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10.1245/s10434-006-9089-6
Annals of Surgical Oncology 14:1397-1404 (2007)
© 2007 Society of Surgical Oncology
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Original Article

Methylation and Gene Silencing of the Ras-Related GTPase Gene in Lung and Breast Cancers

Makoto Suzuki, MD1,2, Hisayuki Shigematsu, MD2, David S. Shames, MD2, Noriaki Sunaga, MD1, Takao Takahashi, MD2, Narayan Shivapurkar, PhD2, Toshihiko Iizasa, MD1, John D. Minna, MD2, Takehiko Fujisawa, MD1 and Adi F. Gazdar, MD2

1 Department of Thoracic Surgery, Graduate School of Medicine, Chiba University, Chiba, Japan
2 Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, TX, USA

Correspondence: Address correspondence and reprint requests to: Makoto Suzuki, MD; E-mail: smakoto{at}faculty.chiba-u.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: RRAD, a small Ras-related GTPase, is highly expressed in human skeletal muscle, lung, and heart. Although loss of expression of RRAD in breast cancer cells has been reported and it may act as an oncogene, the mechanism of silencing is unknown.

Methods: We examined (1) mRNA expression of RRAD in lung and breast cancer cell lines using RT-PCR and (2) methylation status of lung and breast cancers.

Results: Loss of RRAD expression was found in 14 of 20 (70%) NSCLC cell lines, 11 of 11 (100%) SCLC cell lines, and 8 of 10 (80%) breast cancer cell lines; expression was not affected in normal bronchial and mammary epithelial cells. Treatment of 23 expression-negative cell lines with a demethylating agent restored expression in all cases. We developed a methylation-specific assay from the analysis of bisulfite sequencing of the 5' region of RRAD in expression-negative and positive cell lines, which resulted in good concordance between methylation and expression. Primary lung and breast cancers showed hypermethylation in 89 of 214 (42%) and 39 of 63 (62%) cases, respectively. RRAD hypermethylation correlated with smoking history and poorer prognosis in lung adenocarcinomas.

Conclusions: We conclude that epigenetic silencing of RRAD is a frequent event in lung and breast cancers, and analysis of it may provide novel opportunities for prognosis and therapy of these cancers.

Key Words: RRAD • Methylation • Lung cancer • Breast cancer


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
RRAD, a member of a family of Ras-related GTPases, was initially identified by means of subtraction cloning and is overexpressed in the skeletal muscle of a subset of humans with type-2 diabetes.1 It is expressed normally in heart and lung.1 RRAD cycles between GTP- and GDP-bound states and is stimulated by interaction with a GTPase-activating protein present in the cytoplasm.2,3 Overexpression of RRAD inhibits glucose uptake in cultured muscle and fat cells, indicating that it is a negative regulator of glucose uptake.4 Expression of RRAD has been shown to increase with increasing differentiation of human osteoblasts.5 However, the precise function of RRAD, especially in malignant tumors, remains unknown.

In melanoma cells RRAD acts to increase serum-stimulated DNA synthesis, whereas nm23-H1 blocks this effect.3 By immunohistochemical analysis in breast cancers, Tseng and collaborators demonstrated that RRAD is frequently expressed in non-malignant breast tissue, while expression is usually lost in corresponding carcinomas.6 However, when RRAD was present in carcinomas it was associated with higher grade, larger size, and extensive axillary nodal involvement. In a RRAD-transfected breast cancer cell line with no nm23-H1, the authors found increased cell growth, markedly increased soft-agar colony formation, and increased tumor growth rate in nude mice.6 The interaction between RRAD and nm23-H1 appeared to play a significant role in control of tumor cell growth, and they suggested that RRAD may act as an oncogene in breast cancer.6 However, the reason many cancer tissues have less expression than corresponding nonmalignant breast tissues remains unclear.

Epigenetic modification through DNA methylation of the CpG island in the 5' region of genes is the major mechanism that induces transcriptional inactivation of tumor suppressor genes (TSGs).7 DNA methylation of several TSGs has been reported in various tumors, including lung and breast cancers.8–15 Recently, we analyzed global changes in the gene expression profiles of an NSCLC cell line after treatment with the demethylating agent 5-aza-2'-de-oxycytidine (5-Aza-CdR) (unpublished data) and identified downregulation of RRAD and restoration of expression after treatment with the demethylating agent. Moreover, the fibroblast cell line MDAH041 showed loss and restoration of expression of RRAD after treatment with 5-Aza-CdR.16 This gene is located at chromosome 16q22, and loss of heterozygosity in this region is frequent in lung and breast cancers.17–19 In this study, we examined mRNA expression and methylation status of RRAD in lung and breast cancer cell lines and the methylation status of primary tumors.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines and Tumor Samples
We used for this study 31 lung cancer cell lines (20 NSCLC and 11 SCLC cell lines) and 10 breast cancer cell lines that had previously been established by us.20,21 Cell cultures were grown in RPMI-1640 medium (Life Technologies Inc., Rockville, MD) supplemented with 5% fetal bovine serum and incubated in 5% CO2 at 37°C. Cell lines established at the National Cancer Institute have the prefix NCI and those established at the University of Texas Southwestern Medical Center have the prefix HCC. Non-malignant human bronchial epithelial cells (NHBEC) and nonmalignant human mammary epithelial cells (NHMEC) were cultured as reported previously,9 and normal trachea RNA was obtained from Clontech (Palo Alto, CA).

Primary malignancies were obtained at the hospitals of the University of Texas Southwestern Medical Center and Chiba University Hospital, Japan, after obtaining Institutional Review Board approved signed consent. Samples were immediately frozen and stored at –80°C until used.

Reverse Transcriptase-PCR Assay
A reverse transcriptase-PCR (RT-PCR) assay was used to examine RRAD mRNA expression. Total RNA was extracted from the samples with Trizol (Life Technologies) following the manufacturer’s instructions. The RT reaction was run with 4 µg total RNA, deoxyribonuclease I and the SuperScript II First-Strand Synthesis using the oligo(dT) primer System (Life Technologies). Aliquots of the reaction mixture were used for the subsequent PCR amplification. The forward PCR amplification primer of RRAD was 5'-TTTACAAGGTGCTGCTGCTGGG-3 ', and the reverse primer was 5'-TGCCGCTGATGTCTCAATGAAC-3'. These sequences cross an intron, but we confirmed that genomic DNA was not amplified using these primers (GenBank accession number NM_004165; forward nucleotides 395–416; reverse nucleotides 807–828). The housekeeping gene GAPDH was used as an internal control to confirm the success of the RT reaction. The forward primer for GAPDH amplification was 5'-CACTG GCGTCTTCACCACCATG-3', and the reverse primer was 5'-GCTTCACCACCTTCTTGATGTCA-3'. These primer sequences were identical to the human target genes as confirmed by BLAST searches. PCR products were analyzed on 2% agarose gels. NHBEC, NHMEC, and normal trachea were used as normal controls for RT-PCR.

5-Aza-2'-Deoxycytidine (5-Aza-CdR) Treatment
A total of 23 tumor cell lines with RRAD hypermethylation and no gene expression were incubated in culture medium with 5-Aza-CdR at a concentration of 4 µM for 6 days. Medium changes were made on days 1, 3, and 5. The cells were harvested and RNA was extracted on day 6.

DNA Extraction and Methylation-Specific PCR
Genomic DNA was obtained from cell lines, primary tumors, and nonmalignant cells by digestion with proteinase K (Life Technologies) and subsequent phenol/chloroform (1:1) extraction.22 The DNA methylation pattern in the CpG island of RRAD was determined using the method of methylation-specific PCR (MSP), as reported by Herman and collaborators.23 Primer sequences of RRAD for the methylation reaction were 5'-GGTTGTAGTAGTAGCGGCGGCG-3' (forward) and 5'-ATCTACAACCGCCCCGACCCCG-3' (reverse) and for the unmethylated reaction were 5'-GTTGTAGTAGTAGTGGTGGTG-3' (forward) and 5'-TCTACAACCACCCCAACCCCA-3' (reverse). Briefly, 1 µg of genomic DNA was denatured by NaOH and modified by bisulfite. The modified DNA was purified with a Wizard DNA purification kit (Promega, Madison, WI), treated with NaOH to desulfonate, precipitated with ethanol and resuspended in water. PCR amplification was carried out with bisulfite-treated DNA as a template using specific primer sequences for the methylated and unmethylated forms of the gene. DNA from peripheral blood lymphocytes (n = 10) from healthy nonsmoking subjects was used as a negative control for methylation-specific assays. DNA from lymphocytes of a healthy volunteer, used as a positive control for methylated alleles, was treated with Sss1 methyltransferase (New England BioLabs, Beverly, MA) and then subjected to bisulfite treatment. Water blanks were included with each assay. Results were confirmed by repeating the bisulfite treatment and MSP for all samples.

Map of 5' franking Region of RRAD and Bisulfite DNA Sequencing
The locations of the CpG dinucleotides, MSP amplicon (RMSP), and bisulfite DNA sequencing (RBSSQ) in the RRAD 5' region are shown in Fig. 3AGo. Bisulfite-treated DNA of RBSSQ was amplified by means of PCR using the primers 5'-TTGGTGGGGG TGGATAGATA-3' (forward) and 5'-CCTCCCCCA ACCCCCAAAT-3' (reverse). These primers were designed to exclude binding to any CpG dinucleotide to ensure amplification of both methylated and unmethylated sequences. PCR products were cloned into plasmid vectors using the Topo TA cloning kit (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. Subclonal bisulfite sequencing of each clone for the RRAD promoter using human mesothelial cells has previously been reported by us.10 We decided to mix up five clones in this study. Five positive clones were purified using the Wizard Plus miniprep kit (Promega), then sequenced using the Applied Biosystems PRISM dye terminator cycle sequencing method (Perkin-Elmer Corp., Foster City, CA). This 5' region included the MSP primer sites and amplicon and encompassed 30 CpG dinucleotides.


Figure 3
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  		FIG. 3. Representative examples of MSP assay for RRAD in cell lines and primary tumors. Lung cancer (T), matched nonmalignant lung tissue (N), breast cancer (T), matched nonmalignant breast tissue (N). RRAD M RRAD-methylated form, RRAD U RRAD-unmethylated form, M size marker, POS positive control (artificially methylated DNA), NEG negative control (water blank). A visible band indicates amplification of methylated form. Because of contamination of normal tissues, either the unmethylated band only or both the methylated and unmethylated bands were present in primary samples. Each underlined "T" and "N" is patient matched.

 
Data Analysis
Statistical differences between groups were examined using Fisher’s exact test, the chi-square test, and the Mann-Whitney test. Survival was calculated from the date of initial diagnosis until death or the date of the last follow-up (censored). Survival was analyzed according to the Kaplan-Meier method, and differences in survival distributions were evaluated by means of the log-rank test. The Cox proportional hazards model was applied for multivariate analysis. A P value of less than .05 was defined as statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of RRAD in Cell Lines and the Effect of 5-Aza-CdR Treatment
Expression of RRAD in cell lines was examined by means of RT-PCR, and representative results are shown in Fig. 1AGo. RRAD expression was present in NHBEC, normal trachea, and NHMEC. In contrast, loss of RRAD expression was observed in 14 of 20 (70%) NSCLC cell lines, 11 of 11 (100%) SCLC cell lines, and 8 of 10 (80%) breast cancer cell lines. A total of 14 NSCLC cell lines, 3 SCLC cell lines, and 6 breast cancer cell lines that showed loss of expression of the RRAD gene were cultured with 5-Aza-CdR (4 µM, 6 days); RRAD expression was restored after treatment in all of these 23 cell lines tested (Fig. 1BGo).


Figure 1
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  		FIG. 1. A,B Representative examples of RT-PCR for RRAD expression in lung and breast cancer cell lines (A) and the effect of 5-Aza-CdR treatment on RRAD-negative cell lines (B). Treatment with 5-Aza-CdR restored expression of RRAD in four cell lines. Expression of the housekeeping gene GAPDH was run as a control for RNA integrity. M size maker, NEG negative control (genomic DNA), – before 5-Aza-CdR treatment, + after 5-Aza-CdR treatment.

 
Bisulfite Genomic DNA Sequencing
Using methylation-independent primers, we amplified and sequenced the 5' region of the RRAD gene to select the MSP primer sequences (Fig. 2Go). The sequence size of 331 bp contained 30 CpG sites and included exon 1. The translation start site was in exon 1. The percentage G+C was 70.7%, and the observed CpG was 0.79. Thus, this region satisfied the criteria for a CpG island.24 Four NSCLC cell lines (NCI-H1299, HCC95, NCI-H515 and NCI-H2122) and one SCLC cell line (NCI-H211) that showed loss of RRAD expression and also had this expression restored by 5-Aza CdR treatment were methylated moderately at the 30 CpG sites. Two breast cancer cell lines (HCC1954 and HCC202) that showed a loss of expression that was restored by 5-Aza CdR treatment were fully methylated. In contrast, NCI-H358 and NCI-H2347 NSCLC cell lines that showed RRAD expression, lymphocytes from a healthy volunteer, and two nonmalignant lung tissues from the lung cancer patients were only partially methylated. Based on the results of the sequence of the bisulfite-treated DNA, we developed a MSP assay that can distinguish methylated and unmethylated DNA and correlates with mRNA expression. The sizes of methylated and unmethylated alleles are 146 bp and 144 bp, respectively (GenBank accession number U46165; methylated, forward, nucleotides 1722–1743; reverse, nucleotides 1841–1867; unmethylated, forward, nucleotides 1723–1743; reverse, nucleotides 1841–1866).


Figure 2
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  		FIG. 2. Bisulfite sequence analysis of 5' region of RRAD gene. The location and methylation status of CpG dinucleotides in the region for the bisulfite genomic DNA sequence (RBSSQ) of the RRAD gene (GenBank accession number U46165, 1600-1925). Plus number indicates the nucleotide position from the transcription start site and the arrow indicates the transcription start site (TSS). Thin vertical lines and the numbers indicate positions of CpG dinucleotide in the RBSSQ. The horizontal closed bars between numbers indicate the positions of CpG dinucleotide included in MSP primers (RRADMF and RRADMR). At least five independent clones were screened for each cell line and nonmalignant tissue. Open circles indicate unmethylated CpG sites, circles with diagonal lines indicate partially methylated CpG sites, and filled circles indicate fully methylated CpG sites. Plus (+) indicates positive for MSP or RT-PCR, and minus (–) indicates negative for MSP or RT-PCR NS non-small cell lung cancer, S small cell lung cancer, B breast cancer, NML nonmalignant lung tissue, M MSP using methylated-specific primers, U MSP using unmethylated-specific primers, ND not done.

 
DNA Methylation in Cell Lines
Results of measurements of DNA methylation of RRAD in cell lines are detailed in Table 1Go, and representative examples are illustrated in Fig. 3Go. DNA methylation was absent in DNA from peripheral blood lymphocytes from volunteers (n = 10), NHBEC, and NHMEC. DNA methylation was found in 13 of 20 (65%) NSCLC cell lines, 11 of 11 (100%) SCLC cell lines, and 8 of 10 (80%) breast cell lines. All 23 cell lines that showed loss of expression and had expression restored after 5-Aza-CdR treatment revealed methylation of RRAD. Only the methylated or unmethylated forms of the gene were present in 38 of 41 (93%) cell lines, and both forms were present in the other 3 cell lines that showed loss of expression. The concordance between loss of gene expression and DNA methylation of RRAD was 95% in NSCLC cell lines and 100% in both SCLC and breast cancer cell lines. These data established the validity of the MSP assay as a convenient and specific method to examine RRAD gene methylation status and its relationship to gene expression.


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  		TABLE 1. Methylation and expression of RRAD in tumor cell lines
 
DNA Methylation of RRAD in Primary Tumors
We next determined the methylation status of RRAD in primary tumors with the MSP assay. Methylation measurements of RRAD in primary tumors and normal tissues are detailed in Table 2Go and Table 3Go, and representative examples are illustrated in Fig. 3Go. For 114 resected cases in which corresponding nonmalignant lung and breast tissues were available, RRAD methylation was present in 7 (6%). In all cases, the corresponding tumors had RRAD methylation. RRAD methylation was a tumor-specific event in lung cancers (P < .0001) and breast cancers (P < .0001) when compared with corresponding adjacent nonmalignant tissues.


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  		TABLE 2. Methylation of RRAD in lung cancers
 

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  		TABLE 3. Methylation of RRAD in breast cancers
 
Of 214 NSCLCs and 6 SCLCs, 84 (39%) and 5 (83%), respectively, were found to be methylated. There were no significant associations with gender, age, histological types, or stage. RRAD methylation was associated with smoking history (P = .001). More frequent RRAD methylation was shown by SCLCs than by NSCLCs (P = .04). In adenocarcinoma cases (n = 115), RRAD was methylated in 14 of 55 (25%) never smokers and in 29 of 60 (48%) smokers (P = .01). Furthermore, RRAD methylation was significantly associated with a poor prognosis in adenocarcinoma cases, as estimated using the log rank test (P = .01) (Fig. 4Go). Cox proportional hazard regression analysis was done to determine whether RRAD methylation is an independent prognostic factor (Table 4Go). Adenocarcinoma cases with RRAD methylation had a significantly poorer prognosis than those without RRAD methylation (hazard ratio=2.23, P = .03). There was no relationship between prognosis and the RRAD methylation of any other NSCLCs or SCLC. Of 63 breast cancers, 39 (62%) showed RRAD methylation. However, there were no significant associations with age, stage, estrogen receptor, or progesterone receptor status (Table 3Go).


Figure 4
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  		FIG. 4. Kaplan-Meier curve of overall survival for 115 adenocarcinoma cases. Patients with methylation of the RRAD gene had poorer survival than those without methylation, with a statistically significant difference (P = .01) by the log-rank test.

 

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  		TABLE 4. Univariate and Multivariate statistics of the prognostic value of gender, age, smoking history, stage, and RRAD methylation for overall survival in lung adenocarcinomas (n=115)
 
Correlation between RAS Gene Status and RRAD in NSCLC Cell Lines
The Ras mutational status of 12 NSCLC cell lines had been previously examined by direct sequencing, and 3 tumor cell lines had Ras gene alterations. Three cell lines (75%) showed RRAD methylation in Ras gene altered cell lines (NCI-H157, NCI-H460, NCI-H1299 and NCI-H2122), and 7 cell lines (88%) showed RRAD methylation in Ras wild-type cell lines. Thus, there was no correlation between Ras gene status and RRAD methylation in tumor cell lines (P = .6).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Based on the results of microarray analysis before and after treatment with a demethylating agent in lung cancer cell lines, we speculated that the RRAD gene may be downregulated by methylation in lung cancers. In this study, we demonstrated that inactivation of RRAD mRNA expression occurs in lung and breast cancer cell lines and is correlated with methylation of the gene. RRAD expression was restored after treatment with a demethylating agent. These results indicate that methylation was the likely mechanism by which RRAD mRNA expression was suppressed. The concordance between methylation and loss of gene expression and the sequencing studies after bisulfite treatment strongly support this concept. To our knowledge, this is the first report to examine the loss of RRAD expression in lung cancer cells and demonstrates that the silencing of the RRAD was due to methylation in lung and breast cancer cells.

We developed a MSP assay to conveniently examine RRAD methylation in primary tumor samples. The tumors showed relatively frequent methylation of RRAD. Methylation frequencies were very high in SCLC tumors and breast cancers and more modest in NSCLC tumors. In our previous study, frequencies of methylation of p16 and APC were significantly higher in smokers than in never smokers.25 The higher frequency of RRAD methylation in smokers suggests that smoking may affect the methylation of RRAD in lung cancers. We also studied the prognostic significance and found that RRAD methylation was a negative prognostic factor in lung adenocarcinomas. These correlations between RRAD methylation and clinical parameters suggest that RRAD methylation may play a role in lung cancer pathogenesis.

Tseng et al. reported two types of deregulation of the RRAD gene in breast cancers. One was downregulation of the gene found in 73% of breast cancers, and the other upregulation in a small number of tumors with its high expression correlated with tumor-invasive characteristics.6 In our series, 80% of breast cancer cell lines lost RRAD expression, and 62% of tumors showed aberrant methylation. Thus, our data is consistent with Tseng’s with regard to frequent downregulation of RRAD in breast cancers. However, the reason for upregulation of the gene in less-frequent advanced breast cancers in their data, as an oncogenic effect, remains unclear. Thus, the exact role of the RRAD gene regarding the carcinogenesis of breast cancer should be addressed in future work.

In conclusion, our results demonstrated methylation of the RRAD gene in human lung and breast cancers as a molecular pathway underlying the inactivation of this gene in these cancers. Smoking history correlates with methylation of RRAD in lung cancers and RRAD methylation may affect survival of patients with lung adenocarcinomas. These findings are of biological interest for study of the pathogenesis of cancer and are of clinical importance.


   ACKNOWLEDGMENTS
 
This work was supported by grants from the University of Texas Specialized Program of Research Excellence in Lung Cancer (NCI P50CA70907) and Early Detection Research Network (5U01CA8497102), the Gillson Longenbaugh Foundation, and the Emphasis Research Project by expenditure at the discretion of the president of The Chiba University in 2005.

Received for publication December 13, 2005. Accepted for publication March 1, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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