肿瘤（ tumor ） 在致瘤因素作用下，细胞基因失去对细胞增殖、分化和死亡的正常调控，导致组织细胞不断增生而形成的新生物。 良性肿瘤（ benign tumor ）

1. Cancer Etiology1. Introduction 2. Chemical Factors in Carcinogenesis 3. Physical Factors in Carcinogenesis4. Viral Oncogenesis5. Genetic Predisposition

2. Introduction 肿瘤（tumor） 在致瘤因素作用下，细胞基因失去对细胞增殖、分化和死亡的正常调控，导致组织细胞不断增生而形成的新生物。 良性肿瘤（benign tumor） 恶性肿瘤（malignant tumor）

3. 肿瘤发病率和死亡率 • 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。 • 《2010中国卫生统计年鉴》： • 2009年中国恶性肿瘤成为首位死因。 • 每年新发癌症病人约200万，死亡人数约150万。 • 肺癌、肝癌、结直肠癌、乳腺癌、膀胱癌死亡率及其构成明显上升。 • 肺癌成为我国首位恶性肿瘤死亡原因。

5. Hallmarks of cancer(Weinberg, Cell, 2000) Figure 1. The Hallmarks of Cancer. This illustration encompasses the six hallmark capabilities originally proposed in our 2000 perspective. The past decade has witnessed remarkable progress toward understanding the mechanistic underpinnings of each hallmark. (Hanahan D, Weinberg RA. Hallmarks of Cancer: The Next Generation. Cell 2011, 144:646)

6. Hallmarks of cancer(Weinberg, Cell, 2011) . (Hanahan D, Weinberg RA. Hallmarks of Cancer: The Next Generation. Cell 2011, 144:646)

7. The Hallmarks of Cancer • Self-sufficiency in growth signals • Cancer cells do not need stimulation from external signals (in the form of growth factors) to multiply. • Insensitivity to anti-growth signals • Cancer cells are generally resistant to growth-preventing signals from their neighbours. • Tissue invasion and metastasis • Cancer cells can break away from their site or organ of origin to invade surrounding tissue and spread (metastasis) to distant body parts. • Limitless reproductive potential • Non-cancer cells die after a certain number of divisions. Cancer cells escape this limit and are apparently capable of indefinite growth and division (immortality). • Sustained angiogenesis • Cancer cells appear to be able to kickstart this process, ensuring that such cells receive a continual supply of oxygen and other nutrients. • Evading apoptosis • Apoptosis is a form of programmed cell death, the mechanism by which cells are programmed to die after a certain number of divisions or in the event they become damaged. Cancer cells characteristically are able to bypass this mechanism.

8. Deregulated metabolism • Most cancer cells use abnormal metabolic pathways to generate energy, a fact appreciated since the early twentieth century with the postulation of the Warburg hypothesis, but only now gaining renewed research interest. • Evading the immune system • Cancer cells appear to be invisible to the body’s immune system. • Unstable DNA • Cancer cells generally have severe chromosomal abnormalities, which worsen as the disease progresses. • Inflammation • Recent discoveries have highlighted the role of local chronic inflammation in inducing many types of cancer. • (Hanahan, D.; Weinberg, R. A. (2011). "Hallmarks of Cancer: The Next Generation". Cell144 (5): 646–674. doi:10.1016/j.cell.2011.02.013 )

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

10. 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 10

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13. Initiation • Irreversible genetic damage: A necessary, but insufficient prerequisite for tumor initiation • Activation of proto-oncogene, inactivation of a tumor suppressor gene, and etc 13

14. 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 14

15. 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 15

16. 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 16

17. 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 17

18. Gene-environmental interactions • The metabolism of xenobiotics by biologic systems • Individual variation • The competition between activation and detoxication • The alteration of genes by xenobiotics 18

19. 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. 19

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21. (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 21

22. 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 22

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24. 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 24

25. 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 25

26. ---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: 26 MNNG N-Methyl-N-nitroso-N'-nitroguanidine

27. ---Bifunctional alkylating agents 27

28. Indirect Chemical Carcinogensand Their Phase I Metabolic derivatives 28

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

30. 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 30

31. 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 31

32. 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 32

33. 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 33

34. Translesion DNA synthesis 34

35. 1.DNA damage blocks the progression of the replication fork. 2.PCNA plays a central role in recruiting the TLS polymerases (translesion DNA synthesis) and effecting the polymerase switch from replicative to TLS polymerase (low stringency DNA polymerases). 3. TLS polymerases carry out TLS, either singly or in combination, past different types of DNA damage. 4.Such regulation must ensure that (1) the specialized polymerases act only when needed, and (2) that polymerases act only at the right location in DNA. 5.TLS evolved in mammals as a system that balances gain in survival with a tolerable mutational cost, and that disturbing this balance causes a potentially harmful increase in mutations, which might play a role in carcinogenesis. 35

36. Classification of TLS polymerases 36

37. Characteristics of TLS polymerases • They operate at low speed, low processivity and with low fidelity. • Their active sites adopt a much more open structure than replicative polymerases, they are less stringent and can accommodate altered bases in their active sites. • Y-family polymerases lack a 3’-5’ exonuclease activity, which is an integral part of all replicative polymerases and performs a proofreading function. • Each Y family polymerase differs in substrate specificity. • All the Y-family polymerases are localized in the nucleus, and during S phase, • polη, ι, and Rev1 relocate to replication factories with the polymerase sliding • clamp PCNA, and other proteins associated with DNA replication. • There are three examples of TLS reactions in which a specialized DNA • polymerase bypasses its cognate DNA lesion with higher efficiency and higher • fidelity than any other polymerase in the cell: • ---Polh and the UV light-induced CPD (cyclobutane pyrimidine dimers); • ---Polk and benzo[a]pyrene-guanine (major tobacco smoke-induced DNA lesion); • ---Polh and cisplatin-GG (an adduct produced by a drug used in cancer chemotherapy). 37

38. 1. Polη • Polη was discovered as the protein deficient in the variant form of the skin cancer-prone genetic disorder xeroderma pigmentosum (XP). • Most XP patients are deficient in the ability to remove UV photoproducts from their DNA by nucleotide excision repair (NER), but about 20% have problems in replicating their DNA after UV irradiation because of defectiveness of polη gene. • Polη carrys out TLS past CPD (cyclobutane pyrimidine dimers) photoproducts generated by exposure to sunlight. XP variant cells have an elevated UV-induced mutation frequency. 2. Polκ Polκcan carry out TLS past DNA containing benzo[a] pyrene-guanine adducts. 3. Rev1 Rev1 has a restricted DNA polymerase activity that is confined to the incorporation of one or two molecules of dCMP regardless of the nature of the template nucleotide. • Rev1 interacts with multiple TLS polymerases, notably Polη, Polκ, Polι, Polλ, and the REV7 (subunit of Polζ). • Rev1 protein may be specifically involved in polymerase switching during TLS. 4. Polι 5. Polζ is a heterodimer containing the Rev3 catalytic subunit and the Rev7 regulatory subunit. 38

39. Cellular responses evoked by DNA damaging agents are very complex events • Responses may triggered by the signals originated from: genomic and mitochondrial DNA damage malfunction of signaling molecules endoplasmic reticulum stress others • Networks between different signaling pathways • Cellular responses are the comprehensive and integrated consequences 39

40. 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

41. Hormone-related cancer • Estrogen and breast cancer • Endometrial cancer: Estrogen replacement therapy • Ovarian cancer: follicle stimulating hormone • Prostate cancer and androgen • Vaginal adenocarcinoma: in utero diethylstilbestrol (DES) exposure

42. 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

43. Physical factors in carcinogenesis

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

45. Ionizing radiation (IR) • Penetrate cells, unaffected by the usual cellular barriers to chemical agents • IR: a relatively weak carcinogen and mutagen • The initial critical biologic change is damages to DNA • It takes place in a matter of the order of a microsecond or less

47. Electromagnetic fields (EMF) Remains controversial: • Minimal increase in relative risk of brain tumor and leukemia in electric utility workers • Also relatively increased risk for acute lymphoblastic leukemia by EMF exposure during pregnancy or postnatally • However, some studies lend no support for this proposition

48. Ultraviolet (UV) • Sunlight and skin cancer • Well established for basal and squamous cell cancers • Some controversy remains for melanoma • Nonmelanoma skin cancers are the most common cancer in the US (45%) • Usually occurs at the age of 50 – 60

49. Sunlight spectrum and wavelength • UVA (320-400) • photocarcinogenic • weakly absorbed in DNA and protein • active oxygen and free radicals • UVB (290-320) • overlaps the upper end of DNA and protein absorption spectra • mainly responsible through direct photochemical damage • UVC (240-290) • not present in ambient sunlight • low pressure mercury sterilizing lamps • experimental system

50. Shielding us from the sun • Ozone: shorter than 300 nm cannot reach the earth’s surface • UVA and UVB: only a minute portion of the emitted solar wavelengths ( 0.0000001%) • Skin: • melanin pigment • keratin layers