Epigenetic silencing of the 3p22 tumor suppressor DLEC1 by promoter CpG methylation in non-Hodgkin and Hodgkin lymphomas

Background Inactivaion of tumor suppressor genes (TSGs) by promoter CpG methylation frequently occurs in tumorigenesis, even in the early stages, contributing to the initiation and progression of human cancers. Deleted in lung and esophageal cancer 1 (DLEC1), located at the 3p22-21.3 TSG cluster, has been identified frequently silenced by promoter CpG methylation in multiple carcinomas, however, no study has been performed for lymphomas yet. Methods We examined the expression of DLEC1 by semi-quantitative reverse transcription (RT)-PCR, and evaluated the promoter methylation of DLEC1 by methylation-specific PCR (MSP) and bisulfite genomic sequencing (BGS) in common lymphoma cell lines and tumors. Results Here we report that DLEC1 is readily expressed in normal lymphoid tissues including lymph nodes and PBMCs, but reduced or silenced in 70% (16/23) of non-Hodgkin and Hodgkin lymphoma cell lines, including 2/6 diffuse large B-cell (DLBCL), 1/2 peripheral T cell lymphomas, 5/5 Burkitt, 6/7 Hodgkin and 2/3 nasal killer (NK)/T-cell lymphoma cell lines. Promoter CpG methylation was frequently detected in 80% (20/25) of lymphoma cell lines and correlated with DLEC1 downregulation/silencing. Pharmacologic demethylation reversed DLEC1 expression in lymphoma cell lines along with concomitant promoter demethylation. DLEC1 methylation was also frequently detected in 32 out of 58 (55%) different types of lymphoma tissues, but not in normal lymph nodes. Furthermore, DLEC1 was specifically methylated in the sera of 3/13 (23%) Hodgkin lymphoma patients. Conclusions Thus, methylation-mediated silencing of DLEC1 plays an important role in multiple lymphomagenesis, and may serve as a non-invasive tumor marker for lymphoma diagnosis.


Introduction
Epigenetic silencing of tumor suppressor genes (TSGs) by promoter CpG methylation and histone modification has been widely recognized as one of the major causes of tumorigenesis including hematological malignancies [1,2]. Aberrant methylation of TSG is frequently detected even in the early stage of tumorigenesis, suggesting its potential as tumor biomarker for early detection and therapeutic targeting.
In this study, we examined the expression and methylation status of DLEC1 in lymphoma cell lines and tissues, and evaluated its potential as a tumor marker for the early detection of hematologic tumors.  [46][47][48]. Cell lines were maintained in RPMI1640 or Dulbecco's Modified Eagle's Medium supplemented with 10% fetal calf serum (FBS) (Invitrogen, Paisley, Scotland) and 1% streptomycin/penicillin at 37°C in 5% CO 2 . HL cell lines were treated with 5 μM of 5-aza-2 0 -deoxycytidine (Aza) (Sigma) for 3 days, or further treated with 100 nmol/L trichostatin A (Cayman Chemical Co., Ann Arbor, MI, USA) for additional~16 h as described previously [15,49], and L428 and KM-H2 cell lines were treated with Aza for 6 days.

Cell lines and tumor samples
Normal peripheral blood mononuclear cells (PBMC), lymph node samples, different types of lymphoma samples, as well as sera from healthy individuals and HL patients were collected as previously described, and the reliability and quality of all the studied samples in this study have been confirmed before [41,47,50]. The study was approved by Johns Hopkins Medicine Institutional Review Board. Normal adult tissue RNA samples were purchased commercially (Stratagene, La Jolla, CA, USA or Millipore-Chemicon, Billerica, MA, USA). DNA and RNA were extracted from lymphoma cell lines and primary lymphomas using TRIzol reagent (Invitrogen) as previously described [51,52].
All primer sets were previously tested for not amplifying any unbisulfited DNA. Amplified BGS products were cloned and 5 to 10 colonies were randomly chosen for sequencing.
The promoter and exon 1 region of DLEC1 is a typical CpG island, with a T/C SNP site at CpG#29 (Figure 2A). MSP analysis showed that DLEC1 promoter methylation was frequently detected in 3/6 DLBCL, 1/2 PTCL, 5/5 BL, 7/7 (one weak) HL, and 2/3 NL cell lines ( Figure 2B; Table 1), well correlated with its silencing or reduction. DLEC1 methylation was further verified in detail by BGS analysis of 61 CpG sites within the CpG island. CpG sites of DLEC1 examined were heavily methylated in BL cell lines Rael, Raji, and in the HL cell line L428, but rarely in all normal PBMC samples, which confirmed the MSP data ( Figure 2C). These results indicate that DLEC1 silencing by promoter methylation is a critical event in lymphomagenesis.

Silencing of DLEC1 could be reversed by pharmacologic demethylation
To further confirm whether promoter methylation mediates the loss of DLEC1 expression in lymphoma, lymphoma cell lines with methylated and reduced DLEC1 were treated with the DNA methylation inhibitor, Aza, alone or combined with the HDAC inhibitor trichostatin A (TSA). After Aza treatment, DLEC1 expression was significantly induced in cell lines Rael, L428, L1236 and KM-H2. MSP analysis showed increased unmethylated alleles. Similar results were obtained using a combination treatment of Aza and TSA (Figure 3). Demethylation of the DLEC1 promoter was further confirmed by BGS. These results confirmed that promoter CpG methylation directly mediates DLEC1 silencing in lymphomas.
Unmethylated bands were detected in all samples due to the inevitable inclusion of non-tumor cells in the analysis. Furthermore, the reduction or silencing of DLEC1 expression was observed in 6 out of 7 (86%) NL tumors as measured by RT-PCR.
Furthermore as a pilot study, we examined DLEC1 methylation in serum samples of 13 HL patients with sera from 20 healthy individuals as controls. ANKRD30A is a normally methylated gene, thus used as an internal control for validating genomic DNA integrity of these samples, in addition to unmethylated DLEC1. Results showed that DLEC1 methylation was detected in 3/13 (23%) sera from HL patients ( Figure 4D) but not in any normal sera.

Discussion
DLEC1 is located in the commonly deleted region 3p22-21.3 [3,18]. A cluster of TSGs, including RASSF1A, BLU, and CACNA2D2, has been identified within this region. Silencing of RASSF1A, BLU, as well as DLEC1 by promoter CpG methylation had been extensively identified in multiple human cancers. Notably, promoter methylation of several 3p22-21.3 TSGs, such as RASSF1A and BLU, has also been identified in lymphomas [50,55], suggesting that TSGs in this region are frequently susceptible to epigenetic disruption during lymphoma pathogenesis.
Here, we report that DLEC1 is frequently silenced by promoter methylation both in lymphoma cell lines and primary lymphomas, but seldom in normal lymph nodes and PBMCs. Pharmacologic demethylation could reactivate DLEC1 expression, suggesting that epigenetic mechanism including DNA methylation and histone modification mediates DLEC1 transcriptional silencing. As primary tumor tissues usually contain infiltrating   DLBCL, which needs to be further confirmed by large sample size. Our results suggest that epigenetic silencing of DLEC1 is important for lymphoma pathogenesis. Remarkably, DLEC1 is specifically methylated in sera of HL patients, suggesting its potential as an epigenetic biomarker for the non-invasive diagnosis of lymphomas.

Conclusions
This study identifies the frequent epigenetic inactivation of DLEC1 in various lymphomas and demonstrates its potential as a non-invasive tumor marker for the detection of lymphomas. More molecular studies on the tumor suppressive functions of DLEC1 in lymphoma pathogenesis are needed.