Epigenetic modulation of three-dimensional telomeric architecture by hypomethylating agent 5-aza-2'-deoxycytidine in luminal and triplenegative breast cancer cells

Abstract

Introduction: Genomic instability is a hallmark of cancer and is strongly associated with telomeric dysfunction. Telomeres,
structures that protect the ends of chromosomes, when shortened or spatially disorganized, can lead to chromosomal
fusion, deoxyribonucleic acid (DNA) breakage, and aberrant chromosome segregation. Objective: This study evaluated
the impact of the hypomethylating agent 5-aza-2′-deoxycytidine (5-aza-dC) on the three-dimensional reorganization of
telomeres in luminal A (MCF7) and triple-negative (DU4475) breast cancer cell lines, focusing on the relationship between
telomeric architecture and genomic instability. Methods: Cells were treated with 5-aza-dC (10, 20, 30, and 50 μM) for
72 hours. Analysis was performed using Q-FISH and TeloView®, assessing the following parameters: number of telomeres,
telomere aggregates, signal intensity (length), spatial distribution, and nuclear volume. Statistical evaluation was performed using analysis of variance (ANOVA) with Bonferroni post-test (p<0.05). Results: The assays demonstrated that
5-aza-dC induced statistically significant alterations in telomeric parameters, particularly at concentrations of 30 and
50 μM. In MCF7, there was a reduction in the number of telomeres (from 58.4 standard deviation ±6.1 to 42.2±4.8; p<0.001)
and aggregates (from 8.3±1.2 to 4.1±0.9; p<0.0001), indicating reduced genomic instability. In DU4475, nuclear volume
decreased (up to 40%; p<0.0001), and changes in the spatial distribution (p<0.01) suggested nuclear reprogramming and
impact on 3D genome organization. Conclusion: 5-aza-dC promotes 3D telomeric reorganization associated with the
reduction of classical signs of genomic instability. This reinforces the role of telomeres not only as markers of cellular aging
but also as epigenetic sensors of chromosomal stability in breast tumors, including luminal and triple-negative cell lines.