Unit 3 - Cell Biology of Cancer Flashcards
function of CTLA-4
brake on T-cell activation
functions to regulate T-cell activation
cancer cells benefit from reduced T-cell activation
MAb vs CTLA-4 releases the brake, allowing enhanced T-cell killing of tumour cells
PD-I
required for T-cell activation
acting through a different mechanism PD-I also acts as a brake on tumour-directed cells
MAb vs PD-I also ‘releases the brake’, allowing enhanced T-cell killing of tumours
use of MAbs
- MAb vs CTLA-4
- MAb vs PD-I
treatment with MAb has led to dramatic clinical outcomes - remissions and cures of metastatic cancers
- releases brake ⇒ enhanced T-cell killing of tumour cells
- releases brake ⇒ enhanced T-cell killing of tumour cells
CAR T-cell therapy
Chimeric Antigen Receptor
T-cells (specialised WBCs) are isolated from a patient and a custom designed gene, that expresses a new cells surface molecule that recognises the tumour and activates the T cell to kill it, is introduced into cells
cells containing the gene are grown in culture to prepare an inoculum
CAR T-cells are infused back into patient
T-cells target cancer cells for killing
MOA of CAR T-cell therapy
what is cancer
a disease that originates at the cellular level but tumoue function as complex tissues that integrate multiple cellular functions and mechanisms to promote tumour survival and growth
what is needed to identify the cellular origin of tumours
histology
how do cellular properties change as cancer develops/progresses
acquisition of adaptive phenotypes through mutation and genome instability couples with recruitment and modification of non-cancer cells to form tumour microenvironments
⇒ for diagnosis and prognosis + understanding therapeutics, knowledge of the cellular basis of cancer is good pragmatic knowledge (personalised therapy)
6 Hallmarks of Cancer
- sustained proliferative signalling
- evading growth suppressors
- activating invasion and metastasis
- enabling replicative immortality
- inducing angiogenesis
- resisting cell death
metastasis
migration of tumour cells from primary tumour to secondary sites
responsible for 90% of cancer deaths
how do cells spread
via blood, lymph and through proximity
where might secondary tumours form
lung, bone, liver, brain
lymph nodes
what are secondary tumours
tumours of primary tissue irrespective of tumour site
e.g. breast cancer within liver
histochemistry can identify tumour type and aid design of treatment
invasion-matastasis cascade - 7 steps
- localised invasion
- intravasation (into circulation)
- transport
- arrest (in a secondary location)
- extravasation (out of circulation and into tissue - colonisation)
- proliferation
- colonisation
utilise mechanisms related to pathways of embryonic development and wound healing
malignancy
penetration of tumour c ells beyond basement membrane id definitive of malignancy
EMT
epithelial → mesenchymal transition
change in phenotype
properties of epithelial cells
polygonal morphology
network of cell-cell junctions
apical-basal polarisation
limited mobility/motility
mesenchymal cells - properties
migratory
variegated morphology/spindle shaped
loosely organised
present in connective tissue/stromal tissue e.g. fibroblasts
key components of EMT
expression of embryonic transcription factors e.g. Snail, Slug, Twist, Zeb 1/2
loss of e-cadherin function
loss of tight junctions
acquisition of motility through CT
protease secretion
growth factor receptor expression
EMT - change in markers
Epithelial cells express epithelial markers and do not express mesenchymal markers
Twist - down regulation of epithelial cell markers and upreg of mesenchymal markers
anchorage-dependent signalling
E-cadherin
functions as a cell adhesion molecule
Maintains epithelial cell phenotype by signalling cell-cell interactions via IC domain
loss leads to dysregulation of β-catenin, a transcription factor regulated by localisation in the cell
β-catenin
integrated into cadherin-actin adherens junctions complexes
a normal component of Wnt signaling pathway
upon loss of cell adhesion it translocates to nucleus to activate TCF/LEF family transcription factors - loss causes cell to move into a different phenotypic state
regulated by molecular association e.g. E-cadherin and APC and by inhibitors e.g. ICAT (inhibition of β-catenin and TCF4)
cytoplasmic levels are maintained through ubiquitin-dependent proteolysis via the β-catenin destruction complex
mutation/misexpression correlated with cancer progression
familial adenomatous polyposis
proliferation of polyps in colon
1 in 30,000
APC gene
function = regulation of β-catenin through the proteolytic pathway
tumour suppressor gene
autosomal dominant mutations
maintains epithelial cell phenotype in colonic crypts
integrates cellular architecture, motility with cell cycle regulation and gene expression
also functions in mitosis and loss contributes to CIN
(cells live for 4 days)
transcription factors and metastasis
especially embryonic TFs
regulate differentiation and de-differentiation
Tcf/Lef, Slug, Snail
cell surface receptors and metastasis
EGF
E-cadherin
motility regulating proteins and metastasis
GTPases, PI3K and PIP3
cytoskeleton proteins
EC proteases
matrix metalloproteases break down EC matrix providing space to move
mesenchymal type cells
progression of EMT
invasion-metastasis cascade
LOCALISED INVASION
EMT
motility
proteases
invasion-metastasis cascade
INTRAVASION
EMT
invasion-metastasis cascade
TRANSPORT
physical transport in circulation
invasion-metastasis cascade
ARREST
physical occlusion/adherence
invasion-metastasis cascade
EXTRAVASION
motility
proteases
invasion-metastasis cascade
PROLIFERATION
growth regulation
growth factor receptors
invasion-metastasis cascade
COLONISATION
vascularisation
overview of invasion-metastasis cascade
utilises mechanisms related to pathways of embryonic development and wound healing via EMT
the Hayflick limit
somatic cells have limited doubling potential
how do some cells have limitless replicative potential
cells relieved of senescence pathways
e.g. p53, Rb mutations
undergo crisis after some number of doublings
about 50 for human cells
crisis is associated with chromosome damage due to erosion of telomeres (tips)
where can telomeres be found
at the termini of chromosomes
sequence element iterated at telomeres
a repetitive sequence element is iterated for 5-40 kb in mammals
TTAGGG
3’ single strand extension of G-strand, 2-3 repeats, 20-30 in us
nicks in C-strand every 2-3 repeats
Partially fully stranded, partially nicked
what makes telomere structure distinctive
unique chromatin composition and topological arrangement
T-loop shields terminus from exposure
shelterin complex of chromatin proteins also shield terminus (DNA ends are recognised by the cell as damage so this configuration of the telomeres shields the 3’ end of the chromosome and encases it in this chromatin complex)
function of telomeres
REPLICATION OF 5’ ENDS
DNA replication = 5’ → 3’ direction and is initiated by a primer
the extreme 5’ end cannot be primed and requires another mechanism for replication
telomerase provides this mechanism
which end needs to be extended
DNA is melted by a DNA helicase - stabilised by RPA protein
DNA always requires extension of a 3’ hydroxyl - 5’ to 3’
Gap leads to shortening of chromosome in a round of DNA replication - cause of crisis
By extending the 3’ end, the loss of 5’ material doesn’t matter
CARRIED OUT BY TELOMERASE
function of telomerase
telomere replication is mediated by the enzyme telomerase
describe structure of telomerase
ribonucleoprotein enzyme containing
- hTERT reverse transcriptase
- hTR RNA template
what does telomerase do and when is it active
adds nucleotides to 3’ end of chromosomal DNA
telomerase is selectively active in germ line and limited cells types
it is NOT active/has limited activity in most somatic cells and telomeres thus shorten throughout the replicative life of a cell lineage
Protein vs RNA activity
what can they do together
protein - enzymatic activity
RNA - template activity
together they can polymerase a template into sequence onto the end of a DNA fragment
life span of cells - impact of telomerase activity
Limited life span of cells - cells eventually become senescent because chromosomes were undergoing damage
This is because telomerase is selectively active in germ cells and not expressed in most somatic cells, so telomere erosion is occurring
Chromosomes are shorter in older people
function of telomeres
suppression of recombination
free DNA ends are recognised as damage by cells
non-homologous recombination can be induced at breaks
telomeres are specially packaged to prevent recognition of chromosome ends as DNA breaks
what happens to broken chromosomes
they will often undergo fusion with themselves after DNA replication or with another chromosome
Fusion events produce chromosomes with 2 centromeres - during mitosis, a chromosome with 2 centromeres can attach to opposite poles of the mitotic spindle and be pulled in opposing directions and ultimately be broken
Improperly segregate chromosome fragments
Fusion, bridge formation and mitosis, breakage, formation
Severe TOXIC GENOTYPIC STRESS ON THE CELLS
Ultimately destined to die but some cells with broken chromosomes can mend them and survive
telomerase is essential for
unlimited growth of most cancer cells
4 targeted approaches - telomere-based therapeutics
hTERT inhibitors
template antagonists
telomere disruptors - DNA
telomere disruptors - shelterin complex
hTERT inhibitors
direct enzyme inhibition
slow telomere erosion
template antagonists
oligonucleotides complementary to RNA template
GRN183L in clinical trials
telomere disruptors - DNA
G-quadriplex promoters alter telomere structure
inhibit telomerase and may uncap
RHPS4 in preclinical development
telomere disruptors - shelterin complex
potential route to telomere uncapping
difference between normal somatic cells and cancer cells
While normal somatic cells do not express telomerase, cancer cells DO
They are successful because they have adapted a strategy
unusual configuration of telomeres
G-quartet
atypical base pairing between guanine residues in a square format
double looped G quartet structure containing 4 bp strands
Target of drug development - nucleic acid inhibitors disrupt G quartet structures
gene therapy - telomere-based therapeutics
virus dependent on telomerase expression to selectively kill cancer cells
telomelysin in trials
Synthetic virus is constructed which is cytotoxic but ONLY IN PRESENT OF TELOMERASE, so normal cells would not be affected
immunotherapy - telomere-based therapeutics
hTERT is processed and presented by MHC
induce immune cells that attack presenting cells - telomerase vaccine
Proteins present in cytoplasm and in human cells are digested by MHC, and presented on cell surface (immune recognition of cell process)
Cancer cells would express something on their cell surface
as part of combination therapy - telomere-based therapeutics
hTERT inhibition is slow but could be a factor in combo therapy (Imetelstat)
Long term - cancer cells can be severely inhibited
EMT - 7 steps
- loss of e-cadherin function
- dysregulation of β-catenin pathway
- loss of tight junctions
- acquisition of motility
- transcription factor expression
- protease secretion
- growth factor receptor expression
telomeres and cellular lifespan
somatic cells have limited replicative potential - lack of telomerase expression
tumour cells reactivate telomerase expression to support limitless replicative potential
telomeres manage and protect chromosome ends
telomerase reverse transcriptase (TERT) maintains ends by addition of telomere repeats
telomere structure, shelterin complex protects ends from recognition as DNA termini
cancer cell specificity provides target of opportunity for therapeutics
what are solid tumours and what do they require
organ systems requiring vasculature for survival
tumours arise in highly vascularised regions
cells locared > 0.2 mm from vessel do not grow
hypoxia leads to necrosis in tumour cores
tumours actively promote angiogenesis
how to recruit vascular tissue
key molecule
capillaries are formed from endothelial cells
VEGF - vascular endothelial growth factor - key molecule involved in angiogenesis
other important angiogenic factors
what do cancer cells secrete
VEGF - but it is immobilised in ECM
how to activate VEGF
MMPs, Matrix Metabolic Proteases, (MMP-9) proteolyse ECM and give riseto angiogenic swithc
MMPs can be produced by inflammatory mast cells and macrophages - co-opting normal cell functions for tumorigenesis
balancing angiogenesis - what are its inhibitors and where are they found
normally tightly regulated - development and wound healing
ECM contains inhibitors of angiogenesis - thrombospondin-I (Tsp-I), fragments of ECM proteins
other circulating proteins inhibit angiogenesis - IFN, interleukins, TIMP-2
inhibitors of angiogenesis in ECM
Tsp-I
fragments of ECM proteins
inhibitors of angiogenesis - circulating proteins
IFN
interleukins
TIMP-2
how are tumours successful
they evolve a complex of mechanisms that tip the balance toward local angiogenesis and metabolic permissiveness
anti-angiogenic therapies
requirement of angiogenesis for tumour formation makes this a very active area of therapeutic development
alone they are limited in effect on survival - marginal improvements
combination strategies now being undertaken
summary of role of angiogenesis in tumour progression
enabling characteristic - tumour promoting inflammation
inflammatory responses play decisive roles at different stages of tumour development, including
initiation
promotion
malignant conversion
invasion
metastasis
immune cells that infiltrate tumours engage in an extensive and dynamic crosstalk with cancer cells
induction of angiogenesis - production of MMP by macrophages
genome instability - what products of inflammation may be mutagenic
ROS and RNI (rxn to cytokines)
how is proliferative signalling induced
induced by cytokines released in inflammation
pro-survival (anti-apoptotic) signalling - how are they induced
can be induced by cytokine pathways
nature of tumours
organs with differentiated cell compartments and functions
parenchyma of tumour
core of neoplastic epithelial cells - carcinoma
stroma of tumour
surrounding/supporting mesenchymal cells
describe cellular structure of tumour
Surrounded by stromal tissue
Vasculature, endothelial cells, pericytes surround vessels
Then there are infiltrating immune cells
Cancer associated fibroblasts - type of cells that are migratory through the cancer
Contribute to vitality of tumour
inflammatory cells
contribute proteases that resist invasion
cytokines
activate VEGF
pericytes
in communication with the endothelial cells that stabilise the induced vasculature
cancer-associated fibroblasts
secrete multiple growth factors that contribute to epithelial cell growth as well as growth of other cells
cancer stem cells and tumours
common constituent of many if not most tumours
CSCs - how do they work
defined operationally through their ability to efficiently seed new tumours upon inoculation into recipient host mice
what is unique about cells with properties of CSCs
more resistant to various commonly used therapeutic treatments
many have bona fide stem cell like characteristics - ability to transdifferentiate into endothelial-like cells (vasculature) recently documented in glioblastomas
glioblastomas and CSCs
CSCs have the ability to transdifferentiate into endothelial-like cells (vasculature) - recently documented in glioblastomas
model of solid tumour stem cells based on breast cancer
reprogramming energy metabolism - warburg effect of cancer cells
cancer cells depend on glycolysis (rather than ox phos in mitochondria)
glycolysis is typical in anaerobic conditions
what does the warburg effect allow
tumours to be visualised by 18F-deoxyglucose
may aid growth in hypoxic environments - HIF I pathways (cellular response to hypoxia is mediated by HIF I - Activating glycolytic activity through HIF I pathway in addition to helping cells in a low O2 environment, the glycolytic pathway produces lots of biosynthetic intermediates - positive feature for tumour cells to increase conc of metabolic intermediates to allow for increased overall metabolism of tumour cells)
may provide richer range of biosynthetic precursors for increased overall metabolism
potential application of glycolytic inhibitors e.g. 2-deoxyglucose now in clinical trials, glucose transport inhibitors
HIF I pathways
cellular response to hypoxia is mediated by HIF I
Activating glycolytic activity through HIF I pathway in addition to helping cells in a low O2 environment, the glycolytic pathway produces lots of biosynthetic intermediates - positive feature for tumour cells to increase conc of metabolic intermediates to allow for increased overall metabolism of tumour cells
cancer depends on
genetic variety - a positive role for genome instability in tumour formation - diversity of genome and phenome provide a positive role for tumour development by creating more opportunity for tumours to adapt
mutation and aneuploidy thus play direct roles in tumour progression throughout the developemnt of the tumour
⇒ tumour cells are adapted to their ‘ad hoc’ niches - with attendant ‘achilles heels’ e.g. oncogene dependence
epigenetic mutation
non-sequence dependent alterations in gene function
activation/silencing
Chromosome associated proteins that are associated with specific - propagated from one cell to another
aneuploidy
aberrant chromosome numbers
consequence of defects in chromosome segregation
aneuploidy and cancer
aneuploidy is causative of cancer
low levels of aneuploidy
promote tumorogenesis
high levels of anueploidy
do not promote tumorogenesis
too disruptive
aneuploidy leads to
increased rates of mutagenesis through enhanced recombination and defective DNA damage repair
critical players in generation of aneuploidy and in cancer therapeutics
mitosis and mitotic spindle formation
spindle poisons, novel antimitotic drugs
vinblastine/vinca alkaloids
taxol and taxanes
epithilones
Eg5 inhibitors
therapeutic potential
MCQ
MCQ - inhibitors of telomerase
MCQ - inflammatory mechanisms promote tumour establishment by, for example
