Cancer 2

Question 1

How do we use RNA as a target for anti-cancer drugs

Answer 1

The RNA in cancer cells is different to normal cells (as they are producing different proteins). We can subject this to antisense therapy which targets a section of the cancerous mRNA which blocks the mRNA from being transcribed into a cancerous protein

Question 2

What are the advantages of antisense therapy

Answer 2

Advantages

• Same effect as an enzyme inhibitor or receptor antagonist without having to be physically designed to match the ligand. Just has to be an antisense to the mRNA
• Highly specific to where the oligonucleotide is 17 nucleotides or more (as only 16 oligonucleotides can ever be in the same exact sequence as another in the whole genome)
• Smaller dose levels required compared to inhibitors or antagonists for the binding sites
• Potentially less side effects as its so highly specific

Question 3

What are the disadvantages of antisense therapy

Answer 3

Disadvantages

• ‘Exposed’ sections of mRNA must be targeted
• The instability and polarity of oligonucleotides (several nucleotides in a row) (pharmacokinetics)
• Short lifetime of mRNA oligonucleotides and there is poor absorption across cell membranes due to polarity issues

Question 4

What is miRNA

Answer 4

Micro-RNA (miRNA)

Short segments of double stranded RNA

Recognised by enzyme complex RISC in the cell to produce single stranded RNA - a small interfering or small inhibitory RNA (siRNA)

This siRNA binds to complementary region of mRNA

mRNA is cleaved by enzyme complex – undergoes degradation. The antisense sequence is now bound to your risc complex which can go on to bind to the sense sequence on the mrna which then leads to degredation of thr mrna – no gene product synthesised

Question 5

What are the advantages of miRNA as an anti cancer therapy

Answer 5

Advantages

siRNAs have potential to be used in gene therapy

Greater efficiency in silencing mRNA than conventional antisense therapy because of its stability

One siRNA could lead to cleavage of many mRNA molecules

Question 6

What are the disadvantages of miRNA as an anti cancer therapy

Answer 6

Disadvantages

siRNAs need to be metabolically stable (need advantageous pharmacokinteics)

Difficult to reach target cells

Devise a mechanism to ensure entry to target cells

Question 7

What are many of the hallmark processes driven by

Answer 7

Many of the processes behind the hallmarks of cancer are driven by signal transduction and the activity of tyrosine kinases. Thus, these are new therapeutic targets

Question 8

What is the basic signal transduction process

Answer 8

Signal => reception => (amplicication) => transduction => response

Receptor proteins bind “signals” i.e. drugs & endogenous ligands with high affinity

Conformational changes in the structure of the receptor protein then convert the external signal into one or more intra-cellular signals.

This process is known as signal transduction

Question 9

What can be a signal for signal transduction

Answer 9

Osmolarity change, temperature, growth factor etc.

Question 10

What is the basic structure of a tyrosine kinase receptor

Answer 10

Receptors with an extra-cellular and intra-cellular domain

Intracellular domain capable of phosphorylating tyrosine residues in target proteins.

Question 11

What does the activation of a tyrosine kinase receptor do

Answer 11

This can initiate a signalling cascade leading to changes in expression of genes that is important for:

Proliferation
Evasion of apoptosis
Induction of angiogenesis
Generating metastasis  
(can also encourage protumor inflammation)

Question 12

What are the 3 essential components of receptor tyrosine kinases

Answer 12

Ligand binding site (extracellular domain)

Transmembrane domain (α helix) (within the cell membrane)

Domain with tyrosine kinase activity (cytosolic) (inside the cell)

Question 13

How are tyrosine kinase receptors activated

Answer 13

Ligand binds to the receptor site and induces association between adjacent RTKs (usually via dimerisation)

This causes activation of their kinase activity - the internal domains are joined together

They phosphorylate tyrosine residues in other molecules

Once the kinase receptor is phosphorylated, the phospho-tyrosine groups (are able to phosphorolate their own proteins) can act as binding sites for signalling proteins by attracting other molecules which need to be phosphorylated to induce activity

Question 14

What happens once the tyrosine kinase activity on the receptor is activated

Answer 14

Each phosphorylated tyrosine region can bind a signalling protein

These molecules may also become phosphorylated and act go on to act as further binding sites

Phosphorylation cascade (kinases) inducing downstream signalling

Reversed by enzymes called phosphatases – negative feedback loop - this process is often inhibited in cancers

Question 15

Describe the downstream signalling of the MAPK (ERK) signalling

Answer 15

Something binds to the receptor tyrosine kinase and activates it

This leads to RAS being phosphorylated

Leads to RAF, MEK1 and MEK2, ERK1, ERK2 and ERK5 being phoshphorylated all in turn

ERK1, ERK2, ERK5 are executer kinases which, when active, become transcription factors themselves or go on to activate other transcription factors which start gene expression changes leding to proliferation, prevention of apoptosis etc…

Question 16

As a summary, what are the 2 ways in which kinases can be used as drug targets

Answer 16

The modulation of kinase activity can be
achieved through several strategies, including:

Disruption of ligand-receptor interactions

Inhibition of kinase (phosphorylation) activity (block ATP binding)

Question 17

Give an example of a drug which disrupts kinase ligand-receptor interactions

Answer 17

Bevacizumab

This drug binds to a growth factor vascular endothelial growth factor (VEGF) thus preventing its binding to receptors (RTK)

This interferes with tumour blood vessel development.

First line treatment for colorectal cancers

Question 18

Which receptor does tratuzumab (herceptin) target in the treatment of breast cancer

Answer 18

Trastuzumab (Herceptin) targets the HER2 receptor, which is over expressed in (5-20%) of all late stage breast cancers

Herceptin prevents binding of EGF (ligand) to the HER2 receptor by binding to the ligand and thus prevents signal transduction as the ligand can no longer activate the receptor

However its use is limited to cancers which test as HER2 +, since it only works on tumours in which this protein is over expressed.

Needs to be used in conjunction with a diagnostic test for HER2 overexpression to ensure its not used where its not needed

Question 19

Give an example of a drug which inhibits kinase activity by blocking ATP binding

Answer 19

Imatinib mesylate (2001)
Protein tyrosine kinase inhibitor through inhibiting ATP binding to the bcr/abl fusion protein (a super active kinase). This prevents any phosphate being available for the transduction of the cancerous cells signal

Chronic myeloid leukaemia, acute lymphoblastic leukaemia and intestinal stromal tumours

Question 20

Give a second example of a drug which inhibits kinase activity by blocking ATP binding

Answer 20

Gefitinib (2002)
prevents ATP binding to EGFR (which stops the receptor geting hold of any phosphates which allow it to phsophorylate itself and other molecules) – useful in cancers with high EGFR expression

Question 21

Give a brief outline of the the Epidermal growth factor receptor (EGFR) becomes active

Answer 21

Binding of EGF causes receptor dimerisation and activation of enzyme activity

Dimerisation means that the active site on each half of the receptor dimer catalyses the phosphorylation of the tyrosine residues in the other half

If dimerisation does not occur then phosphorylation cannot take place and signalling is inhibited

Important because EGFR is overexpressed in many solid tumours (ie lung cancers)

Question 22

Summary:

How do we inhibit rtk signalling

Answer 22

We can inhibit RTK signalling in several ways using targeted therapies

Prevent ligand/receptor binding – blocks the initiation of the signal
Inhibit kinase activity – blocks intracellular transduciton of the signal and therefore associated response e.g uncontrolled proliferation, evasion of apoptosis etc (hallmarks of cancer)

Question 23

What do phosphorylated tyrosine residues on receptors act as

Answer 23

The phosphorylated tyrosine residues on receptors act as binding sites for signalling proteins

Question 24

Give an example of a targeted therapy which doesnt involve rtks

Answer 24

Disruption of oestrogen-receptor interaction

Breast cancer can be oestrogen-dependent

Oestrogen secretion can be reduced by surgery

An alternative to surgery is use of tamoxifen

• Competitive inhibition of oestrogen binding to its receptor with a higher affinity but ellicits no effect by the receptor
• FDA approved for advanced breast cancers and primary breast cancer

Inhibits expression of oestrogen regulated
genes

Question 25

Give 2 examples of drugs used to inhibit MAPK signalling

Answer 25

. Dabrafenib and vemurafenib

Question 26

When can Dabrafenib and vemurafenib be used

Answer 26

Used in the treatment of melanoma

Only suitable for patients with a mutation in the B-Raf gene

Approximately 50% of melanomas harbour B-Raf mutations

This mutation makes RAF-ERK signalling constitutively active

Question 27

What is the most common B-Raf mutation

Answer 27

Diagnostic test is required to establish this

Most common mutation (90% of cases) is V600E (amino acid in position 600)

Question 28

What part of the MAPK kinase cascade do dabrafenib and vemurafenib work on

Answer 28

The Raf section. In melanoma it can signal on its own without input from ras or the receptor. By turning it off by preventing it from binding phosphorous it cant signal downstream

Question 29

What do all cancer cells posess

Answer 29

Associated inflammation

Inflammation can support all aspects of cancer:

Initiation - immune cells can release reactive oxygen species which goes on to damage dna which increases the number of mutations
Growth - immune cells can produce growth factors
Metastasis - immune cells can often be used to breqk down and remodel tissue in normal wound healing. These processes can be hijacked to allow for tumors to form and spread
Response to therapy - can alter all therapies work

Question 30

What cells are asscoiated with tumors which arent actually tumor cells

Answer 30

Blood vessels, stromal cells, fibroblasts, an extracellular matrix, immune cells (trying to fight the tumor but often end up becoming a part of the tumor….)

Question 31

What are the two ways in which we can target inflammation in cancer

Answer 31

Inhibit pro-tumour inflammation

Promote anti-tumour inflammation

Question 32

Which inhibitors use the approach of inhibiting pro-tumour inflammation

Answer 32

MAPK inhibitors

Blocking certain pathways prevents pro-tumourigenic inflammatory signalling within the tumour

Question 33

How does the checkpoint approach to promote anti-tumour inflammation
work (and give examples of therapy)

Answer 33

T cell immune responses (anti-tumour) are controlled through on and off switches, so called ‘immune checkpoints’

These protect the body from potentially damaging immune responses (hyper responses)

We have developed drugs to target these immune checkpoints

This illicits anti-tumour T-cell-mediated immune responses (which leads to a hyperactive immune response which cause side effects)

e.g. anti-CTLA4 and anti-PD1 or PDL1 therapy

Question 34

What does CTLA4 do

Answer 34

Its the master switch (checkpoint) for T cell activation is CTLA4

CTLA4 is a receptor found on the surface of T cells and its activation inhibits T cell function. If we block it, it enables the activation of what it was inhibiting

When used as therapy - anti-CTLA4 monoclonal antibody (ipilimumab) – binds to CTLA4

This blocks CTLA4 signalling, enabling an anti-tumour T cell response

Question 35

What is the PD-1/PD-L1 pathway and how does it work

Answer 35

PD-1 receptor and its ligand (PD-L1) are expressed on the surface of dendritic cells and macrophages

They are inhibitory factors, which act as a check-point for the dendritic-cell mediated T cell response (and are used to control it)

Therapy = monoclonal antibodies targeted against PD1 and PD-L1 (Nivolumab) – blocks the PD-1 signal hence inhibiting the inhibitor (we enable the t-cell response)

This releases the inhibitory checkpoint enabling a T-cell response, T-cell proliferation and therefore anti-tumour activity

Question 36

What is the role of the pharmacists role in cancer treatment

Answer 36

Know about the disease and how thr medicines work to treat the cancer

Know how to communicate (and make complicated things simple). Always ask to see if the patient understands what were saying to them