Promoter methylation of tumor suppressor genes and microRNAs engaged in TP53 network in acute promyelocytic leukemia

Abstract

Acute promyelocytic leukemia (APL) is one of the subtypes of acute myeloid leukemia carrying t(15;17), and constitutes 10 to 15% of adult AMLs. One of the mechanisms of gene inactivation is hypermethylation of promoter-associated CpG islands. Cancers are characterized by global hypomethylation with locus-specific hypermethylation and hence silencing of tumor suppressor genes. Apart from tumor suppressor genes, microRNA, a class of non-coding RNA measuring 19-25 nucleotides, with tumor suppressive function is also found to be inactivated by DNA methylation in hematological malignancies. microRNAs repress target gene translation and hence expression by binding to 3'-untranslated region of corresponding mRNA. Because TP53 mutation is frequently involved in solid cancer carcinogenesis but is rarely found in APL, TP53 network may be dysregulated through epigenetic inactivation of tumor suppressor gene/miRNAs engaged in TP53 tumor suppressor network.

This thesis aimed to study DNA methylation of tumor suppressor genes and miRNAs engaged in TP53 tumor suppressor network in APL. Overall survival (OS) and event free survival (EFS) of patients with or without candidate gene/miRNA hypermethylation were compared to examine their prognostic significances.

Promoter methylation of DAPK1, p14ARF, miR-34a, miR-34b/c and miR-605 were studied in 10 normal bone marrow samples, NB4 cell line and 60 APL primary samples at diagnosis by methylation-specific PCR (MSP). DAPK1, miR-34a, miR-34b/c and miR-605 were completely unmethylated in normal bone marrow samples but completely methylated in NB4. Treatment of NB4 by 5'-Aza-2'-deoxyctidine (5-azadC) resulted in promoter demethylation together with re-expression of DAPK1, miR-34a, miR-34b/c and miR-605. Promoter methylation of DAPK1, p14ARF, miR-34a were absent while miR-34b/c and miR-605 methylation were detected in 43% and 10% APL samples respectively. However, methylation of miR-34b/c and miR-605 bore no prognostic significance. Overexpression of miR-34b in NB4 resulted in inhibition of proliferation. In short, methylation of DAPK1, miR-34a, miR-34b/c and miR-605 is associated with gene/miRNAs silencing. miR-34b/c is frequently methylated whereas miR-605 is methylated in small number of APL patients. miR-34b/c is a tumor suppressive miRNA in APL. Methylation of miR-34b/c may contribute to APL leukemogenesis.