Lecture 15 - The angiogenic switch and altered energy production and intermediary metabolism

Question 1

Carcinomas: what are they?

Answer 1

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

Question 2

Lack of oxygen (hypoxia) and metabolic waste products can limit cancer cell growth

Answer 2

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)

Question 3

Transcription factor HIF-1α: why does hypoxia affect it, what does it target, what happens to it in normoxia and hypoxia

Answer 3

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

Question 4

Tumours vascular supply

Answer 4

Tumours require new vasculature to grow

Question 5

Tumour vasculature

Answer 5

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.

Question 6

Angiogenic switch: what is it, what does it result in, what causes it, and what are the specific examples?

Answer 6

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

Question 7

Angiogenic activators (VEGF (HIF-1 target), FGFs, PDGF, EGF, and LPA): how do they all interact?

Answer 7

EGF (epidermal growth factor) upregulates VEGF (vascular endothelium growth factor), FGF (fibroblast growth factor) and interleukin-8

LPA upregulates VEGF levels

Question 8

Angiogenic inhibitors: how do they all interact?

Answer 8

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).

Question 9

Angiogenic factors

Answer 9

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.

Question 10

Angiogenesis inhibitors in the treatment of human cancer

Answer 10

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

Question 11

Drugs that target endothelial cells directly

Answer 11

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?)

Question 12

Drugs that block the angiogenesis signalling cascade

Answer 12

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

Question 13

Drugs that target endothelial cells directly

Answer 13

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

Question 14

Alternatives to angiogenesis

Answer 14

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.

Question 15

Glucose and its potential role in cancer

Answer 15

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

Question 16

Warburg effect

Answer 16

Aerobic glycolysis - Pyruvate is mostly converted into lactate but will be minimally aerobically metabolised

Question 17

Citric acid cycle in tumour cells

Answer 17

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

Question 18

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?

Answer 18

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

Question 19

What form does glutamate take when not used in biosynthesis?

Answer 19

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)

Question 20

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?

Answer 20

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)

Question 21

Warburg effect/glutaminolysis advantages for cancer cells

Answer 21

• 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

Question 22

What causes the reprogramming of intermediary metabolism in cancer cells?

Answer 22

Oncoproteins and tumour suppressor proteins regulate the expression and/or activity of multiple enzymes that regulate metabolic flux.

Question 23

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?

Answer 23

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

Question 24

What enzymes affect glucose metabolism in response to (in)activation of TSGs/oncogenes

Answer 24

• 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

Question 25

Targeting metabolic enzymes as a strategy to block biosynthesis or induce energy stress.

Answer 25

Not clinically used yet, toxicity on cancerous cells not prevented from harming normal cells

watch leccy ???

Question 26

Key concepts

Answer 26

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.