Hypermethylation-mediated silencing of RASD1 drives multiple myeloma pathogenesis

TL;DR

This study finds that RASD1 is silenced in multiple myeloma due to hypermethylation, linking this to worse outcomes and suggesting RASD1 acts as a tumor suppressor.

Contribution

The novel contribution is identifying RASD1 as a tumor suppressor silenced by hypermethylation in multiple myeloma.

Findings

RASD1 mRNA and protein are significantly downregulated in multiple myeloma patients.

RASD1 promoter hypermethylation is associated with adverse clinical features in MM.

Demethylating agents restore RASD1 expression and increase apoptosis in MM cells.

Abstract

The role of Ras-related dexamethasone-induced 1 (RASD1) in multiple myeloma (MM) pathogenesis remains unclear. This study investigated the expression profile, clinical significance, and epigenetic regulation of RASD1 in MM.

Bone marrow samples were collected from 26 newly diagnosed patients with MM and 8 healthy controls. RASD1 messenger RNA (mRNA) and protein expression were analyzed using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry, respectively. DNA methylation status was assessed via methylation-specific PCR (MSP). The U266 MM cell line was treated with the demethylating agent decitabine (DAC) to evaluate its effects on RASD1 expression and apoptosis.

RASD1 mRNA and protein expression were significantly downregulated in patients with MM compared to healthy controls (P < 0.001). Low RASD1 mRNA levels correlated significantly with advanced DS stage, anemia, hypercalcemia, and elevated M-protein concentrations (P < 0.05). The receiver operating characteristic curve indicated that RASD1 mRNA expression was a robust discriminator between patients with MM and healthy individuals (area under the curve = 0.882, sensitivity = 100%, specificity = 75%). MSP analysis revealed RASD1 promoter hypermethylation in patients with MM, whereas controls exhibited hypomethylation. Treatment of U266 cells with DAC restored RASD1 expression and significantly increased apoptosis compared with controls (12.08% vs. 5.04%, P < 0.01).

RASD1 is frequently silenced in MM through promoter hypermethylation. This epigenetic inactivation is associated with adverse clinical features and enhanced cell survival, supporting a tumor suppressor role for RASD1 in MM pathogenesis.

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Introduction

Multiple myeloma (MM) is a malignant neoplasm of clonal plasma cells and represents the second most common hematological malignancy [1]. Despite therapeutic advances, including the introduction of novel agents and stem cell transplantation, MM remains largely incurable due to the inevitable development of relapsed or refractory disease [2]. A deeper understanding of its molecular pathogenesis is crucial for identifying new therapeutic targets.

Ras-related dexamethasone-induced 1 (RASD1) is a member of the Ras superfamily of small G-proteins. Unlike its well-characterized oncogenic counterparts (e.g., K-Ras, H-Ras), RASD1 exhibits context-dependent roles in cancer and can function as a tumor suppressor, exerting growth-inhibitory effects in lung adenocarcinoma and breast cancer cells [3, 4]. Conversely, it may promote progression in malignancies such as osteosarcoma [5]. Evidence regarding the role of RASD1 in hematological cancers is limited and contradictory. Some studies suggest that RASD1 suppresses B-cell proliferation [6], whereas others indicate that it promotes growth in B-cell acute lymphoblastic leukemia [7].

Epigenetic silencing via promoter hypermethylation represents a key mechanism for inactivating tumor suppressor genes. In MM, Nojima et al. reported that RASD1 hypermethylation was associated with dexamethasone resistance [8]. However, the clinical relevance of RASD1 expression and methylation in a well-defined cohort of patients with MM remains underexplored.

This study aimed to comprehensively evaluate RASD1 in MM. The mRNA and protein expression were analyzed in patient samples and correlated with clinical parameters, while the role of DNA methylation in regulating RASD1 expression was investigated. Furthermore, the functional consequences of demethylation on RASD1 expression and cell survival were examined in vitro.

Materials and methods

Patient samples and cell culture

Bone marrow aspirates and biopsy tissues were obtained from 26 newly diagnosed patients with MM and 8 healthy controls at the Affiliated Hospital of Zunyi Medical University between June 2019 and October 2020, after obtaining written informed consent. The study was approved by the Ethics Committee of the Affiliated Hospital of Zunyi Medical University (KLLY-2019-209), and was conducted in accordance with the Declaration of Helsinki. All patients with MM were diagnosed according to the International Myeloma Working Group criteria [9]. Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll density gradient centrifugation, and CD138^+^ plasma cells were subsequently purified from patient PBMCs using magnetic-activated cell sorting. The human MM cell line U266 was obtained from the American Type Culture Collection, and was maintained in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum.

RNA extraction and quantitative real-time PCR (RT-qPCR)

Total RNA was extracted from PBMCs, CD138^+^ cells, and U266 cells using TRIzol reagent, following the manufacturer's protocol. Complementary DNA (cDNA) was synthesized using a reverse transcription kit. RT-qPCR was performed with SYBR Green I Master Mix on a Cobas z480 system. GAPDH served as the endogenous control. The primer sequences for RASD1 were: Forward, 5′-CCA CCG CAA GTT CTA CTC CAT-3′; Reverse, 5′-CCA GGA TGA AAA CGT CTC CTG T-3′. Relative gene expression was calculated using the 2^(-ΔΔCt) method [10].

DNA extraction and methylation-specific PCR (MSP)

Genomic DNA was extracted using a commercial kit, and bisulfite conversion was performed with the EZ DNA Methylation-Gold™ Kit. MSP was conducted using primers specific for methylated (M) and unmethylated (U) alleles of the RASD1 promoter, as previously described, with modifications [8].

• Methylated Forward: 5′-TTTCGTAGTAGCGTGGATC-3′
• Methylated Reverse: 5′-GTAATCCGAACTCGAACTTT-3′
• Unmethylated Forward: 5′-AGTTTTTGTAGTAGTGTGGATT-3′
• Unmethylated Reverse: 5′-CTCATAATCCAAATCAAACTTT-3′

Immunohistochemistry (IHC)

Formalin-fixed, paraffin-embedded bone marrow biopsy sections were stained with an anti-RASD1 antibody, following previously described protocols [11]. Staining was evaluated using an immunoreactivity score (IRS), calculated by multiplying the score for the percentage of positive cells (0 = 0%, 1 = 1–25%, 2 = 26–50%, 3 = 51–75%, 4 = 76–100%) by the staining intensity score (0 = negative, 1 = weak, 2 = moderate, 3 = strong). An IRS > 4 was defined as high expression.

Scoring was performed independently by two board-certified pathologists who were blinded to the sample groups (patients with MM vs. healthy controls). Discrepancies, observed in ≤ 10% of cases, were resolved by consensus.

Cell treatment and apoptosis assay

U266 cells were treated with 50 μM decitabine (DAC) for 48 h. Apoptosis was measured by flow cytometry using Annexin V-YF647A/PI staining, according to the manufacturer's instructions [12].

Statistical analysis

Data were analyzed using SPSS version 18.0. Group comparisons were performed using Student's t-test or the chi-square test. Receiver operating characteristic curve analysis was performed to assess the diagnostic value of RASD1 mRNA expression. A P value < 0.05 was considered statistically significant.

Results

RASD1 expression is downregulated in MM

RASD1 mRNA expression was significantly reduced in bone marrow samples from patients with MM (0.42 ± 0.31) compared to healthy controls (1.28 ± 0.67, P < 0.001, Fig. 1a). Immunohistochemistry was employed to assess RASD1 protein expression. The results revealed high RASD1 expression in 7 out of 8 (87.5%) healthy controls, with only one case showing low expression. In contrast, high expression was observed in only 6 out of 26 (23.1%) patients with MM, while the remaining 20 cases exhibited low expression. Statistical analysis demonstrated that RASD1 protein expression was significantly reduced in patients with MM compared to healthy controls (P < 0.001, Fig. 1b, c).Fig. 1a Relative RASD1 mRNA levels (mean ± SD) in patients with MM versus healthy controls (HC), normalized to GAPDH (***P < 0.001). b Quantification of RASD1 protein expression (IRS scores, mean ± SD) in patients with MM versus HC (***P < 0.001). c Representative IHC images (400× magnification); Panels A and B: high RASD1 expression in HC bone marrow biopsies; Panels C and D: low RASD1 expression in bone marrow biopsies from patients with MM

Clinical correlations of RASD1 expression

Reduced RASD1 mRNA expression was significantly associated with advanced DS stage (P = 0.01) [13], anemia (P = 0.04), hypercalcemia (P = 0.03), and elevated M-protein levels (P = 0.04, Table 1). Similarly, low RASD1 protein expression was correlated with hypercalcemia (P = 0.04) and hyperglobulinemia (P = 0.04). Table 1. Correlation between RASD1 mRNA Expression and Clinical Characteristics in Patients with MMClinical CharacteristicTotal (n)RASD1 Expressionχ ^2^PHighLowGender Male166100.0160.9 Female1046Age (years) < 60185132.8210.09 ≥ 60853DS Stage Stage I & II10375.9870.01 Stage III1679ISS Stage Stage I & II10282.341.13 Stage III1688Anemia Yes209113.8690.04 No615White Blood Cell Normal249150.1220.72 Abnormal211Platelet Normal15690.0350.85 Abnormal1147Serum Corrected Calcium (mmol/L) ≤ 2.75191454.3980.03 > 2.75725Serum Phosphorus Normal13670.650.42 Abnormal1349M-protein (g/L) < 309273.3130.04 ≥ 3017710

RASD1 is silenced by promoter hypermethylation

MSP analysis revealed a high frequency of RASD1 promoter methylation in patients with MM, whereas samples from healthy controls were predominantly unmethylated (Fig. 2a). To functionally validate this mechanism, U266 cells were treated with DAC. This treatment significantly restored RASD1 mRNA expression (11.18 ± 1.98 vs. 1.04 ± 0.06; P < 0.001; Fig. 2b) and markedly increased apoptosis (12.08% vs. 5.04%, P < 0.01; Fig. 2c, d) compared to controls.Fig. 2a MSP gel electrophoresis showing methylation (M) and unmethylation (U) status in representative samples. b RASD1 mRNA expression in U266 cells following DAC treatment. c Quantification of apoptosis rates (mean ± SD) in U266 cells (**P < 0.01). d Representative flow cytometry plots of apoptosis (Annexin V-YF647A/PI staining); Total apoptosis: 5.04% in the control group and 12.08% in the DAC-treated group

Discussion

Multiple myeloma (MM) remains an incurable plasma cell malignancy despite advances in novel therapeutic agents [1, 2]. The emergence of drug resistance and clonal evolution continues to pose major clinical challenges, highlighting the need for new molecular targets and biomarkers [13]. The present study provides compelling evidence that RASD1 acts as a tumor suppressor gene epigenetically silenced through promoter hypermethylation in MM.

Epigenetic alterations, particularly DNA methylation, have emerged as critical drivers of MM pathogenesis and treatment resistance [14]. Recent studies have demonstrated that aberrant DNA methylation patterns not only silence tumor suppressor genes but also reshape the bone marrow microenvironment [15]. In this context, investigation of RASD1 is particularly relevant because it is located on chromosome 17p11.2, a region frequently altered in hematological malignancies [16].

Significant downregulation of RASD1 expression was observed at both transcriptional and translational levels in patients with MM compared to healthy controls. This finding aligns with recent reports identifying RASD1 as a potential tumor suppressor in other malignancies. For instance, a 2024 study by Ren M. et al. demonstrated that RASD1 restoration inhibits tumor growth in solid tumors through modulation of the MAPK pathway [17].

Mechanistically, DNA hypermethylation was identified as a direct regulator of RASD1 silencing in MM. Treatment with DAC reversed RASD1 expression and induced apoptosis, while MSP analysis confirmed promoter hypermethylation in primary patient samples. These findings are consistent with the current understanding of epigenetic regulation in MM, wherein DNA methyltransferase inhibitors have shown promise in reversing aberrant methylation patterns [18]. Notably, MSP is a qualitative assay; therefore, future studies using quantitative methylation techniques would further delineate the methylation burden of the RASD1 promoter in MM.

Clinically, low RASD1 expression was significantly correlated with established poor prognostic indicators, including advanced DS stage, anemia, and elevated M-protein levels [19, 20]. This clinical relevance is particularly important in the context of recent efforts to develop molecular biomarkers for risk stratification in MM [21]. The association with aggressive disease features suggests that RASD1 methylation status may serve as a valuable biomarker for identifying high-risk patients.

This study has several limitations. First, the sample size was small (26 patients with MM and 8 healthy controls), which limits the generalizability of the conclusions. Second, functional experiments relied on a single MM cell line (U266), and the DAC-induced increase in apoptosis was modest. Third, MSP is a qualitative assay that does not quantify methylation burden. Finally, external validation using publicly available datasets was not conducted. These limitations highlight the need for future studies incorporating larger sample sizes, additional cell lines, quantitative methylation assays, and independent validation to confirm the present findings.

Despite these limitations, the findings provide a foundation for future research. Recent advances in single-cell epigenomics and multiomics approaches present important opportunities to further elucidate the role of RASD1 in MM pathogenesis [22]. Additionally, combining epigenetic therapies with existing MM treatments represents a promising therapeutic avenue that merits further investigation [23].