CANCER; Lecture 4, 5 and 6 - Biological basis of cancer therapy, External factors controlling division and behavior of normal and cancerous cells, Invasion - regulation of cell migration Flashcards
What are the most common cancers?
Lung, breast, bowel, prostate and stomach
What are the 4 main anti-cancer modalities?
Surgery, radiotherapy, chemotherapy and immunotherapy
Which types of genetic mutations can cause cancer?
Chromosome translocation Gene amplification (copy number variation) Point mutations within promoter or enhancer regions of genes Deletions or insertions Epigenetic alterations to gene expression Can be inherited
What are the 2 types of systemic therapy for cancer?
Cytotoxic chemotherapy and targeted therapies
What are the types of cytotoxic chemotherapies?
1) Alkylating agents 2) Antimetabolites 3) Anthracyclines 4) Vinca alkaloids and taxanes 5) Topoisomerase inhibitors
What are the types of targeted therapies for cancer?
Small molecule inhibitors and monoclonal antibodies
How does cytotoxic chemotherapy work?
Given IV (occasionally orally); works systemically, non-targeted: affects all rapidly dividing cells in the body
When can you use cytotoxic chemotherapy?
Post-op = adjuvant; pre-op = neoadjuvant; monotherapy/combnation and with curative/palliative intent
What are alkylating agents?
Add alkyl groups to guanine residues in DNA and then it cross-links DNA strands and prevents DAN from uncoiling at replication -> then triggers apoptosis; encourages mispairing
What are psuedo-alkylating agents?
Add platinum to guanine residues in DNA, triggering the same mechanism of death as alkylating agents
Name some alkylating agents.
Chlorambucil Cyclophosphamide Dacarbazine Temozolomide
Name some psuedo-alkylating agents.
Carboplatin, cisplatin and oxaliplatin
What are the side effects of pseudo/alkylating agents?
Hair loss (not carboplatin) Nephrotoxicity Neurotoxicity Ototoxicity (platins) Nausea Vomiting Diarrhoea Immunosuppression Tiredness
How do anti-metabolites work?
Masquerade as purine/pyrimidine residues, leading to inhibition of DNA synthesis, breaking of ds-DNA and apoptosis -> block DNA transcription/replication. Can be purine/pyrimidine analogues, also folate antagonists (inhibit dihydrofolate reductase, required to make folic acid)
Give examples of anti-metabolites
Methotraxate** 6-mercaptopurine Fludarabine (purine) 5-fluorouracil Capecitabine Gemcitabine (pyrimidine)
What are the side effects of anti-metabolites?
Hair loss (alopecia) - not 5-fluorouracil or capecitabine Bone marrow suppression causing anaemia, neutropenia and thrombocytopenia Increased risk of neutropenic sepsis (and death) or bleeding Nausea and vomiting (leading to dehydration) Mucositis and diarrhoea Palmar-plantar erythrodysesthesia (PPE) Fatigue
How do anthracylcines work?
Inhibit transcription and replication by intercalating nucleotides within DNA/RNA strand; block DNA repair and create DNA-damaging and cell membrane damaging oxygen free radicals
Give examples of anthracyclines.
Doxorubicin and epirubicin
What are the side effects of anthracyclines?
Cardiac toxicity (arrhythmias, heart failure) - probably due to damage induced by free radicals Alopecia Neutropenia Nausea Vomiting Fatigue Skin changes Red urine (doxorubicin = ‘the red devil’)
What are vinca alkaloids and taxanes?
Derived from natural sources; inhibit assembly (VA) or disassembly (T) of mitotic microtubules causing dividing cells to undergo mitotic arrest
What are the side effects of taxanes and vinca alkaloids?
Nerve damage: peripheral neuropathy, autonomic neuropathy Hair loss Nausea Vomiting Bone marrow suppression (neutropenia, anaemia etc.) Arthralgia (severe pain in a joint without swelling or other signs of arthritis) Allergy
What are topoisomerase inhibitors?
Topoisomerases are responsible for uncoiling DNA; preventing torsional strain on DNA during replication and transcription -> induce temporary single strand (topo1) or double strand (topo2) breaks in the phosphodiester backbone of DNA -> protect free ends of DNA from aberrant recombination. TII alter binding of complex to DNA and allow permanent DNA breaks
Give examples of Topoisomerase inhibitors?
Topotecan (topo1), irinotecan (topo1), etoposide (topo2)
What are the side effects of topoisomerase inhibitors?
Irinotecan = acute cholinergic type syndrome (diarrhoea, abdominal cramps, diaphoresis (sweating) - they are therefore given atropine) Hair loss Nausea Vomiting Fatigue Bone marrow suppression
What are the routes to apoptosis?
Summarise the side effects and treatment methods of anti-metabolites vs. anthracycline vs alkylating agent
What are the resistance mechanisms that form in cells to survive the chemotherapy?
What are the targeted therapies for cancer?
- Non-cytotoxic and seek to manipulate what we know about cancer cells ->
- mainly involved monoclonal antibodies and small molecule inhibitors ->
- in monogenic cancers the signalling can be cut
- for others, parallel pathways and feedback cascades are activated -> so dual kinase inhibitors are created
What are the 6 (+ 4 new ones) hallmarks of cancer (SPINAP DIE U)?
- Self-sufficient
- Pro-invasive/metastatic
- Insensitive to anti-growth signals
- Non-senescent
- Anti-apoptotic
- Pro-angiogenic
- Dysregulated metabolism
- Inflammation
- Evades immune system
- Unstable DNA
How are cancer cells insensitive to anti-growth signals?
Tyrosine kinase linked receptors are associated with 50% of human malignancies -> Over-expression of receptors/ligand or constitutive receptor activation
Which receptors are over expressed in cancers?
HER2 -> amplified and O-E in 25% breast cancers; EGFR - O-E in breast and colorectal and PDGFR - in gliomas (brain); all lead to upregulation of kinase cascade and signal amplification
Which ligands are over expressed in cancers?
VEGF -> prostate, kidney and breast; also leads to upregulation of kinase cascade and signal amplification
Which receptors undergo constitutive receptor activation in cancer (ligand independent)?
EGFR - lung; FGFR - head and neck cancers, myeloma
What are the different suffixes for monoclonal antibodies and what do they mean (with examples)?
What is the difference between humanised and chimeric antibodies?
What is the function of monoclonal antibodies?
Neutralise the ligand , prevent receptor dimerisation and cause internalisation of receptor -> activate Fcgamma receptor-dependent phagocytosis or cytolysis induced complement-dependent cellular cytotoxicity (CDC) or Ab-dependent cellular cytotoxicity (ADCC)
Give examples of monoclonal antibodies in oncology
Bevacizumab (binds and neutralises VEGF) and Cetuximab (targets EGFR)
What do small molecule inhibitors do?
Bind to kinase domain within the cytoplasm and block auto-phosphorylation and downstream signalling
What is Bcr-abl?
- Translocation in CML patients which creates its own fusion protein Bcr-abl ->
- enzyme that drives over-production of white cells
How do small molecule inhibitors work - especially with CML patients?
- Glivec (SMI) targets ATP binding region with kinase domain on c-ABL
- which inhibits the kinase activity of ABL1.
- By acting on receptors either externally/internally, targeted therapies block cancer hallmarks without toxicity observed with cytotoxics
Give examples of small molecule inhibitors that inhibit receptors.
Erlotinib (EGFR) Gefitinib (EGFR) Lapatinib (EGFR/HER2) Sorafenib (VEGFR)
Give examples of small molecule inhibitors that inhibit intracellular kinases.
Sorafenib (Raf kinase) Dasatinib (Src kinase) Torcinibs (mTOR inhibitors)
What are the dis/advantages of monoclonal antibodies and small molecule inhibitors?
What are the resistance mechanisms to targeted therapies?
Mutations in ATP-binding domain (e.g. BCR-Abl fusion gene and ALK gene, targeted by Glivec and crizotinib respectively) Intrinsic resistance (herceptin is effective in 85% of HER2+ breast cancers, suggesting other driving pathways) Intragenic mutations Upregulation of downstream of parallel pathways (that lead to cell proliferation)
What are anti-sense oligonucleotides?
- Single stranded, chemically modified DNA-like molecule 17-22 molecules in length ->
- causes complementary nucleic acid hybridisation to target gene hindering translation of specific mRNA ->
- recruits RNase H to cleave target mRNA ->
- good for undruggable targets
What is RNA interference?
Single stranded complementary RNA -> lagged behind anti-sense technology in cancer therapy mainly -> compounds have to be packaged to prevent degradation
What is the major obstacle to target approach for cancer?
Tumour heterogeneity
How was b-RAF targeted in cancer treatment?
How did immune modulation help in cancer treatment?
Via programmed cell death 1 ->
What is nivolumab?
Anti-PD1 antibody -> In treatment-refractory melanoma, non-small cell lung cancer (NSCLC), and renal cell carcinoma (RCC) Saw overall response rates of 31% in melanoma (cf the usual 5-15%) Median survival of 16 months (phase I trial)
Why would you sequence tumours genetically before starting therapy?
Depends on reliable methods – currently not being done (risk of false negative results) Used to provide treatment as well as prognostic information Concentrate on particular pathways for certain cancers? Circulating biomarkers, tumour cells or DNA
What are the new therapeutic avenues that are present in cancer?
Nanotherapies – delivering cytotoxics more effectively Virtual screening technologies to identify “undruggable” targets Immunotherapies using antigen presenting cells to present “artificial antigens” Targeting cancer metabolism
What is required for cell-ECM adhesion?
- Cell requires to be bound to ECM to be fully competent for responding to soluble growth factors -
- suspended in agar = few cells entered S phase;
- if able to stick to small patch which didn’t allow them to spread fully, then only a small proportion of cells proliferated and
- if the patch was larger then almost all cells started proliferating
What are integrins?
- Most important matrix receptor ->
- about 10 alpha and 8 beta subunits forming more than 20 known combo which specifically binds short, specific peptide sequence (often found in more than one ECM molecule)
How are the integrins linked intracellularly?
- Via actin-binding proteins to the actin cytoskeletons ->
- APART from a6, b4 complex found in epithelial hemidesmosomes which are linked to the cytokeratin cytoskeleton ->
- complexes cluster to form local adhesions (most) or hemidesmosomes (a4-b6).
- Some integrins bind to more than one ECM molecule
How are integrins activated into high affinity state?
- Low affinity can be switched on by inside out signal -> once they bind to matrix, you get other changes taking place;
- ligand binding also causes change in conformation, legs separate and cytoplasmic signalling molecules can then bind which allows signalling to take place (outside-in).
What is the effect of ECM on phenotype of cells?
- Eg: Mammary epithelium was grown in a gel matrix ->
- one set containing type 1 collagen and the other containing basal membrane proteins (laminins).
- The ones in type 1 collagen, formed balls of cells that were clumpy and loosely associated with each other;
- the ones in laminins organised themselves into organoids with polarised epithelium, and produced milk proteins ->
- ECM has a profound effect on phenotype of cells
What are the different types of cell-cell junctions in epithelia?
Junctions arranged as continuous belts (zonula) or as discrete spots (macula)
What is contact-induced spreading of epithelial cells?
- Stable junctions formed between cells ->
- allows epithelial cells to be able to promote the type of behaviour that will enable them to form a coherent epithelium
How does cell adhesion affect proliferation?
- When Ca is removed (many cell-cell junctions are Ca dependent) and cells are still at high density, although they touch each other the junctions aren’t forming ->
- this led to increased MAPK activation, decreased level of p27KIP1 (inhib of prolif in cell cycle) and HIGH prolif.
- When Ca reintroduced, junctions reformed and MAPK inactivated, increased p27KIP1 and LOW prolif.
- Also tried it with adhesion blocking Ab which had the same effect
How are adherens junctions organised?
- Master junctions of cells ->
- control formation of other junctions ->
- consists of cadherin which binds to similar molecules on adjacent cells ->
- IC binds to b catenin which is associated with a-catenin,
- which links to actin cytoskeleton
What is the function of beta-catenin in the cell?
- Adenomatous polyposis coli gene product is a protein involved in degradation of b-catenin ->
- rapidly degraded in cytoplasm by APC, but if any accumulates in cytoplasm,
- associates with LEF-1 to form complex that acts as a transcription factor,
- which enters the nucleus and influences gene expression and prolif.
- In APC, the mutation reduces efficiency of degradation of b-catenin leading to accumulation of beta-catenin in cytoplasm,
- so more association with LEF-1 so more prolif
What is the difference in layering of normal vs cancer cells?
What are examples of proto/oncogenes?
What is the incidence of Ras mutation in certain cancers?
30% of all cancers
How do cancers undergo uncontrolled proliferation?
Loss of density dependency and anchorage dependence -> doesn’t need these signals as one of the signals downstream is permanently turned on
What is the difference between benign and malignant cells?
Benign - too much prolif but contained in one place.
Malignant - poorly differentiated cell and invade surrounding tissues
How does a primary carcinoma cell metastasise?
- Cell-cell adhesion must be down regulated, cells must be motile;
- degradation of ECM must take place (MMP levels increased) in order to migrate through basal lamina and interstitial ECM ->
- carcinoma cell-cell adhesion is an indicator of how differentiated the primary tumour is and indicates invasiveness and prognosis
What is the metastatic process?
x
What are the steps of tumour progression?
- Use MMP’s to break down the basement membranes;
- when benign tumours reach this point they become malignant
How does metastasis occur?
- Break basement membrane,
- migrate through stroma,
- reach lymphatic/blood vessels,
- get out from the vessels,
- invade and become metastatic
What are the different types of tumour cell migration?
- Stratified classification ->
- ameboid = autonomous cell breaks off and metastasises elsewhere.
- Mesenchymal = single/multi cells migration one after the other.
- Cluster = lots of cells migrating together (with coordination).
- Multi-cellular strand/sheets = many cells move together.
- This process is actually a normal process during embryo stage where they move from one side to another, when differentiating
What do tumour cells mimic in metastasis?
- Morphogenic events -> Vascular sprouting: one cell pulling the others, which opens up the blood cell;
- very rapid,
- don’t respect neighbours
- very disorganised movement
What is the process of cell movement?
- Fillopodia move towards new anchor,
- then lamellipodia bring more bits of the cell,
- to form tight contact (new adhesion),
- and move upwards,
- deattach and move to the next anchor
How are g and f actin involved in cell movement?
- Actin cytoskeleton is framework of cells, which supports body and tells it how to change ->
- can be polymerised.
- When signal is detected in other direction, then actin filaments are broken down and built up on the other side instead of turning cell the whole way round ->
- actin is polarised which differentiates which proteins attach to each side
How are filaments organised and structured in the cell?
Lamellopodium has criss-crossed fibres
How are actin filaments remodelled?
If nucleation isn’t wanted then sequestration can occur
How does nucleation occur?
- Arp2-3 complex mimic the actin to build up the filament;
- monomers are difficult to join, so the complex picks up monomers and stabilises them, so they can then continue to increase
How does elongation of actin occur?
- Profilin comes in an adds onto the +ve end of the filament forming actin-profilin complex which lets elongation occur.
- Thymosin sequesters the monomer and keeps it there, preventing elongation of the monomer
How do capping and severing of actin occur?
- Capping -> prevents addition of monomers, with capping protein, so then chain depolymerises from the back (some bind to +ve and some to -ve end).
- Severing - clips all filaments into shorter bits, forming minifilaments, which also need to be capped as otherwise they will continue to grow
How do actin functions cooperate to generate filaments?
- Filament ->
- monomer binds to GTP and becomes active, which can be elongated then severed ->
- leads to annealing, capping or grows again
How do actin cross-link and bundle together?
Certain proteins bundle in different ways -> filamin makes particular angles
How do actin functions cooperate to organise filaments?
Some allow motor proteins to come in and stabilise the proteins
How does branching of actin occur?
Always at 70” angles -> Arp2/3c initiates filament growth which occurs at 70”
How does the gel-sol transition occur?
By actin filament severing -> rigid structure of cross-linked proteins and a severing protein comes and forms a looser structure
At which parts of the cell movement cycle does actin participate?
What is lamellae protrusion?
Highly energetic process
What are filopodia involved in in the cell movement cycle?
Filaments make a force that can force the membrane up -> bundling proteins increase it; with retraction operating at the bottom to prevent over growth
What are the different cell shapes and how is the actin cytoskeleton organised?
Villi are not filopodia but look similar; stereocilia are similar also.
What signalling mechanisms regulate the actin cytoskeleton?
What molecules control the actin cytoskeleton?
Can be upregulated in different tumours
What are the 3 different members of the Rho family of small GTPases?
- Cdc42 = filopodia formation;
- Rac = pancake due to protrusion of lamellipodia;
- Rho = activate extreme contraction and thick filaments (stress fibres)
How does signalling from small GTPases regulate actin cytoskeleton and motility?
What is the role of small GTPases on cell migration?
