Cancer Etiology

1. Cancer Etiology Jimin Shao Department of Pathology and Pathophysiology

2. Cancer Etiology • 1. Extrinsic Factors （Environmental Factors） • Chemical Factors • Physical Factors • Cancer-associated Pathogens • 2. Intrinsic Factors • Inherited Cancer • Tumor Genetic Susceptibility • Immunity • Hormones • Metabolism • Psychological factors • others

3. DNA replication Errors Contribute to Cancer Risk • 致癌基因突变除了一直以来认为的遗传和环境因素，Bert Vogelstein 2017统计模型表明还需要注意第三种因素，不可避免的DNA复制突变。Tomasetti C, Li L, Vogelstein B. Stem cell divisions, somatic mutations, cancer etiology, and cancer prevention. Science. 2017 Mar 24;355(6331):1330-1334. • 从全球涵盖48亿人的69个国家收集海量数据，对32种癌症类型的发病率和相关性进行全面的研究。他们用数据证明了随机DNA复制错误在癌症中的主要作用：28.9％的癌症相关突变归因于环境因素，5％来自遗传因素，66.1％则是源于DNA复制随机错误。他们用数据证明了随机DNA复制错误在癌症中的主要作用：28.9％的癌症相关突变归因于环境因素，5％来自遗传因素，66.1％则是源于DNA复制随机错误。 Bert Vogelstein , AACR 2017 annual meeting Fig. 3 Etiology of driver gene mutations in women with cancer. For each of 18 representative cancer types, the schematic depicts the proportion of mutations that are inherited, due to environmental factors, or due to errors in DNA replication (i.e., not attributable to either heredity or environment).The sum of these three proportions is 100%.

4. Chemical Carcinogenesis • Multi-stage Theory of Chemical Carcinogenesis • Classification of chemical carcinogens • Mechanisms of Initiation in Chemical Carcinogenesis • Types of DNA Damage Induced by Ultimate Carcinogens • DNA Repair and Carcinogenesis

5. Multi-stage Theory of Chemical Carcinogenesis Carcinogensisis a 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---Genetic events Chemical Carcinogens (Direct and Indirect Carcinogens) • Irreversible genetic damage; a necessary, but insufficient prerequisite for tumor initiation • Activation of proto-oncogene, inactivation of a tumor suppressor gene, and etc • Promotion ---Epigenetic events Tumor promoters • Selective expansion of initiated cells, which are at risk of further genetic changes and malignant conversion • Tumore 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. e.g. Murine skin carcinogenesis model: A single dose of polycyclic aromatic hydrocarbon (PAH, initiator) Repeated doses of croton oil (promoter) • Chemicals capable of both initiation and promotion are called complete carcinogens: such as benzo[a]pyrene and 4-aminobiphenyl.

7. 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 • Progression --- Genetic and epigenetic events • Expression of malignant phenotype; 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

8. Classification of chemical carcinogens 1. Based on structure (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

9. Polycyclic aromatic hydrocarbons (PAH) Aromatic amines (AA) Nitrosamines (NA) Aflatoxin (AF)

10. 2. 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: metabolic activation by cellular enzyme(members of the cytochrome P450 family) to form the DNA-reactive metabolite benzo[a]pyrene, • 2-acetylaminofluorene, • benzidine, • Aflatoxin B1, B2.

11. Direct-acting carcinogen • Indirect-acting carcinogen • Ultimate carcinogen （procarcinogen）

12. (2) Epigenetic carcinogens • Promote 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 12

13. Mechanisms of Initiation in Chemical Carcinogenesis • 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 • Epigenetic changes • Activation of oncogenes; inactivation of tumor suppressor genes, etc 13

14. (1) Direct Chemical Carcinogens • Alkylating agents are electrophilic compounds with affinity for nucleophilic centers in organic macromolecules. • 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. • such as Nitrogen and sulfur mustard, mitomycin,cis-platinum • [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.]

15. ---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):

16. ---Bifunctional alkylating agents

17. (2) Indirect Chemical Carcinogens and Their Phase I Metabolic derivatives

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

19. 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; 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 • Alkylation (small adducts), • Bulky aromatic-type adducts, • Oxidation, • Dimerization, • Deamination

20. Typical products of DNA damage and/or related structural perturbations in DNA. • a single strand break (SSB), • abasic (apurinic or apyridiminic, AP) site. • Base mismatches (MM). • oxidation agents (OX – the same site as above and C8 of purines) • alkylation agents (ALK – endocyclic nitrogen atoms with N7 of guanine representing the most reactive site, exocyclic substituents of guanine), • deamination (DA). • pyrimidine cyclobutane dimers (products of DNA damage by UV light), and an intrastrand crosslink of two neighboring guanines by cancer therapeutics cisplatin.

21. DNA Repair and Carcinogenesis • 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.

22. 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 • 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

23. 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 23

24. Physical factors in carcinogenesis

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

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

27. AACR CANCER PROGRESS REPORT 2016

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

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

30. Structure of RNA Oncovirus • Retroviruses: • ssRNA viruses • Reverse transcriptase • Oncogenes

31. Genome of RNA Oncovirus and Gene Products

32. 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

33. Replication of RNA Oncovirus

34. 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

35. 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 (requirement of a helper virus) Examples: RSV (v-src); Abelson murine leukemia virus (v-Abl) 35

36. 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 vicinity of a proto-oncogene (c-myc) Examples: lymphoid leukosis virus;

37. 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. Genome of HTLV

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

39. 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 39

40. 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 40

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

42. Intrinsic factors in carcinogenesis • Inherited Cancer • Tumor Genetic Susceptibility • Immunity • Hormones • Metabolism • Psychological factors • others

43. Tumor Genetic Susceptibility • Some individuals are at increased risk of certain cancers because they inherited a cancer-predisposing genetic mutation. • e.g. RB gene (retinoblastoma), APC gene (CRC), P53 gene(~50% cancers), etc. • Tumor susceptibility genes(DNA repair genes, Tumor suppressor genes, Cytochrome P450 family, etc). • Inherited/familial cancer syndromes (遗传性肿瘤综合征） • the 17 known cancer-predisposing genetic mutations.

45. 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

46. 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 and 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

47. Some higher risks for certain types of cancer • Inflammation and Cancer：for example, ulcerative colitis and Crohn disease increase an individual’s risk for colorectal cancer six fold. • 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. • Type 2 Diabetes Mellitus and Cancer: • Those 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. • it is not well established how type 2 diabetes increases cancer risk. • Similar to obesity, type 2 diabetes increases levels of insulin and causes persistent inflammation. • How changes in energy balance promote cancer is an area of intense research investigation. 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.

48. 思考题： 简述环境化学致癌因子分类及其致肿瘤机制。 简述肿瘤病毒分类及其致肿瘤机制。 肿瘤遗传易感性的概念,举例说明。