Oncogenes and Cancer Class: Molecular Biology, Graduate Institute of Basic Medical Sciences Source: “Genes VII” by Benjamin Lewin, Oxford University Press, 2000

2 Changes When a Cell Becomes Cancer Cell Immortalization Cancer cells are normal cells that have lost their growth control Types of changes:[Fig 28-1] >Immortalization: indefinite growth >Transformation: deviation from normal growth requirements and constrains; independent of anchorage and serum growth factors, not inhibited by density/contact (grow into focus) >Metastasis: invasion of normal tissues In vitro culture, normal cells  Senescence and cease of growth  Crisis Immortalization after surviving crisis; growth characters change and establishment of “cell line” Immortalized cells are “non-tumorigenic; still depend on anchorage & growth factor; density-dependent inhibition; cytoskeleton changes MonolayerAneuploid

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4 Changes When a Cell Becomes Cancer Cell Transformed Cell Lines Derived from tumors More changes in transformed than immortalized cell lines >Grow in much less restricted conditions >Reduced growth factor dependence >Less anchorage dependence: round-up vs. spread out >Forms foci instead of monolayer >Tumorigenic [Fig 28-2; Normal and transformed fibroblast cell lines] Heterogeneous basis for cancer cell formation

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6 Changes When a Cell Becomes Cancer Cell Multiple Genetic Changes Changes lead the conversion of normal cells  transformed cells: Multiple genetic changes: 6-7 events over years Factors (carcinogens) that increase the conversion: Initiate/Promotesuggest stages in cancer development Genes that cause transformation: Oncogenes (100+) Viral oncogenes and cellular counterparts (proto-oncogenes) Gain-of-function or Activated Tumor suppressor genes (~10) Loss-of-function or Inactivated How are oncogenes activated and tumor suppressor gene inactivated?

7 Subjects to Be Covered Transforming viruses carry oncogenes Retroviral oncogenes’ cellular counterparts Mutational activation of Ras proto-oncogenes Insertion, translocation, or amplification Oncogenes & signal transduction cascades Growth factor receptor kinases and cytoplasmic tyrosine kinases Oncoproteins may regulate gene expression Tumor suppressor RB controls the cell cycle p53 suppresses growth or triggers apoptosis Immortalization and transformation

8 Transforming Viruses Carry Oncogenes Transformation may result from tumor virus infection thus “oncogenes” >Polyomavirus/dsDNA/6Kb/ T antigen /Early viral gene/Inactivate tumor suppressor gene >Human papillomavirus/dsDNA/8Kb /E6 & E7 genes /Early viral genes/Inactivate tumor suppressor gene >Adenovirus/dsDNA/37Kb /E1A & E1B genes /Early viral genes/Inactivate tumor suppressor gene >Retrovirus(acute)/ssRNA/6-9Kb /Individual genes /Cellular origin/Activate oncogenic pathway Transformation occurs in non-permissive infection (vs. productive infection in permissive hosts) [Fig 28-4]

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10 Transforming Viruses Carry Oncogenes Common mechanism of DNA tumor virus transformation Early genes with oncogenic potential Integration of viral oncogenes into host genomes Oncogene proteins always interact with host cellular proteins [Cell transformation by polyomavirus/adenovirus; Fig 28-5 ] Polyoma & SV40 produce T-antigens early in infection T-antigen has transforming activity Papillomaviruses produce E6 & E7 oncoproteins EBV immortalized human B lymphocytes EBV oncogene unknown Retroviruses transfer vertically & horizontally

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12 Transforming Viruses Carry Oncogenes Common mechanism of DNA tumor virus transformation Retroviruses transfer vertically & horizontally[Fig 28-6] Integration of viral genome in germ line  vertical transfer Reverse transcription needed for virus w/ RNA genome Types of retroviruses: Non-defective tumor retrovirusesLeukemia viruses No viral oncogene; viral activation of cellular proto-oncogene Acute transforming tumor retroviruses Captured new genes in the form of oncogene (cellular origin) [Cellular gene in transforming retroviruses; Fig 28-7 ] Rare event, cannot replicate by itself and need helper virus Cellular genes might promote growth of transduced cells Other oncogenic mechanism also present.HIV-1

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15 Subjects to Be Covered Transforming viruses carry oncogenes Retroviral oncogenes’ cellular counterparts Mutational activation of Ras proto-oncogenes Amplification, insertion, or translocation Oncogenes & signal transduction cascades Growth factor receptor kinases and cytoplasmic tyrosine kinases Oncoproteins may regulate gene expression Tumor suppressor RB controls the cell cycle p53 suppresses growth or triggers apoptosis Immortalization and transformation

16 Retroviral Oncogenes’ Cellular Counterpart Oncogenes of some retroviruses[Fig 28-8] The normal cellular sequence itself is not oncogenic Difference between oncogenes & proto-oncogenes: cell type, quantity; quality, or both Changes in v-oncogenes could be very small Retrovirus capture of the proto-oncogene (c-)  oncogene (v-) 30+ c-onc genes identified; Most existing oncogene been identified? Rare event, and can be complex; Non-random Direct evidence that v-oncogene accomplishes transformation: Conditional-lethal mutant of v-src gene Oncogenes arise by activation of cellular or proto-oncogene is important to animal cancer Human cancer too?Most human cancers do not involve virus

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18 Subjects to Be Covered Transforming viruses carry oncogenes Retroviral oncogenes’ cellular counterparts Mutational activation of Ras proto-oncogenes Amplification, insertion, or translocation Oncogenes & signal transduction cascades Growth factor receptor kinases and cytoplasmic tyrosine kinases Oncoproteins may regulate gene expression Tumor suppressor RB controls the cell cycle p53 suppresses growth or triggers apoptosis Immortalization and transformation

19 Mutational Activation of Ras Proto-oncogenes Transfection assay[ Fig 28-9 ] Nude mouse test Transforming DNA isolated only from tumorigenic cells Properties of transforming genes: have closely related sequences in normal cells  Mutation theory mutation of normal genes created transforming genes may have counterparts in v-oncogenes carried by transforming virus  Repertoire of proto-oncogenes is limited Oncogenic variants of c-ras gene are found from various tumors Family of ras oncogenes: N-ras, H-ras, K-ras Single base mutation is suffice “Hot spots”/non-randomPositions 12 and 61

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21 Mutational Activation of Ras Proto-oncogenes Quantitative changes (amplification or over-expression) of c-ras gene can also transform normal cells ras protein[Fig 28-10] is a monomeric guanine nucleotide-binding protein has intrinsic GTPase activity interconverts between active and inactive ras proteins Constitutive activation of ras may be oncogenic Mutations that create oncogenic ras inhibition of GTPase activity

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23 Subjects to Be Covered Transforming viruses carry oncogenes Retroviral oncogenes’ cellular counterparts Mutational activation of Ras proto-oncogenes Amplification, insertion, or translocation Oncogenes & signal transduction cascades Growth factor receptor kinases and cytoplasmic tyrosine kinases Oncoproteins may regulate gene expression Tumor suppressor RB controls the cell cycle p53 suppresses growth or triggers apoptosis Immortalization and transformation

24 Amplification, Insertion, or Translocation Genomic changes (amplification, insertion & translocation) that cause proto-oncogene activation: Amplification: c-myc, c-abl, c-myb, c-erbB, c-K-ras, mdm-2 presence of known oncogenes in amplified region amplification of same oncogenes in many cancers Insertion: insertion of retrovirus LTR over-expresses c-myc [Insertion of ALV activates c-myc gene; Fig ] Translocation: [reciprocal translocation by illegitimate recombination; Fig ] immunoglobulin or TCR gene and c-myc oncogene Increased c-myc expression after translocation c-myc coding sequences are unaltered in all cases

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27 Amplification, Insertion, or Translocation Evidence of oncogenic potential of c-myc gene: Transgenic mice carrying c-myc that: linked to B lymphocyte enhancer  lymphoma under mouse mammary tumor virus LTR  various cancers Translocation can generate hybrid oncogenes & human cancers [CML & Philadelphia chromosome; Fig ] c-abl gene on chromosome 9 and bcr gene on chromosome 22 Why is the hybrid bcr-abl protein oncogenic? Activation of ras pathway for transformation

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29 Subjects to Be Covered Transforming viruses carry oncogenes Retroviral oncogenes’ cellular counterparts Mutational activation of Ras proto-oncogenes Amplification, insertion, or translocation Oncogenes & signal transduction cascades Growth factor receptor kinases and cytoplasmic tyrosine kinases Oncoproteins may regulate gene expression Tumor suppressor RB controls the cell cycle p53 suppresses growth or triggers apoptosis Immortalization and transformation

30 Oncogenes & Signal Transduction Cascades How oncogenes work to induce tumors? influence functions connected with cell growth not themselves necessarily code for products that characterize the tumor cells but may direct a cell into a particular pathway switch what are the functions of proto-oncogenes? growth regulator How are these proto-oncogenes changed in transformed cells? [Functions of oncogenes;Fig 28-14] Growth factors & receptors;G protein/signal transduction Intracellular tyrosine kinases;Serine/threonine kinases Signaling;Transcription factors Common features? Capable of triggering general changes in cell phenotype associated with cell growth.

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32 Oncogenes & Signal Transduction Cascades Common features of all oncogene protein functions Capable of triggering general changes in cell phenotypes Possible transformation signal transduction pathway: Growth factors interacts with  activates growth factor receptor (tyrosine kinase)  pass (via adaptor) to Ras  switches to cytoplasmic kinase cascade (serine/threonine kinases)  targeted at transcription factor(s)  widespread changes in pattern of gene expression Multiple signal transduction pathways might be involved Numerous proto-oncogenes code for growth factors

33 Subjects to Be Covered Transforming viruses carry oncogenes Retroviral oncogenes’ cellular counterparts Mutational activation of Ras proto-oncogenes Amplification, insertion, or translocation Oncogenes & signal transduction cascades Growth factor receptor kinases and cytoplasmic tyrosine kinases Oncoproteins may regulate gene expression Tumor suppressor RB controls the cell cycle p53 suppresses growth or triggers apoptosis Immortalization and transformation

34 Growth Factor Receptor kinases and cytoplasmic Tyrosine kinases Protein tyrosine kinases are major class of oncoproteins Transmembrane growth factor receptors Cytoplasmic group of protein kinases How their aberrant forms could be oncogenic? Transmembrane growth factor receptorskinase activity > extracellular N-terminal binds ligand that activates the receptor > intracellular C-terminal contains the kinase activity [Fig 28-15] > v-erb oncogene: truncated proto-oncogene c-erbB > constitutive activation of kinase activity > receptors in development of specific cell type ( myeloid precursor cells ) Cytoplasmic group of protein kinases

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36 Growth Factor Receptor kinases and cytoplasmic Tyrosine kinases Transmembrane growth factor receptors Cytoplasmic group of protein tyrosine kinases: more obscure ( src ; yes; fgr; fps/fes; abl; ros) src: 1 st kinase type oncoprotein & 1 st with tyrosine as target [ Domains of src protein; Fig 28-16] src protein is myristoylated, which is essential for tumorigenicity Major difference between c-src and v-src: kinase activity (~20X) Roles of kinase activity in src function: [Fig 28-17] > cellular phosphorylation targets results inconclusive > state of phosphorylation of src itself How is c-src usually activated? [Fig 28-18] Alternative ways exist for activating c-src

37 Phosphorylation of tyrosine residues 416 and 527 is important.

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40 Subjects to Be Covered Transforming viruses carry oncogenes Retroviral oncogenes’ cellular counterparts Mutational activation of Ras proto-oncogenes Amplification, insertion, or translocation Oncogenes & signal transduction cascades Growth factor receptor kinases and cytoplasmic tyrosine kinases Oncoproteins may regulate gene expression Tumor suppressor RB controls the cell cycle p53 suppresses growth or triggers apoptosis Immortalization and transformation

41 Oncoproteins May Regulate Gene Expression Gene expression alteration is always needed for transformation Oncogenes may code for transcription factors, which may > quantitatively and/or quantitatively altered DNA binding? > alter ability to activate transcription? [Oncogenes and transcription factors; Fig ] Example: a rel gene family is transcription factor NF-B Many stimuli to cells activate NF-B with broad spectrum effects Other transcription factors involved (AP1, jun, fos) Steroid hormone receptor & response elements Ability to bind DNA is also required for transforming capacity

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43 Subjects to Be Covered Transforming viruses carry oncogenes Retroviral oncogenes’ cellular counterparts Mutational activation of Ras proto-oncogenes Amplification, insertion, or translocation Oncogenes & signal transduction cascades Growth factor receptor kinases and cytoplasmic tyrosine kinases Oncoproteins may regulate gene expression Tumor suppressor RB controls the cell cycle p53 suppresses growth or triggers apoptosis Immortalization and transformation

44 Tumor Suppressor RB Controls the Cell Cycle Oncogenes : Gain of functions; dominant over proto-oncogene allele Tumor suppressor genes : Loss of both alleles is tumorigenic Tumor suppressor genes : functions needed for normal cell function loss of function causes tumors best known examples: RB and p53 Retinoblastoma is associated with deletion of q14 of chromosome 13 [Loss of heterozygosity; Fig ] [Nuclear phosphoprotein influences the cell cycle; Fig ] [Cell cycle control and tumorigenesis; Fig ]

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46 G0/G1 phase: nonphosphorylated S phase: phosphorylated by cyclin/cdk Target of Rb: E2F group of transcription factors, which activate genes that are essential for the S phase Rb prevents cells from entering S phase; Released E2F prompts the cell to enter S phase Viral tumor antigens (SV40’s T Ag, Adenovirus E1A and HPV E6) bind specifically to Rb Inactivation of Rb is needed for the cell to cycle, which can be done by cyclic phosphorylation or by sequestering by tumor antigens Over-expression of Rb impeded cell growth

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48 Subjects to Be Covered Transforming viruses carry oncogenes Retroviral oncogenes’ cellular counterparts Mutational activation of Ras proto-oncogenes Amplification, insertion, or translocation Oncogenes & signal transduction cascades Growth factor receptor kinases and cytoplasmic tyrosine kinases Oncoproteins may regulate gene expression Tumor suppressor RB controls the cell cycle p53 suppresses growth or triggers apoptosis Immortalization and transformation

49 p53 suppresses growth or triggers apoptosis >50% of cancers lost p53 or have mutations in p53 gene p53 protein level  in many tumor cells; Oncogene? Mutant protein acted as dominant negative mutants  tetramer Loss of p53: Cell growth advantage; not tissue-specific (many cancers) [Wild type p53 restrains cell growth; Fig 8-24 ] Implication : p53 inhibits normal cells’ capacity of unrestrained growth? Evidence that p53 is indeed a tumor suppressor gene : p53 - mice develop a variety of tumors early in life p53 DNA inhibits transformation by oncogenes in cultured cells Human Li-Fraumeni Syndrome (rare inherited cancer; heterozygous p53 mutation acted as dominant negative or autosomal dominant)

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51 P53 Suppresses Growth or Triggers Apoptosis p53 protects cells from consequences of DNA damages p53  (repair it or destroy if it is unable to repair!) Activation of p53  growth arrest or apoptosis [Fig 28-25] Depends on cell cycle Other molecular activities of p53 [Fig 28-26] p53 can also activate various pathways [Fig 28-27] as a transcription factor Cellular oncoprotein mdm2 inhibits p53 activity [Fig 28-28] forms a negative feedback circuitry How p53 trigger apoptosis? Separable from growth arrest Is p53 function essential for survival?

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56 P53 Suppresses Growth or Triggers Apoptosis How p53 trigger apoptosis? separable from growth arrest at the G1 checkpoint Connection between tumorigenesis & loss of apoptosis apoptosis inhibits tumorigenesis by eliminating tumorigenic cells p53 function is probably not essential for survival p53 - animals Definitive proof the p53 & Rb suppress tumorigenesis still lacking P53 acts as a sensor that integrates information from many pathways that affect the cell’s ability to divide

57 Subjects to Be Covered Transforming viruses carry oncogenes Retroviral oncogenes’ cellular counterparts Mutational activation of Ras proto-oncogenes Amplification, insertion, or translocation Oncogenes & signal transduction cascades Growth factor receptor kinases and cytoplasmic tyrosine kinases Oncoproteins may regulate gene expression Tumor suppressor RB controls the cell cycle p53 suppresses growth or triggers apoptosis Immortalization and transformation

58 Immortalization and Transformation Tumors arise from multiple events: Activation of oncogenes and/or inactivation of tumor suppressor genes Necessary but probably not sufficient to induce tumors! >Normal cells have multiple mechanisms for growth regulation It would be too dangerous otherwise! >Multiple tumor viral genes are needed for transformation Cooperativity between immortalization and transformation Expression of 2 or more oncogenes is needed to convert a normal to tumor cell Tumor antigens of DNA tumor viruses: Binds to Rb: E1A (Adeno), E7 (HPV), T Ag (SV40) Binds to p53: E1B (Adeno), E6 (HPV), t Ag (SV40) Consequence of such bindings loss of tumor suppressors may be a major route in the immortalization pathway

59 Immortalization and Transformation Immortalization: >cellular changes required Established cell lines have usually lost p53 function, suggesting p53 is involved in immortalization process (but probably not sufficient) >may be connected with cell’s inability to differentiate Oncoprotein blocks differentiation may allow a cell to proliferate Continue proliferation may allow mutations to occur Telomerase extends telomeres Telomerase (-) in somatic cells, but (+) in tumor cells Is telomerase essential for tumor formation? and at what stage?

60 Immortalization and Transformation In primary somatic cells: telomere shortening  crisis  p53 activation  growth arrest/apoptosis Telomerase is a critical parameter for immortalization Finite replicative capacity of primary cells as a tumor suppression mechanism that prevents cells from indefinite replication that is needed to make a tumor But telomerase isn’t the ONLY way of supporting immortal state Pathways that control telomerase production in vivo?

61 Summary Oncogenes are gain-of-function genetic modifications associated with “Immortality, Transformation & Metastasis” Proto-oncogene (c-) and viral (v-) oncogenes: oncogenes from DNA tumor viruses: interactions with cellular genes oncogenes from RNA tumor viruses: derived from proto-oncogenes (cell genes) qualitative and/or quantitative differences cellular oncoproteins may be derived from several types of cellular genes growth factor receptors, transcription factors……….. common features: growth regulation Tumor suppressors are loss-of-function mutations that increase cellular proliferation

62 Summary Oncogenes are gain-of-function genetic modifications associated with “Immortality, Transformation & Metastasis” Tumor suppressors are loss-of-function mutations that increase cellular proliferation Nuclear non-phosphorylated Rb sequesters E2F Released E2F (by P-RB) activates genes needed for S phase; Cell cycles proceed p53 can function as dominant negative mutant p53 activity increases in response to DNA damages & other stresses early in cell cycle  pause, repair DNA damages before replication late in cell cycle  causes apoptosis mechanism through which p53 causes cell cycle arrest loss of p53 may be necessary for immortalization

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