Oncogenes and tumour suppressors Flashcards

Question

How can p53 regulate metabolic pathways?

Answer 1

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.

Answer 2

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

Answer 3

Multiple mechanisms! Includes senescence, apoptosis (both nuclear and cytoplasmic p53), autophagy, signalling through mTOR (down regulates growth, up regulates autophagy), decreasing ROS

Answer 4

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

Answer 5

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

Answer 6

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.

Answer 7

A family of Janus (JA) tyrosine kinases (K). There are 4 including Tyk2

Answer 8

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.

Answer 9

Suppressors Of Cytokine signalling proteins. Activated by STATs.

Answer 10

Protein Inhibitor of Activated Stat

Answer 11

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.

Answer 12

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

Answer 13

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

Answer 14

Tyk2, Jak1 and Jak2 are expressed in all tissues; Jak3 is expressed in haematopoietic tissues

Answer 15

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.

Answer 16

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)

Answer 17

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)

Answer 18

As a dimer in a nutcracker formation. Go look at a picture.

Answer 19

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.

Answer 20

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.

Answer 21

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

Answer 22

In sooooo many ways. Loads of regulation this way

Answer 23

Either bind the cytosolic tail of the receptor to block STAT binding Or bind JAK and interfere Depends on the SOCS

Answer 24

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.

Answer 25

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.

Answer 26

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

Answer 27

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.

Answer 28

``` Receptor mutations Activating mutation in JAK Mutation in STAT coding sequence Loss of inhibitory signalling Dysregulation of protein tyrosine phosphatases ```

Answer 29

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

Answer 30

Other tyrosine kinases can phosphorylate STAT e.g. SARC and ABL. Not just JAKs!

Answer 31

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.

Answer 32

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

Answer 33

In the SH2 domain at the surface

Answer 34

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..

Answer 35

Spread out across the STAT as opposed to being concentrated in the SH2 domain. Suggested that acetylation and modification sites may be altered.

Answer 36

Methylation of the promoter and silencing of the promoter - not in the coding region.

Answer 37

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

Answer 38

Drives expression of IL-6 and IL-10 which act on immune cells including macrophages and Tregs. Leads to immunosuppression somehow.

Answer 39

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 Antisense/siRNA Activating protein tyrosine phosphatases

Answer 40

Toxic effects Lack of specificity Inhibition of STAT1 by STAT3 inhibitors (STAT1 is anti proliferative) Delivery

Answer 41

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.

Answer 42

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.

Answer 43

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.

Answer 44

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)

Answer 45

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

Answer 46

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

Answer 47

PTEN (a tumour suppressor). Has the reverse activity of PI3K

Answer 48

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.

Answer 49

Class IA and IB act on PIP(4,5)(2) Class II is thought to act on PI Class III acts on PI

Answer 50

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

Answer 51

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

Answer 52

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.

Answer 53

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

Answer 54

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.

Answer 55

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)

Answer 56

Inherited germ line mutations, somatic mutations, epigenetic and transcriptional silencing, post translational modifications and protein-protein interactions

Answer 57

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

Answer 58

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.

Answer 59

Deletion of p110alpha and p110beta | Treatment with BKM120, a PI3K inhibitor that acts on p110alpha, beta and delta

Answer 60

RTK inhibitors PI3K inhibitors mTOR inhibitors Akt inhibitors (a hub protein, important if there is a loss of PTEN)

Answer 61

``` Pan class I Isoform selective (specific) Rapamycin analogues (rapalogues, inhibit mTOR) Active site mTOR inhibitors dual PI3K/mTOR inhibitors AKT inhibitors ```

Answer 62

Wortmanin. Potent but toxic in animal studies. Less toxic drugs have been developed based on this.

Answer 63

``` 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 ```

Answer 64

An mTOR inhibitor. Inhibits primary and metastatic tumour growth in mice by inhibiting angiogenesis

Answer 65

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

Answer 66

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

Answer 67

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.

Answer 68

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

Answer 69

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.

Answer 70

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.

Answer 71

Cell cycle arrest and other diverse responses e.g. senescence reinforcement (autocrine signalling), wound healing, cellular reprogramming, embryonic development

Answer 72

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

Answer 73

The number of times a cell can duplicate before it goes into senescence.

Answer 74

Related to telomere shortening. Correlates with senescence and the Hayflick limit. The Hayflick limit can be extended by lengthening telomeres

Answer 75

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.

Answer 76

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.

Answer 77

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.

Answer 78

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

Answer 79

Depends on the cell type they are in. In primary genes, Ras activation induces senescence; in immortalised cells Ras activation causes transformation

Answer 80

Are moles. Often contain BRAF mutations but have been induced into senescence. However can go on to form melanomas.

Answer 81

Secrete products that activate immune cels to remove them. Also secrete things to reinforce senescence.

Answer 82

Older people have more senescent cells. Elimination of these cells has been shown to extend healthy life span in vivo. Does senescence cause ageing? Suggested that senescence causes tissue/organ ageing due to impairment of repair mechanisms. This leads to individual ageing.