Cancer misc Flashcards
Borah et al 2015
Studied 23 human urothelial cancer cell lines derived from different tumours. Took genomic DNA from each and TERT promoter was PCR amplified for sequencing. Show that two specific point mutations in the TERT promoter were associated with higher levels of TERT mRNA, TERT protein, telomerase enzymatic activity and telomere length. They further find that elevated TERT mRNA strongly correlated with reduced disease-specific survival in two independent urothelial cancer cohorts. Suggest that TERT promoter mutations provide a particulalry effective mechanism for high level telomerase reactivation in UC. Previous studies that not found any correlation between these promoter mutations and severity of UC - these results suggest that a better marker for pt prognosis may not be the promoter mutation itself, but TERT expression levels which directly determine telomerase activity levels
Yasukawa et al 2020
(1) Did an in vitro kinase assay using recombinant hTERT fragment proteins and CDK1-cyclinB proteins followed by mass spectrometry analysis. They used phosphate-affinity gel electrophoresis to detect the phosphorylation of hTERT incubated with CDK1-cyclinB at a specific site. (2) they then found that phosphorylation at this point occurs more frequently in aggressive cancers - by staining pancreatic caners with anti-249T-P antibodies (3) They then used CRISPR-Cas9 genome editing to introduce substitution mutations at threonine 249 (the specific site) is necessary for hTERT-mediated RNA-dependent RNA polymerase activity. (4) they then demonstarted using Cap Analysis of Gene Expression that TERT phosphorylation at 249 regulates expression of specific genes (FOXO1) associated with cancer cell proliferation and tumour formation (GO analysis) Additional role of CDK1, additional role of hTERT - majority of work here was on cell lines
Muthukkaruppan and colleagues 1982
Compared the behaviour of cancer cells infused into different regions of the same organ. One region was the iris with blood circulation and another was the anterior chamber with no circulation. The cancer cells without blood circulation grew 1-2mm^3 in diameter and then stopped by grew beyond 2mm^3 when placed in an area where angiogenesis was possible
Korsisaari et al (2007)
investigated the role of VEGF in the growth of intestinal benign tumors in Apc+/min mice. In situ hybridization analysis has demonstrated the up-regulation of VEGF in epithelial and stromal cells of adenomas compared with the adjacent normal mucosa. These authors have initiated VEGF blocking treatment with Mab G6–31 (5 mg/kg, once weekly), which blocks both murine and human VEGF, in Apc+/min mice when the animals are 13 weeks old and have found that the inhibition of VEGF reduces vascular density and prevents further growth of intestinal adenomas. The mean lifespan of animals that receive Mab G6–31 treatment is extended by nearly 10 weeks when compared with control treated animals. Interestingly, despite the tumor growth arrest, no reduction has been observed in tumor numbers. These data demonstrate that VEGF signaling is involved in tumor progression, but not in tumor initiation.
Gerber et al 2007
The hum-X VEGF mouse model was then used to compare the activity and safety of a panel of VEGF Mabs with different affinities for VEGF-A. Although in vitro studies clearly showed a correlation between binding affinity and potency at blocking endothelial cell proliferation stimulated by VEGF, in vivo experiments failed to document any consistent correlation between antibody affinity and the ability to inhibit tumor growth and angiogenesis in most animal models. However, higher-affinity antibodies were more likely to result in glomerulosclerosis during long-term treatment. Just shows how much more powerful humanised models can be
Alexandrov et al 2016
They analysed somatic mutations and DNA methylation in >5000 cancers of types for which tobacco smoking confers an elevated risk. The cancers were from both smokers and non smokers. They found the smoking is associated with increased mutation burden lung, larynx, liver and kidney cancers. the generation of increased somatic mutation burden by tobacco smoking appears to be mechanistically complex. Smoking correlates with increases in base substitutions of multiple mutational signatures, together with increases in indels and copy-number changes. The extent to which these distinct mutational processes operate differs between tissue types Although we cannot exclude roles for covariate behaviors of smokers or differences in the biology of cancers arising in smokers compared with nonsmokers, smoking itself is most plausibly the cause of these differences.But original no. of somatic muts in tissues unknown – just happened to be ↑er in smokers than non-smokers?
Merlo et al 1995
They found that p16, a tumour suppressor encoded by CDKN2A has been found to gain de novo methylation in ~20% of different primary neoplasms but not in normal tissues, potentially representing a common pathway of TSG inactivation in human cancers - determned by southern blot in 50 priamry neoplasms
Kerry et al 2017 (Tom Milne’s lab)
They used MLL and AF4 ChIP seq in the human MLL-AF4 SEM cell line. Analysis of MLL-AF4 binding profiles showed enrichment of H2K79me3 (lysine 79 methylation) at the gene targets where MLL-AF4 is bound. We think on a molecular level that MLL-AF4 drives tumorigenesis by binding to genes, recruits a large elongation complex which includes this protein called DOT1L and it is this protein that leaves the H3K79me3 mark. This methylation does something and where not exactly sure what but it seems to be involved in activating genes They treated their SEM cells with a DOT1L inhibitor. They did RNA seq and observed 2,400 downregulated genes, many of which where assoicated with MLL-AF4 targets. Suggested that previously identified MLL-AF4 targets sensitive to DOT1L inhibitors
Martincorena et al 2015
Addresses the number of cancer mutations in phenotypically normal skin. They source of skin was eyelids from pts who had undergone surgery. They then did ultra-deep (NGS) sequencing of 74 cancer relevant genes from 234 biopsies of these phenotypically normal eyelid epidermis speciments from four individuals. The burden of mutations in normal skin in just 74 genes is about five mutations per megabase - this is only a factor of 10 less than seen in squamous cell carcinoma and is comparable to mutation burden seen in cancers of the breast, head and neck.. To determine if these mutations were giving a selective advantage to the normal skin, Martincorena used the ratio of mutant sequence reads that do or do not alter the amino acid. . Six genes—NOTCH1, 2, and 3, P53, FAT1, and RBM10—had an excess of nonsilent mutations, indicating that these genes had been selected for and are therefore presumptive drivers. PThe first five are known drivers for keratinocyte tumors and the last is mutated in other cancers. A seventh gene, FGFR3, had recurrent activating mutations. In total, one-quarter of middle-aged skin contains a mutation in one of these drivers.
If normal skin is carrying somatic mutation of cancer relevant genes - how can we selectively target these in cancer. Maybe it is preferable to act earlier, when the monoclonal sheet of the ‘freshly scrubbed face’ of youth is rarely flecked with preprocancer. Precision medicine aims to treat patients according to their genetic changes in their tumours - would this impact
They only looked at 74 genes in particular - there could be even more - cav
Martincorena et al 2018
Skin on the eyelids was exposed to UV light. They wanted to look at tissue that had not been exposed to such powerful mutagens. They performed targetted gene sequencing of normal oesophageal epithelial from nine human donors of varying age. The mutation rate was lower in the oesophagus than in the skin but there was a strong positive selection of clones carrying mutations in 14 cancer-associated genes. Interestingly, mutations in the cancer driver gene NOTCH1 were more common in normal oesophageal epithelium than in normal cancer. They also noted that the number of mutations increases with age, mainly owing to errors in intrinsic processes such as aberrant DNA replication or repair
Only 9 indivduals - cav
Nicolas et al 2003
They used mouse genetics to see how Notch signalling ablation affects development of SCC. They used the DMBA-TPA mouse model of cutaneous chemical carincogenesis system. They found application of DMBA-TPA to Notch-/- skin results in a dramatic increase in tumour burden. They used a tamoxifen-inducible model of Notch activation as it is embryonic lethal. Only one in 10 of the littermate controls developed a single papilloma. The induced Notch1-/- had an average of 14 tumours in 25 weeks after starting treatment.
Van’t Veer et al (2002)
o They performed microarray analysis of 78 primary breast cancer tumours, and identified a gene expression ‘profile’ of 70 genes that could predict the risk of future distant metastasis, and so could identify in advance which patients would or would not respond to chemotherapy
o The group with a ‘good’ as opposed to a ‘poor’ prognosis did not suffer distant metastases within 5 years Computer software can now compare a patient’s mRNA profile to the gene expression profile from this study in order to predict whether they would benefit from chemotherapy
Cardoso et al 2016
Askes whether this gene expression profiling can be in combination with more traditional histopathological assessment of tumours
- Published a phase III randomised study in the NEJM involving 6693 women with early-stage breast cancer which assessed the clinical risk (by looking at conventional histology) and the genomic risk in parallel. Thus stratified pts into two groups - both low, both high or a lack of concordance.
- These discordant groups were then randomised to recieve chemotherapy or not.
- Aim was to identify which of these predictive parameters is most accurate at identifying those that stand to benefit from chemotherapy.
- Found that there was v little difference between those who recieve chemotherapy and those who dont in terms of survival without metastases in both groups.
- This suggests that a combination of both approaches shows that aroun 40% of pts considered at a high clinical risk may not actually benefit from chemotherapy.
