Prevalence of aberrant methylation of p14ARF over p16INK4a in some human primary tumors

Abstract

The INK4a/ARF locus encodes two unrelated tumor suppressor proteins, p16INK4a and p14ARF, which participate in the two main cell-cycle control pathways, p16–Rb and p14–p53. Methylation of CpG promoter islands has been described as a mechanism of gene silencing. Exon 1 of the p16INK4a gene and the p14ARF promoter gene reside within CpG islands. Therefore, both can become methylated de novo and silenced. It has recently been proposed that the methylation changes in certain genes could be used as molecular markers for the detection of almost all forms of human cancer. Here, we analyzed concomitantly in each tumor sample and normal tissue the methylation status of p16INK4a and p14ARF by methylation-specific PCR (MSP) in 100 breast, 95 colon and 27 bladder carcinomas. A series of clinicopathological parameter were obtained from the medical records of the patients, p14ARF showed a higher rate of hypermethylation than p16INK4a in all three tumor types. p16INK4a and p14ARF aberrant methylation was significantly correlated with poor prognosis clinicopathological parameters of the three tumor types. We conclude that both p16INKa and p14ARF hypermethylation may be involved in breast, colon and bladder carcinogenesis, with special emphasis on the role of the lesser studied p14ARF gene, and that tumors with aberrant methylation in the two genes were associated with worse prognosis.

Introduction

The INK4a/ARF locus (9p21) encodes two unique and unrelated proteins, p16INK4a and p14ARF, which are transcribed from different promoters located in alternative first exons. These are spliced to the same acceptor site in exon 2, which is translated in alternative frames [1]. Both proteins function as tumor suppressors by interpreting responses to hyperproliferative signals and modulating activities of the pRb and p53 pathways. The p16INK4a protein binds both cdk4 and cdk6 and inhibits the catalytic activity of the cdk4–cdk6/cyclin D enzyme complex required for pRb phosphorylation. pRb phosphorylation during G₁ disrupts its association with histone deacetylase and E2F transcription factors, allowing the transcription of genes involved in cell-cycle progression [2]. The p14ARF protein induces both G₁ and G₂ phase arrest [3] in a p53-dependent manner [4]. p14ARF interacts with the Mdm2 oncoprotein and inhibits the nuclear export of Mdm2 by tethering it in the nucleolus [5]. This prevents the Mdm2–p53 association and blocks the Mdm2-induced p53 degradation in the proteosome, thereby stabilizing p53 [6]. Since p14ARF is induced by E2F-1 [7], it is the biochemical link between the pRb and p53 pathways.

CpG islands are G+C rich regions located in, or near, promoters and 5′ regulatory regions [8] of housekeeping genes and are often confined to the coding regions of tissue-specific genes. Not all genes contain CpG islands, but most identified islands are associated with genes [9]. Methylation of CpG promoter islands has been associated with gene silencing [10]. The overall functions of DNA methylation in mammalian cells remain enigmatic, despite the fact that it is essential for development [11]. CpG islands are usually not methylated in the germline and, with some exceptions, in the normal somatic cells [9]. In contrast, widespread methylation of CpG islands occurs on autosomal genes during carcinogenesis [12]. The exon 1 coding sequences of the p16INK4a gene reside within 5′ CpG islands [13] and the p14ARF promoter is a CpG island [14]. Therefore, both can become methylated de novo and silenced.

p16INK4a is commonly inactivated by mutation, deletion and promoter hypermethylation in a wide variety of human tumors [13], [15], [16], [17], [18], [19], [20], [21]. Mutations affecting exon 1β of p14ARF gene have been described in metastatic melanoma and colon cancer cell lines and in one primary colon carcinoma [22], [23]. Deletion inactivation of p14ARF has been reported in human cancers [24], [25], [26], [27], [28], but in these studies p16INK4a was always co-deleted. Only germline deletion of p14ARF-specific exon 1β in a family characterized by multiple melanoma and neural cell tumors [29] has been reported. p14ARF promoter hypermethylation has been described in primary tumors like colorectal, gastric, bladder, lung and breast carcinomas [30], [31], [32], [33], [34], [35].

Aberrant CpG island methylation in human cancer has mainly been studied by a candidate gene approach, focusing on approximately 15 of the 45,000 CpG islands in the genome [36]. Promoter hypermethylation changes may provide a molecular marker system for the detection of the major forms of human cancer, since it has been described genes aberrantly methylated in multiple tumor types, and other genes methylated specifically in others [32], [37]. We examined the methylation status of the p16INK4a and p14ARF genes in three of the most common tumor types in humans in western societies: breast, colon and bladder cancer. We found aberrant methylation of both genes in the three tumor types. p14ARF showed higher promoter hypermethylation than p16INK4a, which points to its role in the tumorigenesis of the tumor types analyzed. Moreover, the tumors harboring p16INK4a or p14ARF aberrant methylation were associated with worse prognosis.