Cancer Etiology

1. Chemical Factors in Carcinogenesis 2. Physical Factors in Carcinogenesis3. Viral Oncogenesis4. Genetic Predisposition

Jimin Shao

shaojimin@zju.edu.cn

Chemical Carcinogenesis

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

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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

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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.

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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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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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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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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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• 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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1. 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) (indirect carcinogen)

(5) Inorganic elements and their compounds: arsenic, chromium,

and nickel are also considered genotoxic agents

Classification of chemical carcinogens

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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 P450 family) benzo[]pyrene, 2-acetylaminofluorene, benzidine, Aflatoxin B1, B2.

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前致癌物（ procarcinogen）

终致癌物（ ultimate carcinogen）

水解，氧化，还原混合功能氧化酶系统 （ CYP450 和 P448 等）

代谢激活间接致癌物

直接致癌物

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(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

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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

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(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 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

Direct Chemical Carcinogens

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Numerous potential reaction sites for alkylation have been identified in all four bases of DNA (not all of them have equal reactivity):

---Monofunctional alkylating agents

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---Bifunctional alkylating agents

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Indirect Chemical Carcinogens and Their Phase I Metabolic derivatives

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BPDE binds DNA covalently, resulting in bulky adduct damage

BPDE intercalates into dsDNA non-covalently, leading to conformational abnormalities

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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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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

DNA Repair

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• 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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• 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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Extended ReadingTranslesion DNA synthesis

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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.

27Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4):Y-family of DNA polymerases.

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Cellular responses evoked by DNA damaging agents are very complex events

• Responses may triggered by the signals originated from: genomic and mitochondrial DNA damages, malfunction of signaling molecules, endoplasmic reticulum stress, and others.• Networks between different signaling pathways; • Cellular responses are the comprehensive and integrated

consequences.

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Gene-environmental interactions

• The metabolism of xenobiotics by biologic systems– Individual variation – The competition between activation and detoxication

• The alteration of genes and epigenetics by xenobiotics

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Physical factors in carcinogenesis

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Physical carcinogens

– Corpuscular radiations

– Electromagnetic radiations

– Ultraviolet lights (UV)

– Low and high temperatures

– Mechanical traumas

– Solid and gel materials

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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

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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

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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

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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

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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

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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

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• 7 complementation groups, with various reduced rates for excision repair

• An 8th, the XP variant, has a defect in replication of damaged DNA (polymerase )

• 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

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Cancer-associated PathogensPersistent 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.

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Viral Oncogenesis

• RNA Oncovirus (Retrovirus)

• DNA Oncovirus

RNA Oncovirus

Rous sarcoma in chickens (RSV): in 1911

Human T-cell lymphotropic virus (HTLV-I,II);

Human immunodeficiency virus (HIV)

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Classification of retrovirus

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Structure of RNA Oncovirus

Retroviruses: • ssRNA viruses• Reverse transcriptase• Oncogenes

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Genome of RNA Oncovirus and Gene Products

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Life cycle1. 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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Replication of RNA Oncovirus

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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

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• 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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•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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•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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• 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

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•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.

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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

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DNA Oncovirus

Papilloma virus: HPV

Polyoma virus

Herpes virus: EBV

Hepatitis B virus

Hepatitis C virus

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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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Gene Map and Function of HPV

ORF Function

E1 Virus proliferationE2 Regulation of transcriptionE5 、 E6 、 E7 Cell transformationL1 、 L2 Encoding capsid proteinE4 Encoding late cytosolic proteinE3 、 E8 Unkown

E5: activates growth factor receptorE6: ubiquitin-mediated degradation of p53E7: binds and inactivates unphosphorylated pRb

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Genome of EB Virus

EBNA (EB virus Nuclear Antigen) EBNA-1 Immortalization of cell EBNA-2 trans-acting transcription activator EBNA-3 Function unknownLP: Leader Protein RNA ProcessingLMP: Latent Membrane Protein Activation of NF-κBTP: Terminal Protein Function unknown

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Genome and Products of HBV

Transforming gene: X gene X protein activates gene transcription via XRE

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Genetic Predisposition • Hereditary Cancer

• Tumor Genetic Susceptibility• Hormones• Metabolism• Immunity• Psychological factors• others

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Some individuals are at increased risk of certain cancers because they inherited a cancer-predisposing genetic mutation.

Tumor susceptibility genes (DNA repair genes, Tumor suppressor genes, Cytochrome P450 family, etc).

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.

Tumor Genetic Susceptibility

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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

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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

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Some higher risks for certain types of cancerInflammation 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.

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.

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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。