Genetic interactors of BRCA2 in BRCA2 deficient

tumorigenesis Shrenik Jain

2

Breast Cancer as a whole

Breast Cancer accounts for 22.9% of all cancers diagnosed worldwide1

Generally affects women more than men in a 100:1 ratio2

It is estimated that  232,340 women will be diagnosed with and 39,620 women will die of cancer of the breast in 20132

Breast Cancer can be caused by a variety of environmental and hereditary factors

Statistics from:1. "World Cancer Report". International

Agency for Research on Cancer. 2008. 2. “Breast Cancer Statistics”. Susan G. Komen

for the cure. 2013.

3

Hereditary Breast Cancer

Approximately 5-10% of all Breast Cancer cases are believed to have a genetic basis3

Mutations in the BRCA gene complex have been identified as a leading cause of hereditary breast cancer

Inherited BRCA gene mutations are responsible for about 5 percent of total breast cancers4

The BRCA gene complex includes tumor suppressor genes BRCA1 and BRCA2

Statistics from:3. “Hereditary Cancer Syndromes”. The University of Texas MD Anderson Cancer Center.4. “BRCA Gene test for breast cancer”. The Mayo Clinic.

4

BRCA2

BRCA2 is a gene in the BRCA complex that acts as a tumor suppressor by performing an essential role in DNA repair

Located on the q arm of the Chromosome 13 in humans

 The human BRCA2 gene contains 27 exons that code for a protein of 3418 amino acids

Experiments have established mice as a suitable model system for the study of BRCA2, despite significant amino acid differences between murine and human genes

BRCA2 has been experimentally verified as essential to the viability of mice ES cells

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The BRCA Paradox

A loss of both copies of BRCA2 leads to cell death

A loss of both copies of BRCA2 leads to uncontrollable cell division

ES cells Mature Tissue Cells

BRCA2BRCA2BRCA2

BRCA2BRCA2BRCA2

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MSCV Retroviral Screening for BRCA2 interactors

MSCV (Murine Stem Cell Virus) was used as a retroviral vector to induce the expression of possible BRCA2 interactors

The vector had two essential qualities:

1. The viral genome was modified to prevent the standard replication of the viral DNA and subsequent lysing of the cell

2. The MSCV LTR acts as an enhancer- increasing or inducing transcription of the gene adjacent to it

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Conditional allele

Knocked-out

HP RT1

HATR; Not viable

Cre Recombinase

Brca2 conditional ES cell (F7) Brca2 deficient ES cell

MSCVretrovirus

HP RT1

Gene X

OR

OR

Brca2 deficient mutant ES cell

Brca2

Brca2

Cre Recombinase

HPRT1

Brca2

HPRT1

Brca2

Brca2

MSCV

MSCV Gene XMSCV

Brca2

MSCV Gene X

Gene X

Screening for genetic interactors in Brca2 Deficient tumorgenesis

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Table 1. Insertion sites and nearby genes of the MSCV-rescued clones

Clonec Chromosome Candidate Gene Comments

1 11qE2 SocS3 Suppressor of cytokine signaling family

3 5qB1 BRE Antiapoptotic, BRCA1/2 interacting, TNFa

4 10qC1 GIPC3 Growth factor signaling, cell adhesion

5 XqA1.3 Araf Activates MEK1

7 7qF3 NFAT Cip  

8 5qE5 PTPN13 Protein tyrosine phosphatase,

9 17qb1 DAXX Enhances fas mediated apoptosis

10 2QE5 ZFP106  

12 6qF3 FKbp4  

22a 8qA1.1 Fbox-25  

22b 12qA1.1 NCoA1 Steroid receptor coactivator-3

25 7qD2 ZFP710  

27 2qH3 NCoA3  

30 7qD2 ZFP710  

Figure provided by Dr. Kajal Biswas

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BRE(Brain and Reproductive Organ Expressed)

I focused my project on one identified interactor- BRE (brain and reproductive organ expressed)

Also known as BRCC4, BRCC45

BRE was found to rescue the lethality of BRCA2 deficient cells

BRE is located on chromosome 2 in humans and chromosome 5 in mice

There are 11 BRE splice variants the largest of which is 1.852 kb in length5

5. “Gene: BRE ENSG00000158019”. Ensemble.org. 2013.

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F7 Clone-3

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

Leve

l o

f B

RE

mR

NA

pre

sen

t

F7 Clone 3d

2.5

2.0

1.5

1.0

0.5

Increased expression of BRE in the rescued clone 3d

Figure provided by Dr. Kajal Biswas

11

Brca cko/++Cre

HAT

Brca cko/ko +BRE+Cre

M15

BRE overexpression partially rescues Brca2 deficient ES cells

Figure provided by Dr. Kajal Biswas

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Generation of BRE overexpressed Transgenic line A transgenic line of BRE overexpressed mice is being

generated

An inducible transgenic construct was prepared for generation of said line

Construct DNA was injected into mice embryos

Theoretically, BRE overexpressed mice should be able to rescue the lethality of BRCA2 deficient mice

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BRE transgenic Construct

Actin Promot

erLac Z Neo

HA-BRE

loxP loxP

Neo Fwd

Bre Rev

Actin Promot

er

HA-BRE

loxP

Cre

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Generation of transgenic mice

Superovulated Female

Gene of interest

Injection of Gene into Fertilized Eggs

Insertion of egg into Psuedopregnant female

Birth of Transgenic Mice

Genotyping

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Genotyping

The inducible BRE transgenic construct was microinjected into pronuclei of mice of mixed genetic background.

Three weeks after the pups were born, a small portion of tail tips (~0.5 inches) was clipped.

DNA was isolated from the tails and analyzed through polymerase chain reaction (PCR) using the primers Neo-Fwd and Bre-Rev to identify transgenic pups.

The PCR products were then separated on 1% agarose gel.

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Genotyping Results

+ve

-v

e

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Future Crosses for Transgenic Mice Line Tg X

BRCA2 KO/+

• Generate KO/+;Tg mice

KO/+;Tg X

KO/+;Tg

• Cross heterozygotes

KO/KO;Tg

• Rescue lethality of BRCA2 null mice with trans gene

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BRE rescued ES cells show higher Cdc25A level after IR

C 0.5 1 2 C 0.5 1 2 Time After IR (Hrs)

Cdc25A

Actin

Brca2cko/- (F7) Brca2-/-;Bre (3d)

• Cdc25A is an oncogene that induces mitosis in cells, and is normally degraded following DNA damage

• However, BRE overexpressed cells show increased levels of Cdc25a despite exposure to Ionizing radiation

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Cdc25a

• Cdc25a (Cell division cycle 25 homologue a) is a protein that regulates the cell at the G1 checkpoint, preceding the S stage

• In healthy cells Cdc25a levels rise and activate CDK2, leading to the continuation of the cell cycle

• However when DNA damage is present Cdc25a is degraded via ubiquitination leading to cell cycle arrest

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BRE up-regulates Cdc25A transcriptionally after IR

F7 F7 IR F7-BRE F7-BRE IR0

0.5

1

1.5

2

2.5

3

3.5

4

Cdc25A RNA level

Cells

Rela

tive m

RN

A level

Figure provided by Dr. Kajal Biswas

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BRE interacts with transcription factor ATF3 to induce transcription of Cdc25a

Bre was found to interact with Cyclic AMP-dependent transcription factor ATF-3 via Co-Immunoprecipitation

ATF3 has multiple known binding sites on the Cdc25a promoter

An experiment was set up to identify the effect of these ATF3 sites on Cdc25a transcription

Cdc25a promoter

Cdc25a promoter

Cdc25a promoter

Cdc25a promoter

Cdc25a promoter

Cdc25a promoter

Luciferase

Luciferase

Luciferase

Luciferase

Luciferase

Luciferase22

Reporter assay using different ATF3 binding site deletion of Cdc25a promoter

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Initial PCR Plan

Cdc25a promoter fragment

Fwd x Cdc Rev

1. Amplify desired Cdc25a promoter fragment via PCR

2.Ligate into luciferase containing plasmid, amplify in bacteria

Amp

Luciferase

3. Cut out Cdc promoter/luciferase, insert into mammalian cell line

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However…

• Initial PCRs were unsuccessful or did not yield sufficient product for ligation

• Electroporation of competent cells failed• The experiment was revised to amplify the Cdc25a

promoter fragment separately, prior to ligation with luciferase

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New PCR Plan

Cdc25a promoter fragment

Fwd x Cdc Rev

1. Amplify desired Cdc25a promoter fragment via PCR

Amp

2. Ligate into Topo-Vector

3. Transduce Plasmid into bacteria, plate bacteria and amplify plasmid

4. Pick colonies and perform colony PCR to identify positives

5. Isolate plasmid, cut out Cdc promoter fragment

Amp Amp

Luciferase

6. Ligate Cdc promoter fragment into luciferase plasmid, finish original plan

A

A

T T

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Results of Colony PCR for FWD6 and FWD8

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Eventual Goals

Place Cdc25a promoter/luciferase in human cell lines and analyze the relationship between number of ATF3 binging sites on promoter to Luciferase expression

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Acknowledgements

1."World Cancer Report". International Agency for Research on Cancer. 2008.

2.“Breast Cancer Statistics”. Susan G. Komen for the cure. 2013

3.“Hereditary Cancer Syndromes”. The University of Texas MD Anderson Cancer Center.

4.“BRCA Gene test for breast cancer”. The Mayo Clinic.

5. “Gene: BRE ENSG00000158019”. Ensemble.org. 2013

Several figures provided by Dr. Kajal Biswas

Research done in the Genetics of Cancer Susceptibility Section, headed by Dr. Shyam Sharan, Mouse Cancer Genetics Program, Frederick National Laboratory

Thank you all for a great summer!