Molecular Basis of Colon Cancer

Question 1

What proportion of colorectal cancer patients have family history ?

Answer 1

25% of colorectal cancer patients have family history

Question 2

What proportion of colorectal cancers have causative mutations been identified for ?

Answer 2

Causative mutations identified in 5-6% of cases

Question 3

What are the main types of colorectal cancer ?

Answer 3

• Familial adenomatous polyposis (FAP)

- Hereditary non-polyposis colon cancer (HNPCC or Lynch syndrome)

Question 4

State the inheritance pattern of FAP.

Answer 4

Autosomal dominant

Question 5

What are the main macroscopic features of FAP ?

Answer 5

1) Large numbers of polyps (100s or more) developing from adolescence onwards
2) 90% of patients also have pigmented lesions in retina (CHRPE)

Question 6

Are polyps cancer ?

Answer 6

No, they are precancerous lesions, some of which can progress to cancer.

Question 7

What is the gene defect responsible for FAP ?

Answer 7

Mutations (mainly nonsense or frameshift ones, resulting in a smaller than normal protein) in the Adenomatous polyposis coli (APC) gene, on chromosome 5 q21-22. This gene encodes 2843 amino acids.

Question 8

Define attenuated FAP.

Answer 8

FAP with lower number of polyps, and occurring at a later age

Question 9

Identify methods to test for gene defects in FAP.

Answer 9

1) Protein truncation test (looking for a shorter protein being produced that normal)
2) Direct sequencing

Question 10

Why do defects in APC predispose to cancer? (i.e. what does APC do ?)

Answer 10

APC is essentially a tumour suppressor gene.
It “indirectly regulates transcription of a number of critical cell proliferation genes”.

APC binds to:
1) Beta-catenin:
Transcription factor. Promotes the transcription of genes that promote cell division.

If beta catenin present at low levels, binds to APC which results in degradation of beta catenin. If beta catenin is not bound to APC, its level rise, gets into nucleus where it can work with other transcription factors to promote expression of genes responsible for cell division. If protein defective (i.e. mutation in APC) and not binding to beta catenin, will get transport to nucleus and over-expression of genes telling cell to divide (positive signal) .

Beta-catenin and APC participate in Wnt signaling. In absence of Wnt signaling, receptor remains inactive but APC is active (results in degradation of beta catenin, inactive transcription factor complex and relevant genes are switched off) . When cells want to divide under normal circumstances, this process is tightly controlled; Wnt signal binds to receptor, stimulates signaling pathway which inactivates APC, which stabilises beta catenin and results in activation of genes responsible for cell division. In the absence of Wnt signal, inactive APC so not degraded beta catenin, so active transcription factor complex, driving these cells to divide (growth factor
independent signaling).

Beta catenin involved in complex connecting actin to adherens junctions (since in the colon, cells held are together tightly in adherens junctions). With mutant APC, the junction us not as tight, so loss of connections between cells. Cells start to divide in disordered way. This can contribute to cancer initiation and progression .

2) Microtubules
Defects in APC also cause chromosome instability (CIN). In normal cell, in mitosis, cells are lined neatly on the microtubules. EB1 attaches chromosomes onto spindles. Both APC and EB1 help that happen. IF APC mutants and cannot attach to EB1, then process becomes defective. If chromosomes cannot attach well, abhorrent cell division, leading to
aneuploidy. Furthermore, spindle has to be oriented within cell in particular direction. If spindle does not, instead of cells growing upwards in single layer, may grow on top of each other.

Question 11

Given that we get two copies of APC, why is it a problem if we inherit a mutant copy of APC from a parent ?

Answer 11

In this case, APC inherited mutation in every cell of body of APC gene. Other copy is fine, preventing cells from dividing. If by chance, one of these cells with already one mutation gets another mutation (loss of functioning copy of the gene), that means cell has no protective working APC, meaning they go on and produce cancer

Question 12

Why is the colon affected by FAP ?

Answer 12

1) That is where APC is expressed
2) Very fast turnover of cells (5 days to move from stem cell to the top of vili where they tend to get lost). That is controlled to some extent by wnT pathway in that it is active towards bottom (i.e. proliferation) and inactive towards top (i.e. no proliferation).

Question 13

Identify extra-intestinal involvement in FAP.

Answer 13

• Jaw cysts
• Sebaceous cysts
• Masses of benign tumours
• Osteomata
• Pigmented lesions of the retina (CHRPE)

Question 14

Define sporadic cancer. Do AFP mutations also occur in sporadic cancers ?

Answer 14

Cancer that occurs in people who do not have a family history of that cancer.
Yes, mutation of APC is also seen in sporadic tumours.

Question 15

Is mutation of APC alone sufficient to cause cancer ?

Answer 15

No, other genetic changes are needed

Question 16

What proportion of all colon cancers are hereditary non-polyposis colorectal cancers (Lynch syndrome) ?

Answer 16

3%

Question 17

State the pattern of inheritance of HNPCC.

Answer 17

Autosomal dominant

Question 18

What cancers is HNPCC associated with ?

Answer 18

Colon cancer

Can be underlying cause of other tumour types eg endometrium, ovarian, small intestine, stomach.

Question 19

What is the main macroscopic feature of HNPCC ?

Answer 19

Low number of polyps

Question 20

What genetic defect(s) are associated with HNPCC ?

Answer 20

Genes which encode proteins involved in DNA mismatch repair:

• MSH2 and MSH3/6 recognize the DNA mismatch. Having done that, they recruit other proteins that are able to cut out mismatch nucleotides and insert the correct ones.
• PMS2 and MLH1 have endonuclease activity, so they can cut out mismatch nucleotides and insert the correct ones.

Repetitive regions are especially susceptible to DNA mismatch, which results in micro-satellite instability (MIN).
When replication machinery tries to replicate these regions, it struggles. If the mismatch repair pathway does not work, it can result in deletion or insertion of extra nucleotides at positions of MINs.

Question 21

Define microsattelite instability.

Answer 21

Predisposition to mutation that results from impaired DNA mismatch repair

Question 22

Identify ways to test for defects in the genes associated with HNPCC.

Answer 22

1) Sample from tumor and identify if it has MINs.
Someone with FAP would have microsatellite stable whereas lymph syndrome would have microsatellite unstable.

2) Once we know unstable, rather than sequencing for different genes, can test which proteins are expressed within tumours and from there, look at potential genes which may be mutated .

Question 23

List the main differences between FAP and HNPCC in terms of:

• Number of polyps
• Mutation rate
• Cancer risk
• Average age of onset
• Lifetime risk (penetrance)

Answer 23

FAP
• Large number of Polyps (because of unregulated pathway)
• Low mutation rate
• High cancer risk because of high number of polyps
• Life-time risk (penetrance) close to 100%

HNPCC
• Low number of polyps
• High mutation rate
• High cancer risk despite low number of polyps (because of high mutation rate)
• Life-time risk (penetrance) approx 80%

• Average Age of onset similar (approx 40)

Question 24

Define penetrance.

Answer 24

“proportion of individuals carrying a particular variant (or allele) of a gene (the genotype) that also express an associated trait”

Question 25

What is a treatment option for FAP and HNPCC ?

Answer 25

Having colon removed.

Question 26

Describe the process of screening for FAP and HNPCC.

Answer 26

• > 50 years screened every 2 years for occult blood (since colon cancer can arise sporadically), and if positive then colonoscopy
• If known FAP/HNPCC in the family- biannual colonoscopy from 25 years
• High to moderate risk- colonoscopy every 5years from age 50-75

Question 27

Explain what it means to be high to moderate risk of FAP/HNPCC.

Answer 27

Close family members are affected

Question 28

Briefly explain the polyp to carcinoma sequence, mentioning the accompanying mutations.

Answer 28

1) Intestinal epithelial lining undergoes mutation in APC, resulting in adenomas (polyps).
2) Mutation in kRAS occurs.
3) Adenoma turns into carcinoma.

Question 29

Identify another mutation which may be involved in sporadic colorectal cancers, besides those already identified for FAP/HNPCC.

Answer 29

EGFR mutation: potential oncogene. Receptor binds to ligand, dimerisation occurs, activation of tyrosine kinase occurs, eventually end results are stimulation of proliferation, angiogenesis, metastasis and inhibition of apoptosis.

If not controlled, protein is over-expressed, leading to ligand dependent signaling and sporadic tumors occur.

Question 30

Identify a potential management for EGFR related sporadic colorectal cancers. How does it work ?

Answer 30

Cetuximab.
Monoclonal antibody, binds to the ligand site of the receptor and blocks dimerisation of protein and sops it from promoting proliferation etc. HOWEVER, Cetuximab only used where wildtype Kras (Kras is often also mutated in the pathway. If block first part of the pathway, that’s good but then still have mutated form of KRAS which will still drive cells to become cancerous)

Question 31

Identify risk factors for colorectal cancer.

Answer 31

• Genetics
• Diet
• Obesity
• Alcohol

Question 32

What about diet increases/decreases the risk of colorectal cancer ?

Answer 32

• High Fibre Diet reduces risk (because toxins, and bile salts etc. spend less time in bowel)
• Fish decreases risk
• Veggies and fruits decrease the risk (presence of anti-oxidants and folate protect DNA from damage)
• High intake of red and processed meats increases risk (meat increases bile production, which can be metabolised by bacteria in fut into secondary bile salts, which can be carcinogenic)
• Cooking at v high temperatures increases risk (Asparagine can be converted into a carcinogen at v high temperature)

Question 33

Why does obesity increase the risk of colorectal cancer ?

Answer 33

1) Intake of potential carcinogens

2) Insulin like growth factors more likely to be synthesised, which have a tendency to stimulate growth

Question 34

What proportion of additional risk do overweight and obese men have of colorectal cancer ?

Answer 34

• Overweight men 25% increased risk

* Obese men 50% increased risk

Question 35

Why does alcohol increase the risk of colorectal cancer ?

Answer 35

1) Lots of alcohol is associated with a low folate diet. Folate is required for synthesis of nucleotides.
2) Alcohol also metabolised into metaldehyde which is a carcinogen

Question 36

What is the increased risk of colorectal cancer if if >5 units per day of alcohol ?

Answer 36

40% increased risk if >5 units per day

Question 37

Identify any potential preventative measures against colorectal cancer.

Answer 37

Aspirin, in low doses for a number of years (less pre-cancerous lesions forming)

Question 38

Why does aspirin prevent colorectal cancer ?

Answer 38

• Aspirin and other NSAID’s inhibit COX-2, which is increased in early stages of colorectal cancer. This COX-2 is associated with increased prostaglandin synthesis, stimulation of proliferation and angiogenesis, and inhibition of apoptosis.

Question 39

What is the problem with the usage of aspirin and other NSAIDs (i.e. COX-2 inhibitors) to prevent colorectal cancer ?

Answer 39

• Increased risk of cardiovascular problems (Prostaglandins regulate blood pressure)

Hence, some COX-2 inhibitors have been withdrawn because of these risks