Cook Lectures: Cancer Bio

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G0

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Metabolically active but low expression of cell-cycle promoting proteins

Days, months, years

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G1

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Largest increase in cell size
E.C. GFs
Chromosomes prepared for duplication

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S

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Chromosome duplication; sensitive to DNA damage

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G2

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Some additional increase in cell size

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M

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1 hr; cell division

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Interphase

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G1 + S + G2 (all but mitosis)

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Transformed cells

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Decreased dep. on GFs

Don't arrest when confluent (space issue)

Don't require anchor

Unlimited proliferation (re-express telomerase)


Highly unstable

Forms tumor in mice

Requires gene change in 5-10 genes

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Immortal cells

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Same as normal but:

Unlimited proliferation (re-express telomerase)

Less stable genome

Don't form a tumor in mice

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Genome instability

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Chromosome replication or repair problems

Errors in chromosome segregation

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Cell fusion experiments: conclusions

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S phase can induce early S phase in G1 but not G2

Mitotic signal (mitosis promoting factor) can induce mitosis in any other phase

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MPF

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Heterodimer: CDK1 +cyclin B
Oscillation of cyclin A and cyclin B activity
Serine/threonine kinase (Cdk1)
Highly conserved

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CDK1+cyclin B

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MPF: active when bound to Cdk1; inactive when cyclin B degraded (happens at start of anaphase)

Histones (condensation), lamins (nuclear membrane breakdown), APC (anaphase-promoting complex) targeted for phosphorylation

Cdk1 protein levels don't change through cell cycle but cyclins do

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Cdk1

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Serine/threonine kinase

Associates with cyclin B as mitotic promoting factor

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Most important determinant of malignancy?

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Mitotic rate

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Cyclins

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Cyclin D: G1 mitogenic response
Cyclin E: G1 to S transition
Cyclin A: S phase
Cyclin B: mitosis

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Cdks govern...

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Transitions
Nature of cyclin determines cdk activity

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Cyclin D

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Mitogenic signal; *only cyclin that responds to extracellular growth factors

GFs --> receptors --> MAPK pathway --> TFs --> transcription Cyclin D

PI3K pathway phosphorylates Cyclin D-cdk4; stabilizes

Proto-oncogene

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Cyclin D/Cdk4 pathway

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Cyclin D transcription + cdk4/6
Inhibits Rb
Relieves Rb inhibition of E2F
E2F active TF
S phase genes get transcribed

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Rb/E2F

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Transcriptional repressor

Cyclin D/cdk4 phosphorylation of Rb relieves repression and S phase genes get transcribed: cdc6, DNA pol, DNA ligase, DHFR, cyclins, E2F

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Retinoblastoma

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3% of childhood cancers

Changes reflective property of eye

Two-hit hypothesis (born heterozygous; second mutation) -- early onset for familial

Some sporadic Rb in older individuals

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c-myc

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TF that controls cell growth (increase in mass)

Sits on promoter; active when phosphorylated

Activated BOTH by PI3K and Ras-MAPK phosphorylation

Stimulates cell growth (mass)

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Cyclins E and A (general)

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Make sure replication happens appropriately (once and only once)

Their transcription is turned on by E2F, turned on by cyclin D

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Cyclin E/cdk2

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Cyclin E can also phosphorylate Rb, inhibiting it and stimulating the G1 --> S transition

Stimulates activation of DNA replication (DNA unwinding)

Stimulates firing: helicase activation, DNA pol activation

Prevents re-licensing and re-replication

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Cyclin cycling regulation

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1. Regulated induction: transcription, translation
2. Regulated destruction: ubiquitination, degradation

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Cul-Roc ligases

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Class of E3 enzyme

Covalent attachment of ubiquitin chain for degradation of cyclins

Regulates cyclins through cell cycle

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Cyclin E destruction

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Self-phosphorylation recognized by cullin docking site

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Cdk regulation

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Activating: cyclin binding, phosphorylation T160 activation loop, Cdk inhibitor destruction (ubiquitination), Cdk de-posphorylation Y15
Inhibiting: cyclin destruction; de-phosphorylation of T160 activation loop; Cdk inhibitor binding, Cdk phosphorylation Y15

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p16/INK4

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p16/INK4 family are CDK inhibitors

When abundant, competes with cyclin D for cdk4/6, inactivating it

Lost in many cancers (tumor suppressor) but not all

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p16/INK4 pathway

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Inhibit Cdk4 --> no inhibition Rb --> so Rb continues to inhibit E2F --> don't get S phase genes or cell prolif

Tumor suppressor

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p27 family

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Inhibit CDK1 and CDK2 (Cyclin B and Cyclins A and E)

Forms ternary complex with cyclin/CDK w/i catalytic pocket

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p27 and skp2

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p27 is tumor suppressor that inhibits cdk1/2
skp2 is inhibitor of p27 that targets p27 for polyubiquitination and destruction
loss of p27 leads to gain of cdk1/2 activity and cell cycling

p21 also in this family

p21, p27, p16 highly expressed in quiescent, scenescent and terminally-differentiated cells

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Cell cycle checkpoint

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Proteins in checkpt are NOT directly involved in executing the processes they regulate

Loss of checkpoint function results in genome instability

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Y15

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Region of Cdk
Phosphorylation results in inactive CDK
De-phosphorylation results in active CDK

Gene: Wee1

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Detector pathway

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Incomplete replication --> detector --> kinase --> kinase --> inhibition of phosphatase (cdc25) that activates cyclin B/cdk1

Or when unattached chromosomes:
kinase --> effector --> inhibition APC

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APC

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Anaphase promoting complex

Ubiquitinates cylcin B and securin -->
Separase freed from securin-->
Separase destroys Cohesin that holds sister chromatids together -->
Separation of sister chromatids

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Cohesin

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Holds sister chromatids together during metaphase

Destroyed by Separase that is activated by APC poly-ubiquitinating securin --> anaphase

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DNA damage: Cdk inhibition

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Kinases activate p53 (active when phosphorylated) and
Inactivate cdc25 (inactive when phosphorylated)
Double inhibition of the mitotic pathway when this happens
Caffeine inhibits this whole pathway but somehow not mutagenic?

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p53

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controls transcription of p21 Cdk inhibitor

Mdm2 normally poly-ubiquitinates p53, resulting in low p53 in cell (but transcribed often)

DNA damage --> mdm2 dissociates from p53 --> p21 --> inhibition of cdk/cyclin

Also activated by heat shock, hypoxia, aberrant signaling, etc.)

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Mdm2

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Ubiquitin E3 for p53

Inhibition of Mdm2-p53 protein-protein interaction results in p53 stimulating p21 that inhibits cycling

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ARF

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Turned on by a stress environment
Blocks Mdm2-dependent p53 degradation

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Tumor-causing viruses

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RNA tumor viruses: normally integrating; carry cellular oncogenes or activate cellular proto-oncogenes

DNA tumor viruses: normally non-integrating; carry viral oncogenes

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HPV

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DNA virus
Induces cell cycle progression in order to support its own replication
Accidental integration of E6/57 genes can be transorming
Targets p53 (E6) and Rb (E7) tumor suppressors

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E6: HPV

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Binds p53
p53 polyubiquitinated
No p21 activation to inhibit cdk/cyclin

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E7: HPV

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Binds Rb, effectively phosphorylating it
No inhibition of E2F TF

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Sporadic cancers...

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"always" lose two critical tumor suppressor pathways

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ARF/p16

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Encoded by the same locus
Often get loss of both ARF and p16

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p53 mutations

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Majority of human cancers have alteration in ARF-Mdm2-p53 pathway

but don't lose multiple pieces of the same pathway

More than 20,000 mutations in p53

50% of all cancers have p53 mutations

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Nutlins

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Small molecules that bind Mdm2 and compete for p53 interaction

Potential theraputic effect that would prevent the elimination of p53

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Hallmarks of cancer

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Sustaining proliferative signaling
Enabling replicative immortality
Evading growth suppressors
Resisting cell death
Activating invasion and metastasis
Inducing angiogenesis

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Progressive stages of cancer

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Abnormal increase in cell number: "clonal population"

Genetic instability: pop becomes increasingly heterogeneous

Changes in cell adhesion and migration

Induction of angiogenesis

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GF-independent activation

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a. Mutation in Ras that blocks GTPase activity is "stuck" in the active state

Ras --> Raf --> Mek --> Erk --> Cdk4/cyclin D

b. Receptor overproduction: sporadic dimerization of tyrosine kinases

c. Mutations that mimic ligand binding

d. Mutations in PI3K pathway

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Proto-oncogenes

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EGFR (Her2) overproduced/un-reg
Ras: GTPase defective
Abl: Bcr-abl fusion
c-myc: TF overproduced
Cyclins, CDK4: over produced
Mdm2: overproduced

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Bcr-Abl

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Translocation between chrom 22 and 9 (Philadelphia)

Creates novel Bcr-Abl oncogene

Gleevec targets specifically

Disrupts normal auto-inhibitory domain of Abl; can't regulate the kinase that activates cyclin D, c-myc pathways

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Gleevec

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Targets Bcr-Abl specifically

Requires second generation drugs

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Induction of angiogenesis (new blood vessels)

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Stimulation of the VEGF receptor in tumors (vascular endothelial growth factor)

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Tumor suppressors

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Rb: deletion
p16/INK: deletion
BRCA1: truncation
ATM: deletion
Chk1/Chk2: mutation
p14/ARF: deletion
p53: mutation/deletion