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1

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

2

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

3

use of MAbs

  1. MAb vs CTLA-4
  2. MAb vs PD-I

treatment with MAb has led to dramatic clinical outcomes - remissions and cures of metastatic cancers

  1. releases brake ⇒ enhanced T-cell killing of tumour cells
  2. releases brake ⇒ enhanced T-cell killing of tumour cells

4

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

5

MOA of CAR T-cell therapy

6

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

7

what is needed to identify the cellular origin of tumours

histology

8

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)

9

6 Hallmarks of Cancer

  1. sustained proliferative signalling
  2. evading growth suppressors
  3. activating invasion and metastasis
  4. enabling replicative immortality
  5. inducing angiogenesis
  6. resisting cell death

10

metastasis

migration of tumour cells from primary tumour to secondary sites

responsible for 90% of cancer deaths

11

how do cells spread

via blood, lymph and through proximity

12

where might secondary tumours form

lung, bone, liver, brain

lymph nodes

13

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

14

invasion-matastasis cascade - 7 steps

  1. localised invasion
  2. intravasation (into circulation)
  3. transport
  4. arrest (in a secondary location)
  5. extravasation (out of circulation and into tissue - colonisation)
  6. proliferation
  7. colonisation

utilise mechanisms related to pathways of embryonic development and wound healing

15

malignancy

penetration of tumour c ells beyond basement membrane id definitive of malignancy

16

EMT

epithelial → mesenchymal transition

change in phenotype

17

properties of epithelial cells

polygonal morphology

network of cell-cell junctions

apical-basal polarisation

limited mobility/motility

18

mesenchymal cells - properties

migratory

variegated morphology/spindle shaped

loosely organised

present in connective tissue/stromal tissue e.g. fibroblasts

19

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

20

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

21

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

22

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

23

familial adenomatous polyposis

proliferation of polyps in colon

1 in 30,000

24

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)

25

transcription factors and metastasis

especially embryonic TFs

regulate differentiation and de-differentiation

Tcf/Lef, Slug, Snail

26

cell surface receptors and metastasis

EGF

E-cadherin

27

motility regulating proteins and metastasis

GTPases, PI3K and PIP3

cytoskeleton proteins

28

EC proteases

matrix metalloproteases break down EC matrix providing space to move

mesenchymal type cells

29

progression of EMT

30

invasion-metastasis cascade

LOCALISED INVASION

EMT

motility

proteases

31

invasion-metastasis cascade

INTRAVASION

EMT

32

invasion-metastasis cascade

TRANSPORT

physical transport in circulation

33

invasion-metastasis cascade

ARREST

physical occlusion/adherence

34

invasion-metastasis cascade

EXTRAVASION

motility

proteases

35

invasion-metastasis cascade

PROLIFERATION

growth regulation

growth factor receptors

36

invasion-metastasis cascade

COLONISATION

vascularisation

37

overview of invasion-metastasis cascade

utilises mechanisms related to pathways of embryonic development and wound healing via EMT

38

the Hayflick limit

somatic cells have limited doubling potential

 

39

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)

40

where can telomeres be found

at the termini of chromosomes

41

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

42

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)

43

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

44

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

 

45

function of telomerase

telomere replication is mediated by the enzyme telomerase

46

describe structure of telomerase

ribonucleoprotein enzyme containing

  • hTERT reverse transcriptase
  • hTR RNA template

47

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

48

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

49

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

50

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

51

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

52

telomerase is essential for

unlimited growth of most cancer cells

53

4 targeted approaches - telomere-based therapeutics

hTERT inhibitors

template antagonists

telomere disruptors - DNA

telomere disruptors - shelterin complex

54

hTERT inhibitors

direct enzyme inhibition

slow telomere erosion

55

template antagonists

oligonucleotides complementary to RNA template

GRN183L in clinical trials

56

telomere disruptors - DNA

G-quadriplex promoters alter telomere structure

inhibit telomerase and may uncap

RHPS4 in preclinical development

57

telomere disruptors - shelterin complex

potential route to telomere uncapping

58

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

59

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

60

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

61

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

62

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

63

EMT - 7 steps

  1. loss of e-cadherin function
  2. dysregulation of β-catenin pathway
  3. loss of tight junctions
  4. acquisition of motility
  5. transcription factor expression
  6. protease secretion
  7. growth factor receptor expression

64

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

65

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

66

how to recruit vascular tissue

key molecule

capillaries are formed from endothelial cells

VEGF - vascular endothelial growth factor - key molecule involved in angiogenesis

67

other important angiogenic factors

68

what do cancer cells secrete

VEGF - but it is immobilised in ECM

69

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

70

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

71

inhibitors of angiogenesis in ECM

Tsp-I

fragments of ECM proteins

72

inhibitors of angiogenesis - circulating proteins

IFN

interleukins

TIMP-2

73

how are tumours successful

they evolve a complex of mechanisms that tip the balance toward local angiogenesis and metabolic permissiveness

74

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

75

summary of role of angiogenesis in tumour progression

76

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

77

genome instability - what products of inflammation may be mutagenic

ROS and RNI (rxn to cytokines)

78

how is proliferative signalling induced

induced by cytokines released in inflammation

79

pro-survival (anti-apoptotic) signalling - how are they induced

can be induced by cytokine pathways

80

nature of tumours

organs with differentiated cell compartments and functions

81

parenchyma of tumour

core of neoplastic epithelial cells - carcinoma

82

stroma of tumour

surrounding/supporting mesenchymal cells

83

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

84

inflammatory cells

contribute proteases that resist invasion

85

cytokines

activate VEGF

86

pericytes

in communication with the endothelial cells that stabilise the induced vasculature

87

cancer-associated fibroblasts

secrete multiple growth factors that contribute to epithelial cell growth as well as growth of other cells

88

cancer stem cells and tumours

common constituent of many if not most tumours

89

CSCs - how do they work

defined operationally through their ability to efficiently seed new tumours upon inoculation into recipient host mice

90

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

91

glioblastomas and CSCs

CSCs have the ability to transdifferentiate into endothelial-like cells (vasculature)  - recently documented in glioblastomas

92

model of solid tumour stem cells based on breast cancer

93

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

94

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

95

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

96

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

 

97

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

98

aneuploidy

aberrant chromosome numbers

consequence of defects in chromosome segregation

99

aneuploidy and cancer

aneuploidy is causative of cancer

100

low levels of aneuploidy

promote tumorogenesis

101

high levels of anueploidy

do not promote tumorogenesis

too disruptive

102

aneuploidy leads to

increased rates of mutagenesis through enhanced recombination and defective DNA damage repair

103

critical players in generation of aneuploidy and in cancer therapeutics

mitosis and mitotic spindle formation

104

spindle poisons, novel antimitotic drugs

vinblastine/vinca alkaloids

taxol and taxanes

epithilones

Eg5 inhibitors

105

therapeutic potential

106

MCQ

107

MCQ - inhibitors of telomerase

108

MCQ - inflammatory mechanisms promote tumour establishment by, for example