Genetic Analysis of Gata3 and Runx1 in Mammary Tumor Formation

by

Nayasta Ademmana Kusdaya

A thesis submitted in conformity with the requirements for the degree of Master of Science

Molecular Genetics University of Toronto

© Copyright by Nayasta Ademmana Kusdaya 2015

ii

Genetic Analysis of Runx1 and Gata3 in Mammary Tumor

Formation

Nayasta Ademmana Kusdaya

Master of Science

Molecular Genetics

University of Toronto

2015

Abstract

Genomic analysis of breast cancer (BC) has revealed the existence of recurrent copy number

losses and mutations in GATA3 and RUNX1. Interestingly, these are predominantly found in

luminal tumors. GATA3 and RUNX1 are transcription factors that help specify mammary

luminal lineages. To test whether BC-associated genetic aberrations of GATA3 and RUNX1

contribute to luminal-like tumorigenesis, I generated cohorts of female mice in which Gata3 and

Runx1 were deleted in mammary epithelium. In addition, I have successfully generated

transgenic mice that express a breast tumor-derived Gata3 mutant. To date, twelve-months old

females with copy number loss of Gata3 and Runx1 are mostly tumor-free (approximately 3%

and 13% of Gata3 and Runx1 conditional heterozygous have developed mammary tumours,

respectively).

iii

Acknowledgments

I sincerely thank my supervisor Dr. Sean E. Egan for the wonderful project, mentorship, advice,

encouragement, writing consultations, and continuous support for my future endeavors. I thank

my supervisory committee members Dr. Barbara Funnell and Dr. Sevan Hopyan for continuous

advice and support on my thesis project. I thank Dr. Eldad Zacksenhaus and his lab members for

advice during lab meetings. I thank Dr. Zhaolei Zhang as the chair of my exam committee. I

thank the following for data contributions: Dr. Maxime Bouchard (McGill University) for

Gata3fl mouse, Dr. Nancy Speck (University of Pennsylvania) for Runx1

fl mouse, Dr. Jessica

Adams (Egan lab) for gene targeting mouse embryonic stem cells and training in the generation

of transgenic mouse and, Jodi Garner and Myra Sohail (SickKids stem cell facility) for stem cell

training and management, Dr. Marina Gertsenstein and Monica Pereira (TCP transgenic core) for

generation of chimeric mice and sperm recovery, Gillian Sleep (TCP Canadian Mouse Mutant

Repository) for sperm collection, Dr. Jennifer Gorman (Woodgett lab) for advice on sick

germline pups, Nathan Schachter (Egan lab) for donating older female mice to Gata3fl and

Runx1fl tumor cohorts, and finally Amanda Loch (Egan lab), Leanne Studley, and Gessica

Raponi for mouse health monitoring. I am grateful to the Hospital of Sick Children Restracomp

and the Canadian Breast Cancer Research Foundation for funding my graduate work.

I thank all of the Egan lab members (past and present): Dr. Jessica Adams for being the best lab

mentor and sharing all resources (too many acknowledgements to list), Nathan Schachter for

mentoring on mouse tumor cohorts and lab techniques, Kelvin Wang for training in cell lineage

tracing techniques and injection help, Nandini Raghuram for responding to my questions and

requests (too many to list), Dr. Rita Quintana for advice on luciferase assay and fluorescent

microscopy, Amanda Loch for helping in mouse colony management and timed matings, Alex

Manno for training in tail clipping and intraperitoneal injection, and finally Dr. Katie Wright,

Natalia Ruiz, and Yeji An for general help and support. I thank all of the Egan lab undergraduate

and high school students for their help and support, especially Katelyn Kozma, Christine Jo, and

Lauren Beck. I thank God, my family (especially Kusdaya Sukada, Gitasanti Kusdaya, and Raka

Andata Kusdaya), as well as members of my community, for their invaluable support. Finally, to

my cousins and best friends, I am truly sorry to have missed your important celebrations and

extremely grateful for your continuous cheer and understanding.

Table of Contents

Acknowledgments .......................................................................................................................... iii

Table of Contents ........................................................................................................................... iv

List of Tables ................................................................................................................................. vi

List of Figures ............................................................................................................................... vii

List of Abbreviations ................................................................................................................... viii

Chapter 1 Introduction .................................................................................................................... 1

1 Introduction ................................................................................................................................ 1

1.1 Mammary epithelium structure and development .............................................................. 1

1.2 Molecular subtypes of human breast cancer and associated genetic alterations ................ 2

1.3 GATA3 ............................................................................................................................... 3

1.3.1 GATA3 protein in transcription factor complex ..................................................... 3

1.3.2 GATA3 in development .......................................................................................... 4

1.3.3 GATA3 in cancer .................................................................................................... 5

1.4 RUNX1 ............................................................................................................................... 7

1.4.1 RUNX1 protein complex in transcription ............................................................... 7

1.4.2 RUNX1 in development ......................................................................................... 9

1.4.3 RUNX1 in cancer .................................................................................................. 11

1.5 Hypotheses ........................................................................................................................ 13

1.6 Objectives ......................................................................................................................... 14

Chapter 2 Materials and Methods ................................................................................................. 19

2 Materials and Methods ............................................................................................................. 19

2.1 Mouse strains and colony maintenance ............................................................................ 19

2.2 Gene targeting and generation of Rosa26Gata3N331fs

transgenic mice ................................ 21

2.3 Drugs ................................................................................................................................. 22

v

2.4 Tumor monitoring, collection, and analysis ..................................................................... 22

2.5 Timed pregnancy, mammary gland collection and immunofluorescence ........................ 23

2.6 In vitro transfection of bi-cistronic cassettes and Western blot ........................................ 25

Chapter 3 Modeling breast tumor-derived GATA3 gene alterations in mouse mammary

epithelium ................................................................................................................................. 26

3 Modeling breast tumor-derived GATA3 gene alterations in mouse mammary epithelium ...... 26

3.1 Modeling GATA3 hemizygosity in mouse mammary epithelium ..................................... 26

3.2 Generation of Cre-conditional Gata3N331fs

transgenic mice .............................................. 27

3.3 Modeling the combination of GATA3N331fs

and copy number loss of GATA3wt

............... 32

Chapter 4 Testing for effects of Runx1 gene alterations on mouse mammary tumorigenesis ...... 42

4 Testing for effects of Runx1 gene alterations on mouse mammary tumorigenesis .................. 42

4.1 Testing for tumor suppressor activity of Runx1 in mouse mammary epithelium ............. 42

4.2 Generation of a construct for transgenic expression of Runx1G141R

: a Runt domain

mutant. .............................................................................................................................. 43

Chapter 5 Discussion and future directions .................................................................................. 49

5 Discussion and future directions .............................................................................................. 49

5.1 Deletion and mutation of Gata3 in mouse mammary epithelium ..................................... 49

5.2 Transactivation ability of Gata3N331fs

................................................................................ 51

5.3 Deletion and mutation of Runx1 in mouse mammary epithelium .................................... 52

5.4 Transactivation ability of Runx1G141R

............................................................................... 53

Bibliography ................................................................................................................................. 57

vi

List of Tables

Table 1. Current report on mammary tumor incidence for Gata3flcohorts. ................................. 33

Table 2. Current report of mammary tumor assay for Rosa26Gata3N331fs

cohorts. .......................... 41

Table 3. Current report of mammary tumor incidence for Runx1fl

cohorts. ................................. 46

Table 4. Cohorts to test for cooperation between Gata3fl

and Pik3caH1047R

or TP53R270H

. .......... 54

Table 5. Cohorts to test for cooperation between Gata3N331fs

and Pik3caH1047R

or TP53R270H

. .... 55

Table 6. Cohorts to test for cooperation between Runx1fl

and Pik3caH1047R

or TP53R270H

. .......... 56

vii

List of Figures

Figure 1. Relative expression levels and roles for GATA3 and RUNX1 in the mammary luminal

cell lineage. ................................................................................................................................... 16

Figure 2 (A-B). Genetic alterations of GATA3 and RUNX1 in breast tumors. ............................ 17

Figure 3 (A-C). Generation of Gata3N331fs targeting construct. ............................................... 34

Figure 4 (A-C). Targeting Gata3N331fs to Rosa26 locus of mouse embryonic stem cells. ....... 37

Figure 5 (A-D). Characterization of Luciferase activity in Rosa26Gata3N331fs

mice. ..................... 39

Figure 6 (A-C). Assembly of Runx1G141R

targeting construct. .................................................... 47

viii

List of Abbreviations

Abbreviation Example Words Full Form

AML AML1 Acute myeloid leukemia

AMP - Amplification of gene copy number

AP-1 - Activator protein 1

ApcMin

- Mouse strain

ATP - Adenosine triphosphate

BC - Breast cancer

BLK - B lymphocyte kinase

BLG BLG-Cre β-Lactoglobulin

Bp - Base pairs

C/EBP C/EBPα CCAAT-enhancer-binding protein

CBF CBF, CBFβ Core binding factor

CCDS CCDS7083.1,

CCDS15674.1,

CCDS42922.1,

CCDS49916.1 Collaborative consensus coding sequence

CCND1 - Cyclin-D1

CD CD24, CD29, CD36,

CD49f, CD53, CD61

Cluster of differentiation

CDH1 - Cadherin-1; the gene that codes for E-cadherin

CDH2 - Cadherin-2; the gene that codes for N-cadherin

cDNA - Complementary deoxyribonucleic acid

CAN - Copy number alteration

Cre - Cre recombinase

CSF1R - The gene that codes for macrophage colony-

stimulating factor 1 receptor (M-CSFR)

Cys Cys-(X)2-Cys-(X)17-

Cys-(X)2-Cys

Cysteine

D336fs GATA3D336fs

An MCF-7-cell-derived frameshift mutation of

GATA3, where residue 336 (aspartic acid) is

changed by out-of-frame amino acids

DAPI - 4',6-diamidino-2-phenylindole

DLC1 - Deleted in Liver Cancer 1

DMEM - Eagle's minimal essential medium

DNA - Deoxyribonucleic acid

E16 - Embyonic day 16

Ear-2 - V-erbA-related protein 2

Ecad - E-cadherin; epithelial cadherin

ECL - Enhanced chemiluminescence

ix

Elf5 - E74-like factor 5 (Ets domain transcription

factor)

EMT - Epithelial-to-mesenchymal transition

EpCAM - Epithelial cell adhesion molecule

ER ERα, K8-CreER Estrogen receptor

ERK - Extracellular signal-regulated kinases

Ets-1 - The gene that codes for ETS DNA-binding

transcription factor 1

ETV6 - The gene that codes for ets variant 6

FBS - Fetal bovine serum

Fl Gata3fl, Runx1

fl Flox; flanked by loxP sites

FLuc - Firefly Luciferase

FN1 - The gene that codes for fibronectin 1

FOG FOG1, FOG2 Friends of GATA

FOX FOXA, FOXC Forkhead box transcription factor

FP - Forward primer

FPD FPD/AML Familial platelet disorder

G141R RUNX1G141R

A breast-tumor derived point mutation of

RUNX1, where residue 141 is changed from

glycine to arginine

G418 - Geneticin

GATA GATA1-6 GATA binding protein

Gcm - Glial cells missing

GFP - Green fluorescent protein

H&E - Hematoxylin and eosin

H1047R Pik3caH1047R

A breast-tumor derived mutation of PIK3CA,

where residue 1047 is changed from histidine to

arginine

HDAC HDAC1/2 Histone deacetylases

HER HER2 Human epidermal growth factor receptor

HETLOSS - Heterozygous copy number loss

HFSC - Hair follicle stem cell

HIF HIF-1α Hypoxia-inducible factor

HIP2K - Homeodomain-interacting protein kinase-2

HOMDEL - Homozygous copy number loss/deletion

HPV - Human papillomavirus

HSC - Hematopoietic stem cell

ID ID1, ID2 Inhibitory domain

IF - Immunofluorescence

Ig IgA1 Immunoglobulin

IL IL3, IL4, IL6 Interleukin

Indel - Nucleotide(s) insertion or deletion

x

Jak Jak-Stat Janus kinase

K K5, K6, K8, K14, K18 Keratin

Kb - Kilo base pair

LEF-1 - Lymphoid enhancer-binding factor 1

LM2 - A subline of human breast cancer cell line MDA-

MB-231 that is metastatic to lung after

transplantation to nude mice.

LY6E - Lymphocyte antigen 6E

M-CSFR - Macrophage colony stimulating factor 1 receptor

MAP2K4 - Mitogen-activated protein kinase kinase

MAP3K1 - Mitogen-activated protein kinase kinase kinase 1

MaSC - Mammary stem cell

MCF-7 - Michigan cancer foundation-7 cell line

MDA-MB-

231

- Human breast cancer cell line that was isolated

from pleural effusions

MDS MDS1 Myelodysplastic syndrome

MEF - The gene that codes for Ets-related transcription

factor 4 (Elf4)

mESC - Mouse embryonic stem cell

MGT - Mammary gland tumor

MLL MLL3 Mixed-lineage leukemia

MMTV MMTV-Cre, MMTV-

PyMT

Mouse mammary tumor virus

MOZ - MYST3; MYST histone acetyltransferase

(monocytic leukemia) 3

MTA3 NuRD(MTA3) Metastasis-associated 1 family, member 3

MTB MMTV-PyMT; MTB;

Gata3+/Tg

MMTV-rtTA mice

MTF - Metal-regulatory transcription factor

MTG MTG8, MTG16 Myeloid translocation gene

MUT - Mutation

N331fs GATA3N331fs

A breast-tumor derived mutation of GATA3,

where residue 331 is changed from asparagine to

lysine, followed by ten out-of-frame amino acids

Neu MMTV-Neu Receptor tyrosine –protein kinase erbB-2 or

human epidermal growth factor receptor 2

(HER2)

NFAT - The nuclear factor of activated T cells

transcription factor

NLS - Nuclear localization signal

NuRD NuRD(MTA3) Nucleosome remodeling deacetylase

P18INK4c - The gene that codes for (INK4) cyclin-dependent

xi

kinase inhibitor 2C (CDKN2C)

p21Cip1

- Cyclin-dependent kinase inhibitor 1; CDKN1A

p27Kip1

- Cyclin-dependent kinase inhibitor 1B; CDKN1B

PAX PAX5 Paired box

PBS - Phosphate buffered saline

PCR - Polymerase chain reaction

PDA - Poorly differentiated adenocarcinoma

PI3K - Phosphoinositide 3-kinase

PIK3CA PIK3CAH1047R

Phosphatidulinositol-4,5-bisphosphate 3-kinase

catalytic subunit alpha; the gene that codes for

p110α

PR - Progesterone receptor

Prl - Prolactin

PU.1 - A transcription factor that binds to purine-rich

DNA sequence 5’-GAGGAA-3’ (PU-box)

PyMT MMTV-PyMT Polyoma virus-middle T

qPCR - Quantitative polymerase chain reaction

R270H TP53R270H

A breast-tumor derived mutation of TP53, where

residue 270 is changed from arginine to histidine

RARRES3 - Retinoic acid receptor responder protein 3

RB1 - Retinoblastoma protein 1

RBPJĸ - Recombination signal-binding protein 1 for J-

Kappa

RNA - Ribonucleic acid

ROSA26 - Reverse orientation splice acceptor 26

RP - Reverse primer

RT-PCR - Reverse transcriptase polymerase chain reaction

RUNX RUNX1-3 Runt-related transcription factor

Sc sc-9009, sc-2060 Santa cruz

SDS-PAGE - Sodium dodecyl sulfate polyacrylamide gel

electrophoresis

Smad Smad3 A homolog of Drosophila protein mothers against

decapentaplegic (MAD) and Caenorhabditis

elegans protein SMA

SOC SOC3/4 Suppressor of cytokine signaling

Stat Stat5 Signal transducer and activator of transcription

SUV39H1 - Suppressor of variegation3-9 homolog 1

T-ALL - T cell acute lymphoblastic leukemia

T47D - A breast cancer-derived cell line isolated from

pleural effusion

TA TA1, TA2 Transactivation domain

TCGAN - The cancer genome atlas network

xii

TCP - The Toronto Centre for Phenogenomics

TCR TCRδE4 T-cell receptor

TG R26TG

loxP-(membrane-targeted tandem-dimer-

Tomato)-STOP-loxP-(membrane-targeted

enhanced green fluorescent protein)

TGF TGFβ Transforming growth factor

Th Th1, Th2 T helper cell

TLE-1 - Transducin-like enhancer protein 1

TP53 - The gene that codes for tumor protein p53

TrkA - Tropomyosin receptor kinase A

UTR 5’UTR, 3’UTR Untranslated region

WAP WAP-Cre Whey acidic protein

Wt Gata3wt

, Runx1wt

Wild type

ZnF ZnF1, ZnF2, ZNF703 Zinc finger

1

Chapter 1 Introduction

1 Introduction

1.1 Mammary epithelium structure and development

The mammary gland is made up of epithelial stromal elements. The stroma or mammary fat pad

is comprised of adipocytes, fibroblasts, blood vessels, macrophages, and other hematopoietic

cells 1-3

. Mammary epithelium is organized into a tree-like structure connected through a central

lumen to the nipple. During embryogenesis, mammary placodes form and each subsequently

sprouts to generate a small ductal tree. Growth of the ductal tree pauses, resuming at puberty.2-4

With the onset of puberty, in mice, at approximately three weeks of age, mammary ducts

elongate and branch in response to estrogen-dependent signaling. When estrous cycle starts,

secondary and tertiary branches as well as rudimentary alveolar structures are formed in response

to elevated progesterone.2-8

During pregnancy, progesterone and prolactin cause milk-secreting

alveoli to grow at ductal tips.3,7-9

Following parturition and weaning, the mammary gland returns

to pubertal-like state through apoptosis and stromal re-organization. This latter process is called

involution.10

In the mouse, mammary epithelial cells originate from embryonic mammary stem cells (MaSCs)

(which are enriched in CD24+ CD29

hi CD49f

hi–sorted population). MaSCs self-renew and give

rise to progenitors that are capable of differentiating into both layers of the ductal tree: luminal

cells and basal cells. Bipotent progenitors (CD49fhi

EpCAM+ K5/6/14

+ K8/18

– ER

– PR

–) give

rise to luminal-restricted (CD49f+ EpCAM

+ K8/18

+ K5/6/14

+) and basal-restricted

progenitors.11,12

Luminal cells terminally differentiate into ductal cells and alveolar cells (CD24+

CD29lo

CD49lo/-

EpCAM+ K8/18/19

+ ER

+ PR

+ GATA3

+ Ecad

+). Basal cells terminally

differentiate into myoepithelial cells (CD24+ CD29

hi CD49

hi K5/14

+) that form the next outer

layer.1-3,11,12

2

1.2 Molecular subtypes of human breast cancer and associated genetic alterations

Breast tumors are thought to arise through transformation of luminal epithelial cell types within

the mammary gland.13

This disease is heterogeneous and commonly classified into subgroups

that share similar gene signatures.11,14,15

Indeed, breast tumors are commonly divided into five

subtypes: luminal A, luminal B, HER2-enriched, basal-like and claudin-low. Luminal subtypes

comprise the majority of breast cancer cases (65%). Luminal A tumors express a gene signature

that is similar to differentiated luminal cells, expressing high levels of ER. Luminal B differs

from luminal A subtype in that tumor cells are less differentiated, more proliferative, less

responsive to hormone therapy, and are associated with a worse outcome.11,14,15

The HER2-

enriched subtype, which involves amplification and/or overexpression of HER2, has a luminal,

although less differentiated gene signature.11,14,15

Basal-like tumor cells resemble cells in basal

layer that lack expression of ER, PR, and HER2. Interestingly, gene expression in these tumors is

related to that seen in luminal progenitor cells.11,14,15

Finally, claudin-low breast cancers express

an epithelial-to-mesenchymal gene signature related to that seen in mammary stem cells or

bipotent progenitors. Another defining feature of this gene signature, as suggested by the name,

is the low expressions of claudins and other genes associated with differentiation of luminal

cells.11,14,15

Each subtype shows a unique prognosis, characteristic response to therapy, as well as

subtype-specific genetic alterations.11,14-17

Recent genome-wide analysis of breast cancer has revealed the existence of recurrent mutations

in PIK3CA, TP53, GATA3, MAP3K1, MLL3, CDH1, RUNX1, and MAP2K4.17

Interestingly,

mutations in GATA3 and RUNX1 transcription factors are almost exclusively found in luminal

tumors (Figure 2A,B).17-20

Yet, whether these mutants play role in luminal-like mammary

tumorigenesis is unknown. Furthermore, while GATA3 and RUNX1 have been associated with

tumor suppressor and oncogenic roles in other cancer, specific roles for GATA3 and RUNX1

mutations in breast tumorigenesis has yet to be defined.21,22

Thus, by studying breast cancer-

associated genetic alterations of GATA3 and RUNX1, for example, in mouse mammary

epithelium we may begin to define how these two genes promote mammary- and subtype-

specific tumorigenesis.

3

1.3 GATA3

1.3.1 GATA3 protein in transcription factor complex

GATA3 is a member of GATA family of transcription factors, which includes GATA1 through

6. Each GATA-family protein contains a conserved Cys-(X)2-Cys-(X)17-Cys-(X)2-Cys Zinc

Finger motif, where X is any amino acid. GATA4, GATA5, and GATA6 have similar protein

structure and tissue-specific expression pattern. They are expressed in the heart, gut, liver,

smooth muscle, urogenitalia, and gonad. On the other hand, GATA3, which is similar to GATA1

and 2 in protein structure, is expressed in hematopoietic lineage cells. Additionally, GATA3 is

expressed in the parathyroid gland, the ear, kidney, and in the mammary gland.23-25

In humans, the GATA3 genomic locus spans 20 kb of sequence on Chromosome 10. It contains

six exons and codes for a 443 amino acid-long protein.23

The transcriptional start and stop sites

are found in exon2 and 6, respectively.23,26,27

The N terminus of GATA3 comprises two

transactivation domains, TA1 (amino acids 31-74) and TA2 (amino acids 132-214). These are

required for transactivation of the TCRδE4 enhancer in T cells.27

Interestingly, these domains are

not highly conserved among GATA transcription factors24

, suggesting that they interact with one

or more specific co-regulators.

Following the TA domains is a nuclear localization signal sequence (NLS; amino acids 249-311)

and an N-terminal Zinc Finger domain 1 (ZnF1).25,27,28

ZnF1 (amino acids 263-287), which is

encoded by exon 4, interacts with co-activators and co-repressors, including FOG2 (friends of

GATA 2). This subsequently stabilizes the interaction between DNA and C-terminal zinc finger

domain (ZnF2). Interestingly, ZnF1 can bind DNA at alternative GATA sites (i.e., GATC, GATT,

and GATG).23,24,27

ZnF1 and ZnF2 are connected by a loop domain containing a KRRLSA motif

for acetylation and phosphorylation.29

Acetylation of GATA3 has been implicated in directing

tissue localization and survival of T cells, while phosphorylation confers stability on

GATA3.30,31

ZnF2 (amino acids 318 - 341), which is encoded by exon5, binds DNA at canonical GATA

binding sites 5’-(A/T)GATA(A/G)-3’. Interestingly, ZnF2 may also bind transcription co-

regulators, such as NFAT, in muscle and T cells and mediate GATA3-homodimerization.23,24

The C terminus of GATA3 contains two basic motifs near ZnF2, YYKLHNINRPL and

4

QTRNRK. The former is required for DNA binding, while the latter mediates homo- and

heterodimerization of GATA proteins.23,24,29,32,33

GATA3 alone may not be sufficient to induce or repress expression of target genes. Accordingly,

transactivation by GATA3 can be enhanced or modulated through interaction with other

transcriptional regulators. For example, GATA3 interacts with FOG1, FOG2, PU.1, or Smad3 to

regulate target genes in T cells. In breast cancer cell lines, GATA3 forms a complex with

FOXA1 and ER to upregulate target genes.29

1.3.2 GATA3 in development

GATA3 is expressed throughout the developing embryo, including in the brain. At post-

embryonic stages, GATA3 expression is predominantly found in T lymphocytes, endothelial

cells, placenta, kidney, adrenal gland, peripheral and central nervous system, and mammary

gland.34

GATA3 is required for development of the parathyroid gland, neurons, kidney, and skin,

where it regulates cell proliferation and differentiation. In the developing mouse parathyroid

gland, Gata3 regulates expression of glial cells missing 2 (Gcm2), a parathyroid-specific gene,

and ultimately promotes survival and differentiation of parathyroid progenitor cells.

Haploinsufficiency of Gata3 is seen in heterozygous knockout mice (Gata3+/–

), leading to

underdevelopment of the parathyroid gland. Indeed significant increase in mortality is seen when

heterozygous mice are restricted to a low-calcium and low-vitamin D diet.35

Gata3+/–

mice also

exhibited malformed sensorineural hair cells in cochlea, with resulting hearing loss.36

Homozygous Gata3 knockout mice (Gata3-/–

), but not Gata3+/–

mice, showed aberrant renal

development due to deregulation of Ret expression in nephric duct formation.35,37,38

In agreement

to these observations, heterozygous mutations or hemizygosity at the GATA3 locus causes

congenital hypoparathyroidism, sensorineural deafness, and renal abnormalities (HDR)

syndrome in humans.23,27,28,39-43

In the hematopoietic system, GATA3 regulates specification of Th2 cells from naive T cells.

This differentiation event occurs downstream of TCR-dependent PI3K, Jagged-Notch-RBPJĸ,

and IL4-Stat6 signaling pathways.2,24,31

In this context, GATA3 upregulates Th2-differentiation

genes and opposes the function of Tbet, a transcription factor promoting an alternative cell fate,

Th1. In fact, GATA3 knockdown in T cells leads to Th1 specification.4 Overexpression of

5

GATA3 in T cells is associated with aberrant lymphoid activity, including allergy44

, eczema45

,

systemic lupus erythematosus (SLE)46

, and T cell acute lymphoblastic leukemia (T-ALL).47

In embryonic mammary gland, Gata3 is expressed in primordial epithelial tree and required for

placode development. Homozygous deletion of Gata3 in epithelial cells (Gata3fl/fl

; K14-Cre)

resulted in failed mammary placode development and loss of nipples tissue.48

In the adult gland,

Gata3 is exclusively expressed in luminal cells, where it is required to specify and maintain

identify of this lineage.4,29

Accordingly, post natal homozygous deletion of Gata3 in mammary

epithelium (Gata3fl/fl

; MMTV-CreF) led to simultaneous depletion and multiplication of luminal

cell layers in the pubertal duct. Ultimately, these mice exhibited severe reduction of epithelial

tree outgrowth. Gata3+/fl

; MMTV-CreF mice were indistinguishable from wild-type animals.

4

Another study looked at conditional homozygous deletion of Gata3 using distinct mammary-

specific Cre transgenic driver (Gata3fl/fl

; MMTV-CreD. These mice showed a similar reduction of

epithelium outgrowth and decreased number of K18+ ERα

+ ductal cells. Interestingly, Gata3

+/fl;

MMTV-CreD heterozygotes showed a similar reduction in ductal outgrowth as well as expansion

of the CD61+ luminal progenitor compartment.

48 Finally, pregnancy-induced homozygous

deletion of Gata3 in mouse mammary luminal cells (Gata3fl/fl

; WAP-Cre) revealed multiple

layers of luminal cell that eventually detached and underwent cell death. These mice also showed

an expansion of the CD61+ luminal progenitor cell compartment, decreased milk protein

expression, underdeveloped lobulo-alveoli, and failed lactation.4,48

As seen during T cell development, Gata3 acts downstream of Jagged-Notch-RBPJĸ and IL4-

Stat6 signaling pathways in mammary epithelium.2 Gata3 cooperates with IL4-Stat6 and Prl-IL2-

Stat5 signaling to induce IL3/IL4 expression, which subsequently regulates expression of ductal

and alveolar cell-differentiation genes.2 Gata3 is thought to facilitate targeting of other luminal-

cell transcription factors in the mammary gland (e.g., ER and FOXA1) and thereby regulate

expression of milk protein-encoding genes, cell adhesion genes (e.g., CDH1), and cell cycle

regulatory genes (e.g., P18INK4c).2

1.3.3 GATA3 in cancer

Aberrant expression of GATA3 is not only found in T cell lymphoma, but also in a subset of

human cancers, including pancreatic, cervical, lung, neuronal, and breast carcinoma.49-52

Overexpression of GATA3 in some of these tumors is associated with transformation. In

6

pancreatic cancer, for example, overexpression of GATA3 is thought to cooperate with TGFβ

pathway activation to promote tumorigenesis.49

In neuroblastoma, GATA3 is responsible for

upregulation of cyclin D1 (CCND1), which promotes cell proliferation and inhibits

differentiation.52

In lung adenocarcinoma, GATA3 is activated downstream of Jagged2-Notch

signaling and it suppresses microRNA-200-dependent EMT and metastasis in this context.51

In

other tumor types, GATA3 is thought to function as a tumor suppressor. For example, in HPV-

induced cervical cancer, GATA3 levels decrease with tumor progression.50

In breast cancer, GATA3 is thought to function as a suppressor of tumor progression. Low levels

of GATA3 are seen in HER2-enriched and basal-like breast cancer, which are known to be

aggressive and metastatic.53

Indeed, ectopic expression of GATA3 in basal-like breast cancer

cells (e.g., MDA-MB-231 and LM2) directly and indirectly represses epithelial-to-mesenchymal

transition (EMT)-associated genes (e.g., CDH1, FOXC1), downregulates prometastatic genes

(e.g., lysyl oxidase, microRNA-29b, ID1/3, KRTHB1, LY6E, RARRES3), and induces expression

of metastatic-suppressing genes (e.g., DLC1, PAEP). Subsequently, xenografts of GATA3-

MDA-MB-231 cells resulted in formation of smaller primary tumors with significantly less lung

metastasis in comparison to xenografts of parental MDA-MB-231.54-57

Similarly, although

GATA3 is highly expressed in luminal subtype tumors, mouse models of luminal breast cancer

(MMTV-PyMT and MMTV-Neu) show loss of Gata3 expression with disease progression.58

Ectopic expression of Gata3 in MMTV-PyMT mice reduced tumor cell dissemination to lungs.58

Additionally, heterozygous copy loss of Gata3 in MMTV-PyMT (MMTV-PyMT; Gata3+/nlslacZ

)

led to earlier tumor onset and less differentiated, more aggressive, tumors with a higher

proportion of luminal progenitor-like cells.59

The Cancer Genome Atlas Network data reveals that 24% of breast cancers show GATA3 gene

alterations. This includes amplifications in 4% of cases, loss of one gene copy number in 11% of

tumors, and mutations in 9% (Figure 2A). Interestingly, tumors with deletion and mutations of

GATA3 are almost all luminal (78% and 97%, respectively).17,19,20

In contrast, tumors with

amplification are distributed across different breast cancer subtypes.17,19,20

Hence, heterozygous

copy number loss and mutations of GATA3 may play a significant role in luminal breast

tumorigenesis, and modeling these in mouse mammary epithelium may help explain the role of

GATA3 in breast cancer.

7

GATA3 mutations are mostly localized within ZnF2 and C-terminal domains (Figure 2A).17-20

Those within ZnF2 domain are more commonly found in luminal B tumors and are frameshift in

nature. These result and truncation at the end of ZnF2. ZnF2 mutations are typically

heterozygous, but sometimes occur together with loss of GATA3wt

or with one extra copy.

Interestingly, western blot analysis in MCF7 and in breast tumors harboring ZnF2 mutations

revealed that truncated GATA3 mutant alleles are expressed at equivalent or higher levels than

GATA3wt

.60-62

A recent study of the GATA3 ZnF2 mutant in MCF7 cells (GATA3D336fs

) showed

that it was resistant to proteasome-mediated and hormone-induced turnover as compared to

GATA3wt

.61

Meanwhile, mutations within C terminus of GATA3 are found in both luminal A

and B subtypes and to a lesser extent in HER2 and basal subtype tumors. Most of these

mutations cause a frameshift, which results in production of a longer protein as compared to

GATA3wt

. Interestingly, C-terminal mutant proteins accumulate to equivalent or lower levels

than GATA3wt

.60-62

Thus, stably expressed ZnF2 mutants may well exert dominant negative

effects, while C-terminus mutants likely behave as null hypomorphic alleles.

Based on these findings, I wish to model ZnF2 frameshift mutations of GATA3, particularly

GATA3N331fs

, which represents a recurrent mutation, found in luminal B tumors, and in some

cases present together with heterozygous copy loss of wild-type GATA3. In some tumors, this

mutation co-occurs with RB1 mutations, also implicated in luminal B tumorigenesis.17,19,20

Hence, by modeling mutations in this residue, I will be able to compare the effect on mammary

tumorigenesis of putative dominant negative GATA3 mutations to GATA3 haploinsufficiency.

1.4 RUNX1

1.4.1 RUNX1 protein complex in transcription

RUNX1 (or AML1) belongs to the Runt-related transcription factor (RUNX) protein family.

This family is composed of RUNX1, 2, and 3. RUNX family members all share a Runt domain

that binds DNA at the consensus nucleotide sequence 5’-TG(T/C)GGT-3’.63,64

All three RUNX

proteins have a similar structure but distinct expression patterns. Runx1 is expressed in many

tissues, including the hematopoietic and nervous system. Runx2 expression is most prominent in

8

osteoblasts. Runx3 is widely expressed, but predominant in the peripheral nervous system and

gastrointestinal tract.64-66

In humans, RUNX1 is found on chromosome 21, and is comprised of twelve exons, numbered 1,

1.1, 1.2, 2, 3, 4, 4.1, 5, 5.1, 5.2, 5.3, and 6. Both Exon1 and 2 contain 5’UTR and ATG

transcriptional start sites. Exon2, 3, 4, and 4.1 encode the Runt domain. Exon5, 5.2, and 6

contain transcriptional stop sites and 3’UTR.64,67,68

Alternative splicing of RUNX1 produces four

distinct isoforms: AML1a (250 amino acids), AML1b (453 amino acids), AML1c (480 amino

acids), and AMLΔN. In many tissues, the major RUNX1 transcript is AML1b, except in spleen

and thymus where AML1c predominates. Expression of AML1a and AMLΔN are generally low

across different tissues.64,69

For simplicity, AML1b will be referred to as RUNX1 in this thesis.

In addition to its ability to bind DNA, RUNX1 binds multiple transcription co-regulators to

control expression of many target genes.64

Hence, RUNX1 includes several functional domains

(Figure 1B). The N-terminal arm (amino acids 1 - 49) binds transcriptional co-regulators, such

as SUV39H1. This domain contains p300-dependent acetylated residues, K24 and K43.

Acetylation of RUNX1 is required for DNA binding, such that mutation of these amino acids

leads to significant attenuation of downstream gene. This domain also includes phosphorylated

residue, S48, which is implicated in cell cycle regulation.64,69

The Runt domain (amino acids 50 - 177) mediates DNA-binding and interacts with other

transcriptional co-regulators, including CBFβ, LEF-1, PU.1, C/EBPα, PAX5, AP-1, STAT,

SUV39H1, HDAC1/2, MEF, and calcineurin. In addition, the Runt domain contains a putative

phosphorylation site, S67, and a putative ATP-binding site, RFVGRSGRG (amino acids 135 -

143).64,66,70,71

The C-terminal end of the Runt domain contains nuclear localization sequence

KITVDGPREPRRHRQKL (NLS; amino acids 167 - 183).66,72

The C-terminus of RUNX1 includes transactivation and inhibitory domains that bind various co-

activators and co-repressors, respectively.64

The first inhibitory domain (ID1; amino acids 208 -

243) binds co-repressors mSin3A and Ear-2.64,66

This is followed by a transactivation domain

(TA; amino acids 243 - 371), which mediates interaction with co-activators, such as MTF and

Ets-1.66,73

Within this domain, a proline-rich motif, PPPY, binds to co-factors YAP, MOZ, Smad

proteins and p300.66

The second inhibitory domain (ID2; amino acids 371 - 411) dictates

transcriptional repression in the absence of CBFβ.64

This domain also mediates homo-

9

dimerization of RUNX1 on regulatory elements, which include multiple RUNX1-binding sites.64

Meanwhile, the third inhibitory domain (ID3) encompasses the last five residues of RUNX1,

VWRPY. This motif is binds cofactors TLE-1 and Hes-1.64,66,74

There are several phosphorylated sites within the Runx1 C-terminal region: S249, S266, T273,

S276, S304, and S423. Residues S249, S266S, T273, and S276 are phosphorylated by ERK

kinase or HIP2K, which subsequently enhances RUNX1 transactivation.64,66,68,75,76

On the other

hand, phosphorylation of S304, S423, as well as S48 in the N-terminus are implicated in

transactivation of cell cycle genes and proteasome-mediated degradation of RUNX1.64,66,75

Transcription regulation by RUNX1 alone is often not enough for robust and proper expression

of target genes.70,71

Accordingly, RUNX1 binds and cooperates with CBFβ and other lineage-

specific co-regulators to control target gene expression. Runx1 and CBFβ often form a

heterodimeric transcription factor complex called core binding factor (CBF). Indeed, the

interaction between Runx1 and CBFβ induces a conformational change in Runx1 that causes the

Runt domain to bind DNA more efficiently. CBF may further recruit coactivators or corepressors

to transactivate or repress target gene transcription, respectively. The interaction between Runx1

and other co-factors can also prevent interaction with CBFβ. For instance, in myeloid cells, the

CBF complex binds to C/EBP and PU.1 to transactivate CSF1R.77,78

When Runx1 binds to Ear-2,

CBF-mediated transactivation of CSF1R is suppressed.77

1.4.2 RUNX1 in development

During embryogenesis, Runx1 is expressed in hematopoietic cells, hair follicle precursors, and in

the nervous system. After birth, Runx1 expression persists in hematopoietic and hair follicle

cells, and is induced in a number of tissues.64,79

Runx1 is required for definitive hematopoiesis, a

process that ensures proper development of adult hematopoietic cells, including the emergence

of adult hematopoietic stem cells (HSCs). At this stage, Runx1 is a downstream target of Notch-

RBPJĸ signaling.80,81

Furthermore, Runx1 is required for differentiation and maintenance of

several hematopoietic lineages. For example, in immature myeloid cells, Runx1 transactivates

myeloperoxidase and neutrophil elastase82,83

, while repressesing myeloid differentiation genes

such as the CD53 receptor, Pim-2 serine protease, and mast cell carboxypeptidase A.64

In

differentiated myeloid cells, Runx1 regulates monocyte genes (e.g., CSF1R and CD36 receptor)

and erythroid genes (e.g., hematopoietic RING finger 1 and AML1-regulated transmembrane

10

protein 1).64,82,83

Runx1 also functions in lymphoid cells. For example, it regulates the T cell

receptor and granzymeB in T cells, as well as IgA1 and BLK tyrosine kinases in B cells.64,73

As

this gene plays such an important role in hematopoietic development, its expression must be very

tightly regulated in this context. In Down Syndrome, the overexpression of RUNX1 caused by

trisomy of chromosome 21, predisposes patients to myeloid leukemogenesis and leads to

quiescence of HSCs.84

On the other hand, haploinsufficiency of RUNX1 or overexpression of

AML1a, which represses the function of AML1b/1c, results in significantly reduced

accumulation of adult hematopoietic progenitor cells, failure in lineage specification, and

predisposes patients to lymphoid leukemia.85-87

Low expression or haploinsufficiency-associated

heterozygous mutations of RUNX1 also lead to familial platelet disorder with predisposition to

acute myeloid leukemia (FPD/AML).63,65,66,70,71,88,89

Mice with homozygous deletion of Runx1

(Runx1-/-

) die as embryos due to failed definitive hematopoiesis. Mutant embryos show

expansion of the HSC population, increased myeloid progenitors, defective lympho-

hematopoiesis, and hemorrhage in the central nervous system.63,65,66,70,71,88,89

RUNX1 also plays a critical role in development of other tissues. In the nervous system, RUNX1

promotes survival and proliferation of embryonic neurons through transcriptional repression of

cell cycle inhibitors p21Cip1

and p27Kip1

. RUNX1 also plays a role in differentiation of sensory

neurons and axonal guidance for a subset of neurons, including those that express the TrkA

receptor protein.64,90

Ectopic expression of Runx1 led to proliferation of cortical neuron

progenitor cells, while deficiency of Runx1 caused premature differentiation of olfactory receptor

neurons.91

Mouse embryos with homozygous Runx1 null alleles (Runx1-/-

) also exhibited massive

neuronal cell death.63,65,66,70,71,88-90,92,93

In skin development, Runx1 is required for homeostasis of

hair follicle stem cells (HFSCs), specifically through activation of the Jak/Stat pathway

throughout the hair cycle. Runx1 is expressed in embryonic mesenchyme and HFSCs to ensure

emergence of adult HFSCs and formation of distinct skin regions.94

In particular, Runx1 is

known to repress the cell cycle inhibitor, p21, within HFSCs such that its deletion blocked cell

cycle progression.21

Runx1 also regulates HIF-1α-associated angiogenesis and development of

skeletal muscles.64

Runx1 also controls mammary epithelium development. Its expression is evident at E16, which

coincides with the time at which primordial ducts start to branch, the nipple forms and lumens

are created.79,95

qPCR analysis showed that Runx1 is expressed throughout adult mammary

11

epithelium development, and peaks at the beginning of pregnancy as well as at the end of

involution. Runx1 is expressed in luminal and basal cells, but relatively higher in the latter.

During pregnancy, however, Runx1 expression decreased and is predominantly found in

myoepithelial cells.96

Runx1 is required for normal function of differentiated myoepithelial cells.

Mice with homozygous deletion of the gene in basal cells (Runx1fl/fl

; K14-Cre) or in a subset of

mammary epithelial cells (Runx1fl/fl

; MMTV-CreD) failed to nurse pups as myoepithelial cell

contraction/milk ejection was impaired.96

Meanwhile, Runx1 was also found to maintain

mammary luminal cell populations, specifically those that express ERα. Homozygous deletion of

Runx1 in a fraction of luminal and myoepithelial cells (Runx1fl/fl

; BLG-Cre) led to a significant

loss of luminal cells.79

Runx1fl/fl

; MMTV-CreD

mice also had a reduced number of ERα+ luminal

cells, and some showed delayed ductal outgrowth.96

Deletion of Runx1 in a fraction of mammary

epithelium (Runx1fl/fl

; MMTV-CreD) also led to upregulation of Elf5, a transcription factor

important for alveolar cell differentiation. Furthermore, the alveolar progenitor compartment

(CD49b+Sca1

-ER

-) was expanded in Runx1

fl/fl; MMTV-Cre

D mice.

96 This suggests that Runx1 not

only maintains ductal (ER+) cell population, but may also antagonizes alveolar (ER

-) cell fate.

1.4.3 RUNX1 in cancer

Genetic alterations of RUNX1 are commonly associated with hematopoietic malignancy. For

example, in leukemia, RUNX1 is often translocated and fused with other cancer-associated

genes, including MTG8, ETV6, MTG16, and MDS1. The fragment of RUNX1 that is retained in

these fusions includes the N-terminus and Runt domain, but not the C-terminal arm. The

chimeric oncoproteins function as transcriptional repressors.64,65,70

Missense mutations of

RUNX1 are also found in leukemia. These mutants often show impaired dimerization and DNA-

binding abilities. Mutations of RUNX1 increase the risk of leukemogenesis, but are not sufficient

to initiate disease. Instead, RUNX1 mutants are thought to cooperate with deregulated HIP2K-

and/or p300 to initiate leukemia.75

Aberrant expression and mutation of Runx1 occur in other cancers. For example, overexpression

of RUNX1 is associated with prostate, colon, ovary, and skin epithelial tumors.22

High-levels of

RUNX1 are indeed required for survival and proliferation of selected skin, ovarian, head and

neck squamous cancer cell lines.22

Also, in support of Runx1 oncogenic roles, carcinogen-

treatment induced fewer and papillomas as well as prolonged tumor latency in Runx1 mutant

12

mice as comparison to wild-type mice. In this study, Runx1 was found to prevent SOC3/4 from

suppressing Jak-Stat signaling and also to act upstream of the cell cycle inhibitor p21.21,22,94

Similarly, Runx1 expression correlates with invasive stage of endometroid and ovarian cancer.97

Runx1 is also implicated as a tumor suppressor, as shown in the mouse gastrointestinal tract.98

ApcMin

mice that additionally harbor deletion of Runx1 developed up to three times more

intestinal tumors as compared to those without deletion of Runx1.98

Like GATA3, RUNX1 is thought to function as a tumor suppressor in the breast.16,17,96,99

Despite

this, homozygous deletion of Runx1 or Runx2 in a fraction of mouse mammary epithelial cells

(i.e., in Runx1fl/fl

; BLG-Cre and Runx2fl/fl

; BLG-Cre), mammary glands were tumor-free at six-

months. When both Runx1 and Runx2 were homozygously deleted (i.e., Runx1fl/fl

; Runx2fl/fl

;

BLG-Cre), one third of mice had pretumorigenic squamous lesions at six-months.96,99

Furthermore, homozygous deletion of Runx1 dramatically reduced the number of ERα+ luminal

cells, a phenotype rescued by deletion of either Trp53 or Rb1.96

Hence, Runx1 deletion may

cooperate with Trp53 and Rb1 deletion to promote luminal-subtype tumor formation.

Furthermore, while higher levels of RUNX1 expression predict worse survival in basal-like

breast tumors79

, low expression of RUNX1 is associated with metastasis and worse prognosis in

overall breast cancer.16,100

RUNX1 mutations occur in 3% of human BCs.17-20

In addition, 1% of breast cancers have

homozygous deletions at RUNX1 and 16% have lost one copy of the gene.17-20

Interestingly,

tumors with mutations and heterozygous loss of RUNX1 are mostly luminal.17-20

Thus, deletion

and mutation of RUNX1 may play role in luminal breast cancer, and modeling these in mouse

mammary epithelium may help explain the role of RUNX1 in breast tumor formation or

progression.

Mutations in RUNX1 typically occur within the Runt, ID1, or TA domains (Figure 2B).17-20

Those within the Runt domain are usually missense, and found in a heterozygous state.17-20

Similar mutations within the Runt domain occur in leukemia and have been shown to decrease

DNA-binding efficiency, reduced transactivation, and enhanced interaction with CBFβ.101,102

Mutations within Runt domain are commonly found in luminal A and B tumors (Figure 2B).17-20

Breast cancer mutations within the ID1 domain usually cause a frameshift, with resulting stop

codon. These mostly occur in luminal A and B subtype tumors.17-20

Mutations within the TA

13

domain mostly generate frameshift alleles, which produce an elongated protein with impaired

TA domain function.101,102

These occur mostly in luminal A breast cancer.17-20

In AML cases,

frameshift mutations within ID1 and TA domains are predicted to bind CBFβ more efficiently, as

they retain the Runt domain.101,102

Thus, it is possible that most Runx1 mutations acquire

dominant-negative properties by binding and sequestering CBFβ away from RUNX1wt

.

Considering mutations in Runt domain predominate over mutations in other motifs, I am

interested in modeling Runt domain missense mutations, particularly those at a conserved

residue G141 (RUNX1G141R

). Missense mutations at residue 141 are relatively frequent, found in

luminal tumors, and in some cases accompanied by heterozygous loss of RUNX1wt

allele. In

addition, some of these tumors also harbor genetic alterations in TP53 and ZNF703, which are

associated with luminal B breast cancer.17-20

Hence, modeling mutations in this residue will

enable comparison to RUNX1 haploinsufficiency on mammary tumorigenesis and may

recapitulate luminal subtype pathology.

1.5 Hypotheses

GATA3: Most GATA3 hemizygous breast tumors (78%) and all breast tumors with

heterozygous frameshift mutations in the GATA3 DNA-binding domain (Znf2) are luminal.17-20

Accordingly, I hypothesize that mice with mammary epithelial-specific deletion of one copy of

Gata3, or expression a frameshift allele Gata3 in the mammary gland will be predisposed to

luminal-like tumors. Copy loss and Znf2 frameshift mutations of Gata3 gene may in part

contribute to tumorigenesis by altering luminal cell lineage commitment in mammary

epithelium. This effect should be enhanced by loss of one or both copies of wild type Gata3.

RUNX1: Hemizygosity and heterozygous missense mutations in the Runt DNA-binding domain

of RUNX1 were also mostly found in luminal tumors (73% and 94%, respectively).17-20

I

therefore hypothesize that mice with deletion of one allele of Runx1 in the mammary gland and

mice ectopically expressing a DNA-binding domain mutant of Runx1 will be predisposed to

luminal-like tumors, in part, due to alterations in luminal cell lineage commitment in mammary

epithelium.

14

1.6 Objectives

The first aim of this thesis involves testing for the effect of breast cancer-associated GATA3 gene

deletion on mammary cell fate specification and tumor formation. To this end, I will cross Cre-

inducible Gata3 null mice (Gata3fl)38

with mammary-epithelial specific Cre transgenics (i.e.,

MMTV-CreNLST

and MMTV-CreA)103,104

, luminal cell-specific and inducible Cre transgenics (i.e.,

K8-CreER)105

, as well as with Cre reporter mice (R26TG

).106

Mice in the Gata3fl/+

; MMTV-

CreNLST

cohort can be used to assess the effects of Gata3 copy number loss on mammary tumor

formation. Mice in the Gata3fl/+

; R26TG

; MMTV-CreA and Gata3

fl/+; R26

TG; K8-CreER cohort

can be used to analyze the effects of Gata3 copy number loss on mammary epithelial cell lineage

commitment.

The second aim of this thesis involves experiments to test for the effect of a breast cancer-

associated GATA3N331fs

allele (i.e., a representative frame shift mutation in the second Zn-finger

domain of GATA3) on mammary mammary epithelial cells. Specifically, I will develop

transgenic mice with a Rosa26-targeted, Tetracycline-repressible, Cre-inducible Firefly

luciferase-T2A-Gata3N331fs

transgene. Once generated, these mice will be crossed with

mammary-epithelial specific Cre transgenics (i.e., MMTV-CreNLST

and MMTV-CreA)103,104

,

luminal cell-specific and inducible Cre transgenics (i.e., K8-CreER)105

, Cre reporter mice

(R26TG

)106

, as well as with Cre-inducible Gata3 null mice (Gata3fl)38

. These can be used to

assess the effects of Gata3N331fs

on mammary epithelial cell lineage commitment and

tumorigenesis, as well as the effect of expressing the Gata3 frame-shift tumor allele in cells that

have only one endogenous copy of wild type Gata3, as in human tumors (as opposed to the effect

of this allele when expressed in the presence of two wild type alleles in the transgenic as made).

The third aim of this thesis involves experiments to test for the effect of deleting one or two

copies of RUNX1 on mammary epithelium. Specifically, I will cross Cre-inducible Runx1 null

transgenic mice (Runx1fl)107

with mammary epithelial-specific Cre transgenics (i.e., MMTV-

CreNLST

and MMTV-CreA)103,104

, luminal cell-specific and inducible Cre transgenics (i.e., K8-

CreER)105

, as well as with Cre reporter mice (R26TG

)106

, to test for the effect of Runx1 gene loss

on cell fate-specification and tumor formation. Mice in the Runx1fl; MMTV-Cre

NLST cohorts can

be used to assess mammary tumor formation. Mice in Runx1fl; R26

TG; MMTV-Cre

A and Runx1

fl;

15

R26TG

; K8-CreER cohorts can be used to analyze the effects of Runx1 gene loss on mammary

epithelial cell lineage commitment. In the future, these mice can also be studied together with

transgenics expressing a DNA-binding Runt domain of Runx1.

As with GATA3, we wish to generate a mouse expressing a tumor-derived mutant allele of

Runx1. To this end, the fourth aim of my thesis is to generate a pTET-bigT Rosa26-targeting

plasmid containing Runx1G141R

(Tetracycline-repressible, Cre-inducible Firefly luciferase-T2A-

Runx1G141R

). This construct can then be introduced to mice and subsequently combined with

MMTV-CreNLST

, MMTV-CreA, or K8-CreER to assess the effects of Runx1

G141R on mammary

tumorigenesis and epithelial cell lineage commitment.

Note: Various Cre transgenic mice are described in this thesis. Many of their relevant properties

are summarized below.

Cre

Strains Cre Expression Notes

MMTV-

CreNLST

Cre is expressed in some mammary epithelial cells

(mostly luminal). Cre expression in other tissues is

less pronounced than in MMTV-CreA mice.

108

This line is used to study effects

of mutations on mammary tumor

initiation and progression.

MMTV-

CreA

Cre is expressed in most mammary epithelial cells

after birth as well as in other tissues, including skin

and hematopoietic cells.109

This line is used to study effects

of mutations on mammary

epithelial cell commitment.

K8-CreER

Cre is expressed in mammary luminal epithelial

stem cells and more committed cells after

Tamoxifen administration.105

This line is used to study effects

of mutations on mammary

epithelial cell

commitment/differentiation.

MMTV-

CreF

Cre is expressed in most mammary epithelial cells

after birth as well as in other tissues, including skin

and hematopoietic cells.109

This line has not been used in this

thesis.

MMTV-

CreD

Cre is expressed in mammary epithelial cells 3

weeks after embryogenesis, and possibly during

embryogenesis.48,109

Its combination with other

mutation can lead to low survival rate. MMTV-

CreD; Gata3

f/fll pups mostly died after birth.

48

This line has not been used in this

thesis.

BLG-Cre

Cre is expressed specifically in mammary alveolar

luminal cells during pregnancy and lactation.109

This line has not been used in this

thesis.

WAP-Cre

Cre is expressed specifically in mammary alveolar

luminal cells during pregnancy and lactation.109

This line has not been used in this

thesis.

K14-Cre

Cre is expressed in primordial and committed basal

epithelial cells during and after embryogenesis. In

addition, its combination with other mutation can

lead to low survival rate. K14-Cre; Gata3f/fll

pups

mostly died after birth (only 1 in 200 survived). 48

This line has not been used in this

thesis.

16

Figure 1. Relative expression levels and roles for GATA3 and RUNX1 in the mammary luminal

cell lineage.

Relative expression levels and roles for GATA3 and RUNX1 in the mammary luminal cell lineage

(adapted from Visvader and Stingl 2014).4,12,48,79,96

GATA3 is expressed in all mammary

epithelial cells except in more committed myoepithelial cells. GATA3 expression is required for

specification of both ductal and alveolar luminal cells by regulating ductal genes (e.g., ESR1 and

FOXA1) and alveolar progenitors.4,48

RUNX1 is expressed in all mammary epithelial cells, except

in more committed alveolar cells. RUNX1 expression is required for specification of ductal

cells.96,99

Black arrows indicate transcriptional activation. Blunt-ended lines indicate

transcriptional repression. Relative levels of gene expression are represented as – (no expression),

+,

++,

+++ (highest expression). Abbreviations: MaSC = mammary stem cell, Lum SC = luminal

stem cell, Lum = luminal, SC = stem cell, P = progenitor, Myo = myoepithelial.

17

Figure 2 (A-B). Genetic alterations of GATA3 and RUNX1 in breast tumors.

Genetic alterations of GATA3 and RUNX1 in breast tumors (adapted from TCGA Breast Invasive

Carcinoma Provisional data from 1104 breast tumor samples).17-20

Abbreviations: BC = breast

cancer, AMP = copy number amplification, HETLOSS = heterozygous copy number loss,

HOMDEL = homozygous deletion/copy number loss, MUT = mutation, CNA = copy number

alteration, Indel = insertion/deletion mutation, LumA = Luminal A subtype, LumB = Luminal B

subtype, HER2 = HER2-enriched subtype, diploid(het) = heterozygous mutation in diploid locus.

18

A) Genetic alterations of GATA3 in breast tumors.17-20

The pie chart on the left shows that

GATA3 is genetically altered in 24% of breast cancers: 4% were by copy number amplification,

11% were by heterozygous copy number loss, 7% were by mutations, and 2% were by a

combination of mutation and copy number changes. Note tumors with copy number

amplification showed equivalent or lower mRNA expression level of GATA3 as compared to

tumors without GATA3 alterations. The schematic GATA3 protein map on the right shows that

most mutations of GATA3 are clustered in ZnF2 domain and C-terminal domain and found in

luminal tumors. ZnF2 mutations at residue 331 (i.e., GATA3N331fs

) were found in luminal tumors

as a heterozygous mutation with or without heterozygous copy number loss of GATA3wt

.

B) Genetic alterations of RUNX1 in breast tumors.17-20

The pie chart on the left shows that

RUNX1 is genetically altered in 21% of breast cancers: 2% were by copy number amplification,

17% were by copy number loss, 2% were by mutations, and 1% was by a combination of

mutation and copy number changes. Note that tumors with copy number amplification showed

equivalent or lower mRNA expression level of RUNX1 as compared to tumors without RUNX1

alterations. The schematic RUNX1 protein map shows that most mutations of RUNX1 are

clustered in Runt, ID, and TA domains and found in luminal tumors. Runt mutations at residue

141 (i.e., RUNX1G141R

) were found in luminal tumors as a heterozygous mutation with

heterozygous copy number loss of RUNX1wt

.

19

Chapter 2 Materials and Methods

2 Materials and Methods

2.1 Mouse strains and colony maintenance

All mouse colonies were maintained at Toronto Centre for Phenogenomics (TCP) in line with the

guidelines from Canadian Council on Animal Care (CCAC). All mice were sacrificed through

carbon dioxide euthanasia according to CCAC guidelines. All mouse cohorts in this thesis

consist of virgin females, except MMTV-CreA and K8-CreER cohorts, which were bred once.

Males were used for breeding.

Pure FVB/NJ wild-type mice were obtained from The Jackson Laboratory (stock number

001800).

Conditional Gata3 null mice (Gata3fl; C57BL/6 background) were obtained from Dr. Maxime

Bouchard at McGill University.38

These mice delete Gata3 in cells where Cre is present.38

After

they were imported to our lab, these mice were backcrossed onto an FVB/NJ background for at

least ten generations. They were genotyped by PCR using published primers FP 5’-

GTCAGGGCACTAAGGGTTGTT-3’, RP 5

’-TGGTAGAGTCCGCAGGCATTG-3

’, and RP 5

’-

TATCAGCGGTTCATCTACAGC-3’ to amplify a 390 bp fragment for the wild-type allele, a

420 bp fragment for the floxed allele, and a 350 bp fragment for the deleted allele.38

Conditional Runx1 null mice (Runx1fl; C57BL/6J background) were obtained from Dr. Nancy

Speck at the University of Pennsylvania.107

These mice delete Runx1 in cells expressing Cre or

their descendants.107

After they were imported to our lab, these mice were backcrossed onto an

FVB/NJ background for at least ten generations. They were genotyped by PCR using published

primers FP 5’- CCCACTGTGTGCATTCCAGATTGG-3

’, RP 5

’-

GACGGTGATGGTCAGAGTGAAGC-3’, and RP 5

’-CACCATAGCTTCTGGGTGCAG-3

’ to

amplify a 203 bp fragment for the wild-type allele, a 275 bp fragment for the floxed allele, and a

310 bp fragment for the deleted allele.107

20

MMTV-CreNLST

mice were provided from Dr. Timothy Lane at the University of California Los

Angeles103

and have been backcrossed onto an FVB/NJ background for at least ten generations in

our lab. These mice express Cre in a fraction of mammary epithelial cells.103,108,110

They were

genotyped by PCR using published primers FP 5’- TCGCGATTATCTTCTATATCTTCAG-3

’

and RP 5’-GCTCGACCAGTTTAGTTACCC-3

’ to amplify a 420 bp fragment, which do not

differentiate between hemizygous and homozygous animals.108

MMTV-CreA mice

104 were obtained from Dr. Tak Mak at University of Toronto and have been

backcrossed onto an FVB/NJ background for at least ten generations in our lab. These mice

express Cre in the majority of mammary epithelial cells, and also in other cell types, including

salivary epithelial cells, skin cells, and lymphocytes as previously described.111

This line was

genotyped by PCR using published primers FP 5’- TCGCGATTATCTTCTATATCTTCAG-3

’

and RP 5’-GCTCGACCAGTTTAGTTACCC-3

’ to amplify a 420 bp fragment, which do not

differentiate between hemizygous and homozygous animals.108

K8-CreER mice were obtained from The Jackson Laboratory (Stock Number 017947; C57BL/6J

background)105

and had been backcrossed onto an FVB/NJ background for at least five

generations in our lab. Following Tamoxifen administration, these mice express Cre in a fraction

of cytokeratin 8+ (K8

+) mammary luminal cells.

105 They were genotyped by PCR using

published primers FP 5’-GCGGTCTGGCAGTAAAAACTATC-3

’ and RP 5

’-

GTGAAACAGCATTGCTGTCACTT-3’, to amplify a 100 bp fragment, which does not

differentiate between hemizygous and homozygous animals.105

R26TG

mice were obtained from The Jackson Laboratory (strain B6.129(Cg)-

Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo

/J, stock number 007576)106

and have been backcrossed

onto an FVB/NJ background for ten generations in our lab. They were genotyped by PCR using

published primers FP 5’-CTCTGCTGCCTCCTGGCTTCT-3

’ and RP 5

’-

CGAGGCGGATCACAAGCAATA-3’, and RP 5

’-TCAATGGGCGGGGGTCGTT-3

’ to amplify

a 330 bp fragment for the wild-type R26 and a 250 bp fragment for the targeted TG allele.106

In this thesis, mice in Gata3fl and Runx1

fl experimental cohorts were maintained on a mixed

genetic background (C57BL/6; FVB/NJ background).

21

2.2 Gene targeting and generation of Rosa26Gata3N331fs

transgenic mice

A Gata3N331fs

cDNA that represents a mouse version of the GATA3N331fs

mutant transcript found

in some human breast tumors (as described in Chapter 3, Figure 3A) was synthesized by

GenScript. In parallel, Firefly Luciferase-T2A (FLuc-T2A) sequence was cloned from (FLuc)-

T2A-RFP Lentivirus vector (provided by Dr. Michael D. Taylor at SickKids) with 5’ NheI and 3’

PstI sites into pCR2.1 vector (Invitrogen, catalogue number 451641). FLuc-T2A and Gata3N331fs

sequences were confirmed by Sanger sequencing at The Centre of Applied Genomics

(SickKids). FLuc-T2A and Gata3N331fs

were ligated at PstI site and cloned into pTet-BigT

vector112

, and then to pRosa26PAm1 vector113

as described in Chapter 3. The pRosa26PAm1-

SA-tTA-TRE-loxP-NeoR-STOP-loxP-FLuc-T2A-Gata3

N331fs construct was linearized at an MluI

site, and linearized construct was purified by phenol-chloroform extraction and ethanol

precipitation as previously described.108

The following experiments were performed by Dr. Jessica Adams from our lab. In three

replicates, 20 μg of MluI-linearized construct was electroporated into 1.0x107 G4 strain mouse

embryonic stem cells (129S6B6F1 background). Electroporation was performed using 800 μL

electroporation buffer (Bio-Rad, catalogue number 1652677) in 4mm cuvette at 6 ms time

constant (Bio-Rad, catalogue number 1652081).114

The gene pulser (Bio-Rad, catalogue number

1652661) was set to 250V, 500uF, and ∞ resistance. Electroporated cells were kept in embryonic

stem cell media (ESCM) overnight and then in G418-ESCM media (200 μg/ml G418) for the

next six days. G418-resistant colonies were isolated into 96-well culture plates.

Genomic DNA from each clone were extracted as previously described115

and screened by PCR

using two sets of primers FP JA7 5’-CGCCTAAAGAAGAGGCTGTG-3’ and RP JA8 5’-

GAAAGACCGCGAAGAGTTTG-3’, as well as FP JA9 5’-GGCAAGCATCTTCCTGCTAC-3’

and RP JA10 5’-CGGCCTCGACTCTACGATAC-3’ to amplify 1.3 kb and 2.0 kb junction

fragments, respectively (Figure 4A).108

Clones with correct gene targeting were expanded (by

Dr. Jessica Adams) and chromosome counted as previously described.114

One of the clones with

diploid chromosomes (clone 5; 40 chromosomes) was submitted for morula aggregation with

CD-1 strain embryos to the transgenic core at TCP.116

22

18 chimeric mice were born with 5 – 40% stem cell contribution, as assessed by coat color. Each

chimera was bred to wild-type FVB/NJ mice, and pups from up to seven litters were monitored

for germ-line transmission. One male germ-line mouse was born from a 25% chimeric female on

her sixth litter. This mouse suffered skin erosion. Subsequently, the mouse was submitted for

sperm cryopreservation and recovery to Canadian Mouse Mutant Repository (CMMR) at TCP.114

Following in vitro fertilization of FVB/NJ eggs and implantation, the healthy Rosa26+/Gata3N331fs

pups were born from Doxycycline diet-fed mothers at the Mendelian ratio. Rosa26Gata3N331fs

mice

were then backcrossed onto an FVB/NJ background for at least five generations. In this thesis,

mice in Rosa26Gata3N331fs

cohorts were maintained on a mixed genetic background (129S6B6F1;

FVB/NJ or 129S6B6F1; C57BL/6; FVB/NJ).

2.3 Drugs

One-gram Tamoxifen (Sigma-Aldrich T5648) was first prepared as stock solution (30 mg/ml) in

DMSO (Sigma-Aldrich D8418) and stored in -20oC. Tamoxifen injection solution (3 mg/ml) was

freshly prepared by diluting Tamoxifen stock solution in sunflower seed oil (Sigma-Aldrich). To

activate K8-CreER without impairing mammary gland development, one dose of 0.5 ml

Tamoxifen injection solution /14-grams mouse (i.e., 1.5 mg Tamoxifen) was intraperitoneally

injected into each female mouse at four-weeks old.105,117

To repress the tTA-activated transgene, mice in Rosa26Gata3N331fs

cohorts were fed with

Doxycycline hyclate-containing pellets, or Dox diet (TD.120769; Teklad), instead of regular diet

(provided by TCP). The composition of this diet predicts that about 2.5 mg of doxycycline would

be consumed by each mouse every day (TD.120769; Teklad). Stock Dox diet was stored at 4oC,

while those in mouse cages were changed every week by the animal attendants at TCP.

2.4 Tumor monitoring, collection, and analysis

Mice in MMTV-CreNLST

cohorts were monitored every day for general health and once every two

weeks for emergence of palpable tumors by animal care attendants at TCP. Mice that were

notified for health concerns or palpable tumors were monitored daily for wellness and prediction

23

of end point by the veterinary technicians at TCP. End point was reached when tumors

developed up to 1700 mm3 in size or were ulcerated. Alternatively, end point was reached when

mice exhibited other poor health characteristics, including lethargy, panting, and distended

abdomen, according to CCAC guidelines.

During necropsy, tumor observations and other abnormalities, including metastasis and

hyperplasia, were recorded. Tumors were dissected and processed as follows. About half of the

tumor bulk was cut with a sterile razor blade and fixed in 10% phosphate-buffered formalin

(Fisher SF984). The remaining tumor bulk was cut into small pieces (approximately 0.5 cm in

diameter) using a sterile razor blade. These pieces were saved for RNA extraction (incubated in

0.5 ml RNALater overnight, QIAGEN 76106) and genomic DNA extraction (snapped frozen in

dry ice). The rest of the tumor was snapped frozen in dry ice for protein analysis. Tumor samples

in RNALater or frozen in dry ice were then stored in the lab at -80oC. Alternatively, the whole

tumor was fixed in 10% formalin if it was small in size (≤ 0.5 cm in diameter).

To determine tumor type by histology, formalin-fixed tumors were embedded in paraffin,

sectioned, and stained with hematoxylin and eosin (H&E) by the pathology core at TCP. H&E-

stained tumor sections were viewed under light microscope for tumor classifications as

previously described.108

To genotype Cre-mediated deletion of Gata3 and Runx1, genomic DNA from snap frozen tumor

samples was purified using a DNeasy kit (QIAGEN 69506) and PCR amplified using genotyping

primers as listed above.

2.5 Timed pregnancy, mammary gland collection and immunofluorescence

To collect mammary glands during pregnancy, MMTV-CreA and K8-CreER cohort females were

mated with FVB/NJ strain males at eight-weeks of age. They were checked for mating plugs

daily, and those with plugs were separated from the males. 12 days after plug was observed,

pregnant-state females were sacrificed for mammary gland collection.

24

To determine gross ductal outgrowth, one mammary gland per mouse (left mammary gland 4)

was spread on a microscope slide (Fisher Superfrost Plus 1255015) and dehydrated in acetone

(Fisher catalogue number 67641) overnight. The glands were stained in hematoxylin solution

(Sigma-Aldrich HHS16) overnight and then fixed in 1% acid alcohol solution (i.e., 1%

concentrated HCl diluted in 70% ethanol) overnight. The glands were incubated in ammonia

water (i.e., 3 ml NH4OH diluted in 1 L ddH2O) for 1 minute, washed in 95% ethanol (diluted in

ddH2O) overnight, and then in 100% ethanol overnight. Finally, the mammary glands were

incubated in toluene (Fisher 108883) for one hour and mounted using Permount (Fisher

SP15500). All steps were done in a fume hood at room temperature. Images were captured on a

dissecting light microscope using a digital camera.

To confirm Cre activity through induction of GFP expression, two mammary glands per mouse

(left mammary glands 3 and 5) were spread on a microscope slide (Fisher Superfrost Plus

1255015) and fixed in 4% paraformaldehyde (i.e., 16% paraformaldehyde diluted in PBS, Life

technologies 28906) overnight at 4oC. GFP expression was viewed under Leica Fluorescence

Stereoscope using Perkin Elmer Volocity software.

To characterize cell fate by immunofluorescence, three GFP+ mammary glands per mouse (right

mammary glands 3, 4, and 5) were fixed in 10% formalin and submitted to the pathology core at

TCP for embedding in paraffin. Antigen retrieval on sectioned glands was performed in sodium

citrate buffer (pH 6) using a pressure cooker (Biocare Medical Decloaking Chamber Pro) that

was set to 125oC for five minutes and 90

oC for ten seconds as previously described.

108 They were

then mounted onto a Tecan FreedomEvo liquid-handling robot for washing, blocking

(DakoCytomation X0909), and incubation with antibodies as previously described.108

Antibodies were used at recommended concentrations: goat Anti-GFP (1/400, Abcam ab6673),

rat Anti-Krt8 (1/100, DSHB TROMA-I), rabbit Anti-Krt14 (1/1000, Cedarlane CLPRB-155),

donkey anti-goat FITC (1/400, Jackson ImmunoResearch), chicken anti-rat Alexa 594 (1/400,

Molecular Probes A21471), goat anti-mouse Cy5 (1/200, Jackson ImmunoResearch). Slides

were mounted using DAPI-containing medium (Dako Fluorescence Mounting Medium S3023).

Images were obtained using Zeiss AxioVision.

25

2.6 In vitro transfection of bi-cistronic cassettes and Western blot

pcDNA3 (as an empty vector), pcDNA3-FLuc-T2A-Gata3N331fs

construct, or pcDNA3-FLuc-T2A-

Runx1G141R

construct was transfected into HEK293T cells using PolyJet kit (SignaGen

SL100688). HEK293T cells were cultured in DMEM containing 10% FBS and 1% Penicillin-

Streptomycin (WISENT 219010XK, 080110, and 450200EL). Cells were harvested 24 hours

after transfection, and lysates prepared in RIPA lysis buffer (Life technologies 89900).

Protein concentrations were determined by Bradford Assay (Bio-Rad 5000006). 30 µg of protein

from each transfection were then run on an SDS-PAGE using NEXT GEL kit (AMRESCO

M255) and transferred to nitrocellulose membrane (Bio-Rad 1620115). Membranes were

washed, blocked, and incubated with antibodies as previously described.108

Antibodies were used

at recommended concentrations: rabbit Anti-Gata3 (1/200, Santa Cruz sc-9009), rabbit Anti-

Runx1 (1 μg/ml, Abcam ab23980), goat Anti-Luciferase (1/2000, Promega G7451), mouse Anti-

beta Actin (1/500, Abcam ab8226), goat Anti-Rabbit IgG-HRP (1/5000, Santa Cruz sc-2004),

donkey Anti-Goat IgG-HRP (1/5000, Santa Cruz sc-2020), and goat Anti-Mouse IgG-HRP

(1/5000, Santa Cruz sc-2060). Finally, membranes were incubated in ECL solution (Amersham

GE RPN2106) and exposed to X-ray film (Amersham Hyperfilm 28906836).

26

Chapter 3 Modeling breast tumor-derived GATA3 gene alterations in mouse

mammary epithelium

3 Modeling breast tumor-derived GATA3 gene alterations in mouse mammary epithelium

3.1 Modeling GATA3 hemizygosity in mouse mammary epithelium

The Cancer Genome Atlas Network data reveal that 20% of breast cancers show GATA3

hemizygous gene loss and/or truncation mutation. Indeed, loss of one copy occurs in 11% of

breast cancer cases, and interestingly, the majority of these tumors (78%) are luminal.17-20

To test

for the effect of GATA3 copy number loss in mouse mammary gland, I generated cohorts that

mimicked this genetic alteration. Specifically, I used conditional Gata3 null mice (i.e., Gata3fl)38

,

in which exon4 of Gata3 is flanked by loxP sites. Accordingly, I bred Gata3fl mice with

mammary epithelial-specific Cre deleter mice to generate cohorts with heterozygous and

homozygous deletion (i.e., Gata3-/+

or Gata3-/-

, respectively). Mice in heterozygous Gata3fl/+

cohorts were used to model breast cancer-associated GATA3 hemizygosity. Mice in homozygous

Gata3fl/fl

cohorts were established as control.

The role of Gata3 in mammary epithelial development and homeostasis has been studied using

Gata3fl mice crossed to mammary-specific Cre deleter strains (e.g., MMTV-Cre

F, MMTV-Cre

D,

WAP-Cre).4,48

Homozygous deletion of Gata3 gene in mammary epithelium of Gata3fl/fl

;

MMTV-CreF mice resulted in simultaneous depletion and multiplication of luminal cell layers as

well as impaired ductal outgrowth at puberty. Mammary epithelium in Gata3fl/+

; MMTV-CreF

mice, on the other hand, appeared to be indistinguishable from mammary epithelium in wild-type

animals.4 Interestingly, it has been reported that mammary epithelium from Gata3

fl/+; MMTV-

CreD heterozygotes showed reduced ductal outgrowth.

48 Finally, pregnancy-induced

homozygous deletion of Gata3 in luminal cells of Gata3fl/fl

; WAP-Cre mice and Doxycycline-

induced Gata3fl/fl

; WAP-rtTA-Cre mice revealed multiple layers of alveolar cells that detached

into the luminal space and died. These mice also showed impaired lobulo-alveoli

27

development.4,48

No such phenotypes were seen in Gata3fl/+

; WAP-Cre and Doxycycline-induced

Gata3fl/+

; WAP-rtTA-Cre).4,48

All of these mice were studied at puberty or as they matured (i.e.,

up to about ten-weeks old).4,48

While it has been shown that heterozygous loss of Gata3

enhances tumor formation in MMTV-PolyomaMT (MMTV-PyMT) transgenic mice59

, it is not

clear whether heterozygous mammary epithelium is tumor prone in the absence of predisposing

oncogenic mutations.

In this thesis, I generated cohort of 31 female mice with Gata3 homozygous null mammary

epithelium (Gata3fl/fl

; MMTV-CreNLST

) and 41 female mice with Gata3 hemizygous mammary

epithelium (Gata3fl/+

; MMTV-CreNLST

). These mice are being monitored for tumor development

until they reach 18-months old. Mice in the Gata3fl/fl

; MMTV-CreNLST

cohort have now reached

13-months of age and mostly remained tumor-free. Two exceptions are one mouse that formed a

mammary tumor at five-months, and another one that developed lymphoma at seven-months.

Mice in the Gata3fl/+

; MMTV-CreNLST

cohort have reached 12-months of age and are also tumor-

free. Mice in control cohorts (i.e., Gata3fl/fl

and Gata3fl/+

) have also remained tumor-free (Table

1).

3.2 Generation of Cre-conditional Gata3N331fs

transgenic mice

GATA3 is mutated in approximately 9% of breast cancers, and these mutations are almost

exclusively found in luminal subtype tumors (97%).17-20

The majority of GATA3 mutations occur

either within the DNA-binding ZnF2 domain or in the C-terminus. ZnF2 mutant protein is

expressed at the same or higher level than wild-type GATA3.60,61

Mutations in the C-terminus,

on the other hand, accumulate to a lower level than GATA3wt

.60,61

These data suggest that stably

expressed ZnF2 mutants may well exert dominant effects in contribution to luminal-like

tumorigenesis, while poorly expressed C-terminus mutants may behave as null or hypomorphic

alleles.

GATA3N331fs

is a recurrent mutation found in luminal B breast tumors.17-20

To test for

transforming effects of this allele, I generated a Cre-conditional and Doxycycline-repressible

transgenic mouse capable of co-expressing Firefly Luciferase (FLuc) and Gata3N331fs

(Rosa26-

tTA-TRE-LSL-FLuc-T2A-Gata3N331fs

). By targeting to the ubiquitously expressed Rosa26 locus

28

and crossing in loxP flanked Gata3, I can manipulate Gata3gene dosage so that Gata3N331fs

dominant (negative) effects can be assessed on its own or in combination with Gata3 copy loss

(as seen in human breast tumors). By incorporating the Tetracycline-repressible (or Tet-Off)

transcriptional system into the transgene, I can assess whether Gata3N331fs

expression is

continuously required to maintain tumor growth, and therefore, whether Gata3N331fs

can be used

as a therapeutic target. Also, by using Cre-inducible system to induce Gata3N331fs

expression, it

can be targeted to virtually any desired tissue or cell type, which, in this context, is mammary

epithelium. Finally, by using a co-translational system to express Firefly Luciferase (FLuc) and

Gata3N331fs

from the same cDNA, I can trace Gata3N331fs

-expressing cells over the course of

tumor development. The FLuc- Gata3N331fs

construct was generated as described below.

Human GATA3 (443 amino acids; CCDS7083.1:

http://www.ncbi.nlm.nih.gov/CCDS/CcdsBrowse.cgi?REQUEST=CCDS&GO=MainBrowse&D

ATA=CCDS7083.1) and mouse Gata3 (443 amino acids; CCDS15674.1:

http://www.ncbi.nlm.nih.gov/CCDS/CcdsBrowse.cgi?REQUEST=CCDS&GO=MainBrowse&D

ATA=CCDS15674.1) share 90% homology in overall amino acid sequence, and 100% identity

for residues within ZnF2. Thus, the asparagine residue at amino acid 331 in human GATA3

corresponds to N331 in the mouse protein. Based on crystal structure analysis, N331 is

positioned between residues that directly contact GATA sequences (e.g., R329, N339, R364).24

Accordingly, frameshift mutation at N331 should disrupt interaction between ZnF2 and DNA.

Breast tumors that harbor GATA3N331fs

mutations typically have one nucleotide inserted at

position 993 of the coding sequence (993insG). This leads to a missense mutation at residue 331

(N331K) and frameshift mutation from residue 332. The resulting mutant protein has a truncated

ZnF2 domain, followed by, 20 amino acids from an alternate reading frame (Figure 3A).17,19,20

In the context of mouse, Gata3, insertion of a G at the same nucleotide position leads to

truncation of ZnF2 followed by two novel residues (Figure 3A). Given the possibility that the 10

amino acid extension at the C-terminus of human GATA3N331fs

may impart neomorphic and

oncogenic properties not recapitulated by the two amino acid extension that would be created

through addition of a single G residue at 993 in the mouse sequence, I have constructed a mouse

human GATA3N331fs

(Figure 3A). In addition, I introduced a neutral nucleotide substitution

(858C>A, which codes for alanine at amino acid 286 in both cases) to generate a novel HindIII

site (Figure 3A). This site will help distinguish mutant and wild-type Gata3 transcripts. The

29

final Gata3N331fs

cDNA sequence, flanked by 5’PstI and 3’ApaI sites, was submitted to

GenScript for synthesis.

GenScript-synthesized Gata3N331fs

cDNA was cloned downstream of Firefly Luciferase sequence

(FLuc) and a translational multicistronic-cleaving sequence from Thoseaasigna virus 2A

(T2A)118,119

(Figure 3C). The FLuc-T2A-Gata3N331fs

bicistronic cassette (i.e., FLuc-T2A-

Gata3N331fs

) was then cloned into pcDNA3. The resulting expression vector was transfected in

HEK293T cells. Western blot analysis of lysates from transfected cultures showed expression of

Firefly Luciferase (62 kDa) and Gata3N331fs

(38 kDa) (Figure 3B). As expected, fusion proteins

(> 62 kDa) were not detected with either FLuc or Gata3 specific antibodies, indicating complete

(or near complete) co-translational cleavage by T2A. The size of Gata3N331fs

(38 kDa) agrees

with ZnF2 frameshift mutant proteins previously characterized.60,61

The bicistronic FLuc-T2A-Gata3N331fs

sequence was then cloned into the pTET-BigT (Tet-Off

vector)112

at 5’NheI and 3’ApaI cloning sites, which are found downstream of SA-tTA-TRE-loxP-

NeoR-STOP-loxP sequence (Figure 3C). The resulting vector, pTET-BigT-FLuc-T2A-

Gata3N331fs

, was cleaved to liberate a 5’PacI-3’AscI fragment, which was cloned into the

Rosa26-targeting vector (i.e., pRosa26PAm1, DM#272)113

(Figure 3C). In the pRosa26PAm1

vector, PacI and AscI cloning sites are flanked by sequences that are homologous to Rosa26

intron1 (Figure 3C). The resulting pRosa26PAm1- SA-tTA-TRE-loxP-NeoR-STOP-loxP-FLuc-

T2A-Gata3N331fs

construct was linearized at MluI before electroporating into mouse embryonic

stem cells (mESC).

MluI-linearized Gata3N331fs

mutant cassette was electroporated into G4 mESC line by Dr. Jessica

Adams (in the Egan lab). The G4 mESC line was derived from a 129S6 x C57BL/6NTac mouse

strain. This line was used because it has been optimized for efficient generation of germline mice

through morula aggregation.116

Correct targeting of Gata3N331fs

cassette into Rosa26 locus would

result in Rosa26 promoter-driven expression of tTA. tTA will then bind Tetracycline response

element (TRE) and induce transcription of downstream genes: NeoR prior to Cre expression, or

FLuc-T2A-Gata3N331fs

following Cre-mediated excision (Figure 4A). 400 clones were selected

and expanded by Dr. Jessica Adams. I isolated genomic DNA from these clones, and performed

two sets of PCR test for homologous recombination at the Rosa26 locus (JA7/JA8 and JA9/JA10

screening primers were used). Of the 400 clones, 164 tested positive in both PCR tests (Figure

30

4B). Aneuploidy is frequently seen in cultured mESC as it can confer a growth advantage.

Unfortunately, aneuploidy also prevents efficient geermline transmission of mESCs.120,121

I

therefore counted chromosomes in fixed cells from 28 correctly targeted mESC clones. Of these,

only three had a normal diploid mouse chromosome number (i.e., 40 chromosomes) in the

majority of cells (≥ 70%) (Figure 4C): clone 5, 18, and 34.

Clone 5 was submitted to the transgenic core at TCP for generation of chimeric mice. In TCP,

mESC clone 5 was aggregated with morula from wild-type CD-1 embryos to produce chimeric

pups that are partially derived from clone 5 mESC. 18 chimeras were generated with 5 – 40%

mESC contribution based on coat colour chimerism. I bred these chimeras with wild-type

FVB/NJ mice for germline transmission. The chimeras were bred to yield up to seven litters,

each comprised of about 20 pups. One black-eyed, agouti-coloured pup was born from one of the

25% chimeric females on her sixth litter (Figure 5A). No more such pups were born from this

chimera in subsequent litters or from any of the other chimeras. Five PCR tests on tail genomic

DNA (using JA12/13/14, JA7/8, JA9/10, JA37/NK1 and FS1/R2 primer pairs) revealed that the

agouti-coloured pup contained the Rosa26-targeted (Gata3N331fs

) allele, while its albino-coloured

littermates did not (Figure 5B). At four-weeks of age, this germline pup was much smaller than

its wild-type littermates (Figure 5A). It also developed a skin erosion below its tail, where the

skin progressively peeled off and bled to end-point before breeding age was reached.

Fortunately, this problem had been previously seen by Dr. Jennifer Gorman (from the Woodgett

lab), who used the same pTET-BigT vector for targeted transgenesis with activated β-catenin.

She ultimately solved this problem by feeding pregnant mothers with Doxycycline (Dox)

containing diet. Indeed, Dr. Gorman speculates that expression of tTA and/or NeoR during

embryogenesis may be toxic, resulting in post-embryonic skin abnormalities. As my single

transgenic pup was approaching end point, I decided to rescue its sperm. This mouse was

submitted to the Canadian Mouse Mutant Repository at TCP for sperm collection. The harvested

sperm was then used to fertilize oocytes from FVB/NJ female mice, which were then transferred

to pseudo-pregnant mothers. These were Dox-fed (through pregnancy and up to weaning). 90

pups were born from these mothers and all appeared healthy. I genotyped these using primer

pairs JA12/13/14 and FS1/R2 (Figure 4A). 49 of 90 pups were heterozygous for the Tet-Off

FLuc-T2A-Gata3N331fs

allele.

31

In the presence of Cre recombinase, loxP-NeoR-STOP-loxP sequences should be deleted

112

followed by Rosa26-driven tTA inducing transcription of FLuc-T2A-Gata3N331fs

(Figure 4A).

However, in the presence of Tetracycline or Tetracycline derivatives, such as Doxycycline

(Dox), tTA should be blocked from binding TRE, thereby preventing transcription of FLuc-T2A-

Gata3N331fs

. To test for Cre-inducible transcription of Gata3N331fs

, I crossed Gata3N331fs

animals

with MMTV-CreNLST

delete line, which expresses Cre in the mammary gland. Western blot

analysis of lysates from mammary epithelium of 14-week old Gata3N331fs

; MMTV-CreNLST

showed expression of Gata3N331fs

(38 kDa) (Figure 5C). Next, to test for Cre-inducible

transcription of FLuc, I crossed Gata3N331fs

animals with the MMTV-CreA deleter line, which

efficiently expresses Cre in the mammary gland and skin.104,108

Eight-week old

Rosa26Gata3N331fs/+

; MMTV-CreA females were injected with Luciferin and imaged for Luciferase

activity. Signals were robustly detected in mammary glands and skin of Rosa26Gata3N331fs/+

;

MMTV-CreA females, but not in Gata3

N331fs females (without Cre) (Figure 5D). Thus, FLuc-

T2A-Gata3N331fs

expression is Cre-dependent as expected. To test for function of the Tet-Off

transcriptional system, Rosa26Gata3N331fs/+

; MMTV-CreA females that previously showed

Luciferase activity, were fed with Doxycycline. Animals fed with Dox for one, two, four and

eight days showed gradual decrement of Luciferase signal (Figure 5D middle and bottom

panels). Finally, to test for reversibility of the Tet-Off transcriptional system, Rosa26Gata3N331fs/+

;

MMTV-CreA females that had been fed with Dox for four consecutive days, were switched to a

normal diet and imaged on day four (after the switch). Indeed, most, if not all, of the Luciferase

signal returned (Figure 5D bottom panel). Therefore, a transgenic line that harbors a

conditional Gata3N331fs

allele that is targeted to Rosa26, Cre-inducible, Doxycycline-repressible,

and co-translated with a Luciferase reporter (i.e., Rosa26-tTA-TRE-LSL-FLuc-T2A-Gata3N331fs

,

or Rosa26Gata3N331fs

) has been successfully generated. Note that in each generation, mothers have

to be on Dox-containing food during pregnancy and lactation to prevent skin erosion in their

Rosa26Gata3N331fs

pups.

32

3.3 Modeling the combination of GATA3N331fs

and copy number loss of GATA3

wt

Breast tumors with GATA3N331fs

or related truncation mutant alleles are usually hemizygous, with

one mutant and one wild-type copy of the gene (i.e., Gata3N331fs/+

). In other, (less frequent) cases,

the wild-type GATA3 gene is lost. Hence, to model breast cancer-associated GATA3 mutation, I

am generating the following mouse cohorts (Table 2): I) Rosa26Gata3N331fs/+

; MMTV-CreNLST

, II)

Rosa26Gata3N331fs/+

; Gata3fl/+

; MMTV-CreNLST

, and III) Rosa26Gata3N331fs/+

; Gata3fl/fl

; MMTV-

CreNLST

. Similar cohorts are also being generated to test effects on cell lineage commitment, in

this case using MMTV-CreA

and K8-CreER deleter lines.

33

Table 1. Current report on mammary tumor incidence for Gata3flcohorts.

The cohort that models breast cancer-associated heterozygous copy loss of Gata3 is highlighted

in pink.

Cohort Purpose Number

of Mice Mice

Dead Mean Age

(months) Mammary

Tumor

Gata3fl/+ negative control 17 0 12 0

Gata3fl/fl negative control 19 0 11 0

Gata3fl/+

; MMTV-

CreNLST model Gata3

+l- in BC

40 2 12 0

Gata3fl/fl

; MMTV-

CreNLST

Test loss-of-function

effects of Gata3 31 2 13 1

34

Figure 3 (A-C). Generation of Gata3N331fs targeting construct.

A) The Gata3N331fs cDNA sequence that was synthesized by GenScript is based on mouse

Gata3. However, the genomic region that is affected by frameshift mutation is based on human

GATA3 cDNA sequence (shown by the dashed lines following amino acid K331) so that the

truncated protein product would be equivalent in size (350 amino acids) to the mutant version of

human GATA3. The neutral point mutation for a new HindIII site was shown by the dashed lines

on amino acid A286.

35

B) Western blot analysis on lysate from pcDNA3-FLuc-T2A-Gata3N331fs-transfected

HEK293T cells showed expressions of mutant GATA3N331fs and Firefly Luciferase, while

those from pcDNA3- or pcDNA3-Gata3wt

-transfected cells did not. β-actin was used as a loading

control.

C) Gata3N331fs targeting construct was cloned using pTet-BigT vector112

and pRosa26PAm1

vector.113

36

37

Figure 4 (A-C). Targeting Gata3N331fs to Rosa26 locus of mouse embryonic stem cells.

A) Schematic representation of mouse Rosa26 genomic locus that is non-targeted, targeted, and

Cre-recombined after targeting. Forward and reverse primers for screening correctly targeted

stem cells are shown in arrows with respect to their annealing sites within the Rosa26 locus and

Gata3N331fs targeting cassette.

B) Gata3N331fs –targeted clones were screened by PCR using primer sets (i.e., JA7/JA8 and

JA9/JA10) that amplify the junction region between Rosa26 promoter and Gata3N331fs

targeting cassette. Abbreviations: ddH2O = distilled water, -ve CTL = negative control, +ve CTL

= positive control, mESC = mouse embryonic stem cells.

C) Correctly targeted clones were further screened by normal, diploid chromosome count (i.e.,

40 chromosomes). Clone 5 was one of the three Gata3N331fs–targeted clones that showed

normal chromosome number and was used for generation of chimeric mice. Abbreviations: WT

= wild-type, Xm = chromosome, N = the number of cells analyzed per clone.

38

39

Figure 5 (A-D). Characterization of Luciferase activity in Rosa26Gata3N331fs

mice.

40

A) One agouti-coloured male pup was born out of all of chimera breeding with FVB/NJ strain

mice. At four-weeks old, this mouse was smaller than littermates.

B) The agouti-coloured male pup inherited Rosa26Gata3N331fs

allele as shown by PCR tests

amplifying regions of the junction between Rosa26 exon1 and Gata3N331fs

targeting cassette

(JA37/NK1), the junction between Rosa26 promoter and Gata3N331fs

targeting cassette

(JA12/JA13), and the junction between FLuc and Gata3N331fs

cDNA (FS1/R2).

C) Western blot analysis on mammary epithelium lysate from Rosa26Gata3N331fs

; MMTV-CreNLST

showed expression of mutant Gata3N331fs

, while those from Rosa26Gata3N331fs

and MMTV-CreNLST

only showed expression of wild-type Gata3. β-actin was used as a loading control.

D) Rosa26Gata3N331fs

mice showed Luciferase signal in the presence of Cre. The signal decreased

as the mice fed on Doxycycline diet (middle panel), but increased again once they returned to

regular diet (bottom panel). Rosa26Gata3N331fs

mice without Cre never showed Luciferase signal

(top panel). Note: MMTV-CreA is known to induce recombination in the mammary and salivary

glands as well as in the skin. Abbrevations: Dox = Doxycycline diet.

41

Table 2. Current report of mammary tumor assay for Rosa26Gata3N331fs

cohorts.

Cohorts that model breast cancer-associated ZnF2 mutation Gata3N331fs

is highlighted in pink.

Cohort Purpose Number

of Mice Mice

Dead Mean Age

(months) Mammary

Tumor

Rosa26Gata3N331fs/+ negative control 15 0 2 0

Rosa26Gata3N331fs/+

;

MMTV-CreNLST

test dominant negative

effects of Gata3N331fs 18 0 2 0

Rosa26Gata3N331fs/+

;

Gata3fl/+

;

MMTV-CreNLST model Gata3

N331fs/+ in BC 1 0 2 0

Rosa26Gata3N331fs/+

;

Gata3fl/fl

;

MMTV-CreNLST model Gata3

N331fs/- in BC 0 0 2 0

42

Chapter 4 Testing for effects of Runx1 gene alterations on mouse mammary

tumorigenesis

4 Testing for effects of Runx1 gene alterations on mouse mammary tumorigenesis

4.1 Testing for tumor suppressor activity of Runx1 in mouse mammary epithelium

The Cancer Genomic Atlas Network data reveals that 20% of breast cancers show deletion or

mutation of RUNX1. This includes loss of one gene copy number in 16%, homozygous deletion

in 1% of tumors and mutation in 3%.17-20

Interestingly, tumors with RUNX1 heterozygous copy

loss are almost all luminal (73%).17-20

To test for the effect of RUNX1 hemizygosity, I generated

mouse cohorts that mimicked this alteration in mouse mammary epithelium. Specifically, I used

conditional Runx1 null mice, in which exon4 is flanked by loxP sites (Runx1fl).

107 In the presence

of Cre, this exon is deleted and Runx1 gene function disrupted. Accordingly, I bred Runx1fl mice

with a mammary epithelium- or cell lineage-specific Cre deleter mice to generate cohorts with

heterozygous and homozygous deletion of Runx1. Mice in heterozygous cohorts will be used to

model breast cancer-associated heterozygous copy number loss, whereas mice in homozygous

cohorts will be used to test for loss-of-function effects of Runx1 on mammary epithelium.

In previous studies, Runx1fl mice and selected mammary- specific Cre deleter strains, (e.g., BLG-

Cre and MMTV-CreD) were used to determine the role of Runx1 in mammary epithelial

development.96,99

Runx1fl/fl

; BLG-Cre mice, in which Runx1 gene was homozygously deleted in

about 15% of mammary epithelial cells, showed reduced ductal outgrowth at puberty. These

mice were tumor-free at 12-weeks old.99

Similarly, Runx1fl/fl

; MMTV-CreD mice, which

expresses Cre in 20% of mammary epithelial cells, were tumor-free at 18-months.96

Interestingly, one third of mice with homozygous deletion of Runx1 and Runx2 (i.e., Runx1fl/fl

;

Runx2fl/fl

; BLG-Cre) had pretumorigenic squamous lesions at six-months, while age-matched

Runx1fl/fl

; BLG-Cre and Runx2fl/fl

; BLG-Cre mice did not.99

Mice with heterozygous deletion of

Runx1 were not described.

43

In this thesis, Nathan Schachter (in the Egan lab) and I generated 34 homozygous and 30

heterozygous Runx1 conditional mutant cohorts using MMTV-CreNLST

(i.e., Runx1fl/fl

; MMTV-

CreNLST

and Runx1fl/+

; MMTV-CreNLST

). These mice are currently under tumor watch, and will be

followed until they reach 18-months. To date, some mice in the Runx1fl/fl

; MMTV-CreNLST

cohort

have already reached reached 13-15 months and are mostly tumor-free. Four mice, however,

developed a poorly differentiated adenocarcinoma (PDA) in the mammary gland at 14, 15, 16,

and 17 months. Two of these also had lung metastasis. Mice in the Runx1fl/+

; MMTV-CreNLST

cohort are also mostly tumor-free, however, some developed mammary tumors between nine and

13-months. Four mice developed PDA mammary tumors at 11, 12, 14 and 15 months. One

developed a mammary papilloma at 9 months. One Runx1fl/+

; MMTV-CreNLST

mouse died from

liver and gastrointestinal tumors at 12 months. Finally, one mouse died with a blood-filled

ovarian cyst at 12-months (Table 3). Mice in control cohorts (i.e., Runx1fl/fl

and Runx1fl/+

) are

tumor-free at 11-months.

4.2 Generation of a construct for transgenic expression of Runx1

G141R: a Runt domain mutant.

RUNX1 is mutated in ~3% of breast cancer s, and these mutations occur almost exclusively in

luminal subtype tumors (94%).17-20

The majority of RUNX1 mutations are found in the DNA-

binding Runt domain. Similar mutations in leukemia lead to decreased DNA-binding efficiency,

reduced transactivation, and enhanced interaction with CBFβ.101,102

Thus, Runt domain

mutations of RUNX1 may exert dominant (negative) effects and contribute to luminal-like

tumorigenesis. RUNX1G141R

is one such Runt-domain mutation.

In most cases, RUNX1 mutations are heterozygous, but in some tumors, the wild-type allele is

also deleted.17-20

Indeed, by crossing Runx1G141R

transgenic mice with Runx1fl mice, it should be

possible to test for dominant and semi-dominant effects of breast cancer mutant alleles. To this

end, I began to assemble a Runx1G141R

mutant cassette for targeting to the Rosa26. In this

cassette, as with Gata3N331fs

(Chapter 3), expression of Runx1G141R

will be Tetracycline-

repressible, Cre-inducible, and co-expressed with a Luciferase reporter (i.e., Rosa26-tTA-TRE-

LSL-FLuc-T2A-Runx1G141R

). Again, I can assess whether Runx1G141R

expression is continuously

required to maintain tumor growth, and therefore, whether Runx1G141R

can be used as a

44

therapeutic target. Finally, by using a co-translational system to express Firefly Luciferase

(FLuc) and Runx1G141R

from the same cDNA, I can trace Runx1G141R

-expressing cells over the

course of tumor development. The FLuc-Runx1G141R

transgenic targeting construct was generated

as described below.

Human RUNX1 (453 amino acids; CCDS42922.1:

http://www.ncbi.nlm.nih.gov/CCDS/CcdsBrowse.cgi?REQUEST=CCDS&GO=MainBrowse&D

ATA=CCDS42922.1) and mouse Runx1 (451 amino acids; CCDS49916.1:

http://www.ncbi.nlm.nih.gov/CCDS/CcdsBrowse.cgi?REQUEST=CCDS&GO=MainBrowse&D

ATA=CCDS49916.1) share 96% identity in amino acid sequence. Indeed, the Runt domains are

100% identical (Figure 6A). Thus, G141 in human RUNX1 corresponds to G141 in the mouse

protein (Figure 6A).96

I generated a mouse version of Runx1G141R

by changing coding nucleotide

421 from a G to a C. In addition, I also introduced a neutral point mutation at coding nuclueotide

432 (G to A), which created a new HindIII site (Figure 6A). This site can be used to distinguish

mutant and wild-type Runx1 transcripts. The final Runx1G141R

cDNA sequence was flanked by

5’EcoRI and 3’XhoI sites and submitted to GenScript for synthesis.

GenScript-synthesized Runx1G141R

cDNA was cloned downstream of FLuc-T2A sequence118,119

(Figure 6C). The expression of FLuc-T2A-Runx1G141R

bicistronic cassette was then cloned into

pcDNA3. The resulting expression vector was transfected in HEK293T cells. Western blot

analysis of lysates from transfected cultures showed expression of Firefly Luciferase (62 kDa)

and Runx1G141R

(48 kDa) (Figure 6B). As expected, fusion proteins (> 62 kDa) were not

detected with either FLuc or Runx1 specific antibodies, indicating complete (or near complete)

co-translational cleavage by T2A.

The bicistronic FLuc-T2A-Runx1G141R

sequence was then cloned into pTET-bigT112

, at 5’NheI

and 3’XhoI cloning sites, which are found downstream of SA-tTA-TRE-loxP-NeoR-STOP-loxP

sequence (Figure 6C). The resulting vector, pTET-BigT-FLuc-T2A-Runx1G141R

, was cut to

liberate 5’PacI-3’AscI fragment, which was cloned into the Rosa26-targeting vector

(pRosa26PAm1, DM#272)113

(Figure 6C). In the pRosa26PAm1 vector, PacI and AscI cloning

sites are flanked by sequences that are homologous to Rosa26 intron1 (Figure 6C). The resulting

pRosa26PAm1-SA-tTA-TRE-loxP-NeoR-STOP-loxP-FLuc-T2A-Runx1

G141R construct was

45

linearized at MluI site and is ready to be electroporated into mouse embryonic stem cells

(mESC).

46

Table 3. Current report of mammary tumor incidence for Runx1fl

cohorts.

The cohort that models breast cancer-associated Runx1 copy number loss is highlighted in pink.

Cohort Purpose

Number

of Mice

Mice

Dead

Mean Age

(months)

Mammary

Tumor

Runx1fl/+

negative control 14 0 10 0

Runx1fl/fl

negative control 16 0 12 0

Runx1fl/+

; MMTV-CreNLST

model Runx1+/-

in BC 30 7 10 4

Runx1fl/fl

; MMTV-CreNLST

model Runx1-/-

in BC 34 4 13 4

47

Figure 6 (A-C). Assembly of Runx1G141R

targeting construct.

48

A) Runx1G141R

cDNA sequence synthesized by GenScript. The breast tumor-derived missense

mutation is flanked by dashed lines at amino acid R141. The neutral point, which introduces a

new HindIII site is also flanked by dashed lines, in this case, at amino acid K144.

B) Western blot analysis on lysate from pcDNA3-FLuc-T2A-Runx1G141R-transfected

HEK293T cells showed expressions of mutant Runx1G141R

and Firefly Luciferase, while those

from control pcDNA3-transfected cells did not. β-actin was used as a loading control.

C) The Runx1G141R

targeting construct was generated using pTet-BigT112

and pRosa26PAm1

vectors.113

49

Chapter 5 Discussion and future directions

5 Discussion and future directions

Recent analysis on the breast cancer genome has revealed recurrent mutation and hemizygous

deletion of GATA3 and RUNX1, both predominantly found in luminal subtype tumors.17-20

While

both transcription factors control development of luminal epithelial cells of the mammary

gland4,48,96,99,122

, the oncogenic effect of mutation or deletion of either gene remains to be

defined. In this thesis, accordingly, I generated cohorts of mice with mutation or deletion of

Gata3, or deletion of Runx1, to test for effects on mammary tumorigenesis.

5.1 Deletion and mutation of Gata3 in mouse mammary epithelium

Cohorts with heterozygous deletion of Gata3 (Gata3fl/+

; MMTV-CreNLST

) are being monitored for

tumor formation up to 18-months. To date, 12-months old mice in this cohort are mostly tumor-

free. If this result holds up, and Gata3 hemizygous mammary epithelium is not tumor prone, this

would fit well with the lack of breast cancer susceptibility in GATA3 hemizygous mutant humans

(GATA3+/-

humans have HDR syndrome - Hypoparathyroidism, sensorineural Deafness, and

Renal dysplasia; MIM 146255). Furthermore, this scenario would suggest that hemizygosity of

Gata3 may well represent a progression rather than initiation event, given that 11% of breast

cancers have lost one copy of the gene, and in mice such a loss enhances tumor formation in the

MMTV-PolyomaMT model.59

To test for this, Nathan Schachter (in the Egan lab) and I are

crossing Gata3fl/+

; MMTV-CreNLST

mice with Pik3caH1047R

and TP53R270H

models. Specifically,

we will test for cooperation between Gata3 hemizygosity and activating mutation of Pik3ca or

loss-of-function mutation of TP53, as seen in some breast tumors (3%). In addition, Dr. Rita

Quintana (in the Egan lab) is performing a Sleeping Beauty mutagenesis screen in Gata3fl/+

;

MMTV-CreNLST

mice to define cooperating events that initiate and enhance tumor development

on a Gata3 hemizygous mutant background (Table 4).

50

In parallel, to determine whether hemizygous deletion of GATA3 contributes to tumorigenesis, in

part, by altering luminal cell lineage commitment, I am currently generating cohorts of female

Gata3fl/+

; R26TG

; Cre mice with luminal-specific and mammary-specific Cre-deleter transgenes

(K8-CreER and MMTV-CreA, respectively). Mammary glands from these cohorts will be

sectioned and subject to immunofluoresence staining to identify cells that have either expressed

Cre, or are descended from cells that expressed Cre, since such cells will delete the Tomato

florescence gene (T) and turn on eGFP (G). Mammary glands will also be stained with

antibodies for Gata3 and other luminal makers (e.g., K8, ERα, Elf5 etc.) as well as for basal

markers (e.g., K14, Sma). In this way, we can compare the lineage and marker expression

profile of eGFP+ cells (Gata3

+/-) to their eGFP

- neighbors that do not express Cre (Gata3

+/+). To

test for the dosage-dependent role of Gata3 in homeostasis, for example, female Gata3fl/+

;

R26mT/mG

; K8-CreERT2

mice can be induced to delete one copy in a fraction of luminal epithelial

cells during pubertal, in the mature adult or during pregnancy.105

On the other hand, females

from the MMTV-CreA cohort can be used to delete one copy of Gata3 in the majority of

mammary epithelial cells during development of the gland.108,111

Based on published data for

homozygous deletion of Gata3 in the mouse mammary gland, I would expect that some

hemizygous mutant cells may show defective cell fate specification or maintenance of the

luminal differentiated fate.123

This effect is not expected to be as strong as the effect of deleting

both copies of Gata3 but it should reveal which Gata3 mutant phenotypes are most sensitive to

loss of only one copy.

To establish similar cohorts for the study of a GATA3 ZnF2 mutation on mammary

tumorigenesis, we can use the conditional Rosa26Gata3N331fs

transgenic mouse generated in my

thesis. Mice with this transgene express Cre-inducible, Doxycycline-repressible Firefly

Luciferase reporter (FLuc) and Gata3N331fs

. Indeed, when Rosa26Gata3N331fs

mice were crossed to

MMTV-CreA transgenic mice, which efficiently express Cre in the mammary gland and skin, the

resulting Rosa26Gata3N331fs/+

; MMTV-CreA females showed luciferase activity in both

compartments. The luciferase signal decreased rapidly after these mice were fed Doxycycline-

containing food, and increased again as they switched to regular food. Importantly, Gata3N331fs

females (without Cre) did not show luciferase activity. Note that in each generation of

Rosa26Gata3N331fs

mice, mothers have to be on Dox-containing food during pregnancy and

lactation to prevent skin erosion in their Rosa26Gata3N331fs

pups. Dr. Jennifer Gorman (in the

51

Woodgett lab) who found and solved this problem speculated that expression of tTA and/or NeoR

during embryogenesis may be toxic, resulting in post-embryonic skin abnormalities.

Rosa26Gata3N331fs

mice can thus be used to study 1) whether Gata3N331fs

exerts dominant

(negative) or loss-of-function effects (similar to copy number loss) on mammary epithelium, 2)

whether Gata3N331fs

is tumor-initiating or requires cooperation with other altered genes for

initiation, 3) whether Gata3N331fs

expression is continuously required to maintain tumor growth,

and therefore, if Gata3N331fs

can be used as a therapeutic target. To answer the first question, I

have crossed Rosa26Gata3N331fs

mice with MMTV-CreNLST

. These mice are currently on tumor-

watch (Table 2 in Chapter 3). I have also crossed these mice to K8-CreER and MMTV-CreA

mice to test for alterations in cell differentiation. To identify cooperating genes, I have crossed

Rosa26Gata3N331fs

; MMTV-CreNLST

mice with Pik3caH1047R

and TP53R270H

models (Table 5), while

Dr. Rita Quintana (in the Egan lab) is performing a Sleeping Beauty screen on Rosa26Gata3N331fs

mice. Finally, if any mice in the mentioned cohorts developed mammary tumors, they will be

fed with a Dox-containing diet to test for tumor regression/oncogene addiction.

5.2 Transactivation ability of Gata3N331fs

To determine whether Gata3N331fs

exert dominant (negative) effects through altered

transactivation ability, I will perform luciferase transcriptional assays. Gata3N331fs

, Gata3wt

, and

Fog2 (a co-factor of Gata3) will be cloned into pcDNA3 vector and co-transfected with the

CCND1 promoter (which contains GATA3 binding sites)-luciferase constructs. Transactivation

will then be compared in transfected HEK293T cells. To date, I have cloned all of the above

constructs, except for pcDNA3-Fog2. Co-transfection with pcDNA3-Fog2 construct will help

determine whether Gata3N331fs

exerts dominant effects, possibly by sequestering co-factors, like

Fog2, away from Gata3wt

. In parallel, I will preliminarily test for transcriptional repression

effects of Gata3N331fs

. A recent study has shown that GATA3 forms a complex with G9A and

NuRD(MTA3) to inhibit the expression of genes implicated in epithelial-mesenchymal transition

of breast tumor progression, including CDH2 and FN1.122

Thus, I will compare the expression of

CDH2 and FN1 in T47D cells (a breast cancer cell line) that are transfected with pcDNA3-

Gata3wt

or pcDNA3-Gata3N331fs

.

52

5.3 Deletion and mutation of Runx1 in mouse mammary epithelium

Mice with heterozygous and homozygous deletion of Runx1 (Runx1fl/+

; MMTV-CreNLST

and

Runx1fl/fl

; MMTV-CreNLST

) are still being monitored for tumor formation up to 18-months. To

date, mice in Runx1fl/fl

; MMTV-CreNLST

cohort are mostly tumor-free, except for four mice that

developed mammary tumors around 14-months of age. Similarly, mice in Runx1fl/+

; MMTV-

CreNLST

cohort are mostly tumor-free, except for seven. Of these, four developed poorly-

differentiated mammary adenocarcinoma around 12-months. The remaining three died with

tumors in other organs, including liver, skin, and ovary (at around 12-months). Thus, deletion of

Runx1 could initiate tumor formation at low frequency, or, through cooperation with other

altered genes. To test for the second possibility, Nathan Schachter (in the Egan lab) and I are

crossing Runx1fl/+

; MMTV-CreNLST

and Runx1fl/fl

; MMTV-CreNLST

mice to Pik3caH1047R

and

TP53R270H

models (Table 6). These crosses are based on the fact that deletion of RUNX1 occurs

together with activating mutation of PIK3CA in 5% and loss-of-function mutation of TP53 in 6%

of breast tumors. Furthermore, Dr. Rita Quintana (in the Egan lab) is also generating Runx1fl/+

;

MMTV-CreNLST

Sleeping Beauty mutagenesis cohort to identify other altered genes that

cooperate with deletion of Runx1. Meanwhile, to determine whether deletion of RUNX1

contributes to breast cancer in part by altering luminal cell lineage commitment, I am generating

similar cohorts as above, using luminal-specific and mammary-specific Cre-deleter lines (K8-

CreER and MMTV-CreA).

To test for transforming effects of a RUNX1 Runt-domain mutation on mammary tumorigenesis,

I generated a Runx1G141R

Rosa26-targeting vector (Rosa26-tTA-TRE-LSL-FLuc-T2A-Runx1G141R

).

In this vector, as with the Gata3N331fs

transgenic knock-in vector described in Chapter 3,

expression of Runx1G141R

will be Cre-inducible, Doxycycline-repressible and co-expressed with a

luciferase reporter. In the future, this vector can be used to generate targeted ES cells and

germline transgenic mice. The resulting line will be used to test whether 1) Runx1G141R

exerts

dominant (negative) transforming properties in mammary epithelium, 2) Runx1G141R

can induce

tumor-initiation or requires cooperation with other oncogenic mutations, and 3) Runx1G141R

53

expression is continuously required to maintain tumor growth, and therefore, if Runx1G141R

can

be used as a therapeutic target.

5.4 Transactivation ability of Runx1G141R

Finally, to determine whether Runx1G141R

functions dominantly to suppress Runx-dependent

transcription, I will perform luciferase transcriptional assays in tissue culture. cDNAs for

Runx1G141R

, Runx1wt

, and Cbfb (a Runx1-binding partner and transcriptional co-factor) will be

cloned into pcDNA3 and co-transfected into HEK293T cells with a M-CSFR promoter-luciferase

construct (which contains Runx1-binding sites). I have generated all of these plasmids, except

for pcDNA3-Cbfb, which will be made soon. By co-transfecting wildtype and mutant Runx1

constructs with, or without, pcDNA3-Cbfb, I will be able to test whether Runx1G141R

exerts

dominant effects by sequestering CBFβ away from Runx1wt

.

54

Table 4. Cohorts to test for cooperation between Gata3fl

and Pik3caH1047R

or TP53R270H

.

The cohort that models breast cancer-associated Gata3 copy number loss is highlighted in pink.

Pik3caH1047R

or TP53R270H

cohorts are separated by a bold line. The TP53R270H

cohort was

generated by Nathan Schachter (in the Egan lab).

Cohort Purpose Number

of Mice Mice

Dead Mean Age

(months) Mammary

Tumor

Gata3fl/+

;

Rosa26Pik3caH1047R/+

;

MMTV-CreTL model Gata3

+/-; Pik3ca

H1047R/+

in BC 0 0 0 0

Gata3fl/+

;

TP53R270H/+

;

MMTV-CreTL model Gata3

+/-; TP53

R270H/+ in

BC 31 6 12 3

55

Table 5. Cohorts to test for cooperation between Gata3N331fs

and Pik3caH1047R

or TP53R270H

.

The cohort that models breast cancer-associated Gata3 ZnF2 mutation is highlighted in pink.

Pik3caH1047R

or TP53R270H

cohorts are separated by a bold line.

Cohort Purpose

Number

of Mice

Mice

Dead

Mean Age

(months)

Mammary

Tumor

Rosa26Gata3N331fs/Pik3caH1047R

;

MMTV-CreTL

test dominant negative

effects of Gata3N331fs

5 0 2 0

Rosa26Gata3N331fs/Pik3caH1047R

;

Gata3fl/+

;

MMTV-CreTL

model Gata3ZnF2/+

;

Pik3caH1047R/+

in BC 0 0 0 0

Rosa26Gata3N331fs/Pik3caH1047R

;

Gata3fl/fl

;

MMTV-CreTL

model Gata3ZnF2/-

;

Pik3caH1047R/+

in BC 0 0 0 0

Rosa26Gata3N331fs/+

;

TP53R270H/+

;

MMTV-CreTL

test dominant negative

effects of Gata3N331fs

0 0 0 0

Rosa26Gata3N331fs/+

;

Gata3fl/+

;

TP53R270H/+

;

MMTV-CreTL

model Gata3ZnF2/+

;

TP53R270H/+

in BC 0 0 0 0

Rosa26Gata3N331fs/+

;

Gata3fl/fl

;

TP53R270H/+

;

MMTV-CreTL

model Gata3ZnF2/-

;

TP53R270H/+

in BC 0 0 0 0

56

Table 6. Cohorts to test for cooperation between Runx1fl

and Pik3caH1047R

or TP53R270H

.

The cohort that models breast cancer-associated Runx1 copy number loss is highlighted in pink.

Pik3caH1047R

or TP53R270H

cohorts are separated by a bold line. The TP53R270H

cohorts were

generated by Nathan Schachter (in the Egan lab).

Cohort Purpose

Number

of Mice

Mice

Dead

Mean Age

(months)

Mammary

Tumor

Runx1fl/+

;

Rosa26Pik3caH1047R/+

;

MMTV-CreTL

model Runx1+/-

; Pik3caH1047R/+

in BC 15 5 6 5

Runx1fl/fl

;

Rosa26Pik3caH1047R/+

;

MMTV-CreTL

model Runx1-/-

; Pik3caH1047R/+

in BC 5 2 6 2

Runx1fl/+

;

TP53R270H/+

;

MMTV-CreTL

model Runx1+/-

; TP53R270H/+

in

BC 31 6 12 3

Runx1fl/fl

;

TP53R270H/+

;

MMTV-CreTL

model Runx1-/-

; TP53R270H/+

in

BC 31 6 12 3

57

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