Oncogenes and tumour Suppressors Flashcards
(12 cards)
How was the first oncogene ID’d? RSV, transformed cells and the origin of viral oncogenes
1st oncogene ID’d by Rous, 1911. Transmissible, filterable agent causing cancers in chickens.
Sarcoma in chicken breast muscle removed, broken up into small chunks+ ground with sand, filtrate collected through fine-pore filter, injected into young chicken-> develops sarcoma.
RSV (rous sarcoma virus)- modified retrovirus w/ src gene transforms cells in culture, as seen w/ a Focus assay. 1 cell infected w/ RSV in cell monolayer w/ contact inhibition (healthy cells only grow until reach+ contact another cell)-> cell transforms, forms a focus (3D clump of cells w/ no contact inhibition).
Transformed cells have: altered morphology, no contact inhibition, anchorage independence (don’t need anchoring to petri dish), immortalization/ indefinite proliferation/ no senescence, reduced requirement for growth factors, high saturation density, increased glucose uptake (Warburg). Transformation requires continuous maintenance by src gene activity- ts src mutants revert to normal morphology under restrictive to
Origin of viral oncogenes: Southern blot analysis/ human DNA restriction digests probed @ lowered stringency w/ v-src show that viruses w/ no src infecting host w/ src result in virions w/ src.
Most oncogenic retroviruses are replication defective. The src gene piggybacks off viruses w/ required exp constructs. Retrovirus integration-> insertion of potent LTR promoter/enhancer at each end.
Proof of cancer as a genetic disease: Ames test, 2 general mechanisms for genes becoming oncogenic, genetic alterations causing cancer and abberrantly expressed growth factor receptors
Cancer is a genetic disease: DNA mutagen exposure in mouse fibroblasts leads to focus formation of morphologically transformed cells that lead to tumours when injected into a mouse host. The Ames test for mutagenicity involves homogenising rat liver, adding test compound, metabolic activation of test compound by rat liver cells, adding them to Salmonella unable to grow w/out added histidine+ count #/bacterial colonies undergoing mutation enabling them to grow without added histidine. Mutagenicity (measured in um per 100 Salmonella revertants) positively correlates w/ tumorigenesis (measured in mg/kg/day giving 50% tumour induction in 24 months).
Cellular genes become oncogenes by 2 general mechanisms: deregulated activity (e.g. mutation) by regulated effectors (kinases, phosphatases, proteases, G proteins) or deregulated/elevated exp of innately active effectors (TFs, some kinases)). Activated endogenous oncogenes found directly by transfection of tumour DNA into normal mouse fibroblasts-> focus injected into mouse-> tumour.
Regulation of src activity, Oncogenic src
Regulation of Src activity: inactive form has Pi CTD (Y527) of kinase C-lobe, holding it to SH2 domain. De-Pi by phosphatase releases kinase CTD from SH2+ kinase N-lobe to SH2 linker moves to reveal src activator binding site on SH3 9allowing activator to bind SH3+ pY of SH2 (initially bound to CTD)-> quasi-active form. Tyr416 of activation loop on kinase C-lobe Pi’d, causing it to swing away from C-lobe-N-lobe junction, revealing catalytic cleft-> active form.
Oncogenic src has chronically Pi’d Tyr ([tyr-p] rises 0.03->0.3%, seen on western blots of normal vs RSV transformed cell lysates) leading to overactive src+ large band of FAK (focal adhesion kinase) leading to a hypermobile cytoskeleton.
genetic alterations causing cancer and abberrantly expressed growth factor receptors
Genetic alterations causing cancer incl small changes (single base change-> missense), indel-> frameshift, nonsense), e.g., APC gene in colon cancer has 1 base deletion-> stop/truncated protein. Also, structural changes: deletion, inversion, duplication, amplification, chromosome translocation
Aberrantly expressed growth factor receptors are often involved in tumorigenesis, e.g., EGFR overexp-> breast/stomach/head+ neck cancers; HER2 overexp-> ~30% breast adenocarcinoma. Some tumour cells exp own growth factor ligands exported to activate own receptors (autocrine signalling) or may have receptor mutations leading to ligand-independent firing. E.g., frequent mutations in exon 11 of KIT gene encoding juxtamembrane domain affects 70-80% GI stromal tumours. (see transmembrane signalling for more)
Oncogenic activation of the EGF signal-> TF activation-> proliferation+ survival. Blocked by Herceptin (MAb), which blocks EGFR/HER2 overexpression (HER2 in breast/stomach/gastro oesophageal junction cancers)
Ras proteins
Ras proteins involved in post-RTK mitogen signalling, connecting receptors to intracellular signalling pathways. Ras mutations are v prevalent in cancer (esp K-ras), implicated in mot pancreatic cancers+ over ½ of thyroid+ colorectal cancers (+ many others). Oncogenic mutations (G12E/V/D, G13D, Q61K) in Ras are associated with GTP binding. Pleiotropic effects of Ras superfam proteins include survival (via FoxO, Bad), metabolism (via mTOR), prolif (Raf-> Mek->erk)+ motility (via cdc42, Rac) (all hallmarks of cancer).
Myc gene, Myc deregulation
Myc gene= e.g., of oncogenic activation by deregulated/ elevated exp of a gene. Myc is a central conduit for upstream oncogenic signalling, w/ SHH/Notch, RTK-Ras, Wnt-beta catenin+ other pathways feeding into it. Myc dereg implicated in many-all cancers. Myc required for cell prolif, w/ exp normally tightly dependent on mitogenic signals. Myc inhibition triggers regression of Kras-G12D lung tumours, leading to death of tumour cells (while healthy cells stop growth/division but don’t die).
Myc deregulation can be by retroviral insertion, mutation of ctrl gene, genomic amplification, chromosomal translocation, persistent stimulation by upstream oncogenic activity, N-Myc amplification (by double-minutes (extra-chromosomal mini chromosomes) correlates w/ poor prognosis, e.g., in neuroblastoma, as can be plotted on a Kaplan-Meier survival plot of neuroblastoma patients of event free survival with low level (<10 copies->usually 6+ years) vs high level (>10 copies->usually <2 years) N-myc amplification. C-myc dereg by chr translocation in non-AIDS Burkitt’s lymphoma.
Philadelphia chromosome (1 line); killing cancer cells and how drugs become ineffective
Philadelphia chromosome: chromosomal translocation between chr 9+22-> bcr-abl oncogene
Killing cancer cells: drug’s ability to target proliferating tumour cells without affecting normal proliferating tissues is expressed as the therapeutic index. Key is to trigger tumour cell apoptosis. Most cases, targeted inhibition of driving oncogene-> tumour cell death rather than growth arrest. Relatively easy to make drugs against enzymes but drug resistance presents challenge, and survival of tumour cells in first-line treatment means second-line treatment required (e.g., erlotinib-> 80% survival, follow with Osimertinib).
Some drugs now ineffective due to: mutations reducing drug binding w/out affecting target activity (“gatekeeper” mutations); compensation I a related pathway; increased genetic instability in tumour cells; drug efflux pumps.
Tumour suppressors, haploinsufficiency, evolution
Cancers arise through clonal accumulation of somatic mutations that inactivate tumour suppressors (cells more fit- coerce stroma-> vascularisation, e.g., therefore dominate)
Haploinsufficiency: studies in 60s/70s (Harris, Krudson)-> 2 copies of tumour suppressor must be inactivated in cancers.
Evolution: didn’t evolve specifically as cancer preventative mechanism- cancer= recent phenomenon, only arises in large, long-lived regenerative organisms w/ sufficient time for clonal accumulation/ combos of somatic mutations necessary to generate rogue cells (70% of human pop to survive over 70 alive now). Near all known TS genes evolutionarily ancient, highly conserved- cancer suppression= re-tasking if pre-existing f(x).
Types of tumour suppressors
caretakers (e.g., BRCA), gatekeepers (wnt pathway, cell cycle checkpoint mutations), stress sensors, landscapers
Tumour suppressors: Caretakers and an example
Caretakers- maintain genomic integrity. Genomes of many adult solid cancers v messy- loss of genome integrity arises as consequence of DNA damage w/ erosion of DNA damage sensing/repair machinery +/failed checkpoints. BRCA: germline mutations in BRCA1/2 -> 70-80% familial ovarian, breast cancers+ confer increased prostate cancer risk. Incidence up to 2.3% in some Ashkenazi Jewish pops. 1+2= unrelated proteins for DNA damage sensing+ genomic integrity maintenance. 1-> repair DS breaks, 2-> homologous recombination. Both also implicated in transcription reg in dev+ possibly other processes. Mutations have v high penetrance as seen in high lifetime risk for cancer. Multimodule proteins w/many partners- forms dimers w/ BARD1 via N-term RING domain, dimer binds damaged DNA+ interacts damage sensing machinery. Generic components of DNA damage response, but inactivation predisposes to cancer in limited tissues- idk why
Tumour suppressors: gatekeepers and 2 e.g.s
Gatekeepers- cell cycle checkpoints+ signal attenuators, ctrling/restraining/attenuating growth/survival/ migration signals. E.g. attenuators/mitogenic/survival signalling- PTEN, NF1; checkpoints-Rb, p21-cip1; APC attenuates Wnt growth factor signalling; attenuation of mitogenic signals inherent in many normal oncoproteins (Ras, Myc).
Familial adenomatous polyposis (FAP) incidence 1 in 10-15k, 95% polyps by age 35, due to inherited inactivation of 1 APC copy (germline); cancer when remaining APC mutationally inactivated, mean age of colorectal cancer in FAP patients 39 yrs- high incidence/penetrance.
Wnt pathway key in tissue dev, stem cell homeostasis. Ligand-induced release of beta-catenin from degradation complex scaffolded by APC; stabilised beta-catenin translocates-> nucleus, promote transcription via Tcf/Lef. Genetic inactivation/APC or stabilising mutations/ beta-catenin-> persistent Tcf/LEF transcription. When ligands disappear, signal must decay- spontaneous instructed feedback reg+ attenuates these signals
Cell cycle checkpoint mutations e.g., E2Fs (~7 TFs) highly conserved, dimerize w/ DP1/2 partners. E2F1-3a= activators, induce genes for S phase progression (Rb inhibits). E2F3b (RB), 4 (Rb, p107, p130),5 (p130) (probably 6 (PcG),7)= repressors/ genes for quiescence +/ differentiation. E2Fs inhibited by ‘pocket proteins’ incl Rb. Rb protein p105RB sequesters E2Fs via pocket- naturally occurring germline mutations in pRB pocket-> hereditary retinoblastoma; highly conserved, 1 of 3 pocket proteins (also p107, p130) of which only pRB= tumour suppressor. E2Fs 1-3a accumulate in G1, but sequestered in inactive, transcriptionally repressive complex by pRB- G1 cyclins inactivate pRB, liberate E2F function. Inhibition also incl HDACs/ chromatin remodelling
Tumour suppressors: stress sensors, Peto’s paradox and landscapers
Stress sensors- p53 ‘guardian of the genome’- TF activated by DNA damage, other stresses. Triggers cytosolic +/ apoptotic effectors. Evolutionarily ancient euk invention. Activated by DNA damage-> ATM/ATR/Chk1/2, inhibited by Mdm2/E3 UQ ligase, etc. P53 or components of its attendant pathways are functionally inactivated in >85% (possibly all) cancers- tumour cells can’t tolerate p53. p53 wild type fibroblasts irradiated w/8Gy (a lot) die, while p53 -ve tolerate DNA damage. P53 also activated by oncogene activation via ARF (oncogene sensor), unfolded protein response, hypoxia, metabolic stress… outputs include cycle arrest (transient or permanent/senescence), apoptosis/necrosis, terminal differentiation, repair/survival. Response depends of cell type+ env, severity+ nature of insult.
Peto’s paradox= bigger animal-> more cells-> higher probability of cancer, with other factors absent- doesn’t match observations with elephants, whales, etc. Naked mole rat p53 protein ½ life 10x that in mammal counterparts (9 min)+ has persistent nuclear localisation- may help explain paradox.
Landscapers- restrain precocious clonal expansion through restrictive impact on somatic env- stromal remodelling, angiogenesis, immune surveillance.
