Genetic and epigenetic analysis of non-small cell lung cancer with NotI-microarrays

This study aimed to clarify genetic and epigenetic alterations that occur during lung carcinogenesis and to design perspective sets of newly identified biomarkers. The original method includes chromosome 3 specific NotI-microarrays containing 180 NotI clones associated with genes for hybridization with 40 paired normal/tumor DNA samples of primary lung tumors: 28 squamous cell carcinomas (SCC) and 12 adenocarcinomas (ADC). The NotI-microarray data were confirmed by qPCR and bisulfite sequencing analyses. Forty-four genes showed methylation and/or deletions in more than 15% of non–small cell lung cancer (NSCLC) samples. In general, SCC samples were more frequently methylated/deleted than ADC. Moreover, the SCC alterations were observed already at stage I of tumor development, whereas in ADC many genes showed tumor progression specific methylation/deletions. Among genes frequently methylated/deleted in NSCLC, only a few were already known tumor suppressor genes: RBSP3 (CTDSPL), VHL and THRB. The RPL32, LOC285205, FGD5 and other genes were previously not shown to be involved in lung carcinogenesis. Ten methylated genes, i.e., IQSEC1, RBSP3, ITGA9, FOXP1, LRRN1, GNAI2, VHL, FGD5, ALDH1L1 and BCL6 were tested for expression by qPCR and were found downregulated in the majority of cases. Three genes (RBSP3, FBLN2 and ITGA9) demonstrated strong cell growth inhibition activity. A comprehensive statistical analysis suggested the set of 19 gene markers, ANKRD28, BHLHE40, CGGBP1, RBSP3, EPHB1, FGD5, FOXP1, GORASP1/TTC21, IQSEC1, ITGA9, LOC285375, LRRC3B, LRRN1, MITF, NKIRAS1/RPL15, TRH, UBE2E2, VHL, WNT7A, to allow early detection, tumor progression, metastases and to discriminate between SCC and ADC with sensitivity and specificity of 80–100%.

[1]  T. Sasaki,et al.  Anti-angiogenic and tumor-suppressive roles of candidate tumor-suppressor gene, Fibulin-2, in nasopharyngeal carcinoma , 2012, Oncogene.

[2]  J. Herman,et al.  Combination epigenetic therapy has efficacy in patients with refractory advanced non-small cell lung cancer. , 2011, Cancer discovery.

[3]  E. Zabarovsky,et al.  RPN1, a new reference gene for quantitative data normalization in lung and kidney cancer , 2011, Molecular Biology.

[4]  Yun Zheng,et al.  Methylation of multiple genes as a candidate biomarker in non-small cell lung cancer. , 2011, Cancer letters.

[5]  George S. Krasnov,et al.  Differential Expression of CHL1 Gene during Development of Major Human Cancers , 2011, PloS one.

[6]  G. Krasnov,et al.  [Novel reference gene RPN1 for normalization of quantitative data in lung and kidney cancer]. , 2011, Molekuliarnaia biologiia.

[7]  Hubert Vesselle,et al.  DNA hypermethylation of tumors from non-small cell lung cancer (NSCLC) patients is associated with gender and histologic type. , 2010, Lung cancer.

[8]  I. Yoshino,et al.  Aberrant methylation in non-small cell lung cancer , 2010, Surgery Today.

[9]  M. Lerman,et al.  Simultaneous down-regulation of tumor suppressor genes RBSP3/CTDSPL, NPRL2/G21 and RASSF1A in primary non-small cell lung cancer , 2010, BMC Cancer.

[10]  P. Laird.  Principles and challenges of genome-wide DNA methylation analysis , 2010, Nature Reviews Genetics.

[11]  L. Montuenga,et al.  Current challenges in lung cancer early detection biomarkers. , 2009, European journal of cancer.

[12]  K. Schuebel,et al.  Concomitant promoter methylation of multiple genes in lung adenocarcinomas from current, former and never smokers. , 2009, Carcinogenesis.

[13]  T. Fearn.  Chemometric space: Particle swarm optimisation , 2014 .

[14]  T. V. Pavlova,et al.  Use of NotI microarrays in analysis of epigenetic and structural changes in epithelial tumor genomes by the example of human chromosome 3 , 2009, Molecular Biology.

[15]  A. G. Stepchenko,et al.  Transcription factor-binding sites responsible for NO-dependent repression of the histone H2B promoter , 2009, Molecular Biology.

[16]  Thomas L. Dunwell,et al.  Epigenetic analysis of childhood acute lymphoblastic leukemia , 2009, Epigenetics.

[17]  E. Zabarovsky,et al.  Downregulation of RBSP3/CTDSPL, NPRL2/G21, RASSF1A, ITGA9, HYAL1, and HYAL2 in non-small cell lung cancer , 2008, Molecular Biology.

[18]  S. Belinsky,et al.  Mining the epigenome for methylated genes in lung cancer. , 2008, Proceedings of the American Thoracic Society.

[19]  L. Hesson,et al.  Evaluation of the 3p21.3 tumour-suppressor gene cluster , 2007, Oncogene.

[20]  J. Minna,et al.  Methods for detecting DNA methylation in tumors: from bench to bedside. , 2007, Cancer letters.

[21]  D. Angeloni.  Molecular analysis of deletions in human chromosome 3p21 and the role of resident cancer genes in disease. , 2007, Briefings in functional genomics & proteomics.

[22]  G. Strathdee.  Epigenetic Markers and Response to Chemotherapy in Cancer , 2007, Disease markers.

[23]  C. Allis,et al.  Epigenetics: A Landscape Takes Shape , 2007, Cell.

[24]  Ian M. Wilson,et al.  Epigenomics: Mapping the Methylome , 2006, Cell cycle.

[25]  S. Belinsky.  Silencing of genes by promoter hypermethylation: key event in rodent and human lung cancer. , 2005, Carcinogenesis.

[26]  S. Belinsky,et al.  Gene-promoter hypermethylation as a biomarker in lung cancer , 2004, Nature Reviews Cancer.

[27]  J. Minna,et al.  Discovery of frequent homozygous deletions in chromosome 3p21.3 LUCA and AP20 regions in renal, lung and breast carcinomas , 2004, Oncogene.

[28]  Gösta Winberg,et al.  RBSP3 (HYA22) is a tumor suppressor gene implicated in major epithelial malignancies. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[29]  A. Protopopov,et al.  Deletion mapping using quantitative real-time PCR identifies two distinct 3p21.3 regions affected in most cervical carcinomas , 2003, Oncogene.

[30]  J. Minna,et al.  Tumor suppressor genes on chromosome 3p involved in the pathogenesis of lung and other cancers , 2002, Oncogene.

[31]  M. Lerman,et al.  Critical tumor‐suppressor gene regions on chromosome 3P in major human epithelial malignancies: Allelotyping and quantitative real‐time PCR , 2002, International journal of cancer.

[32]  A. Protopopov,et al.  NotI subtraction and NotI-specific microarrays to detect copy number and methylation changes in whole genomes , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[33]  Claes Wahlestedt,et al.  NotI flanking sequences: a tool for gene discovery and verification of the human genome. , 2002, Nucleic acids research.

[34]  A. Protopopov,et al.  The RASSF1A tumor suppressor gene is inactivated in prostate tumors and suppresses growth of prostate carcinoma cells. , 2002, Cancer research.

[35]  C. M. Chen,et al.  Dissecting complex epigenetic alterations in breast cancer using CpG island microarrays. , 2001, Cancer research.

[36]  J. Minna,et al.  Overexpression of candidate tumor suppressor gene FUS1 isolated from the 3p21.3 homozygous deletion region leads to G1 arrest and growth inhibition of lung cancer cells , 2001, Oncogene.

[37]  A. Protopopov,et al.  The candidate tumor suppressor gene, RASSF1A, from human chromosome 3p21.3 is involved in kidney tumorigenesis , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[38]  J. Minna,et al.  Epigenetic inactivation of RASSF1A in lung and breast cancers and malignant phenotype suppression. , 2001, Journal of the National Cancer Institute.

[39]  J. Herman,et al.  Predicting lung cancer by detecting aberrant promoter methylation in sputum. , 2000, Cancer research.

[40]  J. Minna,et al.  The 630-kb lung cancer homozygous deletion region on human chromosome 3p21.3: identification and evaluation of the resident candidate tumor suppressor genes. The International Lung Cancer Chromosome 3p21.3 Tumor Suppressor Gene Consortium. , 2000, Cancer research.

[41]  P. Laird,et al.  MethyLight: a high-throughput assay to measure DNA methylation. , 2000, Nucleic acids research.

[42]  J. Herman,et al.  DNA hypermethylation in tumorigenesis: epigenetics joins genetics. , 2000, Trends in genetics : TIG.

[43]  T. Sugimura,et al.  Genetic and epigenetic alterations in carcinogenesis. , 2000, Mutation research.

[44]  A. Protopopov,et al.  NotI clones in the analysis of the human genome. , 2000, Nucleic acids research.

[45]  A. Protopopov,et al.  Analysis of NotI linking clones isolated from human chromosome 3 specific libraries. , 1999, Gene.

[46]  E. Zabarovsky,et al.  Comparative allelotyping of the short arm of human chromosome 3 in epithelial tumors of four different types. , 1999, FEBS letters.

[47]  A. Protopopov,et al.  Identification of new tumor suppressor genes based on in vivo functional inactivation of a candidate gene , 1999, FEBS letters.

[48]  D. Altman,et al.  Statistics Notes: Diagnostic tests 1: sensitivity and specificity , 1994 .

[49]  E. Zabarovsky,et al.  New strategy for mapping the human genome based on a novel procedure for construction of jumping libraries. , 1991, Genomics.

[50]  E. Zabarovsky,et al.  Construction of a human chromosome 3 specific NotI linking library using a novel cloning procedure. , 1990, Nucleic acids research.

[51]  S. P. Yenamandra,et al.  [Technology of analysis of epigenetic and structural changes of epithelial tumors genome with NotI-microarrays by the example of human chromosome]. , 2009, Molekuliarnaia biologiia.

[52]  Esteban Ballestar,et al.  Methyl-DNA immunoprecipitation (MeDIP): hunting down the DNA methylome. , 2008, BioTechniques.

[53]  W. Liang,et al.  TM4 microarray software suite. , 2006, Methods in enzymology.

[54]  W. Liang,et al.  9) TM4 Microarray Software Suite , 2006 .

[55]  Cdm Fletcher,et al.  World Health Organization Classification of Tumours , 2002 .

[56]  S. Naylor,et al.  Deletions of the short arm of chromosome 3 in solid tumors and the search for suppressor genes. , 1997, Advances in cancer research.

[57]  Stanton A. Glantz,et al.  Primer of biostatistics : statistical software program version 6.0 , 1981 .