Clinical Relevance of USP44 Expression and DNA Methylation Status in Breast Cancer Cell Lines, Tumor Tissues, and Circulating Tumor DNA.

Abstract

Purpose: Ubiquitin-specific peptidase 44 (USP44), a deubiquitinating enzyme involved in chromosomal stability and cell cycle control, has been proposed as a potential biomarker and a therapeutic target in various cancers. However, its expression and epigenetic regulation in breast cancer remain understudied. This study investigated USP44 expression and DNA methylation status in breast cancer cell lines, breast cancer tissues and circulating tumor DNA (ctDNA), and their association with clinicopathological features.

Patients and methods: USP44 expression was assessed by quantitative reverse transcription-polymerase chain reaction in cell lines, and by immunohistochemistry in breast tissues including 33 breast cancers and 13 benign breast lesions. DNA methylation status of USP44 was analyzed by pyrosequencing in tissue samples, and by methylation-specific PCR in ctDNA and cell lines. Associations with clinicopathological parameters were statistically evaluated.

Results: USP44 expression varied among breast cancer cell lines, with highest levels in the triple-negative breast cancer MDA-MB-231, corresponding to an unmethylated USP44 promoter. The positive expression rate of USP44 was 69.7% in breast cancer tissues. USP44 expression in breast cancer tissues did not differ significantly with that of benign tissues, but was associated with age ≥60 years and inversely correlated with presence of fibrotic focus. Mean methylation frequency was 43.7 ± 13.2% in breast cancer tissues, and ctDNA methylation was detected in 65.7% of breast cancer patients. Methylation status in ctDNA did not correlate with tissue methylation. Higher tissue USP44 methylation frequency was significantly associated with progesterone receptor positivity, HER2 overexpression and molecular subtype of breast cancer.

Conclusion: USP44 gene expression and DNA methylation status were associated with distinct clinicopathological features in breast cancer. These findings suggest complex epigenetic regulation of USP44 and its biological relevance in breast cancer. Given the limited sample size and exploratory design, further studies in larger independent cohorts are required to validate the potential role of USP44 as an epigenetic biomarker.