Lecture 4: Cancer - Basis of Carcinogenesis I

Question 1

What is a major problem in research and development of new drugs?

Answer 1

Massive attrition rates

Question 2

What is the most common reason for failure during research and development of drugs?

Answer 2

Problems in Phase II – wrong target due to a misunderstanding of the disease

Question 3

What are 4 genomic themes common to all cancers?

Answer 3

1. Non-lethal genetic damage
2. Tumour is formed by colonal damage to a single precursor cell which has then proliferated
3. The main targets of cancer causing mutations are the same: oncogenes (growth promoting), tumour suppressor genes (growth inhibiting), genes that regulate cell death (apoptosis), genes involved in DNA repair
4. Results from an accumulation of mutations

Question 4

1) What causes NEOPLASTIC TRANSFORMATION OF A CELL? 2) How many mutations are required to cause it? 3) What are the 2 ways a person can get these mutations?

Answer 4

1) Non-lethal genetic cumulative damage
2) 6-20
3) i. Inherited mutations (germline) ii. Acquired mutations (somatic) –> e.g. from environmental factors, chemical or can just be random

Question 5

What is Vogelstein’s cascade?

Answer 5

It’s a MOLECULAR MODEL of the PROPOSED EVOLUTION of colorectal cancer starting from benign adenoma —–> carcinoma.

Question 6

What are the 4 stages in Vogelstein’s cascade?

Answer 6

1. Normal colonic epithelium
(APC mutation)
2. Early benign adenoma --> pedunculated polyp
(Mutation of ras gene)
(Mutation of DCC gene)
3. Late benign adenoma --> sessile polyp
(Mutation of p53 gene)
4. Colonic carcinoma

Question 7

Cancers are homogenous/heterogenous

Answer 7

Heterogenous. Remember diagram of ball with lots of different shades of blue.

Question 8

What are the 6 main HALLMARKS OF CANCER?

Answer 8

1. Self-sufficiency in growth signals: e.g. oncogene activation (the focus of this deck of cards)
2. Insensitivity to growth-inhibitory signals: e.g. inactivation of tumour suppressor genes
3. Evasion of apoptosis: eg inactivation of p53 or activation of bcl-2
4. Limitless replicative potential: eg active telomerase expression
5. Sustained angiogenesis: eg over-expression of VEGF
6. Ability to invade and metastasise: eg over-expression of proteases

Question 9

What are the 7th, 8th, 9th and 10th HALLMARKS OF CANCER?

Answer 9

7. Defects in DNA repair: eg leading to genomic instability
8. Altered cellular metabolism: eg switch to aerobic glycolysis (Warburg effect)
9. Avoiding immune destruction: there are several immune escape mechanisms
10. Tumour-promoting inflammation: eg release of cytokines promoting proliferation

Question 10

What is the difference between a PROTO-ONCOGENE, an ONCOGENE and an ONCOPROTEIN?

Answer 10

Proto-oncogene: a normal gene whose product promotes cell proliferation. Only turned on when needed.
Oncogene: an overexpressed proto-oncogene –> continuous signal for cell proliferation
Oncoprotein: the protein encoded by an oncogene which then further promotes cell proliferation

Question 11

What are the 6 proteins involved in REGULATED GROWTH FACTOR SIGNALLING?

Answer 11

(i) Growth factor –> (ii) Receptor Tyrosine Kinase (a.k.a. GF Receptors) –> (iii) G-Proteins –> (iv) Intracellular Kinases –> (v) Transcription Factors –> (vi) Cyclins and CDKs –> PROLIFERATION

Question 12

(i) Over expression of a normal GF results in the development of an a_____e mechanism.

Answer 12

autocrine

*** this mutation by itself doesn’t cause cancer but it increases the risk of the proliferating cells to mutate

Question 13

(ii) What happens when normal GF receptors (RTKs) are over-expressed?

Answer 13

Can cause the overexpression of normal receptors OR a normal amount but they become mutated and abnormal where their kinase activity is always switched on a.k.a they are CONSTITUTIVELY ACTIVE

Question 14

What is the name of a growth factor that we now have inhibitors for when it is over-expressed?
What is one problem with this though?

Answer 14

EGFR. These GFs are smart though and they realise you are blocking their receptors so they find other ways in instead. E.g. even though glioblastomas are known to be mainly caused by EGFR mutations, using this inhibitor doesn’t stop it.

Question 15

(iii) What does the G-protein Ras do?

Describe a mutation in one specific G-protein.

Answer 15

Ras-GTP = active, Ras-GDP = inactive. Ras-GTP activates the P13K and BRAF arms of the kinases (the next downstream stage). However it doesn’t always activate the next step, only when required. Ras has intrinsic GTPase activity which allows it to convert back to the inactive Ras-GDP. It converts back with the help of GAP proteins (e.g. Neurofibromin-1) which bind to the Ras as it converts back to its inactive state.

Mutations = mutations to H-Ras, K-Ras, N-Ras. These changes the GTPase activity of Ras as well as making Ras resistant to GAPs. Overall this means Ras can’t be inactivated :O

*** Can’t make drugs against it - UNDRUGGABLE

Question 16

(iv) Describe 2 mutations of Intracellular Kinases.

Answer 16

1) BRAF mutation. Most common mutation results in production of the oncoprotein BRAF V600E. Responsible for causing 50% melanomas. (Melanoma can be treated with BRAF inhibitor)
2) P13K mutation.
Both result in increased phosphorylation and therefore an increase in the activity of anything downstream to it e.g. TFs —> end result = PROLIFERATION

Question 17

(iv) Describe a mutation in one specific Transcription Factor. What is an example of a cancer in which this mutation occurs?

Answer 17

• MYC. A proto-oncogene that is tightly regulated. The myc region on the chromosome is translocated from chromosome 8 to 14. In cancer MYC is up-regulated and/or over-expressed resulting in transcription of many genes involved in cell growth.
• TFs bind to DNA and either stimulate or suppress transcription in the nucleus. Malfunction in TFs results in the control they have being deregulated –> PROLIFERATION
• Cancer in which this occurs = Burkitt lymphoma
• ** Can’t make drugs against it - UNDRUGGABLE

Question 18

1. The ABL-BCR chimera (aka Philadelphia chromosome) is a mutation arising from what category of proteins? ABL-BCR = an unatural protein
2. What type of cancer does it cause?
3. What is the affect of this mutation?
4. Can it be cured? Why? By what?

Answer 18

1. Intracellular kinases
2. Leukemia
3. The ABL gene mutated so it moves from 9 to 22 chromosome. Result is a tiny chromosome comprising bcl locus and abl oncogene. Result = potent tyrosine kinase activity
4. Yep, leukemia cells (Acute Lymphoblastic Leukemia and (Chronic Myeloid Leukemia) require ABL-BCR tyrosine kinase activity to survive - therefore you can say it has an ONCOGENE ADDICTION. ABL-BCR inhibitors used to block pathway.

Question 19

What is the general role of protein tyrosine kinases

Answer 19

They act as an on/off switch for many cellular functions

Question 20

1. The JAK chimera is a mutation arising from what category of proteins?
2. How does the mutation occur?

Answer 20

1. Intracellular kinases
2. On codon 617 valine is changed to phenalylanine resulting in JAK constantly sending messages to STAT (a TF in the nucleus) which results in proliferation and survival
   * ** Good diagram on Slide 26

Question 21

1. Why are cyclins and CDK4 so important?

2. What does a mutation in these do?

Answer 21

1. In the end these are the two proteins that ultimately cause proliferation - these 2 proteins REGULATE THE CELL CYCLE.
2. Allows a check point in the cell cycle to be passed. Promotes cell cycle progression.
   G1-S = decision to continue into S phase and proliferate (this step is regulated by Cyclin D/CDK4 - therefore mutations in this cause this step to be skipped and progression in the cell cycle)
   G2-M = decision to continue into M phase or apoptose

Question 22

HALLMARK 2
The cyclin dependent kinase inhibitor CDKN2A encodes which 2 very important tumour suppressor proteins?
What does mutation to this CDKN2A do?

Answer 22

p14 (activates p53) and p16 (CDK inhibitor blocking CyclinD-CDK4 phosphorylation of Rb).
Mutation silences p14 and p16 –> lots of proliferation

Question 23

HALLMARK 2
Neurofibromin-1 (NF-1) is a positive/negative regulator of RAS.
What does it code for and what does this protein that it codes for do?
Increasing the GTPase activity inactivates RAS faster/slower.

Answer 23

Negative - therefore NF-1 is a TUMOUR SUPPRESSOR GENE.
It codes for GTPase-activating protein (GAP) that increases the GTPase rate of G-proteins.
Faster.

Question 24

HALLMARK 2
What does neurofibromin-2 (NF-2) code for?
What happens if a person has a mutation in NF-2 meaning they don’t have the protein in which NF-2 encodes.

Answer 24

It codes for neurofibromin-2 a.k.a. MERLIN –> this protein controls cell-cell junctions.
Mutation - cells are unable to ‘feel’ each other & therefore proliferate as they aren’t getting a signal to stop.

Question 25

HALLMARK 2 | What happens if a person has a beta-catenin mutation?

Answer 25

Destruction by APC & cell-cell inhibition (beta-catenin + E-cadherin complex) does not occur.

Question 26

Familial adenomatous polyposis is caused by a _____ mutation on one/two allelles. This progresses into colon cancer if the other allele in a polyp cell gets a _______ mutation.

Answer 26

Germline, one. | Somatic.

Question 27

With Adenomatous Polyposis Coli (APC) a germline mutation in one allele causes ______. In both alleles causes ______.

Answer 27

1: Familial Adenomatous Polyposis 2: Colorectal cancer

Question 28

How does the Wnt / APC / β-catenin pathway work?

Answer 28

B-catenin: involved in cell-cell adhesion + transcription Wnt signals to B-catenin APC = tumour suppressor, marks B-catenin for destruction.

Question 29

What is the beta-catenin/E-cadherin complex? What does a mutation in it cause?

Answer 29

Involves in cell-cell adhesion. | Loss of contact inhibition & therefore proliferation.

Question 30

How does PTEN prevent cells from blocking apoptosis and consequently surviving and proliferating. Is this a common mutation in cancer? What does the mutation cause?

Answer 30

RTK activates P13K which phosphorylates (activates) PIP2 to PIP3. This begins a cascade in which FOXO transcription factors are lost therefore preventing apoptosis. However PTEN dephosphorylates (inactivates) PIP3 to PIP2 therefore preventing the cascade from happening. It NEGATIVELY REGULATES P13K SIGNALLING. Yep, a common mutation in many cancers. Means that PIP3 is not dephosphorylated and therefore always active.

Question 31

HALLMARK 3 Where are BRCA1 and BRCA2 normally expressed? What is there physiological function? What does a mutation in these cause? What are 3 examples of a mutation in these proteins?

Answer 31

Normally expressed in the breast. They repair damaged DNA or if it can't be repaired then apoptosis is stimulated. A mutation means damaged DNA is not repaired and then the cells proliferate and pass on this damage. E.g. BRCA1 mutation, TP53 mutation, MYC amplification

Question 32

a mutation in p53 changes it from a ______ to an _____. Loss of function of p53 is found to some degree in ____% of tumours. What causes Li-Fraumeni syndrome? Can it progress to cancer? Muations also often occur in proteins/genes that regulate p53, e.g. ______.

Answer 32

tumour suppressor gene to an oncogene >50% Germline inheritance of a mutation on one allele (most common is to have a somatic mutation on both alleles). Li Fraumeni syndrome can progress to cancer if the person then gets a somatic mutation on the other allele. MDM2 gene - mutation here results in a deficiency in p53 (MDM2 inhibits it)

Question 33

Cells with mutated/deficient p53 are easier to treat. Chemotherapy can induce _______.

Answer 33

synthetic lethality

Question 34

HALLMARK 3 The BCL-2 protein is a ___-____ protein and inhibits _______. Overexpression of BCL-2 (as seen in many cancers) protects/induces cells to apoptosis.

Answer 34

pro-survival, apoptosis protects

Question 35

HALLMARK 4 What is senescense? What 3 things induce it? What gives the cancer cells its immortality?

Answer 35

Deterioration with age to grow and divide. PERMANENT G1 CELL CYCLE ARREST. Induced by: p53, p16, Rb activation TELOMERASE - it repairs telomeres

Question 36

HALLMARK 3 | What 3 ways does p53 maintain genomic stability?

Answer 36

1. p-53 induced cell cycle arrest 2. p-53 induced senescense 3. p-53 induced apoptosis

Question 37

What is a telomere? Why is it thought of as a clock?

Answer 37

Found on the end of each chromosome, undergoes shortening with each division, counts number of divisions (like a clock) and then undergoes apoptosis when the chromosome gets to a certain length.

Question 38

So far we have looked at the first 4 hallmarks of cancer. All of these are driven by 5 mechanisms. What are they?

Answer 38

1. Point mutations - e.g. EDGF, BRAF, HRAS, JAK, P13K mutations (proliferative signals), p53 (tumour suppressor gene). Leads to increased kinase activity. Leads to increased downstream signalling. 2. Chromosomal translocation - Philadelphia chromosome (alteration - hybrid protein - heightened tyrosine kinase activity) - Myc translocation (over-expression) 3. Activation by gene amplification - V common with the GFRs/RTK - Nothing wrong here, there is just A LOT of them/over-expression and this creates a predisposition for cancer to develop 4. Epigenetic disease - epigenetic = reversible, heritable changes in DNA that occur not due to mutation - cancer cells switch on oncogenes and switch off tumour suppressor genes 5. miRNA - binds to mRNA and blocks translation of important proteins e.g. tumour suppressor genes

Question 39

HALLMARK 1 In Burkitt lymphoma, the Myc-containing segment on chromosome __ is translocated to chromosome __ close to the ___ gene resulting in overexpression of _____ and hectic __________.

Answer 39

8, 14, IgH, Myc, transcriptional activity