An essential role for DNA methyltransferase 3a in melanoma tumorigenesis

Abstract

Abnormal DNA methylation and associated silencing of tumor suppressor genes are common to many types of cancers. Among the three coordinate DNA methyltransferases (Dnmts), Dnmt1 and Dnmt3b were both shown to be important for cancer cell survival and tumorigenesis. However, the relationship between Dnmt3a and tumorigenesis is still largely unknown. Here, we show that inhibition of Dnmt3a expression, by stable transfection of a Dnmt3a-RNA interference (RNAi) construct dramatically inhibited melanoma growth and metastasis in mouse melanoma models. Microarray analysis revealed that genes critical for the tumor immune response, were implicated in the inhibition of melanoma growth. Expression of a cluster of class I and class II MHC genes, class II transactivator (Ciita), as well as a subset of 5 chemokines (Cxcl9, Cxcl16, Ccl12, Ccl4, and Ccl2) were up-regulated. Furthermore, we determined that the promoter IV of Ciita was significantly demethylated in Dnmt3a-depleted tumors. In addition, several known tumor-related genes, which are critical for developmental processes and cell cycle, were confirmed to be misregulated, including TgfB1, Socs1, Socs2, E2F6, Ccne1, and Cyr61. The results presented in this report strongly suggest that Dnmt3a plays an essential role in melanoma tumorigenesis, and that the underlying mechanisms include the modulation of the tumor immune response, as well as other processes.

Introduction

Melanoma, as the most lethal form of skin cancer, is responsible for 80 percent of deaths from skin cancers; only 14 percent of patients with metastatic melanoma survive for five or more years [1].

Abnormal DNA methylation and associated silencing of tumor suppressor genes are common to many types of cancers, including malignant melanoma [2], [3]. Tumor-specific hypermethylation can be a potential second hit [4], by preferentially targeting the wild-type allele of tumor suppressor genes, when the second allele is already mutated. Previous studies have revealed dozens of tumor-related genes, which are inactivated by DNA methylation in melanoma [5], including Rassf1a[6], [7], Rarb[5], [6], Mgmt[8], Cdkn1b[9], Cdkn2a[10], and Pten[11].

DNA methylation patterns are established and maintained by the coordinated action of three Dnmts, Dnmt1, Dnmt3a, and Dnmt3b. Dnmt1 functions exclusively as a maintenance Dnmt [12], [13]. In contrast, Dnmt3a and Dnmt3b are de novo Dnmts [14]. Previous studies have shown that both Dnmt1 and Dnmt3b play important roles in cancer cell survival and tumorigenesis [15], [16]. Overexpression of Dnmt1 and Dnmt3b has been reported in various cancer cells and tissues [17], [18]. Inhibition of the expression of Dnmt1 or Dnmt3b has been reported to significantly inhibit the proliferation of cancer cells and tumorigenesis [19], [20], [21]. However, the relationship between Dnmt3a and tumorigenesis is still largely unknown.

In the current study, Dnmt3a was specifically down-regulated in a mouse B16 melanoma cell line by stable transfection of a Dnmt3a-RNAi construct. Tumor growth and metastasis were evaluated in mice receiving B16 cell transplantation. Changes in gene expression in tumors were examined using microarray analysis and real-time RT-PCR. Additionally, promoter methylation was determined by bisulfite sequencing.