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Keywords:

  • hypermethylation;
  • NSCLC;
  • RGC32;
  • MSP;
  • overall survival

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONFLICT OF INTEREST DISCLOSURES
  7. REFERENCES

BACKGROUND:

Lung cancer is the leading cause of cancer-related deaths worldwide. Epigenetic inactivation of certain genes by aberrant promoter methylation is recognized as a crucial component in the initiation and progression of lung cancer. Response gene to complement 32 (RGC32) has been identified as a cell cycle regulator induced by activation of complements; however, its role in carcinogenesis is still controversial.

METHODS:

The authors examined the methylation status in the promoter region of RGC32 gene in nonsmall cell lung cancers (NSCLCs) using a methylation-specific PCR and correlated the results with clinicopathological features.

RESULTS:

RGC32 methylation was found in 45 of 173 NSCLCs (26.0%) and was related to the gene expression. RGC32 methylation was more frequent in females than in males (P<0.05). RGC32 methylation was not significantly associated with the prognosis of patients; however, when the patients were categorized by TP53 mutational status, the effect of RGC32 methylation on prognosis was significantly different between those with and without TP53 mutations (P = .005 [test for homogeneity]). Specifically, RGC32 methylation was associated with significantly worse survival in the cases with wild-type TP53, whereas it exhibited a better survival outcome in the cases with TP53 mutations.

CONCLUSIONS:

The current findings suggest that methylation-associated down-regulation of RGC32 plays an important role in the pathogenesis of NSCLC, particularly in females. However, further studies with a large number of cases are needed to confirm the authors' findings. Cancer 2011. © 2010 American Cancer Society.

Epigenetic gene silencing constitutes an alternative or complementary mechanism to mutational events in tumorigenesis.1, 2 An emerging picture of genetic and epigenetic changes and their relationship is revealing the biological networks responsible for human cancer. Several genes are commonly the target of promoter methylation in lung cancer.3-5 The predisposition of smokers to the acquisition of epigenetic alterations in key cellular regulatory genes6 suggests that DNA methylation could serve as a biomarker for the earliest stages of preinvasive lung cancer related to tobacco smoking, a major etiological factor. Detailed delineation of methylation alterations are needed for early detection and more effective therapy.

The mammalian cell cycle is regulated by a complex system of inhibitory and stimulatory factors. The aberrant expression of these regulators has been implicated in the pathogenesis of human cancers.7 Response gene to complement 32 (RGC32, recently termed chromosome 13 open reading frame 15 [C13orf15]) was identified as one of the genes induced by complement activation.8, 9 Overexpression of RGC32 was associated with accelerated entry of smooth muscle cells into S phase, with RGC32 acting as an activator and substrate for cyclin dependent kinase 1 (CDK1,formerly CDC2), suggesting that RGC32 is involved in activation of the cell cycle.8, 9 Regarding its proposed oncogenic role,8, 9 RGC32 has been found to be up-regulated in colon, ovarian, and breast cancers.10, 11 However, it has also been reported that RGC32 is down-regulated in many tumors, such as endometrial cancer, invasive prostate cancer, multiple myeloma, and glioblastomas, suggesting that RGC32 acts as a tumor suppressor in certain types of cancers.10 In addition, it has been reported that RGC32 is directly induced by the tumor suppressor TP53, and mediates G2/M cell cycle arrest through interaction with polo-like kinase 1 (Plk1).12 Taken together, these data suggest that RGC32 can form complexes not only with CDK1, but also with Plk1, while playing a more complex role in cell cycle regulation (depending on tumor cell types).10 To understand the role of the RGC32 gene in lung cancer, we investigated the methylation status of the promoter region of the RGC32 gene in nonsmall cell lung cancers (NSCLCs) and analyzed the relationship between RGC32 methylation and the clinicopathological characteristics of the patients.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONFLICT OF INTEREST DISCLOSURES
  7. REFERENCES

Patients and Tissue Samples

Tumor and corresponding, nonmalignant lung tissue specimens were obtained from 173 Korean NSCLC patients who underwent curative resection at the Kyungpook National University Hospital (Daegu, Korea) from January 2003 to July 2007. None of these patients received chemotherapy or radiotherapy before surgery. Informed consent was obtained from each patient before surgery. This study was approved by the Institutional Review Board of the Kyungpook National University Hospital. The clinical and pathological characteristics of the patients are summarized in Table 1. There were 113 males and 60 females with mean age of 63.0 ± 8.6. There were 56 never-smokers and 117 ever-smokers (currentor formersmokers) with mean pack-years of 26.4 ± 25.4. The histologic types of NSCLCs included 56 cases of squamous cell carcinomas (SCCs) and 117 adenocarcinomas (ACs). The pathologic stages included 109 cases at stage I, 29 cases at stage II, and 35 cases at stage IIIA. All of the tumor and macroscopically normal lung tissue samples were obtained at the time of surgery, and rapidly frozen in liquid nitrogen and stored at −80°C until genomic DNA preparation. The macroscopically normal lung tissues were confirmed as normal by hematoxylin-eosin staining. Mutations of the entire coding region (exons 2-11), including exon-intron boundaries of the TP53 gene, were detected using PCR-based direct sequencing. Somatic TP53 mutations were detected in 65 tumors (37.6%). TP53 mutations were significantly more frequent in males than in females (49.6% vs 13.1%), in ever-smokers than in never-smokers (49.6% vs 10.5%), and in SCCs than in ACs (59.3% vs 25.6%; all comparisons, P < .001).

Table 1. Correlation bBetween RGC32 Methylation and Clinicopathologic Features
VariablesMethylation, n (%)P
All subjects (n=173)45 (26.0) 
Age (years)
 ≤63 (n=86)21 (24.4).63
 >63 (n=87)24 (27.6) 
Gender
 Female (n=60)21 (35.0).05
 Male (n=113)24 (21.2) 
Smoking status
 Never (n=56)19 (33.9).10
 Ever (n=117)26 (22.2) 
Histologic types
 Squamous cell ca. (n=56)11 (19.6).19
 Adenoca. (n=117)34 (29.1) 
Pathologic stage
 Stage I (n=109)29 (26.6).82
 Stage II-IIIA (n=64)16 (25.0) 
TP53 mutations
 Negative (n=108)30 (27.8).50
 Positive (n=65)15 (23.1) 

Cell Culture and 5-Aza-2'-Deoxycytidine (5-AzadC) Treatment

Ten human NSCLC cancer cell lines, 6 ACs (A549, H23, H522, H1373, H1793, and H2009), 3 SCCs (H157, H226, and H1703), and 1 large cell carcinoma (H1299) were obtained from the American Type Culture Collection (ATCC, Manassas, VA). All cells were propagated perinstructions from the ATCC. H226 cells were treated with 20 μM 5-AzadC for 3 days, and the culture media were changed daily.

Total RNA Isolation and Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

Total RNA was extracted from the cultured cells or patients' tissues using TRIzol (Invitrogen, Victoria, Australia) according to the manufacturer's instructions. Residual genomic DNA was digested with RNase-free DNase (Invitrogen). First strand cDNA was reverse-transcribed from 2 μg of total RNA in a total volume of 20 μl using oligo(dT) and a SuperScript preamplification kit (Invitrogen). The resulting cDNA was amplified by forward (5′-GCCACTTCCACTACGAGGAG-3′) and reverse (5′-GTGGCCTGGTAGAAGGTTGA-3′) primers under the same condition as described previously.11

Genomic DNA Isolation and Methylation Analysis

Genomic DNA was extracted using a QIAamp DNA Mini Kit (QIAGEN, Valencia, CA). The methylation status of the RGC32 gene was analyzed by a nested methylation-specific PCR (MSP).13 Bisulfite-treated DNA was used to PCR-amplify the RGC32 promoter region -69 to +214 using 5'-GGGTAAATATTTGGGGTTGTAAT-3' and 5'-TTCAACCCTACCAATCCCTTC-3' primers. PCR products from step 1 were diluted at 1:250 and then subjected to the second step of MSP that incorporated unmethylated or methylated primers; methylated, 5'-TCGCGGTTTTAGGGCGGGCGC-3' (forward) and 5'-CCGCTCCCAACACGATCCGCG-3' (reverse); and unmethylated, 5'-TTGTGGTTTTAGGGTGGGTGT-3' (forward) and 5'-CCACTCCCAACACAATCC ACA-3' (reverse). All PCR amplifications were carried out using reagents supplied in a GeneAmp DNA Amplification Kit with AmpliTaq Gold as the polymerase (PE Applied Biosystems, Foster City, CA) on the PTC-100 thermal cycler (MJ Research, Watertown, MA). CpGenome Universal methylated and unmethylated DNA (Chemicon, Temecula, CA) was used as a positive control for the methylated and unmethylated genes, respectively. Each MSP was repeated at least once to confirm the results.

Statistical Analysis

The relationship between the methylation and clinicopathological characteristics was analyzed using a chi-square test or Fisher exact test for categorical variables. Overall survival (OS) was measured from the day of surgery until the date of death or to the date of the last follow-up. Survival estimates were calculated using the Kaplan-Meier method. The differences in OS across different groups were compared using the log-rank test. Hazard ratios (HRs) and 95% confidence intervals (CIs) were estimated using multivariate Cox proportional hazards models, with adjustments for age (≤63 vs >63 years), gender (female vs male), smoking status (never- vs ever-smoker), tumor histology, and pathologic stage (I vs II-IIIA). All analyses were performed using the Statistical Analysis System for Windows, version 9.1 (SAS institute, Cary, NC).

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONFLICT OF INTEREST DISCLOSURES
  7. REFERENCES

MSP and RT-PCR Analysis of RGC32 in Tissue Samples and NSCLC Cell Lines

Long CpG island (CGI) was found in the 1.1-kb 5' flanking region of the RGC32 gene including the first exon (Accession no. AL354833). Intriguingly, this CGI coincided with the 5' end of minimal promoter possessing several Sp1 binding sites and TATAAT signals. Because methylation of a region covering the transcription start site/minimal promoter is associated with expression, we designed the MSP primer pairs covering the proximal CGI with a web-based MethPrimer program. Each specific primer set yielded a single band of the expected size, and representative examples of the MSP analysis are illustrated in Figure 1A. Unmethylated bands were detected in all the samples (ie, both nonmalignant and malignant tissues), thus confirming the integrity of the DNA in these samples. Bisulfite-sequencing of the representative PCR products of RGC32 confirmed their methylation status and showed that all cytosines at non-CpG sites were converted to thymine (data not shown). This excluded the possibility that successful amplification could be attributable to incomplete bisulfite conversion. The frequency of RGC32 methylation was significantly higher in tumor tissues (26.0%) than in the matching nonmalignant lung tissues (10.7%, P = .002), suggesting that its methylation may not be an intrinsic, developmentally programmed event, but a tumor-associated, de novo event.

thumbnail image

Figure 1. (A) Representative results of MSP and RT-PCR analysis in NSCLC patients and (B) human NSCLC cell lines. Upper panel in A and B: the methylation status of RGC32 gene was analyzed by nested MSP. CpGenome Universal methylated or unmethylated DNA (Chemicon) was used as a positive control for the methylated or unmethylated products, respectively. Water was used as a negative control. Lower panel in A and B: expression of RGC32 mRNA was performed by RT-PCR. Amplification of beta-actin was used as an internal loading control. The symbol (−) indicated vehicle alone treatment, whereas (+) indicated the 5-AzadC treatment for 3 days.

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We examined the expression levels of RGC32 mRNA in representative tissue samples. RT-PCR analysis showed low or undetectable levels of RGC32 mRNA expression in tissues with a methylated allele, whereas RGC32 mRNA was expressed at high levels in malignant and corresponding nonmalignant tissues with an unmethylated allele (Fig. 1A). We have further confirmed these results in 10 human NSCLC cell lines. RT-PCR and MSP analysis showed that RGC32 mRNA was absent in the H226 cell line containing the methylated promoter, whereas this mRNA was present in the examined cell lines with unmethylated alleles (Fig. 1B). Subsequently, H226 cells were treated with the demethylating agent 5'-AzadC for 3 days. This process alone failed to induce RGC32 mRNA transcripts, but 5-AzadC treatment succeeded in inducing the re-expression of RGC32 mRNA (Fig. 1B). These results suggest that transcriptional inactivation of RGC32 expression may be caused by promoter methylation of that gene.

Correlation Between RGC32 Methylation and Clinicopathological Features

RGC32 methylation was more frequent in females than in males (35.0% vs 21.2%, P < .05). However, there was no significant association between RGC32 methylation and other clinicopathologic factors such as age, smoking status, histologic type, pathologic stage, or TP53 mutation status (Table 1).

Effects of RGC32 Methylation on Survival Outcomes

Survival outcomes according to clinicopathologic characteristics of the patients are shown in Table 2. There were 47 deaths (27.2%). The estimated 5-year OS for all the patients was 55.2%. Of the clinicopathologic factors, age and pathologic stage were significantly associated with OS: adjusted HR for age >63/≤63 years, 95% CI, 1.14-3.77, P = .02; adjusted HR for stage II-IIIA/I = 3.10, 95% CI, 1.72- 5.57, P < .0001.

Table 2. Overall Survival According to Clinicopathologic Features and TP53 Mutation
VariablesNo. of CasesNo. of Deaths (%)a5-Year OSR (%)bPL-RAdjusted HR (95% CI)P
  • a

    Row percentage.

  • b

    Five-yearoverall survival rate, proportion of survival derived from Kaplan-Meier analysis.

  • c

    Hazard ratios (HRs), 95% confidence intervals (CIs) and their corresponding P-values were calculated using multivariate Cox proportional hazard models, adjusted for other covariates.

  • d

    HRs, 95% CIs, and their corresponding P-values were calculated using multivariate Cox proportional hazard models, adjusted for age, gender, smoking status, tumor histology, and pathologic stage.

Overall17347 (27.2)55   
Age, y
 ≤638618 (20.9)64.021.00 
 >638729 (33.3)46 2.08 (1.14-3.77)c.02
Gender
 Female6011 (18.3)66.161.00 
 Male11336 (31.9)51 2.98 (0.83-10.68)c.09
Smoking status
 Never5610 (17.9)65.301.00 
 Ever11737 (31.6)53 0.58 (0.15-2.21)c.42
Histological type
 Squamous cell ca.5617 (30.4)60.811.00 
 Adenoca.11730 (25.6)51 1.04 (0.55-1.99)c.90
Pathologic stage
 I10921 (19.3)63.00071.00 
 II-IIIA6426 (40.6)44 3.10 (1.72-5.57)c.<0001
TP53 mutations
 Negative10824 (22.2)59.671.00 
 Positive6523 (35.4)52 0.93 (0.48-1.79)d.82

RGC32 methylation was not significantly correlated with the OS of these patients. However, when the patients were stratified according to TP53 mutational status, RGC32 methylation was associated with a worse OS in the patients without the TP53 mutation (adjusted OR = 2.52, 95% CI, 1.05-6.04, P = .04), whereas it was associated with a better OS in the patients with the TP53 mutation (adjusted OR = 0.34, 95% CI, 0.12-1.00, P = .05; PH = 005; Table 3 and Fig. 2). In a multivariate survival analysis using the Cox proportional hazards model, along with the pathologic stage, RGC32 methylation was an independent prognostic factor for patient survival (in the cases without TP53 mutation, P for RGC32 methylation = .04, and P for pathologic stage = .002. In those cases with TP53 mutation, P for RGC methylation = .05, and P for pathologic stage = .01) (Table 4).

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Figure 2. Kaplan-Meier survival curves of NSCLC patients according to RGC32 methylation status. (A) Survival curve of NSCLC total patients, (B) patients without TP53 mutations, and (C) patients with TP53 mutations. Adjusted P values calculated from Cox proportional hazards models are represented.

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Table 3. Association of RGC32 Methylation With Overall Survival in NSCLC Patients
Variables PL-RCrude HR (95% CI)PPHAdjusted HR (95% CI)PPH
  1. Hazard ratios (HRs), 95% confidence intervals (CIs), and their corresponding P-values were calculated using multivariate Cox proportional hazard models, adjusted for age, gender, smoking status, tumor histology, and pathologic stage.

Overall subjects .591.19 (0.63-2.23).58 0.94 (0.49-1.80).84 
TP53 mutationsNegative.082.05 (0.89-4.70).09.062.52 (1.05-6.04).04.005
 Positive.280.57 (0.21-1.59).29 0.34 (0.12-1.00).05 
Table 4. Multivariate Analyses of the Prognostic Values of Various Factors According to TP53 Mutation Status
VariablesAdjusted HR (95% CI)aP
  • a

    Hazard ratios (HRs), 95% confidence intervals (CIs), and their corresponding P-values were calculated using multivariate Cox proportional hazard models, adjusted for age, gender, smoking status, tumor histology, and pathologic stage.

  • b

    AC, adenocarcinoma; SCC, squamous cell carcinoma.

TP53 mutation negative 
 RGC32 methylated/unmethylated2.52 (1.05-6.04).04
 Age, >63/≤63 years2.16 (0.89-5.24).09
 Gender, female/male0.22 (0.03-1.56).13
 Smoking status, ever/never0.47 (0.07-3.27).45
 Histology, AC/SCCb0.85 (0.25-2.84).79
 Pathologic stage, II-IIIA/I4.75 (1.82-12.41).002
TP53 mutation positive 
 RGC32 methylated/unmethylated0.34 (0.12-0.99).05
 Age, >63/≤63 years3.08 (1.18-8.02).02
 Gender, female/male0.29 (0.04-2.07).22
 Smoking status, ever/never0.51 (0.03-8.04).63
 Histology, AC/SCC0.99 (0.41-2.42).98
 Pathologic stage, II-IIIA/I3.08 (1.28-7.43).01

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONFLICT OF INTEREST DISCLOSURES
  7. REFERENCES

The present study has shown that the promoter region of the RGC32 gene was frequently methylated in NSCLCs, and its methylation was related to loss of RGC32 gene expression. These results suggest that aberrant promoter methylation plays an important role in the down-regulation of RGC32 gene expression in NSCLCs. Although RGC32 has been found to be epigenetically silenced in an endometrial cancer cell line,14 the finding presented herein is the first to demonstrate a loss of RGC32 expression via promoter methylation in NSCLC.

It has been reported that RGC32 was up-regulated in ovarian and metastatic breast cancers, whereas it was down-regulated in invasive prostate cancer, multiple myeloma, and drug-resistant glioblastomas.10 In addition, RGC32 can form complexes not only with CDK1, but also with Plk1.12 Therefore, it has been suggested that the effect of RGC32 on the cell cycle depends on the kinase to which it binds in the cell and that RGC32 functions as a tumor promoter or suppressor, depending on the cell types of cancers.10 Notably, we found that inactivation of RGC32 by aberrant promoter methylation exhibits different effects on survival outcomes according to the TP53 mutational status of the tumors. Specifically, RGC32 methylation was associated with a worse OS in the patients without TP53 mutations, whereas it was associated with a better OS in the patients with TP53 mutations (PH for adjusted HRs across the two groups = .005). These findings suggest that RGC32 may have different roles in lung tumorigenesis depending on TP53 mutational status of the tumors. RGC32 exerts a tumor suppressive effect in the tumors with wild-type TP53, but exerts a tumor promoting effect in the tumors carrying TP53 mutations. However, the biologic mechanism for the oppositely directed association of RGC32 methylation with survival outcomes remains to be elucidated. In addition, further studies with large numbers of patients are warranted to confirm our findings.

Another notable finding of the present study is that RGC32 methylation was more frequent in females than in males. In addition, RGC32 methylation was more frequent in never-smokers and ACs than in ever-smokers and SCC, respectively, although neither was statistically significant. These findings suggest that, as mutations in the tyrosine kinase domain of the EGFR gene are common in females, never-smokers, and ACs, RGC32 methylation may be related to other environmental factors rather than tobacco smoking.

RGC32 methylation was found in a patient subset of nonmalignant lung tissues. This finding may derive from contamination of nonmalignant cells with methylated cancer cells. However, because nonmalignant lung tissue specimens were obtained from either the opposite end of resected surgical samples or as distant from the tumor as possible, and were histologically confirmed as normal by H-E stain, the possibility of contamination is extremely low. RGC32 methylation in histologically nonmalignant lung tissues may occur when phenotypically normal lung tissues already harbor epigenetic alterations because the entire field of lung was exposed to a carcinogenic insult, such as cigarette smoking. It has been reported that aberrant promoter methylation frequently occurs in histologically normal-appearing lung tissues, representing a field defect of widespread epigenetic change in lung tissues.15, 16 Considering those data, our finding further supports the field cancerization theory. In addition, the strong concordance of RGC32 methylation in both tumorous and nonmalignant lung tissue suggests that RGC32 methylation may permit the future acquisition and accumulation of genetic and epigenetic changes that lead to malignant transformation.15, 16

Although the current investigation was limited by the small number of samples and the daunting lack of information on protein, this study has shown that the RGC32 gene is frequently down-regulated in NSCLCs and that aberrant promoter methylation is an important mechanism in the deregulation of the gene. In addition, we showed that RGC32 methylation exhibits differential effects on survival outcomes by the TP53 mutational status of the tumors. However, because this is the first study to demonstrate that RGC32 methylation is associated with the survival outcome of lung cancer patients, further studies with larger numbers of patients are warranted to confirm our findings. In addition, the mechanisms underlying this clinically and biologically important finding should be further explored.

CONFLICT OF INTEREST DISCLOSURES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONFLICT OF INTEREST DISCLOSURES
  7. REFERENCES

This work was supported by the Korea Research Foundation Grant, funded by the Korean Government (MOEHRD, Basic Research Promotion Fund) (KRF-2007-E00223).

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONFLICT OF INTEREST DISCLOSURES
  7. REFERENCES