Deregulation of p53 and RB Transcriptional Control Leads to Overexpression of DNA Methyltransferases in Lung Cancer

Abstract

BackgroundOverexpression of DNA 5’-cytosine-methyltransferases (DNMTs), which silence genes including tumor suppressor genes, is involved in many cancers. However, the mechanism of DNMT overexpression remains mostly unclear. ObjectivesThe tumor suppressor p53 and RB proteins, which repress gene transcription, are often inactivated by gene mutation or overexpression of the E3 ubiquitin ligase MDM2 in cancer cells. Therefore, this study aims to examine whether the transcriptional regulation of DNMT1, 3A and 3B genes is controlled by p53/RB/MDM2 pathway in lung cancer using cell and clinical studies. MethodsThe interaction and regulation between p53 or RB with DNMT1, 3A and 3B promoters were detected by promoter activity assay and chromatin immunoprecipitation. Transcriptional repression of DNMT1, 3A and 3B by p53 and RB was confirmed using siRNA-mediated knockdown or overexpression of p53/RB. Immunohistochemistry for p53, RB, MDM2, and DNMTs protein expression and DNA sequencing for p53 mutation were performed on tumor tissues from lung cancer patients. Resultsp53 and RB suppressed the promoter activity and mRNA/protein expression of DNMT1, 3A and 3B through binding to their promoter. The suppression could be attenuated by knocking down of RB and p53. Importantly, co-transfection of both p53 and RB expression constructs showed additional inhibition of DNMT protein expression. In clinical study, functional RB and p53 inversely correlated with DNMT1, 3A and 3B expressions in samples from lung cancer patients (P=0.016~0.024). Lung cancer patients with low RB expression or p53 mutation resulting in DNMTs overexpression showed promoter hypermethylation in multiple tumor suppressor genes. Patients with normal expression of DNMT3A, RB and MDM2 was associated with better prognosis compared to other patients (P=0.049). ConclusionsThis study provides cell and clinical evidence that p53 and RB pathways transcriptionally repress DNMT expression. Normal expression of DNMT3A, RB and MDM2 proteins can be a biomarker for good prognosis in lung cancer.