Flashcards in Oncogenes and tumour suppressors Deck (106):
How many mutations are found in human cancers?
Depends on the cancer type - loads in colorectal cancer as genome is unstable, few in others. A typical tumour contains 2-8 driver mutations, the rest are passengers that give no advantage
How are driver genes classified?
As mountains (often mutated) and hills (mutated less but still frequently). Using a 20/20 rule (in which more than 20 percent of mutations are truncating or inactivating suggest a tumour suppressor whilst more than 20 percent missense mutations in recurrent positions are oncogenes). Found 71 TSGs and 54 oncogenes in a high confidence study
What is haploinsufficiency?
Where one normal copy isn't enough to sustain function. Don't need to lose both copies. Most tumour suppressor genes are haploinsufficient.
What is MutSigCV?
A new approach to identify significantly mutated genes in cancer. Corrects for variation by using patient specific mutation frequency and spectrum as well as gene-specific background mutation rates incorporating expression level (if highly expressed more likely to be mutated) and replication time (when replicated in S phase can effect chance of mutation). Using this method significantly shrinks the list of mutated genes in lung cancer.
How can the Rb pathway be inactivated?
Direct: mutation of the Rb gene/binding and inhibition of Rb by proteins of small DNA tumour viruses.
Indirect: amplification of the gene encoding cyclin D1 (promotes Rb phosphorylation) or loss of the cyclin dependent kinase inhibitors p16 (INK4A)
How is the Rb pathway affected in human cancers?
Inactivated in most human cancers. Depending on the cancer, get different characteristic inactivations at different points (could be oncogenic or tumour suppressive) - this may be due to genetic background of the tissue
How can loss of a tumour suppressor gene occur?
Healthy cell with only one normal copy
Nondisjunction causing chromosome loss
Chromosome loss then chromosome duplication
Describe the E2F family of proteins in humans
9 distinct proteins. 3 activators (1-3a) and 6 repressors (3b-8), though these aren't strict (repressors do some activation etc). E2F1-6 dimerise with DP proteins (of which there are 4). E2F1-5 interact with Rb family and recruit chromatin modifiers and remodelling factors (each E2F has affinity for a different Rb family member). E2F6-8 repress transcription in an E2F independent way
How do different members of the E2F family interact in Drosophila
Only 2 members: E2F1 (activator) and E2F2 (repressor). Can rescue the phenotype of the knock out of one by knocking out the other - functional antagonism
How does E2F induce proliferation and apoptosis?
Through external signals. If there is an increase in E2F signalling, this drives apoptosis. Need the parallel mitogenic signals to turn off the apoptotic pathway so E2F only promotes proliferation. Apoptosis is suppressed by mitogenic signals
How does E2F1 regulate tumour development?
If there is proper growth signalling and no DNA damage, apoptotic potential of E2F1 is repressed. If cells experience DNA damage/lack of signalling, apoptotic activity is released. Knocking out E2F1 results in tumours in mice, a tumour suppressor like property
How does Rb have such a big effect on cellular fate?
Rb interacts directly with chromatin associated factors that regulate transcription, chromatin modification and remodelling. Inactivating Rb changes the transcriptional profile of many genes.
What is the effect on mitosis of loss of Rb?
Causes centromere dysfunction, aneuploidy and chromosomal instability. Could be due aberrant transcription of MAD2, a SAC protein and CENPA?
How does Rb loss cause DNA damage?
If E7 (small DNA virus protein) inactivates pocket proteins, this causes replication stress (E2F regulates expression of DNA replication proteins, nucleotide synthesis genes and DNA damage response and repair genes) and stalling of replication forks at repetitive regions of DNA. This increases DNA damage and loss of heterozygosity and CIN/anueploidy.
How does Rb affect chromosome condensation?
In Drosophila chromatin appears puffy if Rb is deleted. Found that Rb interacts directly with condensing II subunit of CAP-D3 in Drosophila and human cells and is required for condensin accumulation of chromosomes.
Describe the initial evidence surrounding p53
p53 expression levels directly correlated with T-antigen activity
p53 was found to associate with the E1B antigen of adenovirus
Many tumours but not normal tissue showed high p53 levels
Transfection of p53 clones from transformed cell lines could co-operate with HRAS (another oncogene) to transform primary cell cultures
p53 cloned from transformed cell lines could augment the transformed properties of established cell lines
Suggested that p53 was an oncogene
How has p53 shown to be a tumour suppressor?
50% of human tumour cells have p53 loss/mutation
Heritable p53 mutant allele predisposed to cancer
100% of p53-null mice got cancer
Describe the structure of p53
N terminus-TAD1-TAD2-PRD-DNAb--Tet-Basic-C terminus
TAD = transactivating domain
DNAb = DNA binding (cancer hotspot mutations)
Tet = tetramer, has a nuclear export signal
Basic = for non-specific DNA binding (scanning), has nuclear localisation signal
How does p53 bind DNA?
As a tetramer to bi-partite target response elements
What factors affect p53 DNA binding?
Trans: p53 activating stimuli, cellular environment, nuclear p53 levels, post-translational modifications, protein-protein interactions, cofactors and transcription factors, chromatin environment
Cis: non-canonical response elements (RE), canonical tetrameric RE, mismatches in RE number or position, spacer length, sequence context/nearby transcription factor REs, SNPs/CNVs. position of RE to transcription start site
What stimuli does p53 respond to?
Nutrient deprivation (through AMPK phosphorylation), telomere erosion, hypoxia, DNA damage (through ATM/ATR), Ribosomal stress (some ribosomal proteins bind MDM2 as stress indicators), oncogene activation (ARF)
What is the response of p53?
Can trigger apoptosis, cell-cycle arrest, senescence, DNA repair, survival and genomic stability. Can regulate metabolic pathways and autophagy. What it does depends on the modifications that activated it.
How is p53 activated?
Normally, p53 is ubiquitinated and degraded and exported from the nucleus by MDM2. Can be relieved of this and translocated to the nucleus through ARF, Mdmx and modifications of Mdm2 and p53. In the nucleus, modifications of p53 can de-repress it from Mdm2/Mdmx e.g. acetylation or phosphorylation can relieve MDM2/ MDMX inhibition of p53 bound to a response element. Then, p53 can recruit cofactors to aid expression of genes for its response.
How is p53 regulated by Mdm2 and Mdm4?
Mdm4 = MdmX. Mdm2 is an E3 ubiquitin ligase that targets p53 for degradation. Mdm2 and 4 bind p53 and inhibit its transactivation. Mdm4 binds Mdm2, stabilising it and boosting its ligase activity.
How can p53 regulate metabolic pathways?
Can channel glucose into the pentose phosphate pathway by activating TIGAR. This, along with p53 induction of sestrins leads to lower ROS levels and survival
Signalling through AMPK (which detects low glucose) to p53 causes it to activate p21 which induces survival.
How does p53 regulate apoptosis?
Upregulates PUMA which inhibits the binding of BCL-X to p53 in the cytoplasm. p53 then goes to recruit BAX to the mitochondrial membrane directly and inhibit anti-apoptotic proteins
How does p53 suppress tumour growth?
Multiple mechanisms! Includes senescence, apoptosis (both nuclear and cytoplasmic p53), autophagy, signalling through mTOR (down regulates growth, up regulates autophagy), decreasing ROS
What mechanisms exist for p53 gain of function mutations?
Prevent interaction with p53 look a likes
Novel protein interactions (e.g. a new protein or a transcription factor)
Bind different sites
Initiate different transcription
Describe the JAK/STAT signalling pathway
Cytokines bind cytokinR which has constituently associated tyrosine kinases (members of the JAK family such as Tyk2). These phosphorylate themselves and allow STATs to bind. Members of the STAT family bind to the phosphate and are phosphorylated. These then dimerise and are translocated to the nucleus where they up regulate interferon stimulated response element (ISRE) genes.
STATs can also be serine phosphorylated by MAPKs which can affect their localisation
How are STATs down regulated?
SOCs inhibit STATs. These are activated by STATs, so there is a burst of STAT activity which is then down regulated. In cancer, the down regulation doesn't occur.
What are JAKs?
A family of Janus (JA) tyrosine kinases (K). There are 4 including Tyk2
What are STATs?
Signal Transducer and Activator of Transcription. Transcription factors. Are 6 STATs; 2 variants of STAT5 (encoded on different genes - gene duplication) = 7 overall. They can also have splicing variants.
What are SOCs?
Suppressors Of Cytokine signalling proteins. Activated by STATs.
What are PIAS?
Protein Inhibitor of Activated Stat
Describe they cytokine receptors involved in JAK/STAT signalling
Each cytokine binds a specific alpha chain which is associated with a common cytokine receptor gamma©. This then signals through either JAK1 or JAK3 to a different STAT.
Describe the receptor for IL-2 and IL-15
Has a common cytokine receptor gamma© and a specific-to-the-cytokine alpha chain. Also has an IL-2Rbeta chain to make a high affinity complex
Describe the structure of JAK
Has a C terminal JH1 domain which is the kinase domain
Then has a JH2 domain which is the pseudokinase domain (controls kinase activity) where many mutations are found
At the N terminal in domains JH3-7 there is a FERM domain which mediates receptor binding
Where are JAKs expressed?
Tyk2, Jak1 and Jak2 are expressed in all tissues; Jak3 is expressed in haematopoietic tissues
What JAK KOs are viable?
Neither JAK1 (perinatal) or JAK2 (embryonic). JAK3 is but has severe defects in lymphocyte development; Tyk2 is viable and fertile, has defects in cytokine signalling.
What is the structure of STATs?
N terminal can be acetylated and otherwise modified. Then have a CC (coiled coil) domain for nuclear receptor binding and a DNA binding domain. Then have a linker domain and an SH2 domain next to the phosphorylated tyrosine followed by a transactivating domain to bring in other factors, individual to the STAT (gives each STAT a variety of functions)
Where are STATs expressed?
Widely expressed apart from STAT4 which is restricted to mesodermal lineages and T cells where it commits to a Th1 response (Th2 is by STAT6)
How do STATs bind DNA?
As a dimer in a nutcracker formation. Go look at a picture.
What genes does STAT3 bind?
Has a core of 35 binding sites that are always active in any cell type. Include STAT regulatory genes such as up regulating STAT3 expression. SOC3 promotor is bound differentially depended on cell type (found by ChIP-seq)
STAT3 is otherwise strongly cell type specific.
How does STAT3 activate transcription?
It binds DNA at GAS motives and co-operates with other transcription factors and co-factors in transcriptional regulatory modules (TRMs). Include tissue specific and general transcription factors and are responsible for the differing functions of STAT3 in different cell types.
What is the effect of STAT KOs?
Stat1 - loss of type 1 and type 2 IFN responses (downstream of IFNalpha and gamma)
Stat2 - loss of type 1 IFN (complexes with Stat1)
Stat3 - embryonic lethal
Stat4 - impaired NK cell cytotoxicity and Th1 response (no IL-12 signalling)
Stat5a - no mammary gland development or lactogenesis (no prolactin signalling)
Stat5b - loss of growth hormone signalling
Stat6 - no Th2 cell development and loss of IL-4/13 signalling
How are STATs post-translationally modified?
In sooooo many ways. Loads of regulation this way
How do SOCs down regulate STATs?
Either bind the cytosolic tail of the receptor to block STAT binding
Or bind JAK and interfere
Depends on the SOCS
What is the structure and function of SOCS?
8 family members. All have a central SH2 domain and an extended SH2 domain and a C terminal SOCS box. SOCS1 has an NLS. SOCS1 and 3 have a kinase inhibitory region that is a pseudo substrate for JAKs to block their function. The SOCS box interacts with ubiquitinating machinery e.g. Cul5 and an E2 ubiquitin transferase.
How are non-coding RNAs involved in JAK/STAT signalling?
They down regulate the response at many levels. miRNAs are made upon STAT activation that down regulate STAT and JAK expression as well as down regulating inhibitors of STAT and JAK.
How is the JAK/STAT pathway involved in cancer?
STAT3 is constituently activated in 70% of solid tumours (has a role in normal cell growth) and is an important regulator of inflammation and the tumour microenvironment as well as survival
STAT5 is also constituently active
STAT1 has anti tumour and cancer immunosurveillance functions
JAK2 mutations promote myeloproliferative disorders
How mutations in STAT3 cause constituent activation?
A cysteine bridge between the 2 STAT molecules causes constituent dimerisation and therefore constituent activation. Adding this to partially tumourgenic cells in mice leads to aggressive tumours.
How can STAT be constitutively activated?
Activating mutation in JAK
Mutation in STAT coding sequence
Loss of inhibitory signalling
Dysregulation of protein tyrosine phosphatases
Describe some mutations in the cytokine receptor that cause constituent STAT signalling
Tpo - point mutation in which a tryptophan is substituted for a leucine or a lysine at a specific motif is associated with severe myeloproliferative disorders
G-CSFR - truncation mutants promote acute myeloid leukaemia
gp130 - in frame deletions get gain of function mutations
How can STAT be phosphorylated in cancers?
Other tyrosine kinases can phosphorylate STAT e.g. SARC and ABL. Not just JAKs!
What are the consequences of mutations in JAK2?
Too many red blood cells made due to a specific base change in the pseudokinase domain causing constitutive phosphorylation.
In general, JAK2 mutations cause myeloproliferative disorders.
What mutations in STAT3 are seen in hematopoietic malignancies?
Mainly in the SH2 domain (which normally binds the Y-phos on the other STAT or associates with the receptor). Mutated residues are commonly found at the dimerisation interface of the SH2 domain
How is STAT5b mutated in large granular lymphocytic leukaemia?
In the SH2 domain at the surface
How is STAT5 involved in BCR-ABL leukaemia?
Is the central signalling node. STAT5 is phosphorylated by the fusion protein so JAK2 signalling is dispensable. Get elevated target gene expression from STAT5 homodimers and heterodimers with activated STAT3..
How is STAT3 mutated in solid malignancies?
Spread out across the STAT as opposed to being concentrated in the SH2 domain. Suggested that acetylation and modification sites may be altered.
How can SOCS be mutated in hepatocellular carcinoma?
Methylation of the promoter and silencing of the promoter - not in the coding region.
How does activation of STAT3 contribute to tumourigenesis?
Has roles in the regulation of proliferation genes (e.g. myc, cyclin D1), evasion of growth suppression and cell death (Bcl-X), angiogenesis (VEGF), Invasion and metastasis, tumour promoting inflammation
How does STAT3 influence the immune c ell environment?
Drives expression of IL-6 and IL-10 which act on immune cells including macrophages and Tregs. Leads to immunosuppression somehow.
What are the prospects for therapy targeting the JAK/STAT pathway?
Small molecule inhibitors of Stat (hard as aren't enzymes, are transcription factors)
Inhibitors of mutant JAKs
Tyrosine kinase inhibitors (e.g. BCR-ABL)
Deliver a dominant negative STAT somehow
Decoy oligonucleotides for STAT binding
Activating protein tyrosine phosphatases
What are the problems with targeting the JAK/STAT pathway in cancer?
Lack of specificity
Inhibition of STAT1 by STAT3 inhibitors (STAT1 is anti proliferative)
What JAK2 inhibitors have been developed?
Block JAK2 with AZD compounds. Has shown to be tolerated despite JAK2 being important. These drugs can also suppress STAT3 tumorigenesis - could open a new therapeutic door.
How has a STAT3 inhibitor been developed?
Is a non-selective alkylating agent. STAT3 is a flat molecule with no enzymatic activity so is hard to target. Instead, inhibit it by alkylating the protein (and other things), causing toxicity.
What does PI3K do?
Phosphatidylinositol 3 kinase. Phosphorylates phosphatidylinositol at the 3 carbon. If the ring is not phosphorylated at all, this produces PIP(3). If the ring is phosphorylated at other places, get bi and tri phosphates.
Can also act on proteins - is a dual specific kinase.
How is the PI3K pathway involved in cancer?
Highly mutated in many cancers (many components to mutate)
Controls many hallmarks including proliferation, survival, metabolism, invasiveness and genomic stability
Promotes aspects of the tumour microenvironment (angiogenesis, inflammatory cell recruitment)
Several components are deleted/activated
Many potential therapeutic targets, but have to target enzymes that are also active in normal tissue (control things like autophagy)
What does PI3K activity control?
Transmits signals from cell surface to the cytoplasm by generating second messengers which activate more kinase pathways including AKT, PKC, NFkB, JNK/APK resulting in survival and growth
What are the classes of PI3K?
3 classes: class I (A and B), class II and class III. Class I consists of PI3Kalpha, beta and delta (class-IA) and gamma (class-IB). alpha and beta are ubiquitously expressed whilst delta and gamma are mainly in leukocytes. Class I are involved in signal transduction and growth. Class II and III are implicated in vesicular trafficking (e.g. endocytosis control and lysosomal delivery). Class III includes Vps34. Classification is based on substrate and products
How is PI3K down regulated?
PTEN (a tumour suppressor). Has the reverse activity of PI3K
Describe the PI3K pathway which activates AKT?
Signal from growth factors to RTKs. Catalytic subunit p110 is recruited through its regulator subunit p85/p101 which is recruited through adaptors. PI3K is activated and phosphorylates PIPs. These activate AKT which is phosphorylated by PDK1 and mTORC. AKT can then activate NFkB, Mdm2 and inhibit BAD for growth and survival signals.
What does each class of PI3K act on?
Class IA and IB act on PIP(4,5)(2)
Class II is thought to act on PI
Class III acts on PI
Describe the different subunits of PI3K
Class IA: p110 catalytic subunit, associates with one of 5 different regulatory subunits, encoded by 3 different genes and additional splicing isoforms/potentially different start sites
Class IB: p110 catalytic subunit and one of 2 regulator subunits
How can the PI3K pathway be aberrantly activated?
Activating mutations or amplifications of catalytic subunits of PI3Ks
Inactivation of PTEN (deletion, silencing or mutation)
Receptor amplification/mutation (RTKs or GPCRs)
Mutations/amplifications of AKT
What is the structure of p110alpha?
N termal p85 binding domain, then a Ras binding domain, then a C2 region then a helical domain and a kinase catalytic domain at the C terminus. Half the mutations are in the kinase domain, the others are in the helical domain.
Describe the E545K mutation in the PI3K pathway
Is in the helical domain of p85. In wt p85 a conformational change occurs upon activation that then allows binding of Ras and opening of the kinase. In the mutant the protein is always open so there is no need for it to bind a receptor leading to constituent activity
Describe the H1047R mutation in the PI3K pathway
Is in the kinase domain of p85. The context of the cell type in which this mutation arises depends on the kind of cancer that results. Experiments expressing the mutant from different tissues have shown this.
Describe the consequences of mutations in PTEN
A frequently disrupted tumour suppressor. Normally, PTEN antagonises the PI3K pathway to repress growth and survival and promote chromosome stability and DNA repair. Loss increases genomic instability. (These suggest further functions of PTEN in the nucleus)
How can PTEN deficiency arise?
Inherited germ line mutations, somatic mutations, epigenetic and transcriptional silencing, post translational modifications and protein-protein interactions
What is PHTS?
PTEN hamartoma tumour syndrome. Caused by germ line inactivating mutations of PTEN. Benign hamartomas develop in various tissues; increased risk of malignancy. Studies in mice have shown that the condition can be rescued by deleting both p110alpha and p110beta - these are potential therapeutic targets
Describe the structure of PTEN
PIP(2) binding domain at N terminus, then a phosphatase domain (has half of mutations), then a C2 domain (other half of mutations, has a ubiquitination residue) then 2 PEST domains for degradation, then a PDZ domain at the C terminus for protein protein interactions.
How can a PTEN deletion be rescued in mice?
Deletion of p110alpha and p110beta
Treatment with BKM120, a PI3K inhibitor that acts on p110alpha, beta and delta
What are the potential therapeutic targets in the PI3K pathway?
Akt inhibitors (a hub protein, important if there is a loss of PTEN)
What are the classes of PI3K pathway inhibitors?
Pan class I
Isoform selective (specific)
Rapamycin analogues (rapalogues, inhibit mTOR)
Active site mTOR inhibitors
dual PI3K/mTOR inhibitors
What are some pan class I PI3K inhibitors?
Wortmanin. Potent but toxic in animal studies. Less toxic drugs have been developed based on this.
What are the two classes of mTOR inhibitors?
Partial inhibition (includes rapalogues), have a bigger effect on certain mTOR substrates compared to others. Weak inhibition of cap-dependent translation leads to negative feedback and activation of the pathway upstream. Don't block mTORC2 signalling so cell can survive through AKT
Active-site mTOR inhibitors fully block phosphorylation of all known substrates
What is rapamycin?
An mTOR inhibitor. Inhibits primary and metastatic tumour growth in mice by inhibiting angiogenesis
How can interfering with PI3K have indirect anticancer effects?
Can be used to target the immune system and enhance anticancer responses. E.g. by improving circulation to the tumour to aid chemotherapy and immunotherapy/inhibiting angiogenesis promoting cells. Interfering with generation of fibrous connective tissue around tumours. Interfering with survival and homing signals provided by stroma in B cell malignancies. Interfering with immunosuppressive mechanisms to enhance tumour killing
Describe the negative feedback pathway involved in mTOR inhibition
Normally, activation of mTOR results in down regulating signalling through IRS-1 by phosphorylation and targeting for degradation. This is lost if mTOR is inhibited
What is the negative feedback pathway involved in HER2 and PI3K therapies?
FOXO3a is normally excluded from the nucleus through HER2 and PI3K signalling. If this is blocked, FOXO3a moves into the nucleus and causes up regulation of HER3 and IGF1R, causing escape from pathway inhibition.
How can the problem of negative feedback pathways be solved when looking to treat the PI3K pathway?
Combination therapies to block the pathway at multiple steps. However, this increases toxicity. Need to understand feedback better and combine this with knowledge of how therapies affect emerging immunotherapies and cancer vaccines
What do the words primary, immortalised and transformed mean in the context of tumourigenesis in culture?
Primary - can't proliferate forever, limited growth, undergo senescence
Immortalised - 'unlimited growth', sensitive to apoptosis (are easy to kill), non-tumorigenic, loss of senescence
Transformed - 'unlimited growth', sensitive to apoptosis, tumorigenic, loss of contact inhibition, loss of anchorage dependency, form tumours in nude mice.
What signals lead to senescence?
p53-p21 and p16-Rb pathways lead to cell cycle arrest due to a variety of inputs (telomere dysfunction, oxidative stress, oncogene activation, developmental cues, genotoxic stress or DNA damage). Leads to cell cycle arrest and other things.
What is the result of senescence?
Cell cycle arrest and other diverse responses e.g. senescence reinforcement (autocrine signalling), wound healing, cellular reprogramming, embryonic development
What are the markers of senescence?
Stable arrest (remove trigger, arrest stays)
SA-beta-gal (Senescence Associated)
p16 involved in cell cycle arrest
DNA damage may be present
Cell cycle arrest (could also indicate quiescence)
Many markers, none are definitive - have to combine
What is the Hayflick limit?
The number of times a cell can duplicate before it goes into senescence.
What is replicative exhaustion?
Related to telomere shortening. Correlates with senescence and the Hayflick limit. The Hayflick limit can be extended by lengthening telomeres
Describe the structure of telomeres
Have a telomere cap (otherwise blunt end may be interpreted as a DNA break). A long 3' overhang forms a loop which invades back in.
How does telomere shortening induce senescence?
Telomere shortening is recognised as DNA damage and induces apoptosis/senescence to avoid chromosome fusions. Get signalling through p53 to a CDKI p21 which activates Rb. If there is telomere uncapping, also get this pathway triggered. Leads to persistent DNA damage response and senescence.
How does oxygen affect the Hayflick limit?
Atmospheric oxygen levels are toxic to cells. If cells are cultured in low oxygen, the Hayflick limit is delayed. In mouse embryonic fibroblasts, there is an active telomerase, but will still senesce if under oxidative stress.
How do viruses interfere with senescence?
Viral oncoprotein e.g. E1A and E1B from adenoviruses bind Rb and p53 respectively
Large T antigen from SV40 binds Rb and p53
Human papilloma virus E7 and E6 bind Rb and p53 respectively
When can oncogenes induce senescence?
Depends on the cell type they are in. In primary genes, Ras activation induces senescence; in immortalised cells Ras activation causes transformation
What do nevi tell us about senescence?
Are moles. Often contain BRAF mutations but have been induced into senescence. However can go on to form melanomas.
How are senescent cells removed?
Secrete products that activate immune cels to remove them. Also secrete things to reinforce senescence.