Cancer genes

Cancer genes

1. Cancer Genes Mohammed Fathy Bayomy, MSc, MD Lecturer Clinical Oncology & Nuclear Medicine Faculty of Medicine Zagazig University

2. Definition : normal DNA sequences which when altered will initiate or complement process of cellular malignant transformation. Their normal counterparts regulate cellular growth & differentiation in response to environmental signals Classes 1) Oncogenes: arise from activation of normal proto-oncogenes & have growth stimulatory effect 2) Tumour suppressor genes: normally have growth suppressive function on cells. Inactivation of tumor suppressor genes will contribute to neoplastic development 3) Apoptosis genes 4) DNA repair genes

3. Function * Oncogenes & tumor suppressor genes have opposing actions: oncogenes favour cell proliferation & dedifferentiation and inhibit apoptosis (e.g. bcl-2). Conversely, tumor suppressor genes inhibit cellular proliferation, stimulate apoptosis, enhance cellular differentiation & stimulate DNA repair * Normally, there is balance between proto-oncogene & tumor suppressor gene functions, hence stable cell population * Imbalance between oncogene & tumor suppressor gene actions, with predominance of former, results in malignant transformation

4. Complementation of cancer genes * Dysfunction of at least two cancer genes with different functions are required (e.g. myc & ras) * In case of carcinoma of colon, there is mutation of at least 5 cancer genes (APC, ras, DCC, MCC, p53) in addition to other events as mutation of mismatch DNA repair genes & hypomethylation of DNA, both contributing to genetic instability

5. Oncogenes

6. Definition: genes that promote autonomous cell growth in cancer cells Derived by: mutations in proto-oncogenes Characterized by: ability to promote cell growth in absence of normal growth-promoting signals Inheritance: dominant genes (mutation of single allele is sufficient to cause cancer) Mechanism of activation of proto-oncogene to oncogene: * Chromosomal translocation * Gene amplification * Gene mutation

7. Their products: oncoproteins, resemble normal products of proto-oncogenes except that oncoproteins are devoid of important regulatory elements, & their production in transformed cells does not depend on growth factors or other external signals They are classified according to their oncogenic protein products 1) Growth factors 2) Growth factor receptors 3) Signal transduction proteins 4) Transcription factors 5) Cell cycle regulators 6) Antiapoptosis

8. Growth factors Mutations of genes that encode growth factors can make their products oncogenic & cancer cell is stimulated to proliferate by autocrine mechanism Oncogene c-sis encodes beta chain of PDGF (reported in astrocytomas & osteosarcomas) Oncogenes hst-1 & int-2 encode for FGF & are activated in several gastrointestinal & breast tumors Mutations of ras causes overexpression of TGF- α & EGF It is noteworthy that stimulation of cells by growth factors is not sufficient alone to cause cancer. They act as promoting factors through an epigenetic mechanism

9. Growth factor Receptors Mutant receptors: associated with persistent activation of tyrosine kinase activity of their cytoplasmic domain, even without binding of receptor to growth factor  continuous mitogenic signal to nucleus c-erb-B2 (HER-2/neu): reported in adenocarcinoma of breast; ovary & lung arises from mutation that alters single amino acid (valine to glutamine) in transmembrane region of receptor, making receptor constitutively active as kinase c-erb-B1: expressed in squamous carcinoma of lung, EGF receptor loses its binding domain leading to hyperactive truncated protein

10. Signal Transduction Proteins These membrane-bound signal transducers receive signals from receptor-related tyrosine kinase & transmit signal to nucleus by secondary messenger. They belong to two major categories: GTP-binding proteins (ras oncogene) * Mutation of ras is most common oncogene encountered & is observed in 30% of all human tumors of various types (90% of pancreatic cancer, 50% of colon & 30% of lung carcinoma) * Activated by point mutation * Normally there is orderly cycling of ras between guanosine diphosphate (ras-GDP, inactive form) & ras-GTP (active form) * GAP (GTPase activating protein) causes hydrolysis of ras-GTP to its inactive form

11. * With mutation of ras, GAP can not hydrolyse ras-GTP which remains in active state & stimulate another downstream mitogenic signal pathways namely Raf-1 & MAPK (mitogen activated protein kinase) Non-receptor associated Kinase (abl oncogene) * In chronic myeloid leukemia (CML), proto-oncogene (abl) is translocated from its normal location on chromosome 9 to chromosome 22 where it fuses with bcr (break point cluster region) gene * Resulting hybrid gene produce chimeric protein with potent mitogenic tyrosine kinase activity * Chromosome resulting from this reciprocal translocation is called Philadelphia chromosome

12. Transcription factors C-myc * In Burkitt's lymphoma, c-myc is translocated from its normal place on chromosome 8 to chromosome 14 where it is placed close to immunoglobulin heavy chain gene. In this new location c-myc in subjected to stimulation by enhancer element of immunoglobulin gene * Following translocation, c-myc protein is rapidly translocated to nucleus forms heterodimer with another protein (max) * c-myc/max complex activates transcription of several cell-cycle-related genes resulting in cellular proliferation N-myc: amplified in neuroblastoma L-myc: amplified in small cell lung cancer

13. Cell Cycle regulators Orderly progression of various stages of cell cycle is under control of two classes of proteins, namely: cyclins (regulatory units) & cyclin- dependent-kinases (catalytic units). CDKs are expressed continuously, but different cyclins are expressed periodically according to phase of cell cycle In mantle cell lymphoma, translocation t (11; 14)  activation of PRAD-1 gene  overexpression of cyclin-Dl  activates CDK-4  phosphorylation of Rb protein  release of E2F which drives cell to enter S-phase (activates transcription of several genes whose products are essential for progression through S-phase)

14. Antiapoptosis Genes which inhibit apoptosis lead to cell immortalization & cell accumulation, hence favoring neoplastic development In follicular lymphoma translocation t ( 14; 18) in which causes activation of antiapoptotic gene bcl-2

15. Tumor suppressor genes

16. Definition: genes that act as negative regulators, or brakes, of cell cycle & cellular proliferation Inheritance: autosomal recessive traits (mutations in both alleles are required to get disease), change of heterozygous to homozygos state = cancer develops after loss of heterozygosity (LOH) Regard to cancer risk: considered dominant (single mutant allele is associated with significant cancer risk) Classified according to mechanism of action 1) Growth inhibition factor (TGF- β ) 2) Inhibition of signal transduction (NF-1 for ras, APC for β catenin) 3) Augmentation of cell adhesion (E-cadherin) 4) Negative regulators of cell cycle (RB, p53, WT-1, p16) 5) Increase DNA repair (HNPCC, BRCA, MMR genes)

17. Mechanism of loss of function of tumor suppressor gene * Loss of heterozygosity (LOH) in tumour suppressor genes may occur by one of 4 mechanisms which results in loss of remaining normal allele: (1) deletion due to non-disjunction (2) non-disjunction & duplication (3) unequal crossing over (4) gene mutation * Inactivation of their protein products there is loss of tumour suppressor gene function in heterozygous state. This is explained by "dominant negative effect" phenomenon. Protein product of mutant suppressor gene inhibits protein product of wild suppressor gene

18. Retinoblastoma gene (RB) Located in: long arm of chromosome 13 Loss of function of Rb gene: involved not only in retinoblastoma, but also in several other cancers as osteosarcoma, breast, lung and bladder Most retinoblastomas (90%) are sporadic but about 10% are familial Knudson double hit hypothesis: in familial cases, one genetic change (first hit) is inherited from affected parent (germline mutation) & therefore present in all somatic cells. Second mutation (second hit) occurs in one of retinal cells leading to loss of heterozygosity (LOH) & tumor formation. Conversely, in sporadic type of retinoblastoma, both mutations are acquired after birth in somatic cells

19. Product of Rb gene (pRb) * Nuclear phosphoprotein that regulates cell cycle * Normally, it acts as brake of cell cycle by binding with a transcription factor E2F. Thus, in its active hypophosphorylated state pRb prevents cell replication by binding with transcription factor E2F. This binding occurs in special site of Rb molecule called Rb pocket. Any factor which inhibits Rb phophorylation (e.g. p53 action) will keep E2F in bound state with arrest of cell cycle in G1

20. * This braking mechanism is lost with release of E2F from its bound state under 3 conditions 1) Phosphorylation of Rb as a result of ras gene action or loss of p53 gene function 2) Human papilloma virus onocprotein (E7) which binds with Rb pocket 3) Mutation of Rb gene which typically involves the Rb pocket. Under these conditions, E2F is released and induce cyclin E formation and cell enter S phase (cell proliferation)

21. p53 gene Located in: short arm of chromosome 17 Inactivation of p53 gene: the most common gene alteration in human cancer (>50% of human malignant tumors) Product of p53 gene ( p53 ) * Following DNA damage, wild p53 normally causes arrest of cell cycle to allow for DNA repair. If this initial mechanism fails, p53 induces apoptosis to eliminate damaged cells & keep genomic integrity (policeman of DNA or guardian of genome) * Nuclear phosphoprotein, under normal conditions (wild p53) it is concerned with negative control of cell cycle, DNA repair & apoptosis * Wild, p53 is activated following gamma or UV irradiation, chemotherapy or hypoxia

22. * Inhibitory action of wild p53 on cell cycle is mediated through Rb protein * p53  activates gene WAF-1  produce inhibitory protein p21  inhibits cyclin/cyclin-dependent kinase (cycl-Dl/ CDK-4)  hypophosphorylation of pRb  keeps transcription factor E2F in bound state of pRb with arrest of cell cycle in Gl

23. * Results in abnormal protein devoid of its normal function * Characterized by remarkably long half life * There is loss of cell cycle control with unchecked cellular proliferation * Policeman action of p53 is lost  cellular mutations are propagated rather than eliminated. Hence, genetic instability observed in malignant tumors Loss of p53 function (or inactivation) occurs under 3 conditions Mutant p53 gene or inactivated p53 1) Missense mutation, which may be germline (Li- Fraumeni syndrome) or somatic (in several tumors) 2) Inhibitory effect of p53 gene product e.g. MDM2, kind of feedback auto-regulation 3) Inhibitory effect of human papilloma virus oncoprotein products (E6)

24. Apoptosis genes

25. Apoptosis Definition: Apoptosis is defined as a distinctive active, genetically programmed cell death which eliminates unwanted cells. Literally, apoptosis in Greek language means falling of leaves of trees in winter Character * May be physiological or pathological * Resulting from mild injuries * Affects single isolated cells * Nonviable cell constituents shrink & are enclosed by cell membranes,  absence of inflammatory reaction

26. Pathogenesis: enzymes are activated by calcium ions, cytochrome-C, Apaf-1 & ceramide * Enzymatic cleavage of cytoskeleton protein by cysteine proteases (caspases) * Protein cross-linking by transglutaminases * Cleavage of DNA by several agents, including enzymes endonuclease Pathways: two enzymes activation pathways 1) Extrinsic (Death receptor) pathway FAS ligand (soluble or membrane-bound) will bind to FAS cell membrane receptor or cytokine tumor necrosis factor (TNF) will bind to its receptor (TNFR) on cell membrane  activation of membrane-bound protein FADD  caspase activation

27. 2) Intrinsic (Mitochondrial integrity) pathway Increase of mitochondrial permeability  release of calcium & cytochrome-C from mitochondria to cytosol  assembly of apoptotic protease activating factor (Apaf-1)  activates caspases

28. Genetic control of Apoptosis Bcl-2 family members are decision-makers that integrate pro-apoptotic and anti-apoptotic signals to determine whether cell should commit suicide 1) Anti-apoptotic Bcl-2 type proteins * Including Bcl-2, Bcl-xL, Bcl-wL * Two ways of antagonizing death signals - They insert into outer mitochondrial membrane to antagonize channel-forming pro-apoptotic factors  cytochrome c release - Bind cytoplasmic Apaf so that it cannot form apoptosome complex

29. 2) Pro-apoptotic Bcl-2 type proteins * Pro-apoptotic ion channel forming members (Bax, Bak, Bok, Bcl-xs) when they dimerize with pro-apoptotic BH3-only members in outer mitochondrial membrane, they form an ion channel that promotes cytochrome c release rather than inhibiting it * Pro-apoptotic BH3-only proteins (Bad, Bid, Bom/Bim) activates pro-apoptotic family members (Bax) & inactivates anti-apoptotic members (Bcl-2)

30. DNA Repair genes

31. Mutation Definition: any permanent & heritable change in DNA base sequence Classification * Germline mutation: mutation occurs in gamete cells, mutation will be passed on to all cells of body * Somatic mutation: mutation affects somatic cell it will be more restricted & passes to only descendents of that cell lineage in particular organ 1) According to cell type affected 2) According to structural changes * Point mutation: involves single base that substitute one amino acid for another * Deletions or insertions: remove or add one or more bases * Inversion mutation: inverts segment of DNA sequence

32. * Missense mutation: substitutes one amino acid for another thus resulting in abnormal protein * Nonsense mutation: substitutes a stop codon for amino acid codon leading to incomplete or truncated protein * Frameshift mutation: adds or deletes base with shift of base sequence, hence, changing reading frame, resulting in mistranslation of all amino acid sequence beyond mutation * Splicing mutation: will act on splice junction usually causing major changes in m-RNA & encoded protein 3) According to consequence of mutation on encoded protein

33. Pathogenesis * Mispairing of bases ( A-C pair instead of normal A-T pair) during DNA synthesis ( replication error ) or recombination * Slippage of newly synthesized DNA strand during/replication with loop formation is another replication error resulting in copying of bases twice * Loss of purine bases ( depurination ) occurs as a result of effect of body heat and cellular metabolism * De-amination of cytosine leading to C-T substitution 1) Spontaneous mutations

34. * Ultraviolet radiation produce pyrimidine dimers in which adjacent thymine residues in same DNA strand become covalently attached * Exposure to ionizing radiation may produce various lesions such as : base damage (e.g. conversion of thymine to thymine glycol or change of guanine to hydroxy guanine), single or double strand breaks in DNA phosphate-sugar backbone, and cross-links between the two strands of DNA or between DNA & proteins * Chemotherapeutic-agents may cause base damage (alkylating agents), strand breaks (bleomycin) or various adducts or cross-links (cisplatin) 2) Induced mutations

35. DNA Repair Cells have evolved an elaborate array of enzymatic systems to maintain integrity of their genetic material in the face of numerous agents that alter DNA structure or base sequence Cellular repair of macromolecule is known to occur only for DNA, vital molecule for cell survival Tumor suppressor gene p5 3 plays key role in this regard, hence it is called guardian of genome. Thus, with DNA injury, p53 causes arrest of cell cycle to give time for DNA repair enzymes. If DNA lesions are severe & irrepairable, p5 3 eliminates mutant cells by stimulating apoptosis

36. DNA Repair Systems Damaged DNA is reverted to its original state without replacement of constituents Example 1) Damage Reversal * Repair of base damage, by alkyl transferase enzyme , removing methyl & larger alkylating groups from DNA bases * Rejoining single strand break by ligase 2) Mismatch repair (MMR) Four types of mismatch repair genes : hMSH-2 (2p), hMCH-1 (3p), hPMS-1, hPMS-2 During replication, mismatch protein gene products act as "spell checkers" and if there is replication error (erroneous pairing of G-T instead of A-T), this system corrects defect

37. Mismatched lesion is recognized by hMSH-2 protein, or hMSH-2: GTBP/P160 protein heterodimer Removal of mismatch, followed by resynthesis & ligation completes repair process 3) Excision repair A) Base Excision Repair (BER) Limited to small lesions (de-aminated cytosine, single strand breaks Repair patches consist usually of only one or two nucleotides (short patch repair) Base damage is recognized & excised by glycosylase , removal of sugar residue by endonuclease & exonuclease , followed by resynthesis & ligation

38. b) Nucleotide Excision Repair (NER) Involves replacement of long patches of DNA of order of thirty nucleotides (long patch repair) There are 5 steps in NER process, namely: recognition of DNA lesion, incision of damaged strand on each side of lesion, removal of damaged nucleotides, synthesis of new nucleotides, & its ligation 4) Recombination repair Double strand breaks are most difficult to repair due to absence of template strand Unrepaired or misrepaired double strand breaks may lead to serious consequences: end-to-end joining of nonhomologous chromosomes, or formation of new telomeres generating chromosomal rearrangements deletions, use of nonhomologous DNA strands as template results in formation of abnormal base sequence (mutation)

39. Double strand breaks, in view of difficulty of repair, is most common cause of cell lethality following genotoxic agents Repair requires recruitment of homologous or heterologous DNA strands with formation of Holliday junction caused by crossing over of single strands from two adjacent DNA duplexes This junction requires resolution, by enzymatic cutting of crossing point, before repair & duplex separation are complete