Tumour Biology - Mechanisms of Metastasis Flashcards
1
Q
what is metastasis
A
- a metastasis is a tumour spread from its tissue of origin
- metastasis s a multifactorial process (different types of events must happen for a metastasis to occur)
- it is the major cause of death from malignant disease because widespread metastatic disease is difficult to treat
2
Q
where is colorectal cancer mainly metastasised to
A
- The liver
3
Q
correlation between grade of cancer and site of metastasis - what does the tell us about the process of metastasis
A
- As patients progress through the grade, the incidence of metastasis into the lung and liver increases.
- However for metastases to the bone (common site for breast cancer to spread), there is no such correlation.
- basically: patient at any grade of breast cancer can get metastasis to bone, but only at higher grades do we get metastasis to liver & lungs
- This tells us that there must be something different in the way tumour cells metastasise to different organs
4
Q
what are the 4 major sub-types of breast cancer
A
- HR+/HER2-
- HR+/HER2+
- HR-/HER2+
- HR-/HER2-
5
Q
which type of breast cancer is most likely to metastasise to bone
A
HR+/HER2-
- just remember that different cancers have different likelihood of metastasising to different organs
6
Q
What are the key steps in the metastatic cascade and the properties of metastatic cells?
A
1.Vascularisation of the primary tumour (to gain blood supply).
2.Detachment of cancer cells from the primary mass.
- Invasion of the extracellular matrix (e.g. basement membrane).
- Intravasation – cells enter blood vessels.
- Survival in circulation and travel to a secondary site.
- Adhesion to endothelium of the target organ.
- Extravasation – exit from blood vessels.
- Colonisation and survival in the secondary organ, forming micrometastases → macrometastases.
7
Q
what are some factors which will affect the metastatic cascade
A
- many factors affect the metastatic cascade, including supply of oxygen & glucose (energy)
- concentration of O2 is different in normal vs cancerous tissue (O2 levels are much higher in the normal organ vs the tumour)
8
Q
how does O2 concentration differ in cancerous vs normal tissue
A
[O2] is lower in cancerous tissue in all cases of cancer
9
Q
why is the [O2] different in cancerous tissue
A
the simplest explanation for this is: the proliferation of cancerous cells results in the formation of a mass - this means that they are further away from vasculature/supply of O2.
10
Q
why do we get necrotic regions in bigger tumours
A
the bigger the mass of tumour cells, the further away some of the cells are from the supply/vasculature ⇒ less oxygen & nutrients ⇒ they will die ⇒ necrosis
cancerous cells need O2 to proliferate, so they die in hypoxia
11
Q
result of hypoxia-dependant transcriptional factor development
A
hypoxia is important in cancer development & metastasis as the induction of powerful transcriptional factors will lead to multiple changes: (basc the hallmarks)
- immune evasion
- evasion of apoptosis
- regulation of proliferation
- regulation of angiogenesis
- self-sufficiency in growth signals
- genomic instability
- tissue invasion metastasis
- limitless replication potential
- glycolysis
12
Q
how does sufficient supply of oxygen (so in normal cells) prevent angiogenesis
A
- in normoxia we have low concentration of transcriptional factors such as HIF-1⍺ (hypoxia induced factors) because we are oxygenating it via PHD
- oxygenated HIF-1⍺ recruits the protein pVHL
- In normoxia, the interaction between HIF and pVHL causes degradation of HIF-1⍺ through ubiquination
12
Q
how do cancerous cells respond to hypoxia (4 we will focus on in the lecture)
A
- stimulate angiogenesis
- switch from an aerobic to an anaerobic form of energy generation (glycolysis + increase in lactate)
- immune evasion (via lactate)
- tissue invasion metastasis
13
Q
describe how cancer cells cause angiogenesis + so overcome hypoxia
A
- HIF-⍺ (hypoxia induced factor) is stabilised as it can’t be hydroxylated anymore (no oxygen)
- in hypoxia, pVHL is S-nitrosylated and is not recognised by HIF-⍺
- so ubiquination does not take place
- so HIF-1⍺ and HIF-1β form a complex which will result in hypoxia-induced gene expression of raw angiogenic factors such as VEGF ⇒ stimulation of angiogenesis
13
Q
what else can induce expression of HIF-⍺ (oxygen-independent regulation of HIF-1⍺ in cancer) (3)
A
- by inflammation
- cytokines (TNF-a, IFN)
- chemokines (MIF)
- growth factors (PDGF)
- direct HIF-1 activation by pathogens
- siderophores (Ybt, Sal, DFO)
- adhesins (BadA, F1845)
- LPS (E. coli)
- toxins (C. defficile)
- genetic epigenetic activation of HIF-1 expression
- oncogenic pathways (PI2-K)
- mutations
14
Q
why is angiogenesis advantageous for tumour cells (+ overall diagram)
A
angiogenesis = the development of new blood vessels
- so the tumour cells will now have sufficient oxygen supply = promote survival as well as spread
15
Q
describe the capillaries (due to angiogenesis) in normal tissue vs cancerous tissue
A
- Newly developed capillaries in the tumour are different from normal capillaries in the normal tissues.
- Normal Capillaries:
- Smooth, cobblestone appearance
- endothelial cells are tightly connected to each other
- Capillaries in tumours:
- abnormal endothelial cells that partition the lumen
- multiple intercellular openings
- Normal Capillaries:
- In the case of tumour vascularisation, you can see that the capillaries are not as nicely organised (they become torturous).
- This helps tumour cells get inside the vasculature.
16
Q
what is the main source of energy in all cells
A
Glucose (being converted to ATP)
17
Q
metabolism of glucose in normoxia (basically glycolysis)
A
- In glycolysis glucose is taken up by cells and then converted to pyruvate producing ATP (energy).
- Pyruvate is converted to acetyl co-A (and lactate) which enters the Krebs cycles (AKA TCA), this is catalysed by PDH (pyruvate dehydrogenase).
- this results in a sequence of enzymatic events which eventually leads to the production of ATP and NAD+ which enter electron transport chain (ETC)
- the ETC is where most ATP is generated
18
Q
metabolism of glucose in hypoxia (2 main things happen)
A
we get an increase in lactate, how?:
- In the case of hypoxia, you will have HIF-1⍺ stabilized, which will:
- increased production of lactate transporter (MCT4)
- It will increase production of an enzyme called PDK1 (which phosphorylates/inactivates PDH ⇒ can’t dehydrogenate pyruvate).
- As a result of this, in hypoxic conditions, you get pyruvate converted to lactate.
- This inhibits production of Acetyl-CoA and so you get blockage of pyruvate entering the Krebs cycle.
19
Q
is glycolysis an efficient way of generating energy?
A
- Glycolysis is an inefficient way of energy generation as when you block the Krebs cycle the number of ATP molecules produced will be significantly lower.
- But glycolysis allows cells to survive in hypoxic conditions, and then when you have new vasculature formed you will have better supply of glucose and oxygen and so you get switching back to aerobic cycle.
20
Q
why is glycolysis advantageous for cancer cell survival
A
- Upregulation of glycolysis in tumour cells enables tumour cells to outcompete normal cells for scarce glucose supply
- Tumour cells use glycolysis are a major route of energy generation.
- They also upregulate glycose uptake and metabolism:
- They upregulate the glucose transporters which enables them to scavenge very effectively for glucose in the extracellular environment
- They also upregulate the enzymes such as the Hexokinases which are responsible for process of glycolysis.
21
Q
what is the higher production of lactate in hypoxia known as
A
Warburg effect - it is very positive in cancer cells
- The Warburg Effect is defined as an increase in the rate of glucose uptake and preferential production of lactate, even in the presence of oxygen.
- This is what happens in cancer cells
- Whilst this can be stimulated by hypoxia often cancer cells will switch to using glycolysis even under normoxia conditions.
22
Q
what is the advantage of the excess lactate produced by cancer cells due to the Warburg effect (4)
A
Lactate can regulate many things and stimulate different mechanisms which will lead its metastasis:
- tumour micro-environment (stimulate their growth)
- proposal: enhances distribution of tissue architecture and immune cell evasion
- rapid ATP synthesis
- proposal: increases access to a limited energy source
- cell signalling
- proposal: allows for signal transduction through ROS and/or chromatin modulation
- biosynthesis
- proposal: promotes flux into biosynthesitic pathways
23
another way Warburg effect is advantageous (this is from hamzah’s notes, not in the lecture)
- Glycolysis produces lactic acid which will be responsible for the acidification of the tumour microenvironment.
- Normal cells are less able to survive in acidified environment compared to tumour cells.
- This is another contributing factor to the resulting cell death in the immediate vicinity of the cancer.
- Therefore the switch to glycolysis provides increases the invasive potential of the cancer cell.
24
hypoxia will stabilise HIF-1⍺ which will increase lactate levels via expression of MCT4, how is this advantageous in the micro-environment for cancer growth (2)
- the huge excess of lactate will now be transported out of cell via MCT4, lactate extracellularly will :
- suppress the immune response to cancer
- stimulate the conversion of monocytes to M2 macrophages - these will stimulate cancer cell growth & are very important in metastasis
25
lactate will suppress immune response to cancer, which cells in particular will be affected
NK, dendritic, CD8+ & CD4+ T cells
26
what's the first thing that needs to happen for cells to become metastatic
cells have to detach from primary tumour
27
how do cells detach from primary tumour
loss of adhesion - adhesion molecules are down-regulated in metastatic cancer cells
- one of those molecules is E-cadherin, down regulation of this molecule is common, indicating that loss of cell attachment is important for invasion
28
what is the function of E-cadherin
it is a protein which holds epithelial cells together through the formation of specific adhesion complexes called adherens junctions
29
describe the structure of adherens junctions
- cadherin dimer on one cell will make contact with a cadherin dimer on the opposite cell.
- You need some kind of structural support coming from the cell.
- In this case this is from the actin cytoskeleton.
- The actin cytoskeleton is linked to cadherin receptors through specific linker proteins e.g. B-catenin.
- This allows Adherens junctions to function properly.
30
why is β-catenin dangerous in cytoplasm
- Stabilised β-catenin promotes oncogenic transcription.
- If you have excess β-catenin in the cytoplasm you will have β-catenin forming complex with transcription factors resulting in increased expression of cell cycle genes (e.g. MYC) which facilitate growth of tumour cells and facilitate metastatic progression of tumour cells.
- This is why normal cells have tight control over amount of β-catenin present in the cytoplasm.
simplified explanation: it acts as a co-transcription factor ⇒ the formed complex activates the transcription of specific target genes involved in cell proliferation, survival, and other processes e.g. can confer resistance to apoptosis.
31
how do normal cells control cytoplasmic β-catenin levels
- In normal cells, beta-catenin undergoes quick degradation as it is a dangerous protein to have in the cytoplasm.
- The below complex modifies b-catenin and moves to protostomes for degradation.
- There are many proteins in this complex. One of the most important is called APC.
32
how can beta-cetenin degradation be stopped (2)
- APC is frequently mutated in cancers.
- Common example is the mutation of APC/shutdown of APC expression in colorectal cancer (APC shutdown ⇒ no beta-catenin degradation ⇒ free cytoplasmic beta-catenin ⇒ cancer).
- The cancer is caused because b-catenin cannot be degraded so you get excess b-catenin in cytoplasm.
- NB: another way to shut down this degradation machinery is to activate Wnt signalling pathway (basically stabilise the beta-cadherin)
33
what controls invasion and metastasis
Epithelial-mesenchyme transition (EMT) regulates invasion and metastasis
34
what is EMT
- One of the ways tumour cells get rid of the expression of E-cadherin is through a process called EMT.
- EMT is not necessarily a bad thing:
- It is important during embryological development, and important during wound healing.
- But when EMT takes place in tumour cells this is bad as at the end you get decreased expression of e-cadherin and some other adhesion molecules which leads to dissociation of epithelial cells ⇒ cells become more migratory & metastatic.
- it results in a mesenchymal phenotype
35
what factors can induce EMT (2)
- beta-catenin
- SLUG (transcription factors)
36
what is required for tumour cell invasion (2/3)
- required modification of adhesion of tumour cells with extracellular matrix (basement membrane, collagen) = loss of adhesion
- required production of degrading enzymes - matrix metalloproteinases (MMPs)
- MMPs are normal enzyme involved in tissue remodelling
- may be secreted by tumour cells, but also stream (storm is important in tumour progression)
from lecture 1:
- Epithelial cells in solid tumours cells undergo special transcription programmes called EMT (epithelial to mesenchymal transition)
- Loss of cell-cell contacts through changes in expression of adhesion receptors (cadherins, integrins)
- Activation of extracellular proteases
37
what are the different kinds of invasion (2)
- Single cells can separate from the primary tumour and get inside the vasculature.
- However there are some other ways tumour cells can progress to metastatic stage.
- In epithelial tumours you will often see it is not single cells that separate from tumour but a group of cells.
- collective
- single cell motility - 2 types:
- amoeboid
- mesynchymal
38
is cancer spread random
no, it is non-random
- Examples:
- In the case of colorectal cancer, tumour predominantly metastasise to the liver.
- In the case of breast cancer it will predominantly metastasis to brain, bone and liver as well.
39
why do specific types of cancer metastasise to specific locations (2)
- Seed & Soil Hypothesis
- Anatomical & Mechanical Routes
40
what is the seed-and-oil hypothesis (non-random pattern of metastasis (Paget))
- 1889, English surgeon Stephen Paget published the 'seed and soil' hypothesis to explain the non-random pattern of metastasis
- Seed = tumour cells
- Soil = distant site for metastatic cells.
- What he said was that tumour cells end up in specific locations because they find a favourable microenvironment there.
- I.e. the cells can only survive in certain conditions in certain organs.
- At the time not everyone agreed.
- There was an alternative hypothesis was proposed…(Ewing)
41
colonisation and survival at the distant site (Ewing)
James Ewing (1929) proposed that metastasis occurs purely by anatomic and mechanical routes of the circulatory system.
42
who is right, Ewing vs Paget (metastasis & arrest)
- Both of them are right:
- Tumour cells can survive only in organs where they have specific growth factors and specific microenvironmental factors which help their survival. But it is also true that they primarily enter the organ which would be explained by the circulator pattern.
- Also they arrest in the body not only because they have a bigger size but because they have specific type of adhesion receptors on endothelial cells which will capture tumour cells in specific locations.
43
what is arrest in organs (Trapping vs homing)
cancer cells arrest in the body not only because they have a bigger size but because they have specific type of adhesion receptors on endothelial cells which will capture tumour cells in specific locations.
44
what is the metastatic niche
- The seed and soil hypothesis has a further development → The metastatic niche
- Tumour cells, whilst at their primary site, secrete factors which act systemically, modifying the local environment and recruiting host immune cells, facilitating the appropriation of these sites for later colonisation.
