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Cancer Genetics

Gene expression and regulation in cancer

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A successful tumor must aquire six specific capabilities

• become independent of external growth signaling

• become insensitive to external anti-growth signals

• become able to avoid apoptosis• Become capable of indefinite replication • Become capable of sustained angiogenesis • Become capable of tissue invasion and

metastasis

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• The aim of cancer genetics is to understand the multi step mutational and selective pathway that allowed a normal somatic cell to found the population of proliferating and invasive cancer cells.

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Cancer results from alterations in critical regulatory genes that control cell proliferation, differentiation and surveillance

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Cancers can classified as;

• Carcinomas: are derived from epithelial cells

• Leukemias and Lymphomas: from blood cell precursors

• Sarcomas; solid tumor of connective tissue such as muscle, bone, cartilage and fibrous tissue.

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General concepts

• No single mutation can convert a normal cell into a malignant one

• On average six to seven successive mutations are needed to convert a epithelial cell into an invasive carcinoma

• Accumulating all these mutations takes time, so that cancer is mainly a disease of post-reproductive life.

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Stages of tumor development

• Tumor initiation: a single mutated cell begins to proliferate abnormally.

• Tumor progression: additional mutations followed by selection for more rapidly growing cells within the population.

• Tumor invasion and metastasis

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Two ways of making a series of mutations more likely

• Some mutations enhance cell proliferation, creating an expanded target population of cells for the next mutation. The rate of mutation per gene per cell generation is 10-7

• Some mutations affect the stability of the entire genome, at either the DNA or chromosomal level increasing the overall mutation rate.

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Causes of cancer• Carcinogens are substances that cause cancer• Many agents, including radiation, chemicals, and

viruses have been found to induce cancer in both experimental animals and humans. Radiation and chemical carcinogens act by damaging DNA and inducing mutations

• Carcinogens in tobacco smoke, solar UV, aflatoxin of molds

• Tumor promoters: Stimulating cell proliferation rather than cause mutation (hormones, such as estrogen)

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Mutation in two following groups of genes can cause cancer

• Oncogenes : these are genes whose normal activity promotes cell proliferation. Gain of function mutations in tumor cells create forms that are excessively or inappropriately active. The nonmutant versions are called proto-oncogenes.

• Tumor suppressor genes: TS gene products inhibit events leading towards cancer

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Tumor suppressor genes:

• TS genes:TS gene products inhibit events leading towards cancer. Some TS gene products prevent inappropriate cell cycle progression, some promote damaged cells into apoptosis.

• Both alleles of a TS gene must be inactivated to change the behavior of the cell

• TS gene acts as a brake and oncogene act as an accelerator

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The history of oncogenes

• Oncogenes were discovered in 1960 when it was realized that some animal cancer, ( especially leukemias and lymphomas) were caused by viruses(SV40,Papilloma and retroviruses)

• Viral oncogenes were copies of normal cellular genes ,the proto-oncogenes that had become accidentally incorporated into the retroviral particles

• Over 100 oncogenes are currently known15

The history of oncogenes

• In 1970 primary culture of NIH-3T3 mouse fibroblast was transformed by fragmented DNA of human cancer and the human DNA responsible was isolated by constructing a phage genomic library of transformed cell and screening the library for human specific ALU repeat

• The resulted oncogene was resemble to viral oncogene.

• The majority of oncogenes were discovered by this method

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The function of oncogenes• Oncogenes are including: Secreted growth factor(PDGB. EGF) V-sis oncogene encoding the beta subunit of

PDG and is a potent oncogen HST and INT2 are homolog with FGF and are

involved in gastric cancer and malignant melanoma

Cell surface receptors (e.g. ERBB) Component of intracellular signal transduction

systems (e.g. RAS family, ABL)

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The function of oncogenes

DNA binding nuclear proteins, including transcription factors (e.g. MYC, JUN,FOS,)

Components of the network of cyclins, cyclin-dependent kinases and kinase inhibitors that govern progress through the cell cycle (e.g.MDM2)

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Activation of proto-oncogenes

• Activation involves a gain of function • This can be quantitative (an increase in

the production of an unaltered product) or • Qualitative (production of a product as a

result of a mutation, or a novel product from a chimeric gene created by a chromosomal rearrangement)

• These changes are dominant and normally affect only a single allele of the gene

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Activation of proto-oncogenes

• Activation by amplification• Breast cancers often amplify ERBB2 and

sometimes c-MYC• N-MYC is usually amplified in late-stage

neuroblastomas and rhabdomyosarcomas

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Tumor viruses

• E7 inhibit pRb and E6 stimulate p53 degradation

• Sv40 T antigen interact with pRb and p53

• E1A of adenovirus binds to pRb E1B bind to p53.

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Activation of proto-oncogenes

• Activation by point mutation• RAS family genes(H,K, and N) that

mediate signaling by G-protein coupled receptors, are activated in a great variety of tumors. binding of ligand to the receptor triggers binding of GTP to the RAS protein, and GTP-RAS transmits the signal onwards in the cell

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Activation by point mutation

• RAS protein s have GTPase activity and GTP-RAS is rapidly converted to the inactive GDP-RAS

• The mutant RAS protein has reduced GTPase activity, so that the GTP-RAS is inactivated more slowly , leading to excessive cellular response to the signal from the receptor.

• Specific activating point mutations in RAS genes are frequently found in cells from a variety of tumors including colon, lung, breast and bladder cancers.

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Ras oncogenes

• Hras was the first human oncogene that known in gene transfer studies (homolog of Harvey sarcoma virus oncogene)

• Ras oncogene (H,Kand N ras) are involved in 20% of human’s tumors

• Point mutation in at critical position such as subtitution of Valine for glycine at position 12 convert ras proto-oncogene to oncogene

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Ras oncogenes

• The activity of RAS protein is controlled by GTPor GDP binding

• Mutant of RAS are stable in GTP binding state due to resistance of RAS to GAP (GTPase activating protein)

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Activation by a translocation that creates a novel chimeric gene

• This mechanism is rare in carcinomas but common in hematologic tumors and sarcomas.

• The best known example is the philadelphia(ph) chromosome seen in 90% of chronic myeloid leukemia.

• Fusion gene contains 5’part of BCR gene from chromosome 22 and 3’ part of ABL oncogene of chromosome 9 (bcr/abl). The resulted fusion protein has unregulated tyrosine kinase activity with abnormal transforming properties.

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Activation by translocation into a transcriptional active chromatin region

• Activation of MYC oncogene is the central event in Burkitt’s lymphoma

• A characteristic chromosomal translocation t(8;14) is seen in 75-85%of patients. Each of them puts the MYC oncogene close to an immunoglobin locus. Unlike of philadelphia chromosome there is not novel chimeric protein but the expression of MYC oncogene is high. Due to placing the oncogene in an environment of chromatin that is actively transcribed in antibody-producing B-cells.

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Activation by translocation into a transcriptionally active chromatin region

• These translocations are produced by special recombinases involved in immunoglobulin V-D-G gene rearrangement and the translocated MYC gene often contains de novo point mutations induced as part of the mechanisms for generating antibody diversity

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Tumor supressor genes

• Retinoblastoma; An eye tumor of children which develop from retinoblasts

• About 40% of cases are familial.These are inherited as an incompletely penetrant dominant character. Familial cases are often bilateral whereas the sporadic forms are always unilateral.

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Retinoblastomas (p110Rb)

• Knudson noted that the age –of-onset distribution of familial cases was consistent with a single mutation, while sporadic cases followed two-hit kinetics

• He reasoned that all retionoblastomas involved two hits but in familial cases one hit was inherited

• Cavenee typed tumor and blood of Rb patient and explain the hetrozygosity of blood sample for contigous marker

• Loss of hetrozygosity (13q14) 38

Three mechanisms of TS gene mutation

Point mutationDeletion (LOH) TS gene silencing by methylation of

promoter (The most common mechanism)

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Stability of the genome

• Chromosomal instability (CIN): Tumor cells typically have grossly abnormal karyotypes, with multiple extra and missing chromosomes many rearrangements…

• Microsatellite instability (MIN): is a DNA-level instability seen in a few tumors, especially some colon carcinomas

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Chromosomal instability

• Tumor cells lose the spindle checkpoint. This is probably the main source of the many numerical abnormalities.

• Tumor cells pass through the cell cycle despite having DNA damage

• Tumors may also replicate to the point that telomerese become too short to protect chromosome ends (crisis), which leads to all sorts of structural abnormalities

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The spindle checkpoint

• The spidle checkpoint should prevent chromosomal segregation at mitosis untill all chromosomes are correctly attached to the spindle fibers.APC gene is a candidate gene for this checkpoint and APC- / - cells have abnormal mitotic spindles that lead to chromosome instability.APC is involved in polyposis of colon.In the colon CIN is observed even in very early adenomas

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The DNA damage signaling system

• Cells are constantly repairing all sorts of damage to their DNA. The normal response to such damage is to stall the cell cycle until the damage is repaired and a common feature of many cancers is loss of that control

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Telomeres and chromosomal instability

• The end of human chromosomes are protected by a repeat sequence (TTAGGG)n, that is maintained by a special RNA containing enzyme system, telomerase

• Telomerase is present in human germ line but is absent from most somatic tissues and telomere length declines by 50-100 bp with each cell generation

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DNA repair defects • Nucleotid excision repair defects:repairing

single strand breaks due to ionizing radiation (Xeroderma pigmentosum)

• Base excision repair defects; (colon cancer due to defect in the MYH=MYH polyposis)

• Double strand break repair defects: By HR or NHEJ; ATM (AT) , NBS, BRCA1and BRCA2

• Replication error repair defects: HNPCC (MIN)

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Replication error repair defects

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Familial colon cancers

• Familial addenomatos polyposis (FAP or APC): The cause is an inherited mutation in the APC tumor suppresor gene.

• Hereditary nonpolyposis colon cancer (HNPCC) due to replication error repair defect. microsatellite instability (MIN) in HNPCC is general. Mutation in MLH1 and MSH2 cause HNPCC.

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• MYH polyposis mutation in mutY homolog

• Juvenile polyposis : Mutation in SMAD4

• Peutz Jegher syndrome: Mutation in STK11

• Cowden disease Mutation in pTEN

• Breast cancer;Amplification in ERB-B1,2 MYC,int2 and mutation in BRCA1 and BRCA2

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P53• P53 is the gaurdian of the genomes • Almost 50% of tumors have p53 mutations• Cell cycling stalls in cells with damaged DNA .

If the damage is not repairable, apoptosis is triggered . P53 has a crucial role in these processes.

• Aflatoxin B causes G toT mutation in p53 gene in codon 249

• P53 checks G1 to S check point

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P53

• Signals from a whole range of cellular stress sensors, including the damage sensors, lead to phosphorylation and stabilization of p53 .This increases p53-dependent transcription of genes such as p21 that inhibit cell cycling, PUMA and Noxa that control apoptosis

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p53• p53 may be knocked out by deletion, by

mutation or by the action of an inhibitor such as the MDM2 gene product ( which binds p53 and targets it for degradation; MDM2 also binds pRb).

• Constitutional mutations in families with the dominantly inherited Li-Fraumeni syndrome. (multiple primary tumors including sarcoma, renal carcinoma and breast cancer) mutation in codon 245 - 258

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Function of pRb

• In normal cells the gene product a 110 kd nuclear protein is inactivated by phosphorilation and activated by dephosphorilation. Active pRb binds and inactivates the cellular transcription factor E2F, function of which is required for cell cycle progression

• Phosphorilation of pRb is governed by a cascade of cyclins, cyclin-dependent kinases and cyclin kinase inhibitors.

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pRb

• Adenovirus E1A, SV40 T antigen and human papiloma virus E 7 protein can bind to pRb and sequester or degrade pRb, thus favoring cell cycle progression

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Pathway in colorectal cancer

• In FAP one copy of the APC gene is constitutionally mutated

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DNA Methylation

• Overall hypomethylation

• Hyper methylation in TS gene promoters

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Apoptosis and cancer

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Causes of cancer

• Skin cancers due to exposure to carbon in mine personel

• Bladder cancer due to exposure to anylen dye

• Hepatic angiosarcoma in PVC industry

• Lung cancer in silicate fiber industry

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• Helicobacter pilori increases gastric cancer risk 5-6 times

• The breast cancer risk in monozygotic and dizygotic female twins are %17 and 13% respectively.

• The breast cancer risk for a woman that has an affected in her first degree relative is 1.5 to 3 times more than general population risk

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Related with blood group

• There are some relationship between blood group and diseases.

• People with A blood group have 20% risk for gastric cancer compare other blood groups.

• Biochemical agent like slow acetilation ,debrisoquine metabolism have relationship with bladder cancer and GST activity related with lung cancers

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Inbred mice

• Inbred mice are suceptible to some cancers,e.g, C3H for liver and breast cancer and c58 for leukemia

• Viruses can cause breast cancer in mice and this reagent is transfered by milk.

• v-ONC and C-onc and Protooncogen

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Two important factor for oncogenesis

• Genetic susceptibility

• Enviroment agents

• The majority of cancers have not a clear known genetic or environmental causes

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Epidemiologic studies

• In breast cancer, breast feeding especially in young mother prevents from breast cancer

• The frequency of breast cancer in the USA and west European is 8 times more than Japanese's and Chinese wemen

• Gastric cancer has a indirect relation with the socio-economic condition and is frequent in china and japan

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