Cancer Flashcards
What is metaplasia? Give an example
A reversible change in which one adult cell type (usually epithelial) is replaced by another adult cell type
e.g. Barrett’s oesophagus
What is dysplasia?
An abnormal pattern of growth in which some of the cellular and architectural features of malignancy are present
- Pre-invasive stage with intact basement membrane
- Loss of architectural orientation
- Loss in uniformity of individual cells
- Nuclei: hyperchromatic, enlarged
- Mitotic figures: abundant, abnormal, in places where not usually found
Where is dysplasia common?
1) Cervix- HPV infection
2) Bronchus- Smoking
3) Colon- Ulcerative Colitis
4) Larynx- Smoking
5) Stomach- Pernicious anaemia
6) Oesophagus- Acid reflux
What is neoplasia?
An abnormal autonomous proliferation of cells unresponsive to normal growth control mechanisms
What are the characteristics of a benign tumour?
- Do not metastasise
- Encapsulated
- Usually well differentiated
- Slowly growing
- Normal mitoses
When are benign tumours fatal?
Dangerous places: - meninges - pituitary Secretes something dangerous: - insulinoma Gets infected: - bladder Bleeds: - stomach Ruptures: - liver adenoma Torts (twisted): - ovarian cyst
What are the characteristics of a malignant tumour?
- Invade surrounding tissues
- Spread to distant sites
- No capsule
- Well to poorly differentiated
- Rapidly growing
- Abnormal mitoses
What is a metastasis?
A discontinuous growing colony of tumour cells, at some distance from the primary cancer
- They depend on the lymphatic and vascular drainage of the primary site
- Lymph node involvement has a worse prognosis
What is Duke’s staging of bowel cancer?
Describes the progression of the cancer
Dukes A: Growth limited to wall; nodes megative
Dukes B: Growth beyond musc propria; nodes negative
Dukes C1: Nodes positive; apical lymph node negative
Dukes C2: Apical lymph node positive
What are the different types of benign epithelial tumours? Give examples.
- Of surface epithelium = PAPILLOMA
e. g. skin, bladder - Of glandular epithelium = ADENOMA
e. g. stomach, thyroid, colon, kidney, pituitary, pancreas
What is a carcinoma?
A malignant tumour derived from epithelium
What are the different types of carcinoma?
- Squamous cell
- Adenocarcinoma
- Transitional cell
- Basal cell carcinoma
Give an example of a benign soft tissue tumour.
Osteoma
What is a sarcoma?
A malignant tumour derived from connective tissue (mesenchymal) cells
What are the different types of sarcomas?
- Fat = Liposarcoma
- Bone = Osteosarcoma
- Cartilage = Chondrosarcoma
- Striated muscle = Rhabdomyosarcoma
- Smooth muscle = Leiomyosarcoma
- Nerve sheath = Malignant Peripheral Nerve Sheath Tumour
What is leukaemia?
A tumour of white blood cells:
A malignant tumour of bone marrow-derived cells which circulate in the blood
What is lymphoma?
A tumour of white blood cells:
A malignant tumour of lymphocytes (usually) in lymph nodes
What is a teratoma? What are the differences between men and women?
A tumour derived from germ cells, which have the potential to develop into tumours of all three germ cell layers: 1. Ectoderm 2. Mesoderm 3. Endoderm Women → mostly benign Men → All malignant
What is a hamartoma?
A localised overgrowth of cells and tissues native to the organ
- Cells are mature but architecturally abnormal
- Common in children, and should stop growing when they do
e. g. bile duct hamartomas, bronchial hamartomas
What is a benign tumour of glandular tissue?
An adenoma
What is a malignant tumour derived from soft tissue?
Sarcoma
What is the criteria for assessing differentiation of a malignant tumour?
Evidence of normal function still present with production of: - keratin - mucin - bile - hormones Various grading systems for: - breast - prostate - colon No differentiation, ANAPLASTIC carcinoma
What is the most important thing when it comes to assessing the prognosis of cancer?
The stage
What is described by the grade of a tumour?
Describes it’s degree of differentiation
What is described by the stage of a tumour?
Describes how far it has spread
What occurs during M-phase?
Mitosis:
- Nuclear division
- Cell division (cytokinesis)
What occurs during interphase?
Duplication
- DNA
- Organelles
- Protein synthesis
What are the stages of interphase? What happens in each phase?
G₀ - Cell cycle machinery dismantled
G₁ phase (Gap) - Decision point
S phase - Synthesis of DNA / protein
G₂ phase (Gap) - Decision point
What happens during S phase?
- DNA replication
- Protein synthesis: initiation of translation and elongation increased; capacity is also increased
- Replication of organelles (centrosomes, mitochondria, Golgi etc) in case of mitochondria, needs to coordinate with replication of mitochondrial DNA
What is the centrosome? What are it’s functions?
Consists of two centrioles (barrels of nine triplet microtubules)
Functions: microtubules organising centre (MTOC) and mitotic spindle
What are the phases of mitosis?
Prophase Prometaphase Metaphase Anaphase Telophase Cytokinesis
When does condensation of chromatin occur?
Prophase
What is the process (and size) of the condensation of chromatin?
1) Short region of DNA double helix (2nm)
2) “Beads-on-a-string” form of chromatin (DNA wrapped around histones) (11nm)
3) 30nm chromatin fibre of packed nucleosomes
4) Extended scaffold-associated form (Chromatin bound to chromosome scaffold)
5) Condensed scaffold-associated form
6) Chromosome
What is the kinetochore?
The belt of protein around the centromere of a chromosome which microtubules attach to
What happens during prophase?
- Replicated chromosomes condense
- Duplicated centrosomes migrate to opposite sides of the nucleus and organise the assembly of spindle microtubules
- Mitotic spindle forms outside nucleus between the 2 centrosomes
What is the process of spindle formation?
- Radial microtubule arrays (ASTERS) form around each centrosome (microtubule organising centers - MTOC)
- Radial arrays meet
- Polar microtubules form
What occurs during metaphase?
Chromosomes align at the equator of the spindle
Prometaphase: Early prometaphase → late prometaphase
What happens in early prometaphase?
- Breakdown of nuclear membrane
- Spindle formation largely complete
- Attachment of chromosomes to spindle via kinetochores (centromere region of chromosome)
What happens in late prometaphase?
- Microtubule from opposite pole is captured by sister kinetochore
- Chromosomes attached to each pole congress to the middle
- Chromosome slides rapidly towards centre along microtubules
What occurs during anaphase?
- Paired chromatids separate to form two daughter chromosomes
- Cohesin holds sister chromatids together
- Anaphase A and B
What happens during anaphase A?
- Breakdown cohesin
- Microtubules shorter
- Daughter chromosomes pulled toward opposite spindle poles
What happens during anaphase B?
1- Daughter chromosomes migrate towards poles
2- Spindle poles (centrosomes) migrate apart
What occurs during telophase?
- Daughter chromosomes arrive at spindle
- Nuclear envelope reassembles at each pole
- Assembly of contractile ring
What is cytokinesis?
Cytoplasm of both daughter cell condenses
- Acto-myosin ring contracts
- New membrane inserted
- Midbody begins to form
- Chromatin decondenses. Nuclear structures reform
- Interphase microtubule array reassembles
When is the Spindle Assembly checkpoint?
During the transition out of metaphase (prometaphase and metaphase)
- Senses completion of chromosome alignment and spindle assembly (monitors kinetochore activity)
What is required for the spindle assembly checkpoint? When does anaphase occur?
Requires: - CENP-E - BUB protein kinases BUBs dissociate from kinetochore when chromosomes are properly attached to the spindle When all dissociate, anaphase proceeds
What is the normal attachment of microtubules to kinetochores?
Amphelic attachment
One microtubule from each centrosome attaches to one sister chromatid
What is merotelic attachment of microtubules to kinetochores? Does it produce a checkpoint signal? What is the result?
One microtubule from one centrosome attaches to one sister chromatid. Two microtubules from the other centrosome attach to both sister chromatids
No checkpoint signal
Causes chromosome loss at cytokinesis
What is syntelic attachment of microtubules to kinetochores? Does it produce a checkpoint signal? What is the result?
Two microtubules from one centrosome attach to both the sister chromatids
May or may not produce a checkpoint signal
If no checkpoint signal then both sister chromatids will be drawn to the same pole
What is monotelic attachment of microtubules to kinetochores? Does it produce a checkpoint signal?
One microtubule from one centrosome attaches to one sister chromatid
Will produce a checkpoint signal
What is a checkpoint signal?
When a chromatid is not attached to a microtubule, or both are attached to a microtubule attached to the same centrosome a checkpoint signal is generated.
Anaphase cannot proceed until there are no checkpoint signals
What causes aneuploidy?
1) Misattachment of microtubules to kinetochores
2) Aberrant centrosome / DNA duplication
If cell cycle or DNA / centrosome replication causes the daughter cells to have multipolar spindles when cytokinesis occurs the cell can cause aberrant cytokinesis
Why can inducing gross chromosome mis-segregations be used as an anti-cancer therapy?
- Alters microtubule dynamics
- Produces unattached kinetochores
- Causes long-term mitotic arrests
- Can lead to apoptotic cell death
What can go wrong during the cell cycle? What causes these?
- Cell cycle arrest
- at checkpoints (G1 and spindle checkpoint)
- can be temporary (i.e. following DNA repair) - Programmed cell death (apoptosis)
- DNA damage too great and cannot be repaired
- chromosomal abnormalities
- toxic agents
Where are the cell cycle checkpoints? What effects do tumours have on checkpoints?
G₁ checkpoint
G₂ checkpoint
Metaphase (spindle) checkpoint
Tumours block these checkpoints then exit the cell cycle at G₀. They then dismantle cell cycle apparatus (deregulation of cell cycle)
In the absence of stimulus what phase are cells in?
G₀
What is the process of signalling in a cell, causing a cell to exit G₀?
Cell responds to extracellular factors and is modulated by other pathways
- A dimeric ligand bind to the receptor and causes it to form a dimer
- Amino acids are then phosphorylated in the kinase domain causing activation of the receptor
• This causes a conformational change in the receptor; changing it’s activity
• It also creates a docking site for other protein
- Activation of the receptor triggers a kinase cascade and the binding of adaptor proteins
What is the role of c-Myc?
c-Myc is a transcription factor. It stimulates the expression of cell cycle genes. Upregulation of of Myc triggers entry into S-phase
What is the process of growth factor stimulation of signalling pathways?
Mitogenic growth factor (i.e. growth signals from other cells e.g. Hepatocyte Growth Factor released after liver damage) ↓ Receptor protein tyrosine kinase ↓ Small G (GTP-binding) protein (Ras) ↓ Kinase cascade ↓ Immediate early genes (c-Jun, c-Fos, c-Myc- transcription factors) - control the expression of other genes
How does growth factor initiate a response in the cell?
- GF dimer binds to the tyrosine kinase receptor with ligand binding
- Each phosphorylated tyrosine bound to the receptor binds intracellular signalling proteins forming a signalling hub to initiate the cascade
- Grb2 binds to exchange factor Sos which activated Ras which then allows transmission of the signal
What does Herceptin do?
Blocks the action of EGF receptors by blocking ligand binding to the receptor preventing further activation of the receptor
What do adapter proteins bind to on growth factor receptors?
Phosphorylated tyrosines
What are the domains of Grb2?
SH3-SH2-SH3
What does SH3 domains bind to in Grb2?
Proline rich regions
How does Ras bind to growth factor receptors and induce the kinase cascade? Give an example of a factor than can activate Ras
- Cannot bind to the receptor directly- must bind to Grb2
- Ras binds GDP, and when it is activated GTP which allows it to signal the kinase cascade
Activated by exchange factors e.g. Sos
Ras has a lipid modification to bind it to the membrane
What are the different types of oncogenic mutation in Ras? What is the outcome of these mutations?
V12Ras- constitutively active - Glycine → Valine - Prevents GAP binding by preventing inactivation L61Ras- constitutively active - Glutamine → Leucine - Prevents GTP hydrolysis
What does Ras do?
Controls the cell cycle
When Ras is active what process does it induce?
- Activates kinase I (Raf)
- Raf phosphorylates kinase II (MEK)
- MEK phosphorylates kinase III (ERK)
This then:
- Changes protein activity
- Changes gene expression (e.g. c-Myc, cell proliferation)
What is the activity of cyclin-dependent kinases regulated by?
- Interaction with cyclins
- Phosphorylation
What cyclin-Cdk pair cause the cell to enter mitosis?
Cdk 1-mitotic cyclin B
- Forms mitosis promoting factor which is the mitotic checkpoint
What cyclin-Cdk pair trigger DNA replication machinery in the cell?
Cdk-Scyclin
What is the process of formation of Mitosis Promoting Factor
- Cdk1 binds cyclin B to form inactive MPF
- Cdk-activating kinase (CAK) and Wee1 (inhibitory kinase) phosphorylate inactive MPF
- Phosphatase Cdc25 phosphorylates again to produce active MPF
Positive feedback: Active MPF further activates Cdc25 to drive mitosis
Signals from fully attached kinetochores during mitosis cause cyclin B to be degraded. What effect does this have?
- Cdk1 inactivated
- Key substracts dephosphorylated
- Mitosis progresses
What cyclin and Cdk pair are present in the G₁ phase of the cell cycle?
Cdk2 (G1/S-Cdk)
Cyclin E
What cyclin and Cdk pair are present in the S phase of the cell cycle?
Cdk2 (S-Cdk)
Cyclin A
What is the role of cyclin D? What stimulates transcription of this factor?
c-Myc stimulates transcription of cyclin D
Cyclin D bind to Cdk4 or Cdk-6 which allows the cells entry into G₁
What is the timing process of release of cyclins/Cdks throughout the cell cycle?
Cdk4/6-cyclin D - end of G₁ Cdk2-cyclin E - end of G₁ →⅔ of S Cdk2-cyclin A - mid-S → metaphase (mid-mitosis) Cdk1-cyclin B - start of mitosis → anaphase-telophase
What do Cdks do?
They phosphorylate proteins (on Serine or Threonine) to drive cell cycle progression
Cdk1-cyclin B
- MPF
- Phosphorylate subset of proteins during mitosis such as nuclear lamins
- Then disassemble and break down the nuclear envelope
Cdk2-cyclin E
- Phosphorylate proteins which control start of the cell cycle
- e.g. Retinoblastoma protein
What is the role of Retinoblastoma (Rb) protein?
Rb is a “tumour suppressor”
Found at G₀
- Active Rb protein binds to E2F making it an inactive transcription factor
- Cdk4/6-cyclin D and Cdk2-cyclin E causes phosphorylation of pRb to make it the inactive form
- This releases E2F for gene transcription
What are the two families of Cdk inhibitors?
INK4 family:
- G₁ phase CKIs
- Inhibit Cdk4/6 by displacing cycD
CIP/KIP family
- S phase CKIs
- Inhibit all Cdks by bindings to the Cdk/cyc complex
They much be degraded to allow cell cycle progression
Give examples of different oncogenes and the cancer they cause.
- EGFR/HER2 → mutationally activated or overexpressed in many breast cancers
- Ras → mutationally activated in many cancers (inhibitors of membrane attachment)
- Cyclin D1 → overexpressed in 50% of breast cancers
- B-Raf → mutationally activated in melanomas
- c-Myc → overexpressed in many tumours
What treatment is used for HER2-positive metastatic breast cancer?
Herceptin antibody
What are the different tumour suppressors in breast cancer?
- Rb → inactivated in many cancers
- p27ᴷᴵᴾ¹ → underexpression correlates with poor prognosis in many malignancies
Where is cancer incidence lowest in the world? Why is this?
Asia
Diagnosis of cancer is lower due to less advanced medical care
What are the five most common cancers worldwide?
- Lung
- Breast
- Bowel
- Prostate
- Stomach
What are the main anti-cancer treatment modalities?
- Surgery
- Radiotherapy
- Chemotherapy
- Immunotherapy
What types of genetic mutations cause cancer?
- Chromosome translocations
- Gene amplification (copy number variations)
- Point mutations within promoter or enhancer regions of genes
- Deletions or insertions
- Epigenetic alterations to gene expression
- Can be inherited (e.g. BRCA gene)
What are the causes of cancer and they’re relevant proportions?
⅓ of cancer = poor lifestyle choices
⅔ of cancer = random mutations
What are the different types of systemic therapy to treat cancer?
- Cytotoxic chemotherapy
2. Targeted therapies
What are the different types of cytotoxic chemotherapy? How do they work?
- Alkylating agents
- Antimetabolites
- Anthracyclines
- Vinca alkaloids and taxanes
- Topoisomerase inhibitors
“Select” rapidly dividing cells by targeting their structures (mostly the DNA)
What are the different types of targeted therapies?
- Small molecule inhibitors
- Monoclonal antibodies
How is cytotoxic chemotherapy administered? Describe it.
Given intravenously or by mouth (occassionally)
Works systemically
Non “targeted” - affects all rapidly dividing cells in the body
Given post-operatively; adjuvant
- Pre-operatively: neoadjuvant
- As monotherapy or in combination
- With curative or pallative intent
What is the mechanism of action of alkylating agents?
Add alkyl groups to guanine residues in DNA
Cross-link (intra, inter, DNA-protein) DNA strands and prevents DNA from uncoiling at replication
Trigger apoptosis (via checkpoint pathway)
Encourage mis-pairing - oncogenic
What is the mechanism of action of pseudo-alkylating agents? Give examples of drugs
Add platinum to guanine residues in DNA
Same mechanism of cell death as alkylating agents
Examples: carboplatin, crisplatin, oxaliplatin
Give examples of different types of alkylating agents.
- Chlorambucil
- Cyclophosphamide
- Dacarbazine
- Temozolomide
What are the side effects of alkylating and pseudoalkylating agents?
- Hair loss (except carboplatin)
- Nephrotoxicity
- Neurotoxicity (can be permanent)
- Ototoxicity (platinums)
- Nausea, vomiting and diarrhoea
- Immunosuppression
- Tiredness
What is the mechanism of action of anti-metabolites?
Masquarades as purine or pyramidine residues leading to inhibition of DNA synthesis, DNA double strand breaks and apoptosis
- Blocks DNA replication (DNA-DNA) and transcription (DNA-RNA)
- Can be purine (A or G), pyramidine (T/U or C) or folate antagonists (which inhibit dihydrofolate reductase required to make folic acid, an important building block for all nucleic acids - especially T)
Give examples of anti-metabolite cancer treatments. Which one of these is one of the most common chemotherapy drugs?
- Methotrexate (folate)
- 6-mercaptopurine
- Decarbazine
- Fludarabine (purine)
- 5-flurouracil MOST COMMON
- Capecitabine
- Gemcitabine (pyramidine)
What are the side effects of anti-metabolites?
- Hair loss (alopecia)- not 5FU or capecitabine
- Bone marrow suppression causing anaemia, neutropenia and thrombocytopaenia
- Increased risk of neutropenic sepsis (and death) or bleeding
- Nausea and vomiting (dehydration)
- Mucositis and diarrhoea
- Palmar-plantar erythrodysesthesia (PPE)
- Fatigue
What is the mechanism or action of anthracyclines? Give examples of the drug
- Inhibit transcription and replication by intercalating (i.e. inserting between) nucleotides within the DNA/RNA strand
- Also block DNA repair - mutagenic
- They create DNA and cell membrane damaging free oxygen radicals
Examples: doxorubicin, epirubicin
What are the side effects of anthracyclines?
- Cardiac toxicity (arrhythmias, heart failure) - probably due to damage induced by free radicals
- Alopecia
- Neutropenia
- Nausea and vomiting
- Fatigue
- Skin changes
- Red urine (doxorubicin “the red devil”)
What is the mechanism of action of vinca alkaloids and taxanes?
- Originally derived from natural sources
- Work by inhibiting assembly (vinca alkaloids) or disassembly (taxanes) of mitogenic microtubules causing dividing cells to undergo mitotic arrest
What are the side effects of microtubule targeting drugs?
- Nerve damage: peripheral neuropathy, autonomic neuropathy
- Hair loss
- Nausea
- Vomiting
- Bone marrow suppression (neutropenia, anaemia etc)
- Arthralgia
- Allergy
What is the mechanism of action of topoisomerase inhibitors?
- Topoisomerases are required to prevent DNA torsional strain during DNA replication and transcription
- They induce temporary single strand (topo1) or double strand (topo2) breaks in the phosphodiester backbone of DNA
- They protect the free ends of DNA from aberrant recombination events
- Drugs such as anthracyclines have anti-topoisomerase effects through their action on DNA
- Specific topoisomerase inhibitors include Topotecan and irinotecan (topo I) and etoposide (topo II) alter binding of the complex to DNA and allow permanent DNA breaks
What are the side effects of topoisomerase inhibitors?
- Irinotecan: Acute cholinergic type syndrome - diarrhoea, abdominal cramps and diaphoresis (sweating). Therefore given with atropine
- hair loss
- Nausea and vomiting
- Fatigue
- Bone marrow suppression
What are the resistance mechanisms of cancer to treatments?
- DNA repair mechanisms upregulated and DNA damage is repaired
- DNA adducts replaced by Base Excision repair (using PARP)
- Drug effluxed from the cell by ATP-binding cassette (ABC) transporters
What are the advantages and disadvantages on dual kinase inhibitors?
Advantage: Prevents feedback loops (parallel pathways or activation of feedback cascades)
Disadvantage: Increase toxicities
What are the 10 hallmarks of the cancer cell?
- Self-sufficient
- Insensitive to anti-growth signals
- Anti-apoptotic
- Pro-invasive and metastatic
- Pro-angiogenic
- Non-senescent
- Dysregulated metabolism
- Evades the immune system
- Unstable DNA
- Inflammation
Give examples of receptors which are overexpressed in cancer?
HER-2
- Amplified and overexpressed in 25% of breast cancer
EGFR
- Overexpressed in breast and colorectal cancer
PDGFR
- Glioma (brain cancer)
Give examples of ligand which is overexpressed in cancer?
VEGF
- prostate cancer, kidney cancer, breast cancer
Give an example of constitutive (ligand independent) receptor activation in cancer?
EGFR (lung cancer)
FGFR (head and neck cancers, myeloma)
What are the suffixes associated with monoclonal antibodies? Include examples
-momab (derived from mouse antibodies) -ximab (chimeric) e.g. cetuximab -zumab (humanised) e.g. bevacizumb, trastuzumab -mumab (fully human) e.g. panitumumab
What is the action of monoclonal antibodies?
- Neutralise the ligand
- Prevent receptor dimerisation
- Cause internalisation of receptor
- Activate Fcγ-receptor-dependent phagocytosis or cytolysis induces complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC)
Give an example of a monoclonal antibody used in oncology?
- Bevacizumab binds and neutralises VEGF. Improves survival in colorectal cancer
- Cetuximab targets EGFR
What is the mechanism of action of small molecule inhibitors?
- Bind to the kinase domain of the tyrosine kinase within the cytoplasm, and block autophosphorylation and downstream signalling
Where is the BCR-Abl translocation in CML?
Between chromosomes 9 and 22
What is Glivec?
A small molecule inhibitor which targets the ATP-binding region withing the kinase domain
What intracellular receptor does small molecule inhibitor Erlotinib act on?
EGFR
What intracellular receptor does small molecule inhibitor Gefitinib act on?
EGFR
What intracellular receptor does small molecule inhibitor Lapatinib act on?
EGFR
HER2
What intracellular receptor and intracellular kinases does small molecule inhibitor Sorafinib act on?
Intracellular receptor
- VEGFR
Intracellular kinase
- Raf kinase
What intracellular kinase does small molecule inhibitor Dasatinib act on?
Src kinase
What intracellular kinase does small molecule inhibitor Torcinibs act on?
mTOR inhibitors
What effect does inhibiting VEGF have on a tumour?
Alters blood flow to the tumour
What effect does inhibiting AKT have on a tumour?
Blocks apoptosis resistance mechanisms
What is the benefit to using cancer drugs which act of receptors rather than cytotoxic drug?
Less toxicity
What is the main disadvantage to using monoclonal antibodies or small molecule inhibitors?
Resistance
What are the resistance mechanisms to targeted therapies?
- Mutations in ATP-binding domains (e.g. BCR-Abl fusion gene and ALK gene, targeted by Glivec and crizotinib respectively)
- Intrinsic resistance (Herceptin effective in 85% HER2+ breast cancers, suggesting other driving pathways)
- Intragenic mutations
- Upregulation of downstream or parallel pathways
What is the action of Anti-sense oligonucleotides?
- Single-stranded, chemically modified DNA-like molecule 17-22 nucleotides in length
- Complementary nucleic acid hybridisation to target gene hindering translation of specific mRNA
- Recruits RNase H to cleave target mRNA
- Good for “undruggable” targets
How is targeting b-Raf a successful therapy option in cancer?
- Activating mutation of b-Raf identified in 60% of melanomas
- Substitution of glutamic acid for valine (V600E) causes a 500-fold increase in activity
- b-Raf inhibitor (Vemurafenib) showed dramatic Phase 1 activity in melanoma (80% PR or CR)
- Extends life-span by 7 months
- Side effects: arthralgia, skin rash and photosensitivity
How is immune modulation via programmed cell death a successful therapy option in cancer?
- Present on the surface of cancer cells
- Required to maintain T cell activation
- After binding the ligand PDL1, the body’s T cells can no longer recognise tumour cells as foreign
- If either is blocked the immune system is stimulated
- Nivolumab is anti-PD1 antibody
What external influences are detected by cells?
Chemical- hormones, growth factors, ion concentrations, ECM, molecules on other cells, nutrients and dissolved gas (O₂/CO₂) concentrations
Physical- mechanical stresses, temperature, the topography or “layout” of the ECM and other cells
What external factors can influence cell division?
- Growth factors
- Cell-cell adhesion
- Cell-ECM adhesion
What is the process of a cell settling on a culture surface?
- Cell settles on culture surface
- Spreads
- Acquires motility
It is not passive or gravity dependent. Energy is requires to modulate cell adhesion and the cytoskeleton during spreading
What is the requirement of cells to respond to growth factors?
Cells must be bound to ECM (with a degree of spreading) to be fully competent:
- For responding to soluble growth factors
- To begin protein synthesis and proliferation (DNA synthesis)
What are cell-ECM adhesion molecules?
Cells have receptors on their surface which bind specifically to ECM molecules
- these are often linked, at their cytoplasmic domains, the cytoskeleton
- This arrangement means that there is mechanical continuity between ECM and the cell interior
What are integrins?
Heterodimer complexes of α and β subunits that associate extracellularly by their “head” regions. Each of the “tail” regions spans the plasma membrane
- They are the most important ECM receptors
- Recognise short, specific peptide sequences
- More than 20 combinations of α/β known
- Each combination specifically binds a particular peptide sequence
- Peptide sequences are found in more than one ECM molecule
What is peptide sequence RGD found in?
- Fibronectin
- Vitronectin
- Fibrinogen (plus others)
How are integrins bound to the actin cytoskeleton?
Via actin-binding proteins
What does a cluster of integrin complexes form?
- Focal adhesions (mostly)
- Hemidesmosomes
These are involved in signal transduction and bind to specific adhesion molecules on some cells
How are integrins involved in cell signal transduction?
- Integrin complexes binding to ECM can stimulate the complex to produce a signal inside the cell
“outside-in” integrin signalling - A signal generated inside the cell (e.g. hormone binding to receptor) can act on an integrin complex to alter the affinity of an integrin (i.e. alter its affinity for its ECM binding)
“inside-out” integrin signalling
What is “outside-in” signalling in cells?
A cell can receive information about its surroundings from its adhesion to ECM
- e.g. the composition of the ECM will determine which integrin complexes bind and which signals it receives
- this can alter the phenotype of the cell
How do focal adhesions sense the mechanical properties of their surroundings?
The amount of force that is generated at a focal adhesion depends on both the force generated by the cytoskeleton (F cell) and the stiffness of the ECM
Following integrin activation what conformational changes occur with the complex?
- “Inside-out” activation extends the flexed complex (↑ affinity)
- Ligand-binding opens the legs of the complex allowing cytoplasmic signalling molecules to bind (outside-in signalling)
What effect does the ECM have on the phenotype of cells?
Profound effect e.g.
In interstitial matrix (type I collagen)
- mammary epithelium does not differentiate to secretory cells
In basal lamina matrix (basement membrane)
- mammary cells organise into “organoids” and produce milk proteins
What is the ERK MAP kinase cascade required for cyclin D expression?
Growth factor binds to tyrosine kinase receptor
Ras ↓ Raf (MAPKKK) ↓ MEK (MAPKK) ↓ ERK (MAPK) ↓ Gene expression (proliferation)
What is the mechanism of anchorage dependence?
Growth factor receptors and integrin signalling complexes can each activate identical signalling pathways (e.g. MAPK)
- Individually, this activation is weak and/or transient
- Together, activation is strong and sustained
- The separate signalling pathways act synergistically
What are short-term contact interactions between cells?
Transient interactions between cells which do not form stable cell-cell junctions
What are long-term contact interactions between cells?
Stable interactions resulting in formation of cell-cell junctions
What is contact inhibition of locomotion? What does it ensure?
When most non-epithelial cells collide they “repel” one another by paralysing motility at the contact site, promoting the formation of a motile front at another site on the cell, and moving off in the opposite direction
What types of cells for long-term cell-cell contacts by strongly adhering and forming specific cell-cell junctions?
- Epithelial cells (forms layers)
- Endothelial cells (forms layers)
- Neurones (synapses)
What is contact-induces spreading of epithelial cells?
Contact between epithelial cells leads to the mutual induction of spreading so that the total spread area of the contacted cells is greater than that of the sum of the two separated cells
This could result in a stable monolayer
What factors affect cell-cell adhesion?
- Ca²⁺
- Adhesion-blocking antibody
What is the effect of no cell-cell junctions?
- Activated MAPK
- Decreased p27ᴷᴵᴾ¹
- High proliferation
What protein is thought to be the link between cell-cell adhesion and proliferation?
β-catenin
Binds cadherin to α-catenin, which binds to actin. Binds cell to ECM
What effect does the APC gene have on β-catenin?
The product of the APC gene is a protein involved in degradation of β-catenin
- β-catenin is a protein involved in cell-cell adhesion
If APC is mutated then degradation is reduced
= ↑ proliferation
In what circumstances would β-catenin form a complex with LEF-1? What does this complex cause?
β-catenin/LEF-1 complex forms if β-catenin cytoplasmic levels rise as a result of:
- Inhibition of degradation
- Loss of cadherin-mediated adhesion
β-catenin/LEF-1 complex enters the nucleus and influences gene expression, leading to proliferation
Other than the β-catenin/LEF-1 complex what other adhesion-associated signalling pathways are known to influence contact-induced inhibition of proliferation?
- Clustering of cadherins after cell-cell contact is known to alter the activation of small GTPases (e.g. Rac is activated; Rho is inhibited) this can influence proliferation
- Some growth factor receptors are associated with cell-cell junctions. This reduces their capacity to promote proliferation
What happens if cells lose their contact inhibition?
They will:
- Proliferate uncontrollably (lose density dependence of proliferation)
- Are less adherent and will multilayer (lose contact inhibition of locomotion and anchorage dependence)
- Epithelia breakdown cell-cell contacts
- Not Hayflick limited, express telomerase
i. e. Cancer
Other than tumour formation, what is an important consequence of low of contact inhibition of locomotion for the progression of cancer?
Increased invasiveness leads to metastasis
Give examples of parts of the signal transduction pathway where proto-oncogenes can lead to uncontrolled proliferation of cells.
- If the gene coding for a component of a signalling pathway is mutated so that the protein is constitutively active, that pathway will be permanently “on”
- Receptors, signalling intermediates and signalling targets (e.g. transcription factors) are proto-oncogenes
- This can result in loss of growth factor dependence
What is a proto-oncogene?
The normal cellular gene corresponding to the mutant gene which promotes uncontrolled cell proliferation
What oncogenes can arise from proto-oncogene Ras?
V12Ras (Gly12Val mutation)
L61Ras (Gln61Leu mutation
What oncogene arises from proto-oncogene c-Raf?
v-Raf (deletion of regulatory domain)
What oncogene arises from proto-oncogene c-Jun?
v-Jun (deletion of regulatory domain)
What percentage of all cancers are caused by a mutation in Ras? What cancer is this mutation most frequent in?
≈30%
Pancreatic cancer
How does a primary carcinoma cell metastasise?
- Cell-cell adhesion must be down-regulated (e.g. cadherin levels reduced)
- The cells must be motile
- Degradation of ECM must take place; matrix metaloproteinase (MMP) levels increased in order to migrate through basal lamina and interstitial ECM
- The degree of carcinoma cell-cell adhesion is an indicator of how differentiated the primary tumour is, and indicates its invasiveness and the prognosis
What are the steps in tumour progression?
- Homeostasis
- Genetic alterations
- Hyper-proliferation
- De-differentiation (non-reversible)
- Disassembly
- Cell-cell contacts
- Lose polarity
- Invasion
- Increased motility
- Cleavage ECM proteins (allows cells to migrate)
What are the sequential events involved in metastasis?
- Epithelial cells in primary tumours are tightly bound together
- Metastatic tumour cells become mobile mesenchyme-type cells and enter the bloodstream
- Metastatic cells then travel through the bloodstream to a new location in the body
- Metastatic cells exit the circulation and invade a new organ
- Cancer cells lose their mesenchymal characteristics and form a new tumour
What are the different types of tumour cell migration?
- Amoeboid migration
- Mesenchymal (single cell) migration
- Mesenchymal (chains) migration
- Cluster/cohort migration
- Multicellular strands/sheet migration
What is amoeboid cell migration? What cancers does this occur in?
Single, autonomous cell invasion Occurs in: - Lymphoma - Leukaemia - SCLC
What is mesenchymal cell migration? What cancers does this occur in?
Single cell or chains. Single cell opens up the trail and other cells follow Occurs in: - Fibrosarcoma - Glioblastoma - Anaplastic tumours
What is cluster/cohort cell migration? What cancers does this occur in?
Lots of cells migrate together. Requires coordination with neighbouring cells
Occurs in:
- Epithelial cancer
- Melanoma
What is multicellular strand/sheet cell migration? What cancers does this occur in?
Lots of cells migrate together in strands or sheets. Requires coordination between neighbouring cells
Occurs in:
- Epithelial cancer
- Vascular tumours
What genes are upregulated in invasive cells compared to primary tumours?
Genes involved in:
- Cytoskeleton regulation
- Motility machinery
What are the different stimuli that can make a cell move?
- Organogenesis and morphogenesis
- Wounding
- Growth factors/chemoattractants
- Dedifferentiation (tumours)
What specialised structures allow a cell to move?
- Focal adhesion
- Lamellae
- Filopodium
What are filopodia in cells?
Cell structure used for motility
Finger-like projections rich in actin filaments made up of bundles of parallel filaments
What is the function of plaque in cells?
Hold integrins in place and produces force in a cell
- Contains vinculin
