THE INFLUENCE OF NURD1 EXPRESSION ON BRCA2 MUTANT OVARIAN

CANCERMichelle Heeney

Mass Academy of Math and Science at WPI

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.

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.

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 DamageStalled replication forks

Inter/Intrastrand Crosslinks (Cisplatin)Double strand breaks

RAD51

BRCA1

BRCA2

SensorsATM/ATR

DNA End Processing

HomologousRecombination

RAD51

RAD51

P53

(Guillemette, 2013).

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).

RESEARCHABLE QUESTION:

How do BRCA2 mutant ovarian tumors become resistant to chemotherapy independently of genetic reversion?

Preliminary Data:• Knowledge in

PEO1 (parental) and C4-2 (revertant) cells.

• This is will used as a basis for experimentation.

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

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

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.

Do cisplatin resistant BRCA2 mutant cancer cells lose

NURD1 protein expression de novo (on their own)?

HYPOTHESIS:It is hypothesized that NURD1

expression predicts response to chemotherapy specifically in BRCA2

mutant ovarian cancers.

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).

Methods:

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

Methodology:

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

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

C4-2

(Rev

erta

nt)

PEO1 (P

aren

tal)

CR2CR10

NURD1

BRCA2

MCM7

P53

CR12

CR16

CR17

CR24CR18

*

CR19*

CR20

CR21*

CR22

CR23

*Potential BRCA2 Revertant Clone

Revertant Truncated

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.

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.

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.

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.

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.

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