Module 1 Essay Plans Flashcards
Describe the mechanisms of epigenetic regulation
Epigenetic regulation is managed via three main mechanisms: DNA methylation – considered an on/off switch for genes; histone modification – affects the modelling of chromatin and so its accessibility for transcriptional machinery; and miRNA silencing – acts as a ‘dimmer switch’ on gene expression. Together these mechanisms provide a level of control beyond the sequence of bases in DNA.
DNA methylation occurs on the 5’ carbon of cytosine, usually in CpG pairs (where C is cytosine, G is guanine, and p is a phosphodiester bond). CpG pairs are underrepresented in the genome because the cytosine is vulnerable to losing its amide group which, when methylated, causes the cytosine to resemble thymine closely enough that DNA repair mechanisms do not register the change as damage and do not repair it. However, CpG pairs are better preserved in so-called ‘CpG islands’ which are large concentrations of CpG pairs found within gene promoters. Though complex, methylation in the gene body will usually activate the gene, whereas methylation within a gene promoter will silence the gene. This silencing is partially due to steric inhibition of transcriptional co-activator recruitment to the promoter, and partially due to recruitment of methyl-CpG-binding proteins (MBPs) which remodel chromatin to make it inaccessible for transcription.
DNA methylation is carries out by DNA methyl transferase enzymes (DNMTs): DNMT 3a and 3b methylate DNA de novo, whereas DNMT 1 finishes hemi-methylated DNA. Methylation marks are interpreted by reader enzymes, and are actively removed by eraser enzymes.
Histones are proteins around which DNA is wound. Nucleosomes are structures consisting of a core of eight histones, with ~147 base pairs of DNA wound around them, an extra linker histone, and a region of linker DNA connecting to the next nucleosome. Histone modifications can be varied and include: acetylation, methylation, phosphorylation, sumoylation, and ubiquitination, all of which can alter the accessibility of the DNA in the nucleosome for transcription. Easily available, unwound chromatin is known as euchromatin, and is typically found in undifferentiated cells (e.g. embryonic stem cells). Euchromatin is characterised by H3K9 and 14 acetylation, and H3K4 methylation. Heterochromatin refers to tightly wound, inaccessible chromatin, and is usually found in highly differentiated cells. Heterochromatin is characterised by H3K9 and 14 de-acetylation, and H3K27 methylation.
Histone modification is sometimes coupled with DNA methylation. MBPs can recruit histone de-acetylase complexes to make histone tails more suitable for subsequent methylation, and have histone methyl transferase domains which can directly methylate histones. Furthermore, methylated histone tails may recruit DNMTs to methylate associated DNA for long-term gene silencing.
miRNA molecules are small sequences of RNA that are transcribed in order to bind to specific mRNA and prevent its translation, thereby regulating expression of the associated gene. Primary miRNA is transcribed in the nucleus, and cleaved by Drosha into Pre-miRNA. Pre-miRNA exits the nucleus via Exportin 5, and is cleaved by Dicer enzymes, after which it associates with other proteins to form an RNA-induced silencing complex (RISC) which either cleaves target mRNA, or binds to it and prevents its translation by ribosomes.
Describe the role of epigenetic regulation in cancer
Though genetic changes have classically characterised various cancers, epigenetic regulation also plays a significant role. Across most cancers, a general trend of global hypomethylation is seen, leading to genomic instability, expression of aberrant transcripts, and increased oncogene expression. Histone modifications also play a role in cancer: loss of H4K16 acetylation and H4K20 methylation are global changes seen across all cancers.
More localised methylation abnormalities may lead to inactivation or dysregulation of certain cancer-associated genes e.g. death associated protein kinase (DAPK) may be inactivated by hypomethylation, leading to inactivation of pro-apoptotic pathways. The Hox-11 proto-oncogene is converted to an oncogene when hypomethylated, as is seen in leukaemia. OPCML and BRCA1 are tumour suppressor genes, and if their promoters become hypermethylated they become silenced, as seen in ovarian and breast cancer respectively. Methylation may also silence genes that are targets for therapy, leading to resistance.
Methylation also increases the susceptibility of gene sequences to mutation: 50% of p53 mutations in sporadic colon cancer occur at methylated cytosines. Furthermore, methylation increases the chance of pyrimidine dimer formation within the p53 gene sequence in response to UV light – an important step in the pathophysiology of skin cancer. It has also been demonstrated that gene mutations in the lungs of smokers are more likely to occur at methylated CpG sites (where C is cytosine, G is guanine, and p is a phosphodiester bond).
Epigenetic regulation is a promising target for future cancer therapies, and provides a chance to make less toxic therapies which could be personalised depending on an individual’s epigenetic signature. Currently the therapies available targeting epigenetic markings are DNA methyl transferase inhibitors (DNMTi) and histone de-acetylase inhibitors (HDACi). DNMTi are substitute base pairs which are incorporated into DNA upon replication, but cannot be methylated and trap the DNMT enzyme leading to its degradation and preventing copying of epigenetic marks into new DNA – they can be used to treat myelodysplastic syndrome. HDACi bind to the catalytic domain of HDAC and chelate a zinc ion, thereby preventing the enzyme’s action – they can be used to treat t-cell cutaneous lymphoma.
Epigenetic regulation provides promising options for biomarkers as well as treatments. Epigenetic changes occur at 10-100 times greater frequency than mutations in the genome, and so may provide a more incremental and sensitive picture of progression towards cancer that allows earlier diagnosis. Though incremental, epigenetic changes can have pleiotropic effects across the genome, and so are still significant. GSTP1 methylation is an example of a proven epigenetic biomarker: it indicates the presence of prostate cancer.
A significant advantage of epigenetic markers is that they can be obtained from cell-free DNA via a blood sample, which is far less invasive than the biopsies currently used. Furthermore, epigenetic marks may provide evidence of a tumour’s susceptibility to a certain treatment (e.g. BRCA1 methylation associated with better response to PARP inhibitor), or an early indication of the efficacy of a treatment.
Describe the immune therapies available for treating cancer
Immunology’s role in cancer is well established: immune evasion is a hallmark of cancer cells, and autopsies of people with no history of cancer has revealed microtumours that were kept in check by the immune system, and so never developed into cancer. By contrast, increased rates of cancer are seen in patients with immune dysfunction e.g. AIDS or immunosuppressive drugs post-transplant. As the immune system is significant in the pathology of cancer, altering it provides promising options for anti-cancer therapies.
Cytokines have seen some use as stimulators of immune activity. The two currently used cytokine therapies are interferon alpha and IL-2. Interferon alpha normally initiates the anti-viral response by inducing MHC class I expression, mediating dendritic cell maturation, and activating CD8+ cells. IL-2 triggers activation and expansion of CD4= and CD8+ cell populations. Cytokine therapies have been shown to be effective in increasing survival and disease-free survival, but are still largely experimental, have wide-ranging toxic side-effects, and need to be given in large doses.
Vaccination aims to prime the immune system against cancer cells, or against the infection causing them, as many infections are associated with increased risk of cancer (e.g. hepatitis C and hepatocellular carcinoma, HPV and cervical cancer). There are four vaccination strategies in cancer therapy: tumour cell vaccines, dendritic cell vaccines, peptide vaccine, and infection vaccines.
Tumour cell vaccination involves removing cells from a tumour via biopsy, then modifying them to make them more immunogenic, and replacing them in the patient in the hopes of generating an increased immune response. Dendritic cell vaccination involves removing dendritic APCs from a peripheral blood sample, then priming them with tumour antigen and expanding them with IL-4 and GM-CSF, and transplanting them back into the patient to prime effector lymphocytes to respond against the cancer. Peptide vaccination refers to manufacture of a peptide that resembles tumour antigens to trigger an immune response against the cancer.
Vaccination against infection is the most successful of the vaccination therapies, as it involves preventing the infection before it can cause cancer. A successful example is the HPV vaccine; HPV is necessary for the development of cervical cancer, so vaccination has the potential to all but eliminate cervical cancer.
Antibodies can be used in a wide variety of therapies, which can be divided into four main categories of targets: signalling inhibition, delivery of conjugates, direct cell-killing, and inhibition of immunosuppressive pathways.
Signalling inhibition is accomplished via binding of an antibody to a signalling molecule associated with proliferation of cancer cells, thereby preventing its activation and triggering its internalisation and degradation. An example of this is Herceptin (Trastuzumab), an anti-HER2 antibody which may be used in advanced breast cancer to prolong life. Though Herceptin does prolong survival somewhat, only a small proportion of patients are sensitive to the drug (only 20% of breast cancer expresses HER2), within this population only 30% respond to Herceptin, and resistance always arises. Resistance may arise through multiple mechanisms including: loss of expression of HER2, up-regulation of other proliferative receptors to compensate, and mutation of the receptor to prevent Herceptin binding or to induce ligand-independent kinase activity.
Conjugate delivery is used to deliver more targeted chemotherapy by attaching cytotoxic drugs to targeted antibodies. One form of conjugate delivery is antibody-directed enzyme prodrug therapy (ADEPT) which involves giving antibodies with an enzyme attached that converts a prodrug into its cytotoxic active form. The enzyme attaches to cancer cells, then after the unattached enzyme has been cleared from circulation, the prodrug is given, and is only metabolised to the cytotoxic drug at sites where the antibody has attached to tumour cells, thereby limiting the side-effects of chemotherapy.
A simpler approach is to design antibodies which recognise the tumour and trigger opsonisation and phagocytosis, complement activation, and cell lysis. Rituximab is an example of this type of antibody; it is an anti-CD20 antibody used in non-Hodgkin lymphoma, and has a 48% response rate.
The most promising of the antibody-based therapies are those that target and block immunosuppressive pathways. Tumour cell evasion of the immune system is often achieved by activating cell quiescence and apoptosis pathways in lymphocytes that encounter tumour cells. The main immunosuppressive receptors are CTLA-4 and PD1, and blockade of these receptors (with Ipilimumab and Nivolumab respectively) has shown success in stimulating an immune response against cancer cells.
Adoptive immunotherapy strategies include t-cell transfer: extraction and modification of a patient’s own t-cells to adapt to immune evasion by cancer. Extraction of infiltrating lymphocytes is one strategy, which involves separating lymphocytes form a biopsy sample, then culturing and expanding them and re-injecting the expanded population. This aims to amplify the immune response but is difficult: only 30-40% of biopsies yield sufficient lymphocytes for the procedure to work, the process is time-consuming (~6 weeks), and the lymphocytes are difficult to expand in vitro.
Modification of t-cell receptors provides a promising therapy for cancer cells which evade immune detection by losing MHC class I expression. Extraction and altering of a patient’s t-cells to express a chimeric t-cell receptor allows the t-cells to detect tumour cell antigens without interacting with MHC, thereby bypassing this evasion strategy.
Finally, an innovative therapy involves use of a highly specialised subset of lymphocytes – gamma-delta t-cells. These t-cells are highly specialised, very effective at killing tumour cells, have high clonal frequencies, and are differentiated pre-activation, allowing them to respond quickly. They are not used as a mainstream therapy yet, but are a promising prospect.
Describe the regulation of GnRH release, its effects on synthesis and release of gonadotrophins, and therapeutic uses of GnRHR agonists and antagonists
GnRH is a decapeptide molecule released from the hypothalamus which acts on gonadotrophs in the adenohypophysis to stimulate gonadotrophin release. Regulation of GnRH release is complex, and this complexity allows for careful regulation of reproductive hormones. Manipulation of GnRH function is a key element of treatments for multiple conditions.
GnRH release is mediated by negative feedback from gonadal hormones. Hormones involved in feeding also have a profound effect on GnRH release, with PYY increasing it, and ghrelin decreasing it. Leptin is permissive for GnRH release, and is the reason that women with low body fat experience secondary amenorrhea, as leptin is produced in adipocytes.
Kisspeptin has an important role in GnRH release, and accounts at least partially for the differences in reproductive endocrinology between males and females. Kisspeptin administered to men consistently boosts release of gonadotrophins, whereas in women kisspeptin has almost no effect, unless the woman is in the late-follicular stage, at which time kisspeptin hugely boosts gonadotrophin production. Kisspeptin neurons are found in the antero-ventral peri-ventricular (AVPV) nucleus and the arcuate nucleus.
The AVPV nucleus is found in mice and sheep, but not in humans, where a functional equivalent is presumed but has not been discovered. The AVPV nucleus is highly sexually dimorphic, being very large in females, and almost non-existent in males. The key difference between the AVPV and arcuate nuclei is that oestrogen suppresses kisspeptin release from the arcuate nucleus, but increases it from the AVPV nucleus.
Kisspeptin is released from Kndy neurons which also co-express neurokinin B and dynorphin, which are known to help regulate GnRH release. Dynorphin is known to counterbalance kisspeptin by negatively regulating GnRH release (hence why chronic use of opioids leads to infertility – it suppresses GnRH release). Neurokinin B is less understood.
GnRH activates a G-protein coupled receptor in gonadotrophs which activates protein kinase A and C signalling pathways, resulting in MAPK pathway activation. FSH and LH genes are then transcribed and translated. GnRH also induces Ca2+ influx which triggers exocytosis of stored vesicles of FSH and LH. The frequency of pulses of GnRH determines the balance of which gonadotrophin is released: in the early follicular phase the frequency is 1 pulse every 90-120 minutes, which favours FSH release; in the late follicular phase that increases to 1 pulse every 60 minutes which favours LH release. After ovulation the frequency drops to 1 pulse every 3-5 hours which stimulates FSH synthesis and storage, then as the frequency increases back to 1 pulse every 90-120 minutes, FSH is secreted.
GnRHR agonists can be used in IVF to cause de-sensitisation of the pituitary before stimulation of the ovaries. This method of IVF carries an increased risk of ovarian hyperstimulation syndrome (OHSS) because hCG must later be used to mature the oocytes. Alternatively, a GnRHR antagonist can be used to stop gonadotrophin secretion from the pituitary, allowing an agonist to be used to stimulate an endogenous LH surge: this virtually removes the risk of OHSS, but has a slightly lower live birth rate.
GnRHR antagonists may also be used in prostate cancer and breast cancer in pre-menopausal women. In both cases they function to suppress the HPG axis, reducing gonadal steroid production, and thereby limiting the growth of the tumour. Pulsatile GnRH may also be given to restore fertility in cases of hypothalamic amenorrhea.
Describe HPV infection and how it causes cancer. Discuss prevention and management of cervical cancer.
Human papilloma virus (HPV) is a dsDNA virus that infects keratinocytes in the basal layer of the epidermis. HPV is efficient at avoiding immune recognition: it forms non-lytic, non-inflammatory lesions, and antigen-presenting cells (APC) are down-regulated in HPV lesions. HPV is spread by skin-skin contact and may cause both cutaneous and mucosal infections, depending on subtype. Mucosal infections are of the most importance clinically because they have the potential to cause cancer. Most HPV infections are cleared within 6-12 months, but a small percentage of infections persist, and these persistent infections have the potential to cause cervical cancer.
Though of less clinical concern than cervical cancer, HPV can cause non-oncogenic warts. These are mostly caused by subtypes 6 and 11.
HPV infection is a prerequisite, though it is not necessarily sufficient, for cervical cancer. The main cervical cancer-causing HPV subtypes are 16 and 18, which together account for 75% of cervical cancer cases worldwide. Several genes within HPV contribute to cancer developing, including E6 and E7. E6 ubiquitinates and degrades the p53 protein, whereas E7 inactivates and degrades Retinoblastoma protein (pRb). The loss of these two tumour suppressors leads to cell cycle dysregulation and malignancy.
90% of cervical cancer occurs within squamous tissue, and the transformation zone (also known as the squamo-columnar junction) is particularly vulnerable as its thought to be permissive to HPV replication. Cervical cancer is regularly screened for in women between 25 and 65, with 3-yearly screenings between 25-50, and 5-yearly screening between 50-65. HPV is often spread through sexual intercourse, so condoms help, but do not offer total protection as the virus is spread through skin-skin contact.
Vaccination against HPV provides protection against at least the 7 subtypes that account for 90% of cervical cancer worldwide. Vaccination is effective as a prophylaxis but has been shown to be ineffective therapeutically.
Management of cervical cancer depends on its stage: carcinoma in situ can be resected, but later stage cancer may require a radical hysterectomy and lymph node dissection, potentially with chemotherapy or radiotherapy.
Discuss the main causes for anovulatory infertility and how you would investigate them for diagnosis. Describe treatments and explain how to induce ovulation and factors that affect the outcome of treatment.
Anovulatory infertility accounts for 25% of infertile couples. Anovulation is indicated clinically by amenorrhea, which can be divided into primary (the patient never started having periods) and secondary (periods started but have now started). Anovulation is usually caused by disruption of the HPG axis.
Hypothalamic lesions may impair the release of GnRH and disrupt ovulation, but a more common cause of anovulation is functional hypothalamic hypogonadism. This refers to a common (~30% secondary amenorrhea cases) physiological decrease in GnRH release due to low body weight (specifically low body fat, as leptin is permissive for GnRH release), high intensity exercise, or stress. Functional hypothalamic hypogonadism may be easily resolved with weight gain and lifestyle changes, or may require psychotherapy, depending on the underlying cause.
Genetic conditions may lead to hypothalamic dysfunction and subsequent anovulation, e.g. Kallmann syndrome, which is caused by a failure of GnRH neurons to migrate correctly during development, leading to a failure to go through puberty, primary amenorrhea, and anosmia (as olfactory neurons also fail to migrate). Key receptor knockouts may produce a similar failure to go through puberty e.g. TAC3R (Neurokinin B receptor) or Kiss1R knockout (Kisspeptin receptor). In many patients with deficient hypothalamic GnRH release, fertility may be restored using pulsatile GnRH.
Damage to the pituitary, e.g. via infiltrative disease, iron overload, surgery, radiotherapy, or compressive tumour may impair gonadotrophin release, preventing ovulation. Adenomas may also disrupt pituitary function, without necessarily having to compress the pituitary, by secreting high levels of hormones which disrupt gonadotrophin release, e.g. prolactin. Prolactin may be secreted by a prolactinoma, or may be co-secreted by another adenoma e.g. alongside growth hormone in acromegaly. Pituitary dysfunction would be investigated by measuring gonadotrophins and oestradiol, along with measuring prolactin and obtaining an MRI scan of the brain. Management of a prolactin-secreting tumour is with dopamine agonists (e.g. Cabergoline, bromocriptine) and surgery is rarely necessary. Replacement gonadotrophins or GnRH may restore fertility for patients with pituitary damage.
Polycystic ovarian syndrome (PCOS) is defined by the presence of anovulation in conjunction with either clinical (hirsutism) or biochemical (raised LH, raised testosterone) signs of raised androgen production. PCOS is a very common disorder, and accounts for 75% of cases of anovulatory fertility. The pathophysiology of PCOS is not entirely understood, but is thought to arise from the development of multiple ovarian follicles (leading to the ‘polycystic’ appearance on ultrasound), resulting in excessive oestradiol production which suppresses FSH production from the pituitary. As a result, none of the follicles receive sufficient FSH to fully mature.
Fertility may be improved in PCOS by reducing BMI (if it is above 30) and giving medication if there are signs of insulin resistance. Medication may be given to directly improve fertility, with the preferred option being an aromatase inhibitor (e.g. letrozole) which decreases circulating oestrogen by inhibiting aromatisation of testosterone to oestrogen and so allows FSH concentration to rise. Selective oestrogen receptor modulators may also be used (e.g. clomiphene citrate) in order to block the negative feedback effect of oestrogen in the hypothalamus, thereby increasing FSH release. Exogenous FSH may also be given, but is a less safe therapy and so must have its dose carefully titrated up.
Primary ovarian failure (POI) often occurs iatrogenically, as a result of chemotherapy or radiotherapy. In such cases, eggs, embryos, or sections of ovarian cortex can be taken and cryopreserved for later use in IVF treatments. POI may also have an autoimmune cause, or a genetic cause, such as Turner’s syndrome, which is caused by partial or complete loss of an X chromosome in a woman. Turner’s syndrome results in drastically accelerated loss of oocytes, leading to ovarian failure often within a few months after birth. Turner’s syndrome may be diagnosed through amniocentesis, chorionic villus sampling, or ultrasound in utero, or may be diagnosed on clinical exam and testing later in life when the patient fails to go through puberty. Fertility may be restored in these patients using oocyte donation and HRT, which both increases the receptivity of the endometrium to the embryo, and protects against osteoporosis and cardiovascular disease.
