Week 3: Cancer Biology

What was Ras first identified in?

Two RNA tumour viruses isolated from a rat sarcoma: Harvey rat sarcoma virus (v-H-Ras) and Kirsten rat sarcoma virus (v-K-Ras).

What mutations are associated with Ras in tumor cells?

Mutations in codons 12 and 59.

Which Ras isoforms are present in humans?

HRAS, NRAS, and KRAS (with 2 isoforms: KRASA and KRASB).

80-90% similarity in primary sequence

How do we know that RAS is necessary and sufficient for DNA synthesis?

Buffer + Growth cells = cells entered S phase

Anti-RAS antibody + Growth factors = Cells did not enter S phase

Activated RAS protein + no Growth factors = Cells did enter the S phase

What type of molecule is Ras?

Ras is a small GTPase.

Act as molecular switches

How is Ras activated?

GDP → GTP

Ras is activated downstream of receptor tyrosine kinase (RTK) activation, involving the adaptor protein Grb2 and GEF SOS.

What is needed to activate RAS?

Upstream signals triggered by growth factors binding to receptors

What do upstream signals do?

They recruite and activate GEF

What is GEF?

Induce a confirmational change so GDP is removed and replaced by GTP in RAS

What role does GAP play in the Ras pathway?

GAP induces GTP hydrolysis, leading to Ras inactivation.

What is the importance of GTP in Ras signaling?

Ras must bind GTP to become active and signal downstream pathways.

GRB2

Adaptor protein

Recruits SOS protein

Recruited by tyrosine phosphorylation

No enzymatic activity

SH2 and SH3 domain

SH2 binds to TKP domains

SH3 binds to protein rich sequences

What are the key kinases involved in activating Raf?

Serine and tyrosine kinases are involved in the phosphorylation required for Raf activation.

Types of RAF

A- RAF

B- RAF

C- RAF

Sames structure

Horseshoe shape

Different phosphorylation sections

What happens when RAS becomes active?

Binds to RAF

RAF

MAP kinase kinase kinase - Phosphoprylates Mek

Mek

Map Kinase Kinase - Phosphorylates Erk

Erk

Gets translocated to the nucleus

Acts as a transcription factor to regulate gene expression

If it stays in the plasma membrane:

RAS and SOS → negative feedback

What factors influence the consequences of ERK activation in normal cells?

1) Cell type, 2) Ligand/RTK, 3) Transcription factors, 4) Duration of ERK activation, 5) Integration with other MAPKs.

What happens to Raf activity at some phosphorylation events?

Some serine phosphorylation events can suppress RAF activity.

Consequences of ERK activation

Dependent on:

Cell type

Ligand/ RTK

Transcription factors

Duration of ERK activation

Integration with other MAPKs

Oncogenic activation of RAS

Seen in 30% of human tumours

90% of pancreatic K-Ras

100% of PDAC feature K-RAS mutation

60% of Papillary Thyroid H.K, N-Ras

Mutational Hotspots for all 3 RAS

Glycine 12 is most common in KRAS

Glutamine 61 is most common in NRAS

G12, 13 and 161 is even in HRAS

What is the role of G12 in RAS

Renders GTPase domain of Ras insensitive to inactivation of GAP

What is the role of Q61 in RAS

Suppresses the rate of intrinsic RAS GTP hydrolysis and decreases GAP sensitivity

Why is there a difference between different mutations in tumours and hot spots

Different type of RAS might be expressed highly / low depending on the tissue

Different localisation

Difference in sensitivity to mutagenesis

Difference in post translation modification

EACH ISOFORM AND OCOGENIC MUTATION MUST BE CONSIDERED INDIVIDUALLY

Raf - Valine to glutamic acid mutation

30-60% in melanoma

30% in ovarian cancer

Gain of function mutation - permanently turns Raf on.

Why is B-RAF more frequently mutated than the others

B-RAF phosphorylates MEK more efficiently than A-RAF or C-RAF

B- RAF is the main activation of MEK and ERK

CAAX box

Cystine, aliphatic , aliphatic, serine or methionine.

Fundamental for targetting RAS to the membrane.

RAS undergoes Prenylation

Protein Prenylation of RAS

Transfer of farnesyl of geranyl-garenyl added to cysteine.

Done by: Farnyesyl transferase and geranyl-garenyl transferase

Properties and roles of Farnyesyl transferase and geranyl-garenyl transferase

Very hydrophobic

Tether RAS to the membrane and facilitate RAS membrane localisation.

BRAF- targeting uting PLX 4032

PLX4032. Doesnt affect normal cells

ADDITIONAL READING GO AND SEARCH HOW IT WORKS

Transcriptional response to growth factor signalling

Genes can be up or down regulated upon growth factor stimulation

Most are regulated by post phosphorylation events.

Experiment that shows induction and repression of genes is transient

DNA array experiement

3T3 fibroblasts are stimulates with serum. RNA is taken at different time points

Hybridised to array of cDNA

Results of genes switched between minutes - 5+ hours

Order of genes switched on in response to growth factor stimulation

Immediate early: within minutes

Delayed early: within hours

Late: around the onset of S phase

How do we know that early gene expression does not require protein synthesis

Cycloheximide (protein synthesis inhibitor) did not affect early gene expression

How do we know that delayed early gene and late gene expression requires protein synthesis

Cycloheximide inhibited gene expression when it was added

they depend on mRNA.

what is FOS

Immediate early growth factor inducible gene

activated by PMA

MAPK dependent gene expression

Regulation of fos gene expression

Has a DNA motif on fos Promoter known as serum response element

Transcriptional activation requires an addition agent (ternary complex factor) to induce gene expression

ELK-1

Phosphorylated by ERK1 which then stimulates fos gene expression

AP-1 complex

a heterodimer formed with c-FOS and c-JUN

formed by the leucine zipper motif

NF-kB transcription factor complex

can activate 500 genes

work as hetero and homo dimers