DTIC ADA435003: Technologies for Genome-Wide Identification of Stat5 Regulated Genes

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The author hereby certifies that the use of copyrighted
material in the dissertation entitled:

(cid:147)Technologies for Genome-Wide Identification of Stat5 Regulated Genes(cid:148)

beyond brief excerpts is properly acknowledged and used with the permission of the
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the Health Sciences from any damage which may arise from such a copyright violation.

Matthew J. LeBaron
Department of Pathology
Uniformed Services University of the Health Sciences
ii
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1. REPORT DATE 3. DATES COVERED
2003 2. REPORT TYPE -
4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER
TECHNOLOGIES FOR GENOME-WIDE IDENTIFICATION OF
5b. GRANT NUMBER
STAT5 REGULATED GENES
5c. PROGRAM ELEMENT NUMBER
6. AUTHOR(S) 5d. PROJECT NUMBER
5e. TASK NUMBER
5f. WORK UNIT NUMBER
7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION
Uniformed Servicces universsity of the Health Sciences,F. Edward REPORT NUMBER
Herbert School of Medicine,4301 Jones Bridge
Road,Bethesda,MD,20814-4799
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Approved for public release; distribution unlimited
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14. ABSTRACT
Each year more than one million new cases of breast cancer are diagnosed worldwide and an estimated
370,000 women die from breast cancer. Although the vast majority of fatal breast cancer cases involve
metastatic spread of the primary tumor, the formation of metastases is still a poorly understood, complex
process. Identifying the early molecular changes that facilitate metastasis of breast cancer will lead to new
molecular targets for prevention of metastases and improved therapies. Intriguing data from the mentor’s
laboratory show loss of activation of the transcription factor Stat5 during breast cancer progression and
that tumors without active Stat5 have higher histological grade, increased mitotic rate, and unfavorable
prognosis. Furthermore, data developed in the preparation of this dissertation indicate a substantial
growth-inhibitory and pro-differentiation role for Stat5 in mammary epithelial cells. Based on these and
other recent observations, we postulate that loss of Stat5 activation in breast cancer represents a
progression event that leads to dedifferentiation and increased risk of metastatic invasion. Therefore, a
critical analysis of the role of Stat5 in human breast cancer is warranted, including systematic efforts to
identify genes directly controlled by Stat5. The recent completion of the human genome sequence presents
new opportunities for global identification of Stat5 target genes. Work performed in the preparation of this
dissertation has established new methodology to capture, clone, sequence, and validate physiological Stat5
DNA-binding sites in a genome-wide manner. The method can also be used to determine whether Stat5
interacts with a known Stat5-responsive promoter within a given experimental context when coupled with
PCR amplification of the target DNA. Using this methodology, we have demonstrated that glucocorticoids
markedly alter the pattern of chromatin access for Stat5 binding in breast cancer cells. Furthermore, we
also report novel methodology to specifically identify transcripts directly regulated by Stat5, based on
dominant-negative, differential suppression of Stat5 regulated transcripts and large-scale gene chip
analysis. As a result of this work, rapid progress in genome-wide identification of Stat5 target genes is now
possible, as well as molecular mapping of the regulatory role of Stat5 in breast cancer.
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ABSTRACT

Title of dissertation: Technologies for Genome-Wide Identification of Stat5
Regulated Genes

Matthew J. LeBaron, Doctor of Philosophy, 2003

Dissertation directed by: Hallgeir Rui, M.D., Ph.D., Associate Professor, Department
of Pathology

Each year more than one million new cases of breast cancer are diagnosed
worldwide and an estimated 370,000 women die from breast cancer. Although the vast
majority of fatal breast cancer cases involve metastatic spread of the primary tumor, the
formation of metastases is still a poorly understood, complex process. Identifying the
early molecular changes that facilitate metastasis of breast cancer will lead to new
molecular targets for prevention of metastases and improved therapies.
Intriguing data from the mentor(cid:146)s laboratory show loss of activation of the
transcription factor Stat5 during breast cancer progression and that tumors without active
Stat5 have higher histological grade, increased mitotic rate, and unfavorable prognosis.
Furthermore, data developed in the preparation of this dissertation indicate a substantial
growth-inhibitory and pro-differentiation role for Stat5 in mammary epithelial cells.
Based on these and other recent observations, we postulate that loss of Stat5 activation in
breast cancer represents a progression event that leads to dedifferentiation and increased
risk of metastatic invasion. Therefore, a critical analysis of the role of Stat5 in human
breast cancer is warranted, including systematic efforts to identify genes directly
controlled by Stat5.
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The recent completion of the human genome sequence presents new opportunities
for global identification of Stat5 target genes. Work performed in the preparation of this
dissertation has established new methodology to capture, clone, sequence, and validate
physiological Stat5 DNA-binding sites in a genome-wide manner. The method can also
be used to determine whether Stat5 interacts with a known Stat5-responsive promoter
within a given experimental context when coupled with PCR amplification of the target
DNA. Using this methodology, we have demonstrated that glucocorticoids markedly
alter the pattern of chromatin access for Stat5 binding in breast cancer cells.
Furthermore, we also report novel methodology to specifically identify transcripts
directly regulated by Stat5, based on dominant-negative, differential suppression of Stat5
regulated transcripts and large-scale gene chip analysis. As a result of this work, rapid
progress in genome-wide identification of Stat5 target genes is now possible, as well as
molecular mapping of the regulatory role of Stat5 in breast cancer.

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TECHNOLOGIES FOR GENOME-WIDE IDENTIFICATION
OF STAT5 REGULATED GENES

By
Matthew J. LeBaron

Dissertation submitted to the Faculty of the Department of Pathology Graduate Program
of the Uniformed Services University of the Health Sciences in partial fulfillment of the
requirements for the degree of Doctor of Philosophy 2003
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ACKNOWLEDGEMENTS

This dissertation would not have been possible without the wisdom, guidance, and
support of my advisor, Dr. Hallgeir Rui. Words are not sufficient to describe the infinite
dedication, insight, and patience he has displayed working with me the last four years. I
can never begin to thank him enough for all that he has given me and all that I have
learned from the stellar example he set before me.

My success in completing this dissertation is due in no small part to Dr. Jianwu Xie, who
was indefatigable in guiding me through laboratory practices and protocols, answering
my many questions, and providing a role model for me to emulate.

The members of the Rui lab, past and present, were also instrumental in helping me
achieve success in the laboratory. Drs. Marja Nevalainen and Hiroko Yamashita deserve
special note for the immeasurable assistance they gave me, especially as I was starting
out my graduate laboratory career.

I cannot thank Dr. Mary Lou Cutler enough for all that she has done for me throughout
my years at USUHS. She allowed me the opportunity to rotate in her lab, guiding me
through my first laboratory experience with patience, skill, and knowledge. I also extend
my gratitude for the countless hours she spent chairing both my Qualifying Examination
Committee and my Dissertation Defense Committee.

Many thanks to the professors who generously served on my Qualifying Exam
Committee and my Dissertation Defense Committee: Drs. Richard Conran, Gabriela
Dveksler, Phillip Grimley, Elliot Kagan, and Aviva Symes. Thank you for your time,
guidance, and advice.

My parents, James and Luann LeBaron, have always been supportive and encouraging of
everything I have endeavored. Since anything written would be significantly understated
(cid:150) enough said.

To Kristen Catlin-LeBaron, who was with me through this whole journey from the first
day of orientation. It was through her guidance, expertise, and instruction that I was able
to create many of the figures in this dissertation. Thank you from the bottom of my heart
for everything including writing these acknowledgements since I probably would have
skipped it, which would have been wholly inappropriate.

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TABLE OF CONTENTS

BACKGROUND................................................................................................................1

Biology of the Breast......................................................................................................3
Hormonal Influences...................................................................................................3
Biology of Stat Transcription Factors.............................................................................5
Structure......................................................................................................................5
Stat Function...............................................................................................................9
Stat Family Conservation..........................................................................................11
Stat5..........................................................................................................................13
Stat5 Activation........................................................................................................16
Regulation of Stat5 Activation in Mammary Epithelial Cells......................................19
Prolactin....................................................................................................................21
Prolactin Receptor.....................................................................................................22
Janus Kinases (Jaks).................................................................................................25
Molecular Analysis of Signal Transduction.............................................................30
Stat Transcription Factors in Cancer.............................................................................32
Hematopoietic Malignancies....................................................................................33
Solid Tumors.............................................................................................................34
Characteristics of Breast Malignancies.....................................................................37
Role of Prl-Jak2-Stat5 Signaling in Mammary Physiology..........................................39
Clinical Implications of Stat5 Activation in Breast Cancer......................................41
Role of Stat5 in Mammary Epithelial Cell Transformation and Progression...............45
Chromatin Structure/Environment................................................................................47
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Identification of Stat5 Binding Sites.............................................................................50
Messenger RNA evaluation......................................................................................51
Genome Wide Analysis............................................................................................54
RESULTS.........................................................................................................................57

CHAPTER I:.....................................................................................................................58
ROLE OF PRL-JAK2-STAT5 SIGNALING IN MAMMARY EPITHELIAL CELL
DIFFERENTIATION AND GROWTH...........................................................................58

Introduction...................................................................................................................58
Results...........................................................................................................................60
Prolactin-Induced Differentiation of HC11 Mouse Mammary Epithelial Cells
Correlated With Activation of Tyrosine Kinase Jak2...............................................60

Jak2 Antisense Blocked Prolactin-Induced Differentiation of Stably Transfected
HC11 Clones.............................................................................................................63

Construction and Functional Testing of a Dominant-Negative Jak2 Mutant...........65
Adenoviral Delivery of Dn-Jak2 into HC11 Cells Blocked Prolactin-Induced
Differentiation...........................................................................................................68

Disruption of Jak2 Activity was Associated with Inhibition of Stat5a Tyrosine
Phosphorylation in HC11 Cells................................................................................71

Targeted Inactivation of Jak2 in HC11 Cells Resulted in a Hyperproliferative
Phenotype..................................................................................................................73

Targeted Inactivation of Jak2 in HC11 Cells Resulted in Decreased Contact
Inhibition...................................................................................................................75

Inhibition of Jak2 Suppressed Apoptosis of HC11 Cells Induced by Anchorage-
Independent Culture Conditions...............................................................................78

The Hyperproliferative Phenotype Resulting from Jak2 Suppression in HC11 Cells
was Associated with Constitutive Activation of Stat3..............................................80

Discussion.....................................................................................................................83
Jak2 as a Mediator of Prolactin-Induced Differentiation..........................................84
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Evidence That Jak2 Phosphorylates and Activates Stat5 in HC11 Cells..................85
Targeted Inactivation of Jak2 Resulted in Hyperproliferative Phenotype of HC11
Cells..........................................................................................................................86

Materials and Methods..................................................................................................87
Hormones and Antibodies.........................................................................................87
HC11 Cell ex vivo Model of Mammary Epithelial Cell Differentiation..................87
Expression vectors....................................................................................................88
Construction of V5/His Epitope-Tagged Wild-type (Wt) and Dominant-Negative
(Dn) Jak2 Expression Vectors...................................................................................88

Antisense Jak2 Construct and Generation of Stably Expressing HC11 Clones........90
Cell Culture and Transient Transfections.................................................................91
Protein Solubilization, Immunoblotting, and Immunoprecipitation.........................92
Dominant-Negative Jak2 Recombinant Adenovirus.................................................93
Flow Cytometry........................................................................................................93
TUNEL Assay...........................................................................................................94
Anchorage Independent Survival Analysis...............................................................94
Anti-phosphotyrosine-Stat5 Immunocytochemistry.................................................95
CHAPTER II:....................................................................................................................96
DEVELOPMENT OF A METHOD FOR GENOME-WIDE IDENTIFICATION OF
STAT5-CHROMATIN INTERACTION SITES.............................................................96

Introduction...................................................................................................................96
Results/Discussion........................................................................................................99
Initial Cell-free Developmental Experiments...............................................................99
Conceptual Development..........................................................................................99
Antibody Selection/Immunoprecipitation Optimization.........................................101
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