Lecture 7 - Angiogenesis & HIF Flashcards
What is angiogenesis?
development of blood vessels (angiogenesis) is required to deliver oxygen & nutrients to solid tumours
tumour cells distant from blood vessels are liable to die from hypoxia
Hallmark of cancer
Development of blood vessels that tumours need to deliver oxygen and nutrients as well as remove waste products such as lactic acid
Common to find areas of dead cells in centre of tumour – undergone necrosis
Immunohistochemistry – stain tissue section with antibody, look for expression levels of particular protein, in this case HIF (hypoxia inducible factor) – expressed in cells far from blood vessels
Describe how Hypoxia-Inducible Factor (HIF) is Degraded Under Normoxic Conditions
Cells starved of oxygen use HIF to induce genes that promote angiogenesis & haematopoiesis
HIF is synthesized constitutively, but only stable under hypoxic conditions - at normal O2 levels it is hydroxylated on two proline residues & then ubiquitinated by VHL & degraded by proteasomes
Prolyl hydroxylases require O2 as co-substrate – inactive when O2 low
Induction of HIF allow tumours to increase in size that would otherwise be constrained by lack of oxygen at centre of tumour
HIF constitutively synthesised- continuously degraded in presence of oxygen
Synthesised everywhere – only accumulates in hypoxic regions
In presence of oxygen it is in hydroxylated on proline residues by prolyl hydroxylases
Hydroxyprolines recognised by ubiquitin ligase VHL which binds to hydroxylated HIF, polyubiquitinates it and thereby targets it for degradation in proteasome
In low oxygen conditions the prolyl hydroxylases can no longer function because they require oxygen for reaction that they catalyse, so HIF doesn’t get hydroxylated, doesn’t bind VHL and doesn’t get degraded – therefore accumulates
What is VHL Tumour Suppressor?
VHL is part of an E3 ubiquitin ligase that binds hydroxylated proline residues on HIF – it polyubiquitinates HIF, which targets it for degradation
Mutations in VHL gene cause von Hippel-Landau disease, a rare inherited cancer syndrome involving vascular tumours
Kidney cancers are generally highly vascular & usually have somatic VHL mutation or silencing by DNA methylation
Cells with VHL mutation or silencing have active HIF under normoxic conditions, causing inappropriate angiogenesis
Somatic mutation of VHL gene commonly encountered in kidney cancers
Less commonly epigenetic silencing through DNA methylation
When VHL function lost HIF can be expressed and accumulated even in high oxygen conditions
Drives angiogenesis and tumour development
What is PROTAC?
Proteolysis-targeting chimera (PROTAC) technology uses a ubiquitin ligase such as VHL to destroy a chosen target e.g. BRD4
E.g. ARV-771 has a VHL-binding moiety linked to a moiety that binds BET domains
VHL is recruited by a derivative of the hydroxy-proline that it binds in HIF
Ability to act catalytically means that lower doses of PROTAC may be rqrd than inhibitors that function with 1:1 stoichiometry – may reduce off-target binding
Small molecule drugs
Target with ubiquitin ligase to particular protein that needs to be disposed of to combat oncogenic effect
ARV-771 is example of this. Ubiquitin ligase is recruited by a ligand that is a derivative of hydroxyproline that it would bind in HIF – ligand joined by a linker to one of the molecules that was developed as an inhibitor of BRD4 and other BET domain proteins
BRD4 induces MYC expression
BRD4 and other BET proteins bind to acetylated lysines with promoter regions and then recruit pTEFb which phosphorylates pol II and allows escape from promoter pausing, which restricts MYC expression – so BRD4 induces MYC expression
Can see in Western blots that as drug is titrated into cells expression of BRD4 and other BET proteins is suppressed as is MYC
Drugs do not target MYC, but by suppressing BRD4 they inhibit MYC expression
Advantage of this technology means you do not need 1:1 stoichiometry
Explain the potential of PROTAC
Many companies are developing PROTAC drugs against a variety of target proteins
e.g. PROTAC targeting androgen receptor is about to start clinical trials for prostate cancer
PROTAC targeting oestrogen receptor is close to trials for breast cancer
VHL is one of several ubiquitin ligases being exploited
Current small molecule drugs are restricted by necessity to target catalytic sites or sites involved in important interactions
PROTAC technology should be effective wherever the drug binds target protein, as it causes degradation
will allow far more options for binding
Ability of each PROTAC molecule to destroy multiple target molecules should also boost efficacy
PROTAC can bind anywhere on target protein – degrades it
What is HIF?
HIF is composed of HIF-1a or HIF-2a heterodimerized with ARNT (a.k.a. HIF-1b) to form a transcription factor with a bHLH DNA-binding domain
When not hydroxylated, HIF recruits HAT p300 to stimulate transcription
HIF target genes include:-
1. Vascular endothelial growth factor (VEGF) – promotes angiogenesis
2. Erythropoietin (EPO) - increases production of red blood cells
3. Transforming growth factor-a (TGF-a) – mitogen that activates EGF receptor
4. Cyclin D1 gene – stimulates cell cycle & proliferation
5. GLUT1 – imports glucose for fuel
6. Hexokinase 2 (HK2) – promotes glycolysis
7. Pyruvate dehydrogenase kinase (PDK1) – suppresses oxidative phosphorylation
These target genes are also bound & induced by MYC
HIF & MYC induce many of their targets cooperatively
HIF is one of physiological targets for VHL
Transcription factor composed of one molecule of either HIF1a or HIF2a which must dimerise with HIF1b
Basic helix loop helix DNA binding domain which binds and activates a series of important target genes, largely by recruiting HATs eg p300
Through this range of target genes HIF is able to promote at least 3 hallmark features of cancer – angiogenesis, proliferation and deregulation of cellular energetics
These target genes are also direct targets for MYC
Cooperative effects
Describe how HIF & MYC Stimulate Expression of VEGF
VEGF promotes proliferation & migration of endothelial cells that give rise to blood vessels
Acts through cell surface receptors with cytoplasmic tyrosine kinase domains
Promoter of VEGF gene is bound & induced by both HIF & MYC – together they cooperate to stimulate angiogenesis
Shown using cells where a MYC gene is induced by withdrawal of tetracycline, cultured under normoxic (20% O2, no HIF ) or hypoxic (0.1% O2; high HIF) conditions
Model system with MYC trans gene – can be controlled by tetracyclin
When tetracyclin withdrawn, MYC levels go up
Under normoxic conditions levels of VEGF increases in response to induction of MYC
Can also be induced by oxygen withdrawal and the combination of hypoxia and MYC induction leads to further increase in VEGF expression
Describe how it is experimentally shown that Cancers Have Enhanced Glucose Uptake
MYC & HIF both induce expression of GLUT1, a transporter responsible for importing glucose into cells
Glucose uptake is highly elevated in most cancers
Positron Emission Tomography (PET) scans with 18F-fluorodeoxyglucose (FDG) are a sensitive way to detect tumours
Radioactive fluorodeoxyglucose tracer fed to patient
Tumours have unusually high levels of glucose uptake so the tracer accumulates in tumour region and is readily detected.
Sunitinib – inhibitor of VEGF receptor
Tumours no longer visible post therapy
Accumulation is kidneys and bladder – FDG secreted in urine
Describe how HIF & MYC Stimulate Glycolysis
HIF & MYC bind & induce gene encoding hexokinase 2 (HK2), which catalyses the first and often rate-limiting step in glycolysis
HK2 induction stimulates glycolytic metabolism of glucose to generate energy & raw materials for biosynthesis
HK2 catalyses phosphorylation of glucose which is required for it to enter glycolytic pathway
Can be induced by MYC and HIF-1a – combination of both leads to particularly robust induction
Describe how HIF Represses Oxidative Phosphorylation
Glycolysis metabolizes glucose to pyruvate
Pyruvate can be converted to acetyl-CoA by pyruvate dehydrogenase
Oxidative phosphorylation of acetyl-CoA generates high ATP yields via the tricarboxylic acid (TCA) cycle in mitochondria
However, HIF binds & induces PDK1 gene encoding pyruvate dehydrogenase kinase, which inhibits pyruvate dehydrogenase
With pyruvate dehydrogenase inhibited by PDK1, pyruvate is metabolized to lactate instead of entering the TCA cycle
Lactate is secreted
Pyruvate can enter mitochondria and be converted to acetyl-CoA
Secretion of large amounts of lactate is feature of cancer cells
What is the Warburg effect?
Normal non-proliferating cells use oxidative phosphorylation to generate ~36 ATP molecules from each molecule of glucose & only produce lactate if O2 is low
Normal proliferating cells & tumours produce far higher levels of lactate, even when O2 is available – referred to as aerobic glycolysis or the Warburg Effect
Lower ATP yield of aerobic glycolysis is compensated by elevated glucose import & higher glycolytic flux (due to induction of GLUT1 & HK2)
Functional significance of Warburg effect remains under discussion
High uptake of glucose by cancer cells, secretion of lactate, even under aerobic conditions in presence of oxygen
Associated with elevated proliferation
Describe kidney cancer treatment
VHL gene is usually mutated or silenced in kidney cancer
Early-stage (organ confined) kidney cancer is treated by surgery, which is often curative
Kidney cancers that are inoperable or recur after surgery are usually fatal
Kidney cancer does not respond to conventional chemotherapy or radiotherapy
Advanced kidney cancers are treated with one or more of:-
- rapalogs (e.g. everolimus) – esp effective against HIF-1a
- VEGF receptor inhibitors e.g. sunitinib
- Avastin (bevacizumab), a monoclonal antibody that binds VEGF to prevent it activating its receptor
But VEGF is not the only important HIF target & these therapies are rarely curative, although they often prolong survival
HIF-2a is more consistently elevated than HIF-1a in kidney cancer
Small molec drugs have been developed that target HIF-2a & have entered clinical trials
Describe the use of HIF-2a Inhibitors
Structural studies identified a pocket in HIF-2a
small molec PT2385 & derivatives were developed to fit in pocket & disrupt dimerization of HIF-2a with ARNT
Ongoing phase 1 clinical trials in kidney cancer patients show that PT2385 is well tolerated & shows promising effects in delaying tumour progression when combined with immunotherapy using antibody targeting PD-1
Pocket located close to interface with ARNT dimerization partner
Small molecules designed that fit into pocket and prevent dimerization
Co-immunoprecipitation experiment
Cells treated with either vehicle control – solvent they dissolved the drug in or increasing amount of PT2385
Then made lysates of the cells
Used the proteins in immunoprecipitation with an antibody against HIF-2a
Blotted for co-immunoprecipitation of dimerization partner of ARNT with HIF-2a
Amount of binding of HIF-2a to ARNT has been suppressed as concentration of drug increases
