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Potential Loss of Imprinting of Tumor Suppressor Gene RB1 in Triple Negative Breast Cancer
Guojing Xie1, Guangjie Zhang2, Junhao Cui1
1College of Medical Technology, Chongqing Key Laboratory of Sichuan-Chongqing Co-Construction for Diagnosis and Treatment of Infectious Diseases Integrated Traditional Chinese and Western Medicine, Sichuan Key Laboratory of Medical Molecular Testing, Chengdu University of Traditional Chinese Medicine, Chengdu, China.
Objectives:
Triple negative breast cancer (TNBC) is an aggressive subtype with limitations in therapy. Although cyclin dependent kinase inhibitors (CDKi) have been proven in breast cancer, challenges remain in TNBC. Successful inhibition of CDK4/6 relies on intact Rb tumor suppressor (encoded by tumor suppressor gene RB1). However, in addition to gene mutation or deletion, RB1, as an imprinted gene, also has a mechanism of inactivation due to loss of imprinting (LOI). This study aimed to ascertain the imprinting status of RB1 in TNBC.
Methods:
We applied bioinformatic analyses to evaluate methylation differences on the RB1 imprinting control region CpG85 among subtypes of breast cancer. Deregulation of RB1 expression by LOI in TNBC cell lines was further tested by RT-qPCR with stimulation by 5-aza-2-deoxycytidine (DAC) treatment. In addition, RB1 CpG85 methylation levels of circulating cell-free DNA (cfDNA) in plasma was assessed by pyrosequencing in 15 enrolled TNBC patients and 6 non-cancer donors. Survival analysis based on TCGA and GEO databases were performed to explored the potential clinical significance.
Results:
Bioinformatic analysis showed hypomethylation at CpG85 of RB1 in TNBC, which was further confirmed in cell lines. LOI of RB1 affected its transcription. Analysis of cfDNA showed CpG85 was differentially methylated in TNBC patients, with 6/15 patients displaying hypomethylation at cg18481241 and 1/15 at cg03085377 within CpG85. Patients with hypomethylation of these sites correlated with worse overall survival.
Conclusions:
RB1 exhibits potential LOI in TNBCs, laying the groundwork for more precise subtyping and treatment of TNBC patients.
Insights
Triple negative breast cancer (TNBC) shows potential loss of imprinting (LOI) in the RB1 gene, indicated by hypomethylation. This epigenetic alteration in RB1 is linked to poorer survival outcomes in TNBC patients.
Area of Science:
- Oncology
- Genetics
- Epigenetics
Background:
- Triple negative breast cancer (TNBC) is an aggressive subtype with limited therapeutic options.
- Successful therapy with cyclin dependent kinase inhibitors (CDKi) requires intact Rb tumor suppressor (RB1).
- RB1 inactivation can occur through gene mutation, deletion, or loss of imprinting (LOI).
Purpose of the Study:
- To investigate the imprinting status of the RB1 gene in TNBC.
- To evaluate RB1 methylation and its association with gene expression in TNBC.
- To explore the clinical significance of RB1 imprinting in TNBC patients.
Main Methods:
- Bioinformatic analysis of RB1 imprinting control region (CpG85) methylation across breast cancer subtypes.
- RT-qPCR to assess RB1 expression in TNBC cell lines following 5-aza-2-deoxycytidine (DAC) treatment.
- Pyrosequencing of plasma cell-free DNA (cfDNA) for RB1 CpG85 methylation in TNBC patients and controls.
- Survival analysis using TCGA and GEO databases.
Main Results:
- Bioinformatic analysis revealed hypomethylation at RB1 CpG85 in TNBC, confirmed in cell lines.
- Loss of imprinting (LOI) was found to affect RB1 transcription.
- Differential methylation of RB1 CpG85 was observed in TNBC patient cfDNA.
- Hypomethylation at specific CpG85 sites (cg18481241, cg03085377) correlated with worse overall survival.
Conclusions:
- RB1 exhibits potential loss of imprinting (LOI) in triple negative breast cancer (TNBC).
- Epigenetic alterations in RB1 may contribute to TNBC pathogenesis.
- These findings support more precise subtyping and targeted treatment strategies for TNBC patients.
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