[MUSIC PLAYING]
DAVID KOK: Welcome.
I'm Dr David Kok, a radiation oncologist at Peter MacCallum
Cancer Centre, and I'm very pleased to be taking you
through this lesson on the evasion of growth suppression.
We're going to begin with a discussion of cell homeostasis
and the balance between cell proliferation and cell death
before moving on to a discussion of the cell cycle checkpoints
and how they are regulated.
Finally, we'll go on to a discussion of how
dis-regulation of these leads to uncontrolled growth
suppression, which is the second hallmark of cancer.
This diagram represents tissue homeostasis
and how there is a balance between cell proliferation
and growth on one side and cell quiescence
or death on the other side.
Cell quiescence is a term you may not be familiar with
and represents when a cell is alive but unable to replicate.
This is just as important as cell death
in maintaining tissue homeostasis.
The scales between these two sides
are perfectly balanced between stimulatory factors
on one side and inhibitory factors on the other side.
In the normal physiological state,
stimulatory factors do result in cell proliferation.
But this is perfectly balanced by the inhibitory factors
such that at all points in time there
is a one-to-one balance between cell proliferation and cell
death.
Now there are situations when the stimulatory factors
predominate, and this results in sustained proliferative
signalling.
This is a hallmark of cancer, as has been discussed previously
by Dr Vissers.
In this particular lesson, we're going to discuss how removal
of inhibitory factors from this side of the equation can lead
to tipping of the scales further in favour of cellular
proliferation and growth, and this is the evasion of growth
suppression, the second hallmark of cancer.
Let's have a look at how this happens within the cell itself.
So here we have a cell signalling cascade.
It begins with a ligand binding to a surface
receptor on a cell, activating an intracellular domain.
If we zoom out, we can see how this
is occurring at the cell surface,
and then results in a cell signalling
cascade of multiple transducers, eventually
leading to an effector, which enters the nucleus.
This is a transcription factor in this case
and promotes movement through the cell cycle and cell
proliferation.
Therefore, in this particular example,
stimulatory factors are leading to the scales being tipped
in favour of cell proliferation and growth,
and there is a moderate amount of cellular proliferation,
as depicted here on the graph.
Now in our original example, we'd
shown you a single linear example of how a cell
signalling cascade can occur.
But in real life, obviously, it's
much more complex than this.
In this particular example, we've
shown you two parallel pathways, which eventually
lead to multiple different effectors entering the nucleus
and leading to cell proliferation.
This is a good example of how, even though it's
a single signalling molecule, a significant amount of cell
proliferation can occur.
So once again, here the scales are tipped even further
in favour of the stimulatory factors,
and a very significant amount of cellular proliferation is
the result.
So obviously, in the normal physiological state,
then growth cannot continue unabated.
That's why there are a number of natural inhibitory factors
which are designed to reduce this.
Here we have a tumour suppressive protein,
which is illustrated acting on the signal cascade that
we've discussed previously.
Here, this particular tumour suppressor is inhibiting
the actions of the transducers at a very early stage
in the signal cascade.
What this means is that signals are unable to progress down
either the green or the red pathways
and cellular proliferation is controlled.
So what this is doing is balancing the scales in favour
of cell quiescence or death and, as such,
cellular proliferation is kept in check back in a one-to-one
ratio.
So we're now going to spend some time talking about the cell
cycle and how movement through the cell cycle is governed.
You will remember from your basic biology
how the cell cycle is divided into four different phases.
There are the two phases where the cell is at rest--
this is the G1 and the G2 phase.
More importantly are the active phases, the S phase,
or the synthesis phase, and the M phase, or the mitotic phase.
Movement through the cell cycle is
regulated by a very fine number of molecules, these cyclins,
which are indicated along the outside of the diagram here.
In particular, I want to draw your attention
to this area here, cyclin D, which
we're going to spend some time discussing, as well
as the G1/S checkpoint.
The G1/S checkpoint is a very important checkpoint
because it is the stage at which a cell becomes
committed to replication.
Now levels of cyclins fluctuate throughout the cell cycle
in a reproducible and regulated manner.
As you can see in this diagram here,
cyclin D is at its highest level towards the end of G1,
as is indicated on the diagram here.
This is also consistent with the other cyclins.
For instance, you can see cyclin B,
which is indicated at its highest level
just after the synthesis phase.
It is this concentration of cyclins
at each particular point which regulates the movement
through the cell cycle.
So putting this all together now in a real life example, rather
than a generic example, here we have a ligand binding to a cell
surface receptor.
This then activates the RAS MAP kinase pathway,
and ERK enters the nucleus, stimulating the recruitment
of the appropriate cyclins, and progression through the cell
cycle and ultimately, cell proliferation.
We're now getting to spend some time familiarising ourselves
with a few specific tumour suppressive genes
and their proteins.
We've already had a discussion about how tumour suppressive
proteins act by down-regulating or inhibiting the cellular
signalling cascade and therefore reducing proliferation.
We're going to spend some time now talking
about how this actually occurs in the nucleus and some
of the terminology around it.
So here we are in the nucleus of the cell,
and here's a generic transcription
factor indicated bound to the appropriate location on DNA.
The transcription factor recruits RNA polymerase,
which binds to the appropriate site on the DNA,
and travels along the DNA, transcribing this information,
which ultimately gets translated into a tumour suppressor
protein.
Now what we need to understand here is the difference between
a tumour suppressive gene and the tumour suppressor protein
which it produces.
In this particular example, from here to here
is the reading frame of the DNA.
And the information which is in this reading frame is what is
ultimately translated into the tumour suppressor protein.
Therefore, this segment of the DNA is the gene segment
or the tumour suppressive gene which ultimately results
in translation into a tumour suppressive protein.
If we return now to our homeostatic balance,
which we've shown you earlier, we could reconceptualise this
with proto-oncogenes on this side of the scale,
where previously we had stimulatory factors,
as it is the proto-oncogenes genes which encode
the stimulatory factors and on this side,
the tumour suppressor genes which encode the inhibitory
factors.
Let's spend some time now talking about a few
of the specific tumour suppressive genes.
And in particular, we're going to focus on P53, P10,
P16, and retinoblastoma.
All of these tumour suppressor genes tip the scales in balance
of cell quiescence or cell death.
Let's spend some time now focusing on P10.
P10 is a classic tumour suppressor with a fairly simple
action.
Now if we return to this cell signalling cascade which
we showed you earlier, at the time
we'd introduced it to you with a number of generic labels
just so that you could understand the concept.
If we were to remove these labels now,
we could instead replace them with
a specific real-life example.
Here we now have the RAS MAP kinase pathway and in the red,
the PI3 kinase pathway, both of which
have been introduced to you previously by Dr. Vissers.
Now P10 acts on this by inhibiting movement
along the PI3 kinase pathway.
And what it does is, at this stage
where PIP2 gets converted to PIP3,
P10 actually stimulates conversion
of PIP3 back towards PIP2, therefore reducing movement
through the PI3 K cascade and therefore reducing
cell proliferation.
This is a fairly classic tumour suppressor action.
Let's now move on to P53, another classic tumour
suppressor which you'll see quite a lot written about
in the literature.
It's often referred to as the guardian of the genome
because of the wide range of effects it has in suppressing
tumour activity.
What P53 does, it senses genotoxic stress.
And by that, I mean anything which affects
the integrity of the genome.
I've listed some of these here, including oxidative stress,
hypoxia, DNA damage, nutrient deprivation,
oncogene expression, and telomere attrition.
When P53 senses these, then it then halts the cell cycle
and begins a number of different mechanisms
to try and repair this.
For instance, by recruiting DNA repair machinery,
it tries to return DNA back to its original state.
But there are situations where the damage
is too significant to be salvaged,
and so to stop replication of the incorrect information
it can trigger death of the cell the most common way,
through apoptosis.
RB, or retinoblastoma, is another important tumour
suppressor gene with multiple actions within the nucleus.
Here we are in the nucleus, and I
want to introduce to you E2F, which is a transcription
factor bound to DNA, and it has the important regulatory
function in ensuring appropriate movement through the cell
cycle.
We're going to build upon this particular model
over the next few slides but the basic principle
is that E2F recruits RNA polymerase.
RNA polymerase travels along the DNA,
translating that DNA into a number of different proteins
which are required for cell cycle progression.
Now retinoblastoma, or RB, is the molecule
which is indicated here.
RB binds to E2F, and when it is bound to E2F,
it actually stops the DNA transcription machinery
from being able to function.
So here we have this represented by RNA
polymerase, which is unable to bind
and, therefore, there is no transcription which occurs.
Retinoblastoma is able to be deactivated
by a number of different cyclins,
and this plays back into the cell cycle
which we've discussed earlier.
Cyclin D-- which you remember from our earlier
slide was an important factor in allowing progression
through the G1S checkpoint-- is one of the governing molecules
in this particular example.
Cyclin D binds to CDK4 or CDK6, forming a complex.
And this particular complex is then
able to bind to retinoblastoma and, at this point,
the complex phosphorylates retinoblastoma
and allows retinoblastoma to be freed from E2F.
When E2F is therefore freed, its transcription machinery
is able to be activated, and RNA polymerase
is able to perform its function of translating the DNA,
and ultimately leading to the proteins required
for cell cycle progression.
We're now going to talk about the tumour suppressor molecule
P16.
P16 adds one additional layer of complexity
to the cellular signalling mechanism which
we've previously described.
P16 actually forms a complex with CDK4/6.
And when this is complexed, this inhibits the function
of CDK4/6 such that when cyclin D attempts to bind to it,
it is unable to.
Therefore, this also down-regulates
cellular proliferation.
In this particular case, you can see how P16 is acting
as a tumour suppressor as well as RB,
both of these molecules acting as a form of double breaking
on cellular proliferation.
P16 must actually be dissociated from CDK4/6 for cyclin D
to form its complex.
And cyclin D then ultimately phosphorylates retinoblastoma.
This allows it to dissociate and ultimately for RNA polymerase
to form its function and the cell cycle to progress.
So now that we've had a discussion of a few
of the specific tumour suppressor genes,
let's go into a discussion of how tumour suppressor genes can
be inactivated.
So to quickly recap, tumour suppressor proteins are encoded
in the nucleus of a cell.
Here we have RNA polymerase binding to the appropriate site
on the DNA and transcribing that information such that it
ultimately becomes a tumour suppressor protein.
If a mutation were to occur for whatever reason--
and here we have this example of a mutation occurring
of this single base pair in the DNA helix--
then this can significantly change what occurs,
just like the alteration to a single word in a sentence
would completely change its meaning.
So when RNA polymerase comes along now
and performs its transcribing process,
then it produces an aberrant protein.
This aberrant protein then, when it goes to actually perform its
appropriate tumour suppressor function,
is no longer able to do this.
So here we are in our cell signalling cascade.
We have the two different pathways occurring.
We have signal transduction occurring through the green
and the red pathways.
And this aberrant protein, which we had previously introduced,
was having an inhibitory effect on the transducers here,
actually has no particular influence on the pathways.
The effectors enter the nucleus, and cell proliferation
is able to occur unabated.
As a result, the homeostatic balance tips in favour
of the oncogenes, and we have significant amounts of cellular
proliferation, as indicated here.
So in conclusion, tumour suppresor genes, such as P53,
RB, P10, and P16 regulate cell proliferation pathways.
Each cell cycle phase is regulated by protein complexes
consisting of CDKs and cyclins that allow passage
through cell cycle checkpoints.
And mutations in tumour suppressive genes result
in loss of cell cycle checkpoint regulation and, thus,
uncontrolled cell proliferation.
This is the second hallmark of cancer.