Frequent RASSF1A gene promoter hypermethylation in breast cancer [Elektronische Ressource] / presented by Yan Zhang
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Universitätsklinikum Ulm Klinik für Frauenheilkunde und Geburtshilfe (Ärztlicher Direktor: Prof. Dr. Kreienberg) Frequent RASSF1A Gene Promoter Hypermethylation in Breast Cancer Dissertation for the attainment of the Doctoral Degree of Medicine (Dr. med.) at the Faculty of Medicine, University of Ulm, Ulm, Germany Presented by Yan Zhang born in Shenyang, Liaoning Province, P. R. China 2008 Amtierender Dekan: Prof.Dr.Klaus-Michael Debatin 1. Berichterstatter: Prof.R.Kreienberg 2. Berichterstatter: Prof.L.Kinzl Tag der Promotion: 29.05.2008 Content Content Abbreviations……………………………………………………………… III 1 Introduction........................................................................................... 1 1.1 Breast cancer........................................................................................ 1 1.1.1 Overview of breast cancer ................................................................... 1 1.1.2 Clinical classification ........................................................................... 1 1.2 Genetic and expression aberrant of genes in breast cancer............ 3 1.3 Epigenetic alteration in tumorigenesis............................................... 4 1.3.1 DNA methylation................................................................................... 4 1.3.2 Histone modifications .......................................................................... 5 1.3.
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| Publié par | universitat_ulm |
| Publié le | 01 janvier 2008 |
| Nombre de lectures | 25 |
| Langue | English |
Extrait
Universitätsklinikum Ulm Klinik für Frauenheilkunde und Geburtshilfe
(Ärztlicher Direktor: Prof. Dr. Kreienberg)
FrequentRASSF1AGene Promoter Hypermethylation in Breast Cancer Dissertation for the attainment of the Doctoral Degree of Medicine (Dr. med.) at the Faculty of Medicine, University of Ulm, Ulm, Germany
Presented by Yan Zhang born in Shenyang, Liaoning Province, P. R. China 2008
Amtierender Dekan: Prof.Dr.Klaus-Michael Debatin
1. Berichterstatter: Prof.R.Kreienberg
2. Berichterstatter: Prof.L.Kinzl
Tag der Promotion: 29.05.2008
Content
Content Abbreviations III1 Introduction........................................................................................... 1 1.1 ........................................................................................Breast cancer 1 1.1.1 1Overview of breast cancer ................................................................... 1.1.2 Clinical classification ........................................................................... 1 1.2 ............Genetic and expression aberrant of genes in breast cancer 3 1.3 Epigenetic alteration in tumorigenesis ............................................... 4 1.3.1 DNA methylation................................................................................... 4 1.3.2 Histone modifications .......................................................................... 5 1.3.3 Hypo- and hyper-methylation .............................................................. 7 1.3.4 DNA methylation in breast cancer ...................................................... 8 1.4 Ras association domain family 1 A (RASSF1A) gene ....................... 9 1.4.1 Ras association domain family 1 (RASSF1) ....................................... 9 1.4.2 ............................................................................RASSF1A functions 12 1.4.3 Tumor-associated methylation ofRASSF1A.................................... 13 1.5 ...........................................................................The aim of the study 14 2 Material and Methods ......................................................................... 15 2.1 Material ................................................................................................ 15 2.1.1 Chemicals and reagents .................................................................... 15 2.1.2 16Enzymes .............................................................................................. 2.1.3 16Solutions and buffers ......................................................................... 2.1.4 17Kits ....................................................................................................... 2.1.5 ................................................................ 17Equipments and softwares 2.2 Samples ............................................................................................... 19 2.2.1 Tissue samples ................................................................................... 19 2.2.2 ..............................................................................................Cell lines 20 2.3 Genomic DNA extraction ................................................................... 21 2.4 Isolation of DNA, RNA, and Protein from TRIzol-Reagent............... 21 2.5 22 ..........................................................................Bisulfite treated DNA 2.6 Pyrosequencing methylation assay (PMA)....................................... 23 2.6.1 Primer design for pyrosequencing ofRASSF1Apromoter............. 24 2.6.2 PCR amplification and template preparation ................................... 25 2.6.3 ......................................................... 26Sequencing and software used I
2.7 2.7.1 2.7.2 2.8 3 3.1 3.2 3.3 3.3.1 3.3.2 3.3.3 3.3.4 3.3.5 3.4. 3.4.1. 3.4.2 3.5 4 4.1 4.2 4.3 4.4 4.4.1 4.4.2 4.4.3 4.4.4 4.4.5 4.4.6 4.5 4.6 5 6 7
Content
Relative quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) forRASSF1Agene expression ............. 28 Reverse transcription ......................................................................... 28 Realtime reverse transcriptase polymerase chain reaction............ 29 Statistical analyses............................................................................. 30 Results................................................................................................. 31 Frequent methylation ofRASSF1A 31 ......................in breast tissues RASSF1AMethylation status in pair-matched breast samples ...... 34 RASSF1A 35 ...............................methylation and clinical parameters Correlation with age ........................................................................... 38 Correlation with the tumor size ......................................................... 38 Correlation with tumor histological grade........................................ 39 Correlation with Lymph node metastases and recurrence ............. 40 Correlation with hormone receptors ................................................. 40 Demethylation ofRASSF1Ain breast cancer cell line .................... 41 Methylation status ofRASSF1Ain cell lines .................................... 41 Effect of demethylation treatment in cell line MDA-MB-330............ 41 Re-expression ofRASSF1Ain the breast cancer cell line 42 .............. Discussion........................................................................................... 45 Method for methylation study............................................................ 45 RASSF1Amethylation status in paired breast cancer patients...... 46 RASSF1Amethylation level in pair-matched breast tissues .......... 47 RASSF1Apromoter methylation and clinical parameters............... 48 Age ....................................................................................................... 48 Tumor size........................................................................................... 49 Lymph node metastases and recurrence ......................................... 50 Tumor stage and metastasis ............................................................. 50 Histological grade and type ............................................................... 51 Hormone receptors............................................................................. 52 Re-expression of RASSF1A in breast cancer cell line .................... 52 Conclusion .......................................................................................... 53 Summary ............................................................................................. 55 References .......................................................................................... 56 Acknowledgements ............................................................................ 72 II
Abbreviations
AJCC ATM ATP bp BRCA1BRCA2BSP B2M C cDNA COBRA CpG dATPαS DCIS dCTP ddH2O dGTP DNA DNMT dNTP dTTP EDTAEGF ER EtOH FBS g G HDAC HER-2/neuHPLC HR
Abbreviations
American Joint Committee on Cancer Ataxia Telangiectasia MutatedAdenosine Triphosphate Base pairBreast Cancer Gene 1Breast Cancer Gene 2Bisulfite-Sequencing PCR β2 MicroglobulinCytosineComplementary Deoxyribonucleic AcidCombined Bisulfite Restriction AnalysisCytosin-phospho-GuanineDeoxyadenosineαthio-hosptripahet Ductal Carcinomain situDeoxycytidine Triphosphate Double distilled waterDeoxyguanosine Triphosphate Deoxyribonucleic AcidDNA methyltransferase Deoxy-ribonucleoside TriphosphateDeoxythymidine Triphosphate Ethylene Diaminetetraacetic AcidEpidermal Growth FactorEstrogen Receptor Ethyl alcoholFetal bovine serumGramGuanosine Histone Deacetylasev-erb-b2 erythroblastic leukemia viral oncogene homolog 2 High-performance Liquid ChromatographyHormone ReceptorIII
IBC IDC ILC kD LCIS NCI ml mM mRNA MSO MSP Ms-SnuPE MTA NaOH ng OE PCR PMA pmol PPi PR QM-MSP RA domain RASSF1RASSF1ART-PCR SARAHT TE TNM TrisTSA TSGs U
Abbreviations Invasive Breast Carcinoma Invasive Ductal CarcinomaInvasive Lobular Carcinoma Kilodalton Lobular Carcinomain situNational Cancer InstituteMilliliterMilli Molar concentrationMessenger Ribonucleic AcidMethylation-specific Oligonucleotide MicroarrayMethylation-specific PCR Methylation-sensitive Single Nucleotide Primer ExtensionMethylation Target ArraySodium Hydroxide NanogramObserved CpG / ExpectedCpG Polymerase Chain ReactionPyrosequencing Methylation AssayPicomole PyrophosphateProgestogen ReceptorQuantitative Multiplex-methylation-specific PCRRas association domain Ras association (RalGDS/AF-6) domain family 1Ras association (RalGDS/AF-6) domain family 1 isoform A Reverse Transcriptase Polymerase Chain ReactionSav/RASSF/HpoThymidineTris-EDTA Classification Tumor, Nodes, Metastasis-classificationTris (hydroxymethyl) aminomethaneTrichostatin ATumor Suppressor GenesInternational UnitIV
µg
µl
µM
5-Aza-dC
5mC
Microgram
Microliter
Micro molar
Abbreviations
5-aza-2-deoxycytidine
5-methylcytosines
V
1 Introduction
1.1 Breast cancer
1.1.1 Overview of breast cancer
Introduction
Breast cancer is by far the most common form of cancer diagnosed in European women today, accounting for 429 900 incident cases (28.9% of total cancer) in 2006 in Europe, with the continuous increase of early diagnosed cases. Overall, breast cancer (131 900, 7.8% of total cancer death) was the third major cause of cancer death in 2006 in Europe (Ferlay et al. 2007). Because of the ageing of the European population the number of deaths from breast cancer is still rising from 130 000 in 2004 to 132 000 in 2006 in Europe (Ferlay et al. 2007).
1.1.2 Clinical classification
Table 1: The TNM classification of breast cancer(the American Joint Committee on Cancer in collaboration with the National Cancer Institute in 2002)
T- Refers to tumor size pTX: Tumor cannot be assessed pT0: No evidence of primary tumor pTis: in situ, or Paget's disease Carcinomaof the nipple, without a detectable tumor mass pT1: r less(≦2 cm) in greatest dimension Tumor two centimeters o pT2: more than two centimeters (> 2 cm), but less than five centimeters ( Tumor≦5 cm), in greatest dimension pT3:centimeters (> 5 cm) in greatest dimension more than five Tumor T4chest wall or skin (includes inflammatoryTumor of any size, with direct spread to :pcarcinoma and ulceration of the breast skin) N- Refers to lymph node involvement pN0: No regional lymph node metastasis pN1 Metastasisto movable ipsilateral axillary lymph node(s) pN2 Metastasis to ipsilateral axillary lymph nodes fixed to one another or to other structures pN3 Metastasis to ipsilateral internal mammary node(s) M- refers to the extent of metastasis MX: presence of distant metastasis cannot be assessed The M0: distant metastasis No M1: metastasis (includes metastasis to ipsilateral supraclavicular lymph node[s]) Distant
1
Introduction
TNM classification has been traditionally used in the clinical practice to define the disease progress (Table 1).
According to the original cells and the cell growth status, it can be divided to: 1) Non-invasive carcinomas, include ductal carcinoma in situ (DCIS), lobular carcinoma in situ (LCIS), and Pagets disease; 2) Invasive carcinomas, like invasive (infiltrating) ductal carcinoma (IDC, about 75% of all invasive carcinoma), invasive lobular carcinoma (ILC, approximately 5% to 10% of all invasive breast cancer), and other three well recognized types, namely tubular cancers, medullary cancers, and mucinous cancers; 3) Inflammatory carcinoma is a very serious, rapidly spreading type of tumor that accounts for about 1% of all breast cancers; 4) A very small number of breast cancers may arise from the muscle, fat, or connective tissues of the breast. The rare types of sarcoma that occasionally are diagnosed within the breast include angiosarcoma and cystosarcoma phylloides; 5) Breast cancer in special situation, such as breast cancer in men or in pregnancy (http://www.bccancer.bc.ca).
The grading of invasive carcinoma is also important as a prognostic indicator. For the Nottingham-Bloom-Richardson system, according to the percentage of tubule formation, the degree of nuclear pleomorphism, and an accurate mitotic count using a defined field area, three grades of breast cancer histology were defined: grade 1 (the total score is 3-5, well-differentiated), grade 2 (the total score is 6 or 7, moderately-differentiated) and grade 3 (the total score is 8 or 9, poorly-differentiated) (Elston and Ellis 1991).
Steroid hormones, particularly estrogen, have long been linked to mammary carcinogenesis (Fishman et al. 1995). Estrogen receptor (ER) and progestogen receptor (PR) are two members of the steroid hormone superfamily studied in breast cancer. The presence of ER in breast tumors is a predictive marker for response to hormone therapy. However, up to one-third of breast cancers lack ER at the time of diagnosis and a proportion of cancers that are initially ER-positive lose ER during tumor progression (Hortobagyi 1998).
Three disease responsiveness categories were defined according to the steroid hormone receptor (HR): endocrine responsive (the cells express HR), endocrine non-responsive (cells have no detectable expression of HR), and endocrine 2
Introductionresponse uncertain. Features indicative of the last category include low levels of HR immunoreactivity (usually considered as < 10% of cells positive) and lack of PR (irrespective of the expression of ER) (Goldhirsch et al. 2005).
1.2 Genetic and expression aberrant of genes in breast cancer
Human cancer development is a dynamic, complex, progressive and multistep process, involving many genes and gene products that systematically, quantitatively and accumulatively affect a biological network of cellular signaling and functional pathways (Hanahan and Weinberg 2000; Michor et al. 2004). These genetic events lead to gene activation/inactivation through the mechanisms of mutation, amplification and deletion.
It is estimated that 5%10% of all breast cancers in womenare associated with hereditary susceptibility due to mutationsin autosomal dominant genes, such as Breast Cancer Gene (BRCA1andBRCA2),p53, phosphatase and tensin homolog(pTEN),and serine/threonine kinase 11 (STK11/LKB1) (Collaborative Group on Hormonal Factors in Breast Cancer 2001; Claus et al. 1996). Another 15%20% of female breastcancers occur in women with a family history but without anapparent autosomal dominant inheritance pattern, and are probablydue to other genetic factors with environmental influence (Slattery and Kerber 1993). As a heterogeneous disease, many genetic alterations have been detected in sporadic breast cancers. For example, gene amplification at 17q23 (Sinclair et al. 2003) and 20q11-13 (Guan et al. 1996) and chromosome loss at 8p22 (Yaremko et al. 1996), 11q23 (Laake et al. 1999), and 16q22 (Iida et al. 1997) are frequently detected in breast cancer by fluorescent in situ hybridization, comparative genomic hybridization, and loss of heterozygosity studies. Alteration or changes in expression levels have been described for several other genes, includingp53, E-cadherin, andHER-2/neu(Ingvarsson 1999). It is very likely that additional genes, especially some tumor suppressor genes (TSGs) contribute to the sporadic breast carcinogenesis. Determining a "genetic signature" for a tumor may prove to be a very powerful predictor of the aggressive nature of a breast cancer. Now there are nearly 70 genes whose activity patterns may help make such predictions (http://health.nytimes.com/health/guides/disease/breast-cancer/print.html).
3
Introduction
1.3 Epigenetic alteration in tumorigenesis
Mammalian embryonic development is controlled by genetic, epigenetic and environmental mechanisms.During acquired of a progressive appearance of malignant cell behavior, many of the gene changes stem from genetic abnormalities that disrupt coding regions. However, it is becoming clear that epigenetic events, around a gene that lead to inherited alteration of gene expression without affecting the nucleotide sequence of the gene, play a fundamental role in tumor formation and progression. Two epigenetic modifications are commonly used by cells participate in this transcriptional rheostat: DNA methylation and histone modifications (Bird 2002; Yoo and Jones 2006).
1.3.1 DNA methylation
DNA methylation is one well known epigenetic mechanism and is one of the many potential causes for the abnormal growth of cancer cells. The methyl group is located in the fifth position of the ring in some cytosines within cytosine-phospho-guanine dinucleotides (CpG) in the genome of vertebrates (Vanyushin et al. 1970). The pattern of 5-methylcytosine distribution in the genome is unique for each cell type and is established and maintained by several DNA methyltransferases (DNMTs) subsequent to DNA replication in embryogenesis (Bestor et al. 1988).
Cytosine is methylated in the context of CpG dinucleotides, and most CpGs are methylated except for those on CpG islands. Most CpG dinucleotides are unevenly distributed throughout the genome and remain in short stretches or clusters (500-2000 bp), called CpG islands (Laird et al. 1996; Feltus et al. 2006; Vertino et al. 1996). These islands are located in the promoter region, generally kept unmethylated and found in half of all human genes (Baylin 2005). It has been increasingly recognized over the past 4-5 years that the CpG islands of a large number of genes, which are mostly unmethylated in normal tissues, are methylated to varying degrees in human cancers (Yang et al. 2001). In mammals, DNA methylation occurs after replication, when DNA methyltransferase can transfer a methyl group (CH3) from S-adenosyl-methionine to the 5 position of cytosine residues in CpG dinucleotides sequence (Figure 1) (Bird 2002; Dunn 2003; Jones and Takai 2001). With the methylation of CpG islands, endonucleases degrade 4
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