OPCML is a broad tumor suppressor for multiple carcinomas and lymphomas with frequently epigenetic inactivation

Abstract

Background: Identification of tumor suppressor genes (TSGs) silenced by CpG methylation uncovers the molecular mechanism of tumorigenesis and potential tumor biomarkers. Loss of heterozygosity at 11q25 is common in multiple tumors including nasopharyngeal carcinoma (NPC). OPCML, located at 11q25, is one of the downregulated genes we identified through digital expression subtraction.

Methodology/principal findings: Semi-quantitative RT-PCR showed frequent OPCML silencing in NPC and other common tumors, with no homozygous deletion detected by multiplex differential DNA-PCR. Instead, promoter methylation of OPCML was frequently detected in multiple carcinoma cell lines (nasopharyngeal, esophageal, lung, gastric, colon, liver, breast, cervix, prostate), lymphoma cell lines (non-Hodgkin and Hodgkin lymphoma, nasal NK/T-cell lymphoma) and primary tumors, but not in any non-tumor cell line and seldom weakly methylated in normal epithelial tissues. Pharmacological and genetic demethylation restored OPCML expression, indicating a direct epigenetic silencing. We further found that OPCML is stress-responsive, but this response is epigenetically impaired when its promoter becomes methylated. Ecotopic expression of OPCML led to significant inhibition of both anchorage-dependent and -independent growth of carcinoma cells with endogenous silencing.

Conclusions/significance: Thus, through functional epigenetics, we identified OPCML as a broad tumor suppressor, which is frequently inactivated by methylation in multiple malignancies.

Figure 1. Identification of novel splicing variants of OPCML and its expression in normal human tissues.

(A) Genomic organization of the 11q25 locus with the two known genes OPCML and HNT. Transcriptional orientations are shown by curved arrows. (B) Different promoter usage and alternative splicing of OPCML. Alternative mRNA transcripts are shown aligned from 5′ to 3′ on a virtual genome. The 5′-end of OPCML-v1 assembled by ECgene (Genome Annotation for Alternative Splicing, http://genome.ewha.ac.kr/ECgene/) was adapted to this alignment. (C) Left panel: determination of transcription start sites of OPCML transcripts by 5′-RACE. Right panel: expression of OPCML-v1 and v3 in brain and testis by semi-quantitative RT-PCR using 5′-RACE product as the template. Primer pair v1F/R4 amplifies one band that is specific to v1. Primer pair v1F0/R4 amplifies two bands corresponding to v1 and v3, respectively. (D) Expression of OPCML-v1 and v3 in adult tissues by semi-quantitative RT-PCR. Primer pair v1F0/R4 amplifies two bands corresponding to v1 and v3, respectively. The specific and non-specific bands have been confirmed by direct sequencing. (E) Expression of OPCML-v1 and v2 in human normal adult and fetal tissues. Primer pair v1F/R4, v2F/R4 and F3/R2 are specific to the v1-, v2-transcripts, and common exons (exon 2 and 3) of OPCML , respectively. Sk.M., skeletal muscle. (F) Possible transcription of OPCML from other unidentified alternative promoters. Expression of OPCML in normal and tumor cell lines was analyzed by semi-quantitative RT-PCR using primers (F3/R2) specific to common exons (exon 2 and 3) of OPCML. Expression of OPCML-v1 or v2 is indicated as “+”, while downregulation or silencing is indicated as “−”. OPCML-v1 promoter methylation status in each cell line is also shown. M, methylated; U, unmethylated. Transcription of OPCML from unknown alternative promoters was found in some tumor cell lines (underlined) where the OPCML-v1 promoter is methylated and silenced and v2 expression is also silenced.

Figure 2. Epigenetic inactivation of OPCML in multiple tumor cell lines.

(A) Schematic structure of the v1 (NM_002545) CGI, with its exon 1, predicted promoter region, MSP region and BGS region indicated. Each short vertical line represents one CpG site. The v2 promoter is also shown. (B) Validation of the specificity of the MSP system. No signal was detected using the unbisulfited DNA from several tumor cell lines. (C) Representative analyses of OPCML v1 and v2 (NM_001012393) expression by semi-quantitative RT-PCR and methylation status of v1-CGI by MSP in tumor cell lines and normal controls. M, methylated; U, unmethylated. Immortalized normal epithelial cell lines (NE1, NE3, Het-1A, NP69, CCD 841, MLCSV40) and normal epithelial cell lines (HMEC, HMEpC and PrEC-6) with underlined names were used as normal controls. RC170N/h and RC165N/h are telomerase-immortalized benign prostate epithelial cell lines, RC92a/h and RC58T/h/SA#4 are telomerase-immortalized prostate tumor derived cell lines. (D) Expression and methylation of OPCML in glioma cell lines.

Figure 3. High-resolution methylation analysis of the OPCML-v1 promoter by BGS.

A region spanning the promoter with 90 CpG sites was analyzed. Each CpG site is shown at the top row as an individual number. Dense methylation of the v1-CGI was found in ESCC (EC18, EC109) and NPC (C666-1, CNE2, HK1) cell lines, but not in normal esophageal (NE1, NE3) and nasopharyngeal epithelial (NP69) cell lines. Five to 8 colonies of cloned BGS-PCR products from each bisulfite-treated DNA sample were sequenced and each is shown as an individual row, representing a single allele of the CGI analyzed. One circle indicates one CpG site. Dark filled or open circles represent methylated or unmethylated CpG sites, respectively. Δ indicates possible variation of a CpG site to the CpA or CpT dinucleotides. The MSP region in this study and the BGS region studied in the previous report are indicated in frames. The rightmost column is the MSP result of each sample.

Figure 4. Restorations of OPCML-v1 expression by demethylation.

(A) Pharmacological demethylation by Aza (A) and TSA (T) induced the expression of OPCML-v1 but not v2. OPCML expression before and after drug treatment was determined by RT-PCR. (B) Genetic demethylation of the OPCML-v1 CGI also activated its expression. OPCML-v1 expression in HCT116 cells and HCT116 with double knockout of DNMT1 and DNMT 3B (DKO) are shown. (C) Detailed BGS analysis confirmed the demethylation of the OPCML-v1 CGI in DKO cells.

Figure 5. Analysis of homozygous deletion of OPCML in multiple carcinoma cell lines and normal controls.

The abundance of OPCML relative to GAPDH was determined by multiplex differential genomic DNA PCR. The expression of OPCML in each sample is also shown. +, normal expression; −, downregulated/silenced.

Figure 6. OPCML-v1 was also methylated in different primary tumors.

(A) Frequent methylation of the OPCML-v1 CGI in multiple primary tumors as analyzed by MSP. M, methylated; U, unmethylated. Representative results are shown. T, tumors; N, paired non-tumor tissues. Good quality of bisulfited DNA samples of normal NPx (NPx1-4) has been confirmed by Q-MSP for beta-actin . (B) In contrast, no methylation was detected in prostate tumors. EsCa, esophageal carcinoma; HCC, hepatocellular carcinoma; GsCa, gastric carcinoma; BrCa, breast carcinoma; CxCa, cervical carcinoma.

Figure 7. The OPCML-v1 promoter is stress- and p53-responsive.

(A) Locations of transcription factors (HSF, p53, Sp1, E2F, STAT) binding sites in the promoter are indicated. (B) Up-regulation of OPCML-v1 in response to stress treatments is disrupted in tumor cell lines with a methylated promoter. Normal (NE3, HEK293, NE1) and tumor cell lines (Rael, CNE1, C666-1, HK1) were exposed to 42°C heat shock (HS), UV irradiation, or H₂O₂ treatments. OPCML-v1 promoter methylation status in each cell line is shown at the bottom. M, methylated; U, unmethylated. (C) H1299 cells were transfected with different amounts of pcDNA3.1+/TP53 (gift from Dr. Bert Vogelstein) . Expression of OPCML-v1 and v2 was analyzed by semi-quantitative RT-PCR. p53 induced a dosage-dependent upregulation of OPCML-v1.

Figure 8. Ectopic expression of OPCML-v1 inhibits tumor cell growth.

The effect of ectopic OPCML-v1 expression on carcinoma cell clonogenicity was investigated by monolayer colony formation assay (A) and soft agar assay (B). Cells were transfected with pcDNA3.1+/OPCML-v1 or control vector, and selected with G418. (C) Quantitative analyses of colony formation. The numbers of G418-resistant colonies in each vector-transfected control were set to 100%, while OPCML-v1 expressed cells were presented as mean±SD. Three independent experiments were performed in triplicate. The asterisk indicated statistical significant difference (p<0.01).