Cancer (1) Molecular Mechanisms of Cancer

Question 1

Cancer

Answer 1

Disease caused by uncontrolled division of abnormal cells in a part of the body

Or rather - a collection of diseases

Cancer is the 2nd leading cause of death in most developed countries (after cardiovascular disease)

**colorectal cancer generally classified as 1 type, not 2

Question 2

How does cancer develop?

Answer 2

Initial genetic alteration may be
>spontaneous/stochastic/heriditary
>or environmentally induced

Question 3

Carcinogenesis is a multi-step process

Answer 3

E.g. Sequential alterations in colorectal cancer pathogenesis

> Initiation (first “hit”)
>environmental trigger (sporadic)
>spontaneous/stochastic mutations
>germinal mutation (hereditary)

>Progression
>>second "hit" 
>>proto-oncogene mutations
>>accumulation of more mutations 
>>metastasis

Question 4

Altered growth

Answer 4

Erratic growth
>slow or rapid, mitotic figures may be numerous and abnormal

Growth rate of tumours reflects combined influences of
>doubling time of tumour cells
>proportion of tumour cells actually dividing
>Death rate of tumour cells

Selective growth advantage of tumour cells vs healthy counterpart is small

Question 5

Non-selective targeting of cell division

Answer 5

Proliferative pool (sensitive to chemotherapy)
vs 
nonproliferative pool (insensitive to chemotherapy)

Question 6

Nature of gene alterations in cancer

Answer 6

Genetic alterations can come in different flavours

Gene mutations
>point mutations/altered sequence
>frameshift/stop codon mutations/shorter protein

Insertions/Deletions (indels)

Gene amplification

Gene translocation/fusion
>(e.g. BCR/ABL in CML, coming from 22q11/9q34 fusion)

Question 7

Molecular basis of cancer

Answer 7

Inappropriate activation of proto-oncogenes

Inactivation of Tumour suppressor genes

> Driving mutations or other genetic alterations usuually affects several of these pathways

Question 8

Oncogene activation

Induction of self-sufficient growth

Answer 8

Proto-oncogenes
>normal cellular genes encoding proteins mostly involved in
>promoting proliferation or
>suppressing differentiation/apoptosis

Oncogenes:
>genetically altered (often mutated) version
>active in unregulated manner
>independently from growth-promoting signals

Main classes of oncogenes
(overexpression, permanent/constitutive activation)
>growth factors
>growth factor receptors
>signal transduction proteins e.g. protein kinases, GTPases
>transcription factors
>cyclins and cyclin-dependent kinases (cell cycle control)

Question 9

Oncogene activation

Proteins involved in signal transduction

Answer 9

GTP-binding

KRAS > point mutation > colon, lung and pancreatic tumours

HRAS > point mutation > bladder and kidney tumors

NRAS > point mutation > melanomas, hematologic malignancies

Question 10

Oncogene activation

Growth factor receptors

Answer 10

EGF-receptor family

> ERBB1 (EGFR), ERRB2 > overexpression > squamous cell carcinoma of lung, gliomas

Question 11

Oncogene activation

the example of RAS

Answer 11

Upon RAS mutation

> RAS/MAP kinase pathway becomes permanently activated
no more requirement for activating signals from growth factors

Question 12

Oncogene activation

RAS targeting approaches

Answer 12

Salirasib
>blocks membrane association of RAS
>cleaves Famesyl membrane anchor
>shut down signalling

MRTX849 (Mirati Therapeutics)
>covalently binds to codon 12 cysteine in G12C mutant

AZD4785 (Astra Zeneca)
>antisense oligonucleotide targeting of KRAS mRNA
>stop production of new RAS, at some point there will be no more RAS

+ multiple inhibitors targeting downstream targets

Question 13

Where is the best target in any given cancer-driving pathway?

Answer 13

E.g. Alteration of KIT/RAS/MAP kinase pathway in Melanoma

Essentially many targets downstream, and targeting one step in the pathway sometimes causes cancer cell to develop other mutations in response to that selective pressure

Question 14

What about receptors and their ligands?

Answer 14

HER family of trans-membrane receptors
(Receptor tyrosine kinase)
(HER = human epidermal growth factor receptors)

EGFR/HER1
>one of the key growth factor receptors in most tissues

HER2 does not bind to any ligand
>reacts to binding of other ligands on other receptors
>can get heterodimerisation of HER1/HER2, or HER2/HER4 etc and activate the HER2 pathway as well

HER3 and HER 4

All result in dimerisation
>result in activation of PI3K pathway and Ras/Ref/MEK/MAPK pathways

> > key in proliferation, motility, invasiveness, resistance to apoptosis, angiogenesis

Question 15

You can have mutations at receptor level instead of mutations downstream

Answer 15

HER2 amplification
>20% of breast cancers

EGFR amplification
>10% of colorectal cancers (but can have further mutations downstream in KRAS or BRAF)
>10% NSCLC
(non-small-cell lung carcinoma)

EGFR activating mutations
>10% NSCLC

Question 16

Anti-HER targeted approaches

Answer 16

HER2 dimerisation inhibitors
>Trastuzumab
>block dimerisation of HER2 to any of the other receptors
>block downstream signalling pathway of HER2

Anti EGFR blocking antibodies
>Cetuximab
>colorectal cancer patients who dont have a mutation downstream
>Antibody binds in receptor active sites, blocks ligand binding

Anti-ligand blocking antibodies
>block ligand
>e.g. Anti-VEGF

Tyrosine kinase inhibitors
>Erlotinib, Gefitinib, Iapatinib
>bind to ATP pockets and block activity of the receptor

Ligand-toxin conjugates
>binds to receptor, toxin comes so close to cell that it kills the cell
>risky - need very high amplification of receptors or else risk binding to normal cells

Question 17

Tumour suppression inactivation

Answer 17

Loss of sensitivity to growth inhibition and senescence-inducing signals

APC/Beta-catenin gene in cytosol
>Functions as inhibition of signal transduction
>Somatic mutations associated with carcinomas of stomach, colon, pancreas, melanomas
>Inherited mutations associated with familial adenomatous polyposis coli/colon cancer

Mostly (but not always) follow the “two-hit” model
(i.e. both alleles must be inactivated for full impact)
>one normal is enough to keep it in check

Question 18

Tumour suppressor genes - Two major categories

Answer 18

Gatekeepers
>regulate apoptotic pathways (p53, Rb1)
>regulate proliferation (APC)

Caretakers
>BRCA1 (rarely mutated in breast cancers but often methylation), BRCA2, MSH2, MLH1
>involved in DNA repair
>when damage occurs, DNA repair in a correct manner, or else cell will go through cell death
>when dysfunctional, cell does not go through DNA repair

Question 19

Tumour suppressor inactivation

Example of APC

Answer 19

APC gene regulator of Wnt signalling pathway
>controls destruction of beta-catenin (protein that moves into nucleus and is involved in adhesion and transcriptional regulation)

> Destruction complex is inhibited by Wnt-induced signals in normal cells

> When APC loss of function, destruction complex is constitutively inactivated
>constant beta-catenin flooding nucleus and promoting transcription of many proteins involved in proliferation

Question 20

Epigenetics

Answer 20

Inherited changes in phenotype or gene expression
>caused by mechanisms other than chances in underlying DNA sequence

> > modifying structure of chromatin

Question 21

Cancer epigenetics

Answer 21

3 main components of the epigenetic code
>DNA methylation
>Histone modification
>small non-coding RNAs (e.g. miRNAs) that modulate gene expression

Enzymes that apply histone modifications
>called writing enzymes

Enzymes that remove histone modifications
>called erasing enzymes

Enzymes that can bind to DNA once modifications are present
>called reading enzymes

Question 22

A key role for the tumor microenvironment

Answer 22

Targeting the microenvironment so that we can stop tumor growth

> avoiding immune destruction
Inducing angiogenesis

Impact of the microenvironment on tumour cells
>other cells, signalling molecules, cell-cell contacts, cell-matrix contacts, biophysical forces

Question 23

Microenvironment support multiple stages of tumour devleopment, opening the door for multiple targeting avenues

Answer 23

Tumour vasculature

Immune activation*

Altered immune cell recruitment, expansion and depletion

Repolarisation and re-education

Metastasis and/or outgrowth

Question 24

Oncology meets immunology: the cancer-immunity cycle

Answer 24

Early tumour cells normally killed off by immune system

sometimes, tumour cells get past this and escape immune system killing

Question 25

Multiple mechanisms of immune escape in cancer

Answer 25

Tumour-related mechanisms
>loss of tumour antigens
>downregulate MHC or NK ligands
>loss of tumour IFN-gamma responsiveness

Host-related mechanisms
>immune deficiency
>host unable to reach or kill tumour cells

Question 26

Chimeric antigen receptor (CAR) - T cell therapy

Answer 26

Chimeric T-cell receptor
>recognise an antigen on tumour cell surface
>when recognised, activates T cell processes
>mimic MHC activation of T cell

1) Remove blood form PT to get T cells
2) Make CAR-T cells in lab
>insert gene for CAR, so that chimeric antigen receptor is expressed on T cell surface
3) Grow millions of T cells
4) Infuse CAR-T cells into patient
5) CAR-T cells bind to cancer cells and kill them

Key step
>identifying a tumour-specific membrane target (dont want to kill all cells)

Question 27

Immune checkpoint pathways

Answer 27

Lots of co-stimulatory and co-inhibitory interactions between APC and T cell
>Cancer cells take advantage of this mechanism to avoid immune response

> express high amounts of inhibitory co-stimulatory molecules to inhibit the T-cell response even when TCR is activated
block activation of T cell, T cell recognises the antigen but it cannot get activated

> > PDL1 or PDL2 (APC) : PD1 (T cell)
CD80 or CD86 (APC) : CTLA4 (T cell)