Tumour hypoxia

Question 1

What is tumour hypoxia? And what are it’s effect?

Answer 1

It is tumours with reduced oxygen concentration<9mmHG
It will have high metabolic rates
Uncontrolled proliferation
Collapse of vessels and leakiness
Decreased cell nutrients
Increased waste production and necrosis in cells that are >180um away from vessels.

Question 2

What’s the diffusion distance of oxygen?

Answer 2

80-100um

Question 3

What is the effect of cells that are far from blood vessels?

Answer 3

There will be less nutrients and more necrosis and higher metabolic wastes

Question 4

What are the markers used to detect hypoxia and blood vessels?

Answer 4

EF5 -hypoxia

Pecam-blood vessels

Question 5

What are the effects of hypoxia at the molecular level?

Answer 5

Genome changes, clonal heterogenity, clonal selection, post transcription adn translational changes, proteins egradation, proteosome change

Question 6

Can hypoxia be reversible?

Answer 6

Sometimes there is acute hypoxia where the blood vessels that are aberant are shut down but can be reopened.

Question 7

How does hypoxia increase radiotherapy and chemotherapy resistance?

Answer 7

Radio- and chemo therapy resistance occurs becuase of the changes in the DNA damage response, there is no oxygen damage repair cells go on. Drugs can be metabolized by the cells or sequestered because of the acidic environment or proteneonated.

Question 8

How is hypoxia modelled in a lab?

Answer 8

There are chambers that can control oxygen levels by pumping nitrogen. Spheroid mimick hypoxic microenvironment and show the interaction between different factors

Question 9

What are hypoxia inducible factors and how are they regulated and expressed?

Answer 9

They are transcription factors that transcribe hypoxic inducible genes, they are constitutively expressed but degrade in normoxic conditions through it’s hydroxylation.
When there is hypoxia then there will be stabilization of HIFa translocates to nucleus dimerize with HIF1-B and bind to HRE site- sequence is RCGTG

Question 10

What are the functions of the hypoxic inducible genes?

Answer 10

Proliferation, migration/metastasis, metabolism, PH regulation, angiogenesis, stem cell maintenance.

Question 11

Do HIF1A and HIF2A work independently?

Answer 11

No they can target the same genes such PH homeostasis and lipid metabolism. HIF1A regulates glycolysis positively while HIF2A regulates it negatively.

Question 12

What protein regulates HIF stabilization?

Answer 12

VHL binds to HIF and then it’s hydroxylated and ubiquitinated for degradation

Question 13

What is the name of the enzyme that hydroxylates HIF for degradation?

Answer 13

Prolyl hydroxylase, it needs oxygen to do this

Question 14

What other proteins stabilize HIF1A?

Answer 14

Growth factors;insulin, EGF, IGF, FGF

Question 15

Knowing that growth factors can stabilize HIF1A what potential target was effective in inhibiting HIF1A stabilization as shown in past studies?

Answer 15

By inhibiting the PI3K/AKT pathway

Question 16

How does hypoxia increase angiogenesis?

Answer 16

HIF1a increases angiogenesis through proteins such VEGF, other proteins that are needed for the process such as VEGF, angiopoietin 2, collagen. HIF1A transcribes these proteins genes, and it increases blood vessel formation through autocrine signalling. MMPS are released from cells an dreleases pro-angiogenic factors from the stroma. Angiopoietin 2 is released from tumour cells and it increases vascular basement degration and cell migration.

Question 17

What drug is used to treat angiogenesis in tumours? What cancers does it treat?

Answer 17

Monoclonal Antibody against VEGF; Bevacizumab.

CRC, lung cancer, brain cancer

Question 18

What is the optimal treatment if using bevasizumab?

Answer 18

Combination treatment; HIF1a knockout and bevacizumab is most effective

Question 19

What are the pro-angiogenic molecules that are expressed in hypoxic tumour cells?

Answer 19

VEGF, FGF2, ANG1&2, HGF

Question 20

Highlight the steps and what molecules are involved in angiogenesis

Answer 20

VEGF stimulation increases vasodilation and permeability of pre-existing vessels.
ANG2 signalling reduce pericyte coverage
VEGF2 and ANG2 release MMP that break down ECM
Endothelial TIP cell start to migrate forward towards pro-angiogenic stimuli.
Stalk cells follow and proliferate to form vascular lumen via CDC42 and rac.
Blood vessels can be co-opted by tumours

Question 21

How does hypoxia affect proliferation rates and what molecules are involved?

Answer 21

It decreases proliferation rates.
HIF1A increases many growth fcactors such as TGF-B and PDGF which activate MAPK and PI3K pathway, associated with proliferation upon re-oxygenation.
HIF2A prolifate cells through c-myc

Question 22

How does hypoxia effect metabolism?

Answer 22

Metabolism it’s switched to anaerobic pathway, increase lactate production through glycolysis in normoxic conditions, LDHA will convert pyruvate to lactate and will be pumped out. Increase in pentose phosphate pathway(HIF1A) and increase in fatty acid synthesis and less kreb cycle.

Question 23

How is hypoxia involved with pH regulation?

Answer 23

pH regulation, it becomes more acidic. CAIX catalyzes the extracellular hydration of CO2 resulting in bicarbonate and proton that’s pumped back inside through other channels. It’s key in regulating pH in hypoxic cells

Question 24

What genes are involved in regulating pH in hypoxic cells?

Answer 24

MCT1 and MCT4, both pumps lactate out along with protons, but MCT1 can influx lactate and protons depending on the diffusion gradient.
Sodium hydrogen exchanger(NHE1)-pumps sodium in and protons out.
All of these are upregulated in hypoxia.

Question 25

What findings did they find with CAIX knockdown?

Answer 25

It reduces tumour growth

Question 26

How does hypoxia increase metastasis?

Answer 26

It induces EMT (notch)
Activates invasion(MMP)
TAM attracted to hypoxic regions, they direct the tumour cells to vessels
Hypoxia mediates intraversion(MMP)
HIF1A protects from anikoisis(INTRK2)
Hypoxia regulates adhesion to endothelial cells and extraversion(ICAM, VEGF)
Hypoxia fosters Premetastatic niche formation(LOX)
Micrometastatic latency and hypoxia(metastatic suppresor genes)
Hypoxia induces angiogenic switch and upregulates metastatic virulence genes(PGF)
Tumour hypoxic cells release IL6 AND IL8 which may promote self seeding
Cancer stem cells stemness promote stemness(OCT4)

Question 27

How does hypoxia change the stem cell population and how does it do this?

Answer 27

Stem cell fraction increase, HIF1a and HIF2a have been linked to stimulation of CSC IN glioblastoma, it does this by acting on specific signalling pathway sand trascription factors such NOTCH and OCT4 that control stem cell self renewal and multipotency.

Question 28

What are the Cancer stem cell models, explain each one, give an example of them. Which one is the correct model?

Answer 28

Clonal evolution- muation acquired and then selection and expansion of these cells
Classical CSC model- unidirectional conversion CSC to non CSC. Example: Blood tumours
Plastic CSC model- bidirectional conversionof CSC to non CSC. Example of solid tumours
Cancer is heterogenous for three of these models

Question 29

How does hypoxia change immune response and how?

Answer 29

Immune response changes, tumour associated macrophages are attracted to hypoxi regions via chemotatic molecules of VEGF, EMAP11, SDF1 which bind to CXCR4.
TAM increases proliferation through expression of growth factors
It is accumulated at these hypoxic regions and may be trapped through downregulation of chemokine receptors.
Expressed highly by stromal factors
They release machrophage inhibitory factor-CD142, MMP7, and thrombin to promote invasion and metastasis.
Release immunosuppresive factors such as prostaglandin E2 and IL10- which reduce T-cell proliferation.
T cells tend to be in areas of tumours that are less hypoxic

Question 30

Give two examples of ways they can Target hypoxic cells

Answer 30

Using a prodrug that needs to go through multiple rounds of reduction before becoming, in hypoxia the drug can’t be oxidized and return to initial state.
Macrophages isolated from blood transfected with 2 constructs:
HYpoxia regulated E1A/B and promoter element that can be activated with transcription factors found only prostate(TARP). So once the adenovirus is activated in hypoxic region it can produce E1A/B proteins but can only proliferate and be cytotoxic in pancreas, this reduces tumour growth in pancreas,

Question 31

How does HIF make cells immortal? How do stem cells increase chemotheraoy resistance?

Answer 31

By binding to the promoter of HTERT and increasing it’s expression.
Some have ABC glycoprotein transporter pump out the drugs

Question 32

Which protein is strongly involved in metastatic spread??

Outline the main steps of metastasis

Answer 32

HIF1A
Invasion
intraversion
survival in peripheral blood
Organ specific Extraversion and colonization
HIF is involved in all of the steps

Question 33

What processes induce tumour hypoxia generally?

Answer 33

An increase in metabolic demands, increased proliferation and reduced tumour perfusion