My STEM project done at Umass Worcester on BRCA2 mutant ovarian cancer.

1. THE INFLUENCE OF NURD1
EXPRESSION ON BRCA2
MUTANT OVARIAN CANCER
Michelle Heeney
Mass Academy of Math and Science at WPI

2. Introduction
• Cancer encompasses many
diseases which are classified by
being caused by a genetic
mutation.
• The most frequent cause of death
by gynecologic cancer in the US is
ovarian cancer. It is estimated that
throughout the world two hundred
thousand plus women will develop
ovarian cancer annually, and half
of those women will die.
American Cancer Society Facts and Figures, 2013.

3. Introduction Cont.
• Inheriting mutations in the BRCA genes (BRCA1 or
BRCA2) predispose women to breast and/or ovarian
cancer.
• BRCA proteins have an essential role in repairing
damaged DNA.
• The Majority of chemotherapeutic agents act by
causing DNA damage.
• Thus, BRCA mutant ovarian tumors are initially sensitive
to chemotherapy due to their reduced DNA repair
capacity.

4. Introduction:
• DNA damage, which can be Cisplatin, causes signaling to ATM/ATR rectors.
• When the BRCA1 (blue) or the BRCA2 (Green) pathways are blocked the cells are
BRCA1/2 deficient and affected by chemotherapy.
• It has been discovered that BRCA1 mutant cells are resistant by P53 (Green) depletion,
by there is no gene that is linked to BRCA2 mutant cells in a similar way.
DNA Damage
Stalled replication forks
Inter/Intrastrand Crosslinks (Cisplatin)
Double strand breaks
RAD51
BRCA1
BRCA2
Sensors
ATM/ATR
DNA End
Processing
Homologous
Recombination
RAD51
RAD51
P53
(Guillemette, 2013).

5. Introduction Cont.
• However, in the clinic
even BRCA mutant
tumors become resistant
to chemotherapy.
• One Mechanism of
resistance is genetic
reversion.
• In platinum resistant BRCA2 deficient ovarian tumors
only 13/46 had reversion mutations (Norquist, 2011).

6. RESEARCHABLE QUESTION:
How do BRCA2 mutant ovarian tumors
become resistant to chemotherapy
independently of genetic reversion?

7. Preliminary Data:
• Knowledge in
PEO1 (parental)
and C4-2
(revertant) cells.
• This is will used as
a basis for
experimentation.

8. shRNA screen to identify genes that regulate
Cisplatin sensitivity in BRCA2 deficient cells
~116 targets
Lentiviral infection of shRNA pools
(PEO1 cells)
Puromycin selection
Cisplatin treatment
(~99% death with Non-Silencing Control)
Expand, isolate genomic DNA, PCR amplify and
sequence to identify candidates
Validation of Cisplatin resistance
(PEO1 and FA-D1 cells)
24 (h)
1 week

10. • Western Blotting. Dose dependent Cisplatin graphs. NURD1 mRNA levels
(Guillemette, 2013).

11. Preliminary Data (Cont.):
• NURD1 expression does not
significantly associate with
PFS (Progression Free
Survival) and OS (Overall
survival) in all ovarian
tumors.
• NURD1 expression
significantly associates with
improved PFS and OS in
specifically BRCA2-deficient
ovarian tumors.

12. Do cisplatin resistant BRCA2
mutant cancer cells lose
NURD1 protein expression de
novo (on their own)?

13. HYPOTHESIS:
It is hypothesized that NURD1
expression predicts response to
chemotherapy specifically in BRCA2
mutant ovarian cancers.

14. Methodology:
• Cisplatin sensitive PEO1 (human ovarian cancer) cells will be
derived using cycles of cisplatin.
• Resistant clones will be expanded, collected, and lysed to assay for
protein expression by SDS-PAGE.
• The proteins will be transferred onto a nitrocellulose membrane, and
Western blotted with antibodies raised against BRCA2, NURD1, and
MCM7 (loading control).
• Protein expression by chemiluminesence is detected on
radiographic paper by incubating Western blots in secondary
antibodies raised in mouse and/or rabbit that are conjugated to
horseradish peroxidase. With this NURD1 protein expression levels will
be viewed (and if NURD1 is reduced in cisplatin resistant clones).

15. Methods:
• An image of my
methodology to
better understand
how data was
derived.

16. Methodology:
• SDS-PAGE gel loading procedure (Monk, 2013).

17. Results:
A Western blot is a
comparative test for
protein concentration
(this Western blot is for
nuclear protein
concentration).
C4-2 cells are revertant
cells.
~One fourth of Cisplatin
resistant mutant cells
develop resistance by
genetic reversion
(revertant form of BRCA2
in the C4-2 cells).
PEO1 cells are the
parental cells (Cisplatin
resistant clones were
derived from them).
PEO1 cells are still
cancerous, but the cells
have not been exposed
to Cisplatin therapy
NURD1
BRCA2
MCM7
P53
*Potential BRCA2 Revertant Clone
Revertant
Truncated

18. Analysis:
• In C4-2 cells full length BRCA2 is detected migrating lower on the Western blot.
• In C4-2 cells the truncated (mutant) form of BRCA2 is retained.
• In clones CR18, CR19, and CR21 BRCA2 may have undergone genetic reversion confirming
previous findings.
• Western blotting for NURD1 supports the hypothesis.
• Compared to the cisplatin sensitive PEO1 cells NURD1 protein expression is reduced in
Cisplatin resistant clones.
• CR 19, CR22, CR23, CR24 <<< CR10, CR12, CR17, CR22 << CR2, CR14, CR21, < PEO1
• In the resistant clone CR18, which is a potential revertant NURD1 expression remains high.
• Western Blotting for loading control MCM7 reveled fairly equal amounts of protein was run
for each sample.
• Western blotting for the tumor suppressor, P53, was included in analysis because it is
commonly lost in ovarian tumors, especially resistant tumors.
• Total P53 expression was not lost in resistant clones.

19. Conclusion:
• Based on potential Revertant BRCA2 clones roughly 3/12 CR
clones reverted reflecting similar ratios to what was found in
clinic.
• To confirm that CR18, CR19, and CR21 are BRCA2 genetic
revertants we could perform a functional assay to measure
restored homologous recombination (RAD51 focal
accumulation).
• In non-revertant CR clones NURD1 protein expression was
reduced compared to Cisplatin sensitive PE01.
• NURD1 protein expression could serve as a biomarker to predict
whether a BRCA2 mutant cancer cell line will respond to cisplatin
therapy.

20. Extensions:
• Perform Immunofluorescence experiments on Clones CR18, CR19, and CR21
looking at RAD51 foci formation as a functional assay to see if homologous
recombination is restored. C4-2 would serve as positive control and PEO1 cells
would be a negative control.
• Perform densitometry on the Western blot to quantify relation to protein levels.
• The lab has a GFP-NURD1 expression construct that could be used to
ectopically express NURD1 to see if NURD1 re-expression could re-establish
sensitivity to cisplatin in CR clones.
• BRCA2 mutant cells that lose NURD1 protein expression are resistant to
cisplatin. Is there a therapeutic alternative? This can be tested by employing a
small molecule screen using already FDA approved drugs to find molecules
that make these cells sensitive.

21. Appendix:
Western Blot
• This Blot was done
before the major
results and appears to
go against the
literature because all
Cisplatin Resistant (CR)
clones have yet to
revert.
• The same cells were
used in the data on
the earlier slide, this
blot just provides
insight on the timeline
of reversion and when
data collected can
be considered
significant.

22. ACKNOWLEDGEMENTS:
The author wishes to thank her parents for their ongoing support, from providing moral
support to laboratory transportation. The author would also like to thank Shawna
Guillemette and Dr. Sharon Cantor for providing constant aid in experimental design
ideas, laboratory space, material provisions, as well as providing an answer to any
questions asked. Mrs. Maria Borowski was also a phenomenal help over the course of this
experiment, she provided constant support. Without these people this experiment would
not have been able to be conducted.

23. Resources:
• Lab space provided by Doctor Sharon Cantor at Umass Worcester
• Advising, lab training, and preliminary data (including the preliminary data diagrams
seen in this presentation) from Shawna Guillemette
• Advising from Mrs. Maria Borowski
• For labs and studies used please see my full research paper