Christine Flashcards
Cancer
The unregulated growth of abnormal (immature/blast-like) cells, often at inappropriate locations
Hallmarks of cancer
Sustained proliferative signalling Evasion of growth suppressors Avoiding immune destruction Replicative immortality Tumour-promoting inflammation Invasion and metastasis Induction of angiogenesis Genome instability (high frequency of mutations) Resisting cell death Deregulated cellular energetics (e.g. increased aerobic glycolysis)
How are tumours classified?
According to their tissue of origin
Carcinomas
Arise from epithelial cells (~90 % of cancers)
Adenocarcinomas
Arise from glandular tissue e.g. breast
Sarcomas
Arise from connective tissue/muscle
Leukaemias
Blood-derived sarcomas
i.e. a subset of sarcomas
Benign tumours
Cells resemble normal cells but with increased growth - i.e. the cells are abnormal but do not have enough mutations to be cancerous, and can still perform some normal functions
Tend to be localised
Often surrounded by a fibrous capsule
Usually require little treatment but can be surgically removed if required
Malignant tumours
Rapidly grow and divide
Tend to be less well-differentiated than normal cells
High nucleus to cytoplasm ratio with fewer specialised structures
Invade surrounding tissues, making them more difficult to treat (less definition between where tumour ends and normal tissue starts)
Can enter the circulation and grow at a distant site (metastasise)
Oncogene
A gene that has the potential to cause cancer
Mutations in Her2
- Point mutation of Val to Gun in the transmembrane region leads to dimerisation of the receptor in the absence of ligand, resulting in its constitutive activation
- Deletion mutation leading to loss of the extracellular ligand-binding domain causes constitutive activation of the receptor
- Over-expression of Her-2 (by up to 100 fold in many human breast cancers) causes cells to proliferate in the presence of very low concentrations of of EGF
Proposed mechanisms of action of Herceptin
Decreases activation of signalling pathways
Induces downregulation of the receptor
Increases PTEN activation
Induces cell cycle arrest
May increase apoptosis and reduce angiogenesis
May promote antibody-dependent cellular cytotoxicity (ADCC)
Molecular mechanisms of cancer
Cancer cells usually contain 3-7 mutations (Knudson hypothesis - multiple mutations are required for cancer development)
The malignant transformation of a single cell is sufficient to give rise to a tumour - cancer is a “clonal disease”
Any cell in a specific tissue is as likely to be transformed as any other cell of the same type
Benign tissues surrounding malignant tissue often contain all but one of the mutations
Gain of function mutations in oncogenes
Point mutation leading to constitutive activation
Gene amplification leading to an increase in the amount of protein produced
Chromosomal translocation
Retrovirus
Contains reverse transcriptase so can transcribe their RNA into DNA after entering a cell
This retroviral DNA can then be incorporated into the chromosomal DNA of the host cell and be expressed
Oncogenic retrovirus
A retrovirus capable of inducing malignancies in host cells
Structure of c-Src
N-terminal SH3 domain (binds to proline-rich sequences) SH2 domain (binds to phosphorylated Tyr residues) Kinase domain (phosphorylates substrates) C-terminal Tyr
Regulation of c-Src activity
Phosphorylation of the C-terminal Tyr creates an intramolecular binding site for the SH2 domain, resulting in auto-inhibition of the protein through masking of the kinase domain
The action of phosphatases leads to dephosphorylation of the C-terminal Tyr, leading to dissociation of the SH2 domain and activation of c-Src
Her2+ breast cancers
Her2+ cells are associated with a more aggressive tumour phenotype and reduced survival rate (more serious prognosis)
Cells grow faster so tumours are more likely to recur
Detection of Her2+ breast cancers
IHC
FISH (more sensitive)
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. CDKIs
- Inducers of apoptosis e.g. Bad, p53
- DNA repair enzymes (xeroderma pigmentosa)
- Developmental pathways e.g. patched (Hh pathway), Wnt pathway
Cellular responses to p53
Cell cycle arrest (to allow DNA repair before the cell cycle continues) DNA repair Senescence Apoptosis Differentiation
p53 also acts as transcriptional regulator of…
p21 (CDKI, causes cell cycle arrest)
MDM2 (p53 inhibitor, autoregulation)
Bax (pro-apoptotic protein)
CDB3, PRIMA-1
Stabilise mutant p53 and restore its transcriptional function
Nutlin
Inhibits interaction between p53 and MDM2
Pifithrin
Suppresses the endogenous action of p53 in normal tissue in order to reduce the severe side effects associated with chemo/radiotherapy
Ras/MAPK pathway
Essential for cell growth
PI3K/PKB pathway
Essential for cell survival (anti-apoptotic)
Evidence for cancer stem cells
Cancer is a disease of proliferating cells, but most mature cells don’t proliferate
Tumours are often heterogeneous in terms of cellular differentiation, but cancers are clonal
Cancer is caused by the accumulation of mutations in a single cell, but most cells have a finite lifetime and don’t live long enough to acquire >3 mutations
Pluripotent cell
Can differentiate into many different cell types
Stem cells are…
…pluripotent and unspecialised
Where have small numbers of residual stem cells been identified in adult tissue?
Blood (bone marrow) Intestine Skin Muscle Liver Brain
Purpose of stem cells in the bone marrow
Required for normal cell turnover
Purpose of stem cells in the liver and muscle
Involved in healing
Teratoma
Tumour made up of several different types of tissue
Why do stem cells have more opportunity for mutations to accumulate?
They are long-lived and self-renew (proliferate)
How can the heterogeneity of the tumour mass be accounted for?
The asymmetric division of stem cells
Wnt signalling pathway
Proto-oncogene pathway
Leads to the regulation of gene transcription
Controls tissue regeneration in adult bone marrow, skin and intestine
LRP-5/6
Lipoprotein receptor-related protein
Protein components of the beta-catenin destruction complex
Axin
APC
CK1
GSK3
How does the beta-catenin destruction complex degrade beta-catenin?
By targeting it for ubiquitination after phosphorylation by GSK3
Reasons for over-expression of beta-catenin
Mutations in beta-catenin
Deficiencies in the beta-catenin destruction complex e.g. LOF APC
Over-expression of Wnt ligands
Telomeres
The ends of linear chromosomes
TTAGGG repeats
Function of telomeres
“Disposable buffers”
They are truncated during cell division - their presence protects the genes before them on the chromosome from being truncated instead
Telomerase
Promotes telomere lengthening
Expression profile of telomerase
Active in stem cells, germ cells, hair follicles and 90 % of cancer cells
Low/absent in somatic cells
TTAGGG repeats in embryonic/stem cells
3-20 kb
Indefinite replication
TTAGGG repeats in cancer cells
Up to 55 kb Persisten growth (but also chromosome instability - breakage/deletions)
Why are cells with sufficient telomerase activity considered immortal?
They can divide past the Hayflick limit without entering senescence/apoptosis
Why do cancer cells employ telomerases?
So they can maintain their telomeric DNA in order to continue dividing indefinitely (immortalisation)
Hayflick limit
The number of times a normal human cell population will divide before cell division stops
Hedgehog signalling pathway
Transmits signals to embryonic cells required for proper differentiation
Metastasis
The ability of cancer cells to break away from their site of origin (the primary tumour) and travel to and recolonise at distant sites
Cancerous growths at sites far removed from where the primary tumours first arose
What is the fundamental difference between benign and malignant tumours?
Metastasis
What % of deaths from cancer are primary tumours responsible for?
10
Other 90 % from metastases
i.e. metastasis is the most life-threatening aspect of human cancer
Mechanisms employed by tumour cells for carrying out metastasis
Downregulation of E-cadherin expression
Induce surrounding stromal cells to produce MMPs that digest the ECM
Change integrin expression
Epithelial-mesenchymal transition (EMT)
Downregulation of E-cadherin expression
Mechanism of metastasis
E-cadherins are responsible for forming adherens junctions between epithelial cells
Loss of E-cadherin expression decreases the strength of adhesion, therefore increasing tumour cel motility and allowing tumour cells to physically detach from the primary tumour
Induce surrounding stromal cells to produce MMPs that digest the ECM
Mechanism of metastasis
Tumour cells release CSF-1 (colony-stimulating factor-1) to recruit macrophages to the stroma
Macrophages secrete MMPs that catalyse the degradation of components of the ECM (e.g. collagen)
This creates space for tumour cells to move
Changes in integrin expression
Mechanism of metastasis
Integrins are involved in anchoring cells to the ECM
Changes in their expression allow tumour cells to move through the ECM
Intravasation
Degradation of the basal lamina allows cancer cells to move through the basement membrane into the blood or a lymphatic vessel
Why does the lymph system favour metastatic tumour cells?
It is slower flowing than the blood, so there is less stress to harm them
Extravasation
The process of tumour cells leaving their vessel and invading the surrounding tissue
Typically occurs in small capillaries because the tumour cells are large and become trapped
EMT
Epithelial-mesenchymal transition
When epithelial cells assume the shape and transcriptional programme characteristic of mesenchymal cells
The process by which epithelial cells lose their polarity and cell-cell adhesion, gaining migratory and invasive properties to become mesenchymal stem cells
Metastatic tropism
The preference/selection for where a tumour metastases, depending on its organ of origin
Metastatic tropism in breast cancers
Tend to metastasise throughout the body e.g. bone, lungs, liver, brain
Metastatic tropism in prostate tumours
Recolonise in the bone
Metastatic tropism in colon carcinomas
Recolonise in the liver
Theories of metastatic tropism
First-pass organ
Seed and soil hypothesis
First-pass organ theory
Tumour cells recolonise in the first organ they encounter due to trapping in the capillary network
i.e. many breast tumours form metastases in the lungs
Seed and soil hypothesis
Tumour cells recolonise in tissues with similar growth factors to their tissue of origin
or
in tissues that have been ‘prepared’ to receive tumour cells
There is evidence that tumour cells secrete cytokines to ‘prepare’ tissues for recolonisation, creating a “pre-metastatic niche”
MMP inhibitors as a therapeutic approach to metastasis
GM6001
Broad spectrum, reversible MMP inhibitor
Broad spectrum contributed to disappointing clinical performance - dose-limiting muscular and skeletal pain
Anti-tumour and anti-angiogenic activity
Anionic hydroxamic acid motif forms a bidentate complex with the active site zinc
Used in combination with cetuximab (EGFR inhibitor) and celecoxib (COX2 inhibitor)
EMT inhibitors as a therapeutic approach to metastasis
AB-16B5
Humanised monoclonal antibody for clusterin
Clusterin promotes tumour cell migration, invasion and metastasis through stimulation of the EMT pathway
Currently in phase I clinical trials for advanced solid tumours
NM23
Metastatic suppressor (gene, not drug) Reactivation could be a therapeutic approach to metastasis
Angiogenesis
The development/formation of new blood vessels from pre-existing vasculature through extension/remodelling of existing capillaries
Vasculogenesis
De novo blood vessel formation i.e. no existing vasculature
opposite to angiogenesis
Physiological angiogenesis
Occurs naturally during embryogenesis, wound healing, menstrual cycle
Pathological angiogenesis
A hallmark of cancer
Why is angiogenesis essential for tumour progression and metastasis?
Tumour growth is angiogenesis-dependent
Without the formation of new blood vessels, tumours can only grow to a max diameter of 2 mm
Growing tumours stimulate angiogenesis to ensure their own blood supply
How is angiogenesis normally regulated?
Through the production of several pro- and anti-angiogenic factors
Pro = VEGF, bFGF, PDGF
Anti = endostatin, angiostatin, thrombospondin
Angiogenesis is normally suppressed through an excess of inhibitory anti-angiogenic factors - the difference between physiological and pathological angiogenesis lies in the tightly regulated balance of pro- and anti-angiogenic factors
The ‘angiogenic switch’
Occurs when the finely tuned balance between pro- an anti-angiogenic factors is tipped in favour of angiogenesis
This occurs in hypoxic cells
The angiogenic pathway
- Hypoxic/low pH conditions induce the activation of HIF-1 (hypoxia-inducible factor-1). HIF-1 is a tumour cell transcription factor that induces transcription of the VEGF gene. It is stabilised by hypoxia - in normal, well-oxygenated tissue, HIF-1 concentration is kept low by its continual degradation
- Tumour cells release VEGF
- VEGF diffuses into the nearby tissues and binds to VEGFRs on nearby blood vessel endothelial cells
- This activates the endothelial cells to produce enzymes e.g. MMPs that catalyse the degradation of the basal lamina and ECM proteins. This creates a physical space into which the endothelial cells can migrate
- The degradation of the basal lamina/ECM allows the endothelial cells to proliferate/migrate out of their original vessel walls and sprout towards to tumour cell (i.e. towards the source of the pro-angiogenic factor)
- Activation of VEGFR also increases intern expression on the endothelial cell surface, that aid migration by ‘pulling’ the sporting new blood vessel forwards
- The endothelial cells reorganise to form tubules with a central lumen, stabilised by smooth muscle cells and pericytes. PDGF and Ang1 are involved in this maturation of new blood vessels
- The new blood vessels interconnect to form a branched network (anastomosis)
Normal blood vessels
Straight
Branch into successively smaller capillaries to create a widespread network for oxygen and nutrient delivery to the tissue
Tumour vasculature
Tends to comprise a tangle of randomly interconnected vessels that branch erratically, vary in diameter and are generally oversize
Vessels are often weak and ‘leaky’ - there are large pores in the vessel walls that can lead to escape of fluid into the interstitium, leading to painful swelling in/around tumour tissues
The abnormal vessels often have irregular blood flow that can prevent treatment from reaching and attacking tumour cells
Dysfunctional vessels also produce hypoxic/low pH conditions that can prevent the function of immune cells
Current anti-angiogenic approaches
Antisense RNA therapy against bFGF and PDGF
Soluble receptors/monoclonal antibodies e.g. Avastin
Enzyme inhibitors e.g. sunitinib, sorafenib
Activation of tumour suppressors e.g. p53 - up-regulates anti-angiogenic factors and down-regulates pro-angiogenic factors
Avastin
Anti-VEGF monoclonal antibody
Binds to biologically active forms of VEGF and prevents its interaction with VEGFR
Only effective in some cancer types (e.g. metastatic colon, renal, ovarian) - other tumours use different pro-angiogenic factors so Avastin would be ineffective
Most effective when given in combination with cytotoxic drugs, because Avastin is cytostatic - it doesn’t kill the tumour cell, just decreases its growth
Sunitinib, sorafenib
Inhibit VEGFR (but not specific)
Why are the majority of current anti-angiogenic therapies ineffective against established tumours?
Established tumours already have a fully formed network of blood vessels
Current anti-angiogenic treatments are only really useful for preventing blood vessel growth in the first place
Disadvantage of anti-angiogenic therapies
Long-term administration could impair natural wound-healing/menstruation
With Avastin, inhibition of VEGF could lead to an increase/surge in the levels of other growth factors in order to compensate, leading to tumour resistance
Novel anti-angiogenic therapies
Aim to target newly formed blood vessels due to potential molecular differences between established and neovasculature
ANET
Anti-neovascular therapy
Aims to disrupt neovessels, rather than inhibit their formation
Based on the fact that angiogenic endothelial cells are growing cells that would be damaged by cytotoxic agents (just like tumour cells), if the agents are effectively delivered to the cells (vascular targeting) e.g. direct cytotoxic drugs e.g. doxorubicin to endothelial cells
The eradication of endothelial cells would cause a complete cut-off of essential supplies to the tumour cells, leading to indirect but strong cytotoxicity (inhibition of angiogenesis just causes cytostasis)
i.e. the tumour would be eradicated completely
Vascular gene therapy
Correct/alleviate disease through delivery of genes
Requires identification of the appropriate gene and specific delivery to the required area
May be possible with E-selectin, which has endothelial cell-specific expression
Link the promoter region of the E-selectin gene with the gene for dnVEGFR and target to rapidly proliferating cells using retroviruses
Destruction of tumour vasculature (vasculature targeting)
e.g. Combretastatin
Binds to the beta-subunit of tubulin, preventing tubulin polymerisation and microtubule formation
This leads to a ‘ballooning’ of the vasculature endothelial cells, resulting in necrosis of the tumour core
Nanoparticle techology
NGR peptide motif on the surface of the nanoparticle targets it to tumour endothelial cells (binds to CD13)
Nanoparticles contain bortezomib (= proteasome inhibitor)
In some cancers, the proteins that normally kill cancer cells are broken down too quickly by the proteasome
Normalising tumour vessels
Would allow cancer therapies to penetrate the tumour mass and function more effectively
c-Src
Non-receptor Tyr kinase
Activation leads to promotion of cell survival/proliferation
HPV
Human Papilloma Virus
DNA onocovirus
HPV proteins
Can interact with cellular proteins
E5 protein causes prolonged activation of PDGFR
E6 protein inactivates p53 through targeting for ubiquitination
E7 protein competes with pRb for E2F binding
Deficient EGFR signalling
Associated with Alzheimer’s
C-fos
Transcription factor
Forms a heterodimer with c-jun, resulting in the formation of AP-1 complex that binds to DNA leading to the production of cyclin D
Ras as an anti-cancer target
Ras has a fatty acid modification that tethers it to the cell membrane
Inhibitors of this modification were developed but it was found that cancer cells just tethered Ras to the membrane using a different modification
Inheriting one mutated copy of BRCA1
BRCA1 = TSG
60 % probability of developing breast cancer c.f. 2 % probability with 2 wild type alleles
G1 to S phase transition
- Growth factors induce cyclin D expression
- Cyclin D assembles with CDK4/6 to form catalytically active kinase complex
- pRb in unphosphorylated form is bound to E2F (TF), preventing E2F-mediated transcription of genes required for DNA synthesis. Phosphorylation of pRb by cyclin D-CDK4/6 complex causes its dissociation from E2F allowing transcription of enzymes involved in DNA replication, irreversibly committing cells to S phase
- Phosphorylation is initiated by cyclin D-CDK4/6 and is completed by other CDKs e.g. cyclic E-CDK2
Active E2F stimulates its own synthesis as well as that of cyclin E and CDK2 (positive feedback loop)
LOF pRb
Removes E2F inhibition therefore E2F is constitutively active and continuously drives the transcription of genes required for S phase transition
p53
Most critical molecule in cancer
Main detector of DNA damage
Maintains integrity of DNA
How is p53 an exception to the ‘recessive’ rule/”two-hit hypothesis”?
p53 acts as a tetramer
One mutated copy of p53 can lead to LOF of p53 because the incorporation of just one non-functional p53 into the tetramer results in LOF of p53
Where do mutations in p53 generally occur?
In the DNA binding region
Li Fraumeni syndrome
Inherited disorder
Predisposes sufferers to cancer as a result of inheritance of a mutated p53 gene
p53 is inactivated by…
…environmental carcinogens e.g. benzo(a)pyrene in cigarette smoke
aflatoxin
