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Clinical Cancer Research Vol. 8, 2362-2368, July 2002
© 2002 American Association for Cancer Research

Experimental Therapeutics, Preclinical Pharmacology

Clinicopathological Significance of Epigenetic Inactivation of RASSF1A at 3p21.3 in Stage I Lung Adenocarcinoma1

Yoshio Tomizawa, Takashi Kohno, Haruhiko Kondo, Ayaka Otsuka, Michiho Nishioka, Toshiro Niki, Tesshi Yamada, Arafumi Maeshima, Kimio Yoshimura, Ryusei Saito, John D. Minna and Jun Yokota2

Biology Division [Y. T., T. K., A. O., M. N., J. Y.], Pathology Division [T. N., T. Y., A. M.], and Cancer Information and Epidemiology Division [K. Y.], National Cancer Center Research Institute and Thoracic Surgery Division [H. K.], National Cancer Center Hospital, Tokyo 104-0045, Japan; First Department of Internal Medicine, School of Medicine, University of Gunma, Gunma, 371-8511, Japan [Y. T., R. S.]; and Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, Texas 75390 [J. D. M.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: Chromosome 3p is deleted frequently in various types of human cancers, including lung cancer. Recently, the RASSF1A gene was isolated from the 3p21.3 region homozygously deleted in lung and breast cancer cell lines, and it was shown to be inactivated by hypermethylation of the promoter region in lung cancers. In this study, we investigated the pathogenetic and clinicopathological significances of RASSF1A methylation in the development and/or progression of lung adenocarcinoma.

Experimental Design: Association of RASSF1A methylation with clinicopathological features, allelic imbalance at 3p21.3, p53 mutations, and K-ras mutations was examined in 110 stage I lung adenocarcinomas.

Results: Thirty-five of 110 (32%) tumors showed RASSF1A methylation. RASSF1A methylation was dominantly detected in tumors with vascular invasion (P = 0.0242) or pleural involvement (P = 0.0305), and was observed more frequently in poorly differentiated tumors than in well (P = 0.0005) or moderately (P = 0.0835) differentiated tumors. Furthermore, RASSF1A methylation correlated with adverse survival by univariate analysis (P = 0.0368; log-rank test) as well as multivariate analysis (P = 0.032,; risk ratio 2.357; 95% confidence interval, 1.075–5.169). The correlation between RASSF1A methylation and allelic imbalance at 3p21.3 was significant (P = 0.0005), whereas the correlation between RASSF1A methylation and p53 mutation was borderline (P = 0.0842). However, there was no correlation or inverse correlation between RASSF1A methylation and K-ras mutation (P = 0.2193).

Conclusions: These results indicated that epigenetic inactivation of RASSF1A plays an important role in the progression of lung adenocarcinoma, and that RASSF1A hypermethylation appears to be a useful molecular marker for the prognosis of patients with stage I lung adenocarcinoma.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recent advances in the molecular genetics of human cancers have revealed that multiple TSGs3 are involved in human lung carcinogenesis (1) . Chromosome 3p allele loss is a frequent event in the development of lung cancer (2, 3, 4, 5) . Multiple 3p regions have been identified as showing frequent allelic losses in lung and other cancers by detailed allelotyping studies. This suggests that there are several TSGs on 3p (5, 6, 7) . Homozygous deletions have been found at 3p21.3 in lung cancer and breast cancer cell lines, and the 630-kb 3p21.3 homozygous deletion region has been studied extensively (8) . The RASSF1 gene was isolated from this region as a candidate TSG (9, 10, 11) . The RASSF1 locus encodes several major transcripts by alternative promoter selection and alternative mRNA splicing. RASSF1A, one of the several transcripts, encodes a Mr 39,000 predicted peptide with a Ras association domain and a predicted NH2-terminal diacylglycerol binding domain. Transfection and expression of RASSF1A in lung cancer cells resulted in suppression of colony formation, anchorage-independent soft agar growth, and nude mouse tumorigenicity (9 , 10) . Mutations of RASSF1A are uncommon, whereas the lack of expression is common in lung cancer (9 , 10) . RASSF1A was expressed in normal bronchial epithelial cells but was not expressed in 80–100% of small cell lung cancer and 30–60% of NSCLC (9 , 10 , 12 , 13) . A putative promoter region of the RASSF1A gene was highly methylated in lung tumors but not in normal lung tissues, and methylation of this region was associated with reduction of RASSF1A transcription (9 , 10) . It was reported recently that RASSF1A methylation in 107 resected NSCLCs was associated with impaired patient survival (10) . However, because the stage of the disease was not adjusted, it is still unclear whether the RASSF1A methylation is an independent prognostic factor and how RASSF1A methylation correlates with clinicopathological features. Therefore, it is important to examine RASSF1A methylation in a large number and defined subset of primary lung cancers to assess the role of RASSF1A expression in multistage lung carcinogenesis.

Lung cancer is a major cause of cancer-related deaths in the world. In recent years, adenocarcinoma has replaced squamous cell carcinoma as the most frequent histological subtype in lung cancers (14 , 15) . Prognosis of patients is largely dependent on the stage of the disease. However, even in patients with stage I NSCLC, the 5-year survival rate is ~65% (16 , 17) . Because there are only a few prognostic parameters that can estimate survival, it is important to identify a marker for high-risk early stage patients who should benefit from more aggressive treatment approaches.

Here, we investigated the association of RASSF1A methylation with clinicopathological features in 110 cases of stage I lung adenocarcinoma by the MSP method. RASSF1A methylation was detected dominantly in tumors with vascular invasion or pleural involvement, and was observed more frequently in poorly differentiated tumors than in well-differentiated tumors. Furthermore, RASSF1A methylation correlated with adverse survival in patients with stage I lung adenocarcinoma. The results indicated that epigenetic inactivation of RASSF1A plays an important role in the acquisition of aggressive phenotypes in lung adenocarcinoma and that RASSF1A methylation is a marker for prognosis of patients with stage I lung adenocarcinoma.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients and Tissues.
Primary lung tumors and corresponding normal lung tissues were obtained from 110 patients with lung adenocarcinoma. All of the patients underwent curative resection with systematic mediastinal lymph node dissection at the National Cancer Center Hospital, Tokyo, Japan, from December 1986 to December 1996. None of the patients had been treated before operation. All of the cases were pathologically diagnosed as being stage I lung adenocarcinoma according to the Tumor-Node-Metastasis classification of malignant tumors (18) . Detailed information on these patients is summarized in Table 1Citation . The median follow-up period in all of the patients was 101 months. Tumors and normal tissues were frozen and stored at -80°C until DNA extraction. Genomic DNAs were prepared by the method described previously (4) or by a QIAamp DNA Mini kit (Qiagen, Tokyo, Japan).


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  		Table 1 Correlation of RASSF1A methylation with clinicopathological characteristics in patients with stage I lung adenocarcinomas

 
Methylation Analysis of RASSF1A.
The methylation status of the RASSF1A was analyzed by MSP, as described previously (10) , using bisulfite modified genomic DNA (19 , 20) . Briefly, 1 µg of genomic DNA was denatured with 0.2 M NaOH, and 10 mM hydroquinone (Sigma) and 3 M sodium bisulfite (Sigma) were added and incubated at 55°C for 16 h. Afterward, modified DNA was purified using Wizard DNA purification resin (Promega) followed by ethanol precipitation. Treatment of genomic DNA with sodium bisulfite converts unmethylated cytosines (but not methylated cytosines) to uracil, which are then converted to thymidine during the subsequent PCR step, giving sequence differences between methylated and unmethylated DNA. PCR primers that distinguish between these methylated and unmethylated DNA sequences were then used. The primers to detect the methylated form were 5'-GGGTTTTGCGAGAGCGCG-3' (forward) and 5'-GCTAACAAACGCGAACCG-3' (reverse), and the primers to detect the unmethylated form were 5'-GGTTTTGTGAGAGTGTGTTTAG-3' (forward) and 5'-CACTAACAAACACAAACCAAA-3' (reverse). MSP cycling conditions consisted of one incubation of 15 min at 95°C, followed by 35 cycles of a 30 s denaturation at 94°C, 50 s at an annealing temperature (64°C for methylation-specific and 59°C for unmethylated-specific primers), a 30 s extension at 72°C, and a final extension at 72°C for 10 min. PCR products were separated in 2% agarose gels and visualized under UV illumination.

AI Analysis of the RASSF1A Locus.
The following microsatellite loci were examined for AI at 3p21.3: D3S4622/LUCA4.1, D3S4604/LUCA19.1, and D3S4597/P1.5, because these markers were mapped to the regions flanking and close to the RASSF1 gene locus (5) . Primers were end labeled with fluorochromes FAM, HEX, or NED. PCR amplifications were carried out in a 20 µl reaction mixture containing 10 mM Tris HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 10 pmol of each primer, 250 µM of each deoxynucleotide triphosphate, and 0.5 unit of Taq DNA polymerase (Takara, Osaka, Japan). Thirty-five cycles of 94°C (30 s) for denaturation, 55°C (30 s) for annealing, and 72°C (30 s) for extension were performed to amplify DNA fragments. The loading mix was prepared with 12 µl of deionized formamide and 0.5 µl of ABI size standard 400HD (Perkin-Elmer). Each loading sample contained this loading mix plus 0.33 µl each of FAM-, HEX-, and NED-labeled PCR products. The fluorescent signals were laser scanned, and the data were stored electronically by the ABI310 sequencer/genotyper. The intensity of the peak heights was calculated by the Genotyper2.1 software to ascertain the ratios of allele intensities in tumor DNA as compared with the corresponding normal tissue DNA. Normal tissue samples showed small differences in the percentage of allele ratio because of artificial signal variation (SD 5.4%, n = 40). Thus, a signal ratio in the range of 0.76–1.0 could occur even without AI in a tumor because the mean -2 SD per mean +2 SD was 0.76. Therefore, a sample was scored as having AI if the signal ratio was <0.76.

Mutational Analysis of the K-ras Gene.
Codons 12, 13, and 61 of the K-ras gene were examined for mutation by PCR amplification and direct sequencing of PCR products. The primer sets for codons 12 and 13 of the K-ras gene were 5'-GACTGAATATAAACTTGTGG-3' (forward) and 5'-CTATTGTTGGATCATATTCG-3' (reverse), and the primer sets for codon 61 were 5'-TTCCTACAGGAAGCAAGTAG (forward) and 5'-CACAAAGAAAGCCCTCCCCA-3' (reverse), as reported previously (21) . Thirty-five cycles of 94°C (30 s) for denaturation, 55°C (60 s) for annealing, and 72°C (30 s) for extension were performed to amplify DNA fragments for codons 12 and 13 of the K-ras gene. Touchdown PCR was used for the amplification of DNA fragment for codon 61 of the K-ras gene. Touchdown PCR had an initial denaturation step at 94°C for 5 min, which was followed by 10 touchdown cycles of 30 s at 94°C, 1 min at 60°C with a decrease in the annealing temperature by 1°C each cycle, and 30 s at 72°C. This was followed by 25 cycles of 30 s at 94°C, 1 min at 50°C, and 30 s at 72°C, with a final extension at 72°C for 10 min. PCR products were purified for sequencing by a QIAquick PCR Purification kit (Qiagen). Cycle sequencing was performed using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction kit (Perkin-Elmer, Norwalk, CT) and the ABI PRISM 310 DNA Sequencer (Perkin-Elmer).

Mutational Analysis of the p53 Gene.
Ten portions of genomic DNA fragments covering the entire coding region of the p53 gene, between exon 2 and exon 11, were amplified by PCR with p53-specific oligonucleotide primers: 5'-TTGGAAGTGTCTCATGCTGG-3' (forward) and 5'-GTGGTGGCCTGCCCTTCC-3' (reverse) for exon 2, 5'-CCCCTAGCAGAGACCTGTG-3' (forward) and 5'-CCCTCCAGGTCCCCAGC-3' (reverse) for exon 3, 5'-GAGGACCTGGTCCTCTGAC-3' (forward) and 5'-CCAGGCATTGAAGTCTCATG-3' (reverse) for exon 4, 5'-ACTTGTGCCCTGACTTTCAAC-3' (forward), and 5'-CAACCAGCCCTGTCGTCTC-3' (reverse) for exon 5, 5'-AGGGTCCCCAGGCCTCTG-3' (forward) and 5'-AACCCCTCCTCCCAGAGAC-3' (reverse) for exon 6, 5'-GCGCACTGGCCTCATCTTG-3' (forward) and 5'-GCCAGTGTGCAGGGTGGC-3' (reverse) for exon 7, 5'-GGTAGGACCTGATTTCCTTAC-3' (forward) and 5'-TCTCCTCCACCGCTTCTTG-3' (reverse) for exon 8, 5'-GTGCAGTTATGCCTCAGATTC-3' (forward) and 5'-AAACTTTCCACTTGATAAGAGG-3' (reverse) for exon 9, 5'-ATCTTTTAACTCAGGTACTGTG-3' (forward) and 5'-GAGTAGGGCCAGGAAGGG-3' (reverse) for exon 10, and 5'-ACCCTCTCACTCATGTGATG-3' (forward) and 5'-GATGGGGGTGGGAGGCTG-3' (reverse) for exon 11. PCR amplification was carried out in a 25-µl reaction mixture containing 0.6 unit of TaKaRa Ex Taq (Takara) for exons 4 and 5, and AmpliTaq Gold (Perkin-Elmer) for exons 2, 3, and 6–11. Thirty-five cycles of 94°C (60 s) for denaturation, 60°C (60 s) for annealing, and 72°C (90 s) for extension were performed to amplify DNA fragments for exons 4 and 5. Thirty-five cycles (for exons 7 and 10) or 40 cycles (exons 2, 3, 6, 8, 9, and 11) of 94°C (20 s) for denaturation, 58°C (30 s) for annealing, and 72°C (30 s) for extension were performed to amplify DNA fragments. The sizes of PCR products were from 99 to 367 bp. Denatured and reannealed PCR products were analyzed by the WAVE DNA Fragment Analysis System and WAVEMAKER Software 4.0 (TRANSGENOMIC Inc., Omaha, NE). PCR products with detected variant peaks by the WAVE DNA Fragment Analysis System were purified by a QIAquick PCR Purification kit (Qiagen) for sequencing. Cycle sequencing was performed using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction kit (Perkin-Elmer) and the ABI PRISM 310 DNA Sequencer (Perkin-Elmer).

Statistical Analysis.
Fisher’s exact test was used to examine the association of two categorical variables. The survival curves were estimated by the Kaplan-Meier method, and the resulting curves were compared using the log-rank test. A P of <0.05 was considered to be statistically significant. The joint effect of covariables was examined using the stepwise Cox proportional hazards regression model. The stepwise procedure was used to select significant independent variables. The probability of the statistic used to test whether a variable should be included or excluded was 0.2. Statistical analysis was performed using SPSS for Windows statistical package (SPSS Inc., Chicago, IL).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Correlation between RASSF1A Methylation and Clinicopathological Characteristics of Stage I Lung Adenocarcinoma.
One hundred and ten stage I lung adenocarcinomas were examined for RASSF1A methylation by MSP (10) . The site examined by MSP was the one of which the methylation was associated with reduced RASSF1A expression (10) . Representative results are shown in Fig. 1ACitation . RASSF1A methylation was detected in 35 of 110 tumors (32%). We then analyzed the relationship between RASSF1A methylation and clinicopathological characteristics of these patients. The results are summarized in Table 1Citation . RASSF1A methylation was observed preferentially in poorly differentiated subtypes. The difference in frequency between poorly and well differentiated was statistically significant (P = 0.0005), and the difference between poorly and moderately differentiated was near significance (P = 0.0835). RASSF1A methylation was detected dominantly in tumors with vascular invasion (P = 0.0242) or pleural involvement (P = 0.0305). There was no significant correlation of RASSF1A methylation with gender, age, smoking history, T stage, and lymphatic permeation within the tumor.



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  		Fig. 1. A, methylation analysis of RASSF1A in primary stage I lung adenocarcinoma by MSP. U, amplified product with primers recognizing unmethylated sequences; M, amplified product with primers recognizing methylated sequences. NCI-H1299 is used as a positive control for the methylated form. NCI-H2009 is used as a positive control for the unmethylated form. B, AI analysis at 3p21.3 in primary lung adenocarcinoma. Arrows indicate AI at D3S4597/P1.5 (left) and at D3S4604/LUCA19.1 (right) in tumor (No10–22). C, mutational analysis of the p53 gene by the WAVE DNA Fragment Analysis System. Variant peak is indicated by an arrow. The mutation of the p53 gene in this sample (No10–22) was confirmed by sequencing [Arg (AGG) to Met (ATG) at codon 249].

 
Correlation of RASSF1A Methylation with AI at 3p21.3, p53 Mutations, and K-ras Mutations in Stage I Lung Adenocarcinoma.
AI at 3p21.3 was examined in 110 stage I adenocarcinomas by PCR amplification of three polymorphic loci. Representative results are shown in Fig. 1BCitation . The numbers of patients informative for each polymorphic locus were 78 cases (71%) for D3S4622/LUCA4.1, 84 cases (76%) for D3S4604/LUCA19.1, and 71 cases (65%) for D3S4597/P1.5, respectively. The frequencies of AI at these loci were 34 of 78 (44%) at D3S4622/LUCA4.1, 34 of 84 (40%) at D3S4604/LUCA19.1, and 24 of 71 (34%) at D3S4597/P1.5, respectively. In total, 103 of 110 (94%) cases were informative for the allelic status of 3p21.3, and the frequency of AI was 42 of 103 (41%). These tumors were also examined for mutations in exons 2–11 of the p53 gene by the WAVE DNA Fragment Analysis System. Representative results are shown in Fig. 1CCitation . p53 mutations were detected in 39 of 110 cases (35%). Because 2 of 39 cases showed a silent p53 mutation, we counted 37 of 110 as cases with p53 mutation in this analysis. We also examined these 110 tumors for mutations at codons 12, 13, and 61 of the K-ras gene. Mutations in the K-ras gene were detected in 13 tumors (12%): 11 in codon 12, 1 in codon 13, and 1 in codon 61. The frequencies of AI at 3p21.3, p53 mutations, and K-ras mutations in primary lung adenocarcinomas in this study were comparable with those in previous studies (5 , 21, 22, 23, 24) .

We then analyzed the correlation of RASSF1A methylation with AI at 3p21.3, p53 mutations, and K-ras mutations (Table 2)Citation . Among 103 informative cases, 22 of 42 (52%) tumors with AI at 3p21.3 showed RASSF1A methylation, whereas 11 of 61 (18%) tumors without AI at 3p21.3 showed RASSF1A methylation. The correlation between RASSF1A methylation and AI at 3p21.3 was statistically significant (P = 0.0005). RASSF1A methylation was detected in 16 of 37 (43%) tumors with p53 mutations and detected in 19 of 73 (26%) tumors without p53 mutations. The statistical significance of the correlation between RASSF1A methylation and p53 mutation was borderline (P = 0.0842). We found K-ras mutations in 13 tumors. Among the 13 tumors, 11 (85%) tumors did not show RASSF1A methylation, whereas 2 (15%) tumors showed RASSF1A methylation. However, there was no statistically significant correlation or inverse correlation between RASSF1A methylation and K-ras mutation (P = 0.2193).


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  		Table 2 Correlation of RASSF1A methylation with genetic alterations in stage I lung adenocarcinoma

 
Correlation between RASSF1A Methylation and Prognosis of Patients with Stage I Lung Adenocarcinoma.
We analyzed the effect of RASSF1A methylation on patient survival. Kaplan-Meier survival estimates showed that the patients with RASSF1A methylation tended to have a poorer survival than the patients without RASSF1A methylation (Fig. 2)Citation . The difference in the prognosis of these two groups was statistically significant (P = 0.0368; log-rank test). Univariate survival analysis of clinicopathological factors showed that gender was significantly associated with survival, and age and T stage were associated with survival although they did not reach statistical significance (Table 3)Citation . Smoking history, differentiation, vascular invasion, lymphatic permeation, pleural involvement, p53 mutation, K-ras mutation, and AI at 3p21.3 did not account for the adverse survival (Table 3)Citation . Subsequently, a multivariate stepwise Cox proportional hazard regression analysis was performed using all of these clinicopathological and molecular factors. RASSF1A methylation was a significantly unfavorable prognosis factor independent of other clinicopathological and molecular factors (P = 0.032; risk ratio 2.357; 95% CI, 1.075–5.169; Table 3Citation ). Age was also an unfavorable prognosis factor; however, no other factor was considered as being an independent prognostic marker (P > 0.05; Table 3Citation ).



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  		Fig. 2. Kaplan-Meier survival curves for patients with stage I lung adenocarcinoma classified according to RASSF1A methylation and the resulting curves were compared using the log-rank test (P = 0.0368). The tick mark indicates the follow-up period of each patient.

 

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  		Table 3 Overall survival of patients with stage I lung adenocarcinoma in relation to molecular and clinicopathological characteristics

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We demonstrate here that RASSF1A methylation is significantly associated with a poorer survival of patients with stage I lung adenocarcinoma. It was reported recently that RASSF1A promoter hypermethylation in resected NSCLCs was associated with impaired patient survival (10) ; thus, the present study agrees with this report. In the previous study, patients with NSCLCs of various stages and histological types were analyzed (10) , whereas in this study, patients with stage I lung adenocarcinoma were exclusively analyzed. These results suggest that tumors with RASSF1A methylation are more aggressive than tumors without RASSF1A methylation in lung adenocarcinoma. In fact, RASSF1A methylation was more commonly detected in the poorly differentiated type than in the well- or moderately differentiated types, suggesting that RASSF1A methylation is related to the degree of differentiation or the stage of progression in the adenocarcinoma. Furthermore, RASSF1A methylation was detected dominantly in tumors with vascular invasion or pleural involvement. These results also indicate the tumors with RASSF1A methylation are more aggressive than tumors without RASSF1A methylation.

Noguchi et al. (25) have reported histological subtypes for the small size lung adenocarcinoma measuring <=2 cm in the greatest dimension. In this report, type C (LBAC with foci of active fibroblastosis) is considered to be an advanced carcinoma, which is progressed from type A (LBAC) or type B (LBAC with foci of collapsed alveolar structure), and patients with type D (poorly differentiated adenocarcinoma) have a worse prognosis than patients with types A, B, and C. We classified small adenocarcinomas, up to 2 cm in dimension, used in the present study according to Noguchi’s classification (Table 1)Citation . Interestingly, RASSF1A methylation was detected in all 4 of the type D tumors, but not in any of 3 type A tumors, and RASSF1A methylation was more frequently detected in types C and D tumors (9 of 22; 41%) than in types A and B tumors (2 of 11; 18%), although the difference did not reach statistical significance (P = 0.2585; Table 1Citation ). Therefore, it is possible that inactivation of RASSF1A could be involved in a series of morphologically distinct changes in carcinogenesis and progression of lung adenocarcinoma.

The RASSF1A gene is located at chromosome 3p21.3 that frequently undergoes LOH in lung cancers. Recently, it was reported that two of three breast cancers with LOH at 3p21.3 had hypermethylation of the RASSF1A promoter region (26) . We demonstrated here that RASSF1A methylation significantly correlated with AI at 3p21.3 in lung adenocarcinoma (P = 0.0005). These results suggested that the RASSF1A gene is inactivated in accordance with the Knudson two-hit model, including epigenetic mechanisms of gene inactivation (27) . However, there were a number of tumors with RASSF1A methylation without AI at 3p21.3 and with AI at 3p21.3 without RASSF1A methylation. It was also reported that only 6 of 22 NSCLC with 3p21.3 allele loss had RASSF1A methylation (12) . In the cases with retention of 3p21.3 alleles with RASSF1A methylation, both alleles may be inactivated by hypermethylation of the promoter region of the RASSF1A gene. In the cases with AI at 3p21.3 alleles without RASSF1A methylation, other TSGs may be a target of AI. In fact, there are several candidate TSGs at 3p21.3 (8 , 28, 29, 30, 31) .

Recent studies have shown that several genes on chromosome 3p other than RASSF1 are also methylated in lung cancers. The SEMA3B gene located ~60 kb distal to RASSF1 was shown to be inactivated by methylation in lung cancer cell lines (31) . In addition, the FHIT gene at 3p14.2 and the RARß gene at 3p24 have been shown to be inactivated by methylation in primary lung cancers (32 , 33) . Therefore, it is likely that multiple genes on 3p are methylated in lung cancers. It is possible that the methylation of a gene among them correlates more strongly with AI on 3p as well as prognosis than that of RASSF1A. Additional genetic and functional studies should be done on those genes, including RASSF1A, to elucidate the pathogenetic significance of methylation of those genes for the development of lung cancers.

The presence of a Ras association domain in RASSF1A suggests that these proteins may function as effectors of Ras signaling (or signaling of a Ras-like molecule) in cells. Therefore, it was important to analyze the relationship between RASSF1A methylation and K-ras mutation in the current series of tumors. The present study showed that there was no significant correlation or inverse correlation between RASSF1A methylation and K-ras mutation. Therefore, it is possible that inactivation of RASSF1A does not activate K-ras signaling in tumorigenic mechanisms. However, because K-ras mutations were detected in only 13 cases, this negative correlation could be because of the few cases for statistical analysis. In fact, only 2 of 35 (6%) tumors with RASSF1A methylation showed K-ras mutations, implying that there may be an inverse correlation between RASSF1A methylation and K-ras mutation. It was reported that RASSF1C binds Ras in a GTP-dependent manner (11) . Because RASSF1A has the identical Ras associate domain, it is possible that RASSF1A binds to Ras in the same manner as RASSF1C. Therefore, additional investigations in a larger population and functional studies to assess the role of RASSF1A in Ras-dependent growth control will be required.

Multiple TSGs are involved in lung carcinogenesis (1) , and p53 plays a central role in tumor progression (34) . In the present study, we showed that RASSF1A methylation was weakly correlated with p53 mutations. These results suggest that epigenetic inactivation of RASSF1A and p53 mutations play a cooperative role in progression of lung adenocarcinoma. In fact, p53 mutations were also detected more frequently in types C and D tumors (8 of 22; 36%) than in types A and B tumors (0 of 11; 0%; data not shown). However, because RASSF1A methylation but not p53 mutation showed significant correlation with poor prognosis of patients with stage I adenocarcinoma, RASSF1A could play a more dominant role than p53 in the progression of lung adenocarcinoma.

In conclusion, RASSF1A methylation was significantly correlated with vascular invasion, pleural involvement, poor differentiation, and poor survival of patients with stage I lung adenocarcinoma. As expected, there was also a correlation between RASSF1A methylation and AI at 3p21.3. These results indicate that epigenetic inactivation of RASSF1A, which appears to be one of the target TSGs at 3p21.3, plays an important role in the progression of lung adenocarcinoma. Additional investigations will be required to elucidate the importance of RASSF1A methylation as a diagnostic and prognostic marker in the management of early stage lung adenocarcinoma and to elucidate the role of RASSF1A in cell signaling.


   ACKNOWLEDGMENTS
 
We thank Dr. Masatomo Mori, First Department of Internal Medicine, Gunma University, School of Medicine, Gunma, Japan, for his encouragement of this study.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by a Grant-in-Aid from the Ministry of Health, Labor, and Welfare for the 2nd-term Comprehensive 10-Year Strategy for Cancer Control; Grants-in-Aid from the Ministry of Health, Labor, and Welfare and from the Ministry of Education, Culture, Sports, Science and Technology, Japan; National Cancer Institute USA Grants Specialized Programs of Research Excellence P50 CA70907 and R01 CA71618; and the G. Harold and Leila Y. Mathers Charitable Foundation. Back

2 To whom requests for reprints should be addressed, at Biology Division, National Cancer Center Research Institute, 1-1, Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045, Japan. Phone: 81-3-3547-5272; Fax: 81-3-3542-0807. Back

3 The abbreviations used are: TSG, tumor suppressor gene; NSCLC, non-small cell lung cancer; CI, confidence interval; LOH, loss of heterozygosity; AI, allelic imbalance; MSP, methylation-specific PCR; LBAC, localized bronchioloalveolar carcinoma. Back

Received 11/29/01; revised 4/ 2/02; accepted 4/ 3/02.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Kohno T., Yokota J. How many tumor suppressor genes are involved in human lung carcinogenesis?. Carcinogenesis (Lond.), 20: 1403-1410, 1999.[Abstract/Free Full Text]
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