Loss of TIMP-3 Promotes Tumor Invasion via Elevated IL-6 Production and Predicts Poor Survival and Relapse in HPV-Infected Non–Small Cell Lung Cancer
2012; Elsevier BV; Volume: 181; Issue: 5 Linguagem: Inglês
10.1016/j.ajpath.2012.07.032
ISSN1525-2191
AutoresDe‐Wei Wu, Lung‐Hung Tsai, Po-Ming Chen, Ming-Ching Lee, Lee Wang, Chih‐Yi Chen, Ya‐Wen Cheng, Huei Lee,
Tópico(s)Peptidase Inhibition and Analysis
ResumoHuman papillomavirus (HPV) 16/18 E6 oncoprotein is expressed in lung tumors and is associated with p53 inactivation. The tissue inhibitor of metalloproteinase 3 (TIMP-3) is essential for limiting inflammation; therefore, we expected that TIMP-3 loss might induce chronic inflammation, thereby promoting tumor malignancy as well as poor survival and relapse in patients with HPV-infected non–small cell lung cancer. In this study, the loss of TIMP-3 by loss of heterozygosity and/or promoter hypermethylation was more frequent in HPV16/18 E6–positive tumors than in E6-negative tumors. To explore the possible underlying mechanism, E6-negative TL4 and CL1-0 cells were transfected with an E6 cDNA plasmid. A marked decrease in TIMP-3 expression was caused by promoter hypermethylation via increased DNA (cytosine-5-)-methyltransferase 1 (DNMT1) expression. Mechanistic studies indicated that TIMP-3 loss promoted interleukin-6 (IL-6) production, which led to cell invasion and anchorage-independent growth on soft agar plates. Kaplan-Meier and Cox regression models showed that patients with low-TIMP-3/high–IL-6 tumors had shorter overall survival and relapse-free survival periods when compared with patients with high–TIMP-3/low–IL-6 tumors. In summary, loss of TIMP-3 may increase IL-6 production via the tumor necrosis factor α/nuclear factor κB axis, thereby promoting tumor malignancy and subsequent relapse and poor survival in patients with HPV-infected non–small cell lung cancer. Human papillomavirus (HPV) 16/18 E6 oncoprotein is expressed in lung tumors and is associated with p53 inactivation. The tissue inhibitor of metalloproteinase 3 (TIMP-3) is essential for limiting inflammation; therefore, we expected that TIMP-3 loss might induce chronic inflammation, thereby promoting tumor malignancy as well as poor survival and relapse in patients with HPV-infected non–small cell lung cancer. In this study, the loss of TIMP-3 by loss of heterozygosity and/or promoter hypermethylation was more frequent in HPV16/18 E6–positive tumors than in E6-negative tumors. To explore the possible underlying mechanism, E6-negative TL4 and CL1-0 cells were transfected with an E6 cDNA plasmid. A marked decrease in TIMP-3 expression was caused by promoter hypermethylation via increased DNA (cytosine-5-)-methyltransferase 1 (DNMT1) expression. Mechanistic studies indicated that TIMP-3 loss promoted interleukin-6 (IL-6) production, which led to cell invasion and anchorage-independent growth on soft agar plates. Kaplan-Meier and Cox regression models showed that patients with low-TIMP-3/high–IL-6 tumors had shorter overall survival and relapse-free survival periods when compared with patients with high–TIMP-3/low–IL-6 tumors. In summary, loss of TIMP-3 may increase IL-6 production via the tumor necrosis factor α/nuclear factor κB axis, thereby promoting tumor malignancy and subsequent relapse and poor survival in patients with HPV-infected non–small cell lung cancer. Lung cancer is the leading cause of cancer death worldwide, and cigarette smoking is a predominant factor for lung cancer incidence; however, 25% of lung cancer patients are nonsmokers.1Sun S. Schiller J.H. Gazdar A.F. Lung cancer in never smokers—a different disease.Nat Rev Cancer. 2007; 7: 778-790Crossref PubMed Scopus (1127) Google Scholar In fact, lung cancer in nonsmokers ranks as the seventh most common cause of cancer death worldwide, before cancers of the cervix, pancreas, and prostate.1Sun S. Schiller J.H. Gazdar A.F. Lung cancer in never smokers—a different disease.Nat Rev Cancer. 2007; 7: 778-790Crossref PubMed Scopus (1127) Google Scholar In Taiwan, more than 50% of lung cancer patients are nonsmokers, and in female patients, more than 90% are nonsmokers. Unexpectedly, lung cancer has been the leading cause of cancer death in Taiwanese women since 1982. Major clinicopathological, molecular and sex differences in lung cancers between nonsmokers and smokers have been recognized.1Sun S. Schiller J.H. Gazdar A.F. Lung cancer in never smokers—a different disease.Nat Rev Cancer. 2007; 7: 778-790Crossref PubMed Scopus (1127) Google Scholar Because the increasing body of evidence refers only to driver mutations and not HPV infection in lung cancer development, very few studies have found a significant role of HPV infection in non-Taiwanese patients.2Shigematsu H. Lin L. Takahashi T. Nomura M. Suzuki M. Wistuba I.I. Fong K.M. Lee H. Toyooka S. Shimizu N. Fujisawa T. Feng Z. Roth J.A. Herz J. Minna J.D. 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Factors influencing HPV vaccination status and parental attitudes towards vaccine mandates.Vaccine. 2010; 28: 4186-4191Crossref PubMed Scopus (69) Google Scholar, 7Aguayo F. Anwar M. Koriyama C. Castillo A. Sun Q. Morewaya J. Eizuru Y. Akiba S. Human papillomavirus-16 presence and physical status in lung carcinomas from Asia.Infect Agent Cancer. 2010; 5: 20-26Crossref PubMed Scopus (27) Google Scholar Our previous case-control study indicated that human papillomavirus (HPV) 16/18 infection was associated with lung cancer development in female Taiwanese nonsmokers.5Cheng Y.W. Chiou H.L. Sheu G.T. Hsieh L.L. Chen J.T. Chen C.Y. Su J.M. Lee H. The association of human papillomavirus 16/18 infection with lung cancer among nonsmoking Taiwanese women.Cancer Res. 2001; 61: 2799-2803PubMed Google Scholar We further reported that E6 oncoprotein is expressed in HPV-positive lung tumors and promotes tumor growth via inactivation of p53, DDX3, and miR-2188Cheng Y.W. Wu M.F. Wang J. Yeh K.T. 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Human telomerase reverse transcriptase activated by E6 oncoprotein is required for human papillomavirus-16/18-infected lung tumorigenesis.Clin Cancer Res. 2008; 14: 7173-7179Crossref PubMed Scopus (31) Google Scholar, 12Cheng Y.W. Lee H. Shiau M.Y. Wu T.C. Huang T.T. Chang Y.H. Human papillomavirus type 16/18 up-regulates the expression of interleukin-6 and antiapoptotic Mcl-1 in non-small cell lung cancer.Clin Cancer Res. 2008; 14: 4705-4712Crossref PubMed Scopus (37) Google Scholar Therefore, HPV infection conceivably may play a role in lung tumorigenesis, at least in Taiwanese nonsmokers. The tissue inhibitor of metalloproteinase 3 (TIMP-3) gene is located at 22q12.3. TIMP3 is an insoluble 24-kDa glycoprotein that is produced by most cell types and is sequestered at the cell surface, where it is bound by components of the extracellular matrix.13Fata J.E. Leco K.J. Voura E.B. Yu H.Y. Waterhouse P. Murphy G. Moorehead R.A. Khokha R. Accelerated apoptosis in the TIMP-3-deficient mammary gland.J Clin Invest. 2001; 108: 831-841Crossref PubMed Scopus (144) Google Scholar TIMP-3 acts as a tumor suppressor to inhibit tumor growth, invasion, and angiogenesis.14Baker A.H. George S.J. Zaltsman A.B. Murphy G. Newby A.C. Inhibition of invasion and induction of apoptotic cell death of cancer cell lines by overexpression of TIMP-3.Br J Cancer. 1999; 79: 1347-1355Crossref PubMed Scopus (247) Google Scholar, 15Chetty C. Lakka S.S. Bhoopathi P. Kunigal S. Geiss R. Rao J.S. Tissue inhibitor of metalloproteinase 3 suppresses tumor angiogenesis in matrix metalloproteinase 2-down-regulated lung cancer.Cancer Res. 2008; 68: 4736-4745Crossref PubMed Scopus (47) Google Scholar Loss of heterozygosity (LOH) of TIMP-3 has frequently been reported in meningiomas,16Barski D. Wolter M. Reifenberger G. Riemenschneider M.J. Hypermethylation and transcriptional downregulation of the TIMP3 gene is associated with allelic loss on 22q12.3 and malignancy in meningiomas.Brain Pathol. 2010; 20: 623-631Crossref PubMed Scopus (62) Google Scholar secondary glioblastomas,17Nakamura M. Ishida E. Shimada K. Kishi M. Nakase H. Sakaki T. Konishi N. Frequent LOH on 22q12.3 and TIMP-3 inactivation occur in the progression to secondary glioblastomas.Lab Invest. 2005; 85: 165-175Crossref PubMed Scopus (72) Google Scholar and pancreatic endocrine carcinomas,18Wild A. Langer P. Celik I. Chaloupka B. Bartsch D.K. Chromosome 22q in pancreatic endocrine tumors: identification of a homozygous deletion and potential prognostic associations of allelic deletions.Eur J Endocrinol. 2002; 147: 507-513Crossref PubMed Scopus (36) Google Scholar but TIMP-3 LOH has not yet been reported in cervical or lung cancers. Promoter methylation is the key pathway for TIMP-3 inactivation in cancers of the kidney, brain, breast, colon, esophagus, gastric, head and neck, and lung.19Gu P. Xing X. Tanzer M. Rocken C. Weichert W. Ivanauskas A. Pross M. Peitz U. Malfertheiner P. Schmid R.M. Ebert M.P. Frequent loss of TIMP-3 expression in progression of esophageal and gastric adenocarcinomas.Neoplasia. 2008; 10: 563-572Abstract Full Text PDF PubMed Scopus (52) Google Scholar, 20Bachman K.E. Herman J.G. Corn P.G. Merlo A. Costello J.F. Cavenee W.K. Baylin S.B. Graff J.R. Methylation-associated silencing of the tissue inhibitor of metalloproteinase-3 gene suggest a suppressor role in kidney, brain, and other human cancers.Cancer Res. 1999; 59: 798-802PubMed Google Scholar, 21Zochbauer-Muller S. Fong K.M. Virmani A.K. Geradts J. Gazdar A.F. Minna J.D. Aberrant promoter methylation of multiple genes in non-small cell lung cancers.Cancer Res. 2001; 61: 249-255PubMed Google Scholar, 22De Schutter H. Geeraerts H. 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Abnormal TNF activity in TIMP3–/– mice leads to chronic hepatic inflammation and failure of liver regeneration.Nat Genet. 2004; 36: 969-977Crossref PubMed Scopus (235) Google Scholar Our genome-wide analysis revealed a TIMP-3 allelic imbalance at 22q12.3, which occurred more frequently in HPV-infected lung tumors than in non–HPV-infected lung tumors. This preliminary finding prompted us to investigate whether TIMP-3 loss by LOH and/or promoter hypermethylation could play a role in HPV-infected lung tumorigenesis that was mediated by IL-6. The association between TIMP-3 and IL-6 expression in lung tumors was statistically analyzed to determine whether TIMP-3 and/or IL-6 expression could predict overall survival (OS) and relapse-free survival (RFS) in lung cancer patients. Mechanistic studies using HPV E6–positive and –negative lung cancer cells were conducted to elucidate whether HPV E6 could induce TIMP-3 promoter hypermethylation and could promote the capability for cell invasion and anchorage-independent growth on soft-agar plates via elevated IL-6 production. Lung tumor specimens were collected from 165 patients with primary lung cancer at the Department of Thoracic Surgery, Taichung Veterans General Hospital (Taichung, Taiwan), between 1998 and 2004. Patients were asked to submit written informed consent; the study was approved by the Institutional Review Board. The tumor type and stage of each collected specimen were histologically determined according to the World Health Organization classification system. Cancer relapse data were obtained by chart review and confirmed by thoracic surgeons. The TL-1 and TL-4 lung cancer cells were established from patients' pleural effusions, as described previously.8Cheng Y.W. Wu M.F. Wang J. Yeh K.T. Goan Y.G. 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Aliquots of the PCR reactions were then mixed with a size standard and formamide, denatured, and subjected to capillary electrophoresis on a Genetic Analyzer 310 (ABI, Foster City, CA). Collected data were analyzed with GENESCAN software (ABI). Analyses of each marker were repeated independently at least twice and showed a variation of no more than 3% in allelic ratios. Only samples heterozygous for a given locus were regarded to be informative; locus homozygosity and/or microsatellite instability rendered any particular sample noninformative. Samples were considered to show LOH when a peak allele signal from the tumor DNA was reduced by 50% compared to the normal tissue counterpart. Sodium bisulfite–treated genomic DNA was amplified using fluorescence-based real-time methylation-specific PCR37Hino R. Uozaki H. Murakami N. Ushiku T. Shinozaki A. Ishikawa S. Morikawa T. Nakaya T. Sakatani T. Activation of DNA methyltransferase 1 by EBV latent membrane protein 2A leads to promoter hypermethylation of PTEN gene in gastric carcinoma.Cancer Res. 2009; 69: 2766-2774Crossref PubMed Scopus (281) Google Scholar using SYBR Green qPCR MasterMix (Life Technologies, Carlsbad, CA). The methylation status of the TIMP-3 gene was examined using actin as the internal control for DNA quantification. Actin contains no CpG dinucleotides and is not affected by DNA methylation status or sodium bisulfite treatment. The following primers were used: methylated (TIMP-3) forward primer, 5′-TCGGGTTGTAGTAGTTTCGTC-3′ and methylated (TIMP-3) reverse primer, 5′-ACGATAAACCCGAACCAA-3′.actin-forward, 5′-TGGTGATGGAGGAGGTTTAGTAAGT-3′ and actin-reverse, 5′-AACCAATAAAACCTACTCCTCCCTTAA-3′. The bisulfite-treated in vitro methylated DNA (SssI methyltransferase, New England Biolabs) was used as a positive control. Each reaction was performed in triplicate. The bisulfite-treated in vitro methylated DNA was included in each run to serve as the 100% methylated reference for calculating the relative methylation percentages of DNA samples based on the relative 2(−ΔΔCT) quantitation approach.38Kristensen L.S. Mikeska T. Krypuy M. Dobrovic A. Sensitive melting analysis after real time-methylation specific PCR (SMART-MSP): high-throughput and probe-free quantitative DNA methylation detection.Nucleic Acids Res. 2008; 36: e42Crossref PubMed Scopus (147) Google Scholar, 39Treppendahl M.B. Qiu X. Søgaard A. Yang X. Nandrup-Bus C. Hother C. Andersen M.K. Kjeldsen L. Möllgaard L. Hellström-Lindberg E. Jendholm J. Porse B.T. Jones P.A. Liang G. Grønbæk K. Allelic methylation levels of the noncodingVTRNA2-1located on chromosome 5q31.1 predict outcome in AML.Blood. 2012; 119: 206-216Crossref PubMed Scopus (81) Google Scholar Samples were considered to show positive methylation when the percentage of methylation was more than 50%, whereas a finding of less than 50% was considered as negative. Immunohistochemistry was used to detect HPV16/18 E6, IL-6, and TIMP-3 expression. HPV16/18 E6 and IL-6 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The anti–TIMP-3 antibody was obtained from R&D Systems (Minneapolis, MN). The immunohistochemical procedures were conducted as previously described.8Cheng Y.W. Wu M.F. Wang J. Yeh K.T. Goan Y.G. Chiou H.L. Chen C.Y. Lee H. Human papillomavirus 16/18 E6 oncoprotein is expressed in lung cancer and related with p53 inactivation.Cancer Res. 2007; 67: 10686-10693Crossref PubMed Scopus (119) Google Scholar Negative controls were obtained by leaving out the primary antibody. The intensities of signals were evaluated independently by three observers. The immunostaining results for HPV16/18 E6 and IL-6 expression in lung tumors were partially obtained from previous reports.8Cheng Y.W. Wu M.F. Wang J. Yeh K.T. Goan Y.G. Chiou H.L. Chen C.Y. Lee H. Human papillomavirus 16/18 E6 oncoprotein is expressed in lung cancer and related with p53 inactivation.Cancer Res. 2007; 67: 10686-10693Crossref PubMed Scopus (119) Google Scholar, 11Cheng Y.W. Wu T.C. Chen C.Y. Chou M.C. Ko J.L. Lee H. Human telomerase reverse transcriptase activated by E6 oncoprotein is required for human papillomavirus-16/18-infected lung tumorigenesis.Clin Cancer Res. 2008; 14: 7173-7179Crossref PubMed Scopus (31) Google Scholar, 12Cheng Y.W. Lee H. Shiau M.Y. Wu T.C. Huang T.T. Chang Y.H. Human papillomavirus type 16/18 up-regulates the expression of interleukin-6 and antiapoptotic Mcl-1 in non-small cell lung cancer.Clin Cancer Res. 2008; 14: 4705-4712Crossref PubMed Scopus (37) Google Scholar The immunohistochemical staining scores were defined as described previously40Viard-Leveugle I. Veyrenc S. French L.E. Brambilla C. Brambilla E. Frequent loss of Fas expression and function in human lung tumours with overexpression of FasL in small cell lung carcinoma.J Pathol. 2003; 201: 268-277Crossref PubMed Scopus (74) Google Scholar, 41Sung W.W. Wang Y.C. Cheng Y.W. Lee M.C. Yeh K.T. Wang L. Wang J. Chen C.Y. Lee H. A polymorphic_844T/C in FasL promoter predicts survival and relapse in non–small cell lung cancer.Clin Cancer Res. 2011; 17: 5991-5999Crossref PubMed Scopus (53) Google Scholar and the intensities of signals were evaluated independently by three observers. Immunostaining scores were defined as the cell staining intensity (0 = nil; 1 = weak; 2 = moderate; and 3 = strong) multiplied by the percentage of labeled cells (0% to 100%), leading to scores from 0 to 300. A score of more than 150 and include 150 itself were rated as "high" immunostaining, whereas a score of less than 150 was rated as "low." Total RNA was extracted by homogenization in 1 mL of TRIzol reagent, followed by chloroform extraction and isopropanol precipitation. A 3-μg sample of total RNA from lung tumor tissues was reverse transcribed using SuperScript II Reverse Transcriptase (Life Technologies) and oligo d(T)15 primer. The following primer sequences were used for amplification of the TIMP-3 gene: the forward primer, 5′-TGCAACTCCGACATCGTGAT-3′ and the reverse primer, 5′-TCTTCATCTGCTTGATGGTGTAGAC-3′. The TIMP-3 mRNA levels in lung tumors that were higher than the median value were defined as "high," whereas levels lower than the median value were defined as "low." The cells were lysed with lysis buffer containing 0.5% NP-40, 50 mmol/L Tris-Cl (pH 7.5), 1 mmol/L ethylenediaminetetraacetic acid (EDTA), and protease inhibitor cocktail (Roche, Indianapolis, IN). After 3 minutes of lysis, the cell debris was removed by centrifugation, and the protein concentration was determined using a Bradford protein assay kit (Bio-Rad, Hercules, CA). Equal amounts of protein were separated onto sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels and then transferred from the gel onto a polyvinylidene difluoride membrane (PerkinElmer, Norwalk, CT). After blocking, the membranes were reacted with antibody at 4°C overnight, followed by incubation with horseradish peroxidase–conjugated secondary antibody for 1 hour. The blots were observed using an enhanced chemiluminescence kit (PerkinElmer). The TIMP-3–overexpressed plasmid was constructed in pCDNA3.1A (−). RNA interference was performed by expression of small hairpin RNA to target TIMP-3 in lung cancer cell lines. The small hairpin RNA template was constructed from two oligonucleotides with a complementary sequence in the loop region. The following primer sequences were used for construction of the TIMP-3si gene: the forward primer, 5′-GATCGCAAGATCAAGTCCTGCTACTTTCAAGAGAAGTAGCAGGACTTGATCTTGCTTTTT-3′ and the reverse primer, 5′-AGCTAAAAAGCAAGATCAAGTCCTGCTACTTCTCTTGAAAGTAGCAGGACTTGATCTTGC-3′. The NF-κB luciferase construct was kindly provided by Dr. Tsui-Chun Tsou (Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Zhunan, Miaoli, Taiwan). The different concentrations of expression plasmids were transiently transfected into lung cancer cells (1 × 106) using the Turbofect reagent (Fermentas, Glen Burnie, MD). After 48 hours, cells were harvested, and whole-cell extracts were assayed in subsequent experiments. For the luciferase reporter assay, appropriate numbers of cells were transfected with sufficient reporter plasmid, NF-κB-Luc or its derivatives, and either the control vector or the TIMP-3si and TIMP-3 cDNA plasmid. For normalization of transfection efficiency, β-gal was also co-transfected. Transfected cells were harvested at 48 hours posttransfection, and a luciferase assay was performed according to the manufacturer instructions. The luciferase activity was measured with an AutoLumat LB953 luminometer (Berthold, Bad Wildbad, Germany) and normalized with the co-transfected β-gal activity. Chromatin immunoprecipitation (ChIP) analysis was performed as described in a previous report9Wu D.W. Liu W.S. Wang J. Chen C.Y. Cheng Y.W. Lee H. Reduced p21WAF1/CIP1 via Alteration of p53-DDX3 pathway is associated with poor relapse-free survival in early-stage human papillomavirus–associated lung cancer.Clin Cancer Res. 2011; 17: 1895-1905Crossref PubMed Scopus (81) Google Scholar with the following modifications: Immunoprecipitated DNA was precipitated with ethanol and resuspended in 20 μl of ddH2O. Samples were resuspended in 100 μl of ddH2O and diluted 1:100 before PCR analysis. PCR amplification of immunoprecipitated DNA was performed with diluted aliquots, using the primers consisting of the oligonucleotides that encompass the promoter region of TIMP-3. The following primer sequences were used for TIMP-3 ChIP: the forward primer, 5′-GCGCCGGAGGCCAAGGTTGC-3′ and the reverse primer, 5′-CAGTCCCCCAGGCTCCAGCTGC-3′. The PCR products were separated on 2% agarose gels and analyzed using ethidium bromide staining. All ChIP assays were performed at least twice with similar results. For bisulfite sequencing analysis, the endpoint PCR products from the primers (TIMP-3-Forward, 5′-TTGTTATTGGTTTGAGGGG-3′ and TIMP3-Reverse, 5′-TCCCCCAAACTCCAACTAC-3′) were purified with the PCR purification kit (Qiagen, Valencia, CA). The purified PCR products were cloned into the TA cloning vector according to the manufacturer protocol (Yeastern Biotech, Taiwan). For each DNA sample, 5 bacterial clones were randomly selected and their respective plasmids were extracted. The plasmid DNA was subjected to sequencing analysis using an Applied Biosystems automated fluorescent sequencer (Applied Biosystems, Foster, CA) using vector primers according to the manufacturer instructions. Anchorage-independent growth was assayed by the ability of cells to form colonies in soft agar. The bottom agar consisted of growth medium containing 10% fetal bovine serum and 0.75% agarose in 60-mm tissue culture dishes. 500 cells were re-suspended in growth medium containing 10% fetal bovine serum and 0
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