Chromatin insulation orchestrates matrix metalloproteinase gene cluster expression reprogramming in aggressive breast cancer tumors.

BACKGROUND: Triple-negative breast cancer (TNBC) is an aggressive subtype that exhibits a high incidence of distant metastases and lacks targeted therapeutic options. Here we explored how the epigenome contributes to matrix metalloprotease (MMP) dysregulation impacting tumor invasion, which is the first step of the metastatic process. METHODS: We combined RNA expression and chromatin interaction data to identify insulator elements potentially associated with MMP gene expression and invasion. We employed CRISPR/Cas9 to disrupt the CCCTC-Binding Factor (CTCF) binding site on an insulator element downstream of the MMP8 gene (IE8) in two TNBC cellular models. We characterized these models by combining Hi-C, ATAC-seq, and RNA-seq with functional experiments to determine invasive ability. The potential of our findings to predict the progression of ductal carcinoma in situ (DCIS), was tested in data from clinical specimens. RESULTS: We explored the clinical relevance of an insulator element located within the Chr11q22.2 locus, downstream of the MMP8 gene (IE8). This regulatory element resulted in a topologically associating domain (TAD) boundary that isolated nine MMP genes into two anti-correlated expression clusters. This expression pattern was associated with worse relapse-free (HR = 1.57 [1.06 - 2.33]; p = 0.023) and overall (HR = 2.65 [1.31 - 5.37], p = 0.005) survival of TNBC patients. After CRISPR/Cas9-mediated disruption of IE8, cancer cells showed a switch in the MMP expression signature, specifically downregulating the pro-invasive MMP1 gene and upregulating the antitumorigenic MMP8 gene, resulting in reduced invasive ability and collagen degradation. We observed that the MMP expression pattern predicts DCIS that eventually progresses into invasive ductal carcinomas (AUC = 0.77, p < 0.01). CONCLUSION: Our study demonstrates how the activation of an IE near the MMP8 gene determines the regional transcriptional regulation of MMP genes with opposing functional activity, ultimately influencing the invasive properties of aggressive forms of breast cancer.

Epigenetic Profiles of Triple-Negative Breast Cancers of African American and White Females.

Importance: Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype and appears to have disproportionately higher incidence and worse outcomes among younger African American females. Objective: To investigate whether epigenetic differences exist in TNBCs of younger African American females that may explain clinical disparities seen in this patient group. Design, Setting, and Participants: This cross-sectional study used clinical, demographic, DNA methylation (HumanMethylation450; Illumina), and gene expression (RNA sequencing) data for US patient populations from publicly available data repositories (The Cancer Genome Atlas [TCGA], 2006-2012, and Gene Expression Omnibus [GEO], 2004-2013) accessed on April 13, 2021. White and African American females with TNBC identified in TCGA (69 patients) and a validation cohort of 210 African American patients from GEO (GSE142102) were included. Patients without available race or age data were excluded. Data were analyzed from September 2022 through April 2023. Main Outcomes and Measures: DNA methylation and gene expression profiles of TNBC tumors by race (self-reported) and age were assessed. Age was considered a dichotomous variable using age 50 years as the cutoff (younger [<50 years] vs older [≥50 years]). Results: A total of 69 female patients (34 African American [49.3%] and 35 White [50.7%]; mean [SD; range] age, 55.7 [11.6; 29-82] years) with TNBC were included in the DNA methylation analysis; these patients and 210 patients in the validation cohort were included in the gene expression analysis (279 patients). There were 1115 differentially methylated sites among younger African American females. The DNA methylation landscape on TNBC tumors in this population had increased odds of enrichment of hormone (odds ratio [OR], 1.82; 95% CI, 1.21 to 2.67; P = .003), muscle (OR, 1.85; 95% CI, 1.44 to 2.36; P < .001), and proliferation (OR, 3.14; 95% CI, 2.71 to 3.64; P < .001) pathways vs other groups (older African American females and all White females). Alterations in regulators of these molecular features in TNBCs of younger African American females were identified involving hormone modulation (downregulation of androgen receptor: fold change [FC] = -2.93; 95% CI, -4.76 to -2.11; P < .001) and upregulation of estrogen-related receptor α (FC = 0.86; 95% CI, 0.34 to 1.38; P = .002), muscle metabolism (upregulation of FOXC1: FC = 1.33; 95% CI, 0.62 to 2.03; P < .001), and proliferation mediators (upregulation of NOTCH1: FC = 0.71; 95% CI, 0.23 to 1.19; P = .004 and MYC (FC = 0.81; 95% CI, 0.18 to 1.45; P = .01). Conclusions and Relevance: These findings suggest that TNBC of younger African American females may represent a distinct epigenetic entity and offer novel insight into molecular alterations associated with TNBCs of this population. Understanding these epigenetic differences may lead to the development of more effective therapies for younger African American females, who have the highest incidence and worst outcomes from TNBC of any patient group.