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Aberrant Stratifin Overexpression Is Regulated by Tumor-Associated CpG Demethylation in Lung Adenocarcinoma

  • Aya Shiba-Ishii
    Affiliations
    Department of Pathology, Institute of Basic Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Ibaraki, Japan
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  • Masayuki Noguchi
    Correspondence
    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
    Affiliations
    Department of Pathology, Institute of Basic Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Ibaraki, Japan
    Search for articles by this author
Published:February 06, 2012DOI:https://doi.org/10.1016/j.ajpath.2011.12.014
      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.
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      High expression of stratifin is a universal abnormality during the course of malignant progression of early-stage lung adenocarcinoma.
      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.
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      14-3-3Sigma is required to prevent mitotic catastrophe after DNA damage.
      In this manner, SFN induces G2 arrest and allows the repair of damaged DNA. In addition, SFN can bind CDK2 and CDK4, thereby blocking the transition of the eukaryotic cell cycle.
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      Association of the cyclin-dependent kinases and 14-3-3 sigma negatively regulates cell cycle progression.
      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.
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      Downregulation of 14-3-3sigma prevents clonal evolution and leads to immortalization of primary human keratinocytes.
      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,
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      Inactivation of the 14-3-3 sigma gene is associated with 5′ CpG island hypermethylation in human cancers.
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      and this action of SFN is attributed to hypermethylation of the CpG island present in the promoter area of the gene, and not to genetic change.
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      Down-regulation of the tumor suppressor protein 14-3-3sigma is a sporadic event in cancer of the breast.
      reported that down-regulation of SFN does occur sporadically in breast cancers, and a similar study of colorectal cancers has shown that hypermethylation of the SFN promoter area is a rare event.
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      Inactivation of 14-3-3sigma by hypermethylation is a rare event in colorectal cancers and its expression may correlate with cell cycle maintenance at the invasion front.
      On the other hand, many reports also have indicated up-regulation of SFN in cancers of the head and neck, stomach, pancreas, colorectum, and lung.
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      High expression of stratifin is a universal abnormality during the course of malignant progression of early-stage lung adenocarcinoma.
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      14-3-3sigma mediation of cell cycle progression is p53-independent in response to insulin-like growth factor-I receptor activation.
      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.
      • Shiba-Ishii A.
      • Kano J.
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      • Sato Y.
      • Minami Y.
      • Noguchi M.
      High expression of stratifin is a universal abnormality during the course of malignant progression of early-stage lung adenocarcinoma.
      Conversely, Osada et al
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      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.
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      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.
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      Aberrant demethylation of the recoverin gene is involved in the aberrant expression of recoverin in cancer cells.
      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.

      Tissue Specimens

      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.

      DAC Treatment

      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.

      Bisulfite Sequencing

      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.
      • Kano J.
      • Ishiyama T.
      • Iijima T.
      • Morishita Y.
      • Murata S.
      • Hisakura K.
      • Ohkohchi N.
      • Noguchi M.
      Differentially expressed genes in a porcine adult hepatic stem-like cell line and their expression in developing and regenerating liver.
      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.
      Figure thumbnail gr1
      Figure 1In vitro analysis of SFN expression and methylation status using eight cancer cell lines. A: SFN promoter sequence before bisulfite modification. Boldface indicates the primer sequence, and underlining indicates the transcription start site. B: SFN bisulfite sequencing of lung cancer cell lines A549, NCI-H23, PC-14, RERF-LC-KJ, LC-2/ad, Calu-3, Calu-1, and Calu-6. Calu-1 and Calu-6 are squamous cell carcinoma and large cell carcinoma cell lines, respectively. The others are adenocarcinoma cell lines. Filled circles, methylated CpG site; open circles, unmethylated CpG site. C: Real-time RT-PCR for SFN using lung cancer cell lines. β-actin was used as an internal control to normalize the mRNA level.

      Real-Time MSP

      Extraction and modification of DNA were performed as described earlier. Real-time methylation-specific PCR (MSP) was performed as described previously.
      • Iacopetta B.
      • Grieu F.
      • Phillips M.
      • Ruszkiewicz A.
      • Moore J.
      • Minamoto T.
      • Kawakami K.
      Methylation levels of LINE-1 repeats and CpG island loci are inversely related in normal colonic mucosa.
      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.
      • Smith E.
      • Bianco-Miotto T.
      • Drew P.
      • Watson D.
      Method for optimizing methylation-specific PCR.
      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′.

      Immunohistochemistry

      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.
      • Anami Y.
      • Iijima T.
      • Suzuki K.
      • Yokota J.
      • Minami Y.
      • Kobayashi H.
      • Satomi K.
      • Nakazato Y.
      • Okada M.
      • Noguchi M.
      Bronchioloalveolar carcinoma (lepidic growth) component is a more useful prognostic factor than lymph node metastasis.

      Results

      Mutation Status in Human Lung Cancer Cell Lines

      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).
      Figure thumbnail gr2
      Figure 2DAC treatment of Calu-6 and A549. A: SFN bisulfite sequencing of Calu-6 and A549 with or without DAC treatment. The cells without DAC treatment were treated with dimethyl sulfoxide (DMSO) as negative controls. Filled circles, methylated CpG site; open circles, unmethylated CpG site. B: Expression level of the SFN gene in the Calu-6 cell line before and after DAC treatment. Rescue of SFN expression was observed after addition of DAC.

      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).
      • Takai D.
      • Jones P.A.
      The CpG island searcher: a new WWW resource.
      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.
      Figure thumbnail gr3
      Figure 3Methylation status of the SFN promoter in lung adenocarcinoma tissue and its adjacent normal tissues. SFN bisulfite sequencing of 5 lung adenocarcinomas (T) and their adjacent normal tissues (N). Filled circles, methylated CpG site; open circles, unmethylated CpG site.
      Figure thumbnail gr4
      Figure 4Real-time MSP of the SFN promoter using 21 cases of invasive adenocarcinoma and 11 cases of AIS. The percentage of methylated SFN was calculated using the following formula: 100 × methylated reaction/(unmethylated reaction + methylated reaction). Black circles, triangles, and diamonds indicate the newly analyzed samples. Clear circles and triangles indicate the samples used for bisulfite sequencing in .

      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).
      Figure thumbnail gr5
      Figure 5Association between SFN methylation level and expression of the gene. A: Representative SFN IHC. Left panel: invasive adenocarcinoma (SFN positive in cytoplasm), and right panel: is adenocarcinoma in situ (SFN negative). B: Analysis of correlation between SFN mRNA expression and the level of gene methylation in the tumor tissue samples analyzed by real-time MSP in . r indicates the correlation coefficient. C: Box plot of SFN promoter methylation levels in SFN IHC-positive and IHC-negative adenocarcinomas (P < 0.01). D: Correlation of SFN methylation level with tumor pathologic stage. Stage II, III, and IV tumors had significantly lower promoter methylation than stage I tumors (P < 0.01). E: Correlation of SFN methylation level with abnormal expression of p53. No clear relationship was evident. +, positive; −, negative for abnormal p53 expression.
      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.
      SFN expression is induced directly by p53,
      • Hermeking H.
      • Lengauer C.
      • Polyak K.
      • He T.C.
      • Zhang L.
      • Thiagalingam S.
      • Kinzler K.W.
      • Vogelstein B.
      14-3-3 sigma is a p53-regulated inhibitor of G2/M progression.
      and SFN hypermethylation has been detected more frequently in cancers possessing wild-type p53 than those possessing the mutant form.
      • Gasco M.
      • Bell A.K.
      • Heath V.
      • Sullivan A.
      • Smith P.
      • Hiller L.
      • Yulug I.
      • Numico G.
      • Merlano M.
      • Farrell P.J.
      • Tavassoli M.
      • Gusterson B.
      • Crook T.
      Epigenetic inactivation of 14-3-3 sigma in oral carcinoma: association with p16(INK4a) silencing and human papillomavirus negativity.
      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).

      Discussion

      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.
      • Shiba-Ishii A.
      • Kano J.
      • Morishita Y.
      • Sato Y.
      • Minami Y.
      • Noguchi M.
      High expression of stratifin is a universal abnormality during the course of malignant progression of early-stage lung adenocarcinoma.
      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.
      • Shiba-Ishii A.
      • Kano J.
      • Morishita Y.
      • Sato Y.
      • Minami Y.
      • Noguchi M.
      High expression of stratifin is a universal abnormality during the course of malignant progression of early-stage lung adenocarcinoma.
      Promoter demethylation of certain genes such as CD133 (PROM1), CD147 (BSG), and γ-synuclein (SNCG) reportedly is associated with poor prognosis, and can be a marker of tumor malignancy.
      • Hibi K.
      • Sakata M.
      • Kitamura Y.H.
      • Sakuraba K.
      • Shirahata A.
      • Goto T.
      • Mizukami H.
      • Saito M.
      • Ishibashi K.
      • Kigawa G.
      • Nemoto H.
      • Sanada Y.
      Demethylation of the CD133 gene is frequently detected in advanced colorectal cancer.
      • Kong L.M.
      • Liao C.G.
      • Chen L.
      • Yang H.S.
      • Zhang S.H.
      • Zhang Z.
      • Bian H.J.
      • Xing J.L.
      • Chen Z.N.
      Promoter hypomethylation up-regulates CD147 expression through increasing Sp1 binding and associates with poor prognosis in human hepatocellular carcinoma.
      • Ye Q.
      • Feng B.
      • Peng Y.F.
      • Cai Q.
      • Chen X.H.
      • Yu B.Q.
      • Ma J.J.
      • Lu A.G.
      • Li J.W.
      • Wang M.L.
      • Liu B.Y.
      • Zheng M.H.
      Demethylation of the gamma-synuclein gene CpG island in colorectal cancer and its clinical significance.
      These facts suggest that SFN gene methylation in adenocarcinoma of the lung might be a diagnostically useful indicator of tumor invasiveness.
      Although SFN hypermethylation has been detected more frequently in cancers harboring wild-type p53 than in those harboring the mutant form,
      • Gasco M.
      • Bell A.K.
      • Heath V.
      • Sullivan A.
      • Smith P.
      • Hiller L.
      • Yulug I.
      • Numico G.
      • Merlano M.
      • Farrell P.J.
      • Tavassoli M.
      • Gusterson B.
      • Crook T.
      Epigenetic inactivation of 14-3-3 sigma in oral carcinoma: association with p16(INK4a) silencing and human papillomavirus negativity.
      there was no clear association between SFN methylation level and p53 abnormal expression in lung adenocarcinoma. Because similar findings have been reported for oral and vulval cancers
      • Bhawal U.K.
      • Tsukinoki K.
      • Sasahira T.
      • Sato F.
      • Mori Y.
      • Muto N.
      • Sugiyama M.
      • Kuniyasu H.
      Methylation and intratumoural heterogeneity of 14-3-3 sigma in oral cancer.
      • Gasco M.
      • Sullivan A.
      • Repellin C.
      • Brooks L.
      • Farrell P.J.
      • Tidy J.A.
      • Dunne B.
      • Gusterson B.
      • Evans D.J.
      • Crook T.
      Coincident inactivation of 14-3-3sigma and p16INK4a is an early event in vulval squamous neoplasia.
      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.
      • Esteller M.
      • Herman J.G.
      Cancer as an epigenetic disease: dNA methylation and chromatin alterations in human tumours.
      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.
      • Esteller M.
      • Herman J.G.
      Cancer as an epigenetic disease: dNA methylation and chromatin alterations in human tumours.
      • Wu H.
      • Chen Y.
      • Liang J.
      • Shi B.
      • Wu G.
      • Zhang Y.
      • Wang D.
      • Li R.
      • Yi X.
      • Zhang H.
      • Sun L.
      • Shang Y.
      Hypomethylation-linked activation of PAX2 mediates tamoxifen-stimulated endometrial carcinogenesis.
      • Jun H.J.
      • Woolfenden S.
      • Coven S.
      • Lane K.
      • Bronson R.
      • Housman D.
      • Charest A.
      Epigenetic regulation of c-ROS receptor tyrosine kinase expression in malignant gliomas.
      • Cui L.
      • Xu L.Y.
      • Shen Z.Y.
      • Tao Q.
      • Gao S.Y.
      • Lv Z.
      • Du Z.P.
      • Fang W.K.
      • Li E.M.
      NGALR is overexpressed and regulated by hypomethylation in esophageal squamous cell carcinoma.
      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.
      • De Smet C.
      • Loriot A.
      DNA hypomethylation in cancer: epigenetic scars of a neoplastic journey.
      Global DNA hypomethylation is a common event in a variety of cancer types including colorectal cancer, leukemia, ovarian cancer, and lung cancer.
      • Iacopetta B.
      • Grieu F.
      • Phillips M.
      • Ruszkiewicz A.
      • Moore J.
      • Minamoto T.
      • Kawakami K.
      Methylation levels of LINE-1 repeats and CpG island loci are inversely related in normal colonic mucosa.
      • Roman-Gomez J.
      • Jimenez-Velasco A.
      • Agirre X.
      • Castillejo J.A.
      • Navarro G.
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      • Garate L.
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      • Cervantes F.
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      • Heiniger A.
      • Torres A.
      Repetitive DNA hypomethylation in the advanced phase of chronic myeloid leukemia.
      • Menendez L.
      • Benigno B.B.
      • McDonald J.F.
      L1 and HERV-W retrotransposons are hypomethylated in human ovarian carcinomas.
      • Saito K.
      • Kawakami K.
      • Matsumoto I.
      • Oda M.
      • Watanabe G.
      • Minamoto T.
      Long interspersed nuclear element 1 hypomethylation is a marker of poor prognosis in stage IA non-small cell lung cancer.
      In fact, loss of global genomic DNA methylation is one of the earliest epigenetic changes detectable in the process of carcinogenesis.
      • Ehrlich M.
      DNA methylation in cancer: too much, but also too little.
      • Esteller M.
      Epigenetics in cancer.
      In colon cancer, it has been shown that genomic DNA hypomethylation even precedes malignant conversion because it already is apparent in premalignant lesions (benign polyps) of the colon.
      • Goelz S.E.
      • Vogelstein B.
      • Hamilton S.R.
      • Feinberg A.P.
      Hypomethylation of DNA from benign and malignant human colon neoplasms.
      • Feinberg A.P.
      Alterations in DNA methylation in colorectal polyps and cancer.
      • Feinberg A.P.
      • Gehrke C.W.
      • Kuo K.C.
      • Ehrlich M.
      Reduced genomic 5-methylcytosine content in human colonic neoplasia.
      With regard to lung cancer in particular, Saito et al
      • Saito K.
      • Kawakami K.
      • Matsumoto I.
      • Oda M.
      • Watanabe G.
      • Minamoto T.
      Long interspersed nuclear element 1 hypomethylation is a marker of poor prognosis in stage IA non-small cell lung cancer.
      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.
      • Bazhin A.V.
      • De Smet C.
      • Golovastova M.O.
      • Schmidt J.
      • Philippov P.P.
      Aberrant demethylation of the recoverin gene is involved in the aberrant expression of recoverin in cancer cells.
      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
      • Osada H.
      • Tatematsu Y.
      • Yatabe Y.
      • Nakagawa T.
      • Konishi H.
      • Harano T.
      • Tezel E.
      • Takada M.
      • Takahashi T.
      Frequent and histological type-specific inactivation of 14-3-3sigma in human lung cancers.
      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,
      • Costello J.F.
      • Fruhwald M.C.
      • Smiraglia D.J.
      • Rush L.J.
      • Robertson G.P.
      • Gao X.
      • Wright F.A.
      • Feramisco J.D.
      • Peltomaki P.
      • Lang J.C.
      • Schuller D.E.
      • Yu L.
      • Bloomfield C.D.
      • Caligiuri M.A.
      • Yates A.
      • Nishikawa R.
      • Su Huang H.
      • Petrelli N.J.
      • Zhang X.
      • O'Dorisio M.S.
      • Held W.A.
      • Cavenee W.K.
      • Plass C.
      Aberrant CpG-island methylation has non-random and tumour-type-specific patterns.
      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.

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