Modelling cancer Flashcards
What processes are occur during cancer development?
Many. Oncogene/tumour suppressor mutation, angiogenesis, immune system can repress growth, extracellular matrix can promote growth and inhibit movement. Makes cancer very difficult to model.
What are the questions we ask about tumour origins?
What genetic changes occur and whether the order matters
If one or many cells initiate the tumour
If it involves differentiation or dedifferentiation
How carcinogens can effect things
Passenger mutations/driver mutations
How can cancer be investigated in the lab?
From tissue biopsy (small, bad quality DNA)
Adding oncogenes to primary cells
Using cell lines (have been grown for decades, questionable as to what they are)
Genetic data from Cancer Genome Project (see what mutations cancers have in common)
What in vitro model systems can be used to model cancer?
Cells from patients - don’t know if mutations are from patient or from adaptation to growth in culture. Can use to do gene targeting and engineering
Engineered cell lines - express oncogenes. Are they the correct cell type?
Colony forming assays - use blood cells
Primary cells from humans - hard to obtain unless can get from blood or tonsil. High genetic variability
Primary cells from mice
What are the problems with modelling cancer in tissue culture?
Ignores the contribution from the system such as the immune system, ability to metastasise, extracellular matrix, angiogenesis, hormones, growth factors.
How can mice used to model cancer?
Can use primary cells in tissue culture
Can do mouse mimics (where you get a tumour but the process of getting to the tumour is not the same as in humans)
Can do mouse models (where process is the same)
How can we prove a gene product is an oncogene?
Put a transgene into a mouse with a tissue specific promoter and see if get a tumour.
Problems are to do with copy number variations and sites of integration (could be chimeric) - in one study one line of mice had 10 copies of the transgene and died quickly whilst another had only 1 and showed a process more similar to human cancer (however these studies are more difficult to get funding for and are expensive). Also had different tumour phenotypes.
How can translocations be modelled?
Can make conditional (to ensure expression is in the correct cell type), inducible (to ensure translocation ‘occurs’ at the right time) genetically modified mice. Gene dosage should also be considered e.g. reciprocal translocations - which one is important? May reduce gene dosage of a haploinsufficient tumour suppressor
How can Cre-lox be used to model chromosome translocations?
Is tissue specific (express Cre from a tissue specific promoter). Use loxP sites to turn off/turn on (if have a stop cassette that is deleted) gene expression. Having loxP sites on different promoters mimics a translocation in different cell types. Used this to find that Mll:Af9 is an oncogene in myeloid cells but not T cells. Mll:Enl is malignant in T cells.
How can CRISPR-Cas9 be used to model cancer?
Can use to make chromosome translocations by inducing double strand breaks at certain points.
How can oncogene addiction be modelled?
Make an inducible, transient mouse - link transgene to doxycycline dose in a specific tissue. Give mouse doxycycline then withdraw. Tumours have been shown to be addicted to Ras and Myc (tumours apoptose upon withdrawal). Shows that drugs that target oncogenes could cause tumour regression.
How can the necessity of the inactivation of tumour suppressor genes be modelled?
Use KO p53 mice - KO by putting a stop codon in the gene, flanked by loxP cassettes. Irradiate to get a tumour. Reactivate p53 by turning on Cre (e.g. with tamoxifen), find that tumour regresses.
What are tumour initiating cells?
Cell of origin of the tumour, not necessarily responsible for tumour maintenance.
What are cancer stem cells?
Tumour propagating cells, may be distinct from tumour initiating cell. Have reproductive capacity and can self renew. May produce progeny that can’t reproduce
What is the stochastic model of tumour growth?
Different populations in the tumour all contribute to growth
What is the cancer stem cell model for tumour growth?
Only some (one population, the cancer stem cells) of the cells in the tumour contribute to tumour growth, the others don’t.
What are the different strains of immunocompromised mice?
SCID - deletion in DNA PK gene for TCR and Ig rearrangement. Have NK cells
NOD/SCID - non-obese diabetic severe combined immunodeficiency. Also have defects in NK cells. Short lifespan due to reactivation of a provirus
NOD/SCID/beta2 microglobulin null - nearly no NK function, short life span
NSG - no T, B or NK cell activity. No leakiness or tumour incidence.
Describe origination of Acute Myeloid Leukaemia
Found a SCID Leukaemia Initiating Cell (by looking at surface markers). Was able to initiate human haematopoiesis in immunocompromised mice. Got differentiation into leukaemia blasts suggesting a hierarchy for this cancer. Cancer stem cell likely to be a transformed tissue-specific stem cell.
How has BCR-ABL addiction been studied?
In mouse models found that inactivation of BCR-ABL lead to reversion of the leukaemia. Therefore developed drug inhibitors (e.g Imatinib) that were rushed through clinical trials. Found that disease relapsed due to a mutated form of BCR-ABL found in leukaemia stem cells. Now, new inhibitors are being developed and research is being done to find the mechanism of leukaemia stem cell resistance (e.g. expression level, drug efflux, niche environment, not dependent on BCR-ABL)
What models can be used to test new drugs?
Nude mouse xenografts
Patient derived xenografts in NSG mice
Genetically engineered mouse models (may not be truly representative of human disease and may take time to develop a tumour)
Why do people get referred to cancer genetics clinics?
People with family histories of breast, ovary and colon cancers
If have a relative with cancer (especially early onset) increases chance of cancer
Certain mutations carry loads of risk e.g. FAP
What is FAP?
Familial adenomatous polyposis coli. Get polyps throughout the colon and maybe upper GI tract. Average age of cancer development is 15 years of having polyps. Screen for FAP by colonoscopy and do genetic tests to discharge mutation negative individuals. Caused by a mutation in the APC, often truncations in centre of the gene. 2 hit model of tumorigenesis
What is MYH?
Family with 3 colorectal cancers, but markers didn’t segregate with disease as expected (e.g. like in FAP). Found mutations in the APC different to those in sporadic cancer. Found mutation in the BER DNA repair machinery at MYH. Was a missense mutation with a less severe phenotype than FAP
What is Lynch syndrome?
Hereditary non-polyposis colorectal cancer. Autosomal dominant with an early onset, tumours on the right hand side of colon (have to do full colon screen). Also get other cancers (endometrial in women, ovarian, stomach). Is due to mutations in MLH1 or MSH2 (and some rarer mutations) which are involved in mismatch repair
How is Lynch syndrome diagnosed?
In past used Amsterdam criteria - 3 people with colorectal cancer, one below age of 50. These were then relaxed to include other cancers, but didn’t pick up de novo mutations. Now we do micro satellite testing and immunohistochemistry against MSH2 and MLH1. Can have sporadic cancers with these mutations as well esp. MLH1 - test young people with these cancers.
How can cancer be prevented in people with Lynch syndrome?
Surveillance to detect tumours at an early age and remove polyp
Found that people with arthritis get less cancer, theorised to be due to aspirin. Found that giving a high dose of aspirin to people with Lynch syndrome significantly reduces cancer - don’t know how, looking at different doses.
What preventative treatments exist for families with BRCA1 or BRCA2 mutations?
Mastectomy
Oophorectomy (may also reduce the risk of breast cancer)
Birth control pill can reduce risk of ovarian cancer
Lots of screening
At what point do we test for BRCA1/2 mutations?
Look at how much disease on one side of the family and how many unaffected relatives there are. Also look at age of diagnosis - older is more likely to be sporadic. Also must check disease actually is cancer as can be inaccurately reported.
Have to be careful, as these cancers only affect women, so can be passed through men and turn up in a woman with no family history
Can do a Manchester score to determine whether to test, but as testing is cheap now criteria have been widened
How can BRCA1/2 cancers be treated?
By inhibiting PARP - another pathway of DNA repair. Cells need at least one pathway to survive.
How are inherited cancers molecularly classified?
By function - gate keeper genes that regulate cell growth/division (tumour suppressors or photo-oncogenes) OR caretaker genes that help maintain the integrity of the genome (include DNA repair enzymes where mutations lead to genomic instability and increased likelihood of a gatekeeper gene mutation; cause cancer indirectly)
How are inherited cancers clinically classified?
Rare familial cancer syndromes - distinctive phenotypes or clustering of rare cancers
Genetic predisposition to common cancers - no phenotypic markers, familial clustering, early age of onset, multiple primary tumours
Sporadic cases - new muttons for high penetrance genes, high risk polygenic cases
What is pheochromocytoma?
A tumour arising from the adrenal medulla that secretes catecholamine.
Describe the genetics of pheochromocytoma
One third is genetic
25% of sporadic cases may have a gremlin mutation
11 genes are implicated - of which the most frequent are SDHB, SDHD and VHL mutations.
What is paraganglioma?
Can be extra-adrenal - catecholamine secreting tumour arising from sympathetic ganglia
Can be head and neck - non functional tumour arising from parasympathetic ganglia. Similar genetics to pheochromocytoma
Describe the genetics of renal cell carcinoma
Found higher risk of RCC if sibling or parent has it. 3% of patients have a positive family history. Most inherited cases are dominant.
What is multiple endocrine neoplasia 2?
An autosomal dominant cancer. Is a medullary thyroid carcinoma (MTC) and hyperplasia of thyroid C cells. Is a pheomochromocytoma. 30% of sporadic MTC have gremlin RET mutations - incomplete penetrance. Do DNA analysis at 5yrs old and thyroidectomy to prevent thyroid cancer
What are the different types of Multiple Endocrine Neoplasia?
MEN 2A: MTC + pheochromocytoma + presence of parathyroid hormone
MEN 2B: MTC + pheochromocytoma + developmental anomalies
Familial MTC - no pheo
What is the genetic cause of multiple endocrine neoplasia 2?
Inappropriate activation of RET. Can either be in the extracellular domain to cause inappropriate dimerisation (e.g. each monomer has 2 cysteines in which form a cysteine bond in the molecule. If one of these is mutated e.g. to an arginine, then a cysteine bond can form between monomers instead to make a constituent dimer) or intracellular in the tyrosine kinase domain to cause inappropriate activation. Is a protooncogene.
What is Hereditary Papillary RCC type 1?
Renal cell carcinoma. Is very rare dominantly inherited cancer with multiple tumours of incomplete penetrance. Get germ line activation of MET, an RTK (a protooncogene)
How can hereditary papillary RCC type 1 be treated?
MET inhibitors e.g. foretinib (also inhibits other receptors)
What is neurofibromatosis type 1?
An autosomal dominant cancer which occurs from a germ line mutation in the NF1 tumour suppressor gene. Get cutaneous neurofibromas, Lisch nodules and axillary freckling and a 2% risk of pheochromocytoma. Don’t do genetic testing
What is von Hippel-Lindau disease?
An autosomal dominantly inherited disease. Get risk of retinal angiomas, cerebellar haemangioblastomas, clear cell renal cell carcinoma,, pheochromocytoma and other cats and tumours.
How is von Hippel-Lindau disease managed?
Annual eye exam from age 2
2-3 yearly brain scans from 15
Annual abdominal MRI from 16
24hr urine catecholamines
What mutations causes von Hippel-Lindau disease?
Found in VHL, familial pheochromocytoma and recessive polycythaemia. Encodes 2 proteins on 3p25. Found in many sporadic RCC as well.
How was the function of the VHL gene identified?
Has little significant homology. VHL tumours are known to be very vascular. Looked for interacting proteins. Found overexpresssion of hypoxic response genes in tumours and early lesions. Found that pVHL is a ubiquitin ligase that regulates HIF-1 and HIF-2 stability
How can von Hippel-Lindau disease be treated?
With kinase inhibitors targeting hypoxic response proteins such as VEGF
What is Birt-Hogg-Dube syndrome?
Skin fibrofolliculomas on head, neck and upper chest. Lung cysts and renal tumours with variable histopathology. Life time risk of 30% for renal cancer. Have mutations in FLCN in germ line in majority of cases
What does FLCN do
Follicullin. Is a tumour suppressor gene, which is poorly characterised. Found that it is implicated in the mTOR signalling pathway in mice (if inactivated, signalling increased). Has also been linked to TGFbeta signalling and enhanced affect of HIF
What is Reed syndrome?
Hereditary leiomyomatosis and RCC. Is dominantly inherited with incomplete penetrance. Get multiple uterine fibroids and cutaneous leiomyomatosis. Screen for RCC with MRI (tends to be unilateral, solitary and aggressive). Due to mutations in fumarate hydratase (also associated with pheochromocytoma)
How is Reed syndrome monitored?
Predictive testing from 8-10 years
Renal screening by MRI annually
Prompt treatment of tumours with surgical excision
How does the Krebs cycle influence hypoxic signalling?
Fumarate inhibits PHDs which oxidate HIFalpha. This prevents the degradation of HIFalpha by VHL and causes pseudohypoxic signalling. Fumarate can accumulate due to loss of fumarate hydratase.
How are inherited renal cell carcinomas evaluated?
Detailed family history is taken and cancer is confirmed. Histopathology used to see what kinds of cancer are found (e.g. clear/non clear or papillary type ½)
Look for features of a specific syndrome
If a syndromic cause is suspected then testing of genes.
If there are 2 close relatives with RCC (with bilateral/multicentric disease or early onset as an added risk factor) then may suspect non-syndromic inherited RCC - look at chromosomes and do molecular genetic analysis looking at renal cancer gene panel
What is the deterministic/stochastic cancer model?
Each cell in the cancer has a defined chance of replicating the cancer in a different environment. In the right conditions every cell has a chance of replicatingi
What is the cancer stem cell model?
A defined population in the cancer (thought to be small and rare) can replicate the cancer in a different model. Can also replicate the variation in the original cancer. Is accepted as true for haematological cancers; is controversial for some solid organ cancers.
What is the evidence for the cancer stem cell model?
Dick 1997: transferred human tumours to immunocompromised mice and separated cells based on hierarchy. Found that CD34+/CD38- (closer to stem cells) caused leukaemia but CD34+/CD38+ didn’t. Similar experiments have been done for other cancers.
What is the evidence against the cancer stem cell model?
2 papers were published using similar assays to papers in which CSC was ‘proven’. Showed that cancer stem cells weren’t necessarily rare (⅓) and that they would have to be highly frequent
How can the evidence for and against the cancer stem cell model be reconciled?
Relate to normal tissue hierarchy - not all tissues have the same hierarchy. Melanoma is derived from neural cells were there is no tissue hierarchy found.
