Cancer Flashcards
(94 cards)
1
Q
Explain the basis of the bruce ames test
A
- Wild-type His+ salmonella bacteria can synthesise the amino acid histidine.
- This Ames test uses a mutant strain, His- salmonella, that can’t synthesise histidine,
therefore His- salmonella can’t grow in media that lacks histidine. Only a few cells will
spontaneously mutate or ‘revert’ back to wild type His+ and form colonies. - Chemical mutagens increase the rate of reversion. Therefore, the more mutagenic the
chemical the more revertant colonies will grow.
2
Q
What is the modified ames test
A
Problems with the Ames Test:
* Ames Test may not detect all mutagens in e.g. animals.
* May not detect pro-carcinogens
Modified Ames Test:
Pre-incubate test mutagen + post-mitochondrial supernatant of rat liver (SN 9000g) which is rich
in monooxygenases (e.g. Cyt P450
3
Q
How are pro-carnicogens turnt into carcinogens
A
- Rich in enzymes such as the cytochrome p450 monoxygenases.
- Many chemical carcinogens exist as pro-carcinogens and are biotransformed
(metabolised) into active carcinogens in the body by enzymes such as the cytochrome
P450 monooxygenases. This is known as bioactivation.
4
Q
How was the rous sarcoma virus discovered
A
Peyton Rous used the sarcoma from this hen
to show that cancer was transmissible
- Indicated that the tumours were transmitted by an infectious particle
- The infectious practical was small enough to pass through the fine-pore filter -> a virus
- The virus was named the Rous Sarcoma Virus (RSV)
5
Q
What are retroviruses
A
Some RNA viruses are equipped with reserve transcriptase to convert their RNA genomes into
viral DNA (referred to as the provirus). These viruses are known as Retroviruses. e.g. HIV
RNA Tumor Viruses:
* Many RNA viruses → Cancer
* Many of these are retroviruses.
* Oncogenes were first recognized as unique genes from RNA Tumor Viruses → “Viral Oncogenes”.
6
Q
What is the structure of a Avian leukosis retrovirus
A
Structure of a retrovirus virion e.g. Avian leukosis virus (ALV)
* gag encodes for core proteins (the capsid)
* env encodes for coat proteins (the glycoprotein spikes)
* pol encodes for enzymes (reverse transcriptase, integrase,
protease)
* LTR (Long Terminal Repeats) contain promoters & enhancers
that regulate expression of the viral genes
7
Q
What are structural similarities and differences between ALV and RSV
A
- Both Rous sarcoma virus (RSV) and Avian leukosis
virus (ALV) have gag, pol and env genes - RSV genome has an extra gene, src, that encodes
for viral Src kinase, a tyrosine kinase that is similar
to the Src kinase found in mammalian cells.
→ responsible for transformation of cells by RSV
→ src is an oncogene (cancer causing gene
8
Q
What is the difference between c-Src and v-Src
A
- c-Src is a soluble protein - tyrosine kinase → Phosphorylates proteins on tyrosines → Alters their activity
- v-Src is also a soluble protein- tyrosine kinase → Phosphorylates key protein targets in host cell → Transformed Cell.
9
Q
What are the origins of viral oncogenes, especially src
A
- Derived from normal Cellular Genes
- During its evolution, when virus infected target mammalian cell:
→ certain cellular genes (or part of) inserted into viral genomes
→ on passage through the cell, virus retained certain genes
→ gave virus a selective advantage
The viral src gene originates from the cellular host gene
10
Q
Describe the 5 different oncoprotein products
A
A) Some are like soluble tyrosine kinase e.g. v-Src or v-Abl
v-Src - Phosphorylate
key cell targets - Nucleus
Cell growth &
proliferation
B) Some mimic receptor tyrosine kinases e.g. v-Erb-B
v-Erb-B - Phosphorylate
Key cell targets - Cell growth &
proliferation
v-Erb-B: - Truncated form of EGF receptor.
- Constitutively active (doesn’t need EGF)
C) Some act like cellular growth factors that bind and activate receptor tyrosine kinases
e.g. v-Sis (truncated form of platelet derived growth factor)
v-sis - PDGF-R - receptor tyrosine kinase activation - cell growth and proliferation
D) Some act like transcription factors e.g. Fos & Jun, Myc. They locate to nucleus of infected cell and
regulate expression of target genes (whose products are often involved in regulation of cell growth &
proliferation (e.g. Cyclin D1). Often constitutively active or overactive.
v-Fos/v-jun: Complex to form AP1 transcription factor dimer
AP1 → expression of genes involved in cell cycle progression
e.g. Cyclin D1
v-Myc - Regulates genes associated with mitosis
e.g. Cyclin E
E) Some are involved in cell signalling
e.g v-ras oncogene - Codes for a constitutively active version of p21ras (GTP/GDP binding protein)
11
Q
Compare RSV and ALV genomes
A
Rous sarcoma virus (RSV):
* src oncogene
* Transforms cells to a carcinogenic phenotype
Avian leukosis virus (ALV):
* No oncogene
* But is can still lead to cancer. Why?
12
Q
How does ALV induce oncogenic activities in a cell
A
Through insertional mutagenesis
- Viruses such as Avian Leukosis Virus (ALV) lack acquired
oncogenes - Induce their oncogenic actions by integration of their
proviral DNA adjacent to a cellular proto-oncogene
e.g. c-myc proto-oncogene - Transcription of c-myc driven by the strong
constitutively active ALV promoter - Drives cell proliferation
13
Q
What are cellular proto-oncogenes
A
- Code for normal cell proteins e.g. Ras, EGF, EGF Receptor, Fos, Jun.
Proto-Oncogene - Oncogene
13
Q
What are the 5 Basic Mechanisms of Transformation to Oncogenes:
A
1) Promoter Insertion
2) Enhancer Insertion
3) Chromosomal Translocations
4) Gene Amplifications
Viral Mediated
(1 → 4) → Increased Gene Expression
→ Activation To Oncogene
5) Single Point Mutations
(somatic mutation)
→ Altered Codon & Amino Acid
→ Protein with altered structure & activity in the cell
14
Q
What happens during promoter insertion
A
- Certain retroviruses lack oncogenes but can still cause cancer e.g. avian leukemia virus (ALV)
- Viral LTR has promoters & enhancers for efficient gene expression of viral genes.
- During replication, viral DNA is integrated into host genome:
- Integrated viral DNA may rearrange → host cell gene now under control of a Strong Viral Promoter
15
Q
What is enhancer insertion
A
Similarly, viral DNA enhancers can insert upstream or downstream from the gene promoter (e.g. c-myc)
Enhancers in LTR:
* ↑ expression of c-myc gene
* ↑ mitogenesis (cell proliferation).
* Both promoter and enhancer insertion commonly occur in viral-mediated carcinogenesis.
* Insertion of viral DNA → regulate expression of proto-oncogene → → → Oncogene
* Many host genes other than c-myc may be activated in this way.
* Insertion of viral DNA usually occurs at similar position in host genome such that the same genes are affected by
same virus → particular type of cancer.
16
Q
Describe the chromosomal translocation step in oncogene transformation
A
- Many tumor cells have chromosomal abnormalities
- E.g. chromosomal translocations
- Translocation of piece of chromosome from one to another e.g. “Philadelphia
Chromosome”. - This chromosomal translocation occurs in Chronic Myelogenous Leukemia (CML)
- Involves Chromosomes 9 & 22
- End of Chromosome 9 switches places with the end of Chromosome 22.
- Chromosome 9 → long
- Chromosome 22 → short “Philadelphia Chromosome”
- Fused bcr-abl gene
17
Q
How does chronic myeloid leukaemia occur
A
- The bcr-abl fusion gene encodes a hybrid Bcr-Abl
oncoprotein: - Deregulated Abl (tyrosine kinase) activity sends out
strong growth promoting signals - Bcr-Abl oncoproteins are commonly found in
Chronic Myeloid Leukaemia (CML)
Gleevec® (ST1571/imatinib mesylate) - Novartis
- Specifically inhibits BCR-ABL kinase
- Used to treat CML
18
Q
Describe the gene amplifications step in oncogene transformation
A
- Occurs in many cancers.
- Multiple copies of an oncogene are formed
- e.g. Tumors treated with Methotrexate (MTX)
- MTX is an inhibitor of dihydrofolate reductase (DHFR).
- Blocks DNA synthesis.
- Tumor cells become resistant to MTX
- dhfr gene amplified → overcome inhibition by MTX → Up to 400-fold increase in DHFR activity.
- When dhfr gene is amplified → neighbouring DNA (up to 1000 kb) is also amplified → Increased copy
of other (unrelated) neighbouring genes. - Certain cellular oncogenes amplified in this way e.g. c-ras.
19
Q
Describe what happens in the single point mutation step in oncogene transformation
A
e.g. Ras (p21RAS)
Single point mutation of ras (proto-oncogene) → Oncogene.
Single point mutation in codon 12 of ras → rasGly12 → Val12 mutation
→ Ras oncoprotein
→ Continuously ‘on’ (in GTP bound state)
RasGly12 → Val12 mutation leads to Self-Sufficient Growth
- One mutation in ras →→→ Activation.
- All onco-mutations in Ras → Show increased [Ras-GTP]
- Due to: A) Increased affinity for GTP or
B) Decreased rates of GTP hydrolysis.
Oncogenic activation of Ras (e.g. mutation, amplification or upstream dysregulation) is a hallmark of
many human cancers
20
Q
What is the overall MOA of oncogenes
A
Overall mechanism of action of oncogenes:
Act on key intracellular pathways involved in growth control → Increased Mitosis.
21
Q
Name the 4 classes of oncogene and give an example for each
A
CLASS 1: Growth factor related peptides
eg. sis which is a secreted growth factor protein
Class 2: growth factor receptors
eg. erbB: a membrane bound receptor for EGF
Class 3: Intracellular transducers
eg. src: a protein tyrosine kinase located in the cytoplasm
Class 4: Nuclear transcription factors:
eg. myc: transcription factor in the nucleus
22
Q
What is a gain of function mutation
A
A gain-of-function mutation is a genetic lesion that causes the normal product of a gene to be expressed inappropriately (at abnormally high levels) eg. oncogenes
23
Q
outline the two hit theory of cancer causation
A
- Tumour suppressor mutation generates recessive genes.
- Recessive alleles can be inherited.
- Somatic mutation of remaining allele is needed.
24
What are gatekeeper genes and caretaker genes
Gatekeeper genes code for:
* Cell cycle regulatory proteins
* Cell cycle checkpoint proteins
* Proteins that promote apoptosis
→ directly supress proliferation
→loss of gatekeeper genes directly
promotes cancer.
Caretaker genes code for:
* DNA repair proteins
→ maintain genetic stability
→ loss of caretaker proteins indirectly
promotes cancer
25
Describe the location and function of retinoblastoma as a tumour supressor
Location: nucleus
Function: Gatekeeper: Involved in the regulation of
transcription of genes required for a cell to remain
in G0 or to progress to G1 stages of the cell cycle
Cell cycle: G0 → G1 → S
26
How does retinoblastoma progress the cell cycle?
Through phosphorylation of retinoblastoma it is unbound to E2F allowing progression from GI to early S phase
27
Describe the location and function of the p53 tumour supressor
Location: nucleus
Function:
Guardian of the genome
Gatekeeper: Transcription factor involved in the
regulation of genes at the G1 checkpoint for the
control of DNA damage.
If DNA damage is high → increased expression of
p53 → cell cycle arrest to allow for repair of
damaged DNA
G1 checkpoint
28
What do defects in p53 lead to and how can it be inactivated
Defects in p53
→ cellular and genetic instability
"Inactivation" of p53
* e.g. by mutation
→ Cell cycle progresses without DNA repair
→ Accumulation of damaged DNA – Mutations
→ Uncontrolled cell growth.
* Up to 100 mutations found on p53 → each of these can lead to "Inactivation" of p53
* E.g. truncated, nonsense, amino acid mutation, expression
29
Name and explain 5 ways p53 can be inactivated
Deletion of p19: failure to inhibit Mdm2, leads to degradation of p53
Amplification of Mdm2 gene: Mdm2 stimulates degradation of p53
Viral infections: viral proteins inhibit p53 function or trigger degradation
Deletion of C-terminal domain: cannot form and activate tetrameric form
Mutation of DNA binding domain: Cannot bind to DNA and transactivate targets
30
Name two viruses that inactivate tumour supressors
* Human papillomavirus (HPV)
* Epstein Barr virus (EBV)
31
Describe the HPV genome
* Early and late genome expression
Open reading frame: is a portion of a DNA sequence that does not include a stop codon
* Early genes reproduce viral genome (at least 8 Open reading frames (ORFs) )
* Late genes (2 ORFs) encode proteins required to
assemble new viral particles
* LCR – control region (coordinates the expression of
the open reading frames)
32
Give the function of open reading frame (ORF) E5-E8
E5: Transforming Factor – Interferes with GF signalling
E6: Transforming Factor, binds p53 associated with UBE3A
E7: Transforming Factor, binds Rb, LXCXE
E8: Anchorage independent growth promoter
33
Describe the molecular events in HPV-associated neoplasia
HPV-E6 & HPV-E7 work together to promote deregulated cell division
HPV-E6:
* P53 is not normally found to be mutated in HPV-transformed cells.
* HPV-E6 promotes degradation of p53
* HPV-E6 Binds E6AP
binds p53 to form p53-HPV E6- E6AP
E6-E6AP acts as a ubiquitin ligase
p53 degradation
* E6 Removal → cells arrest in G1
* Mutant p53 and E6 removal → cells don’t arrest in G1
E6 causes proteosomal degredation of p53
HPV-E7:
* Binds to Rb, cyclin A, cdk2
* Results in the release of E2F complex from Rb
* E7 acts at the tumour promotion stage
* E6 acts strongly during malignant conversion
* In some HPV+ cervical cancers HPV integrates into host cell genome
Since E2F is liberated, it can progress to S phase of the cell cycle driving tumour progression
34
What is the structure of the epstein barr virus (EBV)
* Member of the Herpesvirus family (HHV-4)
* Enveloped double-stranded DNA virus with a protective nucleocapsid
* Early genes associated with transformation of cells.
* EBNA-3C gene codes for Epstein-Barr virus nuclear
antigen 3C → acts similar to HPV-E7 → disrupts cell cycle
* EBV also has a number of other genes whose products
promote transformation of the infected cell. e.g. LMP1
mimics growth signals → promote immortalization of
infected B cells
35
Describe EPV in terms of transmission, symptoms and infection
* Transmission:
* Primarily saliva (“kissing disease”)
* Infects epithelial cells first, then B cells
* Symptoms of infection:
* Young children: often asymptomatic
* Teens and adults: can cause glandular fever/mononucleosis (mono)
* Infection:
* Initial lytic infection of epithelial cells (and some B cells)
* Lifelong latent infection in B lymphocytes
* Reactivation of lytic infection in B cells is quickly dealt with by immune system
* Latency usually harmless, but can cause mono or contribute to cancer:
* Burkitt’s lymphoma
* Nasopharyngeal carcinoma
36
How do permissive or non-permisive cells differ in viral replication
* Permissive/Non-Permissive cells
* Affects how viral genes are expressed
* Affect the outcome for the host
* Permissive (lytic replication), usually infected
host cells die.
* Non-permissive (viral latency), episomal latency
or integration of viral DNA into host genome.
* Evades the immune system
* Entire viral genome is not necessarily replicated
→ latency programs
37
Describe lytic infection in EBV
* Involves linear DNA
* Mainly in epithelial cells (also B cells→ immune response)
* Leads to viral replication and cell lysis
* Produces and releases more virus
* Linear DNA less likely to integrate into host genome
Initial symptoms
➔ Asymptomatic
➔ Mild sore throat
38
Describe latent infection in terms of EBV
* DNA becomes circular episome
* Establishes episomal latency in B cells
* Minimal gene expression → evades immune system
* Persist lifelong
* Has more potential to integrate and potentially transform the host
genome, but this is rare → different cancers
Latent Symptoms
➔ Asymptomatic
➔ Glandular Fever(“Mono”)
➔ Burkitt’s Lymphoma
➔ Nasopharangeal cancer
39
describe the EBV genomic organisation
* Large dsDNA genome
* About 100 genes, many poorly classified
* Early genes associated with transformation of cells.
* EBNA-3C gene codes for Epstein-Barr virus nuclear
antigen 3C → acts similar to HPV-E7 → disrupts cell cycle
* EBV also has a number of other genes whose products
promote transformation of the infected cell. e.g. LMP1
mimics growth signals → promote immortalization of
infected B cells
40
What is EBV latency and give 1 example for each of the different programmes
* Different stages of latency are associated with different patterns of gene expression
→ known as Latency Programmes 1, 2 & 3
→ lead to different outcomes
* Latency Programme 1 → Burkitt’s Lymphoma
* Latency Programme 2 → Nasopharyngeal Cancer
→ Hodgkin Disease
* Latency Programme 3 → Infectious Mononucleosis (4-6 weeks after infection)
→ More genes expressed than with Latency 1 or 2
→ Detected easier by immune system
→ Reoccurrence not likely
41
What is the difference between vasculogenesis and angiogenesis
* Vasculogenesis: Formation of new vasculature
* embryonic development
* Angiogenesis: Formation of new blood vessels
from existing vasculature
* female reproductive system
* wound healing
42
Name 5 types of angiogenic regulators
* Growth factors
* Cytokines
* Chemokines
* Matrix remodelling enzymes
* Chemotactic compounds
Different regulators are secreted at different stages of
tumour development
43
Give 2 examples each of stimulatory and inhibitory angiogenic factors
Stimulatory
* Vascular endothelial growth factor (VEGF)
* Fibroblast Growth Factor (FGF)
* Platelet Derived Growth Factor (PDGF)
* Transforming Growth Factor (TGF)
* Angiogenin
Inhibitory
* Thrombospondin
* Angiostatin
* Interferons
* Tissue inhibitors of metalloproteinases (TIMPs)
44
What do pro-angiogenic factors do
Tumour cells produce pro-angiogenic factors
→ activates endothelial cells in nearby blood vessels
→ triggers the angiogenic cascade
45
What happens to trigger the angiogenic switch
Hypoxia (low oxygen levels):
e.g. tumour without a blood supply
→ hydroxylation and degradation of HIF-1α are
blocked
Hypoxia-inducible factor-1α (HIF-1α) is
hydroxylated, ubiquitated & degraded at normal oxygen levels
→ HIF-1α is a transcription factor for pro-
angiogenic factors including VEGF
Prolyl hydroxylase (PHD):
* Enzyme
* Catalyses the hydroxylation of HIF-α in the
presence of oxygen (O2) and iron (Fe2+)
VEGF
46
How is the extracellular matrix remodelled during angiogenesis
* For angiogenesis to occur the basement membrane outside existing
vessel wall must be degraded
* Two principle proteolytic systems involved:
* Plasminogen activator system (PA)
* degradation of fibrin, laminin, collagen
* Matrix metalloproteinase system (MMP)
* Extracellular endopeptidases
* Degrade all known components of ECM
* Regulated by tissue-inhibitors of metalloproteinases (TIMPs)
47
How can angiogenesis be treated
Tumour vasculature is immature and “leaky”
→ Can be exploited for treatment
→ interfere with tumour angiogenesis
→ reduce the growth of the tumour
48
Describe the main steps that occur in tumour angiogenesis
1. Hypoxia: Hypoxia induces HIF-1 expression and the consequent release of pro-angiogenic factors, of which VEGF is the most important
2. Proteolytic degradation: Hypoxia also upregulates protease expression leading to basement membrane degradation and pericyte detachment.
3. Tip cell migration: Specialized endothelial cells 'tip cells' - migrate along angiogenic factor gradient
4. Tube formation: Endothelial cells are differentiated into highly proliferative stalk cells which make up the main body of the new vessel.
5. Regulation of vessel size: VEGF stimulates DLL4 secretion which binds to Notch-1 receptors. this down regulates VEGFR supressing proliferation
6. Tumor vascularization: PDGF beta stimulates pericyte attachment and reduces proliferation and VEGF sensitivity. Blood supply stimulates further tumor growth
49
What is tumour metastasis
* Metastasis refers to the development of tumours at
secondary sites far from the original primary tumour.
* Multi-step process, each step is rate-limiting
* Tumour cells
→ Spread into blood vessels or lymphatics
→ Spread into body cavities/CSF
50
How is metastasis inefficient
Luckily metastasis is an extremely inefficient process
* Estimate that <0.1% cancer cells have the capacity to form a higher order tumour
Vast majority of cancer-related deaths are from higher order
tumours (>90%)
51
What are the 5 stages of tumor metastasis (carcinoma)
1. Invasion
2. Intravasation (enters blood vessel)
3. Circulation
4. Extravasation
5. Colonisation of a new site
52
Describe the invasion step of tumour metastasis
* Detachment of metastatic cell from neighbouring
cells in the primary tumour
* Traverse the basement membrane
* Migrate through the extracellular matrix
(ECM) or stroma
* Migrate individually or in a cluster
During invasion changes associated with adhesion are:
* Abnormal expression of cell adhesion molecules (CAMs)
* e.g E-cadherin, I-CAM
* Abnormal glycation of surface glycoproteins
* Matrix metalloproteinase (MMP) activity
* disassemble the basement membrane/ECM e.g collagen
* ↑ MMP-2 and MMP-9 activity in tumours → poor prognosis
* Epithelial-mesenchymal transdifferentiation (EMT) &
transitional stages
This results in diminished adhesion and altered transport properties
53
What is epithelial-mesenchymal transdifferentiation
* Epithelial cells undergo a change in morphology and function to a more mesenchymal phenotype e.g fibroblast-like
* Gain the ability to invade, resist stress and disseminate
* Mesenchymal cells have an increased ability to move
* more actin filaments in mesenchymal that epithelial
* Triggered by hypoxia and metabolic stress
* EMT is not just a single step → transitional stages
* Cell that go through intermediate phenotypes are more successful at forming secondary tumours.
* Each stage has different invasive, metastatic and differentiation characteristics.
* Also different gene expression signatures
54
What happens during the intravasation step in tumor metastasis
* Cancer cells enter the circulation (blood or lymph)
* Active or passive
* Endothelium regulated by factors in
microenvironment
* Nuclear squeezing as cancer cells pass through
endothelium
-> genomic arrangement
-> can increase the metastatic potential.
Events associated with intravasation:
* CAMs (altered integrin expression)
* Macrophages
55
Describe the circulation step in tumour metastasis
* Cancer cells are transported in blood/ lymphatic system
* Environment is harsh
* In the circulations, cancer cells are exposed to immune
cells (T cells, NK cells, macrophages) and cancer cells
must be able to survive them.
* Evasion of the host immune system
* Clusters and neutrophils
* Platelet-shields
* Clusters more likely to survive and metastasise
* Contain stromal cells and immune components from the
original microenvironment enhance survival.
* Neutrophils participate in clusters and supress leukocyte
activation
* Interaction with platelets leads to formation of a coating
shield of platelets around cancer cells prevents detection
by immune cells and protect against physical stress in the
circulation
56
Describe the extravasation step in tumour metastasis
Entrapment in small capillaries
Microvascular rupture vs forced extravasation
* Cluster grows within vessel until the vessel ruptures.
or
* Transendothelial migration due to force in capillary
(→ nuclear squeezing)
* Organ-specific vascular permeability (eg liver
& bone)
* Changes in CAM expression
57
Describe the colonisation step in tumour metastasis
* Migration through ECM
* Mesenchymal to epithelial transition (MET)
* Formation of the metastatic niche
* physical anchorage
* survival signals
* immune surveillance protection
* metabolic requirements.
* Proliferation (anti-apoptosis)
* Angiogenesis
58
What is metastatic dormancy
* Delayed acclimatisation of cancer cells to their
secondary niches
* Responsible for late relapse
* Cross talk between environment and factors
controlling transcription
59
What is metastatic organotropism
A process in which cancers tend to metastasize to specific target organs
60
Besides blood flow, what elses influence metastatic organotropism
* There must be signals that guide the tumour cells to particular sites
* E.g. Bone marrow stromal cells express and secrete the chemokine CXCL12 which attracts prostate
cancer cells which express the chemokine receptor CXCR4.
* The site to which they travel must be particularly hospitable to them
* Secondary site is primed for welcoming the cancer cells → pre-metastatic niche
61
What is pagets 'seed and soil' theory
The ability of a disseminated cancer cell to successfully found
a metastasis depends on whether a distant tissue offers it a hospitable environment to survive and proliferate’
62
What cell changes happen at a metastatic niche
Cell changes at niche:
Fibroblasts → cancer-associated
fibroblasts (CAFs) (ECM
remodeling, tumour cell
recruitment)
Macrophages → M2-like
phenotype (immune suppression,
tumour promotion)
Neutrophils→ N2 phenotype
(immunosuppressive, ECM
degradation, tumour cell trapping)
Endothelial cells → leaky
(promotes extravasation)
63
how is a pre-metastatic niche formed
Pre-metastatic niche is primed by the primary tumour even before metastasis occurs.
Key factors that contribute to the pre-metastatic niche formation:
* Primary tumour-derived Factors
* Tumour-derived secretory factors (TDSFs) e.g. cytokines, growth factors
* Tumour-derived extracellular vesicles (EVs) e.g. exosomes containing mRNA, miRNA and proteins
* Bone marrow derived cells (BMDCs) mobilized by the primary tumour
Influence of these factors:
* TDSFs, EVs and BMDCs activate stromal cells at the niche, prompting remodelling the ECM
* TDSFs, EVs also induce phenotypic changes in niche cells (e.g. fibroblasts, macrophages, endothelial cells, myeloid cells)
→ Changes make the local stromal microenvironment at the prospective metastatic site more hospitable to colonisation
by tumour cells
64
What are the 3 factors that influence metastatic progression
* Tumour microenvironment
* Metastatic-enhancer and metastatic-
suppressor genes
* Epigenetics
65
Describe the tumour micro-enviroment
* Certain microenvironments can supress malignancy while other promote
* Changes in ECM components may promote or inhibit tumour cell motility, invasion or metastasis.
* Components such as proteoglycans, collagens, laminins, fibronectin, elastin, other glycoproteins and proteinases
* Immune environment around primary tumour plays a role in the metastatic potential of the cells.
* Tumour Microbiome
* Metabolically active
* Can alter drug structures and may affect their efficacy
* Both tumour cells and cells in the microenvironment may secrete angiogenic factors and growth factors
* Tumour cells can exploit supporting cells to increase metastatic potential.
* Tumour cells can recruit macrophages which increase tumour cell migration, invasion and intravasation
66
Describe how Metastatic-enhancer and metastatic-suppressor genes affect metastasis progression
Metastasis suppressor genes are commonly mutated
in tumours:
* P53, Rb, CDKN2A (CDK inhibitor 2A),PTEN, PIK3CA
67
Describe how epigenetics affect metastatic progression
* Changes in histone or DNA modifications that regulate
gene activity.
* Heritable yet reversible
* During tumour progression there is often abnormal
activity of enzymes involved in:
* DNA methylation,
* Histone acetylation
* Histone methylation
* Age-related
* Circadian disruptions
68
What is a typical model of cancer progression
Model of cancer progression
* Various stages of dysplasia/malignancy
* Hereditary factors
* Environmental carcinogens
* Oncogene activation
* Tumour suppressor gene inactivation
69
What is APC's significance in colon cancer
The Adenomatous Polyposis Coli (APC) gene:
* Found on chromosome 5
* Codes for the tumour suppressor protein, APC
* Associated with colon cancer
Loss of APC Function:
* Patients develop large numbers of polyps (adenomas)
* Cells in polyps cycle fast
→ Increased risk of accumulating genetic changes
→ Increased risk of colon cancer
Sporadic Colon Cancer
* APC mutations are found in about 60–80% of sporadic colon
cancers → polyps
Familial adenomatous polyposis (FAP)
* 1 in 7000 people
* Inherited mutation of APC gene
* 2nd hit → Loss of APC function → polyps
70
What does a mutated APC protein lead to
* Mutations tend to produce a truncated APC protein that cannot interact properly with -catenin.
* Crucial initiating mutation in >85% colon cancer
Mutated APC:
* ↑↑↑ -catenin → transcription → ↑ proliferation
* Altered interactions between -catenin & E-cadherin →
↓ cell adhesion; ↑ migration; ↓ polarity; altered actin fibres
70
What is the role of the APC protein
APC is expressed in most cells where it functions as a tumour suppressor protein (gatekeeper) helping to control:
* frequency of cell division
* chromosome number in diving cells
* cell attachment within a tissue
APC achieves this through it’s interactions with -catenin
* APC (in a complex with other proteins) acts as a negative regulator of -catenin concentration by tagging it for
proteasomal degradation
* APC supports interactions between -catenin and E-cadherin
-catenin (bifunctional roles)
* Interacts with E-cadherin controlling cell adhesion; migration; polarity; cytoskeleton.
* Transcription of genes that promote cell proliferation e.g. c-MYC
71
What are the steps in colon cancer progression
Accumulation of genetic changes leads to colon cancer
Normal epithelium -> early adenoma -> intermediate adenoma -> late adenoma -> carcinoma
order of genetic alterations is crucial
72
Name 3 main treatments for cancer
* Surgery
* Radiotherapy
* Chemotherapy
73
What are the criteria for anti cancer drugs
* Must kill cancer cells but be non-toxic to normal cells
* Main aim is to block cell growth & division (inhibit DNA synthesis)
* May preferentially affect cells which grow rapidly e.g. gut and bone marrow → side effects
74
For alkylating and related agents give an example and its mechanism of action (MOA)
Carboplatin, cisplatin: intrastrand cross linking of DNA
75
Describe 5 examples of anticancer therapeutics
Methotrexate:
Inhibits DHFR
Microtubule targeting agents:
Blocks mitosis
→ G2/M arrest → apoptosis
Topoisomerase inhibitors:
Inhibit enzymes involved in DNA repair
Synthetic estrogen:
Suppress testosterone
production in prostate cancer
ER antagonists:
Blocks ERs
Aromatase inhibitors:
Blocks estrogen production
Imatinib:
Inhibits Bcr-Abl kinase (CML)
76
What does an ER positive tumour indicate
* ER+: good prognosis, less aggressive tumour
77
How can ER breast cancer be treated
* Can be treated with anti-estrogen therapy
* Selective Estrogen Receptor Modulator (SERM) e.g. Tamoxifen
* Aromatase Inhibitors e.g. Letrozole
* Selective Estrogen Receptor Degraders (SERDs) e.g. Fulvestrant
78
What are genes controlled by the ER transcription complex
Genes controlled by ER transcription complex:
* Cyclin D1 → promotes cell cycle progression (G1→S phase)
* c-Myc → cell proliferation
* E2F transcription factor → cell cycle progression
* Bcl-2 → inhibits apoptosis
79
Describe how a Selective Estrogen Receptor Modulator (SERM) e.g. Tamoxifen treats cancer
* Estrogen Receptor partial antagonist
→ Binds and inhibits Estrogen Receptor-mediated breast cancer growth in people of
all ages.
* Large-scale randomized trials have shown that 5 years of tamoxifen given immediately after surgery for early-stage ER-
positive breast cancer reduces mortality by 28%
80
Describe how an aromatase inhibitor treats cancer
.g. Letrozole
* Blocks generation of estrogen from androgens by aromatase in Post-menopausal Women
81
Describe how Selective Estrogen Receptor Degraders (SERDs) e.g. Fulvestrant treat cancer
* Binds to estrogen receptors (ER) → change ER conformation making it dysfunctional → Promote ER degradation via the
proteasome pathway
* Used for tamoxifen-resistant breast cancer (effective against constitutively active mutant versions of ER)
82
Describe how HER2+ breast cancer can be treated
* About 30% breast cancers → aberrant HER2 expression
* Also known as erbB-2 or neu
* Amplification → constitutively ON growth factor signalling →
Increased proliferation
Trastuzumab (Herceptin®) (FDA 1998; EMA 2000)
* Humanised Monoclonal Antibody (mAb)
* Binds and blocks HER2 receptor signalling
* HER2+ metastatic breast cancer cells
Antibody-drug conjugate: Trastuzumab Emtansine (FDA 2013)
* T-DM1 (Trastuzumab emtansine) aka Kadcyla
* Trastuzumab with emtansine (DM1) attached to it
* Emtansine → microtubule depoymeriser / inhibits microtubule
formation
83
How can Triple Negative Breast Cancer (TNBC) be treated
* ER-, PR-, HER2-
* ~ 15-20% of breast cancers
* Tend to be aggressive, highly metastatic tumours
* Poor prognosis→ Unresponsive to many therapies
TRODELVY (Sacituzumab govitecan) (FDA 2021)
* Antibody Drug Conjugate (ADC)
* Binds TROP-2 receptors (often overexpressed in
cancer cells) → Cell internalises the ADC → The
drug (SN-38) is released → cytotoxic effects
* Survival in patients taking Trodelvy was 12.1
months vs 6.7 months with standard chemo
* Problem: ~ $ 11,000 per dose!!
84
How can angiogenesis be inhibited
Bevacizumab (Avastatin)
* Humanised anti-VEGF mAb
* Neutralises VEGF-A and blocks it from interacting
with its receptor (VEGF is a pro-angiogenic factor)
* Increases survival but usually relapses
85
What is melanoma and how can it be treated
* Melanoma: skin cancer, affecting melanocytes.
* Metastatic melanoma → low survival
* Approx. 50% of melanomas have a mutation in the B-
Raf gene (V600E)
Vemurafenib (PLX4032) FDA:2011; EMA:2012
* Selectively inhibits the mutated V600E BRAF kinase →
reduced signalling through the aberent MAPK pathway
86
What are immune checkpoint inhibitors, give an example
* Immune checkpoints: regulatory pathways that normally prevent autoimmunity
* Exploited by tumour cells to suppress T cell activity
* Immune checkpoint inhibitors block this suppression, reactivating cytotoxic T lymphocytes (CTLs) against tumours
Immune checkpoint inhibitors include:
* PD-1 (expressed on T cells) inhibitors e.g. Pembrolizumab (Keytruda)
* PD-L1 (expressed on tumour cells) inhibitors e.g. Atezolizumab (Tecentriq) Restore T cell activity
87
What is multi-drug resistance
When the tumour is Resistant not only to drug used but to other structurally unrelated drugs
88
Give 4 mechanisms of multi-drug resistance in cancer cells
↑ drug efflux pumps
Altered metabolism
Amplification:
Methotrexate → ↑ DHDR expression (lecture 2
↑ Oncoproteins involved in apoptosis e.g. BCL2
↓ Tumour suppressors involved in apoptosis e.g. BAX
Higher oncoprotein activity involved in GF signalling (lecture 2)
Tumour Microenvironment (lecture 4)
89
Describe Drug efflux pumps, give an example
e.g. P-glycoprotein (P-gp)
* Acts as an energy dependent pump
* Ejects many structurally unrelated anti-cancer drugs from
inside the cancer cell.
* Diminished effect of drug.
* ↑ cancer cell survival
* Tumour cells often express ↑ levels of P-glycoprotein
Just a few of the many substrates of P-glycoprotein
Anthracycline antibiotics
* Daunorubicin
* Doxorubicin
Topoisomerase inhibitors
* Irinotecan
Microtubule-targeting agents
* Vinca alkaloids: Vinblastine & Vincristine
* Taxanes: Paclitaxel & Docetaxel
90
What are some possible ways to combat P-glycoprotein dependent MDR
→ Inhibitors of P-glycoprotein (eg curcumin)
→ Design drugs resistant to P-glycoprotein-
mediated efflux
91
Describe how altered metabolism has an effect in multi-drug resistance
* Phase I: oxidation, reduction, or hydrolysis by enzymes such as cytochrome P450 (CYP)
* Phase II: conjugation by enzymes such as glutathione S-transferases
Cytochrome P450 levels are often altered in cancer:
* Overexpression → reduces efficacy of drugs inactivated by CYP
e.g. Paclitaxel
* Low expression → reduces efficacy of pro-drug that require activation by CYP enzymes
e.g. Cyclophosphamide
Glutathione
* Elevated glutathione → allows cancer cells to neutralise drugs by conjugating them with glutathione →
facilitates their removal from the cell
e.g. Cisplatin
92
Describe how the tumour microenviroment effects multi drug resistance
* Complex network of cells and extracellular components
surrounding tumour cells
* endothelial cells,
* fibroblasts,
* immune cells,
* cytokines,
* growth factors
* extracellular matrix
* tumour microbiome
* Characterized by a low pH, hypoxia, and metabolic
byproducts
* Each of these components or conditions has the potential
to influence the tumour’s response to chemotherapeutics.
E.G. Hypoxia → HIF-1α → ↑ P-glycoprotein → ↑ Drug Efflux
