Cell cycle deregulation in cancer Flashcards
What are the 6 hallmarks of cancer cells?
- Autonomous growth
- Ignore cytostatic signals
- Ignore apoptotic signals
- Stimulate angiogenesis
- Invade and metastasise
- Immortality
What is angiogenesis?
Formation of new blood vessels
What controls normal cell proliferation?
Growth factors (GFs)
What are mitogens? (2)
- Growth factors
- Induce proliferation
How do cancer cells proliferate more than normal? (5)
- Produce growth factors by themselves
- Signal to nearby cells to produce growth factors
- Deregulation in growth factor receptor signalling
- Constitutive activation of signalling downstream from growth factor receptors
- Disruption of negative feedback that prevents proliferative signalling
How can deregulation of growth factor receptor signalling cause uncontrolled cancer cell proliferation? (2)
- Elevated levels of receptors
- Ligand-independent firing (constitutive activation)
What is G0? (2)
- Quiescence
- Can be permanent or transient
What are the stages of the cell cycle? (5)
- G0
- G1
- S
- G2
- Mitosis
What is the restriction (R) point? (2)
- A window in G1 up to a couple of hours before the transition into S phase where the cell decides whether to progress through the cell cycle or revert to G0 based on extracellular signals
- Often deregulated in cancer cells
How can the cell cycle be studied? (3)
- Flow cytometry
- Immunofluorescence
- FUCCI
What is flow cytometry?
Analysis of the cell cycle based on measuring the DNA content
How can immunofluorescence be used to study the cell cycle?
Stain for proteins that are specifically expressed in different phases of the cell cycle
What is BrdU? (3)
- Bromo-2deoxyuridine
- Replaces thymidine during DNA synthesis
- Used to identify cells in S phase
Which marker is used to identify cells in S phase?
BrdU
Which marker is used to identify cells in G2/M?
Cyclin B1
Which marker is used to identify cells undergoing mitosis?
Histone H3
What is histone H3? (2)
- Phosphorylated during chromosome condensation
- Used to identify cells undergoing mitosis
What is the disadvantage of flow cytometry and immunofluorescence?
Can’t be used in vivo because cells need to be fixed
What is FUCCI?
Fluorescence Ubiquitin Cell Cycle Indicator
How does FUCCI work? (3)
- Cdt1 is marked with red fluorescence and is expressed during G1 (low expression to start then increases)
- Geminin is marked with green fluorescence and is expressed during S and G2
- Allows you to observe the dynamics of the cell cycle in living cells
What is the FUCCI transgenic mouse? (2)
- Mouse expressing the red G1 marker was crossed with a mouse expressing the green S/G2/M marker
- Results in a mouse where every somatic cell expressed red or green
What does no/low red fluorescence indicate in the FUCCI system?
Cells in early G1
What does strong red fluorescence indicate in the FUCCI system?
Cells in late G1
What does yellow/orange fluorescence indicate in the FUCCI system?
Cells in G1/S transition (red and green fluorescence)
What does green fluorescence indicate in the FUCCI system?
Cells in S/G2/M
How does the cell cycle stage impact cell fate? (2)
- Cells in early G1 differentiate into endoderm and mesoderm but not neuroectoderm
- Cells in late G1 differentiate into neuroectoderm but not endoderm or mesoderm
What do cells in early G1 differentiate into?
Endoderm/mesoderm
What do cells in late G1 differentiate into?
Neuroectoderm
How does the cell cycle stage impact metastasis? (2)
- Cells in G1 are the most invasive as they express a protease which degrades ECM
- G1 cells migrate first and other cells follow
Why might a drug that blocks the cell cycle be dangerous?
Cells in G1 may be more invasive so blocking cells in G1 would stop tumour growth but would promote metastasis
What are CDKs? (3)
- Cyclin-dependent kinases
- Kinases which drive the progression of the cell cycle
- Require cyclin binding to be active
What are cyclins?
Accessory proteins which form complexes with CDKs to drive the progression of the cell cycle
How do cyclin/CDK complexes control the cell cycle? (2)
- Cyclin binds to CDK to form a complex which activates the CDK
- CDK can then phosphorylate downstream targets which make the cell cycle progress
How is CDK activity regulated? (4)
- Cyclins activate the catalytic activity of the CDK
- Cyclin binding increases substrate recognition of the cyclin/CDK complex
- Phosphorylation of the ATP-binding domain of the CDK causes inactivation
- Binding of CKIs cause inactivation
How is the cyclin/CDK complex removed when it is no longer needed? (2)
- Cyclin is phosphorylated and ubiquitinated
- Causes degradation of cyclin
Which cyclin/CDK complex is expressed in G1?
Cyclin D + CDK 4/6
Which cyclin/CDK complex is expressed in late G1?
Cyclin E + CDK2
When is the R point?
Late G1
What is the R point? (2)
- The point where the cell decides to progress through the cell cycle or revert to G0
- The cell no longer requires growth factors to enter into the cell cycle
Which cyclin/CDK complex is expressed in S phase?
Cyclin A + CDK2
Which cyclin/CDK complex is expressed in S/G2 phase?
Cyclin A + CDC2
Which cyclin/CDK complex is expressed in M phase?
Cyclin B + CDC2
Why can the cell cycle only progress in one direction?
Cyclins are ubiquitinated and degraded as the cell progresses through the cycle so the cycle can’t go backwards
What is a CKI?
CDK inhibitor
What are the 2 families of CKIs?
- INK4
- CIP and KIP
Which cyclin/CDK complexes are present around the R point? (2)
- Before: cyclin D + CDK 4/6
- After: cyclin E + CDK 2
What is the role of cyclin D1? (2)
- Controlled by extracellular signals: growth factors and integrin-mediated ECM anchorage
- Checks if the environment is good for proliferation
How do mutations affect cyclin D1 in cancer? (3)
- Constitutive nuclear localisation
- Impaired degradation
- Activation of oncogenes causes overexpression of cyclin D1
How do oncogenes drive tumour formation?
Cause overexpression of cyclin D1
What does overexpression of cyclin D1 cause in cancer cells? (3)
- Increased cell proliferation
- Increased anchorage-independent growth
- Chemotherapy resistance
What is shRNA? (2)
- An RNA sequence which binds to a target mRNA and causes degradation
- Causes reduced expression of the target protein
What happens to cancer cells when you reduce cyclin D1 expression? (3)
- Tumour grows slower
- Induction of apoptosis
- Not tumour regression
What is overexpression of cyclin D1 associated with in gastric cancer? (3)
- Poor prognosis
- Lower overall survival
- Increased invasion and metastasis
Which cyclins are associated with cancer? (2)
- D1
- E1
What is cyclin E + CDK2 complex required for?
Transition from G1 to S phase
How does High Grade Serum Ovarian Carcinoma (HGSOC) originate and progress? (4)
- Cancer originates in the fallopian tube epithelium (FTE)
- Mutations in p53 occur in early lesions
- Cancer cells migrate, reach the ovary and form the primary tumour
- Cells detach and metastasise
What does overexpression of cyclin E1 cause in ovarian cancer cells? (3)
- Promotes rapid cell growth
- Promotes clonogenic growth
- Promotes loss of contact inhibition
What is overexpression of cyclin E1 associated with in HGSOC? (2)
- Poor patient survival
- Overexpression occurs in early lesions so cyclin E1 is involved in early HGSOC development
How is cyclin E involved in breast cancer? (3)
- Cyclin E is cleaved into a low molecular weight cyclin E which is more stable and has a higher affinity for CDK2
- Causes cell cycle deregulation and chemotherapy resistance
- Cleaved form is only seen in cancer
What is the cleaved form of cyclin E associated with in breast cancer?
Patient’s positive for cleaved cyclin E had a worse prognosis in multiple breast cancer subtypes
What is the role of TGFβ signalling in normal cells?
Prevents progression through the cell cycle
What is the role of TGFβ signalling in cancer cells? (2)
- Early stages: blocks proliferation like in normal cells
- Later stages: promotes invasion and metastasis
What is TGFβ?
Transforming Growth Factor β
How does TGFβ signalling work? (5)
- TGFβ binds to a type II receptor which recruits and phosphorylates a type I receptor
- Type I receptor phosphorylates SMAD2/3 (R-SMADs)
- SMAD2/3 bind SMAD4 and form a complex
- R-SMAD/SMAD4 complexes accumulate in the nucleus and act as transcription factors
- Acts on tumour suppressor genes and tumour promoting genes
What are R-SMADs? (2)
- Receptor-regulated SMADs
- SMAD2/3
How does TGFβ signalling inhibit cell proliferation? (4)
- SMAD complex promotes transcription of p15-INK4B which is a CKI
- Causes inhibition of cyclin D-CDK4/6 complexes so cells can’t reach R point
- Also weakly activates p21-Cip1 (CKI) which inhibits all other CDK complexes
- p27 strongly activated in response to DNA damage in order to stop the cell cycle until damage is repaired
How do mitogens influence TGFβ signalling in normal cells? (4)
- Mitogens bind and activate P13K which activates the Akt/PKB pathway
- Akt phosphorylates p21-Cip1 which causes translocation into the cytoplasm
- Akt phosphorylates p27-Kip1 in the cytosol and prevents nuclear translocation
- Gets rid of CKIs and drives cell cycle progression
What is the effect of Akt/PKB signalling on the cell cycle?
Drives the progression of the cell cycle by reducing nuclear localisation of CKIs
What is associated with nuclear localisation of p27-Kip1 in cancer patients?
Better prognosis
True or false: G0 can be reversible or irreversible
True
True or false: TGFβ promotes cell cycle progression
False
True or false: stem cells in early G1 differentiate into endoderm/mesoderm
True
True or false: cyclin D expression is downregulated in cancer
False
True or false: cells in G2 have increased invasiveness
False
True or false: TGFβ promotes invasion and metastasis
True
True or false: cytosolic p27 localisation correlates with poor prognosis
True
True or false: immunofluorescence can be used to study mitosis
True
How do TGFβ and mitogen signalling differ?
TGFβ inhibits cell cycle progression by inducing CKIs but mitogens promote cell cycle progression by inhibiting CKIs
