Cancer Etiology 1 Chemical Factors in Carcinogenesis 2

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

Chemical Carcinogenesis u Multi-stage Theory of Chemical Carcinogenesis u Classification of chemical carcinogens u Mechanisms of Chemical Carcinogenesis u Types of DNA Damage u DNA Repair

PM 2. 5 and Cancer l l l WHO-IARC: 肺癌发病率和死亡率与空气污染密切相关. IARC于2013年 10月首次认定大气污 染对人类致癌，并视其为普遍和主要的环境致癌物. Outdoor air pollution, mostly by PM 2. 5, leads to 3. 3 million premature deaths per year worldwide (2010), predominantly in Asia. The contribution of outdoor air pollution to premature mortality could double by 2050. Air pollution is associated with many health impacts, including chronic obstructive pulmonary disease (COPD) , acute lower respiratory illness (ALRI), cerebrovascular disease (CEV), ischaemic heart disease (IHD), lung cancer (LC). The mortality attributable to air pollution in China is approximately an order of magnitude higher than that attributable to Chinese road transport injuries and HIV/AIDS, and ranks among the top causes of death. l International Agency for Research on Cancer, World Health Organization. World Cancer Report 2014. Feb 3, 2014. （http: //globocan. iarc. fr） l Lelieveld J, et al. The contribution of outdoor air pollution sources to premature mortality on a global scale. Nature. 2015; 525(7569): 367 -71. l Burnett, R. T. et al. An integrated risk function for estimating the Global Burden of Disease attributable to ambient fine particulate matter exposure. Environ. Health Perspect. 122, 397– 403 (2014). 3

China being the main contributor (1. 36 million per year).

Multi-stage Theory of Chemical Carcinogenesis Initiation ------Genetic events Chemical Carcinogens (Direct and Indirect Carcinogens) Promotion -------Epigenetic events Tumor promoters e. g. 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 5

Carcinogensis is multistep process, involving the multiple genetic and /or epigenetic changes, leading to the activation of oncogenes and the inactivation of tumor suppressors in cells. 6

Initiation • Irreversible genetic damage: A necessary, but insufficient prerequisite for tumor initiation • Activation of proto-oncogene, inactivation of a tumor suppressor gene, and etc 7

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 8

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 tumorsuppressor gene 9

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 protooncogenes and the inactivation of tumorsuppressor genes 10

• 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 11

Classification of chemical carcinogens 1. Based on sturcture (1) Nitrosamines (NA) N-methyl-N’-nitro-N-nitrosoguanidine (MNNG), methyl methanesulfonate (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) (indirect carcinogen) (5) Inorganic elements and their compounds: arsenic, chromium, and nickel are also considered genotoxic agents 12

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2. 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: metabolic activation by cellular enzyme to form the DNA-reactive metabolite (members of the cytochrome P 450 family) benzo[a]pyrene, 2 -acetylaminofluorene, benzidine, Aflatoxin B 1, B 2. 14

p直接致癌物 p间接致癌物 代谢激活 水解，氧化，还原 前致癌物 终致癌物 （procarcinogen） 混合功能氧化酶系统 （ultimate carcinogen） （CYP 450和P 448 等） 15

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

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, etc 17

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/nrc 3185. ] (2) These agents can be either monofunctional or bifunctional. ---Monofunctional alkylating agents have a single reactive group and thus interact covalently with single nucleophilic centers in DNA. such as MNNG ---Bifunctional alkylating agents have two reactive groups, and each molecule is potentially able to react with two sites in DNA. Interstrand DNA cross-link; Intrastrand cross-link. such as Nitrogen and sulfur mustard, mitomycin, cis-platinum 18

---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): 19

---Bifunctional alkylating agents 20

Indirect Chemical Carcinogens and Their Phase I Metabolic derivatives 21

BPDE binds DNA covalently, resulting in bulky adduct damage BPDE intercalates into ds. DNA noncovalently, leading to conformational abnormalities 22

Types of DNA Damage Induced by Ultimate Carcinogens • • • During the course of life of a single cell, the genome is constantly damaged by endogenous and exogenous agents. Damage to DNA impairs essential DNA metabolic processes such as DNA replication, transcription, and recombination and if left unrepaired, leads to events generating chromosomal rearrangements, fusion, deletion, mutations or chromosomal loss. Defective repair or replication errors leads to permanent changes in the genetic information that may pass on to the daughter cells. • • DNA Adduct Formation DNA Break Single Strand Break Double Strand Break DNA Linkage DNA-DNA linkage DNA-protein Linkage Intercalation l l l Bulky aromatic-type adducts, Alkylation (small adducts), Oxidation, Dimerization, Deamination 23

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DNA Repair 英美三位科学家荣获了2015年诺贝尔化学奖，获奖理由是“DNA修复的细胞机制研究”。对不 同的DNA损伤，细胞可以有不同的修复反应。在哺乳动物细胞中发现了四个较为完善的DNA 修复通路，分别是核苷酸切除修复、碱基切除修复、重组修复和错配修复。 • • To ensure genome stability, cells use a global signaling network, namely the DNA damage response (DDR) to sense and repair different types of DNA damage，and coordinates a response that includes activation of transcription, cell cycle control, DNA repair pathways, apoptosis, senescence, and cell death. Despite several repair mechanisms that repair different types of DNA lesions, it is likely that the replication machinery would still encounter lesions that are mis-repaired or not repaired. In this scenario, the cells employ the DNA damage tolerance (DDT) pathway that recruits a specialized low fidelity translesion synthesis (TLS) polymerase to bypass the lesions. DDT is not a repair pathway but a mechanism to tolerate DNA lesions. Paradoxically, DDT process is also associated with increased mutagenesis, which can in turn drive the cell to cancer development. DDT process functions as a double-edged sword guarding the genome. 25

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 • Nucleotide excision repair (NER): – involves recognition, preincision, 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 26

• Double-strand breaks (DSBs) – homologous recombination （HR） – non-homologous end joining (NHEJ): DNA-PK • Postreplication repair – 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 27

Extended Reading Translesion DNA synthesis 28

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. 29

30 Sulfolobus solfataricus P 2 DNA polymerase IV (Dpo 4): Y-family of DNA polymerases.

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. Polκcan carry out TLS past DNA containing benzo[a] pyrene-guanine adducts. 3. Rev 1 has a restricted DNA polymerase activity that is confined to the incorporation of one or two molecules of d. CMP regardless of the nature of the template nucleotide. • Rev 1 interacts with multiple TLS polymerases, notably Polη, Polκ, Polι, Polλ, and the REV 7 (subunit of Polζ). • Rev 1 protein may be specifically involved in polymerase switching during TLS. 4. Polι 5. Polζ is a heterodimer containing the Rev 3 catalytic subunit and the Rev 7 regulatory subunit. 31

Physical factors in carcinogenesis 32

Physical carcinogens – Corpuscular radiations – Electromagnetic radiations – Ultraviolet lights (UV) – Low and high temperatures – Mechanical traumas – Solid and gel materials 33

Ionizing radiation (IR) • Penetrate cells, unaffected by the usual cellular barriers to chemical agents • 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 34

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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 36

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 37

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 38

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 39

Xeroderma pigmentosum (XP) (着色性干皮病) • • • Autosomal recessive disease, 1/250, 000 Obligate heterozygotes (parents): asymptomatic Homozygotes: skin and eyes, even neurologic degeneration Onset at 1 -2 year of age 2, 000 times higher frequency for cancer 30 -year reduction in lifespan • • 7 complementation groups, with various reduced rates for excision repair An 8 th, the XP variant, has a defect in replication of damaged DNA (polymerase h) Groups A and D are very sensitive to UV killing Group C is the largest group, or called the common/classic form, only shows skin disorders, preferentially repairs transcriptionally active genes 40

Cancer-associated Pathogens p. Persistent infection with some pathogens is an important cause of about 20 percent of cancers worldwide. p. This knowledge has enabled the development of new cancer prevention strategies that use medicines and vaccines to eliminate or prevent infection with these agents. 41

Viral Oncogenesis • RNA Oncovirus (Retrovirus) • DNA Oncovirus 42

RNA Oncovirus Rous sarcoma in chickens (RSV): in 1911 Human T-cell lymphotropic virus (HTLV-I, II); Human immunodeficiency virus (HIV)

Classification of retrovirus 44

Structure of RNA Oncovirus Retroviruses: • ss. RNA viruses • Reverse transcriptase • Oncogenes 45

Genome of RNA Oncovirus and Gene Products 46

Life cycle 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 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 47

Replication of RNA Oncovirus 48

Mechanisms of Oncogenesis Induced by RNA Oncovirus • Transducing Retrovirus v-onc • cis-Activating Retrovirus c-onc • trans-Activating Retrovirus tax trans-acting x p 40 tax repressive expression x p 27 rex, p 21 rex 49

• 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) 50

• 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 protooncogene (c-myc) – Examples: lymphoid leukosis virus; 51

• Grow stimulation and two-step oncogenesis – The defective spleen focus-forming virus (SFFV) and its helper, the Friend murine leukemia virus (Fr-Mu. LV) – Induce a polyclonal erythrocytosis in mice – Require the continued viral replication – A mutant env protein gp 55 of SFFV binds and stimulated the erythropoietin receptor, thus inducing erythroid hyperplasia – Fr-Mu. LV or SFFV integration inactivates p 53 52

• 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 (p 40), Rex (p 27, p 21) – 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. Genome of HTLV 53

• Immunodeficiency • AIDS patients have an extraordinary increased rate of developing high-grade lymphomas and Kaposi’s sarcoma (KS) • Probably secondary • However, Tat protein of HIV (the transactivating protein) may induce KS-like lesions in mice. 54

Endogenous retroviruses • Exo or endo: somatic vs germline • 0. 5 -1% mammalian genome is composed of retroviral proviruses • Some properties: – Most are defective – Great variations between species or within – Variable level of expression – Generally not pathogenic – The potential to induce disease is notable 55

DNA Oncovirus Papilloma virus: HPV Polyoma virus Herpes virus: EBV Hepatitis B virus Hepatitis C virus 56

Mechanism of Oncogenesis Induced by DNA Oncovirus Transforming proteins 1. HPV E 6 interact with P 53 E 7 interact with RB 2. Adenovirus E 1 a interact with RB E 1 b 3. Polyoma virus SV 40 Large T interact with RB Py virus Large and Middle T Transcription activators 1. EB virus EBNA-2 and LMP 2. HBV p 28 X protein 57

Gene Map and Function of HPV ORF E 1 E 2 E 5、E 6、E 7 L 1、L 2 E 4 E 3、E 8 Function Virus proliferation Regulation of transcription Cell transformation Encoding capsid protein Encoding late cytosolic protein Unkown E 5: activates growth factor receptor E 6: ubiquitin-mediated degradation of p 53 E 7: binds and inactivates unphosphorylated p. Rb 58

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Genome and Products of HBV Transforming gene: X gene X protein activates gene transcription via XRE 60

Genetic Predisposition • • Hereditary Cancer Tumor Genetic Susceptibility Hormones Metabolism Immunity Psychological factors others 61

Tumor Genetic Susceptibility p. Some individuals are at increased risk of certain cancers because they inherited a cancer-predisposing genetic mutation. p. Tumor susceptibility genes (DNA repair genes, Tumor suppressor genes, Cytochrome P 450 family, etc). p. Not all potentially inheritable causes of cancer have been identified, but if an individual suspects that a relative has a cancer caused by one of the 17 known cancer-predisposing genetic mutations, he or she should consult a physician and consider genetic testing for verification. 62

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Hormones and cancer • Major carcinogenic consequence of hormone exposure: cell proliferation • How to get exposure: contraceptives, hormone replacement therapy, or during prevention of miscarriage • The emergence of a malignant phenotype depends on a series of somatic mutation; Germline mutations may also occur; • Epidemiological studies 64

Hormone-related cancer • • Breast cancer and estrogen Endometrial cancer: Estrogen replacement therapy Ovarian cancer: follicle stimulating hormone Vaginal adenocarcinoma: in utero diethylstilbestrol (DES) exposure • Prostate cancer androgen • Cervical cancer • 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 65

Some higher risks for certain types of cancer p. Inflammation and Cancer：for example, ulcerative colitis and Crohn disease increase an individual’s risk for colorectal cancer six fold. p. Obesity and Cancer: Obesity increases risk for a growing number of cancers, most prominently the adenocarcinoma subtype of esophageal cancer, and colorectal, endometrial, kidney, pancreatic, and postmenopausal breast cancers. It also negatively impacts tumor recurrence, metastasis, and patient survival for several types of cancers. p. Type 2 Diabetes Mellitus and Cancer: üThose with type 2 diabetes are most at risk for developing liver, pancreatic, and endometrial cancers, but also have an increased risk for developing biliary tract, bladder, breast, colorectal, esophageal, and kidney cancers, as well as certain forms of lymphoma. üit is not well established how type 2 diabetes increases cancer risk. üSimilar to obesity, type 2 diabetes increases levels of insulin and causes persistent inflammation. p. Energy balance is a complex dynamic that is not only influenced by calorie consumption and physical activity, but also by other factors such as genetics, diet composition, body weight or body composition, and sleep. How changes in energy balance promote cancer is an area of intense research investigation. 66

思考题： 1. 简述环境化学致癌因子分类及其致肿瘤机制。 2. 简述肿瘤病毒分类及其致肿瘤机制。 3. 肿瘤遗传易感性的概念。 67