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Updated: May 7, 2026
Comprehensive DNA Methylation Analysis Using a Methyl-CpG-binding Domain Capture-based Method in Chronic Lymphocytic Leukemia Patients
Published on: June 16, 2017
Methylation profiling and evaluation of demethylating therapy in renal cell carcinoma
Christopher J Ricketts1, Mark R Morris1,2, Dean Gentle1
1Centre for Rare Diseases and Personalised Medicine, School of Clinical and Experimental Medicine, College of Medical and Dental Sciences, University of Birmingham, Birmingham B15 2TT, UK.
Background:
Despite therapeutic advances in targeted therapy, metastatic renal cell carcinoma (RCC) remains incurable for the vast majority of patients. Key molecular events in the pathogenesis of RCC include inactivation of the VHL tumour suppressor gene (TSG), inactivation of chromosome 3p TSGs implicated in chromatin modification and remodelling and de novo tumour-specific promoter methylation of renal TSGs. In the light of these observations it can be proposed that, as in some haematological malignancies, demethylating agents such as azacitidine might be beneficial for the treatment of advanced RCC.
Results:
Here we report that the treatment of RCC cell lines with azacitidine suppressed cell proliferation in all 15 lines tested. A marked response to azacitidine therapy (>50% reduction in colony formation assay) was detected in the three cell lines with VHL promoter methylation but some RCC cell lines without VHL TSG methylation also demonstrated a similar response suggesting that multiple methylated TSGs might determine the response to demethylating therapies. To identify novel candidate methylated TSGs implicated in RCC we undertook a combined analysis of copy number and CpG methylation array data. Candidate novel epigenetically inactivated TSGs were further prioritised by expression analysis of RCC cell lines pre and post-azacitidine therapy and comparative expression analysis of tumour/normal pairs. Thus, with subsequent investigation two candidate genes were found to be methylated in more than 25% of our series and in the TCGA methylation dataset for 199 RCC samples: RGS7 (25.6% and 35.2% of tumours respectively) and NEFM in (25.6% and 30.2%). In addition three candidate genes were methylated in >10% of both datasets (TMEM74 (15.4% and 14.6%), GCM2 (41.0% and 14.6%) and AEBP1 (30.8% and 13.1%)). Methylation of GCM2 (P = 0.0324), NEFM (P = 0.0024) and RGS7 (P = 0.0067) was associated with prognosis.
Conclusions:
These findings provide preclinical evidence that treatment with demethylating agents such as azacitidine might be useful for the treatment of advanced RCC and further insights into the role of epigenetic changes in the pathogenesis of RCC.
Insights
Demethylating agent azacitidine suppressed renal cell carcinoma (RCC) cell proliferation. This study identifies novel methylated tumor suppressor genes (TSGs) and provides preclinical evidence for azacitidine in treating advanced RCC.
Area of Science:
- Oncology
- Epigenetics
- Cancer Biology
Background:
- Metastatic renal cell carcinoma (RCC) remains largely incurable despite targeted therapies.
- Key pathogenic events in RCC include VHL tumor suppressor gene (TSG) inactivation, 3p TSG alterations, and promoter methylation of renal TSGs.
- Demethylating agents, like azacitidine, show potential for treating advanced RCC, similar to their use in hematological malignancies.
Purpose of the Study:
- To investigate the efficacy of azacitidine in inhibiting renal cell carcinoma (RCC) cell proliferation.
- To identify novel epigenetically silenced tumor suppressor genes (TSGs) in RCC.
- To explore the prognostic significance of gene methylation in RCC.
Main Methods:
- Treatment of 15 RCC cell lines with azacitidine.
- Colony formation assays to assess cell proliferation.
- Combined analysis of copy number and CpG methylation array data.
- Expression analysis of RCC cell lines pre- and post-azacitidine treatment.
- Comparative analysis of tumor/normal tissue gene expression and methylation data.
Main Results:
- Azacitidine suppressed proliferation in all 15 RCC cell lines tested.
- A significant response was observed in cell lines with VHL promoter methylation, and also in some without, suggesting broader epigenetic targets.
- Identified RGS7, NEFM, TMEM74, GCM2, and AEBP1 as candidate methylated TSGs in RCC.
- Methylation of GCM2, NEFM, and RGS7 was associated with patient prognosis.
Conclusions:
- Azacitidine demonstrates preclinical efficacy against advanced renal cell carcinoma (RCC).
- Epigenetic alterations, including gene promoter methylation, play a significant role in RCC pathogenesis.
- These findings support further investigation of demethylating agents for advanced RCC treatment.
