Oncogenes and Cancer

1
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
CellImmortalization

• 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
• Monolayer Aneuploid

3
(No Transcript)
4
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

5
(No Transcript)
6
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 Activated
• Tumor suppressor genes (10)
• Loss-of-function or Inactivated
• How are oncogenes activated and tumor suppressor
  gene inactivated?

7
Subjects to Be Covered

• lt1gt Transforming viruses carry oncogenes
• lt2gt Retroviral oncogenes cellular counterparts
• lt3gt Mutational activation of Ras proto-oncogenes
• lt4gt Insertion, translocation, or amplification
• lt5gt Oncogenes signal transduction cascades
• lt6gt Growth factor receptor kinases and
  cytoplasmic tyrosine kinases
• lt7gt Oncoproteins may regulate gene expression
• lt8gt Tumor suppressor RB controls the cell cycle
• lt9gt p53 suppresses growth or triggers apoptosis
• lt10gt 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

9
(No Transcript)
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

11
(No Transcript)
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
• lt1gt Non-defective tumor retroviruses Leukemia
  viruses
• No viral oncogene viral activation of cellular
  proto-oncogene
• lt2gt 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
• lt3gt Other oncogenic mechanism also present. HIV-1

13
(No Transcript)
14
(No Transcript)
15
Subjects to Be Covered

• lt1gt Transforming viruses carry oncogenes
• lt2gt Retroviral oncogenes cellular counterparts
• lt3gt Mutational activation of Ras proto-oncogenes
• lt4gt Amplification, insertion, or translocation
• lt5gt Oncogenes signal transduction cascades
• lt6gt Growth factor receptor kinases and
  cytoplasmic tyrosine kinases
• lt7gt Oncoproteins may regulate gene expression
• lt8gt Tumor suppressor RB controls the cell cycle
• lt9gt p53 suppresses growth or triggers apoptosis
• lt10gt 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

17
(No Transcript)
18
Subjects to Be Covered

• lt1gt Transforming viruses carry oncogenes
• lt2gt Retroviral oncogenes cellular counterparts
• lt3gt Mutational activation of Ras proto-oncogenes
• lt4gt Amplification, insertion, or translocation
• lt5gt Oncogenes signal transduction cascades
• lt6gt Growth factor receptor kinases and
  cytoplasmic tyrosine kinases
• lt7gt Oncoproteins may regulate gene expression
• lt8gt Tumor suppressor RB controls the cell cycle
• lt9gt p53 suppresses growth or triggers apoptosis
• lt10gt 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
• lt1gt have closely related sequences in normal
  cells
• ? Mutation theory
• mutation of normal genes created
  transforming genes
• lt2gt 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-random Positions 12 and 61

20
(No Transcript)
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

22
(No Transcript)
23
Subjects to Be Covered

• lt1gt Transforming viruses carry oncogenes
• lt2gt Retroviral oncogenes cellular counterparts
• lt3gt Mutational activation of Ras proto-oncogenes
• lt4gt Amplification, insertion, or translocation
• lt5gt Oncogenes signal transduction cascades
• lt6gt Growth factor receptor kinases and
  cytoplasmic tyrosine kinases
• lt7gt Oncoproteins may regulate gene expression
• lt8gt Tumor suppressor RB controls the cell cycle
• lt9gt p53 suppresses growth or triggers apoptosis
• lt10gt 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
  28-11
• Translocation
• reciprocal translocation by illegitimate
  recombination Fig 28-12
• immunoglobulin or TCR gene and c-myc oncogene
• Increased c-myc expression after translocation
• c-myc coding sequences are unaltered in all cases

25
(No Transcript)
26
(No Transcript)
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 28-13
• 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

28
(No Transcript)
29
Subjects to Be Covered

• lt1gt Transforming viruses carry oncogenes
• lt2gt Retroviral oncogenes cellular counterparts
• lt3gt Mutational activation of Ras proto-oncogenes
• lt4gt Amplification, insertion, or translocation
• lt5gt Oncogenes signal transduction cascades
• lt6gt Growth factor receptor kinases and
  cytoplasmic tyrosine kinases
• lt7gt Oncoproteins may regulate gene expression
• lt8gt Tumor suppressor RB controls the cell cycle
• lt9gt p53 suppresses growth or triggers apoptosis
• lt10gt 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.

31
(No Transcript)
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

• lt1gt Transforming viruses carry oncogenes
• lt2gt Retroviral oncogenes cellular counterparts
• lt3gt Mutational activation of Ras proto-oncogenes
• lt4gt Amplification, insertion, or translocation
• lt5gt Oncogenes signal transduction cascades
• lt6gt Growth factor receptor kinases and
  cytoplasmic tyrosine kinases
• lt7gt Oncoproteins may regulate gene expression
• lt8gt Tumor suppressor RB controls the cell cycle
• lt9gt p53 suppresses growth or triggers apoptosis
• lt10gt Immortalization and transformation

34
Growth Factor Receptor kinases and cytoplasmic
Tyrosine kinases

• Protein tyrosine kinases are major class of
  oncoproteins
• lt1gt Transmembrane growth factor receptors
• lt2gt Cytoplasmic group of protein kinases
• How their aberrant forms could be oncogenic?
• lt1gt Transmembrane growth factor receptors kinase
  activity
• gt extracellular N-terminal binds ligand that
  activates the receptor
• gt intracellular C-terminal contains the kinase
  activity Fig 28-15
• gt v-erb oncogene truncated proto-oncogene
  c-erbB
• gt constitutive activation of kinase activity
• gt receptors in development of specific cell type
  (myeloid precursor cells)
• lt2gt Cytoplasmic group of protein kinases

35
(No Transcript)
36
Growth Factor Receptor kinases and cytoplasmic
Tyrosine kinases

• lt1gt Transmembrane growth factor receptors
• lt2gt Cytoplasmic group of protein tyrosine
  kinasesmore 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 tumorigenicity
• Major difference between c-src and v-src kinase
  activity (20X)
• Roles of kinase activity in src function Fig
  28-17
• gt cellular phosphorylation targets -----
  results inconclusive
• gt 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.
38
(No Transcript)
39
(No Transcript)
40
Subjects to Be Covered

• lt1gt Transforming viruses carry oncogenes
• lt2gt Retroviral oncogenes cellular counterparts
• lt3gt Mutational activation of Ras proto-oncogenes
• lt4gt Amplification, insertion, or translocation
• lt5gt Oncogenes signal transduction cascades
• lt6gt Growth factor receptor kinases and
  cytoplasmic tyrosine kinases
• lt7gt Oncoproteins may regulate gene expression
• lt8gt Tumor suppressor RB controls the cell cycle
• lt9gt p53 suppresses growth or triggers apoptosis
• lt10gt 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
• gt quantitatively and/or quantitatively altered
  DNA binding?
• gt alter ability to activate transcription?
• Oncogenes and transcription factorsFig
  28-19
• Example a rel gene family is transcription
  factor NF-kB
• Many stimuli to cells activate NF-kB 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

42
(No Transcript)
43
Subjects to Be Covered

• lt1gt Transforming viruses carry oncogenes
• lt2gt Retroviral oncogenes cellular counterparts
• lt3gt Mutational activation of Ras proto-oncogenes
• lt4gt Amplification, insertion, or translocation
• lt5gt Oncogenes signal transduction cascades
• lt6gt Growth factor receptor kinases and
  cytoplasmic tyrosine kinases
• lt7gt Oncoproteins may regulate gene expression
• lt8gt Tumor suppressor RB controls the cell cycle
• lt9gt p53 suppresses growth or triggers apoptosis
• lt10gt 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 28-21
• Nuclear phosphoprotein influences the cell
  cycle Fig 28-22
• Cell cycle control and tumorigenesis Fig
  28-23

45
(No Transcript)
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 (SV40s 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

47
(No Transcript)
48
Subjects to Be Covered

• lt1gt Transforming viruses carry oncogenes
• lt2gt Retroviral oncogenes cellular counterparts
• lt3gt Mutational activation of Ras proto-oncogenes
• lt4gt Amplification, insertion, or translocation
• lt5gt Oncogenes signal transduction cascades
• lt6gt Growth factor receptor kinases and
  cytoplasmic tyrosine kinases
• lt7gt Oncoproteins may regulate gene expression
• lt8gt Tumor suppressor RB controls the cell cycle
• lt9gt p53 suppresses growth or triggers apoptosis
• lt10gt Immortalization and transformation

49
p53 suppresses growth or triggers apoptosis

• gt50 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)

50
(No Transcript)
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?

52
(No Transcript)
53
(No Transcript)
54
(No Transcript)
55
(No Transcript)
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 cells ability
  to divide

57
Subjects to Be Covered

• lt1gt Transforming viruses carry oncogenes
• lt2gt Retroviral oncogenes cellular counterparts
• lt3gt Mutational activation of Ras proto-oncogenes
• lt4gt Amplification, insertion, or translocation
• lt5gt Oncogenes signal transduction cascades
• lt6gt Growth factor receptor kinases and
  cytoplasmic tyrosine kinases
• lt7gt Oncoproteins may regulate gene expression
• lt8gt Tumor suppressor RB controls the cell cycle
• lt9gt p53 suppresses growth or triggers apoptosis
• lt10gt 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!
• gtNormal cells have multiple mechanisms for
  growth regulation
• It would be too dangerous otherwise!
• gtMultiple 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
• gtcellular changes required
• Established cell lines have usually lost p53
  function, suggesting p53 is involved in
  immortalization process (but probably not
  sufficient)
• gtmay be connected with cells 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 isnt 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

63
(No Transcript)