centrosomes amplification and B23 NPM1

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centrosomes amplification and B23 (NPM1)

• Michal Sheffer

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Overview

• Cell division and centrosome overview
• Centrosome amplification
• B23 (NPM1)

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The cell cycle
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The centrosome

• Small organelle (1µm)
• Consists of two centrioles that are surrounded by
  pericentriolar material (PCM), displaying
  orthogonal orientation
• The centrioles are cylindrical structures made up
  of nine triplet microtubules

Schematic drawing
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The centrosome

• Microtubules organizing center
• Crucial for chromosome segregation and
  cytokinesis
• Determine to position of the cleavage plane
  essential for asymmetric divisions
• Centrosome duplication occur only once and must
  be in coordination with DNA synthesis

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Cell cycle regulation

• Both centrosome duplication and DNA replication
• require the hyper-phosphorylation of RB and the
• activation of CDK2.

Growth Signals
P53
ARF
S phase Entry
P21
Cdk4,6
P
P
Centrosome duplication
P
RB
E2F
E2F
P
RB
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Cell division
Anaphase A - segregation of the sister
chromatids,
Prophase - the duplicated centrosomes migrate
around the nucleus
Prometaphase - nuclear envelope breaks down,
chromosomes moves towards the mitotic spindle
Anaphase B- poles move further apart from one
another, contractile ring begins to assemble and
contract
Telophase - separation of the daughter cell,
nuclear envelope reassemble around the pole of
each daughter cell
Metaphase - sister chromatids are aligned on the
mitotic spindle, facing opposite poles
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Overview

• Cell division and centrosome overview
• Centrosome amplification
• B23 (NPM1)

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Chromosomal instability

• chromosomal instability is a phenotype of most
  solid tumors
• Chromosomal instability is characterized by
  losses or gains of whole chromosomes (aneuploidy)
  as well as chromosome rearrangements.
• Centrosomal abnormalities are one of the
  important causes of chromosomes missegregations.

Aneuploidy Chromosomes (or part of them) are
gained or lost.
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Centrosome aberration in cancer

• Centrosome amplification has been described for
  nearly all types of cancers, including brain,
  breast, colon, head and neck, lung, pancreas,
  prostate, cervix.
• Centrosome amplification was also found in
  low-grade pre-invasive tumors, implying that
  centrosomal abnormalities consist an important
  cause of chromosome instability.

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Centrosome aberration in cervical cancer
Growth Signals
P53
E6
ARF
Apoptosis
S phase Entry
P21
Cdk4,6
E7
P
P
Centrosome duplication
P
RB
E2F
E2F
P
RB
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Centrosome amplification

• The cell fate in Model II is dependent on
  functional P53

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Centrosome amplification and P53

• Loss of P53 results in amplification of
  centrosomes (10-30 of P53-/- mouse embryos
  fibroblasts)
• Amplification was found in cells with deleted
  P21, GADD45 and in cells with overexpressed MDM2
• Involvment of P53 in centrosome amplification is
  probably through an indirect check-point related
  mechanism (using Aurora-A and other mitotic
  kinases)
• E6 overexpression produced centrosome
  amplificationin conjugated with multinucleation,
  indicating unsuccessful cell division.

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Centrosome amplification and RB

• E7 can induce abnormal centrosome numbers by
  centrosome duplication defect

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Centrosome aberration in cervical cancer
HPV triggers two major mechanisms for centrosome
amplification
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Consequences of centrosome amplification

• Multipolar spindleslead to aneuploidy
• Strong positive correlation between centrosomal
  abnormalities and chromosomal abberations in
  tumor cells
• It has not been shown directly that Centrosome
  amplification is a major primary cause of
  aneuploidy
• Impact on asymmetry cell division (?)

P53-/- cells
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Advantages for centrosome amplification in tumors

• Selective pressure of certain aneuploidy cells

• Alternative mechanism for bipolar spindle
  formation, other then centrosomes allow tumor
  cells to divide normaly and still maintain
  genetic instability through occasional multipolar
  divisions.

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Overview

• Cell division and centrosome overview
• Centrosome amplification
• B23 (NPM1)

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Ribosomal Cluster

• Contains many ribosomal proteins, B23.

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Proliferation and metabolism Cluster

• Bub1, mad2, PTTG1(securin) - mitotic spindle
  proteins
• DNMT1 - DNA methylation.
• TTK(Mps1p) - centrosome duplication
• RAN, Nek2 - centrosome localized proteins.
• TOP2A, CKS2 and more - related to cancer

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B23 probe sets

• 200063_s_at is in the ribosomal cluster
• 221923_s_at, 221691_x_at have correlation of 0.5
  with the proliferation cluster.

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Myc and B23

• Myc overexpression and DNA damage lead to
  centrosomes amplification (Sugihara et al, 2004)
• Myc enhances the expression of genes that
  function in ribosome biogenesis and protein
  synthesis, including B23. (Boon et al, 2001)

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B23 Is a Target of CDK2-CyclinE in Centrosome
Duplication (Okuda et al, cell 2000)

• B23 Associates specifically with Unduplicated
  centrosomes
• B23 dissociates from centrosomes by CDK2-cyclinE
  phosphorylation on T199.
• Nonphosphorylated mutant B23 blocks centrosome
  duplication
• CDK2-cyclinA also phosphorylates B23 on T199.
• CDK1-cyclinB phosphorylated B23 on T234 and T237
  in vitro

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The role of B23 in centrosome duplication (Okuda,
Oncogene 2002)
Okuda et al.
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Original Suggested hypothesis in colon cancer
ß-catenin
B23
Myc
APC
Overexpression?
P
Centrosome hyper-amplification
E2F3
CycE
CIN
Mis-segregation of sister chromatids
Bub1/Mad2
?
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B23 and centrosome amplification

• Inactivation of E2F3/overexpression of MEK1
  causes hyper-phosphorylation of B23 which results
  in centrosome amplification (probably by early
  phosphorylation of B23)
• PLK belong to a conserved family of protein
  serine/threonine kinases, localizing to
  sub-cellular structures such as mitotic spindle,
  centrosome. Disruption of the B23 phosphorylation
  site results in centrosome amplification.

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Experimental question

• Does Hyper-phosphorylation of B23 contribute to
  centrosome amplification? Or is it just the
  timing of the phosphorylation that cause the
  amplification?

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Other mitotic proteins involved in centrosome
amplification

• cyclinB1-CDK1
• Cdc25C
• CHK1, CHK2
• Aurora-A (STK)
• TTK
• NEK2
• More

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References

• Centrosome abberations cause or consequence of
  cancer progression? (Nigg, 2002)
• Loss of P53 and centrosome hyperamplification
  (Tarapore, 2002)
• Checking out the centrosome (Kramer, 2004)
• The good, the bad and the ugly the practical
  consequences of centrosome amplification (Sluder,
  2004)
• B23 is a target of CDK2/cyclin E in centrosome
  duplication (Okuda, 2000)
• Cell division (Scholey, 2003)
• The centrosome in normal and transformed cells
  (Wang, 2004)
• Human papillomaviruses and centrosome duplication
  errors modeling the origin of genomic
  instability (Duensing, 2002)
• Inactivation of E2F3 results in centrosome
  amplification (Saavedra, 2003)
• B23 Serine 4 Phosphorylation Mediates Mitotic
  Functions of Polo-like Kinase 1 (Zhang, 2004)
• MEK1 and MEK2 different regulators of the G1/S
  transition (Ussar, 2004)

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