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Hi. Thank you for listening.
My name is Britt Duijndam. In this presentation...
I would like to give you an overview of my PhD project...
where we developed a reporter platform for oestrogen receptor alpha signaling.
Exposure to certain pharmaceuticals or compounds...
can result in tumour development in humans.
Whereas genotoxic compounds can be readily identified in vitro...
we mostly rely on lifetime rodent bioassays for the detection of non-genotoxic carcinogens.
However, this lifetime rodent bioassay has its limitations.
In line with the 3R-principle...
known as the replacement, reduction and refinement of animal testing...
more efforts are being made to develop cell-based assays.
But since the mode of action of these non-genotoxic carcinogens...
is quite complex and diverse, this remains challenging.
Whereas genotoxic compounds mostly rely on DNA damage as their mode of action...
the mode of action of non-genotoxic carcinogens is diverse.
And we wanted to focus on receptor-mediated endocrine modification.
This receptor binding can have various consequences...
such as the induction of cellular processes such as proliferation, apoptosis, homeostasis...
and this can eventually culminate in tumour development.
In this project, we studied one of these involved receptors...
namely the oestrogen receptor alpha.
To evaluate and link this complex ERa signaling pathway to non-genotoxic carcinogenicity...
we made use of an adverse outcome pathway framework.
This AOP is a simplified presentation of a biological process...
which describes the link between the initial chemical interaction...
and the occurrence of adverse effects, by using a series of key events.
When we fill out this AOP for ERa activation in relation to proliferation, it will look like this.
So as a molecular initiating event, the receptor will be bound by a compound...
translocate to the nucleus, where it acts as a transcription factor.
This will lead to the first key event, namely the transcription of target genes...
followed by subsequent key events like cell cycle progression and proliferation...
ultimately culminating in tumour formation.
To integrate this AOP into human risk assessment...
it is essential to carefully define and quantify these key events.
For this purpose, we established fluorescent reporters...
which in combination with advanced live cell imaging...
allow for time- and concentration-dependent assessment of pathway activation.
To monitor the first key event, target activation...
we carefully identify three target genes, namely GREB1, PGR and TFF1.
These targets fit in our AOP because they are inducible by our model compound E2...
which is also known as 17ß-estradiol.
This is the endogenous ligand of the oestrogen receptor alpha...
and also a known human non-genotoxic carcinogen.
Secondly, this induction is completely dependent on oestrogen receptor alpha...
and we have demonstrated that they play a role in E2-induced proliferation.
With BAC-GFP technology, we've coupled these three target genes to a green fluorescent protein.
These constructs make use of an endogenous promoter.
This better reflects the regulation of these targets in the human situation.
Next, we transfected these constructs in human MCF7 cells.
In this way, we developed three different fluorescent reporters.
In combination with advanced live cell imaging...
we can monitor the dynamic of pathway activation in these individual reporters...
on a single-cell level.
When we quantify the GFP expression over time, displayed in the graphs...
the three reporters demonstrate concentration-dependent activation.
However, they show clearly distinct activation dynamic patterns over time.
Now that we have covered the first key event, we move on to the second key event.
To monitor cell cycle progression, we developed another reporter in the MCF7 cells...
which is based on a cell cycle indicator construct.
We named this cell line, or this reporter, we named it MCF7-FUCCI.
This FUCCI reporter makes use of two different genes coupled to two different fluorescent tags.
Geminin coupled to GFP and Cdt1 coupled to RFP.
These genes have distinct expression patterns in the cell cycle.
So based on the expression of the tags...
we can determine in which phase a cell is residing over time.
We employed this FUCCI reporter to look at the link between target activation...
and cell cycle progression.
By using small interfering RNAs...
we knock down the expression of the individual target genes in the FUCCI reporter...
expose the FUCCI reporter to E2 and monitor the cell cycle progression over time.
As expected, cell cycle progression is inhibited after knockdown of these target genes...
which can be seen in the time-lapse movies.
Whereas control cells keep progressing through the cell cycle upon exposure to E2...
cells with knockdown of PGR or TFF1 tend to reside for a longer period in the G1 phase.
Interestingly, cells with knockdown of GREB1 tend to accumulate in the G1-S transition phase.
This is also reflected in the graph...
where we show the fraction of cells in each cell cycle phase at the end of our experiment.
This clearly indicates that these genes have a different role...
in cell cycle progression and proliferation.
Currently, we're further looking into this role of the genes...
by using whole transcriptome next generation sequencing.
As I hopefully demonstrated, we've established a human cell-based reporter platform...
which can provide us more insight in pro-proliferative ERa signaling...
by covering different key events in the pathway.
But can this reporter platform also detect other estrogenic compounds...
besides our model compound E2?
And is it able to discriminate between non-estrogenic and estrogenic compounds?
For this purpose, we used a reference set as defined by the OECD test guideline.
This list of reference compounds...
is composed of a wide variety of non-estrogenic and estrogenic compounds...
ranging from very potent pharmaceuticals to very weak industrial phenolics.
We've exposed our four reporters...
and collected concentration response curves at different time points for each reporter.
In this way, we monitor the different key events of the pathway.
Namely, target activation by monitoring GFP intensity...
FUCCI fractions reflecting cell cycle progression...
and additionally, we determined the total amount of cells to reflect proliferation.
Based on the generated concentration response curves...
we determined the potency of each compound reflected by EC50 value.
When we incorporate these EC50 values...
of each compound for each reporter at a different time point in one single heat map...
we are clearly able to discriminate non-estrogenic from estrogenic compounds...
and we can cluster the estrogenic compounds based on their potency in different clusters.
Finally, we also compared the performance of our reporters...
to other OECD-validated assays.
Based on the EC50 values, our reporters are equally sensitive as other assays.
To summarize, our human cell-based reporter platform...
can monitor ERa pathway activation on a single cell level...
and by additionally monitoring cell cycle progression and proliferation...
we can link activation of the pathway to a potential adverse outcome.
By making use of endogenous regulation, this better reflects the human situation.
Besides that, it can identify various estrogenic compounds with different potencies...
with a similar sensitivity as other OECD-validated assays.
All in all, it represents a weight-of-evidence approach...
which provides a better understanding of ERa pathway activation in the context of proliferation.
In turn, this will yield in improved risk assessment for human health.
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