TRAIL-R3-related apoptosis: Epigenetic and expression analyses in women with ovarian neoplasia

Abstract

Objective

To assess the expression of TRAIL-R3 and the methylation of a CpG island within the TRAIL-R3 promoter both in cystadenoma tumors and primary and metastatic epithelial ovarian carcinoma (EOC).

Methods

RNA was obtained from women with normal ovarian (NO) tissues (n = 18), ovarian serous cystadenoma tumors (n = 11) and EOC (n = 16) using Trizol®. Quantitative PCR (qRT-PCR) was performed to quantify the relative levels of TRAIL-R3. The methylation frequency of the CpG island in the TRAIL-R3 promoter was assessed using the methylation-specific PCR (MSP) assay after DNA bisulfite conversion. The differences between the groups were evaluated using the chi-square, Student's t, ANOVA, Mann–Whitney U, Wilcoxon or Kruskal–Wallis tests as indicated. The survival rates were calculated using the Kaplan–Meier method.

Results

Cystadenoma and metastatic EOC tumors expressed significantly more TRAIL-R3 mRNA than primary EOC tumors. Methylation of the TRAIL-R3 promoter was absent in NO tissues, while hemimethylation of the TRAIL-R3 promoter was frequently found in the neoplasia samples with 45.4% of the cystadenoma tumors, 8.3% of the primary EOC samples and 11.1% of the metastatic EOC samples showing at least partial methylation (p = 0.018). Neither the expression of TRAIL-R3 nor alterations in the methylation profile were associated to cumulative progression-free survival or the overall survival in EOC patients.

Conclusions

Primary EOC is associated to a lower TRAIL-R3 expression, which leads to a better understanding of the complex disease and highlighting potential therapeutic targets. Promoter DNA methylation was not related to this finding, suggesting the presence of other mechanisms to transcriptional control.

Introduction

Epithelial ovarian cancer (EOC) is the seventh most common cancer in women with an estimated 225,500 new cases and 140,200 deaths reported worldwide in 2008 [1]. Despite improvements in surgical management and advances in cytotoxic therapies, EOC still is the most common cause of death among women with gynecologic malignancies. This high death rate is mainly due to late-stage diagnoses (stage III or IV) and a poor prognosis with the present therapies [2], [3]. The poor prognosis is commonly due to drug resistance caused by alterations in the cellular factors that regulate cell survival, differentiation, immunity, DNA repair and apoptosis [4], [5].

Apoptosis, also known as programmed cell death, is fundamental for the development and homeostatic maintenance of complex biological systems [6], [7]. The apoptotic process can be executed intracellularly by the release of a number of factors from the mitochondria (intrinsic pathways) or through the transmembrane death receptors (DRs), which are activated upon binding their cognate ligands (extrinsic pathway) such as TRAIL, a TNF-related apoptosis-inducing ligand that is a type II transmembrane protein. Apoptosis can also be induced by a pathway that involves T-cell mediated cytotoxicity and is dependent on the perforin granzyme [7], [8], [9], [10].

Four homologous human receptors for TRAIL have been identified. TRAIL receptor 1 (TRAIL-R1/DR4/TNFRSF10A) [11] and TRAIL receptor 2 (TRAIL-R2/DR5/TRICK2/TNFRSF10B) [11], [12], [13] are capable of mediating apoptosis. Both TRAIL-R1 and TRAIL-R2 contain an extracellular cysteine-rich domain, a transmembrane domain and an intracellular death domain that can activate classic death signaling and lead to the activation of caspase 8 and caspase 10 [14], [15]. In contrast, neither the decoy receptor (TRAIL-R3/TRID/DcR1/TNFRSF10C), which lacks a cytoplasmic domain [11], [12], [16], nor TRAIL receptor 4 (TRAIL-R4/DcR2/TNFRSF10D), which contains a truncated cytoplasmic death domain [17], transduces an apoptotic signal directly. In this report, the TRAIL receptors are called TRAIL-R1, TRAIL-R2, TRAIL-R3 and TRAIL-R4.

Alternatively, TRAIL can also activate the intrinsic pathway by cleaving the BH3-interacting domain death agonist (BID) that binds to the Bcl-2-associated X protein (BAX) and the Bcl-2 homologous antagonist killer (BAK) [18], [19]. Intriguingly, TRAIL signaling can also induce the activation of NF-κB by phosphorylating of the inhibitor of κB (IκB), which leads to its degradation [20].

Targeted cancer therapies are emerging from investigations examining basic signaling mechanisms and molecular targets capable of inducing apoptosis in tumor cells. Although the activation of the apoptotic machinery by the DR ligands such as TRAIL represents a novel therapeutic strategy, little is known about the relevance of these receptors in ovarian neoplasia. Therefore, the aim of this study was to describe the genetic expression and epigenetic variation of TRAIL-R3 in normal ovarian (NO) tissue, cystadenoma tumors, primary EOC and metastatic EOC. These analyses were performed to evaluate the prognostic significance of TRAIL-R3 for patient survival.