30-01-23 - Molecular Basis of Colon Cancer Flashcards
Learning outcomes
- to define the inheritance patterns of Familial Adenomatous Polyposis, FAP, and Hereditary non-polyposis colon cancer, HNPCC (Lynch syndrome)
- to differentiate between genetic tests for FAP and HNPCC
- to describe the molecular mechanisms underlying FAP and HNPCC
- to identify additional risk factors for colon cancer
- to identify measures which may protect against colon cancer
What % of colorectal cancer patients have family history?
How often are causative mutations identified?
What are the 2 main types of Familial colorectal cancer?
- About 25% of colorectal cancer patients have family history
- Causative mutations identified in 5-6% of cases
- 2 main types of Familial colorectal cancer:
1) Familial Adenomatous Polyposis (FAP)
2) Hereditary nonpolyposis colon cancer (HNPCC or Lynch syndrome)
What is the mode of inheritance Familial Adenomatous Polyposis (FAP)?
What are features of Familial Adenomatous Polyposis (FAP)?
- The mode of inheritance for Familial Adenomatous Polyposis (FAP) is autosomal dominant (inheriting risk of cancer, not cancer itself)
- In FAP, there are a large number of polyps (100s or more) developing from adolescence onwards
- Most colon polyps are harmless, but over time, some colon polyps can develop into colon cancer, which may be fatal when found in its later stages
- 90% patients also have pigmented lesions in retina (CHRPE)
What is the gene defect associated with FAP?
What chromosome is the APC gene found on?
How large is it?
What defects do we typically see in the APC gene in FAP?
Where is the gene commonly affected?
What can cause attenuated FAP?
- The gene defect associated with FAP is in the Adenomatous polyposis coli (APC) gene, which is a tumour suppressor gene
- It is located on Chromosome 5 q21-22
- The APC gene is 2843 amino acids (large gene)
- With FAP, we typically see nonsense (coding for stop codon) or frameshift mutations in the APC gene that result in a truncated (shortened) protein
- There are hotspots at the end of the APC gene which are commonly affected in patients with FAP
- If there are gene defects at the start or end of the APC gene, this can cause attenuated FAP, where there is still a high risk of colorectal cancer but there is less polyps and a later onset of tumours
How can we test for an APC gene defect?
- We can check for an APC gene defect using direct sequencing
- In most tissues one copy of the APC is normal while one is defective.
- We can compare them to see if there has been a mutation
What is the 2-hit hypothesis?
How does the two-hit hypothesis explain why defects in the APC gene predispose to individuals to cancer?
- The Knudson hypothesis, also known as the two-hit hypothesis, is the hypothesis that most tumour suppressor genes require both alleles to be inactivated, either through mutations or through epigenetic silencing, to cause a phenotypic change.
- How does the two-hit hypothesis explain why defects in the APC gene predispose to individuals to cancer:
- A normal person is safe, as they have 2 normal copies of the APC tumour suppressor gene
- If they acquire a mutation by chance in 1 of the copies of the APC gene in 1 cell, then there is likely no consequence, as they are protected by the other copy of the tumour suppressor gene that is still able to work
- In patients with an inherited copy a defective APC gene, if they by chance acquire a mutation in the 2nd copy of the gene, they have no working tumour suppressor gene
- This can result in them being unprotected from this form of cancer
What are 2 functions of the APC gene?
What are the hot spot sections for mutations on the APC gene responsible for?
- 2 functions of the APC gene:
1) Binds Beta-catenin
2) Binds microtubules - The hot spot sections for mutations on the APC gene are responsible for:
1) Catenin binding
2) Microtubule binding
3) EB1 binding - This means these functions of the APC gene will often be affected in the development of FAP
How does the APC gene affect the functioning of beta catenin?
What can happen if the APC gene does not bind to beta-catenin? (in picture)
Describe how Beta-catenin and APC participate in wnt signalling.
How is this pathway affected when there is a defective APC gene?
How does this lead to tumour formation?
- Describe how Beta-catenin and APC participate in wnt signalling (in picture)
- When there is a defective APC gene, part of the pathway (highlighted in photo) remains active irrespective of what is occurring upstream in the pathway
- This results in WNT independent promotion of this part of the pathway, which leads to proliferation of gut stem cells and tumour formation as seen in the polyps in FAP
What effect does beta-catenin have on normal colonic epithelial cells?
What does the lack of beta-catenin binding from the APC gene lead to?
What 4 negative effects does lack of beta -catenin have on colon cells?
What do these 4 things lead to?
- In normal colonic epithelial cells, beta-catenin forms part of the complex (with microtubules) that holds cells together tightly through adherence junctions
- A lack of beta catenin results in a loss of connection between actin and adherence junctions
- 4 negative effects lack of beta catenin binding from the APC gene has on colon cells:
1) Distorted cytoskeleton network
2) Loss of polarity
3) Decreased cell-cell adhesion
4) Aberrant cell migration - These 4 things lead to cancer cell initiation and progression, which results in polyps developing
What is the APC responsible for during cell division?
What occurs if we have APC defects?
- During cell division, APC binds to EB1 and microtubules in the spindle
- If we have APC defects, this can result in chromosome instability (CIN)
- If APC doesn’t bind to EB1, then we get abnormal anchoring of chromosomes to spindle or detachment of chromosomes from spindle
- When the signal to divide comes, chromosomes may migrate abnormally to opposite sides of the spindle
- This can result in chromosomes becoming aneuploidy - the condition of having an abnormal number of chromosomes in a haploid set
Describe the different cell types in the crypts of the colon?
What are the different regions of proliferation in crypts?
How will this system be affected when there is an APC gene defect?
What can these effects combined with lack of beta catenin binding lead to?
- In the crypts of the colon, stem cells are at the bottom that begin to move differentiate and move up the crypt
- At the bottom, the Wnt pathway is active, resulting in cell proliferation
- Further up, the Wnt pathway is inactive, which results in no cell proliferation
- If there is an APC defect, it can cause abnormal proliferation in the Wnt pathway active segment
- It can also affect the Wnt pathway inactive segment, as there can be Wnt independent pathway signalling (seen earlier)
- These combined with the problems from lack of beta-catenin binding can lead to starting to the cancerous pathway
- This is why APC gene defects lead to colon cancer
What are 5 extra-Intestinal involvements of APC gene defects?
- 5 extra-Intestinal involvements of APC gene defects:
1) Masses of benign tumours
2) Jaw cysts
3) Sebaceous cysts
4) Osteomata
* An osteoma (plural: “osteomata”) is a new piece of bone usually growing on another piece of bone, typically the skull.
* It is a benign tumor
5) Pigmented lesions of the retina (CHRPE)
What is the mutation of APC also seen in?
- Mutation of APC is also seen in sporadic tumours
- Sporadic tumours are cancer that occurs in people who do not have a family history of that cancer or an inherited change in their DNA that would increase their risk for that cancer
- Mutation of APC alone is not sufficient to cause cancer
What % of colon cancers are Hereditary Nonpolyposis colorectal cancer (HNPCC/ Lynch syndrome)?
What is the mode of inheritance?
What are 3 features of HNPCC?
- Hereditary Nonpolyposis colorectal cancer (Lynch syndrome) make up approximately 3% of cancers
- The mode of inheritance is autosomal dominant
- 3 features of HNPCC:
1) High risk of colon tumours
2) Can be underlying cause of other tumour types eg endometrium, ovarian, small intestine, stomach
3) Low numbers of polyps
What are 4 gene defects associated with HNPCC mutations?
What are these 4 genes involved in?
- 4 gene defects associated with HNPCC mutations:
1) MLH1 (most common defect)
2) MSH2 (2nd most common defect)
3) PMS2
4) MSH6 - These genes are all involved in DNA mismatch repair
- MSH2 and MHSH3/6 recognise mutated DNA
- PMS2 and MLH1 help to recruit proteins that cut out mutated genes then recruit ligase to stitch together repaired DNA
Which regions are more susceptible to errors?
What can occur if errors are not identified?
What are the 3 steps in the development of MIN?
- Repetitive regions of DNA are more susceptible to errors
- If errors are not identified, this can cause Microsatelite instability (MIN)
- 3 steps in the development of MIN:
1) When replicating a repetitive region, slippage can occur
2) If there is no recognition by DNA mismatch repair systems, this can result in insertion and deletion, leading to MIN
3) If there is MIN, in repetitive regions, some cells may have 10Ts in a row, while some might have 10 Ts in a row
How do we test for a defect that causes microsatellite instability?
What does a greater range in consecutive residues suggest?
- To test for a defect that causes microsatellite instability, we can look at normal DNA and then tumour DNA and see if there is any change in microsatellites in well known positions
- Is there a different in the space of the graph between tumour and normal?
- The greater the range of consecutive t residues for example (used in previous example), the stronger the chance of the patient being microsatellite unstable
How can we use histology to tests for a defect in the DNA mismatch repair genes?
- How we can use histology to tests for a defect in the DNA mismatch repair genes
- We can use antibodies for our 4 target proteins and use a staining pattern of histological sections against those antibodies as a code in order to tell us where to start looking for genetic mutations
- In photo diagram:
- The sample being probed for MLH1 antibodies is dark, meaning there are MLH1 genes present
- The sample being probed for MSH2 is light, meaning the patient the sample is negative for MSH2 and may have a mutation in the MSH2 gene, which gives us a clear idea of where to start looking for this mutation in the patient
What % of colon cancer are cases with familial risk?
What % are FAP and HNPCC? What are most cases?
- 10 to 30% of colon cancer are cases with familial risk
- 2 to 3% of colon cancer cases are HNPCC and less than 1% are FAP
- Most cases are sporadic - Cancer that occurs in people who do not have a family history of that cancer or an inherited change in their DNA that would increase their risk for that cancer
How can we identify different mutations that have occurred in the polyp to carcinoma sequence?
Describe the polyp to carcinoma sequence (in picture)
- In the development from polyps to carcinoma sequence, we can identify and purify polyps and precancerous lesions (non-cancerous lesions - adenomas) and look and see genetically what has happened
- This allows us to look at different mutations in different genes and put them in order of sequence of events
- Describe the polyp to carcinoma sequence (in picture)
What kind of gene is the EGFR gene?
When is the EGFR gene overexpressed?
What 4 events does this result in?
How can this be prevented?
How do we detect defects in EGFR?
- The EGFR gene is an oncogene
- The EGFR is overexpressed in a majority of tumours due to activating mutations
- 4 events this results in:
1) Proliferation
2) Angiogenesis
3) Metastasis
4) Inhibition of apoptosis - This can be prevented using antibodies in cetuximab against EGFR to block signalling to EGFR, meaning it wont be able to bind to its ligand
- We can use histological analysis to detect EGFR mutations
When is cetuximab used for EGFR mutations?
Why is this?
- Cetuximab is only use in EGFR mutations when there is a wildtype KRAS gene
- Tis is because KRAS is an oncogene, so if it is mutated, it is always switched on
- There is no point using monoclonal antibodies to block EGFR activity if later on in the pathway the signal is switched on through the activation of KRAS
