Cancer cell signalling Flashcards
How do cancer cells make different decisions to non-cancer cells
Cells must continually sense and respond to environmental state and adapt according to them e.g Survive, grown + divide, differentiate, die
In cancer, how these cells respond are very different (constantly proliferate)
Why study cancer cells?
Cell signaling is altered in most cancers
- Signaling tuned on and unresponsive to inhibition
- Volume of signaling increased
- Signaling at wrong time or place
Alterations of signaling enable the cell to evade normal regulatory controls
The most fundamental trait of cancer cells involved their ability to maintain chromic proliferation (hallmarks)
Whats the difference between cell signalling and signal transduction?
Cell signaling: The mechanisms that cells use to perceive and adapt to their state and surroundings
Signal transduction: the biochemical process that facilitates information processing by the cell
Signaling pathways have both plasma membrane/cytosolic events and nuclear events and gene-expression
These operate in different time scales…
cell signaling – fast ON/OFF (seconds to minutes), transient changes (minutes-hours)
Gene expression – Slow ON/OFF (minutes to hours), stable changes (hours – years), energetically costly
Three general mechanisms for signal transduction
Lipophilic ligands are able to traverse the cell membrane and typically bind to an intracellular receptor in the cytoplasm or nucleus (e.g steroid hormones, or nitric oxide). These bind to receptors that are internal to the cell.
Gated channels allow the passage of specific ions as in the case for neurons conducting electrical signals
Hydrophilic ligands such as peptides or proteins are unable to cross the membrane and instead bind to extracellular receptor, which is then altered so that information can e passed over the membrane.
Properties of transmembrane receptors
Transduces information
Convert extracellular ligand concentration into intracellular signal
Discriminator domain binds specific ligand - ‘dicscrimintor’ on the extracellular face of the plasma membrane that is able to selectively bind ligands (targets for many different drugs in oncology and other medical fields)
Transmembrane domain anchors in membrane - typically composed of hydrophobic amino-acids that spans the membrane and joins the ligand binding domain to an intracellular effector
Effector domain directly or indirectly linked to intracellular enzymatic activities
Indirect: receptor associates with separate kinase protein
Direct: kinase domain in same protein structure as transmembrane receptor
Properties of RTKs
Receptors with intrinsic Tyr kinase activity
Recent evolutionary development
< 100 tyrosine kinases in mammals (~60 are receptor)
Important target for oncogenic mutations
Diverse ligands (growth factors, insulin)
Large family of proteins with sub-families, classified according to ligand binding
Ectodomains are highly variable according to the ligand, whereas the sequence and structure of the tyr kinase domains is relatively well conserved
These are releatively recent in evolutionary terms because they are generally not found in single cell eukaryotes, they have evolved in concert with the evolution of multicellularity
Give an example of an RTK and how its activated
e.g. EGFR
dimerization and activation
How does dimerization occur?
Different mechanisms for different receptors
Epidermal growth factor receptors best studied
Ligands bind to and stabilize transiently associated homodimers
Monomers of EGRF typically mobile within the plasma membrane.
However, a homodimer (two copies of the protein) is able to form when two molecules encounter one another.
These is achieved by ligands:
having two receptor binding sites,
by ligands existing as homodimers (as is the case with PDGF)
or in case of EGF by inducing a conformationl change that changes the affinity of receptor molecules for one another.
EGFR: This changes the conformation of the receptor molecules, and if present allows the binding of the EGF ligand
This cause stabilization, and idnduces the alloseric conformational changes that result in auto phosphorylation of the tyrosine kinase domains with downstream signalling
RTK mechanisms for intracellular activation
Conformational changes in cytoplasmic domains upon dimerization/activation lead to Two principal mechanisms of intracellular activation :
Auto-phosphorylation and recruitment of signalling proteins
Phosphorylation of scaffold proteins that organizes signalling complexes
How is signalling through receptor tyrosine kinases altered in cancer?
- Mutations may alter propensity for dimerization (red dots)
- Or alternatively complete loss of whole domains (for example ectodomains – which results in higher levels of basal phosphorylation of the intracellular domains). i.e. phosphorylation even in absence of ligand
- Alternatively the proteins themselves may not be altered by mutations, but their expression may be disregulated. This could result from mutations that are not in the coding sequence of the genes, but in promoter, that alter expression levels of the gene and associated proteins
- Alternatively by increasing the production of receptor tyrosine kinases proteins in the membrane, the chance that they will form dimers increases substantially, resulting in constitutive activation.
–Oncogenic versions may lack ectodomains
–Truncated receptors no longer responsive to ligands
–Constitutive activation of signalling(promoter always on)
–Important oncogenic mutations in many cancers e.g. breast
- EGFR: copy number alterations cause amplification of protein expression
–Increases protein expression and chance of receptor firing
–Alterations due to chromosomal or gene amplifications
Explain EGFR aplifications
Copy number amplifications (chromosomal 2-5 amplification) or gene amplifications 5-10 cause over-abundance of EGFR expression, increasing probability of dimerization and firing, even in presence of low levels of EGF ligands
Focal amplifications are common for EGFR but not so common for other growth factor receptors.
Targeted therapies for RTKs
Gefitinib specifically targets tyrosine kinase domains of EGFR (ATP binding site)
Prevents activation of signal transduction
For types of lung cancer with specific EGFR mutations
Although initially effective for patients with non-small-cell lung cancers with EGFR mutations, most relapse due to acquisition of resistance
Emergence of second point mutation in kinase domain is a major cause of resistance (this mutation is caused by selective pressure in tumours) – it is rarely found in patient tumours that have not been treated with gefitinib
Mutations that cause resistance may alter binding topology of ATP binding pockets
And may cause greater affinity for ATP or disrupt the interaction between gefitinib and kinase domain.
Possible solutions include targeting other pathways in addition to EGFR
But also better matching of patients with drug treatments – need to ascertain just what mutations are present in a given tumour prior to drug treatment (personalized medicine).
Cellular functions of cancer related kinases
phosphorylation integral to cell signalling and frequent targets of mutation
play multiple roles
Kinase motifs in receptors are only one cellular location in which kinases work
Mutations in kinases underlie different diseases e.g. cancer
Kinase domain often conserved, but other regulatory motifs in other parts of the protein may regulate the context – substrate specificity or binding to other signal transduction proteins
Explain phosphorylation
Addition (and removal) of phosphate groups – a common mechanism for regulating protein activity
Ser > Thr > Tyr commonly modified in eukaryotes
Kinase writers, Phosphatase erasers
30% human proteins may be phosphorylated
Reminder of importance of phosphorylation in protein regulation
Important in signalling as well as other related processes of proliferation , growth, cell cycle
Remember that phosphorylated or unphosphorylated form may correspond to the activated form.
What are the functional consequences of phosphorylation
Activation/inactivation
Form/hinder protein interactions
Sub-cellular relocalization
Degradation via proteasome
e.g. phosphorylation of serine adds negative charge and changes size of side chain.
Explain the different types of PTMs in signalling
Phosphorylation
Acetylation
Methylation
Ubiquitylation
Apart from the fact PTMs of signalling molecules (e.g. p53) intails combinational complexity, what else makes it very difficult to know how many different post-translationally modified versions exist?
because most techniques such as phosphoproteomics can identify phosphor sites from only a population of molecules – so cannot know what is happening on any one protein
is p53 extensivly modified post-translationally?
Yes
Explain interplay between PTMs and an example
PTMs may promote or antagonize another
Integrate and process multiple upstream signal
Multiple PTMs may target same amino-acid (e.g acetylation or methylation on lysine)
PTM may be required for subsequent modification by another PTM
e.g. CDKs regulate cell cycle transitions;
-phosphorylate Ser/Thr residues on targets -this then recognized by ubiquitin ligase complex, which polyubiquitlates the phosphoprotein
- targeteing it for destruction via proteasome
Features of signalling pathway outputs
Transcriptional regulation key output of signaling pathways
Mediated via modification of transcriptional regulator proteins
May/may not bind directly to DNA
Many oncogenes or tumor suppressors encode transcription factors
What are the transcriptional targets of the Wnt signalling pathway
Wnt pathway activation induces expression of genes that lead to proliferation
Many transcriptional targets of β-catenin/TCF activated
Cyclin D1
- Drives G1 to S phase transitions in cell cycle
Myc
- Proto-oncogene encoding multifunctional transcription factor involved in cell cycle, apoptosis
Explain the dynamics of the transcriptional response
If stimulate cells with growth factors can identify two waves of transcriptional response
Immediate early genes – direct targets of transcription factors from signaling pathways
Delayed early genes – those that require activation via transcription factors that must be synthesized first
And so response from minutes to hours.
Also some genes may come on transiently.
Different profiles of gene expression, different dynamics associated with functions.
Explain how signalling pathways are inter-linked in different ways
Convergence (multiple signals activate Ras)
Divergence (one signal elicits multiple outcomes)
Cross-talk (interaction of signalling pathways)
How complex is the molecular signalling network
In human genome
~1500 signal receptors
~500 kinases
~1500 transcription factors
How many signaling protein molecules per cell?
20,000 protein molecules per cell
EGFRs normal 104, tumor 106
Variation according to pathway, context, cell type
e.g. Ras 104-107 per cell
Axin v.low conc. regulator of Wnt pathway
Local concentrations of signaling molecules may be increased in sub-cellular compartments
And in cancer may be greatly increased number of molecules due to mutations e.g. egfr
What is Wnt?
Wingless (Wg) gene -identified as important developmental gene during Drosophila embryogenesis
Called Int1 when identified in mice (as gene promoting breast cancer)
realised these are homologs - renamed ‘Wnt’
refers to both the pathway and the genes encoding the pathway ligand
Wnt pathways subsequently shown to be universally present across metazoans (animals)
Aberrant Wnt signaling implicated in many different developmental disorders and cancers
Explain Wnt Gene nomenclature
Gene and protein names often derived from phenotype of mutants in model organisms
- Wingless (Wnt in human)
- Armadillo (β-catenin in human)
- Frizzled (Frizzled in human)
- Naked
- Legless
- Pygopus
