邵吉民，教授，病理学与病理生理学系 shaojimin@zju

1. Cancer Etiology1. Introduction 2. Chemical Factors in Carcinogenesis 3. Physical Factors in Carcinogenesis4. Viral Oncogenesis5. Genetic Predisposition 邵吉民，教授，病理学与病理生理学系 shaojimin@zju.edu.cn

2. Introduction • Tumor • Benign tumor • Malignant tumor

3. Cancer Incidenceand 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年国际抗癌联盟（UICC）： • 2008年全世界1270万新增癌症患者，死亡人数760万。 • 全国肿瘤登记中心《2012中国肿瘤登记年报》 • 每年新发肿瘤病例约312万例，每天约8550人; • 每年因癌死亡270万例,居民因癌死亡率13%，即每7-8人中有1人因癌死亡。 • 恶性肿瘤发病:第一位肺癌，其次胃癌、结直肠癌、肝癌和食管癌; • 恶性肿瘤死亡：第一位肺癌，其次肝癌、胃癌、食管癌和结直肠癌； • 中国近20年来癌症呈现年轻化及发病率和死亡率“三线”走高的趋势。癌症种类呈现地域化特点。

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

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

6. 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 6

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8. Initiation • Irreversible genetic damage: A necessary, but insufficient prerequisite for tumor initiation • Activation of proto-oncogene, inactivation of a tumor suppressor gene, and etc 8

9. 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 9

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 10

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 11

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 12

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 13

14. Classification of chemical carcinogens 1. Based on mechanisms • 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[a]pyrene, 2-acetylaminofluorene, benzidine, Aflatoxin B1, B2. 14

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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 16

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 17

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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 19

20. Direct Chemical Carcinogens • (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. • ---Monofunctional alkylating agentshave a single reactive group and thus interact covalently with single nucleophilic centers in DNA (although varied). • such as MNNG • ---Bifunctional alkylating agentshave 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 20

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

22. ---Bifunctional alkylating agents 22

23. Indirect Chemical Carcinogensand Their Phase I Metabolic derivatives 23

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

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 25

26. DNA Repair Repair systems • Direct DNA repair/ Direct reversal : • DNA alkyltransferase (O6-alkylguanine-DNA alkyl transferase) • One enzyme per lesion • Base excision repair (BER) • small adducts, • overlap with direct repair • glycosylase to remove the adducted base 26

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 27

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 28

29. Translesion DNA synthesis 29

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 ofsomatic mutation • Germline mutations may also occur • How to get exposure: contraceptives, hormone replacement therapy, or during prevention of miscarriage • Epidemiological studies

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

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

33. Physical factors in carcinogenesis

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

36. Viral Oncogenesis • RNA Oncovirus (Retrovirus) • DNA Oncovirus 36

37. 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)

38. Classification of retrovirus 38

39. Structure of RNA Oncovirus 39

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

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

42. Replication of RNA Oncovirus 42

43. Mechanisms of Oncogenesis Induced by RNA Oncovirus • Transducing Retrovirus v-onc • cis-Activating Retrovirus c-onc • trans-Activating Retrovirus tax trans-acting x p40tax rex repressive expression x p27rex,p21rex 43

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) 44

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; 45

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 46

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. 47

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

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 49

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 50