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Address reprint requests to Masayuki Noguchi, M.D., Department of Pathology, Institute of Basic Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
We previously have shown the aberrant overexpression of stratifin (SFN, 14-3-3 ς) in lung adenocarcinoma. Although SFN is known to facilitate tumor cell proliferation, the mechanism that underlies its aberrant expression has remained unclear. SFN, the downstream target of p53, often has been reported to be hypermethylated and subsequently silenced in certain cancers; however, its hypomethylation-linked reactivation has not yet been validated. In this study, we investigated the DNA methylation status of the SFN promoter region using 8 lung cancer cell lines and 32 specimens of adenocarcinoma tissue. Real-time methylation-specific PCR analysis showed that although both normal lung tissue and adenocarcinoma in situ bore a completely methylated SFN promoter, the promoter region in almost all invasive adenocarcinomas was at least partially methylated. The expression of SFN and its level of methylation were correlated strongly. Furthermore, statistical analysis revealed that the level of methylation became reduced with progression of the pathologic stage, although no clear relationship between methylation level and p53 abnormality was found. These results suggest that methylation-related silencing of SFN occurs in both normal lung tissues and adenocarcinoma in situ, and that demethylation of the SFN promoter participates in the aberrant expression of SFN in invasive adenocarcinoma cells, independently of p53 alteration. This novel finding might be informative for clarifying the mechanism that underlies the progression of early lung adenocarcinoma.
Despite recent advances in our understanding of the molecular mechanisms of carcinogenesis and the use of multimodal cancer therapy, lung cancer remains one of the major causes of cancer-related death worldwide.
Among the histologic subtypes of lung cancer, adenocarcinoma is the most common, and is increasing in frequency, accounting for almost half of all non-small cell lung carcinomas. The 5-year survival rate is generally <40%, and this high mortality rate is caused primarily by the difficulty in detecting the cancer at an early stage and its rapid progression. However, Noguchi et al
have shown the existence of adenocarcinoma in situ (AIS, Noguchi type A), showing an extremely favorable prognosis, with a 5-year survival rate of 100%. AIS progresses stepwise to early but invasive adenocarcinoma (eIA, Noguchi type C), showing a relatively poor outcome, with a 5-year survival rate of 75%. We previously compared the expression profiles of AIS with those of eIA associated with lymph node metastasis or a fatal outcome, and screened the differentially expressed genes by cDNA microarray.
We searched for the genes showing significantly higher expression in eIA than in AIS, and finally focused on stratifin (SFN, 14-3-3 σ).
SFN belongs to the 14-3-3 family of abundant, widely expressed 28- to 33-kDa acidic polypeptides that spontaneously self-assemble as homodimers or heterodimers. There are seven closely related genes, encoding β, ε, η, γ, τ, ζ, and σ isoforms, which are conserved across mammalian species. They can bind to >100 functionally diverse cellular proteins and thereby play important roles in various cellular processes such as signal transduction, cell-cycle regulation, apoptosis, cytoskeletal organization, and malignant transformation.
14-3-3 proteins regulate enzyme activity and may act as localization anchors, controlling the subcellular localization of proteins. In addition, 14-3-3 proteins can function as adaptors or scaffolds, stimulating protein-protein interaction.
These findings define SFN as a negative regulator of cell-cycle progression. Furthermore, in primary-cultured human epidermal keratinocytes, down-regulation of SFN allows the cells to overcome senescence.
Therefore, functional inactivation of SFN is thought to be linked to carcinogenesis. This hypothesis has been supported by many studies revealing SFN down-regulation in various human malignancies, including cancers of the breast, stomach, colon, liver, prostate, oral cavity, and vulva,
showed that, in breast cancer, SFN mediates cell-cycle progression via the phosphatidylinositol 3-kinase/Akt pathway in a p53-independent manner. These studies suggest that SFN might be a context-dependent gene, and that its functions may vary among organs or tissues. For lung adenocarcinoma in particular, we recently reported that SFN shows higher expression in eIA than in AIS, and functionally facilitates cell proliferation.
found that DNA hypermethylation occurs in small-cell (69%) and non-small cell (6%) lung cancer cell lines, and that SFN expression is regulated by promoter methylation. Even in cancers of the same organ, the expression status of SFN and its functions vary according to histologic type.
Hypermethylation of gene promoters often is associated with transcriptional silencing of tumor suppressors. However, hypomethylation of gene promoters is also a common event in cancer cells, and the two phenomena occur in parallel in a wide variety of cancer types.
These findings indicate that hypomethylation in the SFN promoter may contribute to the aberrant overexpression of SFN in adenocarcinoma cells. However, the molecular mechanisms that underlie DNA hypomethylation in tumorigenesis are poorly understood, and only a few studies have analyzed hypomethylation in primary cancers with the aim of exploring its clinical importance as a molecular marker.
In this study, we assessed methylation levels in the promoter region of SFN in lung adenocarcinoma to clarify whether DNA methylation regulates the expression of SFN during the course of malignant progression.
Materials and Methods
Cell Lines and Culture Conditions
Cell lines A549, PC-14, RERF-LC-KJ, and LC-2/ad were purchased from RIKEN Cell Bank (Ibaraki, Japan), and NCI-H23, Calu-3, Calu-1, and Calu-6 were purchased from the American Type Culture Collection (ATCC; Manassas, VA). A549 was maintained in Dulbecco's modified Eagle's medium/F12 (Life Technologies Corporation, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich Co., St. Louis, MO), and 100 U/mL penicillin and 100 μg/mL streptomycin (Sigma-Aldrich Co.). NCI-H23, PC-14, and RERF-LC-KJ were maintained in RPMI 1640 (Life Technologies Corporation) supplemented with 10% FBS, and 100 U/mL penicillin and streptomycin. Calu-3 was maintained in minimum essential medium (Sigma-Aldrich Co.) supplemented with 10% FBS and 100 U/mL penicillin and streptomycin. LC-2/ad was maintained in RPMI 1640/F12 (Sigma-Aldrich Co.) supplemented with 15% FBS, 100 U/mL penicillin, and streptomycin. Calu-1 was maintained in McCoy's 5a modified medium purchased from ATCC supplemented with 10% FBS, 100 U/mL penicillin, and streptomycin. Calu-6 was maintained in Eagle's minimum essential medium ATCC supplemented with 10% FBS, 100 U/mL penicillin, and streptomycin. All cells were cultured in a 5% CO2 incubator at 37°C.
Thirty-two adenocarcinomas were obtained from patients who had undergone surgical resection at the Department of Thoracic Surgery, Tsukuba University Hospital (Ibaraki, Japan). A small amount of each specimen (tumor and normal region) was embedded directly in Tissue-Tek OCT Compound (Sakura Finetek Japan, Tokyo, Japan) and frozen immediately in acetone and dry ice. The specimens then were stored at −80°C until analysis. All specimens also were fixed with 10% formalin and embedded in paraffin.
The lung cancer cell lines Calu-6 and A549 were treated with culture medium containing 5-aza-2′-deoxycytidine (DAC; Sigma-Aldrich Co.), which had been dissolved in dimethyl sulfoxide. Calu-6 or A549 cells (1 × 106 cells/100-mm dish) were incubated in culture medium with DAC (final concentration, 2 or 10 μmol/L) and without DAC (dimethyl sulfoxide final concentration, 10 μmol/L) for 2 days. The culture medium was changed every day. After cell harvest, RNA was extracted for real-time RT-PCR.
Genomic DNA was extracted by digestion with protease K, followed by use of a QIAamp DNA Mini Kit (Qiagen, Düsseldorf, Germany). For normal lung tissue, the alveolar area was resected using a LM-2000 laser-capture microdissection system (Arcturus Engineering, Mountain View, CA) as described previously.
Briefly, frozen cryostat sections (8-μm thick) of normal lung were stained with Kernechtrot because this allows clearer morphologic discrimination of individual cells and better preservation of nucleic acid quality. Subsequently, the sections were dehydrated in a graded ethanol series and cleared in xylene. After being dried in a vacuum desiccator for 5minutes, alveolar cells were selectively laser-microdissected onto thermoplastic films mounted on optically transparent LCM caps (CapSureMacro LCM Caps LCM0201; Arcturus Engineering) under direct microscopic visualization with a PixCell II laser-capture microdissection system (Arcturus Engineering). Three hundred nanograms of DNA was denatured with NaOH and modified with sodium bisulfite using an ABI methylSEQr Bisulfite Conversion Kit (Applied Biosystems, Foster City, CA) in accordance with the manufacturer's instructions. CpG islands in the promoter region of SFN were identified using CpG Island Searcher. CpG islands were defined using the following criteria: CG, >55%; observed CpG/expected CpG, >0.64; and length, >200. For bisulfite sequencing, modified genomic DNA from five sets of paired samples was subjected to PCR with the following bisulfite sequencing primer set: BS (forward); 5′-CGGTATTGGTTTTAGGTAGTAGTTAG-3′, and BS (reverse); 5′-CCACCACGTTCTTATAAACTACTAA-3′. The region from −242 to −30 was amplified (Figure 1A), and seven clones were sequenced from each of the specimens. The Methyl Primer Express software package v.1.0 (Applied Biosystems) was used to design the bisulfite sequence primers. Because all 11 CpGs were located in the upstream region of the SFN promoter, the transcription initiation site was not included in the amplified region. Because the primers did not contain CpG dinucleotides, methylated and unmethylated sequences were amplified with equal efficiency.
Extraction and modification of DNA were performed as described earlier. Real-time methylation-specific PCR (MSP) was performed as described previously.
The primers for real-time MSP also were designed using Methyl Primer Express (Applied Biosystems). The primers for the methylated reaction were as follows: MSP-SFN (forward); 5′-GGTAGTAGTTAGTTCGTCGTTC-3′, and MSP-SFN (reverse); 5′-AAATTTCGCTCTTCGCAA-3′. The primers for the unmethylated reaction were as follows: unmethylation-specific primer (USP)-SFN (forward); 5′-TTAGGTAGTAGTTAGTTTGTTGTTT-3′, and USP-SFN (reverse); 5′-AACAAATTTCACTCTTCACAA-3′. All samples were analyzed using primer sets for both methylated and unmethylated DNA. The primer set contained 5 CpG dinucleotides of the promoter sequence (3 CpGs in the forward primer, 2 CpGs in the reverse primer). The MSP-SFN (forward) primer was designed to have a cytosine of CpG at the 3′ end because the stringency increases when the positioning is as close as possible to the 3′ end of the primer.
The percentage of methylated SFN was calculated using the following formula: 100 × methylated reaction/(unmethylated reaction + methylated reaction). This yields the percentage of bisulfite-converted input copies of DNA that are fully methylated at the primer hybridization sites. Real-time PCR analysis was performed using SYBR Premix Ex Taq (Perfect Real Time; Takara Bio, Tokyo, Japan), for which no probes were needed. PCR reactions were performed using an ABI 7300 Sequence Detection System (Applied Biosystems) at 95°C for 30 seconds followed by 40 cycles of 95°C for 5 seconds and 60°C for 31 seconds.
Quantitative Real-Time RT-PCR
Total RNA was prepared from 32 frozen lung adenocarcinoma specimens or lung cancer cell lines using an RNeasy Mini Plus Kit (Qiagen), and its quality was evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany). One microgram of total RNA per 20 μL of reaction mixture was converted to cDNA using a high-capacity cDNA Reverse Transcription Kit (Life Technologies Corporation). Quantitative real-time PCR was performed with SYBR Premix Ex Taq (Perfect Real Time; Takara Bio) on a GeneAmp 7300 Sequence Detection System (Life Technologies Corporation) in accordance with the manufacturer's protocol. We used β-actin as an internal control to normalize the mRNA levels between different samples for an exact comparison of gene expression levels. False-positive reactions were checked using no reverse-transcription control samples. Primers used for real-time RT-PCR were as follows: forward; 5′-TCCACTACGAGATCGCCAACAG-3′, and reverse; 5′-GTGTCAGGTTGTCTCGCAGCA-3′.
Sections (4-µm thick) were cut from 10% formalin-fixed, paraffin-embedded blocks. The deparaffinized and rehydrated sections were autoclaved in 10 mmol/L citrate buffer (pH 6.0) at 121°C for 10 minutes for antigen retrieval, then incubated with monoclonal anti-SFN antibody diluted 1:40 (Immuno-Biological Laboratories Co., Ltd., Gunma, Japan) for 30 minutes at room temperature. Subsequently, the sections were incubated with peroxidase-labeled polymer conjugated to goat anti-mouse IgG (DAKO, Carpinteria, CA) for 30 minutes at room temperature. Immunoreactivity was detected with a diaminobenzidine (DAB) substrate kit (Dako Japan, Kyoto, Japan), and the sections were counterstained with hematoxylin. The immunoreactivity was evaluated using two-tier grading: negative (not stained) and positive (partially or diffusely positive). The method of p53 immunohistochemistry (IHC) has been described elsewhere.
To analyze the relationship between the methylation status of the SFN promoter and expression of the gene, bisulfite sequencing and real-time RT-PCR were performed using eight lung cancer cell lines. As shown in Figure 1B, seven of these cell lines, the exception being a large-cell carcinoma cell line, Calu-6, had an unmethylated SFN promoter (Figure 1B). Corresponding to this hypermethylation of the SFN promoter, the expression of SFN was suppressed in Calu-6 (Figure 1C). Next, to determine whether promoter methylation played a role in SFN expression, we examined Calu-6 in more detail by treating it with the demethylation agent DAC. Demethylation of the SFN promoter was observed using bisulfite sequencing (Figure 2A), and rescue of SFN expression was confirmed using real-time RT-PCR (Figure 2B). As a control, A549 also was treated with DAC and then subjected to bisulfite sequencing and real-time RT-PCR, but no alteration of either methylation status or SFN expression was observed (Figure 2A). Conversely, a histone deacetylase inhibitor, trichostatin, had no influence on the level of SFN mRNA (data not shown).
Methylation Status of the SFN Promoter Region in Human Lung Adenocarcinoma and Its Counterpart Normal Lung Tissue
To investigate the methylation status of human lung adenocarcinoma and normal lung tissue, 5 paired samples of tumor and normal tissue were subjected to bisulfite sequencing. The promoter region of the gene was identified using the WWW Promoter Scan (http://www-bimas.cit.nih.gov/molbio/proscan, last accessed August 1, 2011), and CpG islands in the promoter region were identified using CpG Island Searcher (Figure 1A).
For xbisulfite sequencing, modified genomic DNA from the samples was subjected to PCR using bisulfite sequencing primer sets designed to amplify the region of interest. We examined seven clones for each of the samples. The sequencing results showed that most CpGs in normal lung tissues were methylated, whereas those in tumor tissues were sparsely methylated (Figure 3). From the results of bisulfite sequencing, we defined primer sets targeting the sequence around the most frequently methylated site, and used them to perform real-time MSP. Eleven cases of AIS and 21 cases of invasive adenocarcinoma were subjected to real-time MSP (Figure 4). The 5 paired samples subjected to bisulfite sequencing also were included in the group used for real-time MSP. A strong positive correlation between the proportion of methylated CpGs revealed by bisulfite sequencing and the methylation levels revealed by real-time MSP was observed (r = 0.96). As was the case for the results of bisulfite sequencing, tumor tissues showed a significantly lower rate of SFN promoter methylation than the corresponding normal tissues. Interestingly, AIS showed complete methylation in the SFN promoter region, similar to normal lung tissues.
Correlation between Methylation and Expression of SFN and Its Association with Pathologic Tumor Stage
We next determined whether promoter methylation regulates SFN expression in adenocarcinoma tissues. To analyze the expression of SFN, we performed real-time RT-PCR to determine mRNA levels and IHC to determine protein levels using the same samples as those that had been subjected to real-time MSP (Figure 5A). Although all 11 cases of AIS were negative for SFN, 81% (17 in 21 cases) of invasive adenocarcinomas showed positive staining. As shown in Figure 5B, there was a strong inverse correlation between the amount of the SFN transcript and the level of SFN promoter methylation in tumor tissue. Moreover, the IHC-negative group showed a significantly higher level of methylation in tumor tissue than the positive group, indicating that the level of SFN protein also was regulated by promoter methylation (Figure 5C).
We also analyzed the association between the methylation status of tumor tissue and pathologic stage. As shown in Figure 5D, the rate of SFN promoter methylation gradually decreased as tumor stage progressed. Stage II, III, and IV tumors showed significantly lower methylation of the SFN promoter than stage I tumors.
To validate the relationship between SFN methylation status and p53 alteration in lung adenocarcinoma, we performed p53 immunohistochemistry using the same formalin-fixed, paraffin-embedded samples as those used for real-time MSP; however, no clear association between them was noted (Figure 5E).
Recently we have found that SFN shows higher expression in eIA than in AIS. In addition to expression analysis, we also performed functional analysis using specific small-interfering RNA and expression vectors for SFN in the lung adenocarcinoma cell line A549.
This showed that transfection with small-interfering RNA SFN led to a significant decrease of cell proliferation, whereas transfection with SFN expression vectors increased cell proliferation. We also examined the degree of invasiveness after small-interfering RNA–SFN transfection, but no clear correlation was observed. These results indicate that SFN facilitates cell proliferation and induces progression of lung adenocarcinoma. Because blocking of SFN expression was expected to reduce the progression of this tumor, our interest became focused on the underlying mechanisms responsible for regulation of SFN expression.
We preliminarily examined SFN gene amplification using fluorescence in situ hybridization and genomic PCR with genomic DNA from lung adenocarcinoma tissues, but found no amplification in the SFN coding area (data not shown). Subsequently, we performed methylation analysis. In Calu-6, which has a methylated SFN promoter and lacks expression of SFN mRNA, demethylation of the SFN promoter and subsequent rescue of its SFN expression by DAC was confirmed. This suggested that methylation of the SFN promoter regulates the transcription of SFN mRNA. However, because DAC is a universal intervention, any direct correlation between SFN promoter methylation and SFN expression could not be proven definitively. There still remains a possibility that release of SFN expression could be an indirect effect, such as that resulting from demethylation of a transcription factor, rather than a direct effect. Further analysis to clarify the actual function of SFN demethylation therefore is required.
Bisulfite sequencing and real-time MSP revealed that invasive adenocarcinoma had a significantly lower rate of SFN promoter methylation than AIS or the corresponding normal tissues. This indicated that methylation-related silencing of SFN expression was present in AIS and normal lung tissue, and that demethylation of the SFN promoter region occurred during the course of malignant progression from AIS to invasive adenocarcinoma. Because MSP can assess only the methylation status of CpGs that are included in the primer sequence, the results do not represent the entire degree of complexity and CpG site-to-site variability. However, because of the obvious difference of methylation levels between AIS and invasive tumors, we considered that the results of our real-time MSP would precisely reflect the methylation status of the promoter areas.
Moreover, we found a strong inverse correlation between the amount of the SFN transcript and the level of SFN promoter methylation in tumor tissue, suggesting that SFN expression is regulated by alteration of its promoter methylation. Because invasive adenocarcinomas showed lower SFN promoter methylation levels than AIS (Figure 4), a methylation-free promoter status appeared to facilitate SFN expression in invasive adenocarcinoma. We also showed that the level of SFN promoter methylation gradually decreased as tumor stage progressed. The SFN methylation status revealed in the present study was in agreement with our previous study showing that the expression of SFN was higher in invasive adenocarcinoma than in AIS.
the methylation level of SFN does not appear to be associated with abnormal expression of p53. This strongly suggests that SFN promoter demethylation in lung adenocarcinoma occurs independently of p53.
DNA methylation is a dynamic, reversible form of epigenetic regulation that can modify the functionality of numerous genes within the cell. Alterations of the DNA methylation pattern are common in cancer cells. Hypermethylation of CpG islands within certain gene promoter regions inhibits transcription, thus contributing to the silencing of crucial tumor-suppressor genes, such as p16INK4α (CDKN2A), RB, E-cadherin (CDH1), and BRCA1.
On the other hand, hypomethylation of specific loci reactivates the expression of genes with oncogenic potential. For example, in some cancers, hypomethylation-linked activation has been reported for genes such as HRAS, MYC, PAX2, ROS1, and HGALR.
DNA demethylation occurs through two distinct processes: passive demethylation, which results from improper preservation of methylation marks during DNA replication owing to lack of methylation maintenance activity, and active demethylation, which involves a demethylase that actively erases methylation marks from DNA in a replication-independent manner.
showed that global DNA hypomethylation was a marker of poor prognosis in stage IA non-small cell lung cancer by quantifying the methylation levels of long interspersed nuclear element 1 as an indicator of global methylation. Long interspersed nuclear element 1 hypomethylation also strongly is correlated with the hypomethylation of certain genes, such as ΔNp73 in non-small cell lung cancer, suggesting that reduction of gene methylation levels is a passive consequence of global hypomethylation.
In the present study, we showed that demethylation of the SFN promoter occurred during the course of malignant progression of lung adenocarcinoma, and induced subsequent suppression of SFN expression. Although the causes of SFN demethylation are still unclear, there is a possibility that global hypomethylation of genomic DNA may induce passive demethylation, as is the case for the ΔNp73 gene. Although Osada et al
showed that 69% of small-cell lung cancers and 6% of non-small cell lung cancers bore a hypermethylated SFN promoter, such hypermethylation was not observed in adenocarcinoma cell lines. In addition, aberrant CpG island methylation usually is tumor type-specific,
and a tissue type-specific difference in hypomethylation might be linked to the induction of aberrant SFN expression in adenocarcinoma.
In conclusion, we have shown that a CpG-related silencing mechanism participates in the suppression of SFN expression in normal lung tissue, and that demethylation of the CpGs located in the SFN gene promoter regions is involved in the aberrant SFN expression of lung adenocarcinoma cells. Moreover, the level of SFN methylation in tumor cells is associated with pathologic stage, but not with p53 abnormality. Functionally, SFN facilitates cell proliferation and might be one of the enhancers of malignant progression in lung adenocarcinoma. These findings might be considerably informative for helping clarify the mechanism underlying the progression of early lung adenocarcinoma. We are now planning to devise a new system using a mouse model to validate the importance of SFN in pulmonary adenocarcinogenesis in vivo.