Lecture 15 - The angiogenic switch and altered energy production and intermediary metabolism Flashcards
Carcinomas: what are they?
Carcinomas are complex tissues comprising neoplastic epithelial cells and recruited stromal cells (connective tissue cells)
Stromal cells include endothelial cells, pericytes, fibroblasts and various bone-marrow-derived cells (BMDCs), including macrophages, neutrophils, mast cells, myeloid cell-derived suppressor cells (MDSCs) and mesenchymal stem cells
All these different cell types intercommunicate through secreting growth factors
Lack of oxygen (hypoxia) and metabolic waste products can limit cancer cell growth
Beyond a distance of ~100um, oxygen is limited and the environment of the cells becomes too acidic (largely due to lactic acid buildup) resulting in cell death by necrosis (granular appearing cells)
Transcription factor HIF-1α: why does hypoxia affect it, what does it target, what happens to it in normoxia and hypoxia
Hypoxia induces stabilisation of hypoxia-inducible transcription factor 1 - stabilisation of the alpha-subunit
- Target genes such as the vascular endothelial growth factor (VEGF) to induce new blood supply
- Other targets allow the cell to shift more effectively to anaerobic respiration
Normoxia - Proline hydroxylase + O2 hydroxylates HIF-1alpha. Then pVHL recognises and binds with two other proteins and poly-ubiquilates it, sending it to the proteasome for degradation
Hypoxia - stabilisation occurs, more TF HIF-1α present, dimerises (?) with HIF-1β and targets specific genes
Tumours vascular supply
Tumours require new vasculature to grow
Tumour vasculature
The vasculature of tumours is chaotically organized and blood vessels are leaky due to small holes (fenestrations in endothelial cells). This raises the hydrostatic pressure within the interstices of tumours which reduces the distribution of chemotherapy.
Angiogenic switch: what is it, what does it result in, what causes it, and what are the specific examples?
The term given to the point where the number or activity of the pro-angiogenic factors exceeds that of the anti-angiogenic factors, resulting in the angiogenic process proceeding
This gives rise to new blood vessels accompanied by increased tumour growth, metastasis, and potential drug resistance
Angiogenesis is orchestrated by a variety of activators and inhibitors which mediate the angiogenic switch.
Activators:
* VEGFs (RTK)
* FGFs (RTK)
* PDGFB (RTK)
* EGF (RTK)
* LPA
Inhibitors:
* Thrombospondin-1
* The statins - Angiostatin, endostatin, canstatin, and tumstatin
Angiogenic activators (VEGF (HIF-1 target), FGFs, PDGF, EGF, and LPA): how do they all interact?
EGF (epidermal growth factor) upregulates VEGF (vascular endothelium growth factor), FGF (fibroblast growth factor) and interleukin-8
LPA upregulates VEGF levels
Angiogenic inhibitors: how do they all interact?
Thrombospondin-1, which modulates endothelial-cell proliferation and motility
Many inhibitory molecules, such as ‘statins’, are derived from larger proteins that do not affect angiogenesis - ie angiostatin (a fragment of plasminogen that binds ATP synthase and annexin II), as well as endostatin, tumstatin and canstatin (fragments of collagens that bind to integrins).
Angiogenic factors
Interestingly, not only cancer cells but also inflammatory cells that infiltrate the tumour, notably mast cells and macrophages, and the extracellular matrix can be a source of angiogenesis factors.
Angiogenesis inhibitors in the treatment of human cancer
Inhibiting angiogenesis can shrink tumours or prevent their growth in animal models of cancer. This has led pharmaceutical companies to develop and test a number of angiogenesis inhibitors in the clinic. These inhibitors fall into several different categories, depending on their mechanism of action
Some inhibit the angiogenesis signalling cascade, while others inhibit endothelial cells directly or block the ability of endothelial cells to break down the extracellular matrix
Drugs that target endothelial cells directly
Molecules that directly inhibit the growth of endothelial cells - endostatin, the naturally occurring protein known to inhibit tumor growth in animals.
EMD121974 (cilengitide), interferes with integrin αVβ3 binding my mimicking ECM peptide ligands, promotes the destruction of proliferating endothelial cells.
Thalidomide, a sedative used in the 1950s that was subsequently taken off the market because it caused birth defects when taken by pregnant women. Although this drug clearly would not be suitable for pregnant women, its ability to prevent endothelial cells from forming new blood vessels might make it useful in treating nonpregnant cancer patients (mechanism of action?)
Drugs that block the angiogenesis signalling cascade
Included in this category are anti-VEGF antibodies (e.g. bevacizumab/Avastin) that block the VEGF receptor from binding growth factor.
Another agent, interferon-alpha, is a naturally occurring protein that inhibits the production of bFGF and VEGF, preventing these growth factors from starting the signalling cascade. Also, several synthetic drugs capable of interfering with endothelial cell receptors like the VEGFR are being tested in cancer patients.
Interferon-alpha
Anti-VEGF
antibody
SU5416
SU6668
PTK787/ZK 22584
Drugs that target endothelial cells directly
MMP enzymes that catalyze the breakdown of the extracellular matrix is another target.
Because breakdown of the matrix is required before endothelial cells can migrate into surrounding tissues and proliferate into new blood vessels, drugs that target MMPs also can inhibit angiogenesis
Several synthetic and naturally occurring molecules that inhibit the activity of MMPs are currently being tested to see if interfering with this stage of angiogenesis will prolong the survival of cancer patients.
Marimistat
AG3340
BMS-275291
Alternatives to angiogenesis
Vasculogenesis is an alternative mechanism for neovascularisation whereby new vessels form de novo (as during embryogenesis) from circulating endothelial progenitor cells released from the bone marrow
Vascular mimicry is where cancer cells themselves can form channels through which blood circulates. These tumour cell-lined conduits may express endothelial-selective markers and anti-coagulant factors which allow for anastomosis with host endothelium.
Glucose and its potential role in cancer
Cancer cells display increased conversion of glucose into lactic acid (fermentation) even in the presence of oxygen (a.k.a aerobic glycolysis)
More glucose is consumed and its metabolites are diverted into biosynthetic reactions
FDG-PET image - fluorodeoxyglucose is a radioactive glucose analogue, >90% sensitive for the detection of metastases
Warburg effect
Aerobic glycolysis - Pyruvate is mostly converted into lactate but will be minimally aerobically metabolised
Citric acid cycle in tumour cells
High extracellular glutamine concentrations stimulate tumour growth and are essential for cellular transformation
In tumour cells, the citric acid cycle is truncated due to an inhibition of the enzyme aconitase by high concentrations of reactive oxygen species (ROS) generated by impaired OXPHOS
The cells overexpress phosphate-dependent glutaminase and NAD(P)-dependent malate decarboxylase which in combination with the remaining reaction steps of the citric acid cycle from α-ketoglutarate to citrate comprise a new energy-producing pathway:
Glutamine -> glutamate, aspartate, pyruvate, CO2, lactate and citrate
Glutaminolysis: what is it, what is it an example of, what is it catalysed by, what is an example of it, what do transaminases do, and what form does glutamate take when not being actively used?
Removal of nitrogen from AAs and addition of nitrogen to ketoacids
An example of ‘anaplerosis’ - a metabolic pathway that replenishes citric acid cycle intermediates
Aminotransferases or transaminases (α-amino group is transferred to α-ketoglutarate (α-KG) to form glutamate)
Glutamine -> glutamate, aspartate, pyruvate, CO2, lactate and citrate
Transaminases can utilize glutamate as a nitrogen source and preserve the flow of nitrogen towards biosynthetic processes (during AA synthesis)
When not used in biosynthesis, glutamate nitrogen is converted to free ammonium by GDH
What form does glutamate take when not used in biosynthesis?
When not used in biosynthesis, glutamate nitrogen is converted to free ammonium (NH4⁺) by GDH oxidative deamination reaction catalyzed by the bidirectional enzyme glutamate dehydrogenase (GDH)
Glutamate: what does it do, what is it formed by, what is the terminal γ-nitrogen used for, how is NH4⁺ recycled, what is excess NH4⁺ converted to, and what is the remaining carbon skeleton used for?
Acts as a prominent intermediate in nitrogen metabolism and is usually formed by direct deamination of glutamine by glutaminase (GLS)
Glutamine terminal γ-nitrogen is used in the synthesis of pyrimidines, purines, asparagine, hexosamines, and NAD
NH4⁺ can be recycled by the reductive activity of GDH, glutamine synthetase (GS), and carbamoyl phosphate synthetase (CPS)
Excess ammonia can be converted to urea by the urea cycle enzymes
The remaining carbon skeleton left as α-ketoacids from AA deamination can have three different fates: used to fuel the TCA cycle, synthesize glucose/glycogen in liver cells (glucogenic AA), or be converted to acetyl-CoA/acetoacetyl-CoA (ketogenic AA)
Warburg effect/glutaminolysis advantages for cancer cells
- Renders cancer cells better adapted to fluctuating oxygen levels
- Metabolites can be redirected to biosynthetic reactions without affecting energy production
- Reduces levels of harmful reactive oxygen species generated by OXPHOS
- Lactic acid can be used by normoxic cells (cancer and stromal cells) to regenerate pyruvate leading to symbiosis between oxygenated and hypoxic regions of the tumour
- Lactic acid can result in the suppression of immune cells
What causes the reprogramming of intermediary metabolism in cancer cells?
Oncoproteins and tumour suppressor proteins regulate the expression and/or activity of multiple enzymes that regulate metabolic flux.
PKMs: what are they, what do they give rise to, what do they do, how expressed are they, what forms do they exist in, and what use is PKM1 in cancer treatment?
Pyruvate kinase isoenzymes (PKMs)
PKM gene gives rise to two different transcripts by alternative splicing encoding PKM1 and PKM2
Catalyses the final step of glycolysis
PKM2 is overexpressed in nearly all cancers but undetectable in most normal tissue. Induced by the c-Myc oncogene.
PKM2 can exist in a low-activity dimer form or a high-activity tetramer form:
* The low-activity form greatly reduces the conversion of PEP to pyruvate allowing metabolites upstream of pyruvate to accumulate and be channelled into biosynthetic pathways
* In tumour cells, PKM2 is mainly in the dimeric form. The quantification of dimeric PKM2 in plasma and stool is a tool for early detection of tumours and a biomarker during therapy.
Binding phosphotyrosine peptides produced by RTKs inhibits tetramer formation
Forced expression of PKM1 in malignant cells (always tetrameric and hence active) reverses the Warburg effect and blocks tumour formation
What enzymes affect glucose metabolism in response to (in)activation of TSGs/oncogenes
- PKM2
- MYC affect gene expression - stimulate glutaminase (GLS)
- Hypoxia-inducible factor 1 (HIF1) affect gene expression
- p53 affect gene expression - stimulates GLS2 expression
- AKT alters glycolytic proteins post translationally
Targeting metabolic enzymes as a strategy to block biosynthesis or induce energy stress.
Not clinically used yet, toxicity on cancerous cells not prevented from harming normal cells
watch leccy ???
Key concepts
Tumours are complex tissues comprised of neoplastic cells and non-neoplastic stroma
Stroma also contributes to tumour progression
Requirement for neovascularization for tumours to grow. Achieved through angiogenic switch
Angiogenic switch involves a gain in activity of angiogenesis activators such as VEGF and a reduction of inhibitors such as endostatin
Cancer cells display greatly enhanced glycolysis and glutaminolysis
This is in order to maintain a high level of anabolic reactions while generating sufficient ATP even under hypoxic conditions
Both angiogenesis and altered cellular metabolism are being pursued as therapeutic targets.
