Integrative methylome and transcriptome analyses identify LHX3 as an epigenetically downregulated marker with tumor suppressive properties in breast cancer

Breast cancer (BC) remains a major global health concern, with rising incidence and mortality rates. Early diagnosis of BC plays an important role in its prevention. However, number of clinically validated biomarkers that enable accurate and minimally invasive diagnosis are still insufficient. Epigenetic modifications, particularly hypermethylation-mediated inactivation of numerous tumor suppressor genes occur early in tumorigenesis and are detectable even before the clinical onset, making them promising candidates for early diagnosis. Therefore, this study aimed to identify breast cancer-associated epigenetically regulated genes with potential diagnostic and biological relevance. A set of forty genes that displayed both hypermethylation and downregulation in BC were initially identified through bioinformatics analysis of publicly available datasets. Based on Gene Ontology (GO) and Reactome pathway enrichment analyses, eight genes were subsequently shortlisted for experimental analysis. Among these eight genes, LHX3 was significantly downregulated in both MCF-7 and MDA-MB-231 cells and in all tested BC tissue samples. LHX3 promoter hypermethylation was found in 80% of tumor tissues, compared to 32% in matched normal tissues. Aberrant LHX3 promoter methylation was significantly associated with metastasis status (P = 0.0002), with higher methylation in non-metastatic tumors, tumor size ≤20 mm (P = 0.002), and advanced tumor stage (P = 0.005). Given the complex pattern of these associations, findings were interpreted cautiously. Treatment with 5-aza-2’-deoxycytidine reversed promoter hypermethylation and restored LHX3 expression in MDA-MB-231 and MCF-7 cells. Additionally, ectopic expression of LHX3 not only reduced migration and proliferation but also induced cell death in BC cells. Our study provided the first evidence of epigenetically induced silencing of LHX3 and its anti-tumor role in BC. Furthermore, our findings support LHX3 as a candidate epigenetically regulated biomarker with tumor-suppressive properties in BC; however, independent cohort validation, quantitative methylation assays, and stage/subtype-stratified analyses are required before its diagnostic or prognostic utility can be established.