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

Introduction

Breast cancer remains a leading cause of cancer morbidity and mortality worldwide, reflecting marked molecular heterogeneity.¹ In addition to genetic alterations, epigenetic changes such as DNA methylation play a crucial role in breast tumorigenesis and progression and are increasingly leveraged as biomarkers for diagnosis, subtyping, and prognostic stratification.^2,3

Ubiquitin-specific peptidase 44 (USP44) belongs to the deubiquitinating enzyme family involved in the regulation of protein turnover and cell cycle progression.⁴ USP44 has been implicated in chromosome segregation, spindle checkpoint control, and genomic stability.⁴ USP44 plays an important role in maintaining genomic integrity through regulation of the spindle assembly checkpoint by controlling the activity of the anaphase-promoting complex/cyclosome.⁴ Loss of USP44 activity may promote chromosomal instability and aneuploidy, which are key features of breast cancer progression.^4–6 Dysregulated USP44 has been implicated in cancer progression and may modulate cell proliferation, invasion, and apoptosis.^4–6 Various functions and pathways of USP44 on tumorigenesis has been reported in several malignancies, including lung, colorectal, liver, prostate, and esophageal cancer.^5–10 Bioinformatic analyses and experimental studies have previously demonstrated USP44 hypermethylation and functional downregulation in breast cancer, suggesting a tumor-suppressive role.⁵ However, comprehensive clinical evaluations of USP44 expression and methylation in breast cancer patients remain limited. We therefore hypothesized that altered USP44 methylation may contribute to reduced gene expression and may be associated with specific clinicopathological features in breast cancer.

Epigenetic alterations, particularly DNA methylation of CpG islands in gene regulatory regions, often precede transcriptional changes and serve as stable biomarkers detectable in tumor tissues and circulating tumor DNA (ctDNA).^4,11 While promoter hypermethylation typically leads to transcriptional silencing, the relationship between DNA methylation and gene expression is not always linear, particularly in complex tumor microenvironments. To date, only limited data are available regarding the methylation status of the USP44 gene in breast cancer tissues or ctDNA, and its association with clinicopathological features has not been fully elucidated.^5,10,11

Given the emerging interest in therapeutic potential of USP44 for cancer treatment,^4,9 this study investigated the expression and DNA methylation status of the USP44 gene in breast cancer cell lines, breast cancer tissues and ctDNA. Furthermore, we evaluated their associations with clinicopathological characteristics to clarify the potential clinical relevance of USP44 as a biomarker in breast cancer.

Materials and Methods

Patient Samples and Clinicopathological Data

Breast cancer tissue samples were obtained from 33 patients who underwent surgical resection for primary breast cancer at Daegu Catholic University Hospital, Daegu, Republic of Korea from February 2021 to September 2022. Tissue samples of 13 benign breast disease were used as controls. The study was conducted in accordance with the Declaration of Helsinki. All samples were obtained with informed consent, and the study protocol was approved by the Institutional Review Board at the Daegu Catholic University Hospital (CR-20-198). Clinicopathological data, including age, tumor size, histologic grade, lymph node status, hormone receptor status, human epidermal growth factor receptor 2 (HER2) expression, and molecular subtype, were collected from medical records and pathology reports. Molecular subtypes were defined according to immunohistochemical profiles. A subset of patients included in the present study was derived from a previously reported cohort investigating ctDNA methylation markers in breast cancer.¹² While our previous publication focused on ctDNA methylation of USP44 and its clinical relevance, the current analysis represents a distinct investigation focusing specifically on USP44 expression and DNA methylation status and its correlation with clinicopathological parameters.

Cell Lines and Cell Culture

Human breast cancer cell lines representing breast cancer subtypes of luminal A (MCF-7), luminal B (BT-474), HER2-positive (SKBR3) and triple-negative subtype (MDA-MB-231) were used to assess USP44 expression and methylation status in vitro. All cell lines were obtained from the Korean Cell Line Bank (Seoul, Republic of Korea) and cultured under standard conditions according to the supplier’s recommendations.

USP44 Expression Analysis

USP44 expression in breast cancer cell lines was analyzed using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). RNA was extracted from cultured cells using High Pure RNA isolation kit (Roche, Mannheim, Germany) according to the manufacturer’s protocol. RT-PCR was performed using the Roche LightCycler 480 II system (Roche, Mannheim, Germany). For PCR of USP44 transcripts, forward primer was 5’-TTGGCTCGGATGATGAAC-3’ and reverse primer was 5’- AGTGAGTGTGACTGAAGAAT-3’. The level of ALCAM transcripts was analyzed using an ND-2000 spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE, USA). All qRT-PCR experiments were performed in triplicate biological replicates with technical duplicates. Relative USP44 expression levels were normalized to GAPDH as the internal reference gene using the 2^−ΔΔCt method.

USP44 expression in breast cancer tissues was evaluated using immunohistochemistry (IHC). Immunohistochemical staining was independently evaluated by two pathologists blinded to clinicopathological data. Tissue sections were stained with an anti-USP44 antibody (LS-C336416, LSBio), and expression was assessed semi-quantitatively based on staining intensity (0–3) and the proportion of positive tumor cells (0–3). Staining intensity was scored as 0 (negative), 1 (weak), 2 (moderate), or 3 (strong), and the proportion of positive cells was scored as 0 (< 1%), 1 (1–10%), 2 (11–50%), or 3 (51–100%) according to the percentage of stained tumor cells. An immunoreactivity score (IRS) was calculated by multiplying intensity and proportion scores, yielding a total score ranging from 0 to 12. IRS score of 0–6 was categorized as USP44-negative and 7–9 as -positive or according to predefined scoring criteria (Figure 1).

Figure 1 Representative immunohistochemical expression of USP44 in breast cancer tissues. The left panel shows negative USP44 expression, whereas the right panel shows positive USP44 expression in tumor cells.

DNA Methylation Analysis

Genomic DNA was extracted from breast cancer tissues, ctDNA, and cell lines. The methylation status of the USP44 gene in tissue samples was analyzed by pyrosequencing using PyroGold reagent kit and PyroMark ID (Qiagen, Hilden, Germany) following the methods described in our previous study.¹³ Pyrosequencing analysis included evaluation of 4 CpG sites located within the USP44 promoter region. The average methylation percentage across analyzed CpG sites was calculated and used for the statistical analysis. To assess the methylation status of USP44 in ctDNA and cell lines, methylation-specific PCR (MSP), was performed following the methods described in our previous study.¹⁴ MSP products were analyzed by agarose gel electrophoresis and visualized by ethidium bromide staining, and the bands were classified as methylated or unmethylated. Data with the comparative Ct method were used to compare relative methylation values.¹⁴

Statistical Analysis

Associations between USP44 expression or DNA methylation status and clinicopathological variables were analyzed using appropriate statistical tests, including the chi-square test or Fisher’s exact test for categorical variables and t-tests for continuous variables. The association between DNA methylation status and USP44 expression in tissues was analyzed using t-test or the nonparametric Mann–Whitney U-test. Correlation between DNA methylation status and USP44 expression in cell lines was analyzed using correlation analysis. A p-value < 0.05 was considered statistically significant. Statistical analyses were performed using SPSS version 25.0 (IBM Corp., Endicott, NY, USA). Because this was an exploratory observational study with a limited available cohort, formal sample size calculation was not performed. Statistical findings should therefore be interpreted cautiously, particularly for subgroup analyses.

Results

Cell Line Analysis of USP44 Expression and Methylation

In breast cancer cell lines, USP44 expression was markedly higher in MDA-MB-231 cells compared with the other types of breast cancer cell lines (p < 0.001) (Figure 2). MSP analysis demonstrated an unmethylated pattern of the USP44 gene in MDA-MB-231 cells, whereas weak methylation was observed in the other types of breast cancer cell lines, indicating an inverse relationship between methylation and expression in vitro (p < 0.001) (Figure 3).

Figure 2 USP44 expression in breast cancer cell lines. Relative USP44 mRNA expression levels were quantified by qRT-PCR in breast cancer cell lines representing luminal A (MCF-7), luminal B (BT-474), HER2-positive (SKBR3), and triple-negative subtype (MDA-MB-231). Expression levels were normalized to GAPDH as the internal reference gene using the 2−ΔΔCt method. Data are presented as mean ± standard deviation from three independent experiments.

Figure 3 USP44 methylation in breast cancer cell lines. USP44 methylation was analyzed in breast cancer cell lines representing luminal A (MCF-7), luminal B (BT-474), HER2-positive (SKBR3), and triple-negative subtype (MDA-MB-231) by methylation-specific PCR (MSP). MSP products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining, with bands classified as methylated or unmethylated.

Association Between USP44 Expression in Breast Cancer Tissues and Clinicopathological Features of Breast Cancer Patients

USP44 expression was detected in 69.7% of breast cancer tissues and 76.9% of benign breast tissues. There was no significant difference in USP44 expression between malignant and benign tissues (p = 0.729).

In the analysis of the relationship between USP44 expression in breast cancer tissues and clinicopathological characteristics, positive USP44 expression rate was higher in older than 60 years compared to younger than 60 (p = 0.035) (Table 1). Negative USP44 expression was significantly associated with the presence of fibrotic focus (p = 0.017). USP44 expression in breast cancer tissues was not significantly associated with other pathological parameters, including tumor size, histologic grade, lymph node involvement, hormone receptor status, HER2 status, or molecular subtype.

Table 1 Association Between USP44 Expression in Breast Cancer Tissues and Clinicopathological Features of Breast Cancer Patients

DNA Methylation Status of USP44 in Breast Cancer Tissues and ctDNA

The mean methylation frequency of the USP44 gene in breast cancer tissues was 43.7 ± 13.2%. Analysis of ctDNA revealed a positive methylation rate of 65.7% for the USP44 gene. There was no association between USP44 methylation frequency of tissues of breast cancer patients and methylation status in ctDNA of those patients (p = 0.536). No significant correlation was observed between USP44 DNA methylation status and USP44 expression in breast cancer tissues (p = 0.229).

Association Between USP44 Methylation in Breast Cancer Tissues and Clinicopathological Features of Breast Cancer Patients

DNA methylation frequency of the USP44 gene in breast cancer tissues was significantly higher in positive progesterone receptor (PR) expression and positive HER2 overexpression (p = 0.039 and p = 0.012, respectively) (Table 2). In addition, DNA methylation status of the USP44 gene was significantly associated with molecular subtype of breast cancer (p = 0.023). In particular, the frequency of USP44 methylation in basal-like subtype was lower than that of the other types.

Table 2 Association Between USP44 Methylation in Breast Cancer Tissues and Clinicopathological Features of Breast Cancer Patients

Discussion

In this study, we investigated the clinical relevance of USP44 expression and DNA methylation in breast cancer cell lines, breast cancer tissues and ctDNA, and their associations with clinicopathological features. We observed an inverse relationship between USP44 expression and promoter methylation across breast cancer subtypes, which is consistent with previous studies suggesting epigenetic regulation of USP44.^7,9,15 In addition, our results demonstrate that USP44 expression and its DNA methylation patterns are associated with specific clinicopathological features. These findings both extend and refine existing preclinical and bioinformatic evidence implicating USP44 as a potential biomarker in breast cancer.^5,16 Furthermore, while our previous publication focused on ctDNA methylation of USP44 and its clinical relevance for disease monitoring and tumor characteristics,¹² the current study is novel in that it comprehensively evaluates USP44 DNA methylation status and integrates both expression and epigenetic regulation analyses to better elucidate the biological and clinical significance of USP44 in breast cancer.

Our study showed USP44 mRNA expression varied markedly among breast cancer cell lines, with the highest levels in the triple-negative breast cancer cell line, MDA-MB-231 and reduced expression in luminal and HER2-positive models. This pattern was also consistent in breast cancer tissues. Furthermore, USP44 expression inversely correlated with methylation status, suggesting an epigenetic influence on USP44 expression in vitro. Previous work using TCGA datasets and functional assays reported USP44 hypermethylation and downregulation in breast cancer, consistent with a putative tumor-suppressive role.⁵ In this study, demethylation increased USP44 expression and suppressed cell proliferation, migration, and invasion, indicating that epigenetic silencing may contribute to aggressive tumor behavior.⁵ Our in vitro data are in line with this inverse relationship, although the lack of a statistically significant direct correlation between methylation and expression of USP44 in primary tissues suggests additional regulatory mechanisms in vivo.

Despite the lack of a significant difference in overall USP44 expression between malignant and benign tissues in our study, certain clinicopathological variables exhibited significant associations. Specifically, older patients (≥ 60 years) and negative fibrotic focus were associated with positive USP44 expression. These findings suggest the complex interplay between aging and tumor microenvironment on USP44 biology. Given the essential role of USP44 in maintaining chromosomal stability through spindle assembly checkpoint regulation, age-related decline in mitotic fidelity and epigenetic drift may contribute to reduced USP44 activity.^4,17 This provides a biologically plausible link between aging, genomic instability, and increased cancer susceptibility. Experimental studies have demonstrated that loss or dysregulation of USP44 leads to chromosome mis-segregation and aneuploidy, phenotypes that are also hallmarks of cellular aging.^17,18 In parallel, aging-associated epigenetic drift, particularly promoter hypermethylation of tumor suppressor genes, may contribute to reduced USP44 expression in aged tissues. Although direct clinical evidence linking USP44 expression to chronological aging remains limited, these findings suggest that age-related decline in USP44-mediated mitotic surveillance may represent a mechanistic link between aging, genomic instability, and tumorigenesis, including breast cancer.

In the present study, loss of USP44 expression was significantly associated with the presence of fibrotic focus, a histopathologic feature linked to aggressive tumor behavior, hypoxia, and poor prognosis in breast cancer. Fibrotic focus reflects an activated tumor microenvironment characterized by stromal remodeling, increased angiogenic signaling, and focal hypoxia, all of which impose mitotic and replicative stress on tumor cells.^19,20 USP44 is a key regulator of chromosomal stability and its downregulation has been associated with chromosomal instability and tumor progression in multiple cancer types.⁴ Reduced USP44 expression in fibrotic focus–positive tumors may therefore indicate impaired mitotic surveillance in biologically aggressive cancers enriched for hypoxic and fibrotic microenvironments. Although a direct mechanistic link between USP44 and fibrotic focus has not previously been reported, our findings suggest that USP44 loss may contribute to the genomic instability and microenvironmental adaptation underlying fibrotic focus formation, supporting its potential role as a marker of adverse tumor biology.

In this study, USP44 methylation was significantly associated with hormone receptor and HER2 status, as well as molecular subtype of breast cancer. Higher USP44 methylation frequency was observed in PR-positive tumors and in cancers with HER2 overexpression, suggesting that epigenetic silencing of USP44 may preferentially occur in biologically defined subgroups rather than uniformly across breast cancer. Notably, USP44 methylation was significantly less frequent in basal-like tumors compared with other molecular subtypes. In previous studies, basal-like breast cancers are typically characterized by profound genomic instability driven by mechanisms other than epigenetic silencing.^21–23 In this regards, reduced USP44 methylation in this subtype may reflect alternative pathways of gene dysregulation. In contrast, luminal and HER2-enriched tumors may rely more heavily on epigenetic modulation of genes involved in cell-cycle control and chromosomal stability, including USP44.^24,25 The significant associations of tissue methylation with PR and HER2 status further suggest that USP44 epigenetic regulation may be linked to distinct molecular phenotypes, raising the possibility that methylation profiling could complement existing molecular subtyping in therapeutic decision-making. These findings are consistent with emerging evidence that DNA methylation signatures can stratify tumor subtypes and reflect distinct underlying oncogenic pathways.^26–28 Furthermore, our findings align with broader evidence that epigenetic alterations, including DNA methylation of regulatory regions, can precede and modulate transcriptional changes in complex tumor environments. Promoter hypermethylation is typically associated with gene silencing; however, the methylation–expression relationship in heterogeneous tumors is multifactorial and may differ across genomic contexts and cellular subtypes.^27,29,30

Recent work on breast cancer ctDNA markers suggests that while circulating methylation signatures may capture tumor-specific epigenetic alterations, their clinical utility for disease monitoring remains to be fully established.³¹ The limited concordance between methylation findings in tumor tissue and circulating tumor DNA may reflect biological heterogeneity, variable ctDNA shedding, and technical sensitivity differences between tissue-based pyrosequencing and plasma-based methylation-specific PCR.^32,33 Similar discrepancies have been reported in recent studies evaluating circulating epigenetic biomarkers in breast cancer, highlighting the challenges of translating tissue methylation markers directly into liquid biopsy applications. Nonetheless, the detection of USP44 methylation in a substantial proportion of ctDNA samples suggests potential for non-invasive monitoring, albeit requiring larger validation cohorts and optimized assays.

Several limitations should be acknowledged. First, the study cohort was relatively small and derived from a single institution, which may limit statistical power and generalizability. Prospective validation in larger and independent cohorts with extended follow-up is needed to determine whether USP44 status predicts disease recurrence, metastasis, or clinical outcomes. Second, no functional experiments were performed to directly confirm the biological consequences of USP44 methylation in breast cancer cells. Functional studies to elucidate the mechanistic consequences of USP44 methylation and expression in tumorigenesis and treatment response will further refine its clinical utility. Additionally, integration of USP44 biomarker data with genomic and transcriptomic profiling could enhance precision stratification in future studies. Third, an independent validation cohort was not available. Therefore, the present findings should be interpreted as exploratory and hypothesis-generating.

Conclusion

USP44 expression and methylation show distinct clinicopathological associations in breast cancer. These findings provide preliminary evidence that epigenetic regulation of USP44 may contribute to breast cancer biology. However, because of the exploratory nature of this study and limited sample availability, our findings should be interpreted cautiously. Further multicenter validation studies and mechanistic investigations are required before clinical application of USP44 as a biomarker or therapeutic target.