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
Wnt functions during embryogenesis
Determination of body axis (which way is up?)
- Injecting Wnts into frog embryos causes axis duplication
Cell fate decisions
- Stem cell renewal and differentiation
Cell proliferation
- Wnt signaling activates proteins that promote cell-cycle
Cell migration
- Promotes epithelial mesenchymal transition (EMT)
What are mutations in the Wnt pathway called?
Dickkopf mutations (have a look at Hikasa et al 2013)
Explain the evolutionary conservation of signalling
Pathways are conserved across animal evolution
Model systems can be used to study cancer signaling pathways
Many cancer signaling pathways are important regulators during embryogenesis and development
Wnt, Notch, Hedgehog, TGF-Beta
Cell signaling evolved 100s of millions of yrs ago as a need for cells in multicellular organisms to be able to communicate in orde to build tissues, organs and bodies
Cells are constantly talking to one another,;
Production of tissues requires careful regulation of how much and how rapidly cells are proliferating and differentiating; this must be carefully coordinated as a whole
Explain Aberrant Wnt signalling and disease
Wnt signaling activity kept within homeostatic range by regulatory processes
Over (or under) -activity leads to disease
Most cancers have elevated levels of Wnt signalling activity
Some neurodegenerative diseases have decreased activity
Diseases affected by changes in Wnt signalling
Cancer(s)
-Wnt regulates stem cell homeostasis in the gut; inappropriate activation leads to colorectal cancer
Neural tube defects
-Neural tube does not close (e.g. spina bifida)
Bone density defects
-Mutations that promote Wnt signaling lead to high bone density and vice-versa
Tooth agenesis
-Dental abnormalities in which teeth fail to develop
Diabetes
-Mutations in Wnt transcription factor link to elevated risk for type II diabetes
Wnts involved in many different diseases - why?
Perhaps because regulate such fundamental biological processes as cell differentiation, proliferation?
Which are intrinsic to all biological processes..
Explain the different types of Wnt signalling pathways
“Canonical”
β-catenin dependent Wnt signaling
Embryogenesis; differentiation and proliferation
Important role in many cancers (e.g. colorectal)
“Non-canonical”
Planar Cell Polarity
β-catenin independent Wnt signaling
Tissue patterning, cell polarization and migration
Neural tube defects (e.g. spina bifida)
Metastasis? - its thought that non-canonical pathways may be important in this
Canonical is most well studied
Talk about B-catenin dependent and planar cell polarity as these have documented roles in cancer cells.
How distinct are these pathways, since components are shared and there is cross-regulation between canonical and non-canonical pathways.
Explain the pinciple of Wnt signalling
Activation of signalling protects a transcription factor from degradation
i.e. signalling turns off a degradation process
Potentially an expensive mechanism, since cell must continually produce the transcription factor
May allow for rapid response since production of new protein not required
B-catenin is the TF, Wnt ligand turns off the degradation process, this allows for rapid response as new proteins don’t need to be made
Explain canonical Wnt signalling
Highlight important components and mechanisms in this pathway
Key factor that it is really the reconfiguration of destrution complex that regulates the levels of B-catenin and signaling in the cell
Continual turnover of B-catenin in the cell.
Nuclear accumulation of B-catenin
Features of Wnt ligands
Small secreted glycoproteins with specific lipid modifications to target to membrane
May function as short or longer range morphogens through paracrine and/or autocrine mechanisms
19 Wnt genes in human genome encoding Wnt proteins (conserved gene family)
Which canonical, which are non-canonical?
Explan the different Wnt receptors
‘Frizzled’ family receptors, 7 transmembrane domain proteins - type of G-protein-coupled-receptor (GPCR)
Wnt ligands form trimeric complex with Frizzled receptor and co-receptor LRP5/6
Wnts may also bind to certain RTK type receptors, to activate other pathways
Receptor type and context play an important role in outcome (rather than the ligands themselves)
From these and other data, a picture emerges of various Wnts binding to different classes of receptors. The outcome of signaling is then regulated by receptor context and is not intrinsic to a particular Wnt.
Explain negative fedback in the Wnt pathway
Dickkopf (Dkk) proteins are Wnt antagonists (through binding LRP receptor)
Dkk are transcriptional targets (turned on) by canonical Wnt signaling
Dkks provide negative feedback to regulate Wnt activity
Mechanism is conserved over 500m yrs (Dkks found in cnidarians- sea urchins?)
Dkk expression turned off in colorectal cancer (turned off) i.e. tumour suppressor
(Niers 2006)
Negative feedback is important for many signalling pathways, this is one way Wnt signalling is controlled
Explain inhibiting inhibators in terms of Wnt signalling (drug stratagy)
drug strategy for bone disorders?
Wnt signalling promotes bone formation
Dkk1 and sclerostin bind Wnt co-receptors and inhibit signalling, could be drug targets (Ke et al, 2012)
Recessive mutations of sclerostin associated with increased bone mass
Romosozumab is an anti-sclerostin antibody that promotes bone formation used as a treatment for osteoporosis (prescribed since 2019)
EXplain the therapeutic stratagy for maintaining b-catenin destruction
Axin and the destruction complex is normally inactivated / or degraded by activation of Wnt signalling
tankyrase: an enzyme that works to degrade Axin through attachment parsylation (addition of ADP-ribose units)
Tankyrase can be inhibited - this preservs the destruction complex (stabalising Axin) and preventing β-catenin accumulation (peterson 2009)
Most Wnt pathway components are not very druggable
Thus some excitement at the discovery of a molecule that could prevent the inactivation of the destruction complex following Wnt activation
Explain how b-catenin is a multi-functional protein
Signalling function:
Central effector of Wnt signalling pathway
Structural function:
Component of cell-cell junctions (adherens junctions)
There are different pools of B-catenin in the cell that have different functions
EXplain b-catenins function in the nucleus
β-catenin imported to nucleus
Binds to TCF transcription factors and releases inhibitory factors
TCFs are a family of DNA-binding proteins that provide specificity of DNA-binding
A classical means of measuring Wnt signaling is using a transcriptional reporter consisting of TCF binding regions linked to luciferase (this is known as top flash)
Transcriptional targets of Wnt signalling
Wnt pathway activation induces expression of genes that lead to proliferation
Many transcriptional targets of β-catenin/TCF activated
Cyclin D1 (CCND1):
Drives G1 to S phase transitions in cell cycle
Myc:
Proto-oncogene encoding multifunctional transcription factor involved in cell cycle, apoptosis
Explain Wnt and colon cancer progression
Wnt signaling plays a key role in the development and homeostasis of the villus epithelium
Activation of Wnt signaling (through APC mutations (APC is involved in the destruction complex )) is an early ‘gatekeeper’ event in development of colon cancer
APC loss may occur with initial germline mutation then additional somatic mutation ‘two-hit’ hypothesis
Diagram (Davies et al 2005)
Explain B-catenin and colon crypt biology
Normal circumstances:
Bottom of crypt; Stem cells present, b-catenin: Tcf/Lef target genes induced by Wnt, cells are proliferatinh, undifferentiated progenators as you move up
as move up towards the intestinal lumen, b-catenin: Tcf/Lef turns off and this reasults in cell cycle arrest of differentiated cells
In APC or b-catenin mutations, where b-catenin: Tcf/lef is tuned off normaly, it stays on, creating a progenator like phoenotype and accumulation at site of future polyp formation, this results in the failure for the mutant cell to continue outward migration towards the intestinal lumen
NB: Wnt activity decreases naturally towards the epithelial layer, this ISNT shut off when theres APC or b-catenin mutations
Examples of oncogenic b-catenin mutations
Stabilizing mutations of β-catenin delete codon encoding phosphorylation site
Allow β-catenin escape destruction via proteasome
Occurrence is usually mutually exclusive of APC mutations
Examples of Adenomatous Polyposis Coli (APC) (TSG) in cancer
Destruction complex component
300kDa protein; multi-domain protein
Frequent truncation mutations colorectal cancer (~60%)
“gatekeeper” tumor suppressor (two hit…)
Loss of APC tumor suppressor leads to carcinoma through β-catenin/ Wnt signaling
Germline/(somatic mutations v.freq. colorectal cancer)
Truncations remove important binding domains allowing β-catenin accumulation
APC mutations aren’t complete knockouts - when the mutation cluster region is mutated you get truncations in many CRCs
Explain an example of a comprehensive analysis of colorectal cancers
Genome (exome) sequencing and gene-expression analysis of 276 colorectal tumours (the cancer genome atlas, Nature 2012)
showed that out of the tumours studied, Wnt signalling was affected in 92% of cases (the highest of the signalling pathways tested)
Check ref/image
Different experimental systems in cancer signalling
Clinical samples
- Human tumour sample (e.g. biopsy)
- Human biofluid sample (e.g. blood plasma, urine), may pick up a signal from the tumour in the plasma
Model organism
-Genetic model of human cancer or xenograft
Cultured human cells
- Immortalised cells from tumours (harbour defined mutations and capable of indefinite proliferation)
- Primary cells from tumours (derived from individual tumours)
- Cultured cells come in different forms: Use as a 2d culture, grow as tumour spheres (cells ageegate to form a clump of cells that mimics a tumour), organoids (miniature versions of organs)
Questions/problems with the different experimental systems in cancer signalling
Disease relevance? - is a mouse model the same?
Ease of experimental manipulation?- human cultured cells make this easier
Sample complexity and heterogeneity? - individual tumours are very heterogenous, meaning between patients the tumour profile could be very different
Explain experimental pertubation of signalling
Addition of ligand (e.g. that activates pathway)
Genetic deletion (e.g. knock-out) to remove key proteins e.g. a Tumour suppresser protein
Pharmacological (e.g. small molecule inhibitor)
Mutant kinase + ATP analog, Mutant kinase is created with mutation in ATP binding pocket of selected kinase, so that normal function of the kinase is not disrupted, but which allows binding of an ATP analog that can be given to the cell. i.e. kinase can be very precisely and selectively switched off
Need perturbation before can study signalling
Explain experimental activation/inhibition of Wnt signalling
Wnt ligand
Wnt conditioned media (from cells expressing Wnt ligand)
Recombinant, purified Wnt protein
Pharmacological inhibition
Inhibition of GSK3B (kinase that phosphorylates β-catenin) using lithium
Genetic
Knock-out cells
e.g. β-catenin knock-out / mutant and wild-type alleles
RNAi
Not all the same, since affect proximal or distal components of the pathway
Specificity (e.g. lithium – other cellular effects?). Also GSK3B involved in other processes
How can Wnt signalling output be measured
Topflash system links TCF binding domains to reporter luciferase
Introduce into cells of interest as plasmid
Measure Topflash/Fopflash ratio
Links TCF (T cell factor) a TF to a reporter gene e.g. leuciferase (Luc)
e.g. Zhang 2008
looked at protein Osx
Authors believed Osx was an inhibitor of Wnt signalling
They used different cells with different combinations of Wnt3A (ligand), Osx and Dkk1 (also a negative regulator for Wnt signalling, this addition of Dkk1 showed further inhibition when Osx was added, further confirming/showing Osx is an inhibator
Explain genome wide RNAi screens
Use short RNA molecules (siRNA) to inhibit expression of specific genes
Libraries of siRNA molecules have been created to target all genes in genome
RNAi screens proceed by introducing all of these siRNA molecules into cells, and observing the effect on a phenotype of interest
Applied in many different organisms to study many different processes, including signalling
Explain identifying signaling pathway components using RNAi screens
Detect activating and inhibitory regulators
Detect regulators acting that different levels and through different mechanisms
Does not provide mechanistic information
Pros/cons:
False positives, false negatives
Identify known components is good
Example of an RNAi screen for Wnt pathway regulators
DasGupta 2005
Screened 22k RNAs in Drosophia cells for alterations to Topflash activity - Identified 238 regulators
Limitations: provides genetic, does not clarify mechanisms
Also, many may be drosophila-specific proteins (95% drosophila genome)
Features of protein-protein interactions
Protein-protein interactions critical component of pathways
Differing properties
- Permanent/transient
- Obligate/non-obligate
Influenced by
- Post-translational modifications
- Sub-cellular location
- Rate of synthesis and/or degradation
Explain the use of Yeast wo-hybrid for detecting interacting proteins
An assay performed in yeast cells to test whether two proteins interact
Can be applied to proteins from any organism
Can be scaled up to test 1000s of proteins
Has been used to map 1000s of human protein-protein interactions
Explain how yeast two-hybrid works
- Fuse test proteins X and Y to activation and binding domains of transcription factor to make two hybrid - proteins
- Reporter gene
–Gene that allows yeast to grow when turned on
–Only active when Activation and Binding domains interact
- If test proteins X and Y interact
–Transcription factor is reconstructed
–reporter gene is on
–yeast cells grow
Explain large scale yeast two-hybrid
Test many protein combinations?
Make libraries of all proteins fused to activation or binding domains
Screen combinations by mating yeast strains
Crossed-strains that grow indicate protein-protein interaction
Can be broadly applied (e.g. test interactions between human proteins)
Applying Yeast two-hybrid to the MAPK signalling pathway:
Yeast two-hybrid used to identify >600 interactions pertaining to the MAPK signalling pathway (from 86 ‘baits’)
Identifies existing an novel protein-protein interactions
RNAi knock-down analysis can then be used to identify which proteins regulate MAPK signalling
explain Integrated multi-omics of oncogenic β-catenin signaling
Derivative cell-lines expressing either mutant or wild-type Beta-catenin
Wild-type
E-cadherin associated /membrane localized
Mutant
Nuclear localization, enhanced Wnt activation
Elevated EMT markers
Ewing 2018 CHECK OUT PAPER
Reference for how data mining can be used to identify new proteins that regulate b-catenin
Looked at EMT markers in mutant vs WT b-catenin cells
Ayati 2015
identrifieds antagonist for Wnt signalling called ELF3
Paper that showed ELF3 is an antagonist of Wnt/b-catanin signalling
Gingras 2016
looked at mRNA levels usinh western blots
showed it was a TSG and mutations associated with ampullary cancer
Paper showed ELF3 is associated with good prognosis in CRC
Liu 2019
Representative ELF3 staining pattern (High or Low ELF3) in 86 human CRC tissue microarray cores
IHC scores of ELF3 staining in 86 paired human CRC and adjacent normal tissues
Kaplan–Meier survival curve shows significant association between low levels of ELF3 and poor survival in CRC patients
