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Keywords:

  • ring finger protein 180;
  • epigenetic alteration;
  • diagnosis;
  • prognosis;
  • gastric cancer

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. FUNDING SOURCES
  7. REFERENCES

BACKGROUND:

By using genome-wide methylation screening, the authors identified ring finger protein 180 (RNF180) as preferentially methylated in cancer. This study was undertaken to clarify its structure and functional role in gastric cancer.

METHODS:

The transcription start site and core functional promoter region of RNF180 were revealed by 5′ rapid amplification of cDNA ends and luciferase activity assays. Promoter methylation was detected by combined bisulfite restriction analysis and bisulfite genomic sequencing. Cell growth was detected by colony formation assay, apoptosis by annexin V assay, and RNF180 target genes by cDNA microarray.

RESULTS:

The authors revealed the transcription start site of RNF180 gene and identified the functional core promoter region (−202/+372) in the CpG island, which could be silenced by in vitro methylation assay. RNF180 was silenced in 6 of 7 gastric cancer cell lines and significantly down-regulated in primary gastric cancers compared with adjacent normal tissues (P = .001). Loss of gene expression was associated with promoter methylation. Re-expression of RNF180 suppressed cell growth (P < .001) and induced apoptosis (P < .05), which were mediated by up-regulating the antiproliferation regulators MTSS1 and CDKN2A and the proapoptotic mediator TIMP3. Promoter methylation of RNF180 was detected in 76% (150 of 198) of primary gastric cancers and 55% (11 of 20) of intestinal metaplasia, but in none of 23 normal gastric tissues. Methylated RNF180 DNA was detected in the plasma of 56% of gastric cancer patients, but not in healthy controls (P = .003). Patients with low or loss of RNF180 expression had significantly poorer overall survival.

CONCLUSIONS:

RNF180 is a novel potential tumor suppressor in gastric carcinogenesis and has potential clinical utility as a biomarker for gastric cancer patients. Cancer 2012;. © 2011 American Cancer Society.

The aberrant hypermethylation of CpG islands is strongly associated with the inactivation of tumor suppressor genes and represents a major etiological factor in many human malignancies.1-3 Aberrant CpG island hypermethylation of tumor suppressor genes accumulates during cancer development.4, 5 Recent progress on tumor suppressor genes identification by genome-wide DNA methylation screening has uncovered the mechanisms of cancer progression and new molecular biomarker and therapeutic targets. By using the genome-wide methylation-sensitive arbitrarily primed polymerase chain reaction (PCR),6 we identified RNF180 (ring finger protein 180) as a novel preferentially methylated gene in cancer.7

RNF180 is a ring finger protein that shares the RING finger and the coiled-coil domain.8 Coexistence of these domains is identified in a tripartite motif protein family.9 Genes belonging to this family are implicated in a variety of biological processes, including cell growth, differentiation, and tumorigenesis.10-12 However, the role of RNF180 in human cancer has not been studied.

In this study, we first uncovered the gene structure of RNF180 transcript variants and recognized the functional core promoter region within the CpG island. Having revealed that the transcription repression was mediated by promoter methylation, we studied the biological functional and the molecular basis of RNF180 in gastric cancer as a potential tumor suppressor. We further investigated the frequency of RNF180 promoter methylation in primary gastric cancers and precancerous tissues, and explored the feasibility of detecting methylated RNF180 DNA in the plasma of gastric cancer patients. The potential relation of RNF180 protein expression with patient clinicopathologic features and survival data was also investigated.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. FUNDING SOURCES
  7. REFERENCES

Subjects and Sample Collection

Gastric biopsies including 198 gastric tumors, 20 noncancer gastric mucosa with intestinal metaplasia, and 23 normal gastric tissues were obtained during endoscopy according to a standard protocol from Prince of Wales Hospital, Hong Kong. The adjacent normal tissue was subsequently verified by histology without tumor infiltration. Plasma samples were collected from 32 gastric cancer patients and 64 health donors. Tumor was staged according to the American Joint Committee on Cancer TNM System. All subjects gave informed consent, and the study protocol was approved by the Clinical Research Ethics Committee of the Chinese University of Hong Kong.

Gastric Cancer Cell Lines

Seven gastric cancer cell lines (AGS, Kato III, MKN28, MKN45, N87, SNU1, and SNU16) were purchased from the American Type Culture Collection (Manassas, Va). They were cultured in RPMI-1640 medium (Gibco BRL, Rockville, Md) supplemented with 10% fetal bovine serum (Gibco BRL) and incubated in 5% CO2 at 37°C.

RNA Extraction, Semiquantitative Reverse Transcriptase PCR, and Real-Time PCR Analyses

Total RNA was isolated using TRIzol Reagent (Invitrogen, Carlsbad, Calif), and cDNA was synthesized using Transcriptor Reverse Transcriptase (Roche, Indianapolis, Ind) followed by semiquantitative reverse transcriptase (RT)-PCR and real-time PCR analyses.13-15

Mapping of the Transcription Start Site (5′ Rapid Amplification of cDNA Ends)

The transcription start site of the promoter region of RNF180 gene was determined by 5′ rapid amplification of cDNA ends using the GeneRacer Kit (Invitrogen). Normal human stomach RNA sample was purchased commercially (Ambion, Austin, Tex). The mRNA cap structure was dephosphorylated and removed with tobacco acid pyrophosphatase. The dephosphorylated and decapped RNA was ligated with the GeneRacer RNA oligonucleotide (Invitrogen). The cDNAs for rapid amplification of cDNA ends were synthesized using the GeneRacer and RNF180 gene specific primer. A dilution of the cDNAs for rapid amplification of cDNA ends was amplified using nested PCR with the GeneRacer 5′ nested primer and RNF180 gene-specific nested primer. The PCR product was cloned into pCR2.1 vector (Invitrogen) and sequenced. The transcription start site, defined by the represented 5′ ends, was numbered +1.

Identification of a Functional Promoter Region in RNF180

Seven constructs of RNF180 promoter, covering regions from −564 bp to +372 bp relative to the transcription start site, were cloned and ligated into a reporter gene vector, pGL3-basic, at XbaI-SacI sites (Promega, Madison, Wis). Transient transfection was performed in MKN28 and AGS cells using FUGENE 6 (Roche). The promoter activity was measured using the Dual-Luciferase Reporter Assay System (Promega) after 48 hours of transfection. Assays were carried out in 3 independent triplicates.

Construction of RNF180 Expression Vectors and Transfection

The RNF180 expression vectors were generated by PCR cloning with a pcDNA3.1 TOPO TA Expression Kit (Invitrogen). cDNA corresponding to the open reading frame of RNF180 transcript was obtained by RT-PCR amplification of normal human stomach RNA (Ambion). PCR aliquots were subcloned in the pcDNA3.1 TOPO vector. Clones were screened and sequenced using vector-specific primers.

Colony Formation Assay

Gastric cancer cell line AGS (2 × 105/well) was transfected with 0.5 μg RNF180-expressing or empty vector (pcDNA3.1) using FuGENE 6 (Roche). Transfected cells were selected with G418 (0.4 mg/mL) (Merck, Darmstadt, Germany) for 2 weeks. Colonies were fixed with methanol/acetone (1:1), stained with Gentian Violet, and counted.14-16

Cell Apoptosis Assay

Cell apoptosis was determined by dual staining with annexin V:fluorescein isothiocyanate (V:FITC) and propidium iodide (Invitrogen). After 48 hours of transfection, annexin V:FITC and propidium iodide were added to the cellular suspension, and sample fluorescence of 10,000 cells was analyzed by flow cytometry (BD Pharmingen, San Jose, Calif).16

cDNA Expression Array

Gene expression profiles in AGS stably transfected with pcDNA3.1-RNF180 or pcDNA3.1 empty vector were analyzed by the Human Cancer PathwayFinder RT2 Profiler PCR Array containing 84 functionally well-characterized genes involved in human tumorigenesis (SABiosciences, Frederick, Md). Gene expression changes ≥1.5-fold or ≤1.5-fold were considered to be biological significance.15

DNA Mutation Analysis

Mutation of RNF180 coding sequences were determined by direct DNA sequencing (Applied Biosystems, Foster City, Calif). Sequence homologies were analyzed using the BLAST program of the National Center for Biological Information database.

In Vitro DNA Methylation Assay

The functional promoter construct was treated with 160 μM S-adenosyl methionine (New England BioLabs, Ipswich, Mass) in the presence (methylated) or absence (mock-methylated) of 6 U of M.SssI (CpG) methylase per microgram of DNA at 37°C overnight. Aliquots of purified constructs were digested with HpaII (New England BioLabs) to confirm the methylation status. The methylated promoter construct was transfected into MKN28 and AGS cells, and the promoter activity was determined by luciferase reporter assay.

Sodium Bisulfite Modification, Combined Bisulfite Restriction Analysis, and Bisulfite Sequencing

Genomic DNA was extracted using the DNeasy Tissue Kit (Qiagen, Valencia, Calif) and then was treated with sodium bisulfite for 16 hours using the EZ DNA methylation Kit (Zymo Research, Hornby, Canada). Hot start PCR with the bisulfite-treated DNA was performed with a 301bp PCR product spanning promoter region −207bp to 92bp relative to the transcription start site of RNF180. This region contained 43 CpG dinucleotides and 6 BstUI restriction sites. PCR products were digested with BstUI (New England BioLabs). The DNA digests were separated in 10% nondenaturing polyacrylamide gels.15 Cloned bisulfite genomic sequencing was performed to confirm the methylation status identified by combined bisulfite restriction analysis.15, 16

Demethylation Treatment

Cells were seeded in 100 mm dishes (1 × 105 cells/mL). After 24 hours, cells were treated with 2 μM of the DNA demethylating agent 5-Aza (Sigma-Aldrich, St Louis, Mo) for 5 days.

Detection of Methylated RNF180 DNA in Plasma

Blood samples were centrifuged at 1600 × g for 10 minutes at 4°C, and the plasma portion was recentrifuged at 16,000 × g for 10 minutes. Genomic DNA was extracted from 800 μL of plasma using a QIAamp DNA Blood Mini Kit (Qiagen) and was then digested with 100 U of BstUI, a methylation-sensitive restriction enzyme, at 60°C for 16 hours. The methylation level of RNF180 promoter in plasma DNAs was assessed by specific quantitative methylation-specific PCR.13, 17 RNF180 primers and probe were designed on the functional promoter region (−234/−144) using a Taqman MGB (minor groove binding) probe (Applied Biosystems) (Fig. 1A).

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Figure 1. RNF180 CpG island methylation analysis in gastric cancer cell lines is shown. (A) The region of combined bisulfite restriction analysis and bisulfite genomic sequencing (BGS) spans the promoter region from −207 to +94. Cleavage of the polymerase chain reaction (PCR) product with BstUI digestion sites is marked with arrows. (B) Combined bisulfite restriction analysis of the RNF180 in gastric cancer cell lines is shown. (C) Cloned bisulfite sequencing analysis of the RNF180 promoter effect on gastric cancer cell lines is shown. The open circles indicate unmethylated and closed circles indicate methylated CpG sites. (D) RNF180 expression in the gastric cancer cell lines after pharmacological reversal of DNA methylation by 5-Aza was examined by reverse transcriptase PCR. COBRA, combined bisulfite restriction analysis; M, methylated; TSS, transcription start site; U, unmethylated.

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Immunohistochemistry

Formalin-fixed paraffin-embedded archive tissues of 149 primary gastric cancers were arranged in tissue array blocks. Immunohistochemistry was performed using anti-RNF180 antibody (1:500) (Sigma-Aldrich). The cytoplasmic expression of RNF180 was assessed by assigning the average intensity of positive tumor cells (0, none; 1, weak; 2, intermediate; 3, strong).

Statistical Analysis

The results were expressed as mean ± standard deviation. Mann-Whitney U test was performed to compare the variables of the 2 sample groups. The association between patient characteristics and RNF180 methylation status was analyzed by the chi-square test. Cox models were constructed to estimate the relative risks of death associated with RNF180 expression. The Kaplan-Meier method was used for univariate survival analysis, and the log-rank test was used to compare the difference in survival curves. All analyses were performed using SPSS software (version 13.0, Chicago, Ill). P values <.05 were considered statistically significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. FUNDING SOURCES
  7. REFERENCES

Characterization of the Promoter Region and Transcript Variant of Human RNF180 Gene

The human RNF180 gene was predicted based on the information from the cDNA library in the AceView browser (http://www.ncbi.nlm.nih.gov/IEB/Research/Acembly), which suggested that RNF180 might contain alternative transcript variant and promoter. However, this library did not provide cDNA clones in stomach. To better define putative promoter regions for RNF180, we performed 5′ rapid amplification of cDNA ends from normal human stomach tissue and generated a major PCR product of 427 bp (Fig. 2A). This revealed the transcription start site of RNF180 locates at Chr11:63,497,387 (Fig. 2B), but not at Chr11:63,497,427 as predicted by the University of California Santa Cruz human genome database (http://genome.ucsc.edu/). To determine the transcript variant of the RNF180 gene in humans, the cDNA for rapid amplification of cDNA ends was subjected to PCR amplified from the 3′ end and sequencing, which confirmed the existence of the RNF180 transcript (1779 bp) (Fig. 2C).

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Figure 2. Genomic structure is shown of RNF180 transcription start site (TSS) and transcript variant. (A) Gel image of 5′ rapid amplification of cDNA ends analysis of RNF180 locus exon 1 starting transcripts is shown. (B) Nucleotide sequence of the 5′ rapid amplification of cDNA ends products is shown. (C) The transcript variant (GenBank accession: NP_001107033) contains RING finger and transmembrane domain.

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Silencing or Down-Regulation of RNF180 in Gastric Cancer Cell Lines and in Primary Gastric Cancers

RNF180 transcript was silenced or down-regulated in 6 of 7 (86%) gastric cancer cell lines, except MKN45 (Fig. 3A). In contrast, RNF180 expression was readily detected in normal gastric tissues (Fig. 3A). We then compared RNF180 expression in 9 paired gastric tumors and their adjacent nontumor tissues. As determined by real-time RT-PCR, levels of RNF180 mRNA were significantly lower in tumor samples than in the surrounding noncancerous tissue (P = .001, Fig. 3B). The results of immunohistochemistry analysis on these 9 paired tissues samples confirmed that RNF180 protein was silenced or down-regulated in the cancer cells compared with surrounding noncancer tissues (Fig. 3C). However, down-regulation of RNF180 mRNA was not observed in other gastrointestinal cancer types such as colon and liver (data not shown).

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Figure 3. Down-regulation of RNF180 mRNA and protein level in gastric cancer. (A) mRNA expression of RNF180 was determined by conventional reverse transcriptase polymerase chain reaction (RT-PCR). (B) RNF180 mRNA expression in 9 pairs of human gastric tumors and corresponding normal counterparts by real-time RT-PCR is shown. (C) Representative images of RNF180 protein expression by immunohistochemistry are shown.

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Characterization of the Core Promoter Region Driving RNF180 Gene Transcription

To elucidate whether promoter methylation mediates RNF180 silencing, we first characterized the core promoter sequence driving RNF180 gene transcription. A typical CpG island was revealed to span the transcription start site to exon 1 of the RNF180 gene by CpG Island Searcher (http://cpgislands.usc.edu/) (Fig. 4A). Seven DNA constructs covering the region from −564 to 372 of the RNF180 promoter were cloned, as this region resides in a CpG island with 65.5% of GC content, and the observed/expected CpG ratio is 0.904 (Fig. 4A). These constructs were transfected into AGS and MKN28 cells. The maximal promoter activity for RNF180 was found in a 574-bp region (−202 to 372) as determined by luciferase reporter activity assay (Fig. 4B).

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Figure 4. Functional promoter mapping and in vitro DNA methylation analysis are shown. (A) The location of CpG dinucleotides (vertical bars) of RNF180 are shown. (B) RNF180 functional promoter mapping using luciferase reporter assay in AGS and MKN28 cell lines is shown. (C) In vitro methylation of the RNF180 functional promoter sequence (−202/+372) inhibits promoter activity. Error bars indicate standard deviation. COBRA, combined bisulfite restriction analysis; BGS, bisulfite genomic sequencing.

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Promoter Methylation Directly Mediates RNF180 Transcription Silencing In Vitro

To evaluate the effect of promoter methylation in regulating the transcription silencing of RNF180, the core functional promoter region (−202 to 372) was methylated in vitro by treatment with SssI (CpG) DNA-methyltransferase (Fig. 4C). In vitro methylation of the promoter construct drastically reduced its transcriptional activity both in AGS (139-fold reduction) and in MKN28 cell lines (30-fold reduction) when compared with their unmethylated constructs (Fig. 4C).

Promoter Methylation of RNF180 Was Correlated With Transcriptional Silencing in Gastric Cancer Cell Lines

To further elucidate the silencing of RNF180 by epigenetic changes, RNF180 methylation status was examined in 7 gastric cancer cell lines by combined bisulfite restriction analysis. Six cell lines with silenced (AGS, KatoIII, MKN28, N87, and SNU1) or decreased (SNU16). RNF180 expression displayed fully methylation or partial promoter methylation, respectively, whereas no methylation was detected in the expressed cell line (MKN45) (Fig. 1A, B). The methylation status detected by combined bisulfite restriction analysis was validated and confirmed by bisulfite genomic sequencing (Fig. 1C), in which dense methylated CpG sites were detected in methylated cell lines (KatoIII, MKN28), but no methylated residue was found in an unmethylated cell line (MKN45) (Fig. 1C).

Demethylation Treatment With 5-Aza Restored RNF180 Expression

All 6 methylated gastric cancer cell lines were treated with DNA methyltransferase inhibitor (5-Aza). The treatment resulted in restoration of RNF180 expression in all cell lines examined (Fig. 1D), confirming that promoter methylation directly contributed to the RNF180 silencing in gastric cancer cell lines.

Genetic Deletion or Mutation of RNF180 Was Not Detected in Gastric Cancer Cell Lines

Genetic deletion and mutation analyses of RNF180 coding exons by DNA direct sequencing did not show any homozygous deletion or mutation in 7 gastric cancer cell lines. Only a single nucleotide polymorphism (intron 1 −196; C>G) was found in 4 cell lines (KatoIII, MKN28, MKN45, and SNU16).

RNF180 Suppressed Tumor Cell Growth

The frequent silencing of RNF180 in gastric cancer cell lines suggests that RNF180 is likely a tumor suppressor. We thus examined the growth-suppressive effect through ectopic expression of RNF180 in AGS cells that showed no RNF180 expression. Ectopic expression of RNF180 was confirmed by RT-PCR (Fig 5A). Re-expression of RNF180 significantly decreased the number of colonies (Fig. 5B).

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Figure 5. (A) Functional assay of RNF180 in gastric cancer AGS cells is shown. (A) Re-expression of RNF180 in stably transfected AGS cells was confirmed by reverse transcriptase polymerase chain reaction. (B) Colony formation assay is shown. Upper panel shows the representative dishes of transfection with pcDNA3.1, RNF180 expression vector. Quantitative analyses of colony numbers (%) are shown in the lower panel as values of mean ± standard deviation. (C) Fluorescence-activated cell sorting (FACS) using apoptosis assay labeled with annexin V:FITC-A fluorescence staining is shown. Upper panel shows the FACS histograms of transfection with pcDNA3.1, RNF180 expression vector. Quantitative analyses of annexin V-positive cells (%) are shown in the lower panel as mean ± standard deviation.

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RNF180-Induced Apoptosis

We examined the contribution of apoptosis to the observed growth inhibition in AGS cells derived by RNF180. Apoptosis was investigated using 2-color fluorescence-activated cell sorting analysis. Our results indicate the induction of significantly more apoptotic cells among RNF180-transfected AGS cells than among vector-transfected AGS cells (up to 43% of vector controls, P < .05) (Fig. 5C).

Identification of Genes Modulated by RNF180

To gain insights into the molecular mechanisms, we performed cDNA expression array to identify RNF180 modulated downstream targets. When compared with vector control cells, re-expression of RNF180 transcript up-regulated the antiproliferation and proapoptotic key regulators, including the antiproliferation regulators MTSS1 (metastasis suppressor 1) and CDKN2A (cyclin-dependent kinase inhibitor 2A), as well as the proapoptotic regulator TIMP3 (TIMP metallopeptidase inhibitor 3) (Table 1).

Table 1. Differential Expressed Genes Induced by RNF180 Re-expression in Gastric Cancer AGS Cells
GenBank Accession #Gene NameSymbolFold ChangeGene Function
NM_014751Metastasis suppressor 1MTSS15.48Antiproliferation
NM_000077Cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4)CDKN2A2.49Antiproliferation, proapoptosis
NM_000362TIMP metallopeptidase inhibitor 3TIMP31.60Proapoptosis

RNF180 Promoter Methylation in Primary Gastric Cancers and in Gastric Mucosa With Intestinal Metaplasia

We next examined the RNF180 methylation status in 198 primary gastric cancers, 20 gastric specimens containing foci of intestinal metaplasia (IM), a preneoplastic lesion, and 23 normal gastric tissues using combined bisulfite restriction analysis. Frequent methylation was detected in gastric cancers (150 of 198, 76%), but there was less in IM (11 of 20, 55%), and none in 23 normal gastric tissues (Fig. 6A1). Further detailed bisulfite genomic sequencing analysis confirmed methylated promoter alleles in tumors, whereas normal gastric tissues showed no methylation (Fig. 6A2). There was no correlation between the methylation of RNF180 and clinicopathologic features.

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Figure 6. RNF180 promoter methylation and survival of gastric cancer patients are shown. (A1) Promoter methylation status of RNF180 in primary gastric tumors, intestinal metaplasia (IM) tissues, and normal gastric mucosa was determined by combined bisulfite restriction analysis. (A2) Cloned bisulfite genomic sequencing analysis of the RNF180 promoter on primary tumors and normal gastric mucosa is shown. (B1) Kaplan-Meier estimates of patient survival with gastric cancers, according to RNF180 protein expression are shown. RNF180 immunopositive indicates moderate or high RNF180 expression, and immunonegative indicates weak or no expression. (B2) The Kaplan-Meier curve of gastric cancer patients is further stratified into the stage I-III and the late stage IV. P values were calculated by the log-rank test.

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Detection of Methylated RNF180 DNA in Plasma of Gastric Cancer Patients

Because of the high frequency of RNF180 methylation noted in primary gastric cancers, we investigated whether RNF180 methylation could be used as a biomarker for gastric cancer. The plasma samples of 32 gastric cancer patients and 64 normal subjects were tested for methylated RNF180 DNA using quantitative PCR with primers flanking on the RNF180 core promoter region. By using the cutoff value of 5.1, 56% (18 of 32) of plasma samples from cancer patients had RNF180 methylation, whereas RNF180 methylation was not detected in the plasma of 64 normal controls. Moreover, all patients with methylation detected in the plasma DNA had methylation in the corresponding tumors. We determine the specificity and sensitivity using receiver operating characteristic (ROC) curve analysis. The ROC curve yield an area of 0.685 (95% confidence interval [CI], 0.54 to 0.84). At the cutoff value of 2.2, the specificity was 91% and the sensitivity was 63% in discriminating gastric cancer patients from control subjects. Stepwise logistic regression analysis further indicated that detection of RNF180 methylation in plasma could be a potential noninvasive marker for gastric cancer (P < .0005). The odds ratio for subjects with RNF180 being associated with gastric cancer was 16.1 using a cutoff value of >2.2 (95% CI, 5.3-48.6).

RNF180 Is a Predictor of Poor Outcome in Gastric Cancer Patients

We evaluated RNF180 protein expression by large-scale immunohistochemistry on tissue microarrays containing 149 gastric cancer samples. Immunoreactivity of RNF180 was predominantly localized in the cytoplasm (Fig. 3C). Loss or weak stain was noted in 54% (81 of 149) of gastric cancers. The association of RNF180 expression and clinicopathologic characteristics including clinical outcome was analyzed and showed that no or weak RNF180 expression was found more frequently in patients with advanced TNM stages (Table 2). However, there were no correlations between RNF180 down-regulation and age, sex, Helicobacter pylori infection, Lauren type, and tumor differentiation (Table 2).

Table 2. Clinicopathological Features of RNF180 Expression in Gastric Cancer
FeaturesNumberRNF180 ExpressionP
No/ WeakModerate/ High
  1. Abbreviation: SD, standard deviation.

Age, y, mean ± SD65.3 ± 13.163.7 ± 14.067.2 ± 12.0.15
Sex    
 Male7133 (46%)38 (54%).19
 Female4426 (59%)18 (41%) 
Helicobacter pylori infection
 Positive5524 (44%)31 (56%).23
 Negative4123 (56%)18 (44%) 
Lauren type    
 Intestinal5424 (44%)30 (56%).06
 Diffuse3523 (66%)12 (34%) 
 Mixed155 (33%)10 (67%) 
Differentiation    
 Poor4724 (51%)23 (49%).61
 Well/moderate4822 (46%)26 (54%) 
TNM stage    
 I42 (50%)2 (50%).05
 II84 (50%)4 (50%) 
 III289 (32%)19 (68%) 
 IV2417 (71%)7 (29%) 
TNM stage    
 I-III4015 (38%)25 (63%).01
 IV2417 (71%)7 (29%) 

In univariate Cox regression analysis (Table 3), loss or down-regulation of RNF180 was associated with a significantly increased risk of cancer-related death (relative risk [RR], 2.23; 95% CI, 1.409-3.519; P = .001). After the adjustment for potential confounding factors, multivariate Cox regression analysis showed that RNF180 down-regulation was a predictor of poorer survival of gastric cancer patients (RR, 2.13; 95% CI, 1.11-4.08; P = .02) (Table 4). As shown in the Kaplan-Meier survival curves, gastric cancer patients with loss of or low RNF180 expression had significantly shorter survival (median, 1.05 years) than others (median, 2.70 years) (P = .0016, log-rank test) (Fig. 6B1). After stratified by tumor staging, the overall survival of patients with loss of or low RNF180 expression was significantly shorter than that of other gastric cancer patients for stage I to III, but not for stage IV (Fig. 6B2).

Table 3. Univariate Cox Regression Analysis of Potential Poor Prognostic Factors for Gastric Cancer Patients
VariableHazard Ratio (95% CI)P
  1. Abbreviation: CI, confidence interval.

Sex  
 Male0.48 (0.31-0.75).001
 Female1.00 
 Age1.00 (0.98-1.02).86
Helicobacter pylori infection  
 Negative1.11 (0.68-1.82).68
 Positive1.00 
Lauren type  
 Diffuse1.62 (0.96-2.73).07
 Mixed1.96 (0.99-3.86).05
 Intestinal1.00 
Differentiation  
 Well0.33 (0.10-1.11).07
 Poor1.57 (0.94-2.64).09
 No0.90 (0.12-6.64).90
 Moderate1.00 
TNM stage  
 I0.24 (0.06-1.04).06
 II0.20 (0.06-0.69).01
 III0.42 (0.23-0.78).006
 IV1.00 
RNF180 expression  
 No/weak2.23 (1.41-3.52).001
 Moderate/high1.00 
Table 4. Multivariate Cox Regression Analysis of Potential Poor Prognostic Factors for Gastric Cancer Patients
VariableHazard Ratio (95% CI)P
  1. Abbreviation: CI, confidence interval.

Sex  
 Male0.23 (0.11-0.49).0005
 Female1.00 
Age0.97 (0.94-1.00).03
TNM stage  
 I0.19 (0.04-0.78).02
 II0.31 (0.09-1.08).07
 III0.91 (0.45-1.80).77
 IV1.00 
RNF180 expression  
 No/weak2.13 (1.11-4.08).02
 Moderate/high1.00 

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. FUNDING SOURCES
  7. REFERENCES

In this study, we first identified the RNF180 transcript variant, and defined the transcription start site and functional promoter region in normal human stomach (Fig. 2). This transcript variant encoded a RING-finger domain and a hydrophobic transmembrane region in its C-terminal end. Having identified the transcription start site of the RNF180 gene in stomach, we evaluated RNF180 expression and found that RNF180 was frequently absent or down-regulated in gastric cancer cell lines in vitro, and was also significantly decreased in primary gastric cancers compared with their adjacent nontumor tissues in vivo at both mRNA and protein levels. However, this down-regulation was not identified in other digestive cancers, suggesting that RNF180 would be a candidate tumor suppressor in gastric cancer development.

Hypermethylation of CpG islands in the promoter region of genes associated with gene silencing becomes crucial to the development of gastric carcinogenesis.14-16, 18 Thus, identification of the functional core promoter region of the RNF180 gene in the CpG island is essential. In this regard, we characterized the core functional promoter region of the RFN180 promoter located within the CpG island near the transcription start site (−202/+372) with maximal promoter activity among the 7 regions investigated (Fig. 4).

We demonstrated that the absent or very lowly expression of RNF180 is inversely correlated with hypermethylation of this core promoter region. Bisulfite sequencing of the RNF180 promoter region showed dense methylation in gastric cancer cell lines and primary cancers, but no methylation in the normal gastric tissues. The silencing of RNF180 can be reversed by pharmacological demethylation. This was further confirmed by in vitro methylation assay in the RNF180 core promoter region. In addition, no genetic deletion or mutational inactivation of RNF180 was found in all tumor cell lines examined, indicating that promoter hypermethylation is the predominant mechanism for the silencing or down-regulation of the RNF180 gene in gastric cancer.

We studied the tumor suppressive effect of RNF180 in a gastric cancer cell line. The full-length human RNF180 constructs were transfected into the silenced gastric cancer cell line AGS. Restoration of RNF180 in AGS cells significantly inhibited colony formation and induced cell apoptosis. These results suggested for the first time that RNF180 functions as a potential tumor suppressor in gastric cancer. RNF180 belongs to the member of the RING-finger protein superfamily. Other reports have shown that members of the RING finger protein family are involved in cell growth and tumorigenesis,19 and several of them are aberrantly down-regulated in tumor cells acting as tumor suppressors.20-22 Our results have demonstrated the importance of RNF180 as a novel potential tumor suppressor in gastric cancer. We demonstrated the molecular mechanisms through which RNF180 exerts the tumor suppressor effect in gastric cancer and observed that induction of RNF180-mediated apoptosis occurs via up-regulating proapoptotic mediators TIMP3 and CDKN2A. Consistent with our result, recent studies showed that TIMP3 and CDKN2A trigger apoptosis in human melanoma and colon cancer cells.23, 24 The antiproliferative ability exerted by RNF180 in gastric cancer cells is at least associated with up-regulation of the antiproliferation regulators MTSS1 and CDKN2A, which have been reported to suppress tumor growth of certain types of human cancers in vitro and in vivo.25, 26 Thus, activation of these proapoptotic mediators and antiproliferation regulators by RNF180 may offer the explanation for the growth inhibition in gastric cancer cells.

We further determined the clinical application of RNF180 in gastric carcinogenesis in vivo and found that RNF180 hypermethylation was detected in the majority (76%, 150 of 198) of gastric cancer tissues, but not in normal controls, indicating that RNF180 methylation is a common event in gastric cancers. In addition, RNF180 promoter methylation was also found in 55% of intestinal metaplasia, a precancerous lesion of gastric cancer, suggesting that aberrant promoter methylation of RNF180 is an early event in gastric carcinogenesis and may be involved in the initiation of cellular transformation from normal cells into tumors. Having observed the high frequency of RNF180 methylation in gastric cancer, we conducted experiments designed to test the potential for detection of methylated RNF180 DNA in the plasma samples as a noninvasive biomarker. It was recognized that RNF180 methylation was detected in 56% of the plasma samples from gastric cancer patients, but not in any of the plasma samples from normal controls. The methylation in plasma samples was also detected in their corresponding tumor tissues and revealed that the specificity and sensitivity of detecting RNF180 methylation in the plasma DNA are 91% and 63%, respectively. This finding raised the possibility that RNF180 methylation can be used as a potential noninvasive biomarker in plasma for gastric cancer. However, the sensitivity of this single marker is relatively low. In this connection, further studies are warranted to find new biomarkers and refine the best panel of biomarkers for simultaneous detection of gastric cancer.

Recognizing the biological functions of RNF180, the inactivation of this gene by promoter methylation would favor tumor progression and a worse outcome. In this connection, we examined the influence of RNF180 protein expression on outcome of gastric cancer patients. Among our panel of 149 primary gastric cancers, loss or weak stain of RNF180 protein was noted in 54% (81 of 149) of gastric cancers. More importantly, we demonstrated that reduction of RNF180 protein accumulation was significantly associated with poorer survival independent of patient characteristics, particularly in patients with early stage gastric cancer. Thus, in keeping with the biological function of RNF180 identified in the present study, the inactivation of this gene by promoter methylation would favor tumor progression and a worse outcome. This provided additional evidence for the role of RNF180 as a novel candidate tumor suppressor gene in the pathogenesis of gastric cancer.

In conclusion, we have uncovered the transcriptional start site and the promoter region of RNF180 gene and determined that methylation in a core functional region of the promoter is strongly correlated with RNF180 transcriptional suppression, a frequent event in human gastric cancers and their precancerous lesions. Our results suggest that RNF180 is a potential tumor suppressor, with key roles of suppressing cell proliferation and inducing apoptosis. RNF180 methylation may serve as a potential noninvasive plasma biomarker for the detection of gastric cancer, and the lack of RNF180 expression may predict poor outcome for gastric cancer patients.

FUNDING SOURCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. FUNDING SOURCES
  7. REFERENCES

The project was supported by a Research Grants Council grant (473008), a 973 national program fund (2010CB529305), an RFCID (08070172), a Chinese University of Hong Kong Group Research Scheme (3110043), and a Chinese University of Hong Kong Focused Investments Scheme (1903026).

CONFLICT OF INTEREST DISCLOSURES

The authors made no disclosures.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. FUNDING SOURCES
  7. REFERENCES