Checkpoint controls Flashcards
What processes does the genetic material of the daughter cells depend on? (2)
- Faithful DNA replication in S phase
- Proper allocation of the replicated DNA to daughter cells in M phase
What are checkpoints? (2)
- Surveillance mechanisms to monitor each step of the cell cycle progression
- Cell cycle is halted if the previous step hasn’t been completed correctly
What are the 5 checkpoints in the cell cycle?
- Restriction (R) point
- G1/S checkpoint
- S phase checkpoint
- G2/M checkpoint
- Spindle assembly checkpoint
What is the G1/S checkpoint? (2)
- DNA damage checkpoint before the cell enters S phase
- DNA damage = can’t enter S phase
What is the S phase checkpoint? (2)
- DNA damage checkpoint during DNA replication
- DNA damage = DNA replication is paused for repair which extends the time in S phase
What is the G2/M checkpoint? (2)
- DNA damage and synthesis checkpoint before the cell enters mitosis
- Cell can’t enter M phase until the whole genome has been replicated without damage
What is the spindle assembly checkpoint? (2)
- Checkpoint during mitosis to make sure that the chromosomes are correctly assembled on the mitotic spindle during metaphase
- Anaphase blocked if chromosomes not assembled correctly
What happens to the checkpoints in cancer cells?
Inactivated
What regulates the restriction (R) point?
pRb (retinoblastoma gene)
What is the function of pRb? (2)
- Inactive pRb in the hyperphosphorylated form allows cells to go through the R point
- Active pRb in the hypophosphorylated form blocks cells going through the R point
When is pRb active?
When it is hypophosphorylated
When is pRb inactive?
When it is hyperphosphorylated
What happens to pRb as a cell moves through the R point? (3)
- pRb is unphosphorylated during early/mid G1
- Presence of mitogens upregulates cyclin D, cyclin D+cdk4/6 complex starts to phosphorylate pRb = hypophosphorylated, pRb still active
- Cyclin E is upregulated at the R point, cyclin E+cdk2 complex which hyperphosphorylates pRb = inactive
How does pRb phosphorylation control progression through the R point? (3)
- Un/hypophosphorylated pRb in early/mid G1 binds to E2Fs
- Hyperphosphorylated pRb dissociates from E2Fs
- E2Fs are released and cause transcription of genes which allow entry into S phase
What are E2Fs?
Transcription factors that promote transcription of genes which mediate the G1/S transition
How is the action of E2Fs terminated? (2)
- Cyclin A+cdk2 complex is upregulated during G1/S transition which inhibits E2F transcriptional activity
- E2Fs are ubiquitinated and degraded when S phase transition is complete
What positive feedback mechanisms drive rapid advance and irreversibility of the cell cycle through the R point? (3)
- E2Fs promote cyclin E expression = more cyclin E+cdk2 = hyperphosphorylation of pRb which releases more E2Fs
- E2Fs promote E2F expression
- Cyclin E+cdk2 phosphorylates p27kip1 (CKI) causing degradation = more cdk2 activity = more hyperphosphorylation of pRb
What are the mechanisms of pRb inactivation? (3)
- RB1 gene mutations causing complete loss of pRb/pRb loss of function
- De-regulated pRb phosphorylation
- Interaction with viral proteins
Why does pRb inactivation cause cancer?
Absence of pRb activity means there is nothing to inhibit E2Fs so they drive transcription of G1/S transition genes and cause proliferation
How does deregulation of pRb phosphorylation cause cancer?
If pRb is always in the hyperphosphorylated form it can’t bind to E2Fs and block entry into S phase
How does interaction with viral proteins cause inactivation of pRb? (2)
- E7 protein produced by HPV displaces E2F from pRb
- pRb can’t inhibit E2Fs even when hypophosphorylated which leads to uncontrolled proliferation in cervical cancers
Which cyclins are often overexpressed in tumours? (2)
- Cyclin D
- Cyclin E
Why are cyclins not ideal as drug targets? (2)
- No kinase activity (most of the drugs target kinases)
- Intracellular localisation
Which cell cycle proteins could be targeted for cancer treatment? (2)
- CDKs (kinases)
- CDK inhibitor drugs are in clinical trials
What is Palbociclib? (2)
- CDK4/6 inhibitor
- Inhibits breast cancer cell growth in vitro with no effect on normal cells
What is an example of a CDK inhibitor drug?
Palbociclib
What is the problem with using CDK inhibitor drugs?
All patients with metastatic disease eventually develop resistance
What are CDK4/6 inhibitors approved to treat?
Hormone receptor-positive breast cancers
How does resistance to CDK4/6 inhibitor drugs develop? (3)
- Activation of upstream regulators of CDK4/6
- Inactivating mutations in pRb to allow cell cycle progression regardless of cyclin D+CDK4/6 activity
- Downstream activation of cyclin E+CDK2
What abnormalities are observed in CDK4/6 inhibitor resistant cells? (2)
- Increased myc expression
- Increased cyclin E+cdk2
How does resistance to CDK4/6 inhibitors happen involving myc? (4)
- Cells increase expression of myc
- Myc promotes cyclin E expression
- Increased cyclin E+cdk2 activation
- Causes resistance
What is the solution to CDK4/6 inhibitor resistance?
CDK2/4/6 inhibitors to inhibit downstream cdk2 as well as 4/6 e.g. PF3600
What is an example of a CDK2/4/6 inhibitor?
PF3600
What is a PDX? (3)
- Patient-derived xenograft
- Tumour sample taken from the patient and injected into a mouse
- Test drugs on the mouse to see what works
What is the disadvantage of PDX?
Animal has to be immunocompromised otherwise will reject the patient sample
How are CDK4/6 inhibitors used in combination therapy? (3)
- P to T regime (constant treatment with Palbociclib then 24hr Taxol treatment)
- T to P regime (24hrs of Taxol then constant Palbociclib)
- T to P regime inhibits cancer cell growth a lot more than P to T/P or T alone
What is Taxol?
A chemotherapy drug
How can resistance to CDK inhibitors be overcome? (2)
- Combination therapy
- Make inhibitors to additional targets
What is the DNA damage response (DDR)? (2)
- Complex network of signalling pathways which monitor DNA integrity and activate cell cycle arrest/DNA repair in the case of DNA damage
- If repair is successful cells carry on, if unsuccessful cells undergo apoptosis
What happens to DDR in cancer? (2)
- DDR inactivation is a hallmark of cancer, contributes to increased genomic instability
- Some DDR has to remain intact to maintain enough genomic stability to allow the cancer cells to grow/survive
What are the 2 main regulators of DDR?
Kinases ATM and ATR
What activates ATM in DDR?
DNA double strand breaks (DSB)
What is the role of ATM in DDR?
Acts in the G1/S checkpoint to prevent cells with damaged DNA entering S phase
What activates ATR in DDR?
DNA single strand breaks (SSB) at stalled replication forks (replicative stress)
What is the role of ATR in DDR?
Acts in the S phase and G2/M checkpoints to prevent entry into mitosis before DNA replication is complete/if there is DNA damage
What is replicative stress (RS)? (2)
- Stalling/slowing of replication fork progression and/or DNA synthesis during replication
- Stress response when cells are forced to replicate faster than usual
Why do cancer cells need DDR?
Cancer cells always under replicative stress which can lead to DNA damage so need DDR to allow them to keep growing
How could DDR be targeted to treat cancer?
DDR inhibitors e.g. CHK1 inhibitor
What is CHK1?
Cell cycle checkpoint kinase 1
Which cancer is associated with myc overexpression?
Neuroblastoma
How could CHK1 inhibitors be used to treat cancer? (4)
- Neuroblastoma cells with myc overexpression show upregulation of CHK1
- CHK1 inhibitor reduces tumour volume for a certain time
- Limited efficacy as a monotherapy, combination is required
- Toxicity also a problem
What are the 3 members of the myc family?
- c-Myc
- N-Myc
- L-Myc
How can myc overexpression be induced in cancer? (3)
- Gene amplification
- Chromosomal translocation
- Pro-virus integration
How does myc cause cancer?
Mutations cause overexpression of normal myc protein which causes transcription of genes involved in proliferation
What is bHLH? (2)
- Basic DNA binding domain followed by amino acid sequences forming α-helix, a loop and a second α-helix
- Group of transcription factors
What family of molecules does myc belong to?
bHLH family of transcription factors
How do bHLH proteins work? (2)
- Form dimers with eachother and bind to gene promoters in DNA
- Can promote or inhibit expression
What is the function of the myc-max dimer? (2)
- Promotes proliferation
- Inhibits differentiation
What is the function of the mad-max dimer? (2)
- Inhibits proliferation
- Promotes differentiation
What broadly determines whether a cell is differentiating or proliferating?
Balance between myc and mad
What are the actions of myc on the cell cycle? (3)
- Myc-Max promotes expression of cyclin D2 and CDK4
- Myc-Miz1 suppresses transcription of CKIs p15, p21, p27
- Myc promotes degradation of p27
Is myc alone sufficient to drive proliferation? (3)
- Myc expressed as a fusion protein with the oestrogen receptor
- Addition of oestrogen/tamoxifen drives myc translocation into the nucleus
- Tamoxifen addition induces transition from G0 to G1/S = myc alone is enough to drive proliferation
What are the 2 types of myc inhibitors?
- Direct
- Indirect
How do direct myc inhibitors work? (2)
- Prevents formation of the myc-max dimer
OR - Stops the myc-max dimer binding to DNA
How do indirect myc inhibitors work? (2)
- Prevents myc transcription
OR - Targets the transactivation function of myc
How does the PDAC mouse model using Cre/LoxP and tetracycline work? (5)
- Cre is a DNA recombinase that cleaves DNA at LoxP sites
- Cre is under the control of Pdx1 promoter (pancreas-specific) and is upstream of tTA
- In the pancreas, Cre cleaves DNA at LoxP sites preceeding tTA, causes tTA expression
- tTA drives expression of reporter genes and myc
- Dox inhibits tTA action
What is PDAC?
Pancreatic ductal adenocarcinoma
What were the results from the PDAC Cre/LoxP mouse model? (2)
- Inhibition of myc expression from doxycycline treatment causes tumour regression
- Significant decline in proliferation and induction of autophagy
What is Mycro3?
Direct myc inhibitor (prevents dimerisation of myc-max)
What is 18F-FDG? (2)
- Labelled version of glucose that can be imaged by PET/CT scan
- Used to visualise tumours as they have a higher glucose uptake compared to normal tissues
What was the effect of myc inhibitors in mice with human PDAC tumours? (3)
- Inject human PDAC cells into mice
- One group of orthotopic injection, other heterotopic
- Myc inhibitor reduced tumour growth in both groups
What is orthotopic injection?
Injection of cells into the location where they should be (i.e. injecting PDAC cells directly into the pancreas)
What is heterotopic injection?
Subcutaneous injection
Why are pancreatic tumours hard to treat?
Known as very fibrotic (surrounded by ECM) so hard for drugs to penetrate
How does TGFβ signalling prevent cell cycle progression? (2)
- Strongly increases levels of p15 which inhibits cyclin D-CDK4/6
- Weak induction of p21 which inhibits all other cyclin-CDK complexes
How does TGFβ counteract myc activity? (2)
- Prevents expression of myc
- Prevents myc binding to CKI gene promoters = CKIs are expressed, CDK4/6 and CDK2 are inhibited, pRb is not phosphorylated
How can TGFβ signalling be inactivated in cancer? (4)
- Smad 4 loss of function
- Smad 2 loss of function
- Smad 3 loss of expression
- TGFβ receptor expression/activity lost
What is the problem with targeting TGFβ to treat cancer? (2)
- TGFβ starts as tumour suppressing
- Becomes tumour promoting as the cancer progresses
How is TGFβ tumour promoting? (4)
- Overexpression of myc counteracts the inhibition activity TGFβ
- Hyperactivation of PI3K/AKT pathway inhibits the cytostatic activity of TGFβ
- TGFβ signalling causes expression of cytokines and growth factors which increases proliferation in cancer cells
- TGFβ promotes expression of EMT regulating genes = invasion
What is EMT?
Epithelial to mesenchymal transition
What compounds can be used to target TGFβ signalling? (4)
- Antisense oligonucleotides which prevent expression of TGFβ ligand
- Neutralising antibodies which block the ligand/receptor interaction
- Antibodies that sequester ligands
- TGFβ kinase inhibitors
What is the issue with TGFβ inhibitors?
They aren’t specific for the pro-oncogenic responses and inhibit all TGFβ induced effects
What is p53?
Transcription factor that causes expression of genes which prevent cell growth and promote apoptosis
What are the most common p53 mutations in cancer?
Mutations in the DNA-binding domain so p53 can’t bind to DNA and cause transcription of downstream targets
What is the action of p53? (4)
- Rapid increase in p53 levels in response to DNA damage
- Key element in the cell cycle checkpoints
- Promotes cell cycle arrest and DNA repair
- Cell either continues to proliferate or goes into senescence/apoptosis
What happens to p53 in the absence of stress?
p53 is ubiquitinated by mdm2 and degraded by the proteosome
How does p53 respond to DNA damage? (5)
- p53 expression is not regulated at the level of transcription because mRNA levels remain constant
- ATM and ATR are activated in response to DNA damage
- ATM and ATR phosphorylate p53 so it can’t bind to mdm2
- ATM also phosphorylates mdm2 causing inactivation
- p53 levels accumulate
What happens to p53 in the presence of growth factors? (3)
- Growth factors promote mdm2 expression and phosphorylate mdm2 at a different site causing activation
- Mdm2 binds to p53 promoting ubiquitination and degradation
- p53 can’t stop the cell cycle
How does p53 stop the cell cycle?
Upregulates p21 which inhibits most cyclin/CDK complexes
True or false: myc activation is necessary and sufficient to drive cell cycle progression
True
True or false: treatment with myc inhibitors didn’t affect tumour growth in mice
False
True or false: p53 is regulated at the transcriptional level
False
True or false: TGFβ signalling is inactivated in some cancers
True
True or false: E2F transcription factors promote the entry into S phase
True
True or false: cyclins are easy to target pharmacologically and inhibitors are used in clinical trials
False
True or false: pRb is activated by phosphorylation at the R point
False
True or false: pRb can be inactivated in cancer by interaction with viral proteins
True
What is an indication of the existence of cancer stem cells?
Cell heterogeneity
