus (IE8) that establishes a TAD boundary, partitioning nine MMP genes into two inversely regulated clusters. This MMP expression architecture was linked to shorter relapse-free (HR = 1.57 [1.06–2.33]; p = 0.023) and overall survival (HR = 2.65 [1.31–5.37]; p = 0.005) in TNBC patients. CRISPR-mediated IE8 disruption reshaped the MMP expression landscape, suppressing the pro-invasive MMP1 while inducing the anti-tumorigenic MMP8, leading to reduced invasion and collagen degradation. Furthermore, this MMP signature predicted DCIS progression to invasive carcinoma (AUC = 0.77, p < 0.01). Our study reveals that a single insulator element near MMP8 orchestrates the regional regulation of MMP genes with opposing roles, directly impacting the invasive behavior of aggressive breast cancers.
