1
Q
Define epigenetics.
A
Epigenetics is the study of hereditable changes in gene expression that do not involve a change in the primary DNA sequence.
The epigenome is changed, while genome stays the same.
2
Q
Describe the epigenetic modification types and how they are linked to cancer.
A
- Epigenetic changes are established during differentiation and are maintained throughout cycles of cell division, allowing cells to have distinct identities with the same genetic sequence.
- Modifications will regulate what part of the genetic information can be accessed by cellular machinery by altering the structure of chromatin.
- They include –> DNA methylation, histone modifications and miRNA regulation.
- Failure in maintaining these epigenetic marks will lead to activation or inhibition of potentially key signalling pathways - all cancers show epigenetic aberrations – usually in genes that are involved in the classical hallmarks of cancer: cell cycle, signal transduction, apoptosis, DNA repair, senescence and metastasis.
- The fact that these epigenetic modifications are reversible makes them an attractive target for cancer therapy.
3
Q
Describe the mechanism of DNA methylation and how they can cause silencing.
A
- Occurs on the 5’ position of cytosine, mainly at CpG islands (areas rich in CpG dinucleotides) usually near promoters.
- 3-4% of CpG islands are methylated.
- Catalysed by DNA methyltransferases. (DNMT) which transfer methyl group from S-adenyl methionine cytosine.
- They can be pharmacologically reversed (e.g. by DNMT inhibitors) – useful in therapy as we can switch TSGs back on.
- Can turn a gene ‘on’ (by unmethylation) or ‘off’ (by methylation) on CpG islands
- —-> In normal cells: methylation of CpG islands is associated with gene silencing – such as in imprinting and X chromosome inactivation (occurs during development).
- —-> In normal cells: CpG islands are generally unmethylated (allowing transcription) and CpG non islands are methylated (also allowing transcription).
- Silencing –methylation will cause the recruitment of methyl binding domain MBD proteins that interact with HDAC and chromatin remodelling enzymes that cause the coiling of DNA –> no transcription.
4
Q
What are the three hypotheses behind aberrant regulation of DNA methylation in cancer?
A
- There are constitutional methylation errors that are occurring in the germ-line. There are some claims that DNA methylation can be inherited transgenerationally. This may mean that you inherit an increased risk of cancer.
- DNA methylation occurs after DNA damage – cancerous cells are exposed to damage and that during the repair process there’s ‘seeding’ of aberrant DNA methylation. Evidence: if you introduce a double strand break and let the cell repair it, you’ll see methylation changes.
- Stem cells – in embryonic stem cells there are ‘bivalent’ patterns of both active and repressive marks at the promoter region of developmentally important genes. These are established by polycomb group of proteins and these same genes are often hypermethylated in cancers.
5
Q
Explain the mechanism of histone modification.
A
- Histones can undergo post-translational modification on their N-terminal tail.
- These modifications can affect the electrostatic repulsion of the histones – leading to either a condensed state of chromatin or a more open conformation, this can determine whether a gene is expressed or not.
- These modifications include methylation, acetylation, and phosphorylation.
- Acetylation is mediated by histone acetylases or histone deacetylases on lysine residues. HATs will relax chromatin allowing transcription and HDACs will coil up chomatin causing transcription be switched off.
6
Q
How is methylation related to histone modifications?
A
- Unmethylated histone – open conformation, acetyl groups are present – genes are expressed.
- Methylated histone – allows recruitment of proteins to DNA, called methyl domain binding proteins (MBD) and they act as a platform for recruitment of further complexes – including histone deacetylases which will catalyse removal of acetyl group leading to further modification of the tails.
- - Histone methyltransferases will also be recruited and will methylate the histone tail, changing electrostatic repulsion – closed histone state and transcriptional repression.
- - HP1 also plays a part in spreading the repression along the other histones.
7
Q
How does miRNA play a role in epigenetics?
A
- Small, ~22nt non-coding RNA that regulates gene expression via the post-transcriptional silencing of target genes.
- They bind complementary to their target genes (sequence-specific) and affect their transcription or target them for degradation.
- They are part of RNAi, which acts as a host defence mechanism.
8
Q
How can miRNA be affected in cancers?
A
- Some microRNAs have been found to undergo silencing in cancers causing the activation of the target genes they used to silence.
- e.g. let-7 and miR15/16 play important roles in down-regulating RAS and BCL2 oncogenes (respectively), and their silencing has been found in cancer cells.
- A decrease in expression of miR-125b1, a miRNA that functions as a tumor suppressor (shown to decrease proliferation and promote apoptosis), was observed in prostate, ovarian, breast and glial cell cancers.
9
Q
List some of the alterations in epigenetic mechanisms seen in cancers.
A
- Widespread hypomethylation is seen in cancers, leading to genomic instability..
- Hypermethylation of TSGs – it has been reported in the Rb, BRCA and p16 genes.
- Deregulation of histone modification patterns – leads to aberrant silencing of TSGS. e.g. EZH2 is overexpressed in several cancers, leading to hypermethylation.
- Deregulation of miRNAs – there’s repression of tumour suppressor miRNAs (shown above) and activation of oncogenic miRNAs – e.g. miR-21 which targets PTEN.
10
Q
What are two classes of SMI’s approved for use that target epigenetic modifications?
A
i. DNMT inhibitors – Azacytidine and Decitabine: used in treatment for high-risk MDS, CMML and AML.
ii. HDAC inhibitors – Vorinostat: approval in US for treatment of advanced cutaneous T-cell lymphoma
11
Q
Describe the mechanism of action of azacytidine and decitabine and outline their issues.
A
- Have hypomethylating activity - they are both two modified analogues of deoxycytidine.
- The only difference in these is that Decitabine is the 2’deoxy form meaning that it can only be incorporated in DNA, while Azacitidine can be incorporated in both DNA and RNA.
- They are incorporated into DNA as a false base and they are recognised as a methylated base and covalently link the DNMT causing it to be sequestered so it can no longer act.
- There’s evidence that they may be best administered at lower doses – they have cytotoxic activity (can cause DNA instability) – but they also have a biological optimum at where they best demethylate. Transient low doses have been shown to be just as effective.
- These drugs have a number of problems – toxicity, short half-lives, hard to deliver, there are currently no markers to tell which drugs are needed.
- -The hypermethylation returns once treatment is stopped meaning that constant administration is need.
12
Q
How is decitabine useful on drug-resistant tumours?
A
Decitabine has been shown to reactivate the MLH1 gene, a gene commonly associated with hereditary nonpolyposis colorectal cancer.
- MLH1 is necessary for platinum induced cell death and is silenced by methylation.
- The loss of MLH1 gene is associated with the chemo-resistance of tumours.
- In addition, decitabine has shown to re-sensitise drug resistant xenografts to cytotoxic drugs such as carboplatin.
- Stratification biomarkers – analysing methylation of MLH1 in plasma DNA and you can stratify patients on methylation status to predict response to carboplatin.
13
Q
How is a MGMT considered a biomarker for Temozolomide (TMZ) response?
A
- TMZ is an alkylating agent.
- MGMT is the DNA repair gene is responsible for the repair of DNA damage caused by the chemotherapeutic TMZ.
- MGMT can be silenced by methylation of its CpG islands near promotor.
- Patients with methylated MGMT would have better response to TMZ and survive longer.
- MGMT could be used as biomarker for prognosis.
14
Q
Explain how targeting tumour stem cells via EZH2 is a possible future cancer therapy.
A
- Tumour stem cells are one of major reasons why conventional chemotherapy fails.
- If we target stem cells, we can cause tumour regression and even maybe a cure.
- Expression profiling on tumour stem cells shows that they overexpress NOS targets (marker for stem cells) and polycomb complex which is involved in repressing transcription.
- PRC2 is a protein polycomb complex that catalyses protein methylation of lysine residues on histone proteins.
- –> It is made up of three main proteins including EZH2, which has histone methyltransferase – making it a druggable target for stem cells.
- Knock down of EZH2 using siRNA methods we see a reduction in its target H3K27me3 –> less of a repressive mark.
- Many genes in cancer, including TSGs are silenced by mechanisms associated with the H3K27me3.
- We also saw a reduction in number of side population stem cells and reduction in their ability to grow in an anchorage independent manner and reduction in ability to form tumours.
- EZH2 is frequently overexpressed in a wide range of tumour types, it is a driver of metastasis and is also expressed in platinum resistant tumours.
- This shows that EZH2 could a potential anti-cancer target. miRNAs can be used to selectively repress oncogenes in humans, e.g. miR-101 targets EZH2.
