CpG Island Methylator Phenotype In Human Colorectal Cancer

Peter W Laird, Associate Professor
Surgeryuniversity Of Southern California

Grant 5R01CA118699-02 from National Cancer Institute, IRG: EPIC

Abstract: Human colorectal cancer arises as a consequence of both genetic and epigenetic alterations, including promoter CpG island hypermethylation. A subset of colorectal tumors has been described to have an unusually high number of hypermethylated CpG islands, leading to the definition of a distinct phenotype, referred to as "CpG Island Methylator Phenotype", or "CIMP". The long-term objective of this proposal is to study the association between CIMP status and molecular, demographic, and histopathologic features, and environmental risk factors, using colorectal cancer samples collected through the Cooperative Family Registry for Colorectal Cancer Studies (Colon CFR), an NCI-supported consortium intended as a resource to promote collaborative and interdisciplinary studies in the genetic epidemiology of colorectal cancer. We have recently published an improved DMA methylation marker set and analysis technology with which CIMP can be efficiently defined with high accuracy in archival colorectal cancer specimens. We propose to 1) estimate the association between CIMP status and age, sex, family history, race and country of origin, using 4,943 population-based colorectal cancer samples collected through the Colon CFR, 2) estimate the association between CIMP status and tumor location, grade, invasive margin, lymphocytic infiltration, direct spread, lymph node spread, venous spread and type of residual adjacent polyp, if present, and 3) estimate the association between CIMP status and selected risk factors, both genetic and environmental/lifestyle factors, including somatic mutations in BRAF, germline mutations in the MMR genes, smoking history, red meat and alcohol intakes, dietary folate intake, folate metabolic enzyme polymorphisms and history of hormone use. This study will contribute to our understanding of the etiology of CIMP, and its relationship to other molecular and histopathologic features of colorectal cancer. Colorectal cancer involves changes to genes that control cell growth and division. These changes can be structural, as in the case of genetic mutations, or they can reflect an alteration in how actively the gene is being used, referred to as an epigenetic change. This study will investigate how some colorectal tumors acquire an unusually high number of epigenetic changes, with the long-term goal of using this knowledge to block or reverse these types of deleterious changes

Project start date: 2007-09-01

Project end date: 2012-07-31

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Suppression Of Neoplasia By DNA Hypomethylation

Peter W Laird, Associate Professor
University Of Southern California Department Of Contracts And Grants Los Angeles, Ca 90033

Grant 2R01CA075090-05A1 from National Cancer Institute, IRG: MGN

Abstract: We have developed a mouse model system, using hypomorphic alleles of the major DNA methyltransferase Dnmt1, in which we can assess the role of DNA methylation in oncogenesis and investigate various mechanisms by which DNA methylation and/or Dnmt1 may affect the cancer process. We have recently shown that combinations of Dnmt1 hypomorphic alleles can achieve complete genetic suppression of ApcMin/+ induced polyp formation, suggesting that sufficient levels of Dnmt1 expression are required for intestinal polyp development. This is further supported by our observation that intestinal tumorigenesis in mismatch-repair deficient Mih1-/- mice is also suppressed by low levels of Dnmt1. However, lymphomagenesis is increased in these same mice, suggesting that modulation of Dnmt1 levels can have opposing effects on oncogenesis in vivo. The molecular basis for this strong effect of Dnmt1 levels on various models of oncogenesis is not understood. We have recently shown in a tissue-culture model system that Dnmt1 deficiency causes a reduction in methylation-dependent genetic events, such as methylcytosine deamination, and a reduction in methylation-dependent epigenetic events, such as transcriptional silencing by promoter CpG island hypermethylation. In this competing continuation, we propose to 1) expand our analysis of the effects of in vivo modulation of DNA methylation on cancer model systems and 2) analyze the influence of DNA methylation and/or methyltransferases on mutation frequencies in vivo, and 3) analyze sequence features affecting de novo methylation in ES cells. In summary, we propose to continue to investigate the causal effects of DNA methylation in cancer and to analyze both genetic and epigenetic mechanisms by which DNA methylation and/or DNA methyltransferases could affect oncogenesis.

Keywords: DNA methylation, carcinogenesis, enzyme activity, methyltransferase, molecular oncology, neoplastic transformation, tumor suppressor gene, 5 methylcytosine, enzyme deficiency, gastrointestinal epithelium, gene deletion mutation, gene frequency, gene mutation, genetic promoter element, loss of heterozygosity, neoplasm /cancer genetics, tumor progression, gene targeting, laboratory mouse, nucleic acid sequence, polymerase chain reaction, southern blotting, tissue /cell culture

Project start date: 1997-08-15

Project end date: 2007-12-31

2R01CA075090-05A1 (2003): $365625

Environmental Epigenomics

Peter W Laird, Associate Professor
University Of Southern California Department Of Contracts And Grants Los Angeles, Ca 90033

Grant 5R21ES011672-03 from National Institute Of Environmental Health Sciences, IRG: ZES1

Abstract: Recent investigations of the joint effects of genetics and environmental exposures on human health have focused on the role of germline polymorphisms in DNA sequences, including single nucleotide polymorphisms (SNPs) and differences in the number of nucleotide repeats. Although such mutations and their interactions with environmental exposures clearly play a role in human diseases, a growing body of evidence indicates that epigenetic changes, especially changes in patterns of DNA methylation, are important contributors to the pathogenesis of human disease. DNA methylation adds a new dimension to the understanding of gene-environment interactions. DNA methylation is uniquely positioned as both an additional source of genetic modification of the response to environmental influences, and is also a potential biomarker of environmental exposure. Despite the evolving evidence for the importance of DNA methylation in human disease pathogenesis, little is known about the environmental determinants of methylation or how gene polymorphisms influence the patterns of epigenetic changes following exposure. The need for a better understanding of the interactions between environmental exposures, polymorphisms and DNA methylation is clear. In this application, the investigators propose to take the next steps beyond studies of the effects of environmental exposures and genetic variation on human diseases, by integrating information on genomic methylation patterns. The broad objectives of this planning application are to develop a framework that will foster a collaborative interdisciplinary research program to study environmental and genetic determinants of DNA methylation patterns and to understand the influence of these epigenetic changes on human disease occurrence in the context of germline DNA variation. The collaborations to be built during the 3-year planning period bring together experienced investigators to develop and utilize evolving biostatistical, molecular, genetic, and epidemiologic methods. At the end of the planning period, they intend to submit an application to establish a Center for Environmental Epigenomics, to be affiliated with the Environmental Genome Project.

Keywords: DNA methylation, environment related neoplasm /cancer, environmental exposure, gene environment interaction, gene mutation, genetic polymorphism, genome, clinical research

Project start date: 2002-08-01

Project end date: 2005-03-31

5R21ES011672-03 (2004): $243750

5R21ES011672-02 (2003): $243750

1R21ES011672-01 (2002): $243750

DNA Methylation Markers In Esophageal Adenocarcinoma

Peter W Laird, Associate Professor
University Of Southern California Department Of Contracts And Grants Los Angeles, Ca 90033

Grant 5R01CA001815-05 from National Cancer Institute, IRG: ZRG1

Abstract: In recent years, it has become clear that molecular events leading to oncogenesis include not only genetic mutations, but also epigenetic alterations, such as the DNA methylation of promoter CpG islands. We have shown that DNA methylation changes are both causal contributors to neoplasia, and potential biomarkers for cancer, including adenocarcinoma of the esophagus. We have developed a sensitive, quantitatively accurate, automated DNA methylation analysis technique, called MethyLight, with which we have shown that DNA methylation profiles can discriminate between normal squamous mucosa of the esophagus, intestinal metaplasia, and dysplasia. These preliminary results suggest that DNA methylation markers could eventually be exploited as clinical tools in the early detection of esophageal adenocarcinoma and/or in risk assessment in surveillance programs. Since the 1970s, incidence rates for esophageal and gastric cardia adenocarcinomas have risen substantially, particularly among white males in the United States. Reasons for the increase of these tumor types are not well understood. We have conducted a case-control study to determine the role of smoking, alcohol use, body size characteristics, and other risk factors in the etiology of adenocarcinoma of the esophagus and gastric cardia. Here we propose to use automated MethyLight technology to generate extensive methylation profiles for tissue samples retrieved from this population-based case-control study. This will allow us to identify superior methylation markers for further development and testing as clinical tools in the early detection of esophageal adenocarcinoma, and for the differentiation of Barrett s cases that are at high risk for further progression. We will also investigate whether factors known to increase the risk of adenocarcinoma of the esophagus, increase the risk of DNA methylation in normal tissue, or in the resulting adenocarcinoma.

Keywords: DNA methylation, adenocarcinoma, biomarker, esophagus neoplasm, Barretts esophagus, disease /disorder proneness /risk, early diagnosis, longitudinal human study, neoplasm /cancer diagnosis, clinical research, human genetic material tag, human subject, human tissue, interview, polymerase chain reaction, questionnaire

Project start date: 2002-09-26

Project end date: 2008-08-31

5R01CA001815-05 (2006): $357033

5R01CA001815-04 (2005): $365625

5R01CA001815-03 (2004): $365625

5R01CA001815-02 (2003): $365625

1R01CA001815-01 (2002): $365625

CpG Island Methylator Phenotype In Human Colorectal Cancer

Peter W Laird, Associate Professor
Surgeryuniversity Of Southern California

Grant 5R01CA118699-02 from National Cancer Institute, IRG: EPIC

Abstract: Human colorectal cancer arises as a consequence of both genetic and epigenetic alterations, including promoter CpG island hypermethylation. A subset of colorectal tumors has been described to have an unusually high number of hypermethylated CpG islands, leading to the definition of a distinct phenotype, referred to as "CpG Island Methylator Phenotype", or "CIMP". The long-term objective of this proposal is to study the association between CIMP status and molecular, demographic, and histopathologic features, and environmental risk factors, using colorectal cancer samples collected through the Cooperative Family Registry for Colorectal Cancer Studies (Colon CFR), an NCI-supported consortium intended as a resource to promote collaborative and interdisciplinary studies in the genetic epidemiology of colorectal cancer. We have recently published an improved DMA methylation marker set and analysis technology with which CIMP can be efficiently defined with high accuracy in archival colorectal cancer specimens. We propose to 1) estimate the association between CIMP status and age, sex, family history, race and country of origin, using 4,943 population-based colorectal cancer samples collected through the Colon CFR, 2) estimate the association between CIMP status and tumor location, grade, invasive margin, lymphocytic infiltration, direct spread, lymph node spread, venous spread and type of residual adjacent polyp, if present, and 3) estimate the association between CIMP status and selected risk factors, both genetic and environmental/lifestyle factors, including somatic mutations in BRAF, germline mutations in the MMR genes, smoking history, red meat and alcohol intakes, dietary folate intake, folate metabolic enzyme polymorphisms and history of hormone use. This study will contribute to our understanding of the etiology of CIMP, and its relationship to other molecular and histopathologic features of colorectal cancer. Colorectal cancer involves changes to genes that control cell growth and division. These changes can be structural, as in the case of genetic mutations, or they can reflect an alteration in how actively the gene is being used, referred to as an epigenetic change. This study will investigate how some colorectal tumors acquire an unusually high number of epigenetic changes, with the long-term goal of using this knowledge to block or reverse these types of deleterious changes

Project start date: 2007-09-01

Project end date: 2012-07-31

1R01CA118699-01A2 (2007): $502520

DNA Methylation Markers In Ovarian Cancer

Peter W Laird, Associate Professor
University Of Southern California Department Of Contracts And Grants Los Angeles, Ca 90033

Grant 5R01CA096958-05 from National Cancer Institute, IRG: CONC

Abstract: Epithelial ovarian carcinoma is the leading cause of death from gynecologic cancer in the US. Approximately 75% of patients present with advanced stage disease, for which the five-year survival rate remains below 30%, whereas the five-year survival rate for stage I disease is 93%. Therefore, it is anticipated that effective methods of early detection of ovarian cancer would substantially reduce overall mortality rates for this disease. Despite much effort, there are currently no reliable procedures for the early detection of ovarian cancer available. In the past decade, a substantial amount of evidence for the occurrence of extensive alterations of DNA methylation patterns in cancer cells has accumulated. We and others have recently shown that these abnormal DNA methylation patterns can be detected in tumor-derived DNA in the serum and plasma of cancer patients. We propose to use a sophisticated automated methylation analysis technology that we have developed, called MethyLight, to screen a large panel of genes to identify markers specific for ovarian cancer, compared to non-neoplastic ovarian tissue. This will allow the correlation of methylated markers in ovarian tumors with clinicopathological features, and with response to chemotherapy and overall survival. Subsequently, we can use the information obtained in this screen of tissue samples to develop markers for the detection of ovarian tumor-derived DNA in the serum of patients with existing or recurring disease. Finally, we propose to use the most promising biomarkers to evaluate the capacity to detect preclinical relapse of disease, as a function of time before clinical diagnosis of relapse.

Keywords: DNA methylation, biomarker, diagnosis design /evaluation, early diagnosis, neoplasm /cancer genetics, ovary neoplasm, genetic marker, cell line, clinical research, female, histology, human subject

Project start date: 2002-09-24

Project end date: 2008-08-31

5R01CA096958-05 (2006): $317759

5R01CA096958-04 (2005): $325406

5R01CA096958-03 (2004): $325406

5R01CA096958-02 (2003): $325406

1R01CA096958-01 (2002): $325406

SUPPRESSION OF NEOPLASIA BY DNA HYPOMETHYLATION

Peter W Laird, Associate Professor
University Of Southern California Department Of Contracts And Grants Los Angeles, Ca 90033

Grant 5R01CA075090-04 from National Cancer Institute, IRG: BIOL

Abstract: Adapted from ). The long-term goal of Dr. Laird s laboratory is to determine the role of DNA methylation in cancer. 5-Methylcytosine DNA methylation can mediate epigenetic effects through alterations in gene expression as well as genetic effects through an increased mutation rate of 5-methylcytosine. Dr. Laird recently showed that polyp formation in the ApcMin/+mouse model could be almost completely prevented by lowering the levels of functional DNA methyltransferase (MTase) expression and thus the levels of DNA methylation. The goal of the proposed study is to elucidate the mechanism by which reduced DNA MTase levels suppress intestinal polyp formation in ApcMin/+ mice. There are four specific aims. 1) Determine whether the tumor-suppressive effect of DNA hypomethylation is restricted to intestinal neoplasia or whether it is a general characteristic of ApcMin/+ -induced tumors. He will address this by determining the effect of DNA hypomethylation of ApcMin/+ -induced tumors in the pancreas. 2) Test the hypothesis that DNA hypomethylation suppresses polyp formation by reducing the frequency of 5-methylcytosine mutation events. He will test this hypothesis using mutation assays in tissue culture and in vivo. 3) Test the hypothesis that lower levels of DNA MTase impair DNA hypermethylation of tumor-suppressor genes. He will use a novel quantitative assay to track CpG island hypermethylation in microdissected normal epithelium and adenomas from mice with different levels of DNA MTase. 4) Test the hypothesis that DNA hypomethylation suppresses intestinal polyp formation by decreasing the rate of loss of heterozygosity of the Apc gene. He proposes to use a tissue culture selection assay to determine the effect of DNA methylation on chromosome stability.

Keywords: DNA methylation, enzyme activity, intestine neoplasm, methyltransferase, molecular oncology, neoplastic transformation, tumor suppressor gene, 5 methylcytosine, adenomatous polyp, gastrointestinal epithelium, gene deletion mutation, gene frequency, gene mutation, loss of heterozygosity, neoplasm /cancer genetics, genetic strain, laboratory mouse, nucleic acid sequence, tissue /cell culture

Project start date: 1997-08-15

Project end date: 2002-05-31

5R01CA075090-04 (2000): $290657

5R01CA075090-03 (1999): $282261

5R01CA075090-02 (1998): $274952

1R01CA075090-01 (1997): $275881

5R01CA075090-09 (2007): $346679

5R01CA075090-08 (2006): $357033

5R01CA075090-07 (2005): $365625

5R01CA075090-06 (2004): $365625

IN VIVO ANALYSIS OF THE WILMS´ TUMOR SUPPRESSOR GENE

Peter W Laird, Associate Professor
Whitehead Institute For Biomedical Res
9 Cambridge Ctr
cambridge, Ma 021421479

Grant 5F32CA009097-03 from National Cancer Institute, IRG: BIOL

5F32CA009097-03 (1993): $32500

Molecular Characterization of MSI-H Colorectal Cancer by MLHI Promoter Methylation, Immunohistochemistry, and Mismatch Repair Germline Mutation Screening. Cancer Epidemiol Biomarkers Prev. 2008 Nov; 17( 11): 3208-3215. PMID: 18990764

Campan M, Weisenberger DJ, Trinh B, Laird PW.

MethyLight. Methods Mol Biol. 2009; 507: 325-37. PMID: 18987824

McIntosh M, Anderson G, Drescher C, Hanash S, Urban N, Brown P, Gambhir SS, Coukos G, Laird PW, Nelson B, Palmer C.

Ovarian cancer early detection claims are biased. Clin Cancer Res. 2008 Nov 15; 14( 22): 7574. Epub 2008 Oct 23. No abstract available. PMID: 18948385

Lopes EC, Valls E, Figueroa ME, Mazur A, Meng FG, Chiosis G, Laird PW, Schreiber-Agus N, Greally JM, Prokhortchouk E, Melnick A.

Kaiso contributes to DNA methylation-dependent silencing of tumor suppressor genes in colon cancer cell lines. Cancer Res. 2008 Sep 15; 68( 18): 7258-63. PMID: 18794111

Weisenberger DJ, Trinh BN, Campan M, Sharma S, Long TI, Ananthnarayan S, Liang G, Esteva FJ, Hortobagyi GN, McCormick F, Jones PA, Laird PW.

DNA methylation analysis by digital bisulfite genomic sequencing and digital MethyLight. Nucleic Acids Res. 2008 Aug; 36( 14): 4689-98. Epub 2008 Jul 15. PMID: 18628296

Anglim PP, Galler JS, Koss MN, Hagen JA, Turla S, Campan M, Weisenberger DJ, Laird PW, Siegmund KD, Laird-Offringa IA.

Identification of a panel of sensitive and specific DNA methylation markers for squamous cell lung cancer. Mol Cancer. 2008 Jul 10; 7: 62. PMID: 18616821

Byun HM, Choi SH, Laird PW, Trinh B, Siddiqui MA, Marquez VE, Yang AS.

2'-Deoxy-N4-[2-(4-nitrophenyl)ethoxycarbonyl]-5-azacytidine: a novel inhibitor of DNA methyltransferase that requires activation by human carboxylesterase 1. Cancer Lett. 2008 Aug 8; 266( 2): 238-48. Epub 2008 May 21. PMID: 18499340

Kang GH, Lee S, Cho NY, Gandamihardja T, Long TI, Weisenberger DJ, Campan M, Laird PW.

DNA methylation profiles of gastric carcinoma characterized by quantitative DNA methylation analysis. Lab Invest. 2008 Feb; 88( 2): 161-70. Epub 2007 Dec 24. PMID: 18158559

Houshdaran S, Cortessis VK, Siegmund K, Yang A, Laird PW, Sokol RZ.

Widespread epigenetic abnormalities suggest a broad DNA methylation erasure defect in abnormal human sperm. PLoS ONE. 2007 Dec 12; 2( 12): e1289. PMID: 18074014

Liu Z, Choi SW, Crott JW, Keyes MK, Jang H, Smith DE, Kim M, Laird PW, Bronson R, Mason JB.

Mild depletion of dietary folate combined with other B vitamins alters multiple components of the Wnt pathway in mouse colon. J Nutr. 2007 Dec; 137( 12): 2701-8. PMID: 18029487

Tsou JA, Galler JS, Siegmund KD, Laird PW, Turla S, Cozen W, Hagen JA, Koss MN, Laird-Offringa IA.

Identification of a panel of sensitive and specific DNA methylation markers for lung adenocarcinoma. Mol Cancer. 2007 Oct 29; 6: 70. PMID: 17967182

Siegmund KD, Connor CM, Campan M, Long TI, Weisenberger DJ, Biniszkiewicz D, Jaenisch R, Laird PW, Akbarian S.

DNA methylation in the human cerebral cortex is dynamically regulated throughout the life span and involves differentiated neurons. PLoS ONE. 2007 Sep 19; 2( 9): e895. PMID: 17878930 [PubMed]

Tsou JA, Galler JS, Wali A, Ye W, Siegmund KD, Groshen S, Laird PW, Turla S, Koss MN, Pass HI, Laird-Offringa IA.

DNA methylation profile of 28 potential marker loci in malignant mesothelioma. Lung Cancer. 2007 Nov; 58( 2): 220-30. Epub 2007 Jul 30. PMID: 17659810

Chen H, Xia M, Lin M, Yang H, Kuhlenkamp J, Li T, Sodir NM, Chen YH, Josef-Lenz H, Laird PW, Clarke S, Mato JM, Lu SC.

Role of methionine adenosyltransferase 2A and S-adenosylmethionine in mitogen-induced growth of human colon cancer cells. Gastroenterology. 2007 Jul; 133( 1): 207-18. Epub 2007 Apr 11. Erratum in: Gastroenterology. 2007 Nov;133(5):1747. PMID: 17631143

Feng Q, Hawes SE, Stern JE, Dem A, Sow PS, Dembele B, Toure P, Sova P, Laird PW, Kiviat NB.

Promoter hypermethylation of tumor suppressor genes in urine from patients with cervical neoplasia. Cancer Epidemiol Biomarkers Prev. 2007 Jun; 16( 6): 1178-84. PMID: 17548682

Kerr KM, Galler JS, Hagen JA, Laird PW, Laird-Offringa IA.

The role of DNA methylation in the development and progression of lung adenocarcinoma. Dis Markers. 2007; 23( 1-2): 5-30. Review. PMID: 17325423

Widschwendter M, Fiegl H, Egle D, Mueller-Holzner E, Spizzo G, Marth C, Weisenberger DJ, Campan M, Young J, Jacobs I, Laird PW.

Epigenetic stem cell signature in cancer. Nat Genet. 2007 Feb; 39( 2): 157-8. Epub 2006 Dec 31. PMID: 17200673

Wong HL, Byun HM, Kwan JM, Campan M, Ingles SA, Laird PW, Yang AS.

Rapid and quantitative method of allele-specific DNA methylation analysis. Biotechniques. 2006 Dec; 41( 6): 734-9. PMID: 17191619

Sodir NM, Chen X, Park R, Nickel AE, Conti PS, Moats R, Bading JR, Shibata D, Laird PW.

Smad3 deficiency promotes tumorigenesis in the distal colon of ApcMin/+ mice. Cancer Res. 2006 Sep 1; 66( 17): 8430-8. PMID: 16951153

Ogino S, Odze RD, Kawasaki T, Brahmandam M, Kirkner GJ, Laird PW, Loda M, Fuchs CS.

Correlation of pathologic features with CpG island methylator phenotype (CIMP) by quantitative DNA methylation analysis in colorectal carcinoma. Am J Surg Pathol. 2006 Sep; 30( 9): 1175-83. PMID: 16931963

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