Oncogenes/ TSGs/ apoptosis Flashcards
Templeton et al 2014
Meta-analysis. Looked at 16 studies comprising 11, 056 pts. There was sig association between overexpression of RTKs and decreased 5 year OS in women with breast cancer. Worst overall survival was seen with overexpression f FGFR and EGFR/HER1. Before this study, the HER2 overexpression was known to have an impact on prognosis but other GF receptors were not. Limitation - no correction could be made for other prognostic factors, such as tumour grade - so actually, it remains unknow whether overexpression of the RTK described here are truly independent adverse prognostic features
Konduri et al 2016
Identified EGFR fusions as novel therapeutic targets in lung cancer. To determined the frequency of EGFR mutations in lung cancer, they analysed data from ~10,000 clinical cases. Fusion events were detected in 5 patients with metastatic lung cancer. NGS revealed this fusionwa swas commonly a fusion of the EGR-RAD51 fusion (where RAD51 is a protein involved in DNA damage responses). This could have just been a passenger mutation. They confirmed this fusion was oncogenic in vitro - They transfected NR6 cells (which lack the endogenous EGFR) with EGFR-RAD51 and this significantly increased colony formation of NR6 cells in soft agar when compared to control transfected cells. In cell culture - went on to show that a variety of existing EGFR inhibitors were able to target EGFR-RAD51 - inhibiting proliferation of EGFR-expressing cells. However, these effects were just seen in vivo - still could be a passenger mutation and not actually contribute to oncogenesis. Colony formation and proliferation also does not necessarily mean cancer
Bang et al 2017
phase I study involving patients with HER2-positive advanced-stage solid tumours. Both breast or gastric or other carnomas that overexpress HER - looked at 60 pts. In this study, 12% of patients (7 of 60) had a partial responses and 50% (30 of 60) had stable disease. The majority of those who had stable disease or better (70%) had disease progression on previous HER2-targeted therapies. Ex vivo analyses of peripheral blood mononuclear cell samples confirmed the ability of margetuximab to support an enhanced level of ADCC compared with trastuzumab.
Mulcahy 1985
they microinjected the protein products of the RAS oncogene into NIH 3T3 cells. Following this, the recipient cells were able to initiate DNA synthesis in the absence of added serum. They further showed that NIH 3T3 cells that were induced to divide through addition of serum, were not able to enter S phase following microinjection of anti-RAS antibodies. This demonstrated a requirement of RAS for S phase transition
Filmus et al 1994
“demonstrated this when a relationship was seen between activated RAS and overexpression of cyclin D1 in epithelial cells from the rat intestine.
o In their study, they transfected intestinal cells with an inducible RAS expression vector.
o They treated these with anti-sense cyclin D1 oligonucleotides and this led to a reduction in rate of cell proliferation.
o This led to the conclusion that RAS-induced cyclin D1 contributed to the higher rate of proliferation caused by the RAS oncogene in intestinal epithelial cells “
Bar-Peled et al 2013
First, they treated human embryonic kidney cells expressing FLAG-tagged RagB with a chemical crosslinker and identified via mass spectrometry protiens that coimmunoprecipitate with FLAG-RagB. Allowed them to identify GATOR complex (with two subcomplexes GATOR1 and GATOR2 that were coexpressed with Rags. Then used RNAi in HEK cells and Drosophila S2 cells to examine the function of each GATOR component in amino acid sensing by mTORC1 and dTORC1 respectively. Depletion of proteins in mTORC2 or their Drosophila orthologues strongly blunted the aa-indced mTROC1 and dTORC1 activation. GATOR1 had the opposite and prevented the inactivation of mTORC1 and dTORC1 normally caused by aa deprivation. This further allowed them to determine that RNAi knockdown of GATOR2 following GATOR1 inhibition had no effect of dTORC1 activity - showed that GATOR2 is upstream of GATOR1. They then invetsigated the role GATOR1 plays in human tumours. They looked at pubically available data from TCGA - found a subset of glioblastomas and ovarian cancers with nons or frameshift mutations or truncating deletions in genes encoding GATOR1 proteins (DEPDC5 and NPRL2). THey used Cosmic resources to identify human cancer lines with deletions in DEPDC5, NPRL2 and NPRL3. They then found htat in these lines mTORC1 signalling was hyperactive and insensitive to aa deprivation. When DEPDC5 and Nprl2 were reintroduced into the cancer cell lines lacking them, the mTORC1 pathway regained sensitivity to aa regulation.
Yao et al 2011
Did a phase 3 study of everolimus. Randomly assigned 410 pts with pancreatic neuroendocrine tumours to receive everolimus or placebo. Primary end point was progression-free survival. The median progression-free survival was 11 months in everolimus vs 4.6 months w placebo (P<0.001). This represented a 65% reduction in the estimated risk of progression or death. There was a low rate of severe adverse effects
Colman et al 2009
Look at rhesus macaques. Had a control group and a CR group. Looked at a total of 76 monkeys. With the CR animals they did a 30% reduction in food intake. Followed them for 20 years. Incidence of neoplasia was reduced by 50% in animals undergoing CR comapred to controls.
Descamps et al 2005
A previous study of alternate-day fasting, which was performed in middle-aged mice for a total of 4 months, also found that fasting reduced the incidence of lymphoma, bringing it from 33% (for control mice) to 0% (in fasted animals). There was no difference in overall food consumption between the ADF and AL mice - this effect was not due to CR. 25 mice total
Short duration of study - did fasting prevent or simply delay tumour inset?
Evan et al 1992
They looked at Rat-1 fibroblasts (these have the property of being dependent on mitogens for their signalling). Overexpressed c-myc in Rat-1 fibroblasts constitutively via preparation of recombinant retroviruses. They found that the growth curves of all cells was very similar, irrespective of whether the cells were constiutively expressing c-myc or in they were controls. This indicated that constituive c-myc expression has no effect on the ability of Rat-1 cells to slow growth in low serum levels. However, Evan then looked at the cell cycle distribution of these cells following withdrawal from serum for 48h. They examined the growth of WT Rat-1 cells and Rat-1/myc cells cells by DNA content BrdU incorporation by flow cytometry. They found that the control cells were not proliferating and had DNA content that was equivalent to G1 phase. In contrast, they found that the c-myc cells, althoguh not increasing in cell number - 45% of these were in S phase. Therefore, the cells were proliferating but without increase ub cekk number. Time-lapse video microscopy revealed cells were dying by apoptosis - this was rather unexpected
Serrano et al 1997
One of the mutant alleles of HRAS is G12V, Transduced human, mouse embryonic and rat embryonic fibroblasts with a retrovirus encoding HRAS G12V or a control virus. They observed that introducing G12V abolished the increase in cell number that we see in the control culture - i.e. the cells stopped proliferating. They then used a propidium iodide-BrdU flow cytometry method to determine DNA content per cell as well as newly synthesized DNA in the fibroblasts. It was seen that there was a significant decrease in the S phase fraction following introduction of the mutant RAS allele. Even on day 1 after tarnsductoin we can see a decrease frim 31% ti 5.4% and by 3 days we can see the S phase fraction is almost completely gone. Therefore, mutant RAS in these cells led to cell cycle arrest, rather than proliferation
How this was working at a molecular level. They did Western blotting (without a loading control - caveat - could have had unequal amounts of protein loaded) of lysates from fibroblasts transudced with retrovirus encoding HRAS G12V or control retrovirus. They found a pretty dramatic increase in p53 levels in all three types of fibroblasts following G12V transfection. Conversely, there is also a decrease in cyclin A abundance. Rb decreases and cyclin A decreases - interpretted as dephos of Rb. This suggests that induced of mutant RAS is halting the cell cycle through a combination of activation of p53 and dephos of Rb.
Harrington 1994
Dual hit hypothesis
Montes de Oca Luna et al 1995
It was already known that p53 heterozygote mice have elevated cancer incidence. You can breed these p53-/+ mice to generate p53 -/- mice - these are v cancer prone and die mostly from lymphomas by 6 months. In contrast, if we knock out Mdm-2 - we find that this is an embryonic lethal mutation. The surprising result ehre however is that if we combine the Mdm-2 KO and the p53 KO mice - these are viable. This astonishing result is a clear piece of evidence that the major function of Mdm2 is to limit the activity of p53 (whats happening in the Mdm2 KO mice alone is that they are dying of massive p53 induced apoptosis)
Bartkova et al 2005
They transfected osteosarcoma cells with inducible cyclin E or E2F1 (overexpression drives cells into S phase and so is a way of mimicking downstream effects of oncogene activation). In both cases, immunofluorescence microscopy showed that there was upregulation of DNA damage markers stained with antibodies - these were gamma-H2AX, Ser15-phosphorylated p53 or phosphorylated Chk1. Induction of DNA damage associated markers as a consequence of induction of cyclin E and E2F1.
They used an osteosarcoma cell line - this is already a tumour cell
Xin Lu et al 2001 (Oxford)
ASPP1 was identified in a genome database search for proteins with homology to the previously isolated protein called 53BP2/Bbp - which had previously been shown to interact with p53. They UV-irradiated MCF-7 cells and immunoprecipitated ASPP1 and ASPP2.Western blot analysis of the immunocomplexes showed that p53 interacts with ASPP1 and ASPP2. Saos-2 cells were transfected with a p53 expression plasmid or a control plasmid together with cell surface marker CD20. FACS analysis showed that the expression of p53 caused about 17% apoptosis among the transfected cell population (Figures 2A and 2B). Expression of ASPP1 or ASPP2 in the absence of p53 caused a small fraction of the cells to die by apoptosis. In contrast, coexpression of p53 with ASPP1 or ASPP2 caused 50% of the transfected cells to die of apoptosis. ASPPs seemed to enhance the apoptotic functions of p53.
In 2003, they reported the identification of the third member of the p53-regulating protein family-iASPP (inhibitory member of the ASPP family). They knocked down the gene function using antisense RNA in human cell lines and saw an increase in apoptosis. This finding indicated that the newly identified ASPP protein had an inhibitory effect on apoptosis, earning it the name iASPP
