Biology of Cancer

What are 5 ways cancer cells different to normal cells?

Loss of: Anchorage dependence, GF dependence, Contact inhibition, density inhibition, replicative senescence.

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What is the relationship between the dosage of a carcinogen and the delay in tumour formation

Inversely proportional, meaning the greater the dose the shorter the delay

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What is a possible explanation for the reason there is a delay between tumour formation and carcinogen exposure?

It suggests multiple independent hits genetically are required to produce a tumour (this is also seen in viral tumour viruses and the need for co-factors)

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What is the function of telomeres

During DNA replication they fix the ‘end replication defect’, however limited replication is also a result of telomere shortening.

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How does telomere shortening cause limited replication in normal cells & what is the result of this shortening

With each replication telomeres get shorter, at a certain length DNA is flagged as damaged and senescence and apoptosis occur via tumour suppressor networks

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What are the tumour suppressor networks involved in telomere induced senescence / apoptosis?

p16INK4a / Rb and p53 / p21cip1

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What is the name of the enzyme that maintains telomere length

Telomerase / TERT reverse transcriptase. Synthesis telomere repeats from a short RNA template

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What % of human tumours express telomerase and what is the significance of this?

~85%. Meaning these tumour are able to constantly add telomere repeats and lose this senescence / apoptotic pathway of telomere shortening

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Tumour cells achieve telomerase activity by reactivating expression, however a minority of tumour cells achieve long telomeres without reactivating this enzyme, what is the name of this pathway?

ALT pathway

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Name for a gene that contributes to cancer and is derived from a cellular protocol-oncogene

Oncogene

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Do oncogenes have a dominant or recessive effect

Dominant

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Name 4 ways in which oncogenes create cancer whereas proto-oncogenes do not

1. Overexpression. 2. Inappropriate expression. 3. Deregulation of protein product. 4. Altered specificity / function (translocations can create a whole new protein altogether which has a different function to the original pronto-oncogene)

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What are the normal functions of protooncogenes

Cell growth / proliferation, regulation of apoptosis, differentiation and cellular life span

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PDGF = and is released by what cell type

Platelet derived GF, platelets

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How many different genes are there for PDGF

4 (A->D increasing in size)

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Which of the 4 PDGFs are ubiquitous

A, B and C

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What is the expression pattern of PDGF-D

Highest expression in: heart, pancreas and ovary. None is seen in brain, lung and muscle

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All PDGFs have a conserved region of how many amino acids? And what residues do they all contain

100aa, cysteine residues allowing them to form disulphide bonds

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PDGF form dimers to be ligands, how many isoforms of PDGF ligand are there?

5

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Which two PDGF genes only form homodimers

C and D

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What are the biological functions of PDGF? (3)

Embryonic development (kidney, blood vessels, lung, CNS), Adult blood vessels, Wound healing (fibroblasts, WBCs, SMCs)

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Sis oncogene derives from a PDGF chain / gene. Which PDGF chain

B

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Sis oncogene is an example of what type of expression?

Inappropriate

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EGF genres all contain a same core motif of how many amino acids, how many cysteine residues do they all have and how many peptide loops does this develop?

100aa, 6 cysteine, 3 loops (through the formation of disulphide bonds)

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Are EGF proteins transmembrane?

Yes

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What are the biological functions of EGF (2)

Wound healing (fibroblasts, regeneration of epidermis), in viva involved in embryonic development

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TGF-Beta =

Transforming growth factor Beta

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Are TGF-beta and TGF-alpha in the same family?

No, TGF-alpha is part of the EGF family

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What are three proteins that are part of the TGF-beta family?

TGF-beta, Activin A, BMPs

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How many TGF-beta isoforms are there and which is the most ubiquitous

5 and B1

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Describe the steps of biosynthesis of TGF-beta

1. Complex of TGF-beta, signalling sequence and latency-associated protein. 2. The signal sequence is removed in the Golgi apparatus. 3. LAP-TGF-beta complex forms a homodimer with another complex via disulphide bonds. Homodimer is released into ECM

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What are the two methods of activating TGF-B from its inactive form sequestered by LAP and held in the matrix at LAPs ECM binding sites?

1. Plasmin cleaves TGF-b from LAP. 2. LAP has an RGD sequence that binds to integrin (transmembrane protein that anchors cells to ECM). As the cell moves there is a mechanical pull that frees TGF-b

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What is the significance of the mechanical activation of TGF-beta in tumorigenesis?

Matrix surrounding tumours is different to that of normal cells, and the mechanical activation opportunity presents much more widely in tumour cells.

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What effect does TGF-beta have on cell proliferation?

Can be both pro or anti (but mostly anti), dependent on the growth environment. Slows down in fibroblasts but promotes NK cells

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As well as cell proliferation what are other biological activities of TGF-beta

Cell adhesion (increased integrin, ECM proteins and inhibition of ECM degradation), cell differentiation, tissue repair

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FGF =

Fibroblast growth factor

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How many FGF genes are there in humans and how many families are they divided into

22 genes, 7 families

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What are the two favourable integration sites of MMTV (mouse mammary tumour virus)

Wnt-1 and FGF3

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Once a ligand has activated a receptor, signalling is then regulated by (4)?

Affinity of ligand to receptor, duration of interaction, internalisation of receptor, receptor degradation (turn off) or cycling back (sustained)

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What is a receptor tyrosine kinase (RTKs)

A catalytic receptor that has an enzymatic region (which is a tyrosine kinase) that is activated upon a ligand binding. Has extra cellular, transmembrane and intracellular domains.

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From ligand binding to Sh2 domain containing protein recruitment, what are the steps that occur on an RTK?

1. Ligand binding promotes receptor dimerisation. 2. Dimerisation and therefore conformational change -> kinase activity which trans/autophosphorylates leading to activation. 3. Proteins which contain Sh2 domains bind to phosphorylated tyrosine

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Name 4 groups of RTKs

EGFR, FGFR, PDGFR, HGFR

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How many known RTKs and oncogenes are there known in humans

58 RTKs, 29 oncogenes

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What was the first discovered RTK

EGFR

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EGFR is also known as

Erb and HER

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How many types of EGFR are there?

4

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EGF forms homo or heterodimers, what are the most common?

Homodimer of 1, 1:4, 2:3, 2;4

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Which EGFR lacks an extra cellular domain and which lacks an intracellular domain?

2 - extracellular, 3 - intracellular

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HER2 mutations you do not see truncations / mutations like other EGFR receptors, what do you see?

Over expression / amplification

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Ertibux / cetuximab block what region of the EGFR

The receptor site (extracellular)

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Gefitinib, erlotinib, lapatinib target what region of the EGFR receptor

Kinase terminal

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Herceptin is what?

Monoclonal Ab that selectively targets the extracellular domain of HER2

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How many subunits of a PDGFR are there and what are they called?

2, alpha and beta. (There are differences in the downstream signalling of each receptor) can form homo or heterodimers

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Which PDGFs bind to alpha PDGFR

A, B, C

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Which PDGFs bind to beta receptor

B and D

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Which PDGF ligand dimer can interact with all PDGF receptors

PDGF-BB

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What would be the effect of an activating point mutation in a kinase domain of an RTK

Receptor will be able to phosphorylate without an external signal -> constant phosphorylation

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Amplification of PDGFR alpha is seen in which cancers

Glioblastoma

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Activating point mutation of PDGF alpha R is seen in which cancer

GIT, stromal tumours

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Translocation involving the PDGFBR is seen in which cancers

Chronic myelomonocytic leukaemia

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Translocation involved the PDGFB ligand is seen in which cancers

Dermatofibrosarcoma

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How many FGFR genes are there?

4

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What is required for FGF binding to the FGFR

Heparin / heparin sulphate (this is also required for the dimerisation of FGFR)

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Name of a drug that has been shown to reduce tumour growth and metastasis by its action on FGF

Thalidomide

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What GF binds with strong affinity to a receptor encoded by the c-met oncogene

HGF

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What intrinsic kinase activity does TGFbR have

Serine / threonine therefore a RSTK

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How many high affinity TGFbR are there and which two have an intracellular Serine threonine domain

3, 1 and 2 have kinase domains, 3 is a proteoglycan

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What are the steps of ligand binding to TGFbR receptor -> intracellular signalling

1. TGFb binds to TGFbR3 which then presents TGFb to TGFbR2. Or 1b. TGFb binds to TGFbR2 directly. 1. Binding induces TGFbr2 to recruit TGFbR1. 3. R2 then phosphorylates and activates R1. 4. R1 then phosphorylase intracellular proteins

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TGFb inhibits epithelial, endothelial and haematopoietic cell proliferation, therefore what would you expect to see in tumour cell

Virtually all epithelial derived tumours become resistant to growth inhibition by TGFb

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TbRII gene is downregulated in what two cancer types

Breast and Lung

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TbRII is mutated in what two cancer types?

Colon and pancreas

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TbRII protein is not expressed in what cancer type?

Prostate

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V-src has several mutations including the deletion of Tyr527, what is the relevance of this?

Phosphorylation of Tyr527 inhibits kinase Src activity, no terminal = no inhibition

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Src family of tyrosine kinases share how many homologous domains and what does each domain recognise

3. 2 = phosphotyrosine binding site, 3 - prolin-rich motif binding site

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SH2 has two recognition domains to allow for specificity. What are these?

1 - phosphotyrosine binding domain, 2 - binding site for amino acid side chain

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Fos, jun and myc are ‘immediate early’ genes as they are switched on by GF even in the presence of what?

Protein synthesis inhibitor cycloheximide

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SH2 domain binds to

Phosphorylated tyrosines

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SH3 domains bind to

PXXP - proline rich motifs

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What are the three major MAPK in animals

Erk, Jnk, p38

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What do MAPK regulate (4)

Proliferation, apoptosis, differentiation, migration

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To become activate MAPK need phosphorylation of what residues?

Serine and threonine and therefore need a dual specificity kinase

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Describe the steps from EGF binding to R to SOS recruitment?

EGF binds to EGFR - dimerisation, activation, phosphorylation of tyrosine. Tyrosine-P recruits SH2 domain protein Shc. Shc is phosphorylated and recruits Grb2 SH3 domain. Grb2 binds PPP domain on SOS. SOS is a GEF for Ras

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What is a GEF

Guanine exchange factor. Catalyses conversion of GDP -> GTP

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What is Ras, what is it’s active form

An oncogenic protein that is bound to the plasma membrane with intrinsic GTPase activity. Active form is when GTP bound

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What % of pancreatic tumours have a Ras mutation

90%

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To switch of Ras what enzyme is required

GAPs (GTPase activating proteins) Ras will then hydrolyse GTP back to GDP

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What is the key effector protein of Ras

Raf

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What is Raf

A kinase

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What are the 3 closely related proto-oncogenes of Ras

H-ras, K-ras (4a, 4b) N-ras

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What are the two domains that make up Raf

Ras binding domain and a serine/threonine kinase domain

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How many isoforms are there of Raf

3

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What is the structure of inactive Raf

Raf folded in the cytoplasm with 14-3-3 protein bound to phosphorylated shrines in both the kinase and Ras domain

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How is Raf activated?

GTP-Ras recruits Raf to plasma membrane, Ras binds to ras binding domain, De-phosphorylation of Ras binding domain. Raf then becomes phosphorylated at additional residues (by PAL, SRC of Raf), leading to activation of kinase domain.

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What protein is Rafs target and what is its function

MEK, which is a dual specificity kinase

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What is MEKs target

Erk

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What happens after the phosphorylation of Erk

Erk translocates the nucleus where it acts as a s/t kinase for >50 substrates including cyclin d1

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As well as growth factor signalling, what other mechanism leads to Erk activation

Downstream of integrin engagement. Following a focal adhesion, proteins are recruited inside the cell into an adhesion plaque, some proteins in this plaque are kinases which can phosphorylate Raf...

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Without either GF and integrin mediated adhesion, what occurs in a normal cell

The signalling of Erk is not strong or sustained enough that the cell does not progress into G1

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Some non-receptor tyrosine kinases can be bound to what kinase

JAK

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Describe the steps of JAK-STAT signalling

Cytokine binding to receptor triggers activation of JAKs through conformational changes, JAKS then trans/auto phosphorylate and then phos the R at a tyrosine residue. Phos-(y) recruits sH2 domain protein STAT. STAT then phosphorylted by JAK

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What is the unique response element for STAT1 homdimer

GAS

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What is the URE for STAT3 homodimer

SIE/GAS E

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What is the URE for Stat1:2 heterodimer

ISR E

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How do SOCs suppress cytokines signalling

They are unregulated in response to STAT:DNA binding, they bind to activated JAKs and inhibit catalytic effect via -ve feedback

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P38 and Jnk are activated following what kind of signal

Stress (Rac (also a GTPase) -> PAK -> MEKK1...)

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How many genes and isoforms are there of Jnk

3 genes, 10 isoforms

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What does Jnk phosphorylate

AP-1 complex (made up of c-jun and c-fos, jun is what’s phosphorylated)

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What does p38 phosphorylate

MEF2 and dimer of ATF-2 and c-jun

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Strong ATF-2 expression is seen in what tumour typw

Melanoma

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What is R-SMAD

Regulatory SMAD, sits at the TGFbR either SMAD2 or SMAD3

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What is co-SMAD

SMAD4

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Describe TGFb signalling

Once the R has been activated. R-Smad and co-sad form an oligomer which migrates to the nucleus and recruits other gene proteins to activate transcription

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What are some examples of DNA binding factors for r-smad:co-sad complexes

FAST and OAZ which can target TF such as AP-1

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What are target genes for the TGFbeta signalling

Fibronectin, collagen type 1, MMP-2

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Ras mutations are present in what % of human cancers

30%

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Raf mutations are present in what % of melanomas

60%

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What codons are most Ras mutants recognised

12, 13 and 61 (GTP binding sites of ras) - these tend to prevent Ras from hydrolysing GTP and therefore is constantly on

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B-raf is frequently mutated in 60% melanomas, what is one hotspot

V600E (majority of mutations are in the kinase domain rather than regulatory). Allows RAF to be always active even as a monomer

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Why are tumour cells with RAF or Ras mutations Anchorage independent

Normal cells require both GF and integrin signalling. However with sustained GF signalling, this produces enough Erk that Anchorage isn’t required. WHy transformed cells can grow on soft agar and normal cannot

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What is meant by oncogene addiction

The observation that a tumour cell, despite multiple genetic alterations will exhibit dependence on a single oncogenic pathway

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What are the three popular methods for inhibiting kinases (in order of most popular to least)

ATP competitive inhibition, substrate competitive inhibition, non-competitive inhibition

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What is the target for Imatinib/Gleevec

BCR-Abl, substrate binding

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What are 4 mechanisms in which cancers cells develop resistance to biologics

Over express PDGFRB, promote WT raf diners, n-ras mutates, cancer cells reactivated by stromal HGF

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What is the target for PLX4032 / vemurafenib

RAF - interaction inhibitor

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Resistance emerges to which EGFR inhibitor due to a mutation at T790M

Erlotinib

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What is the name of a promising drug in mutated Ras targeting

Cpd12

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Outcome of shh signalling is dependent on what?

Concentration and duration of signal

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Describe signalling pathway of Shh in presence of Shh

Shh binds to PtcR (co-receptors Cdo/boo), Shh inhibits Ptc binding to Smo, Smo is liberated, proteins of intracellular complex are no longer proteolytically cleaved and GliA are able to translocations the nucleus

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What are Shh target genes e.g.

Bcl-2, cyclind1, Snail, Gli1

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Activation of Shh occurs in the

Primary cilia

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How many Gli transcription factors are there and which are activating which are repressive

3, Gli1 = A, Gli2 and 3 = R

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Mutations in Ptc are seen in what diseases?

BCC, Gorlin-gotz syndrome (nevoid BCC), medulloblastoma, rhabdomyosarcomas

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What is SUFU

Protein involved in the cleavage of Glis

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What % of BCC are due to LOF ptch 1 and GOF smo

90% - Ptch1, 10% - smo

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What are the effects of Shh signalling within the developing cancer cell

Proliferation (cyclinD1,D2, Myc, wnt, hes 1), survival (Bol-2), EMT

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What are the effects of hh signalling in tumour environment?

Angiogenesis (vegf), immune escape, stromatolites myofibroblasts (cancer promoting fibroblasts that encourage metastasis)

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What is juxtacrine signalling?

Both ligand and receptor are membrane bound, seen in notch signalling

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How many notch receptors are there in mammals

4

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What are the intracellular domains of Notch

PEST sequence (ICD stability), TAD (transcriptional active domain), NLS (nuclear localisation signals)

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What are the mammalian ligands in notch

DLL 1,4,3 and JAG 1 and 2

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Steps of notch signalling to formation of NICD

DLL ligand binds to receptor, cleavage by TACE and gamma secretase, liberate NICD, NICD enters nucleus and modulates transcription

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How does NICD modulate transcription

No NICD, proteins part of this signalling CSL complex on dNA bind bound to co-repress or. NICD displaces co-depressor and binds to cSL recruit CoAa and mastermind like 1. This activates target genes including Hes repression genes

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What are the three modes of notch signalling

Lateral inhibition, lineage decision, inductive signalling

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Lateral inhibition occurs in the ear, do the hair cells produce the ligand or receptor

Ligand, support cells have > receptor

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In lineage / binary signalling - what does a daughter cell inherit to inhibit notch signalling

Numb

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Notch signalling is a tumour promoter in what cancers and what is the effect

T-all and melanoma (proliferation and survival), breast and colon (snail and slug)

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Notch signalling is a tumour suppressor for what type of cancer

Skin

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What % of T-all patients have notch mutations

50%

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T-all is the result of what translocation

T(7;9) truncated notch from 9 moves to C7 TCRb promoter -> increased NICD

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What mutations are seen in notch signalling (4)

Heterodimerisation domain (increased NICD activity), PEST inactivity (no degradation of NICD), Ikaros (lymphoid normally inhibits notch R), FBW7 (normally removes nicd from nucleus)

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Notch signalling increases levels of what TF that drive EMT

Twist, snail, slug, ZEB1/2

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Mutations in wnt is the leading cause of what type of cancer

Colon (mutations in tumour suppressor APC)

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How many Wnts and how many FzR are there

19wnts, 10 frizzled receptor

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What are the three key pathways in Wnt signalling

1. Canonical (wnt-beta catenin), JNK (planar polarity), wnt/ca2+

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Describe the steps of the Wnt-b catenin pathway

Wnt bind to Fz (with co-factors LRP5/6, lgr5 and rspondin), binding modulates Dsh. Dsh inhibits destruction complex. Destruction complex normally target b-catenin for ubiquitination meaning inhibition of this complex allows beta-c to enter the nucleu

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What does the canonical pathway regulate

Cell proliferation, survival and fate

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JNK pathway regulates what?

Cell shape, orientation and migration

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Is wnt signalling outcome more dependent on the Wnt or the fzR

Receptor

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Biology of Cancer

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