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Down-Regulation of DUSP6 Expression in Lung Cancer

2009; Elsevier BV; Volume: 175; Issue: 2 Linguagem: Inglês

10.2353/ajpath.2009.080489

1525-2191

Koji Okudela, Takuya Yazawa, Tetsukan Woo, Masashi Sakaeda, Jun Ishii, Hideaki Mitsui, Hiroaki Shimoyamada, Hanako Sato, Michihiko Tajiri, Nobuo Ogawa, Munetaka Masuda, Takashi Takahashi, Haruhiko Sugimura, Hitoshi Kitamura,

Ubiquitin and proteasome pathways

Our preliminary studies revealed that oncogenic KRAS (KRAS/V12) dramatically suppressed the growth of immortalized airway epithelial cells (NHBE-T, with viral antigen-inactivated p53 and RB proteins). This process appeared to be a novel event, different from the so-called premature senescence that is induced by either p53 or RB, suggesting the existence of a novel tumor suppressor that functions downstream of oncogenic KRAS. After a comprehensive search for genes whose expression levels were modulated by KRAS/V12, we focused on DUSP6, a pivotal negative feedback regulator of the RAS-ERK pathway. A dominant-negative DUSP6 mutant, however, failed to rescue KRAS/V12-induced growth suppression, but conferred a stronger anchorage-independent growth activity to the surviving subpopulation of cells generated from KRAS/V12-transduced NHBE-T. DUSP6 expression levels were found to be weaker in most lung cancer cell lines than in NHBE-T, and DUSP6 restoration suppressed cellular growth. In primary lung cancers, DUSP6 expression levels decreased as both growth activity and histological grade of the tumor increased. Loss of heterozygosity of the DUSP6 locus was found in 17.7% of cases and was associated with reduced expression levels. These results suggest that DUSP6 is a growth suppressor whose inactivation could promote the progression of lung cancer. We have here identified an important factor involved in carcinogenesis through a comprehensive search for downstream targets of oncogenic KRAS. Our preliminary studies revealed that oncogenic KRAS (KRAS/V12) dramatically suppressed the growth of immortalized airway epithelial cells (NHBE-T, with viral antigen-inactivated p53 and RB proteins). This process appeared to be a novel event, different from the so-called premature senescence that is induced by either p53 or RB, suggesting the existence of a novel tumor suppressor that functions downstream of oncogenic KRAS. After a comprehensive search for genes whose expression levels were modulated by KRAS/V12, we focused on DUSP6, a pivotal negative feedback regulator of the RAS-ERK pathway. A dominant-negative DUSP6 mutant, however, failed to rescue KRAS/V12-induced growth suppression, but conferred a stronger anchorage-independent growth activity to the surviving subpopulation of cells generated from KRAS/V12-transduced NHBE-T. DUSP6 expression levels were found to be weaker in most lung cancer cell lines than in NHBE-T, and DUSP6 restoration suppressed cellular growth. In primary lung cancers, DUSP6 expression levels decreased as both growth activity and histological grade of the tumor increased. Loss of heterozygosity of the DUSP6 locus was found in 17.7% of cases and was associated with reduced expression levels. These results suggest that DUSP6 is a growth suppressor whose inactivation could promote the progression of lung cancer. We have here identified an important factor involved in carcinogenesis through a comprehensive search for downstream targets of oncogenic KRAS. Oncogenic mutations of KRAS occur very early in carcinogenesis of the lung and affect even premalignant lesions.1Kitamura H Kameda Y Ito T Hayashi H Atypical adenomatous hyperplasia of the lung. Implications for the pathogenesis of peripheral lung adenocarcinoma.Am J Clin Pathol. 2000; 113: 610-620Google Scholar, 2Okudela K Hayashi H Ito T Yazawa T Suzuki T Nakane Y Sato H Ishi H KeQin X Masuda A Takahashi T Kitamura H K-ras gene mutation enhances motility of immortalized airway cells and lung adenocarcinoma cells via Akt activation: possible contribution to non-invasive expansion of lung adenocarcinoma.Am J Pathol. 2004; 164: 91-100Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar It is therefore likely that additional genetic and/or epigenetic alterations are necessary for lung neoplasms to develop into advanced form of cancers. The inactivation of putative tumor suppressors, p53 and p16INK4A/RB protein, and activation of telomerase, are generally accepted as crucial to the promotion carcinogenesis.1Kitamura H Kameda Y Ito T Hayashi H Atypical adenomatous hyperplasia of the lung. Implications for the pathogenesis of peripheral lung adenocarcinoma.Am J Clin Pathol. 2000; 113: 610-620Google Scholar, 3Sharpless NE DePinho RA Telomeres, stem cells, senescence, and cancer.J Clin Invest. 2004; 113: 160-168Crossref PubMed Scopus (425) Google Scholar A recent study, however, reported that these alterations are not enough to provide primary bronchial epithelial cells with a fully malignant phenotype in vitro, 4Sato M Vaughan MB Girard L Peyton M Lee W Shames DS Ramirez RD Sunaga N Gazdar AF Shay JW Minna JD Multiple oncogenic changes (K-RAS (V12), p53 knockdown, mutant EGFRs, p16 bypass, telomerase) are not sufficient to confer a full malignant phenotype on human bronchial epithelial cells.Cancer Res. 2006; 66: 2116-2128Crossref PubMed Scopus (226) Google Scholar and suggested further alterations would be necessary for the progression of lung cancer. DUSP6, dual-specificity phosphatase 6, is a putative negative feedback regulator for the RAS-ERK pathway, playing physiologically important role(s) in the maintenance of cellular homeostasis in response to growth factors.5Li C Scott DA Hatch E Tian X Mansour SL Dusp6 (Mkp3) is a negative feedback regulator of FGF-stimulated ERK signaling during mouse development.Development. 2007; 134: 167-176Crossref PubMed Scopus (221) Google Scholar, 6Ekerot M Stavridis MP Delavaine L Mitchell MP Staples C Owens DM Keenan ID Dickinson RJ Storey KG Keyse SM Negative feedback regulation of FGF signalling by DUSP6/MKP-3 is driven by ERK1/2 and mediated by Ets factor binding to a conserved site within the DUSP6/MKP-3gene promoter.Biochem. 2008; 412: 287-298Crossref Scopus (155) Google Scholar, 7Muda M Boschert U Dickinson R Martinou JC Martinou I Camps M Schlegel W Arkinstall S MKP-3, a novel cytosolic protein-tyrosine phosphatase that exemplifies a new class of mitogen-activated protein kinase phosphatase.J Biol Chem. 1996; 271: 4319-4326Crossref PubMed Scopus (323) Google Scholar, 8Muda M Theodosiou A Rodrigues N Boschert U Camps M Gillieron C Davies K Ashworth A Arkinstall S The dual specificity phosphatases M3/6 and MKP-3 are highly selective for inactivation of distinct mitogen-activated protein kinases.J Biol Chem. 1996; 271: 27205-27208Crossref PubMed Scopus (312) Google Scholar, 9Keyse SM Dual-specificity MAP kinase phosphatases (MKPs) and cancer.Cancer Metastasis Rev. 2008; 27: 253-261Crossref PubMed Scopus (375) Google Scholar, 10Warmka JK Mauro LJ Wattenberg EV Mitogen-activated protein kinase phosphatase-3 is a tumor promoter target in initiated cells that express oncogenic Ras.J Biol Chem. 2004; 279: 33085-33092Crossref PubMed Scopus (45) Google Scholar, 11Owens DM Keyse SM Differential regulation of MAP kinase signalling by dual-specificity protein phosphatases.Oncogene. 2007; 26: 3203-3213Crossref PubMed Scopus (649) Google Scholar Disruption of this feedback loop could therefore result in neoplastic, and even malignant, transformation by providing cells with enhanced growth activity. Actually, a series of previous studies reported the involvement of DUSP6 down-regulation caused by the hypermethylation of its promoter in the progression of pancreatic cancers.12Furukawa T Yatsuoka T Youssef EM Abe T Yokoyama T Fukushige S Soeda E Hoshi M Hayashi Y Sunamura M Kobari M Horii A Genomic analysis of DUSP6, a dual specificity MAP kinase phosphatase, in pancreatic cancer.Cytogenet Cell Genet. 1998; 82: 156-159Crossref PubMed Scopus (70) Google Scholar, 13Furukawa T Sunamura M Motoi F Matsuno S Horii A Potential tumor suppressive pathway involving DUSP6/MKP-3 in pancreatic cancer.Am J Pathol. 2003; 162: 1807-1815Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 14Furukawa T Fujisaki R Yoshida Y Kanai N Sunamura M Abe T Takeda K Matsuno S Horii A Distinct progression pathways involving the dysfunction of DUSP6/MKP-3 in pancreatic intraepithelial neoplasia and intraductal papillary-mucinous neoplasm's of the pancreas.Mod Pathol. 2005; 18: 1034-1042Crossref PubMed Scopus (118) Google Scholar, 15Furukawa T Molecular genetics of intraductal papillary-mucinous neoplasms of the pancreas.J Hepatobiliary Pancreat Surg. 2007; 14: 233-237Crossref PubMed Scopus (13) Google Scholar, 16Xu S Furukawa T Kanai N Sunamura M Horii A Abrogation of DUSP6 by hypermethylation in human pancreatic cancer.J Hum Genet. 2005; 50: 159-167Crossref PubMed Scopus (118) Google Scholar To our knowledge, however, there have been only a few reports investigating the significance of DUSP6 in other types of cancers including lung cancers.4Sato M Vaughan MB Girard L Peyton M Lee W Shames DS Ramirez RD Sunaga N Gazdar AF Shay JW Minna JD Multiple oncogenic changes (K-RAS (V12), p53 knockdown, mutant EGFRs, p16 bypass, telomerase) are not sufficient to confer a full malignant phenotype on human bronchial epithelial cells.Cancer Res. 2006; 66: 2116-2128Crossref PubMed Scopus (226) Google Scholar, 17Chen HY Yu SL Chen CH Chang GC Chen CY Yuan A Cheng CL Wang CH Terng HJ Kao SF Chan WK Li HN Liu CC Singh S Chen WJ Chen JJ Yang PC A five-gene signature and clinical outcome in non-small-cell lung cancer.N Engl J Med. 2007; 356: 11-20Crossref PubMed Scopus (793) Google Scholar Our preliminary study revealed that oncogenic KRAS (KRAS/V12) dramatically suppressed growth of immortalized airway epithelial cells whose p53 and RB proteins were inactivated by viral antigens. This seemed a novel event different from so-called premature senescence that is induced by p53 or RB pathway,3Sharpless NE DePinho RA Telomeres, stem cells, senescence, and cancer.J Clin Invest. 2004; 113: 160-168Crossref PubMed Scopus (425) Google Scholar and suggested there might exist novel tumor suppressors downstream of oncogenic KRAS. Thus, we have been interested in identifying such suppressors. Also, our recent study demonstrated that insulin-like growth factor-binding proteins 2 and 4, downstream targets of oncogenic KRAS, suppress the growth of cancer cells in a negative-feedback manner and that disruption of this feedback loop, through their promoter’s hypermethylation, is possibly involved in the progression of lung cancers.18Sato H Yazawa T Suzuki T Shimoyamada H Okudela K Ikeda M Hamada K Yamada-Okabe H Yao M Kubota Y Takahashi T Kamma H Kitamura H Growth regulation via insulin-like growth factor binding protein-4 and -2 in association with mutant K-ras in lung epithelia.Am J Pathol. 2006; 169: 1550-1566Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar It is worth noting that growth suppressors lie hidden downstream of the signal transduction pathway of an oncogene. Thus, we were prompted to search for downstream targets of oncogenic KRAS comprehensively, to further identify potential growth suppressors. Among genes whose expression was modulated by oncogenic KRAS, this study has focused on DUSP6, and elucidated its role in the KRAS-induced growth suppression and in carcinogenesis of the lung. An immortalized human airway epithelial cell line (16HBE14o, Simian virus 40-transformed human bronchial epithelial cells), described by Cozens et al,19Cozens AL Yezzi MJ Kunzelmann K Ohrui T Chin L Eng K Finkbeiner WE Widdicombe JH Gruenert DC CFTR expression and chloride secretion in polarized immortal human bronchial epithelial cells.Am J Respir Cell Mol Biol. 1994; 10: 38-47Crossref PubMed Scopus (794) Google Scholar was kindly provided by Grunert DC (California Pacific Medical Center Research Institute) via Kaneko T (Yokohama City University School of Medicine). A subclone of 16HBE14o cells, described as NHBE-T in this paper, was used for experiments. Immortalized airway epithelial cell lines (HPL1D and HPL1A, Simian virus 40-transformed human small airway epithelial cells) were established by Masuda et al.20Masuda A Kondo M Saito T Yatabe Y Kobayashi T Okamoto M Suyama M Takahashi T Takahashi T Establishment of human peripheral lung epithelial cell lines (HPL1) retaining differentiated characteristics and responsiveness to epidermal growth factor, hepatocyte growth factor, and transforming growth factor beta1.Cancer Res. 1997; 57: 4898-4904PubMed Google Scholar Human lung cancer cell lines (A549, H358, and H1299) and the human embryonic kidney cell line (HEK293T) were purchased from American Type Culture Collection (ATCC, Manassas, VA). Human lung cancer cell lines, LC2/ad, Lu134A, Lu140, and PC9, were obtained form Riken Cell Bank (Tsukuba, Japan), and Immuno-Biological Laboratories Co. (Gunma, Japan), respectively. Human lung cancer cell lines, TKB1, TKB2, TKB4, TKB5, TKB6, TKB7, TKB8, TKB12, TKB15, TKB16, TKB17, and TKB20 were kindly provided by Kamma H (Kyorin University School of Medicine, Tokyo Japan). The cells were cultured and grown in DMEM (Sigma Aldrich, St. Louis, MO) (NHBE-T, HPL1D, HPL1A, A549, H358, PC9, LC2/ad, TKB1, TKB2, TKB4, TKB5, TKB6, TKB7, TKB8, TKB2, and H1299) or RPMI1640 medium (Sigma) (Lu130, Lu134A, Lu140, HEK293T, TKB12, TKB15, TKB16, and TKB17) supplemented with 10% heat-inactivated fetal bovine serum (Sigma), 100 units/ml of penicillin (Sigma), and 100 μg/ml of streptomycin (Sigma). Cells (2.5 × 105) were seeded onto a 10-cm culture dish (Iwaki, Tokyo, Japan), and grown to a semiconfluent state for 5 to 7 days. The cells were counted, and 2.5 × 105 cells were seeded again onto a 10-cm dish. Several passages were repeated in the same manner. The sum of population doublings (PDLs) at each point, was calculated by the formula ΣPDLn = log2 (countn/2.5 × 105) + ΣPDLn−1. Cells (1.0 × 104 or 5.0 × 104) were seeded onto a 10-cm culture dish (Iwaki), and grown for optimal period (8 to 14 days). The cells were fixed with methanol and Giemsa-stained, and colonies visible in scanned photographs were counted. Cells (1.25 × 104) were grown in 1 ml of DMEM-based 0.3% agar (agar noble; Becton Dickinson, Sparks, MD) containing 10% fetal bovine serum in a 3.5-cm culture dish (Iwaki) for 5 weeks. The agar was fixed with a buffered 4% paraformaldehyde solution, and colonies visible in scanned photographs were counted. cDNA encoding wild-type KRAS (KRAS/G12) and mutated KRAS (KRAS/V12) were PCR-amplified (PrimeSATR HS DNA polymerase, Takara Bio Inc., Kyoto, Japan) with primers having BamH1 restriction sites (forward (F), 5′-CTCCGCGGATCCAAGCTTGCTGAAA-3′ and reverse (R), 5′-AGGGGCGGATCCTCATTACATAA-3′; the BamH1 restriction sites are in italics). Plasmid vectors, pcDNA3.1-KRAS/G12 and -KRAS/V12 described previously,2Okudela K Hayashi H Ito T Yazawa T Suzuki T Nakane Y Sato H Ishi H KeQin X Masuda A Takahashi T Kitamura H K-ras gene mutation enhances motility of immortalized airway cells and lung adenocarcinoma cells via Akt activation: possible contribution to non-invasive expansion of lung adenocarcinoma.Am J Pathol. 2004; 164: 91-100Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar were used as templates. The amplified fragments were digested with BamH1 (Takara), and inserted into pQCXIH retroviral provirus vectors (BD Clontech, Palo Alto, CA). cDNA encoding DUSP6 variant 2 (Gene Bank Accession # BC003143) was PCR-amplified (PrimeSATR HS DNA polymerase, Takara) with primers (F, 5′-GCTCGACCCCCATGATAGATACG-3′ and R, 5′-CATTCCAGCAAGGAGGGATGTGG-3′), using cDNA reverse-transcribed (SuperScript III, Invitrogen, Carlsbad, CA) from NHBE-T-derived polyadenylated RNA as a template. After the adenylation of 3′ terminals with TaqDNA polymerase (Takara), the amplified fragment was subcloned into the pT7Blue vector (Novagen, Darmstadt, Germany). The DUSP6 cDNA was PCR-amplified (PrimeSATR HS DNA polymerase, Takara) again using pT7Blue/DUSP6 as a template with mismatch primers having BamH1 restriction sites (F, 5′-GATTGCGGATCCGGCATGATAGATACGCTCAG-3′ and R, 5′-TCCAGCAAGGAGGGATCCGGGGTCTTTCAC-3′; BamH1 restriction sites are in italics). After digestion with BamH1 (Takara), the fragment was inserted into pQCXIP (BD Clontech). A dominant negative mutant of DUSP6 that had lost its phosphatase activity (DUSP6/C293S: Cysteine at position-293 was converted to Serine)8Muda M Theodosiou A Rodrigues N Boschert U Camps M Gillieron C Davies K Ashworth A Arkinstall S The dual specificity phosphatases M3/6 and MKP-3 are highly selective for inactivation of distinct mitogen-activated protein kinases.J Biol Chem. 1996; 271: 27205-27208Crossref PubMed Scopus (312) Google Scholar was generated using a site-directed mutagenesis kit (Stratagene, La Jolla, CA) with mismatch primers (5′-GTCTTGGTACATAGCTTGGCTGGCATTAG-3′ and 5′-CTAATGCCAGCCAAGCTATGTACCAAGAC-3′: mismatch bases are in italics). A retroviral provirus vector (psiDKI, Takara) expressing siRNA targeting two positions of DUSP6 mRNA (5′-CCAACCAGAATGTATACCA-3′ [+1103 to +1121] and 5′-GAACTGTGGTGTCTTGGTA-3′ [+855 to +873]) and the same sized siRNA with scramble sequences were constructed according to the manufacturer’s instruction (Takara). Candidate promoter regions of −1539 to +24, −1030 to + 24, and −539 to + 24 at the DUSP6 gene locus (NC_000012) (position of the first base (A) of the start codon [ATG] was determined as + 1), were PCR-amplified (PrimeSATR HS DNA polymerase, Takara) with primer sets, F-1539, 5′-GGTCTTGCGGGAGGACTT-3′ and r + 24, 5′-CACGGGTCTGAGCTATCT-3′; F-1030, 5′-CAAACAGACCTGGGCCTTTA-3′ and r + 24, 5′-CACGGGTCTGAGCTATCT-3′; F-511, 5′-GAGACGCTCGCTGTTTGTATC-3′ and r + 24, 5′-CACGGGTCTGAGCTATCT-3′, using genomic DNA extracted from non-tumor parts of surgically resected lung as a template. After adenylation of the 3′ terminals with TaqDNA polymerase (Takara), the products were subcloned into pT7Blue (Novagen). They were then cut out with HindIII (Takara) and KpnI (Takara), and inserted into pGL4.1 (Promega, Madison, WI). The accuracy of all of the constructs was verified by DNA sequencing (Dye-deoxy DNA sequencing kit, Amersham Life Science, Piscataway, NJ). The pQCXIP(H)-based expression vectors and pDKsi based-knockdown vectors designed as described above and the pCL10A1 retrovirus packaging vector (Imgenex, San Diego, CA) were cotransfected into HEK293T cells with Lipofectamine 2000 reagent (Invitrogen). At 24 hours after the transfection, conditioned medium was recovered as a viral solution. Desired genes were introduced by incubating cells with the viral solution containing 10 μg/ml of polybren (Sigma) for 24 hours. Cells stably expressing desired genes were selected with 1.0 μg/ml of puromycin (or 500 μg/ml hygromycin B or 1000 μg/ml of neomycin [G418]) (Invitrogen) for 3 days. The pooled clones were used for biological analyses and expression profiling. Gene expression in the empty vector-, KRAS/G12-, and KRAS/V12-transduced NHBE-T cells was comprehensively evaluated with an U133A human gene chip microarray (Affymetrix, San Diego, CA) according to the manufacturer’s recommendations. In brief, total RNA was extracted from the cells immediately after completion of the selection (5 days post-transduction), and was reverse transcribed with Super Script II reverse transcriptase (Invitrogen). Complementary RNA was transcribed in vitro using T7 RNA polymerase (Invitrogen) and concomitantly labeled with a fluorescent dye. The labeled complementary RNA was fragmented by acetic acid treatment, and hybridized to the cDNA array. The hybridized signals were scanned with a Gene Chip Scanner 3000 (Affymetrix), and the signal values of each probe were globally normalized according to manufacture’s recommendation. It was checked that differences in signal values of house keeping genes including β-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase among the transfectants were no more than 1.5-fold. Probes showing an absence call were excluded. Then, a hierarchical clustering analysis (Ward’s Method) was performed with computer software (GeneSpring, Agilent technology, Palo Alto, CA). Probes (= genes) whose signal values were elevated or reduced more than fivefold by the KRAS/V12 transduction in comparison with both the empty vector transduction and the KRAS/G12 transduction were extracted by using GeneSpring (Agilent Technology). First-strand cDNA was synthesized from total RNA using the SuperScript First-Strand Synthesis System according to the protocols of the manufacturer (Invitrogen). The cDNA generated was used as a template in real-time PCR with SYBR Premix EXTaq (Takara) and run on a Thermal Cycler DICE real-time PCR system (Takara). The primer set used for the detection of DUSP6 was F, 5′-AACAGGGTTCCAGCACAGCAG-3′; R, 5′-GGCCAGACACATTCCAGCAA-3′. That for ACTB was F, 5′-TGGCACCCAGCACAATGAA-3′; R, 5′-CTAAGTCATAGTCCGCCTAGAAGCA-3′. The means and standard deviations of the copy number of DUSP6 normalized to the value for ACTB mRNA were statistically obtained from triplicate reactions. The cells grown to subconfluence were solved with extraction buffer, as described elsewhere.2Okudela K Hayashi H Ito T Yazawa T Suzuki T Nakane Y Sato H Ishi H KeQin X Masuda A Takahashi T Kitamura H K-ras gene mutation enhances motility of immortalized airway cells and lung adenocarcinoma cells via Akt activation: possible contribution to non-invasive expansion of lung adenocarcinoma.Am J Pathol. 2004; 164: 91-100Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar After centrifugation, supernatants were recovered as protein extracts. The extracts were mixed with equal volumes of 2×SDS buffer, and then boiled. The samples were subjected to SDS-polyacrylamide gel electrophoresis, and transferred onto polyvinylidene difluoride membranes (Amersham). The membranes were incubated with nonfat dry milk in 0.01 M/L Tris-buffered saline containing 0.1% Tween-20 to block non-immunospecific protein binding, and then with 0.1 μg/ml of primary antibody against either KRAS (Santa Cruz, Santa Cruz, CA), DUSP6 (Abnova), ERK (Cell Signaling Technology, Beverly, MA), phosphorylated ERK (Cell Signaling Technology), or β-actin (Sigma). After washing with 0.01 M/L Tris-buffered saline containing 0.1% Tween-20, the membranes were incubated with animal-matched horseradish peroxidase-conjugated secondary antibodies (Amersham). Immunoreactivity was visualized with the enhanced chemiluminescence system (ECL, Amersham). Cells were treated either with 10 μmol/L of 5-azacytidine (AZA, Sigma) for 72 hours by exchanging the medium everyday or with 300 ng/ml of trichostatin A (TSA, Wako, Osaka, Japan) for 24 hours. In addition, cells were treated also with AZA for 48 hours and then with a combination of AZA and TSA for an additional 24 hours. Genomic DNA was extracted from cultured cells as described previously,2Okudela K Hayashi H Ito T Yazawa T Suzuki T Nakane Y Sato H Ishi H KeQin X Masuda A Takahashi T Kitamura H K-ras gene mutation enhances motility of immortalized airway cells and lung adenocarcinoma cells via Akt activation: possible contribution to non-invasive expansion of lung adenocarcinoma.Am J Pathol. 2004; 164: 91-100Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar and subjected to bisulfate conversion treatment using a MethylEasy DNA bisulfate modification kit (Human Genetic Signatures, Macquarie Park, Australia) according to the manufacturer’s instructions. The region from −960 to −266 of the DUSP6 gene, including the promoter locus, was PCR-amplified (TaqHS DNA polymerase, Takara) with three sets of primers (F1, 5′-GAGTTGGGTTTTTAAAGTGGTAAATA-3′ and R1, 5′-CAAAACACATAAACCAAAACACTTC-3′; F2, 5′-GAAATATGAGATAATTGAAGTGTTTTGG-3′ and R2, 5′-CAACTCCTCAATAAATACAAACAAC-3′; F3, 5′-TGTTTGTATTTATTGAGGAGTTGTTT-3′ and R3, 5′-CTACCCAAATAATTTTTATTCCTCC-3′), and subcloned into pT7Blue (Novagen). Eight clones were randomly chosen, and nucleotide conversions at CpG sites were searched for, by DNA sequencing (Dye-deoxy DNA sequencing kit, Amersham). The region from +544 to +627 in intron 1 of the DUPS6 gene, reported to be highly methylated in pancreatic cancers,16Xu S Furukawa T Kanai N Sunamura M Horii A Abrogation of DUSP6 by hypermethylation in human pancreatic cancer.J Hum Genet. 2005; 50: 159-167Crossref PubMed Scopus (118) Google Scholar was PCR-amplified (TaqHS DNA polymerase, Takara) with primers specific for either methyl (F, 5′-GTAGGGGTCGCGAATCGCGC-3′ and R, 3′-ACCGCCGTAACCCGCAACCG-3′) or un-methyl DNA (F, 5′-GTAGGGGTTGTGAATTGTGT-3′ and R, 5′-AACCACCAATACCCACAACCA-3′)16Xu S Furukawa T Kanai N Sunamura M Horii A Abrogation of DUSP6 by hypermethylation in human pancreatic cancer.J Hum Genet. 2005; 50: 159-167Crossref PubMed Scopus (118) Google Scholar using bisulfate-converted DNA as a template. The pGL4.1-based vectors bearing the 5′-untranslated region of the DUSP gene (described in the section Plasmid Construction) and pGL4.7-TK Renilla (Promega, Madison) were co-transfected into NHBE-T cells with Lipofectamine 2000 (Invitrogen). After 24 hours, cells were solved, and luminosity was measured using a Dual Luciferase Reporter Assay system (Promega) on a luminometer (TD-20/20, Turner BioSystems, Sunnyvale, CA). The luminosity derived from each pGL4.1-based vector was corrected with that from pGL4.7-TK Renilla, to normalize the transfection efficiency. Luminosity relative to that of an empty pGL4.1 vector was determined as promoter activity. Cells were incubated in the ordinal medium (DMEM with 10% fetal bovine serum) containing 10 μmol/L of bromodeoxyuridine (BrdU, Sigma) for 45 minutes, thereafter fixed with 4% paraformaldehyde, and then treated with 1N HCl to denature DNA. Incorporated BrdU was visualized by immunocytochemistry with a specific antibody against BrdU (Becton Dickinson, San Jose, CA) using an EnVSION detection system (DakoCytomation, Dako, Ely, UK). A thousand or more cell nuclei were counted, and the percentage of positively labeled cells was determined as a pulse-labeling index. Cells were harvested by trypsinization, fixed with cold 70% ethanol, and treated with 10 mg/ml of RNase A (Sigma). The cells were suspended in 50 μg/ml of propidium iodide. DNA content was measured with a flow cytometer (Epics Elite; Beckman Coulter, Fullerton, CA). All cases examined were of lung cancer patients who underwent surgical resection at Kanagawa Cardiovascular and Respiratory Disease Center Hospital between 2001 and 2008. Informed consent for research use was obtained from all of the subjects providing materials. One hundred and seventy-two cases (adenocarcinomas [ADC, 107], squamous cell carcinomas [SQC, 51], and large cell carcinomas [LCC, 14]), were examined for KRAS codon 12 mutations by restriction endonuclease-mediated selective PCR21Ward R Hawkins N O'Grady R Sheehan C O'Connor T Impey H Roberts N Fuery C Todd A Restriction endonuclease-mediated selective polymerase chain reaction: a novel assay for the detection of K-ras mutations in clinical samples.Am J Pathol. 1998; 153: 373-379Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar as described below. Twenty-two cases (ADC,18Sato H Yazawa T Suzuki T Shimoyamada H Okudela K Ikeda M Hamada K Yamada-Okabe H Yao M Kubota Y Takahashi T Kamma H Kitamura H Growth regulation via insulin-like growth factor binding protein-4 and -2 in association with mutant K-ras in lung epithelia.Am J Pathol. 2006; 169: 1550-1566Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar LCC,3Sharpless NE DePinho RA Telomeres, stem cells, senescence, and cancer.J Clin Invest. 2004; 113: 160-168Crossref PubMed Scopus (425) Google Scholar and SQC1Kitamura H Kameda Y Ito T Hayashi H Atypical adenomatous hyperplasia of the lung. Implications for the pathogenesis of peripheral lung adenocarcinoma.Am J Clin Pathol. 2000; 113: 610-620Google Scholar) were found to have a mutated KRAS (12.8%). The cases with KRAS mutations and 74 cases of non-small cell lung cancer without a KRAS mutation, a total of 96 cases, were used for further analyses. ADCs were classified into histological subtypes according to the criteria of the World Health Organization.22Travis WD Brambilla E Muller-Hermelink HK Harris CC World Health Organization Classification of Tumors. Pathology and Genetics of the Lung, Pleura, Thymus and Heart. IARC Press, Lyon2004Google Scholar If the case involved a mixed type, the major component was described as the subtype in the present study. Genomic DNA was extracted from fresh frozen lung cancer tissues or paraffin sections by methods described previously.2Okudela K Hayashi H Ito T Yazawa T Suzuki T Nakane Y Sato H Ishi H KeQin X Masuda A Takahashi T Kitamura H K-ras gene mutation enhances motility of immortalized airway cells and lung adenocarcinoma cells via Akt activation: possible contribution to non-invasive expansion of lung adenocarcinoma.Am J Pathol. 2004; 164: 91-100Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 21Ward R Hawkins N O'Grady R Sheehan C O'Connor T Impey H Roberts N Fuery C Todd A Restriction endonuclease-mediated selective polymerase chain reaction: a novel assay for the detection of K-ras mutations in clinical samples.Am J Pathol. 1998; 153: 373-379Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar For detection of KRAS codon 12 mutations, the RESMS PCR described by Ward et al was partially modified. Briefly, the reaction mixture, a final volume of 5 μl, containing 0.5 μl of 10 ng/μl DNA solution (1 ng/μl), 0.5 μl of 10×PCR buffer (1×), 0.4 μl of each 2.5 mmol/L dNTP (0.2 mmol/L), 0.04 μl of 50 μmol/L primes (three primer sets described by Ward et al, each 0.4 μmol/L), 0.025 μl of TaqHS DNA polymerase (Takara), 0.4 μl of BstN1 (New England BioLabs, Northbrook, IL) (0.8 unit/μl), and 2.935 μl of distilled water (final volume, 5 μl), was PCR-amplified on a Thermal Cycler Dice (Takara) with the following program; 94°C for 2 minutes (one cycle), 92°C for 20 seconds, 60°C for 3 minutes (35 cycles), held at 4°C. The product was resolved by electrophoresis in 5% agar. A representative result is shown in supplementary Figure S1 (see supplemental Figure S1 at http://ajp.amjpathol.