Download V9: Cell cycle, CDKs and cancer

V9: Cell cycle, CDKs and cancer
Active CDK1-cyclin complexes phosphorylate
more than 70 substrates during G2 and
early mitosis to trigger e.g.
-  centrosome separation,
-  Golgi dynamics,
-  nuclear envelope breakdown and
-  chromosome condensation!
In humans, the CDK family is composed
of 13 members that interact with at least
29 cyclins or cyclin-related proteins.
Today: role of CDKs during transcription + relation to cancer.
Malumbres, Barbacid, Nature Rev. Cancer 9, 353 (2009)
9. Lecture WS 2010/11
Cellular Programs
1
Transcription by Polymerase II in Drosophila
The 3 main phases of the transcription cycle are known as
initiation, elongation and termination.
During transcription initiation, a transcription-competent RNA polymerase
complex forms at the promoter and the DNA template is aligned in the active site of
the polymerase.
The active site is where nucleotides are paired with the template and are joined
processively during elongation to produce the RNA transcript.
Termination of transcription involves release of the RNA transcript and the
dissociation of the transcription complex from the DNA template.
Saunders et al. Nat. Rev. Mol Cell Biol 7, 557 (2006)
9. Lecture WS 2010/11
Cellular Programs
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Elongation
Regulation of transcription occurs
-  at the level of RNA polymerase recruitment to the promoter and
-  at the level of elongation.
RNA polymerase II (Pol II) transcription elongation is divided into 3 distinct stages:
(1) promoter escape,
(2) promoter-proximal pausing, and
(3) productive elongation.
Saunders et al. Nat. Rev. Mol Cell Biol 7, 557 (2006)
9. Lecture WS 2010/11
Cellular Programs
3
C-terminal tail of RNA Polymerase II
The C-terminal domain (CTD) of
the largest subunit of RNA
polymerase II (Pol II), Rpb1,
consists of tandem heptapeptide
repeats.
This C-terminal domain distinguishes Pol II from the other two eukaryotic RNA
polymerases.
The number of repeats that exactly match the consensus sequence varies among
species.
Saunders et al. Nat. Rev. Mol Cell Biol 7, 557 (2006)
9. Lecture WS 2010/11
Cellular Programs
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Modifications of C-terminal tail of Pol II
The CTD can be modified by
phosphorylation (mostly at Ser2
and Ser5), glycosylation, and cis/
trans isomerization of prolines.
CDK8 phosphorylates the CTD at
Ser5 and possibly Ser2.
Peptidyl-prolyl isomerases (e.g. yeast Ess1 and mammalian PIN1) can alter the
conformation of the CTD, and thereby regulate CTD phosphorylation and the
binding of other protein factors to Pol II.
Modification of the CTD is important for the coordination of transcription events.
Different modification states of the CTD are characteristic of different
transcriptional stages.
Saunders et al. Nat. Rev. Mol Cell Biol 7, 557 (2006)
9. Lecture WS 2010/11
Cellular Programs
5
Role of C-terminal tail of Pol II during elongation
During the transition from transcription initiation to elongation, Pol II changes from
a hypophosphorylated form to a hyperphosphorylated form.
The level of Ser5 phosphorylation peaks early in the transcription cycle and
remains constant or decreases towards the 3‘ end of the gene.
By contrast, Ser2 phosphorylation predominates in the gene body and towards the
3‘ end of the gene.
Saunders et al. Nat. Rev. Mol Cell Biol 7, 557 (2006)
9. Lecture WS 2010/11
Cellular Programs
6
Transcription initiation
Before transcription initiation, a pre-initiation complex (PIC) forms at the
promoter, consisting of Pol and several general transcription factors (GTFs).
The GTFs position Pol II near the transcription-start site (TSS) and dictate the
precise location of transcription initiation.
The general transcription factor TFIIH is needed for the structural remodelling of
the PIC.
11-15 base pairs around the TSS are unwound to form an ‘open complex‘ that
allows the single-stranded DNA template to enter the active site of Pol II.
Saunders et al. Nat. Rev. Mol Cell Biol 7, 557 (2006)
9. Lecture WS 2010/11
Cellular Programs
7
Promoter escape: elongation stage 1
Productively elongating Pol II can transcribe the full length of a gene in a highly
processive manner without dissociating from the template DNA or releasing the
nascent RNA product.
This is possible after promoter escape during which the polymerase breaks its
contacts with promoter-sequence elements and at least some promoter-bound
factors and simultaneously tightens its grip on the nascent RNA.
Saunders et al. Nat. Rev. Mol Cell Biol 7, 557 (2006)
9. Lecture WS 2010/11
Cellular Programs
8
Promoter escape – formation of an early elongation
complex
The unwinding of promoter DNA to create a transcription bubble begins at a fixed
position, ~20 base pairs downstream from the binding site of the TATA-box-binding protein
(TBP).
The upstream bubble edge (vertical dashed line) remains fixed until the completion of
promoter escape, whereas the downstream edge expands together with transcription.
The initially transcribing complex (ITC) cycles through several rounds of abortive
initiation, releasing large amounts of 2–3-nucleotide-long RNA transcripts (red).
Saunders et al. Nat. Rev. Mol Cell Biol 7, 557 (2006)
9. Lecture WS 2010/11
Cellular Programs
9
Escape commitment
After synthesis of the first 4 nucleotides, the B-finger of TFIIB (orange) and a
switch domain (dark blue oval) of Pol II (large blue oval) stabilize the short RNA,
reducing abortive initiation.
This transition to a metastable transcription complex is termed escape
commitment.
Saunders et al. Nat. Rev. Mol Cell Biol 7, 557 (2006)
9. Lecture WS 2010/11
Cellular Programs
10
Action of Polymerase II in Drosophila
After 5 nucleotides are added, the nascent RNA collides with the B-finger of
TFIIB, inducing stress within the ITC.
This can cause increased abortive initiation, strong pausing, or transcript
slippage, if the nucleotides at the 3′ end of the RNA–DNA hybrid interact weakly,
and probably contributes to the rate-limiting step of promoter escape.
Saunders et al. Nat. Rev. Mol Cell Biol 7, 557 (2006)
9. Lecture WS 2010/11
Cellular Programs
11
Promoter escape
Stress from the growing transcription bubble and the production of a 7-nt-long RNA trigger
collapse of the transcription bubble, providing the energy to remodel the transcription
complex.
The B-finger is ejected from the RNA-exit tunnel and TFIIB is released from the
transcription complex. The RNA–DNA hybrid is at its full length of 8–9 base pairs and can
make contacts with protein loops near the RNA-exit tunnel.
Abortive initiation ceases, as does the need for ATP hydrolysis, and transcript slippage is
markedly reduced, all indicating that the transcription complex has changed into an early
elongation complex (EEC).
Saunders et al. Nat. Rev. Mol Cell Biol 7, 557 (2006)
9. Lecture WS 2010/11
Cellular Programs
12
Transitioning to the pause region
Following promoter escape, the RNA remains stably bound in the transcription
complex, but has a tendency to undergo transcript slippage, backtracking and
arrest until about +30.
This phase is often accompanied by transcriptional pausing near the promoter.
Progress depends on stimulation by appropriate signals.
Consequently, this stage serves as a checkpoint for regulation.
The details how this step is regulated are subject of very active current research.
Saunders et al. Nat. Rev. Mol Cell Biol 7, 557 (2006)
9. Lecture WS 2010/11
Cellular Programs
13
Pol II binding profiles
ChIP-chip assays were carried out with 2–4 h Toll10b embryos using antibodies
that recognize both the initiating and the elongating forms of Pol II.
y axis: enrichment ratios of Pol II.
(a–d) Binding patterns across genes that are repressed in Toll10b embryos. All
four genes show high Pol II signals near the transcription start sites. At some
genes, such as tup (a), Pol II is tightly restricted to this region, whereas at other
genes, including sog (c) and brk (d), Pol II is also detected at lower signals
throughout the transcription unit.
Zeitlinger et al. J. Nat. Gen. 39, 1513 (2007)
9. Lecture WS 2010/11
Cellular Programs
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(e,f) Pol II is uniformly distributed across the transcription units of genes that are
actively transcribed. The stumps gene (e) is specifically activated in
mesodermal precursor cells, whereas RpL3 (f) is a highly expressed ribosomal
gene.
(g,h) No Pol II binding is found at many genes that are inactive during
embryogenesis. The eyeless (ey) gene (g) is expressed during eye
development at larval stages but not in the early embryo. Likewise, the torso
(tor) gene (h) is active only during oogenesis but not in the early embryo.
Zeitlinger et al. J. Nat. Gen. 39, 1513 (2007)
9. Lecture WS 2010/11
Cellular Programs
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Cell cycle, CDKs and cancer
Marcos
Malumbres,
Madrid
Mariano
Barbacid
Malumbres, Barbacid, Nature Rev. Cancer 9, 353 (2009)
9. Lecture WS 2010/11
Cellular Programs
16
Timeline: cell cycle regulation and cancer
Malumbres, Barbacid, Nature Rev. Cancer 9, 353 (2009)
9. Lecture WS 2010/11
Cellular Programs
17
Cell cycle, CDKs and cancer
Three basic cell cycle defects are mediated by mis-regulation of CDKs:
-  unscheduled proliferation
-  genomic instability (GIN)
-  chromosomal instability (CIN)
Tumour-associated mutations in human tumor cells often deregulate CDKcyclin complexes, leading either to continued proliferation or unscheduled reentry into the cell cycle.
Do tumour cells use CDKs in the same way as „normal“ cells, or do they show
different patterns of CDK activity?
Indeed, some tumour cell lines display a selective dependence on interphase
CDKs.
Malumbres, Barbacid, Nature Rev. Cancer 9, 353 (2009)
9. Lecture WS 2010/11
Cellular Programs
18
Cell cycle models
Malumbres, Barbacid,
Nature Rev. Cancer 9, 353 (2009)
9. Lecture WS 2010/11
Cellular Programs
19
a The diagram depicts the S. cerevisiae cell cycle and 3 models of the mammalian cell
cycle. In the currently accepted model based on biochemical evidence (classical' model),
each of the main events that take place during interphase (G1, S and G2) is driven by
unique CDKs bound to specific cyclins. The essential' cell cycle is based on genetic
evidence indicating that CDK1 is sufficient to drive proliferation of all cell types up to mid
gestation as well as during adult liver regeneration. The specialized' cell cycles are based
on the unique requirements of specialized cell types for specific CDKs as indicated.
b Unscheduled cell proliferation requires aberrant mitogenic signalling driven by either
excessive exogenous signalling (for example, growth factors and nutrients) or endogenous
oncogenic mutations. Other mutations affecting mitogenic breaks (for example, tumour
suppressors and negative regulators) that decrease the threshold for mitogenic signalling
also contribute to unscheduled proliferation. Oncogene-induced cell cycling provokes DNA
replication stress that is sensed by the DNA replication checkpoints. Failure in this control
mechanism results in an increased mutation rate and ultimately may lead to genomic
instability. During mitosis, defects within the spindle assembly checkpoint induce
deregulation of CDK1 activity that may result in abnormal chromosome segregation. All
these defects converge in deregulation of CDK activity, eventually leading to tumour
development.
Malumbres, Barbacid, Nature Rev. Cancer 9, 353 (2009)
9. Lecture WS 2010/11
Cellular Programs
20
Effects of gene deletions during development
Malumbres, Barbacid, Nature Rev. Cancer 9, 353 (2009)
9. Lecture WS 2010/11
Cellular Programs
Mice lacking
CDK2 and CK4
die at birth.
21
Figure legend
The genotypes of the various CDK mutant strains used in this analysis are indicated. Wildtype CDKs are indicated in bold font. Ablated CDKs appear in normal font. The arrows
indicate the extent to which each of the mouse strains develops with the indicated CDK
content. Stop signs indicate the developmental stage at which the strains are no longer
viable. The main defects responsible for loss of viability are also indicated. Briefly, mice
expressing all interphase CDKs but not CDK1 do not progress beyond the two-cell embryo
stage. Mice expressing CDK1 but no interphase CDKs, progress up to mid gestation
(embryonic day (E)12.5–E13.5). Mice lacking CDK4 and CDK6 develop until late
embryonic development (E16.5–E17.5), whereas those lacking CDK4 and CDK2 die at
birth. Finally, mice lacking CDK6 and CDK2 develop to adulthood and have a normal life
span. Except for embryos lacking CDK1, none of the mutant embryos or mice display cell
cycle defects except in those cell types indicated in the yellow boxes. P, postnatal day.
Malumbres, Barbacid, Nature Rev. Cancer 9, 353 (2009)
9. Lecture WS 2010/11
Cellular Programs
22
Key molecules involved in mitogenic progression
In red:
active
molecules
Malumbres, Barbacid, Nature Rev. Cancer 9, 353 (2009)
9. Lecture WS 2010/11
Cellular Programs
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Figure legend
The anaphase-promoting complex/cyclosome (APC/C)–CDC20 (cell division control 20)
complex targets cyclin A (CycA) and NEK2 for degradation by ubiquitylation in a spindle
assembly checkpoint (SAC)-independent manner. In the presence of unaligned
chromosomes, separase is kept inactive by securin and CDK1–cyclin B. Under these
conditions, sister chromatids are held together by cohesins. After complete bipolar
attachment of chromosomes to the mitotic spindle, cyclin B and securin are also
ubiquitylated by APC/C–CDC20, but in a SAC-dependent manner. Ubiquitin-dependent
degradation of these proteins inhibits CDK1 leading to the activation of separase, which in
turn cleaves cohesins and releases sister chromatids, hence facilitating the metaphase-toanaphase transition. APC/C–CDH1 also targets cyclin A and cyclin B to keep low levels of
Cdk1 activity during mitotic exit and the following G1 phase. Other APC/C–CDH1
substrates involved in mitotic progression include CDC20, TPX2, forkhead box protein M1
(FOXM1), aurora kinase A (AURKA), AURKB and PLK1. Additional APC/C–CDH1 targets
involved in the control of DNA replication, such as SKP2, CDC6 or geminin, are omitted for
clarity. Tumours with chromosomal instability are characterized by molecular signatures in
which most of these molecules (in bold) are overexpressed. Active proteins are
represented by red boxes.
Malumbres, Barbacid, Nature Rev. Cancer 9, 353 (2009)
9. Lecture WS 2010/11
Cellular Programs
24
Current clinical trials with CDK inhibitors
Malumbres, Barbacid, Nature Rev. Cancer 9, 353 (2009)
9. Lecture WS 2010/11
Cellular Programs
25
CDK inhibitors in clinical tests
CDK inhibitors are likely to produce certain toxicities by also affecting the
proliferation of those cells that require specific interphase CDKs to maintain
tissue homeostasis.
The first generation of CDK inhibitors (e.g. flavopiridol, UCN-01) did not show
significant clinical advantages.
Some expected toxicities for CDK4 (diabetes), CDK2 (sterility) or CDK6 (mild
anemia) inhibitors may be acceptable for adult cancer patients.
On the other hand, small molecule against CDK1 and possibly against CDK7
may result in general toxicities.
Therefore, promiscous CDK inhibitors often display high toxicities in early
clinical trials.
Malumbres, Barbacid, Nature Rev. Cancer 9, 353 (2009)
9. Lecture WS 2010/11
Cellular Programs
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