Tumour suppressor genes

• What are tumour suppressor genes?

  Genes which normally function to restrict growth
• How do tumour suppresor genes lead to cancer?

  • Recessive loss of function mutations - can be sporadic and germline (inherited)
  • e.g. Could inherit one inactive copy of a TSG and one functioning copy, but could develop a sporadic mutation that seizes the working copy
• What are the main classes of tumour suppressor genes?

  • Growth/development suppressors, e.g. TGFB, patched1
  • Cell cycle checkpoint proteins, e.g. pRb, p53
  • Cell cycle inhibitors, e.g. CDKI, p16
  • Inducers of apoptosis, e.g. Bax, p53
  • DNA repair enzymes, e.g. Xeroderma pigmentosa
  • Developmental pathways, e.g. patched (Hh pathway)
• What do inactivating mutations in tumour suppressor genes tend to occur in?

  Cancer

  • Deletions result in no protein (inactive)
  • Point mutations result in protein with altered function (nonsense)
• How does mutations in BRCA1/2 increasing susceptibility to further mutations?

  • BRCA genes are responsible for DNA repair
  • Mutations in BRCA genes prevents DNA repair due to defective recombination --> destabilises the genome
  • Genome becomes more susceptible to further mutations
• What is retinoblastoma?

  Retinoblastoma (RB) TSG encodes the protein pRb - prevents excessive cell growth by inhibiting cell cycle progression until cell is ready to divide

  
  

  pRb positively regulates (checks cell cycle)

  • Histone deacetylases - recruited by pRb, repressing gene transcription
  • Polycomb proteins - interacts with pRb, repressing specific target genes

  
  

  pRb negatively regulates (halt cell cycle)

  • E2F TF - pRb inhibits E2F until it is phosphorylated (inactivated), causing gene expression
  • HPV (E7 protein) - pRb binds to E7 rather than E2F, allowing gene expression of E2F targets
• Describe the pRb restriction point of the cell cycle

  1. Growth factor stimulation
  2. Cyclin D1 levels rise
  3. Cyclin D/CDK4 complex
  4. CDK4 phosphorylates pRb
  5. pRb releases E2F - drives it through transcription phase
  6. Without pRb = no sequestration of E2F = constitutively active E2F driving cell cycle

  
• How can TAp73 be used to replace p53 activity in p53-deficient cells?

  • MDM2 also inhibits p53 family member, TAp73
  • Targeting MDM2 will free up TAp73
  • TAp73 can then upregulate DNA repair enzymes and apoptosis
• What is an example agent used that targets MDM2-TAp73?

  Use a proteolysis targeting chimera (PROTAC) which targets MDM2 to elicit cell death in TP53-mutant-triple-negative breast cancer cells via. activation of TAp73
• What factors trigger stabilisation/activation of p53?

  • DNA damage
  • Hypoxia/anoxia
  • Loss of trophins
  • Oncogenes

  
• What are some examples of p53 mutations?

  p53 is inactivated by carcinogens

  
  

  Lung cancer

  • Benzo(a) pyrine in cigarette smoke is metabolised in the liver producing a potent mutagen
  • Causes G > T transversions in DNA
  • Hot spots in p53 at R175, R248, R273

  Liver cancer

  • Aflatoxin (fungal metabolite)
  • Causes G > T transversions
  • Hot spot in p53 at R249
• What agents target p53?

  • Advexin - adenoviral delivery of WT p53
  • CDB3/PRIMA-1 - stabilises mutant p53 and restores transcriptional function-
  • MDM2 inhibitors, e.g. nutlins mimic p53
  • Pifithrin - suppresses endogenous p53 in normal tissue to reduce susceptibility to chemotherapy/radiation induced apoptosis
  • Onyx 015/H101 - oncolytic adenovirus that replicates specifically in cancer cells
• What is adenomatous polyposis coli (APC)?

  • APC is a tumour suppressor gene encoding APC protein
  • APC protein acts as an negative regulator of the Wnt signalling pathway - controls beta-catenin concentrations and interacts with E-cadherin, which are involved in cell adhesion
  • Defects in this - autosomal dominant pre-malignant disease characterised by adenomatous polyps (mainly in the epithelium of the large intenstine) that usually progresses into colon cancer when left untreated

  
• What is familial adenomatous polyposis (FAP)?

  • Genetic condition characterised by precancerous intestinal polyps affecting the gastrointestinal tract
  • Polys are usually benign, excessively grow, encapsulated, and sit on lining of large intestine
  • Polyps typically occur in the intestine or rectum but inviduals with FAP also have an increased chance of developing cancer in other organs due to metastasising, e.g. the stomach, small intestine, and the pancreas

  
• What are examples of tumour suppressor genes associated with cancer predisposition?

  Autosomal dominant inheritance of cancer predisposition (one mutated copy is enough to predispose)

  But both alleles of the genes must be inactivated for tumour to develop (e.g. develop a secondary somatic mutation)

  • APC - precancerous intestinal polyps = increased risk of colon cancer
  • BRCA1 - hereditary breast–ovarian cancer syndrome = 60% probability of inheriting breast/ovarian cancer compared to 2% with two WT alleles
• What is breast cancer type 1/2 susceptibility protein (BRCA1/2)?

  • BRCA1 and BRCA2 are tumour suppressor genes involved in DNA double-strand break (DSB) repair via. homologous recombination (HR)
  • HR requires undamaged template DNA as reference to repair the DSB - potential for aggressive chromosomal rearrangement and loss of genetic information that leads to cell death (mutations)
  • Mutations in the BRCA genes increases the probability of inheriting a breast-ovarian cancer syndrome
• Why are breast tissue and ovarian tissue likely to harbour mutations?

  • Involved in the monthly menstrual cycle in women - e.g. uterus lining shedding, hormone-induced breast tissue changes
  • These tissues undergo frequent turnover and production of reactive oxygen species (ROS) which can cause DNA damage
  • Requires changes within the cell to respond to this - these changes can harbour mutations
• Summarise the mechanism of action of PARP inhibitors to target BRCA-mutation cells


  PARP repairs SSBs via. base-excision repair

  BRCA repairs DSBs via. HR

  Synthetic lethality of PARP inhibitors

  • Non-cancer normal cells with functional BRCA have intact HR DSB repair pathways - compensates for inhibited PARP SSB repair
  • Cancer cells, already deficient in BRCA and HR DSB repair, are now also impaired in PARP SSB repair - accumulate unrepaired DNA damage, particularly DSBs which are toxic
  • Selective killing of cancer cells with BRCA mutations while normal cells live (have HR repair)

  
• What is p53?

  • TP53 tumour suppressor gene encodes for tumor protein p53 (or p53) - tetrameric transcriptional regulator (TF)
  • Present in healthy resting cells at low levels, complexed to MDM2 inhibitor protein
  • Stress signals inhibit MDM2 - allows release and activation of p53 which binds mutated/damaged DNA (result of stress) and directs either repair or apoptosis - essentially regulating DNA repair and cell division
  • Evolved to prevent tumour development by upregulation of anti oxidant genes to counteract ROS-mediated damage
• What is the mechanism of action of p53?

  Normal cells

  • p53 binds a complex of MDM2 and MDM4/MDMX - complex has E ligase activity that recruits ubiquitin
  • Ubiquitin degrades p53 - kept at low levels in the cell

  DNA damage (stress)

  • p53 is released from complex (unstable by itself)
  • Binds to damaged DNA (stabilised)
  • Transcription of p53 targets - e.g. cell cycle arrest, DNA repair, apoptosis

  
• What are p53-independent activities of MDM2 and MDM4/MDMX?

  Expression increased in cancer

  • Stimulate cell cycle progression
  • Inhibition of DNA repair
  • Enhancement of tumour cell invasiveness
• What is the significance of the tetramerisation domain in p53?

  Allows p53 to form a tetramer (4 monomers) - forms an active/functional TF complex to drive gene transcription

  When there is 1 mutant allele

  • High probability of incorporating a mutated monomer into the tetramer
  • Stops p53 complex working
  • This is because p53 is more stable (compared to two WT alleles) - p53 won't be degraded as easily
  • Patients with 1 mutated copy generally predipose to cancer

  
• What is the significance of p53 status?

  • Chemotherapeutic agents and radiation rely on inducing apoptosis for their cytotoxic effects
  • However, lack of p53 often makes tumour cells resistant due to a lack of functional apoptotic pathway
  • p53 is rarely mutated in cervical cancer - but polymorphism affects susceptibility to HPV E6-mediated degradation
  • Research focused on restoring p53 function
• Describe ONYX-015/H101 mechanism of action

  E1A region = targets pRb

  E1B region = targets p53

  • Oncolytic adenoviruses selectively infect and lyse (due to replication) cancer cells
  • ONYX-015 = E1B55KD-deleted adenovirus - deleted E1B55KD gene means that E1B55KD protein cannot inactivate p53 and p53 can now undergo viral replication
  • As p53-deficiency is common in many tumors, ONYX-015 exploits this deficiency and targets only cancer cells and not normal cells
  • H101, related to Onyx 015, clinically approved (2005) for treating head and neck cancer in combination with chemotherapy

  
• Which region of p53 experiences the most mutations?

  DNA-binding region - key region for p53 interaction with DNA, thus mutations here are beneficial

  • Essential to p53's ability to regulate gene expression (for cell cycle regulation, apoptosis, DNA repair etc.) - mutations here directly impair this ability, leading to a loss of its tumor suppressor function
  • Complex 3D structure crucial for DNA interaction - mutation hotspots because they are critical for maintaining the domain's structure and function