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Pharmacologic inhibition of epigenetic modifications, coupled with gene expression profiling, reveals novel targets of aberrant DNA methylation and histone deacetylation in lung cancer

Oncogene volume 26, pages 2621–2634 (2007)Cite this article

Abstract

Lung cancer is the leading cause of cancer-related deaths in the United States due, in large part, to the lack of early detection methods. Lung cancer arises from a complex series of genetic and epigenetic changes leading to uncontrolled cell growth and metastasis. Unlike genetic changes, epigenetic changes, such as DNA methylation and histone acetylation, are reversible with currently available pharmaceuticals and are early events in lung tumorigenesis detectable by non-invasive methods. In order to better understand how epigenetic changes contribute to lung cancer, and to identify new disease biomarkers, we combined pharmacologic inhibition of DNA methylation and histone deacetylation in non-small cell lung cancer (NSCLC) cell lines, with genome-wide expression profiling. Of the more than 200 genes upregulated by these treatments, three of these, neuronatin, metallothionein 3 and cystatin E/M, were frequently hypermethylated and transcriptionally downregulated in NSCLC cell lines and tumors. Interestingly, four other genes, cylindromatosis, CD9, activating transcription factor 3 and oxytocin receptor, were dominantly regulated by histone deacetylation and were also frequently downregulated in lung tumors. The majority of these genes also suppressed NSCLC growth in culture when ectopically expressed. This study therefore reveals new putative NSCLC growth regulatory genes and epigenetic disease biomarkers that may enhance early detection strategies and serve as therapeutic targets.

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References

  • Abrahamson M, Alvarez-Fernandez M, Nathanson C-M . (2003). Cystatins. Biochem Soc Symp 70: 179–199.

    Article  CAS  Google Scholar 

  • Ai L, Kim W-J, Kim T-Y, Fields CR, Massoll NA, Robertson KD et al. (2006). Epigenetic silencing of the tumor suppressor cystatin M occurs during breast cancer progression. Cancer Res 66: 7899–7909.

    Article  CAS  Google Scholar 

  • Belinsky SA . (2004). Gene-promoter hypermethylation as a biomarker in lung cancer. Nat Rev Cancer 4: 1–11.

    Article  Google Scholar 

  • Belinsky SA, Klinge DM, Stidley CA, Issa J-P, Herman JG, March TH et al. (2003). Inhibition of DNA methylation and histone deacetylation prevents murine lung cancer. Cancer Res 63: 7089–7093.

    CAS  PubMed  Google Scholar 

  • Belinsky SA, Nikula KJ, Palmisano WA, Michels R, Saccomanno G, Gabrielson E et al. (1998). Aberrant methylation of 16INK4a is an early event in lung cancer and a potential biomarker for early diagnosis. Proc Natl Acad Sci USA 95: 11891–11896.

    Article  CAS  Google Scholar 

  • Bignell GR, Warren W, Seal S, Takahashi M, Rapley E, Barfoot R et al. (2000). Identification of the familial cylindromatosis tumour-suppressor gene. Nat Genet 25: 160–165.

    Article  CAS  Google Scholar 

  • Bird A . (2002). DNA methylation patterns and epigenetic memory. Genes Dev 16: 6–21.

    Article  CAS  Google Scholar 

  • Bottone FGJ, Martinez JM, Collins JB, Afshari CA, Eling TE . (2003). Gene modulation by the cyclooxygenase inhibitor, suldinac sulfide, in human colorectal carcinoma cells. J Biol Chem 278: 25790–25801.

    Article  CAS  Google Scholar 

  • Cameron EE, Bachman KE, Myohanen S, Herman JG, Baylin SB . (1999). Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer. Nat Genet 21: 103–107.

    Article  CAS  Google Scholar 

  • Cassoni P, Sapino A, Marrocco T, Chin B, Bussolati G . (2004). Oxytocin and oxytocin receptors in cancer cells and proliferation. J Neuroendocrinol 16: 362–364.

    Article  CAS  Google Scholar 

  • Chen H, Pan Y-X, Dudenhausen EE, Kilberg MS . (2004). Amino acid deprivation induces the transcription rate of the human asparagine synthetase gene through a timed program of expression and promoter binding of nutrient-responsive basic region/leucine zipper transcription factors as well as localized histone acetylation. J Biol Chem 279: 50829–50839.

    Article  CAS  Google Scholar 

  • Chen RZ, Pettersson U, Beard C, Jackson-Grusby L, Jaenisch R . (1998). DNA hypomethylation leads to elevated mutation rates. Nature 395: 89–93.

    Article  CAS  Google Scholar 

  • Clyde-Smith J, Silins G, Gartside M, Grimmond S, Etheridge M, Apolloni A et al. (2000). Characterization of RasGRP2, a plasma membrane-targeted, dual specificity Ras/Rap exchange factor. J Biol Chem 275: 32260–32267.

    Article  CAS  Google Scholar 

  • Cui H, Cruz-Correa M, Giardiello FM, Hutcheon DF, Kafonek DR, Brandenburg S et al. (2003). Loss of IGF2 imprinting: a potential marker of colorectal cancer risk. Science 299: 1753–1755.

    Article  CAS  Google Scholar 

  • Digel W, Lubbert M . (2005). DNA methylation disturbances as novel therapeutic target in lung cancer: preclinical and clinical results. Crit Rev Oncol Hematol 55: 1–11.

    Article  Google Scholar 

  • Dutta R, Sens DA, Somji S, Sens MA, Garrett SH . (2002). Metallothionein isoform 3 expression inhibits cell growth and increases drug resistance of PC-3 prostate cancer cells. Prostate 52: 89–97.

    Article  CAS  Google Scholar 

  • Eickoff B, Ruller S, Laue T, Kohler G, Stahl C, Schlaak M et al. (2000). Trichostatin A modulates expression of p21waf1/cip1, Bcl-XL, ID1, ID2, ID3, CRAB2, GATA-1, hsp86 and TFIID/TAFII31 mRNA in human lung adenocarcinoma cells. Biol Chem 381: 107–112.

    Google Scholar 

  • Espada J, Ballestar E, Fraga MF, Villar-Garea A, Juarranz A, Stocker JC et al. (2004). Human DNA methyltransferase 1 is required for maintenance of the histone H3 modification pattern. J Biol Chem 279: 37175–37184.

    Article  CAS  Google Scholar 

  • Esteller M, Sanchez-Cespedes M, Rosell R, Sidransky D, Baylin SB, Herman JG . (1999). Detection of aberrant promoter hypermethylation of tumor suppressor genes in serum from non-small cell lung cancer patients. Cancer Res 59: 67–70.

    CAS  PubMed  Google Scholar 

  • Evans HK, Wylie AA, Murphy SK, Jirtle RL . (2001). The neuronatin gene resides in a micro-imprinted domain on human chromosome 20q11.2. Genomics 77: 99–104.

    Article  CAS  Google Scholar 

  • Feinberg AP, Tycko B . (2004). The history of cancer epigenetics. Nat Rev Cancer 4: 1–11.

    Article  Google Scholar 

  • Fiegl H, Millinger S, Mueller-Holzner E, Marth C, Ensinger C, Berger A et al. (2005). Circulating tumor-specific DNA: A marker for monitoring efficacy of adjuvant therapy in cancer patients. Cancer Res 65: 1141–1145.

    Article  CAS  Google Scholar 

  • Fraga MF, Ballestar E, Villar-Garea A, Boix-Chornet M, Espada J, Schotta G et al. (2005). Loss of acetylation at Lys16 and trimethylation at Lys20 of histone H4 is a common hallmark of human cancer. Nat Genet 37: 391–400.

    Article  CAS  Google Scholar 

  • Frommer M, Mc Donald LE, Millar DS, Collis CM, Watt F, Grigg GW et al. (1992). A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc Natl Acad Sci USA 89: 1827–1831.

    Article  CAS  Google Scholar 

  • Garber ME, Troyanskaya OG, Schuens K, Petersen S, Thaesler Z, Pacyna-Gengelbach M et al. (2001). Diversity of gene expression in adenocarcinoma of the lung. Proc Natl Acad Sci USA 98: 13784–13789.

    Article  CAS  Google Scholar 

  • Gius D, Cui H, Bradbury CM, Cook J, Smart DK, Zhao S et al. (2004). Distinct effects on gene expression of chemical and genetic manipulation of the cancer epigenome revealed by a multimodality approach. Cancer Cell 6: 361–371.

    Article  CAS  Google Scholar 

  • Hai T, Wolfgang CD, Marsee DK, Allen AE, Sivaprasad U . (1999). ATF3 and stress response. Gene Exp 7: 321–335.

    CAS  Google Scholar 

  • Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB . (1996). Methylation-specific PCR: A novel assay for methylation status of CpG islands. Proc Natl Acad Sci USA 93: 9821–9826.

    Article  CAS  Google Scholar 

  • Higashiyama M, Taki T, Ieki Y, Adachi M, Huang CL, Koh T et al. (1995). Reduced motility related protein-1 (MRP-1/CD9) gene expression as a factor of poor prognosis in non-small cell lung cancer. Cancer Res 55: 6040–6044.

    CAS  PubMed  Google Scholar 

  • Jenuwein T, Allis CD . (2001). Translating the histone code. Science 293: 1074–1080.

    Article  CAS  Google Scholar 

  • Jones PA, Baylin SB . (2002). The fundamental role of epigenetic events in cancer. Nat Rev Genet 3: 415–428.

    Article  CAS  Google Scholar 

  • Karpf AR, Peterson PW, Rawlins JT, Dalley BK, Yang Q, Albertsen H et al. (1999). Inhibition of DNA methyltransferase stimulates the expression of signal transducer and activator of transcription 1, 2, and 3 genes in colon tumor cells. Proc Natl Acad Sci USA 96: 14007–14012.

    Article  CAS  Google Scholar 

  • Kim MS, Blake M, Baek JH, Kohlhagen G, Pommier Y, Carrier F . (2003). Inhibition of histone deacetylase increases cytotoxicity to anticancer drugs targeting DNA. Cancer Res 63: 7291–7300.

    CAS  PubMed  Google Scholar 

  • Kim T-Y, Zhong S, Fields CR, Kim JH, Robertson KD . (2006). Epigenomic profiling reveals novel and frequent targets of aberrant DNA methylation-mediated silencing in malignant glioma. Cancer Res 66: 7490–7501.

    Article  CAS  Google Scholar 

  • Kuerbitz S, Pahys J, Wilson A, Compitello N, Gray TA . (2002). Hypermethylation of the imprinted NNAT locus occurs frequently in pediatric acute leukemia. Carcinogenesis 23: 559–564.

    Article  CAS  Google Scholar 

  • Maecker HT, Todd SC, Levy S . (1997). The tetraspanin superfamily: molecular interactions. FASEB J 11: 428–442.

    Article  CAS  Google Scholar 

  • Maruyama R, Sugio K, Yoshino I, Maehara Y, Gazdar AF . (2004). Hypermethylation of FHIT as a prognostic marker in nonsmall cell lung carcinoma. Cancer 100: 1472–1477.

    Article  CAS  Google Scholar 

  • Mazieres J, He B, Xu Z, Lee AY, Mikami I, Reguart N et al. (2004). Wnt inhibitory factor-1 is silenced by promoter hypermethylation in human lung cancer. Cancer Res 64: 4717–4720.

    Article  CAS  Google Scholar 

  • Nuovo GJ, Plaia TW, Belinsky SA, Baylin SB, Herman JG . (1999). In situ detection of the hypermethylation-induced inactivation of the p16 gene as an early event in oncogenesis. Proc Natl Acad Sci USA 96: 12754–12759.

    Article  CAS  Google Scholar 

  • Pequeux C, Breton C, Hagelstein M-T, Geenen V, Legros J-J . (2005). Oxytocin receptor pattern of expression in primary lung cancer and in normal lung. Lung Cancer 50: 177–188.

    Article  Google Scholar 

  • Ramaswamy S, Ross KN, Lander ES, Golub TR . (2003). A molecular signature of metastasis in primary solid tumors. Nat Genet 33: 49–54.

    Article  CAS  Google Scholar 

  • Rhee I, Bachman KE, Park BH, Jair K-W, Yen R-WC, Schuebel KE et al. (2002). DNMT1 and DNMT3b cooperate to silence genes in human cancer cells. Nature 416: 552–556.

    Article  CAS  Google Scholar 

  • Robertson KD . (2002). DNA methylation and chromatin – unraveling the tangled web. Oncogene 21: 5361–5379.

    Article  CAS  Google Scholar 

  • Sato N, Maehara N, Su SG, Goggins M . (2003). Effects of 5-aza-2′-deoxycytidine on matrix metalloproteinase expression and pancreatic cancer cell invasiveness. J Natl Cancer Inst 95: 327–330.

    Article  CAS  Google Scholar 

  • Schrump DS, Nguyen DM . (2005). Targeting the epigenome for the treatment and prevention of lung cancer. Semin Oncol 32: 488–502.

    Article  CAS  Google Scholar 

  • Somech R, Izraeli S, Simon AJ . (2004). Histone deacetylase inhibitors – a new tool to treat cancer. Cancer Treat Rev 30: 461–472.

    Article  CAS  Google Scholar 

  • Suh YH, Kim WH, Moon C, Hong YH, Eun S-Y, Lim JH et al. (2005). Ectopic expression of neuronatin potentiates adipogenesis through enhanced phosphorylation of cAMP-response element-binding protein in 3T3-L1 cells. Biochem Biophys Res Commun 337: 481–489.

    Article  CAS  Google Scholar 

  • Topaloglu O, Hoque MO, Tokumaru Y, Lee J, Rativitski E, Sidransky D et al. (2004). Detection of promoter hypermethylation of multiple genes in the tumor and bronchoalveolar lavage of patients with lung cancer. Clin Cancer Res 10: 2284–2288.

    Article  CAS  Google Scholar 

  • Trompouki E, Hatzivassillou E, Tsichritzis T, Farmer H, Ashworth A, Mosialos G . (2003). CYLD is a deubiquitinating enzyme that negatively regulates NF-κB activation by TNFR family members. Science 424: 793–796.

    CAS  Google Scholar 

  • Wang L, Baiocchi RA, Pal S, Mosialos G, Caligiuri M, Sif S . (2005). The BRG1- and hBRM-associated factor BAF57 induces apoptosis by stimulating expression of the cylindromatosis tumor suppressor gene. Mol Cell Biol 25: 7953–7965.

    Article  CAS  Google Scholar 

  • Yan C, Lu D, Hai T, Boyd DD . (2005). Activating transcription factor 3, a stress sensor, activates p53 by blocking its ubiquitination. EMBO J 24: 2425–2435.

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported by NIH Grants K22CA084535 and R01CA114229 (KDR). We thank Bert Vogelstein and Lei Xiao for providing cell lines, Mick Popp and Li Liu for assistance with the microarray analysis, Martha Campbell-Thompson for providing tumor samples and Michael Kilberg for critical reading of the paper. We also thank Xose Bustelo, George Mosialos and Hansoo Lee for providing plasmids.

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  1. Department of Biochemistry & Molecular Biology, UF-Shands Cancer Center Program in Cancer Genetics, Epigenetics and Tumor Virology and Genetics Institute, College of Medicine, University of Florida, Gainesville, FL, USA

    S Zhong, C R Fields, N Su, Y-X Pan & K D Robertson

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  1. S Zhong
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Correspondence to K D Robertson.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).

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Zhong, S., Fields, C., Su, N. et al. Pharmacologic inhibition of epigenetic modifications, coupled with gene expression profiling, reveals novel targets of aberrant DNA methylation and histone deacetylation in lung cancer. Oncogene 26, 2621–2634 (2007). https://doi.org/10.1038/sj.onc.1210041

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