Oncogenes and Cancer Class: Molecular Biology, Graduate Institute of Basic Medical Sciences Source:...

Oncogenes and Cancer

Class: Molecular Biology, Graduate Institute of Basic Medical Sciences

Source: “Genes VII” by Benjamin Lewin, Oxford University Press, 2000

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Changes When a Cell Becomes Cancer CellImmortalization

Cancer cells are normal cells that have lost their growth controlTypes 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 CrisisImmortalization 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

Monolayer Aneuploid

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Changes When a Cell Becomes Cancer CellTransformed 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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Changes When a Cell Becomes Cancer CellMultiple Genetic Changes

Changes lead the conversion of normal cells transformed cells:Multiple genetic changes: 6-7 events over 20-40 years

Factors (carcinogens) that increase the conversion:Initiate/Promote suggest stages in cancer development

Genes that cause transformation:Oncogenes (100+)

Viral oncogenes and cellular counterparts (proto-oncogenes)

Gain-of-function or ActivatedTumor suppressor genes (~10)

Loss-of-function or InactivatedHow are oncogenes activated and tumor suppressor gene

inactivated?

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Subjects to Be Covered

<1> Transforming viruses carry oncogenes<2> Retroviral oncogenes’ cellular counterparts<3> Mutational activation of Ras proto-oncogenes<4> Insertion, translocation, or amplification<5> Oncogenes & signal transduction cascades<6> Growth factor receptor kinases and cytoplasmic tyrosine

kinases<7> Oncoproteins may regulate gene expression<8> Tumor suppressor RB controls the cell cycle<9> p53 suppresses growth or triggers apoptosis<10> Immortalization and transformation

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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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Common mechanism of DNA tumor virus transformationEarly genes with oncogenic potentialIntegration of viral oncogenes into host genomesOncogene proteins always interact with host cellular proteins

[Cell transformation by polyomavirus/adenovirus; Fig 28-5]

Polyoma & SV40 produce T-antigens early in infectionT-antigen has transforming activity

Papillomaviruses produce E6 & E7 oncoproteinsEBV immortalized human B lymphocytes

EBV oncogene unknown

Retroviruses transfer vertically & horizontally

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Common mechanism of DNA tumor virus transformation

Retroviruses transfer vertically & horizontally [Fig 28-6]Integration of viral genome in germ line vertical transferReverse transcription needed for virus w/ RNA genomeTypes of retroviruses:<1> Non-defective tumor retroviruses Leukemia viruses

No viral oncogene; viral activation of cellular proto-oncogene<2> 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 virusCellular genes might promote growth of transduced cells

<3> Other oncogenic mechanism also present. HIV-1

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<1> Transforming viruses carry oncogenes<2> Retroviral oncogenes’ cellular counterparts<3> Mutational activation of Ras proto-oncogenes <4> Amplification, insertion, or translocation<5> Oncogenes & signal transduction cascades<6> Growth factor receptor kinases and cytoplasmic tyrosine


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Retroviral Oncogenes’ Cellular Counterpart

Oncogenes of some retroviruses [Fig 28-8]The normal cellular sequence itself is not oncogenicDifference between oncogenes & proto-oncogenes:

cell type, quantity; quality, or bothChanges in v-oncogenes could be very smallRetrovirus 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-randomDirect evidence that v-oncogene accomplishes transformation:

Conditional-lethal mutant of v-src geneOncogenes arise by activation of cellular or proto-oncogene is important to animal cancerHuman cancer too? Most human cancers do not involve virus

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<1> Transforming viruses carry oncogenes<2> Retroviral oncogenes’ cellular counterparts<3> Mutational activation of Ras proto-oncogenes <4> Amplification, insertion, or translocation<5> Oncogenes & signal transduction cascades<6> Growth factor receptor kinases and cytoplasmic tyrosine


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Mutational Activation of Ras Proto-oncogenes

Transfection assay [Fig 28-9]Nude mouse test

Transforming DNA isolated only from tumorigenic cellsProperties of transforming genes:<1> have closely related sequences in normal cells

Mutation theory mutation of normal genes created transforming genes<2> 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 tumorsFamily of ras oncogenes: N-ras, H-ras, K-ras

Single base mutation is suffice“Hot spots”/non-random Positions 12 and 61

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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 proteinhas intrinsic GTPase activityinterconverts 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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<1> Transforming viruses carry oncogenes<2> Retroviral oncogenes’ cellular counterparts<3> Mutational activation of Ras proto-oncogenes<4> Amplification, insertion, or translocation<5> Oncogenes & signal transduction cascades<6> Growth factor receptor kinases and cytoplasmic tyrosine


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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-2presence of known oncogenes in amplified regionamplification of same oncogenes in many cancers

Insertion: insertion of retrovirus LTR over-expresses c-myc

[Insertion of ALV activates c-myc gene; Fig 28-11]

Translocation:[reciprocal translocation by illegitimate recombination; Fig 28-12]

immunoglobulin or TCR gene and c-myc oncogeneIncreased c-myc expression after translocation

c-myc coding sequences are unaltered in all cases

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Amplification, Insertion, or Translocation

Evidence of oncogenic potential of c-myc gene:Transgenic mice carrying c-myc that:

linked to B lymphocyte enhancer lymphomaunder mouse mammary tumor virus LTR various cancers

Translocation can generate hybrid oncogenes & human cancers[CML & Philadelphia chromosome; Fig 28-13]

c-abl gene on chromosome 9 and bcr gene on chromosome 22Why is the hybrid bcr-abl protein oncogenic?

Activation of ras pathway for transformation

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Oncogenes & Signal Transduction Cascades

How oncogenes work to induce tumors?influence functions connected with cell growthnot themselves necessarily code for products that characterize the tumor cells

but may direct a cell into a particular pathway switchwhat are the functions of proto-oncogenes? growth regulatorHow are these proto-oncogenes changed in transformed cells?

[Functions of oncogenes; Fig 28-14]Growth factors & receptors; G protein/signal transductionIntracellular tyrosine kinases; Serine/threonine kinasesSignaling; Transcription factors

Common features? Capable of triggering general changes in cell phenotype associated with cell growth.

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Oncogenes & Signal Transduction Cascades

Common features of all oncogene protein functionsCapable 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 involvedNumerous proto-oncogenes code for growth factors

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Growth Factor Receptor kinases and cytoplasmic Tyrosine kinases

Protein tyrosine kinases are major class of oncoproteins<1> Transmembrane growth factor receptors<2> Cytoplasmic group of protein kinases

How their aberrant forms could be oncogenic?

<1> Transmembrane growth factor receptors kinase 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)

<2> Cytoplasmic group of protein kinases

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Growth Factor Receptor kinases and cytoplasmic Tyrosine kinases

<1> Transmembrane growth factor receptors<2> Cytoplasmic group of protein tyrosine kinases:more obscure

(src; yes; fgr; fps/fes; abl; ros)

src: 1st kinase type oncoprotein & 1st with tyrosine as target

[Domains of src protein; Fig 28-16]src protein is myristoylated, which is essential for tumorigenicityMajor 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

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Phosphorylation of tyrosine residues 416 and 527 is important.

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Oncoproteins May Regulate Gene Expression

Gene expression alteration is always needed for transformationOncogenes may code for transcription factors, which may

> quantitatively and/or quantitatively altered DNA binding?> alter ability to activate transcription?

[Oncogenes and transcription factors;Fig 28-19]

Example: a rel gene family is transcription factor NF-BMany stimuli to cells activate NF-B with broad spectrum effectsOther 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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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 functionloss of function causes tumorsbest known examples: RB and p53

Retinoblastoma is associated with deletion of q14 of chromosome 13

[Loss of heterozygosity; Fig 28-21]

[Nuclear phosphoprotein influences the cell cycle; Fig 28-22]

[Cell cycle control and tumorigenesis; Fig 28-23]

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G0/G1 phase: nonphosphorylatedS 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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p53 suppresses growth or triggers apoptosis

>50% of cancers lost p53 or have mutations in p53 genep53 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 lifep53 DNA inhibits transformation by oncogenes in cultured cellsHuman Li-Fraumeni Syndrome (rare inherited cancer; heterozygous p53 mutation acted as dominant negative or autosomal dominant)

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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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P53 Suppresses Growth or Triggers Apoptosis

How p53 trigger apoptosis?separable from growth arrest at the G1 checkpoint

Connection between tumorigenesis & loss of apoptosisapoptosis inhibits tumorigenesis by eliminating tumorigenic cells

p53 function is probably not essential for survival p53- animalsDefinitive 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

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Immortalization and Transformation

Tumors arise from multiple events:Activation of oncogenes and/or inactivation of tumor suppressor genesNecessary but probably not sufficient to induce tumors!

>Normal cells have multiple mechanisms for growth regulationIt would be too dangerous otherwise!

>Multiple tumor viral genes are needed for transformationCooperativity 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 bindingsloss of tumor suppressors may be a major route in the immortalization pathway

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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 differentiateOncoprotein blocks differentiation may allow a cell to proliferateContinue proliferation may allow mutations to occur

Telomerase extends telomeresTelomerase (-) in somatic cells, but (+) in tumor cells

Is telomerase essential for tumor formation? and at what stage?

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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?

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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 genesoncogenes from RNA tumor viruses: derived from proto-oncogenes (cell genes)qualitative and/or quantitative differencescellular 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

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Summary

Oncogenes are gain-of-function genetic modifications associated with “Immortality, Transformation & Metastasis”

Tumor suppressors are loss-of-function mutations that increase cellular proliferationNuclear non-phosphorylated Rb sequesters E2FReleased E2F (by P-RB) activates genes needed for S phase; Cell cycles proceed

p53 can function as dominant negative mutantp53 activity increases in response to DNA damages & other stresses

early in cell cycle pause, repair DNA damages before replicationlate in cell cycle causes apoptosis

mechanism through which p53 causes cell cycle arrestloss of p53 may be necessary for immortalization

Oncogenes and Cancer Class: Molecular Biology, Graduate Institute of Basic Medical Sciences Source:...

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