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Cancer Etiology 1. Introduction 2. Chemical Factors in Carcinogenesis 3. Physical Factors in Carcinogenesis 4. Viral Oncogenesis 5. Genetic Predisposition shaojimin@zju.edu.cn

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2 Tumor Benign tumor Malignant tumor Introduction

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3 Cancer Incidence and Mortality Siegel R, Naishadham D, Jemal A. Cancer statistics, 2012. CA Cancer J Clin. 2012;62(1):10-29. 1,638,910 new cancer cases and 577,190 deaths from cancer are projected to occur in USA in 2012. One in 4 deaths in USA is due to cancer. 2010 UICC 2008 1270 760 2012 312 8550 ; 270 , 13% 7-8 1 : ; 20

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4 History of Cancer Research Kiberstis P, Marshall E. Cancer crusade at 40. Celebrating an anniversary. Introduction. Science. 2011;331(6024):1539.Cancer crusade at 40. Celebrating an anniversary. Introduction.

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Chemical Carcinogenesis Multi-stage Theory of Chemical Carcinogenesis Classification of chemical carcinogens Mechanisms of Chemical Carcinogenesis DNA Damage Induced by Ultimate Carcinogens DNA Repair

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66 Multi-stage Theory of Chemical Carcinogenesis Initiation -----------Genetic events Chemical Carcinogens (Direct and Indirect Carcinogens) Promotion ------- Epigenetic events Tumor promoters Murine skin carcinogenesis model: A single dose of polycyclic aromatic hydrocarbon (PAH, initiator) Repeated doses of croton oil (promoter) Malignant conversion Progression ------ Genetic and epigenetic events

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88 Initiation Irreversible genetic damage: A necessary, but insufficient prerequisite for tumor initiation Activation of proto-oncogene, inactivation of a tumor suppressor gene, and etc

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99 Promotion Promotion: Selective expansion of initiated cells, which are at risk of further genetic changes and malignant conversion Promoters are usually nonmutagenic, not carcinogenic alone, often do not need metabolic activation, can induce tumor in conjuction with a dose of an initiator that is too low to be carcinogenic alone Chemicals capable of both initiation and promotion are called complete carcinogens: benzo[a]pyrene and 4- aminobiphenyl

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10 Malignant conversion The transformation of a preneoplastic cell into that expresses the malignant phenotype Further genetic changes Reversible The further genetic changes may result from infidelity of DNA synthesis May be mediated through the activation of proto-oncogene and inactivation of tumor- suppressor gene

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11 Progression The expression of malignant phenotype, the tendency to acquire more aggressive characteristics, Metastasis Propensity for genomic instability and uncontrolled growth Further genetic changes: the activation of proto- oncogenes and the inactivation of tumor- suppressor genes

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12 Activation of proto-oncogenes: Point mutations: ras gene family, hotspots Overexpression: Amplification Translocation Loss of function of tumor-suppressor genes: usually a bimodal fashion Point mutation in one allele Loss of second allele by deletion, recombinational event, or chromosomal nondisjunction

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13 Gene-environmental interactions The metabolism of xenobiotics by biologic systems Individual variation The competition between activation and detoxication The alteration of genes by xenobiotics

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14 Classification of chemical carcinogens 1. Based on mechanisms (1)Genotoxic carcinogen (DNA-reactive) Direct-acting: intrinsically reactive N-methyl-N-nitro-N-nitrosoguanidine (MNNG), methyl methanesulfonate (MMS), N-ethyl-N-nitrosourea (ENU), nitrogen and sulfur mustards Indirect-acting: require metabolic activation by cellular enzyme to form the DNA-reactive metabolite (members of the cytochrome P450 family) benzo[ ]pyrene, 2-acetylaminofluorene, benzidine, Aflatoxin B1, B2.

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16 (2) Epigenetic carcinogens Promotes cancer in ways other than direct DNA damage/ do not change the primary sequence of DNA Alter the expression or repression of certain genes and cellular events related to proliferation and differentiation Promoters, hormone modifying agents, peroxisome proliferators, cytotoxic agents, and immunosuppressors Organochlorine pesticides, [saccharin], estrogen, cyclosporine A, azathioprine

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17 2. Based on sturcture (1) Nitrosamines (NA) MNNG, MMS (direct carcinogen) (2) Polycyclic aromatic hydrocarbons (PAH) Benzo(a)pyrene (indirect carcinogen) (3) Aromatic amines (AA) 2-acetylaminofluorene, benzidine (indirect carcinogen) (4) Aflatoxin (AF) (5) Inorganic elements and their compounds: arsenic, chromium, and nickel are also considered genotoxic agents

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19 Mechanisms of Initiation in Chemical Carcinogenesis (1) DNA damages: Pro-carcinogen metabolic activation (Phase I and II) Ultimate carcinogen (electrophiles) Interaction with macromolecules (nucleophiles) DNA damage, mutations, chromosomal aberrations, or cell death (2) Epigenetic changes (3)Activation of oncogenes; inactivation of tumor suppressor genes

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20 (1) Alkylating agents are electrophilic compounds with affinity for nucleophilic centers in organic macromolecules. [Fu D, Calvo JA, Samson LD. Balancing repair and tolerance of DNA damage caused by alkylating agents. Nat Rev Cancer. 2012 Jan 12;12(2):104-20. doi: 10.1038/nrc3185.] (2) These agents can be either monofunctional or bifunctional. ---M onofunctional alkylating agents have a single reactive group and thus interact covalently with single nucleophilic centers in DNA (although varied). such as MNNG ---B ifunctional alkylating agents have two reactive groups, and each molecule is potentially able to react with two sites in DNA. Interstrand DNA cross-link: the two sites are on opposite polynucleotide strands; Intrastrand cross-link: on the same polynucleotide chain of a DNA duplex. such as Nitrogen and sulfur mustard, mitomycin, cis-platinum Direct Chemical Carcinogens

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21 Numerous potential reaction sites for alkylation have been identified in all four bases of DNA (not all of them have equal reactivity: MNNG N-Methyl-N-nitroso-N'-nitroguanidine ---Monofunctional alkylating agents

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22 ---Bifunctional alkylating agents

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23 Indirect Chemical Carcinogens and Their Phase I Metabolic derivatives

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24 BPDE binds DNA covalently, resulting in bulky adduct damage BPDE intercalates into dsDNA non-covalently, leading to conformational abnormalities

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25 Types of DNA Damage Induced by Ultimate Carcinogens DNA Adduct Formation DNA Break Single Strand Break Double Strand Break DNA Linkage DNA-DNA linkage DNA-protein Linkage Intercalation Bulky aromatic-type adducts, Alkylation (small adducts), Oxidation, Dimerization, Deamination

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26 Repair systems Direct DNA repair/ Direct reversal : DNA alkyltransferase (O 6 -alkylguanine-DNA alkyl transferase) One enzyme per lesion Base excision repair (BER) small adducts, overlap with direct repair glycosylase to remove the adducted base DNA Repair

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27 Nucleotide excision repair (NER): involves recognition, preincision, incision, gap-filling, and ligation, large distortions strand specific, the transcribed strand is preferentially repaired xeroderma pigmentosum (XP): NER deficiency Mismatch repair (MMR) transition mispairs are more efficiently repaired (G-T or A-C) than transversion mispairs microenvironment influences efficiency similar to NER involves the excision of large pieces of the DNA

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28 Double-strand breaks (DSBs) homologous recombination non-homologous end joining (NHEJ): DNA-PK Postreplication repair a damage tolerance mechanism occurs in response to replication of DNA on a damaged template the gap either filled through homologous recombination with parental strand or insert an A residue at the single nucleotide gap

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29 Translesion DNA synthesis

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30 Hormones and the etiology of cancer Major carcinogenic consequence of hormone exposure: cell proliferation The emergence of a malignant phenotype depends on a series of somatic mutation Germline mutations may also occur How to get exposure: contraceptives, hormone replacement therapy, or during prevention of miscarriage Epidemiological studies

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31 Hormone-related cancer Breast cancer and estrogen Endometrial cancer: Estrogen replacement therapy Ovarian cancer: follicle stimulating hormone Prostate cancer and androgen Vaginal adenocarcinoma: in utero diethylstilbestrol (DES) exposure

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32 Other hormone-related cancers Cervical cancer: OC use might increase the risk, still a lot complicating factors Thyroid cancer: the pituitary hormone thyroid stimulating hormone (TSH) Osteosarcoma: incidence associates with the pattern of childhood skeleton growth; and hormonal activity is a primary stimulus for skeleton growth

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33 Physical factors in carcinogenesis

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34 Physical carcinogens Corpuscular radiations Electromagnetic radiations (EMF) Ultraviolet lights (UV) Low and high temperatures Mechanical traumas Solid and gel materials

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36 Viral Oncogenesis RNA Oncovirus (Retrovirus) DNA Oncovirus

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RNA Oncovirus Retroviruses: ssRNA viruses Reverse transcriptase Oncogenes Rous sarcoma in chickens (RSV): in 1911 Human T-cell lymphotropic virus (HTLV-I,II) Human immunodeficiency virus (HIV)

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38 Classification of retrovirus

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39 Structure of RNA Oncovirus

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40 Genome of RNA Oncovirus and Gene Products Genome of Human T-cell Leukemia virus (HTLV)

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41 Life cycle 1.Receptor binding and membrane fusion 2.Internalization and uncoating 3.Reverse transcription of the RNA genome to form double-stranded linear DNA 4.Nuclear entry of the DNA 5.Integration of the linear DNA into host chromosomal DNA to form the provirus 6.Transcription of the provirus to form viral RNAs 7.Splicing and nuclear export of the RNAs 8.Translation of the RNAs to form precursor proteins 9.Assembly of the virion and packaging of the viral RNA genome 10.Budding and release of the virions 11.Proteolytic processing of the precursors and maturation of the virions

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42 Replication of RNA Oncovirus

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43 Mechanisms of Oncogenesis Induced by RNA Oncovirus Transducing Retrovirus v-onc cis-Activating Retrovirus c-onc trans-Activating Retrovirus tax trans-acting x p40 tax rex repressive expression x p27 rex, p21 rex

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44 Oncogene transduction Acutely transforming in vivo and in vitro Transform cells by the delivery (transduction) of an oncogene from the host cell (v-onc) to a target cell Cause the formation of polyclonal tumors Most of this group of viruses are replication defective (the requirement of a helper virus) Examples: RSV (v-src); Abelson murine leukemia virus (v-Abl)

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45 Insertional activation Long latent periods, Less efficient Do not induce transformation of cells in vitro Usually are replication competent No oncogenes Tumors are usually monoclonal Provirus (LTR) is found within the vincity of a proto- oncogene (c-myc) Examples: lymphoid leukosis virus;

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46 Grow stimulation and two-step oncogenesis The defective spleen focus-forming virus (SFFV) and its helper, the Friend murine leukemia virus (Fr-MuLV) Induce a polyclonal erythrocytosis in mice Require the continued viral replication A mutant env protein gp55 of SFFV binds and stimulated the erythropoietin receptor, thus inducing erythroid hyperplasia Fr-MuLV or SFFV integration inactivates p53

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47 Transactivation HTLV-1 and 2 Like cis-activation group: replication competent, carries no oncogene, induces monoclonal leukemia, and latent Like transducing group: can immortalize cells in vitro, has no specific integration site Unique 3 genomic structure: the X region; Encodes at least three proteins: Tax (p40), Rex (p27, p21) Tax is the focus Transactivate the viral LTR, results in a 100- to 200-fold increase in the rate of proviral transcription Transactivate cellular enhancers and promoters, including genes for IL-2, granulocyte-macrophage colony-stimulating factor (GM-CSF), c-fos, and others.

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48 DNA Oncovirus Papilloma virus Polyoma virus Adenovirus Herpes virus: EB virus Hepatitis B virus

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49 Mechanism of Oncogenesis Induced by DNA Oncovirus Transforming proteins 1. HPV E6 interact with P53 E7 interact with RB 2. Adenovirus E1a interact with RB E1b 3. Polyoma virus SV40 Large T interact with RB Py virus Large and Middle T Transcription activators 1. EB virus EBNA-2 and LMP 2. HBV p28 X protein

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50 Gene Map and Function of HPV ORF Function E1 Virus proliferation E2 Regulation of transcription E5 E6 E7 Cell transformation L1 L2 Encoding capsid protein E4 Encoding late cytosolic protein E3 E8 Unkown E5: activates growth factor receptor E6: ubiquitin-mediated degradation of p53 E7: binds and inactivates unphosphorylated pRb

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51 Genome and Products of HBV Transforming gene: X gene X protein activates gene transcription via XRE

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52 Genetic Predisposition Hereditary Cancer Tumor Genetic Susceptibility --- Tumor susceptibility genes (Cytochrome P450 family, DNA repair genes, Tumor suppressor genes, etc) Immunity Hormones and metabolism Psychological factors others