TRANSCRIPTIONAL REGULATION OF ESTROGEN RECEPTOR ALPHA
TARGET GENES BY HEXAMETHYLENE BISACETAMIDE-INDUCIBLE GENE
1 (HEXIM1) AND ITS ROLE IN MAMMARY GLAND DEVELOPMENT AND
BREAST CANCER
by
NDIYA OGBA
Submitted in partial fulfillment of the requirements
For the degree of Doctor of Philosophy
Dissertation Adviser: Dr. Monica M. Montano
Department of Pharmacology
CASE WESTERN RESERVE UNIVERSITY
January, 2010
CASE WESTERN RESERVE UNIVERSITY
SCHOOL OF GRADUATE STUDIES
We hereby approve the thesis/dissertation of
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candidate for the ______degree *.
(signed)______(chair of the committee)
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(date) ______
*We also certify that written approval has been obtained for any proprietary material contained therein. TABLE OF CONTENTS
Title page i
Signature sheet ii
Table of contents 1
List of Tables 4
List of Figures 5
Acknowledgements 8
List of abbreviations 9
Abstract 15
CHAPTER I: Introduction, review of literature and statement of purpose
Introduction 17
Review of literature 18
Statement of purpose 47
CHAPTER II: HEXIM1 regulates 17β-estradiol/Estrogen Receptor-α- mediated expression of cyclin D1 is modulated by P-TEFb in mammary cells
Abstract 50
1 Introduction 51
Materials and methods 54
Results 60
Discussion 70
Acknowledgments 74
Figures and Tables 76
CHAPTER III: HEXIM1 modulates vascular endothelial growth factor expression and function in breast cancer cells
Abstract 97
Introduction 98
Materials and methods 100
Results 106
Discussion 115
Acknowledgments 119
Figures 120
CHAPTER IV: Summary and future directions
Summary 133
Future directions 136
2 Concluding remarks 154
BIBLIOGRAPHY 155
3 LIST OF TABLES
Table II-1. Primer sequences used for transgenic mice
genotyping 93
Table II-2. Primers used for reverse transcriptase PCR
(RT-PCR) reactions 94
Table II-3. miRNA oligonucleotide sequences used for
HEXIM1 knockdown experiments 95
Table II-4. Primers used in chromatin immunoprecipitation
(ChIP) experiments 96
4 LIST OF FIGURES
Figure I-1. Structures of Estrogen Receptors alpha and beta
with identified activation function regions 21
Figure I-2. Structure of HEXIM1 44
Figure II-1. Increased HEXIM1 expression inhibits estrogen-
regulated mammary gland morphogenesis due to
changes in proliferation and apoptosis 76
Figure II-2. Increased HEXIM1 expression inhibits E2-induced
cyclin D1 expression and serine 2 phosphorylation
of RNAP II in mouse mammary gland 78
Figure II-3. Increased HEXIM1 expression does not inhibit c-Myc
expression in mouse mammary gland 80
Figure II-4. HEXIM1 regulates E2-induced cyclin D1 expression in
breast epithelial cells 81
Figure II-5. Increased HEXIM1 expression inhibits E2-induced
pS2 expression in breast epithelial cells 83
Figure II-6. Increased HEXIM1 expression leads to increase
in HEXIM1 occupancy on DNA of ER target genes 85
Figure II-7. Effect of increased HEXIM1 expression on E2-
dependent recruitment of ERα and P-TEFb (cyclin T1)
to ER-responsive genes 87
Figure II-8. Increased HEXIM1 expression inhibits E2-induced
P-TEFb activity and recruitment of serine 2
5 (hyperphosphorylated) RNA polymerase II to the
coding region of ER-responsive genes 89
Figure II-9. Increased HEXIM1 expression inhibits E2-induced
P-TEFb activity 91
Figure II-10. Proposed model for HEXIM1 action on ERα and
P-TEFb at ER-responsive genes, pS2 and CCND1,
in mammary cells 92
Figure III-1. Increased HEXIM1 expression inhibits E2-induced
transcription of VEGF via ERα in a P-TEFb-
independent manner in breast cancer cells 120
Figure III-2. Increased HEXIM1 expression inhibits E2-induced
VEGF expression in ERα-expressing MDA-MB-231
cells 122
Figure III-3. Increased HEXIM1 expression inhibits E2-induced
VEGF mRNA expression under hypoxia that
correlates with a decrease in E2-induced HIF-1α
expression 123
Figure III-4. Increased HEXIM1 expression inhibits E2-induced
recruitment of HIF-1α to VEGF Hypoxia Response
Element 125
Figure III-5. HEXIM1 modulates VEGF and HIF-1 expression and
vascularization in mouse mammary gland 127
6 Figure III-6. Expression of HEXIM1 C-terminus mutant enhances
carcinogen-induced mammary tumorigenesis and
correlates with increased vascularization of tumors 129
Figure III-7. HEXIM1 C-terminus mutant inhibits P-TEFb activity
and does not affect proliferation in mammary gland 131
Figure III-8. Model: HEXIM1 regulates VEGF expression via ER
HIF-1 to modulate angiogenesis and tumorigenesis 132
Figure IV-1. Summary model of HEXIM1 mechanism of action in
breast cancer 137
7 ACKNOWLEDGEMENTS
I would like to take this opportunity to thank so many people who have guided me during this dissertation process. I was very privileged to work under the supervision and guidance of Dr. Monica Montano. Thank you very much,
Monica, for looking out for my interests and giving me so many opportunities to advance as a graduate student and scientist and helping me grow as a person.
I also want to give many thanks to all the members of my thesis committee, Dr. Yu-Chung Yang, Dr. George Dubyak, Dr. Koh Fujinaga and Dr.
Noa Noy. I am thoroughly grateful for giving me your time, guidance and advice throughout the dissertation process. I am also indebted to the department of
Pharmacology for giving me the opportunity to gain an education here and will always be grateful for all I have learned through my interactions with the faculty, students and staff during my time here at Case.
The support from my lab mates, friends and family cannot really be put into words, so all I will say is thank you. There are so many of you, so I will save the trees and not name everyone. Just know that I thank God for putting all of you in my way so that we could get to know, learn from and support each other. I wish you all Godspeed on your journey and keep up a good attitude when you are able. Lastly, to my dear parents, thank you for being my heroes and supporting my dreams.
8 LIST OF ABBREVIATIONS
7SK snRNA Ubiquitous, non-coding small nuclear RNA
AF Activation Function
AI Aromatase inhibitor
AR Androgen receptor
AP1 Activator protein 1
AhR Aryl hydrocarbon receptor
AIB Amplified In Breast cancer
AKT V-Akt murine thymoma viral oncogene homolog 1
AML Acute myeloid leukemia
BPA Bisphenol A
BRCA1 Breast cancer-1 early onset
CD Cluster of differentiation molecule
CK Cytokeratin
CR Coiled-coil region
CBP Cyclic AMP response element binding protein
CDK Cyclin dependent kinase
9 CLP-1 Cardiac lineage protein-1
CRE Cyclic AMP response element
CSC Cancer stem cell
CTD Carboxy terminal domain
CYP2D6 Cytochrome P450 2D6
CARM1 Co-activator-associated arginine methyltransferase-1
ChIP Chromatin immunoprecipitation assay
COBRA1 Cofactor of BRCA1
DBD DNA binding domain
DPN Diarylpropionitrile
DRB 5,6-dichloro-1-b-D-ribofuranosylbenzimidazole
DMBA 7,12-dimethylbenz[a]anthracene
DRIP Vitamin D-interacting protein
DSIF DRB-sensitivity inducing factor
E1 Estrone
E2 17-beta estradiol
E3 Estriol
10 EP300/p300 E1A-binding protein 300
ERα Estrogen Receptor alpha
ERβ Estrogen Receptor beta
ECM Extracellular matrix
EGF Epidermal growth factor
ERE Estrogen response element
ERK Extracellular signal-regulated kinase
EDG1 Estrogen down-regulated gene 1
EGFR Epidermal growth factor receptor
FDA Food and drug administration
FCP1 TFIIF-interacting CTD phosphatase
GR Glucocorticoid receptor
GTF General transcription factor
GPR30 G-protein couple receptor 30
HAT Histone acetyltransferase
HDM2 Human double minute-2
HIF Hypoxia inducible factor
11 HRT Hormone Replacement Therapy
HSP Heat shock protein
HAND1 Heart and neural crest derivatives expressed 1
HDAC Histone deacetylase
HMBA Hexamethylene bisacetamide
HEXIM1 Hexamethylene bisacetamide-inducible protein 1
HIV-LTR Human Immunodeficiency Virus long terminal repeat
IGF-1 Insulin-like growth factor 1
IGF-1R Insulin-like growth factor 1 receptor
Kd Dissociation constant
MEC Mammary epithelial cell
MAPK Mitogen-associated protein kinase
MMTV-LTR Mouse mammary tumor virus long terminal repeat nM nanomolar
NF-κB Nuclear factor kappa B
NLS Nuclear localization signal
NPM Nucleophosmin
12 NCoR Nuclear receptor corepressor
NELF Negative elongation factor
PR Progesterone receptor
PARP-1 Poly(ADP)-ribose polymerase 1
PCAF p300/CBP-associated factor
PI3K Phosphatidylinositol-3-kinase
PLGA Poly(lactic-co-glycolic acid)
PPAR-γ Peroxisome proliferation activated receptor gamma
P-TEFb Positive transcription elongation factor b
REA Repressor of ERα activity
RTK Receptor tyrosine kinase
RNAP II RNA polymerase II
SP1 Specificity protein 1
SNP Single nucleotide polymorphism
SRC Steroid receptor complex
SERM Selective Estrogen Receptor Modulator
SMRT Silencing mediator for retinoid and thyroid hormone
13 receptor
STAT Signal Transducer and Activator of Transcription
TFF1 Trefoil factor 1
TRAP Thyroid receptor-associated protein
VEGF Vascular endothelial growth factor
VEGFR Vascular endothelial growth factor receptor
14 Transcriptional Regulation Of Estrogen Receptor Alpha Target Genes By
Hexamethylene Bisacetamide-Inducible Gene 1 (HEXIM1) And Its Role In
Mammary Gland Development And Breast Cancer
Abstract
by
NDIYA OGBA
Breast cancer is the second leading cause of death in women in the United
States, making the search for more therapeutic targets in breast cancer etiology very significant. This aim of this thesis was to further elucidate the function of hexamethylene bisacetamide-inducible protein 1 (HEXIM1) in estrogen (E2)- mediated signaling and mammary gland development and tumorigenesis. In previous work, our laboratory has shown that HEXIM1 inhibits breast cell growth and estrogen receptor-alpha (ERα) transcriptional activity. Other studies have shown that HEXIM1 inhibits the activity of positive transcription elongation factor b (P-TEFb) with functional consequences for gene expression. However, it is not clear whether HEXIM1 regulation of all genes is P-TEFb-dependent and how the
P-TEFb-inhibitory function of HEXIM1 ties into mammary gland development or tumorigenesis—which is the main focus of the work described therein.
15 We demonstrate that HEXIM1 regulates E2-driven transcriptional activity of
ERα and P-TEFb within the context of some ERα target genes. P-TEFb inhibition correlated with a decrease in expression of ERα target genes, cyclin D1 and pS2. Cyclin D1 plays a major role in the cell cycle and tumorigenesis. In the mammary gland, we found that increased HEXIM1 expression decreased ductal branching, an E2/ERα-driven process and decreased cyclin D1 expression.
Additionally, recent work in our laboratory uncovered a novel role for
HEXIM1 during heart and vascular development via the regulation of the vascular endothelial growth factor (VEGF). VEGF is a cytokine that regulates the formation of blood vessels and it represents a major target for tumor therapy. In this thesis, we demonstrate that HEXIM1 regulates VEGF expression in a P-
TEFb-independent manner in breast epithelial cells by decreasing E2/ERα- induced expression of hypoxia-inducible factor 1 alpha (HIF-1α), a potent inducer of VEGF. In the mammary gland and during tumorigenesis, HEXIM1 plays a role in regulating HIF-1α and VEGF expression and related-angiogenesis.
Overall, the studies provide insight into the role of HEXIM1 in regulating
E2/ERα gene expression in breast cancer cells and in the mammary gland.
Ultimately, we hope that the collective of this work demonstrates therapeutic potential for HEXIM1 given its anti-proliferative and anti-angiogenic functions in vivo.
16 CHAPTER I
INTRODUCTION
In 1896, George Beatson surgically removed the ovaries of a breast cancer patient and observed a decrease in her tumor mass, providing evidence that supported a role for hormones in breast cancer progression [1, 2].
Subsequently, when Ernest Starling coined the word, “hormone” in 1905 for
“chemical messengers in the bloodstream that coordinate growth activities in different parts of the body,” it set the stage for many fundamental discoveries about the nature and mechanism of action of hormones [3]. Presently, we are still uncovering novel aspects of endocrine action and how they contribute to physiological and pathological processes.
Within the last ten years, two cases worthy of note have crossed over the lines of scientific investigation into mass media debate. These include the widely publicized termination of the hormone replacement therapy (HRT) trials with estrogen and progestin by the National Institute of Health and the British Medical
Research Council in 2003 due to the high incidence of stroke and cardiovascular disease and increased risk for breast cancer [4]. More recently, due to advisory reports from the Food and Drug Administration (FDA) and other organizations, the endocrine disruptor, bisphenol A (BPA) is taking center stage as a risk factor for developmental defects in unborn infants and young children and increased risk for carcinogenesis later in life, though this recommendation is not without controversy due to conflicting reports [5, 6].
17 On the other hand, current knowledge about the specific molecular mechanisms by which hormones direct their activities has given rise to effective targeted therapies including the use of drugs like tamoxifen in breast cancer, which target the effects of the estrogen hormone and its cognate receptor [7]. To understand why hormones—with a focus on estrogens—have such an essential impact on pathophysiology and present as an important therapeutic target, it is perhaps best to start by elaborating on its role in normal physiology and development.
REVIEW OF LITERATURE
A closer look at estrogen and its molecular activities
A. Estrogen, Estrogen-like compounds and Estrogen receptors: The basics
Estrogens are a group of steroid hormones synthesized in ovarian cells and that are converted from a testosterone intermediate to a more potent form,
17-beta estradiol (E2) by aromatase [8]. In women with active menstrual cycles, the ovaries generate E2 daily, which is then converted to metabolites, estrone
(E1) and estriol (E3), which have weaker estrogenic activity [9, 10].
Although estrogens are generated locally, they circulate systemically and are essential for a variety of physiological processes in men and women. Some of these processes include the development and maintenance of secondary
18 sexual organs and estrogens exert a variety of biological effects in cardiovascular, musculoskeletal, immune and central nervous systems [8, 9].
Estrogens carry out their biological effects through the estrogen receptors
(ER) alpha (α) and beta (β) [11, 12]. The ER is a member of the nuclear receptor superfamily that binds steroids and other ligands, functioning as a ligand- inducible transcription factor [13]. E2 binds and activates ERs in the nucleus of cells to carry out its genomic activities [14].
Estrogens also activate GPR30, an orphan G-protein coupled receptor to potentiate rapid E2 signaling and some genomic activity [15]. In addition, estrogens initiate non-genomic effects via the interaction of E2-bound ER with cell signaling molecules that activate the mitogen-activated protein kinases
(MAPKs) and phosphatidylinositol-3-kinase/Akt (PI3K-Akt) pathways [9]. Some reports have shown that E2 activates ERs in the plasma membrane and modulates the interaction between of ERs and membrane-associated proteins to also activate the MAPK and PI3K-Akt pathways and trigger cell processes including proliferation and apoptosis [16-18].
The ER is made up of functional domains including: 1.) the A/B domain, which contains activation function-1 (AF-1); 2.) the C domain or DNA binding domain (DBD) which contains two DNA-binding zinc-finger-motifs and a dimerization domain; 3.) the D domain or hinge region, which contains a nuclear localization signal; 4.) the E domain, which contains the ligand-binding site, a second dimerization domain and the AF-2 and 5.) the F-domain, which appears
19 to optimize the transcriptional activity of the receptor [13, 19]. Both ERα and ERβ are highly homologous (~97% amino acid identity) in the DBD but differ significantly in the other domains and these differences are thought to dictate the variation in their affinities for ligands and subsequent transcriptional activities [19-
21] (See Figure I-1 for ERα and ERβ structures comparing domains).
ERα and ERβ have similar high affinities for E2 in the nanomolar (nM) range with a dissociation constant (Kd) of approximately 0.1nM [22]. Other compounds exhibit estrogenic activity including phytoestrogens and environmental estrogenic compounds. Phytoestrogens are plant-derived non- steroidal compounds that are structurally similar to estrogens and include compounds like resveratrol, coumestrol and genistein [23]. Environmental estrogenic chemicals or xenoestrogens are man-made and include bisphenol A
(BPA) and polychlorinated hydroxy biphenyls. Both phytoestrogens and environmental estrogens compete with E2 for binding to ERs, show selectivity for binding to ERα and ERβ, and activate ERs within concentrations ranging from 1 to 1000nM [22]. Both phytoestrogens and environmental estrogens are also thought to cause transient and persisting effects on pubertal development that lead to pathological physiologies manifested in obesity and breast cancer [24,
25].
E2 regulates the expression of ERs in a tissue- or cell-dependent context and target tissues can be categorized as either classical or non-classical E2 target tissues [26]. Classical target tissues have high ERα expression and
20
Figure I-1. Structure of Estrogen Receptors alpha and beta with identified
Activation Function regions (marked in red). ERβ differs significantly from
ERα in the N-terminus region, the ligand binding and hinge domains and these have been suggested to contribute to variations in their function.
Figure I-1 adapted from Jordan VC et al, 2007 J Clin Oncol 25:5815 [27].
21 respond to E2 with increased transcription of ER target genes and they include the mammary gland, uterus, placenta, liver, cardiovascular system, central nervous system (CNS) and bone. Non-classical target tissues on the other hand typically have high ERβ expression and include the prostate epithelium, ovarian follicles, lung, intestinal epithelium and muscle [26]. Consequently, both ERα and
ERβ have distinct non-redundant roles in reproductive tissues and immune, skeletal, cardiovascular and central nervous systems [10].
The physiological functions of the ERs in these tissues are illuminated in mice models. ERα-null animals present with retarded mammary duct development, reduced follicle number in the ovaries and are insensitive to estrogen in the uterus [12]. ERβ knockout (KO) mice have normal mammary ductal development and respond normally to estrogens in the uterus. However,
ERβ KO mice also have reduced ovarian follicle numbers, and present with neurodegeneration in the central nervous system and prostate hyperplasia in male ERβKO mice [9, 12].
Approximately 70% of primary breast tumors in human females are ERα- positive and hormone-dependent, and ERα expression serves as a valuable marker for predictive response to anti-estrogen therapy [28]. Compared to ERα expression, ERβ expression levels are significantly lower in most ERα-positive breast cancers and many studies support a protective role for ERβ in breast and prostate cancers. Several studies have also shown that ERβ is a marker for good
22 prognosis and disease-free survival, particularly in response to tamoxifen treatment [29, 30].
B. Estrogen-driven genomic signaling and Estrogen Receptors
In the absence of ligand, ERs are localized in the nucleus [2, 14] and histone deacetylases (HDACs) generate condensed chromatin at ER target genes, which keep ER target gene promoters in a repressed state [21]. Upon estrogen binding to the ER ligand-binding domain (LBD), ERs dimerize and bind estrogen response elements (EREs) in target genes [2, 9, 13, 16]. In terms of ER dimerization in response to ligand, ERs form either ERα or β homodimers or
ERα/β heterodimers [31-33]. Studies have shown that ERα homodimers and
ERα/β heterodimers bind EREs with similar affinity (~ 2 nM), which is about four times greater than the DNA binding affinity of ERβ homodimers [31].
The consensus ERE is a 13-base pair inverted repeat sequence
(GGTCAnnnTGACC), where “n” represents a random nucleotide, although many
EREs have variations from the consensus [9, 16]. ERs can also bind ER target genes indirectly via the cyclic AMP-like response element (CRE) or specificity protein 1 (SP1), nuclear-factor kappa B (NF-κB), and activator protein 1 (AP-1) transcription factors [34, 35]. At the AP-1 binding site on some gene promoters,
ERβ is known to have repressive functions [26]. ERβ overexpression has also been shown to inhibit ligand-induced ERα transcription in breast cancer cells.
The inhibitory effect of ERβ was attributed to the AF-1 domain in ERβ and the
23 converse was not found to be true for the effect of ERα overexpression on ligand-induced ERβ transcription [36].
Once ER is ligand-bound, 4 of the 12 alpha (α) helices that comprise the
LBD of ERα are rearranged to form a hydrophobic cleft with docking sites for coactivators and most coactivators associate with ER at the AF-2 domain in a ligand-dependent manner [9, 14, 37]. There are two main classes of ER coactivators that have been identified and one class includes members of the steroid receptor complex (SRC)/p160 family: SRC-1, transcriptional intermediate factor 2 (TIF2/SRC-2) and Amplified In Breast cancer 1 (AIB1/SRC3) [9, 16]. The p160 coactivators interact with and recruit other coactivator proteins including
E1A-binding protein 300/cAMP response element binding protein (EP300 or p300/CBP), p300/CBP-associated factor (PCAF), histone acetyltransferases
(HATs) and co-activator-associated arginine methyltransferase-1 (CARM-1), which either bind directly to ER or associate with other coactivators [2, 12, 38-
40].
The other class of coactivators includes the Vitamin D-interacting protein
(DRIP) or thyroid receptor-associated protein (TRAP) or Mediator complex, which has been shown to contain approximately 15 subunits, some of which are similar to those found in the mammalian Mediator complex integral for RNA polymerase
II-mediated transcription [41-43]. The interaction of the DRIP/Mediator complex with ligand-bound ER can be dependent on or occur independently of an LXXLL motif found within the DRIP subunits [41, 42]. Although both p160 proteins and the DRIP/Mediator complex are important for ER transcription, they exhibit
24 reciprocal recruitment patterns to ER target genes and the DRIP/Mediator complex is thought to promote the formation of the pre-initiation complex for subsequent rounds of transcription initiation [44, 45].
The traditional view that E2/ER and its coactivator complexes binds to and remains associated with target gene DNA as long as estrogen was present conflicted with other findings that suggested a transient association of ER and
AIB1, due to hyperacetylation of AIB1 by CBP/p300, which correlated with a decrease in hormone-induced gene activation [46]. Using chromatin immunoprecipitation (ChIP), several groups have elegantly demonstrated the role of various coregulators in the assembly of ER transcription complexes with details on the specific order and timing of ER and cofactor occupancy as they cycle on and off of some ER target genes and the correlation to gene expression under continuous estrogen stimulation [47, 48].
ERα is also phosphorylated on serine residues within the AF-1 domain in response to E2 and ERα phosphorylation is thought to enhance coactivator recruitment and ER-mediated transcription [49, 50]. Phosphorylated ERα serine residues include including Ser104, Ser106 and Ser118 and thus far, the extracellular signal-regulated kinases 1 and 2 (ErK1/2) MAPK appear to be involved in Ser104 and 106 phoshorylation [51]. Additionally, phosphorylated forms of ERα have been detected in some cases of ERα positive breast cancer
[52, 53].
25 Conversely, structural studies have shown that α-helix 12 in the AF-2 domain of ERα undergoes conformational changes unique to the presence of either agonists or antagonists which, in the case of antagonists like tamoxifen and raloxifene, allows the ER to associate with corepressors [14, 37]. Some of these include nuclear receptor corepressor 1 (NCoR1) and silencing mediator for retinoid and thyroid hormone receptor (SMRT or NCoR2) and Repressor of ERα
Activity (REA) which bind to ERα and inhibit its transcriptional activity [14, 37,
54]. Other mechanisms of corepressors include interfering with mRNA processing of ER target genes, inhibition of ER dimerization and ER DNA binding
[28]. In addition, ER is ubiquitinated and rapidly degraded in an E2-dependent manner by the 26S-proteasome [55]. It is generally thought that a combination of the aforementioned mechanisms comes into play to fully inhibit ER transcriptional activity.
C. The transcription cycle: the relationship between RNA polymerase II, P-
TEFb and Estrogen Receptor α
The occupancy of RNA polymerase II (RNAP II) on DNA of ER target genes is also an indicator of transcriptional activity [56, 57]. Because a part of this thesis focuses on ERα and its transcriptional activity, in this section I will summarize the transcription cycle with a focus on RNAP II and the phosphorylation events that are significant for the efficient transcriptional activation of ERα target genes.
26 At the center of transcription is RNAP II, composed of 12 subunits that are conserved throughout eukaryotes [58]. Once general transcription factors (GTFs) are bound to conserved sequences on the target genes, RNAP II is recruited to the start site of transcription and transcription is initiated. This process involves the separation of DNA strands and a disruption of interactions between RNAP II and the promoter [58].
Although the specific mechanisms are not well understood, it is thought that the recruitment of GTF, transcription factor IIH (TFIIH), prevents premature transcription arrest. Subsequently, phosphorylation of the largest subunit of
RNAP II, Rbp1, on the carboxy terminal domain (CTD) by TFIIH-associated kinase, Cdk7, stimulates promoter clearance and transitions RNAP II into the elongation phase of transcription [56].
The mammalian RNAP II CTD contains about 52 heptapeptide repeats of this consensus sequence, YSPTSPS, and the critical phosphorylation events take place on the second and fifth serine residues (Ser2 and Ser5). Studies have shown that the Ser5 phosphorylated form of RNAP II associates with promoter- proximal regions of transcribed genes, whereas the Ser2 RNAP II form associates mostly with the 3’ region of genes, implicating both forms in initiation and elongation phases of transcription, respectively [56]. The RNAP II CTD is phosphorylated on Ser5 by Cdk7 in TFIIH [59].
The elongation factor implicated in the phosphorylation of RNAP II CTD is the positive transcription elongation factor b (P-TEFb), composed of cyclin-
27 dependent kinase 9 (CDK9) and its cyclin T partner. Treatment of mammalian cells with nucleotide analog, 5,6-dichloro-1-b-D-ribofuranosylbenzimidazole
(DRB) inhibits transcription via inhibition of CDK9 activity [56]. To transition to the elongation phase, P-TEFb phosphorylates negative elongation factor, DRB- sensitivity-inducing factor (DSIF) to relieve transcription pausing, and the CTD of
RNAP II on Ser2 [56, 60].
The hyperphosphorylated form of RNAP II (Ser2 RNAP II) is important for the interaction of RNAP II with RNA maturation factors including the capping enzyme, splicing factors and proteins involved in transcript termination and inhibition of P-TEFb is linked to abortive mRNA processing [57, 61-63]. Studies have shown that the phosphorylation of both Ser 2 and Ser5 in RNAP II CTD is required for ER complex cycling off of ER target genes [48]. P-TEFb activation is not required for the transcription of all genes [64], but studies from our laboratory and from other groups have demonstrated that it is necessary for E2-induced ER transcription of some ER target genes [65-67].
Additionally, RNAP II occupancy on DNA does not always correspond with gene expression. Negative transcription elongation factors, negative elongation factor (NELF) and DSIF bind to RNAP II and cooperatively repress gene expression [56]. NELF has been shown to inhibit ERα transcription by stalling
RNAP II on gene promoters [68]. Upon E2 stimulation, RNAP II is phosphorylated on Ser2 and Ser5 by P-TEFb and TFIIH respectively. RNAP II phosphorylation is sufficient to overcome the transcriptional elongation-mediated repression by
NELF and DSIF and phosphorylated RNAP II associates with regions
28 downstream of the promoter of ER target genes despite the concurrent association of NELF and DSIF with gene promoters [65].
Recent studies have revealed that Ser7 also is phosphorylated and correlates with optimal RNAP II transcriptional elongation activity. However, additional studies are necessary to identify the kinase involved [69].
The termination phase of the transcription cycle is the least well understood of all the phases, but it is known that TFIIF-interacting CTD- phosphatase, FCP1, dephosphorylates RNAP II CTD and unphosphorylated
RNAP II is thought to be recycled for another round of transcription [56, 57, 70,
71].
Although E2 induces the transcription of target genes through ERs, other factors can also activate ERs and induce transcriptional activation of ER target genes. I will briefly review some of these factors and their mechanism of action.
D. Ligand-independent ER transcription
Factors other than estrogens including insulin-like growth factor-1 (IGF-1) and epidermal growth factor (EGF) can activate ERs by stimulating the phosphorylation of the receptor and initiate ligand-independent ER-mediated transcription [9, 16]. However, the ER transcriptional complex can significantly differ at the ER target gene when the stimulus is IGF-1 compared to E2 [72]. In addition, it has been shown that E2 and IGF-1 differentially regulate the expression of ER target genes, cyclin D1 and c-myc, to cooperatively stimulate breast cancer cell proliferation [73].
29 During mammary gland development, IGF-1 and E2 are required for ductal morphogenesis [74]. Overexpression of both IGF-1 receptor (IGF-1R) and members of the epidermal growth factor receptor (EGFR) family including HER2 are linked to the development of mammary cancers and associated with antiestrogen resistance in both ER positive and negative breast cancers [74, 75].
E. Membrane-associated Estrogen Receptors and non-genomic E2 signaling
Another facet of estrogen action includes rapid, nongenomic mechanisms, which involve estrogen-bound ER interacting with and activating signal cascades at the cell membrane or non-ER estrogen-bound proteins in the plasma membrane [2, 12, 76]. These pathways include the activation of MAPK and
PI3K/Akt signaling [35].
Thus far, the only member of the G-protein coupled receptor family that has been linked to the action of E2 is G-protein coupled receptor 30 (GPR30) [15,
77]. GPR30 has been also localized to the endoplasmic reticulum and mediates
E2-driven EGFR and MAPK signaling [78, 79]. GPR30 is widely expressed in a highly tissue and cell-specific manner in the male reproductive tract, prostate and adipose tissues and skeletal and cardiac muscles. GPR30 is also overexpressed in breast and ovarian cancers and is thought to be indicative of an aggressive phenotype [15, 80].
One way that the paths of genomic and non-genomic E2 signaling events converge is elucidated through the interaction of ERα with Signal Transducer and
30 Activator of Transcription (STAT) factors, which belong to a family of transcription factors that mediate cell proliferation and differentiation [81]. A recent report showed that ERα interacts with STAT proteins, 5b and 5c, which requires EGFR activity and activates STAT-mediated transcription [81]. E2 also induces the expression of ER target genes, cyclin D1 and c-myc via an increase in the recruitment of STAT5b, ERα, and phosphorylated RNAP II to cyclin D1 and c- myc promoters [81].
In animal mammary tumor models and human breast cancer, overexpression of HER2 is associated with more aggressive phenotypes and resistance to antiestrogen therapy [82]. Since HER2 acts in part via MAPK and
PI3K-Akt pathways, through which E2 also mediates some effects, a better understanding of how genomic and nongenomic ER signaling mechanisms converge is likely to yield new therapeutic targets in ER-positive breast cancers
[83].
While the estrogen signaling genomic and non-genomic pathways contribute to pathophysiological processes in breast cancer, they also play major roles in mammary gland development. The following section will expand on the role of estrogens and the estrogen receptors in mammary gland development.
Estrogen, Estrogen Receptors and Mammary Gland Development
All processes during mammary gland development are hormonally regulated and receptors for estrogen and progesterone are present in mammary
31 epithelial cells (MECs) of neonatal humans and rodents [84]. In addition, the transcriptional activity of ER changes during differentiation of mammary epithelial cells depending on whether it is associated with coactivators or corepressors [8].
Histomorphological changes during pubertal development of the mammary gland have been extensively characterized in mice so major phases associated with
ERs in development will be described using this model.
At birth, the mammary tissue consists of an epithelial rudiment or anlage embedded in stromal tissue [12]. At puberty, which occurs at approximately 4 weeks of age, ovarian hormones including estrogen induce the outgrowth of ductal structures, which become enlarged and form terminal end buds (TEBs).
The TEB is a major site of mitosis and allows for ductal elongation and eventual filling out of the mammary fat pad. Ductal elongation is induced by estrogen and growth hormone [13]. The outermost layer of the TEB is made up of a single layer of cap cells that lack polarity and are loosely adherent with each other. As the duct develops, cap cells can also migrate inward toward the lumen and become body cells that eventually make up the ductal epithelium [84].
Initially, it was thought that stromal ERα expression was sufficient to promote mammary gland growth [85], but recent studies in mice with an epithelial specific ablation of ERα in the mammary gland showed that epithelial ERα and not stromal ERα is essential for ductal branching and elongation [86].
Another hormone necessary for TEB formation is progesterone but it appears to be involved in the development of tertiary side branches and
32 lobuloalveolar development once the effects of estrogen and growth hormone have taken place [13, 87]. Furthermore, ERα knock-out mice do not undergo normal ductal development [13, 88] and ovariectomy-abrogated ductal growth can be restored by the introduction of exogenous estrogens [89]. On the other hand, ERβ or progesterone receptor (PR) knockout mice develop full ductal epithelial structures during puberty [12].
Other hormones including prolactin and luteinizing hormone and their cognate receptors come into play to regulate the development of the mammary gland during pregnancy and its subsequent involution [84].
Estrogens and breast cancer
A. Classification, progression and risk factors
Classification and progression: The exact etiology of breast cancer is unknown but strong determinants include a lifetime exposure to estrogens and hereditary factors [90]. Breast cancer involves a progression through defined pathological and clinical stages starting with ductal hyperproliferation, subsequent in situ and invasive carcinomas and finally, metastatic disease [90].
Histologically, they are classified as either low- or high-grade tumors as the tumors progress and typically, ER positive breast cancers correlate with ductal carcinomas whereas, loss of ER expression with increased HER2 expression is characteristic of invasive phenotypes [91-93].
33 Additionally, breast cancer is a heterogeneous disease with subtypes that present with varied molecular and clinical phenotypes, as well as varied therapeutic sensitivity [28, 94]. Currently, five molecular breast cancer subtypes include normal breast-like, luminal A (ERα positive, low histological grade), luminal B (low ERα expression, high histological grade), HER2 (amplification and overexpression of ERBB2 gene) and basal-like or triple-negative breast cancers
(negative for ERα, PR and HER2) [93, 94].
Luminal A breast cancers are responsive to antiestrogen therapy, but sensitivity decreases in luminal B subtypes due to lower ERα expression levels and possible increased HER2 expression [93]. In the HER2 subtype which accounts for 30% of all breast cancers, HER2 or ERBB2, a member of the
Epidermal Growth Factor Receptor (EGFR) family, is overexpressed and there is a significant correlation between HER2 overexpression and decreased survival of breast cancer patients [95]. HER2 overexpression also inversely correlates with
ER expression and correlates with a lack of response to endocrine therapy and chemotherapeutic agents. Trastuzumab, a monoclonal antibody against HER2, was developed and approved by the FDA in 1998 for the treatment of HER2 overexpressing cancers in the U.S. [93, 95].
BRCA1 (breast cancer-1, early onset) is a tumor suppressor gene important in the maintenance of chromosome stability, activation of cell-cycle checkpoints, and DNA repair. BRCA1 has also been shown to interact with and inhibit ERα transcription [96]. In basal-like breast cancers, BRCA1 germline mutations frequently occur. Additionally, basal-like cancers express high levels of
34 basal markers including cytokeratin (CK) 5 and 6, EGFR and extracellular matrix
(ECM) receptor, α6β4 integrin [93, 97]. Basal-like breast cancers generally have poor prognosis because they are aggressive and do not respond to antiestrogens or anti-HER2 antibodies [96]. Current therapeutic strategies include use of DNA- damaging agents to induce double-stranded DNA breaks (for example: anthracyclines and etoposide) and kinase inhibitors including imatinib to curb
EGFR signalling [97].
Risk factors: Epigenetic mechanisms linked to decreased ER expression in breast cancer include ER mutations, deletions, loss of heterozygosity, hypermethylation of ER promoters, and irregular chromatin remodeling via HDAC activation [98]. Additionally, variations in gene sequence or single-nucleotide polymorphisms (SNPs) in the ERα gene are known to contribute to increased risk for breast cancer [76].
Although estrogens have been shown to have contrasting effects on breast cancer in different studies [99, 100], it is widely accepted that breast cancer occurs as a result of the combined influence of genetic susceptibility and exposure to carcinogens, environmental and endogenous estrogens [101]. Other risk factors include early menarche (menstruation begins at < 12 years), late menopause (menopause occurs after 55 years), post-menopausal obesity, hormone-replacement therapy and alcohol consumption [99, 101].
Conversely, it is thought that early-full term pregnancy and multiparity decrease lifetime breast cancer risk in women of all ethnic groups depending on
35 the breast cancer subtype and except when they are carriers of breast cancer susceptibility genes including BRCA1 [94]. Paradoxically, pregnancy is associated with a transient increase in breast cancer risk in young women (under age 35 or 45). The prognosis is worse 2 to 5 years after women give birth, even after prognostic determinants have been accounted for including ER, progesterone receptor (PR), HER2 and p53 expression [94, 102]. Suggested risk factors that may account for this include infrequent mammography screening, elevated levels of insulin and melatonin during pregnancy, and the largely unexplored contribution of the mammary gland stromal compartment and cancer stem cells [94].
Several studies are making associations between distinct stem cell populations and the involvement of the tumor microenvironment at each stage of tumorigenesis [103, 104]. The cancer stem cell (CSC) hypothesis states that cancers are driven by cellular components that have stem cell properties including self-renewal, tumorigenicity and multi-lineage differentiation capacity
[105]. CSCs are also thought to contribute to resistance to chemotherapy in breast cancer [90, 105]. Identification of CSC surface markers including cluster of differentiation molecule 44 and 24 (CD44 and CD24) has created a broader classification scheme of breast cancer subtypes based on their stem cell lineage and ER expression is significantly absent in CSCs [105]. Thus far, studies on
CSCs have been done in mice, but a recent study on human breast tumor samples indicated that CD44 expression was increased in tumors post- chemotherapy in ER positive invasive carcinomas. However, no change in CD44
36 expression was observed in triple negative breast cancers before or after chemotherapy, suggesting that increased CSC proliferation contributes to resistance to chemotherapy [106].
The tumor microenvironment contains adipocyte, vascular, myoepithelial, fibroblasts and immune cells, all of which have been shown to influence tumorigenesis [107]. Myoepithelial cells generally exert negative effects on tumor cell growth, invasion and angiogenesis, and fibroblasts have been demonstrated to be tumor-promoting cells [108]. Although ER expression is typically decreased in metastatic breast cancer, during tumorigenesis E2 and ER play significant roles in regulating angiogenesis and inducing chemokine expression in endothelial cells to drive tumor growth [8, 109].
B. Estrogen-associated molecular targets in breast cancer and current therapeutic strategies
Based on what is known about the determinant factors that influence breast cancer progression, several therapeutic strategies are being developed or are currently being used to treat ER positive or estrogen-responsive breast cancers and a few of them are outlined below.
Selective Estrogen-Receptor Modulators (SERMs): Tamoxifen was the first
SERM to be developed and used in clinical applications, mostly in the treatment of breast cancer [21]. Tamoxifen competes with E2 for binding to ER and it has agonist or antagonist-like effects on ER depending on the target gene and the cell context [21]. As an antagonist, it binds ERα and the receptor recruits
37 corepressors and HDACs to target gene promoters in mammary cells [48].
However, in endometrial and bone cells, tamoxifen binds ERα and induces the recruitment of coactivators, thereby exhibiting estrogen-like activity. Other
SERMs include raloxifene, which has bone-protective effects and is inhibitory on breast cancer cell growth, without any growth-promoting activity in the uterus
[21]. Coregulators are thought to play a major role in the gene- and cell-context dependent functions of SERMs [27, 76].
However, approximately 40 to 60% of tumors that are ER positive and PR negative are not responsive to antihormonal therapy due to metabolic and intrinsic or acquired resistance [27]. Genetic variations in the cytochrome P450 enzyme 2D6 (CYP2D6) that converts 4-hydroxytamoxifen to active metabolite, endoxifen, can contribute to metabolic mechanisms of SERM resistance [27].
Intrinsic resistance occurs when ER-positive breast cancers are initially unresponsive to tamoxifen treatment and acquired resistance can occur when changes in ER signal transduction pathways convert inhibitory SERMs into growth stimulatory signals [110].
Aromatase inhibitors (AIs): Conversion of androgen to estrogens occurs in adipose tissue, muscle, liver and the breast tumor in post-menopausal women
[35]. AIs act by inhibiting the enzyme, aromatase, and preventing the formation of estrogens from precursor molecules, androgens, androstenedione and testosterone. Current AIs approved by the FDA for use as adjuvant treatment with tamoxifen and other chemotherapeutic agents include exemstane, anastrazole and letrozole [35].
38 Cyclin D1 and cyclin dependent kinase inhibitors: Cyclin D1 is a member is the family of D-type cyclins that associate with a variety of cyclin dependent kinases (CDKs), including Cdk4 and Cdk6, and are involved in the regulation of the G1-S phase of cell cycle division in mammals [111]. Mice lacking cyclin D1 are small and have defects in mammary gland and retinal development [112].
Overexpression of cyclin D1 in the mammary gland leads to hyperplasia and carcinogenesis in mice, and cyclin D1 is implicated in both ER- and HER2- mitogenic signaling [95, 112, 113]. The regulation of cyclin D1 transcription by E2 and ERα is known to occur through CRE or AP-1 sites because a classical ERE is absent in the cyclin D1 promoter [114].
Cyclin D1 is also overexpressed in approximately 40% of invasive breast cancers, and ERα expression positively correlates with high cyclin D1 expression levels in some breast cancers [115]. There are cases where cyclin D1 overexpression does not necessarily correlate with increased proliferation in breast cancer [113], which could indicate that the tumors are dependent on other genes for proliferation [116]. However, it has been widely demonstrated that cyclin D1 is a good prognostic factor associated with better patient outcome especially for ER-positive patients [117].
The growth-promoting activities of cyclin D1 are linked to associated-
CDKs. Other transcription factors like P-TEFb carry out their function through
CDKs. Thus, CDKs represent a therapeutic target for breast cancer. Flavopiridol is one CDK inhibitor that has been developed and extensively studied.
Flavopiridol is a synthetic flavone that inhibits several kinases including CDK9 in
39 P-TEFb, in a dose-dependent manner and has been reported to decrease cyclin
D1 expression in breast cancer cells [118]. In acute myelogenous leukemias, flavopiridol has proven to be effective when used in combination with other therapeutic agents [119]. However, in breast cancer, the utility of flavopiridol is yet to be realized and clinical trials with flavopiridol in combination with adjuvant therapies are currently in progress.
Histone deacetylase (HDAC) inhibitors: Histone proteins organize DNA into nucleosomes, which are repeating structures of chromatin [120]. The acetylation status of histones is dictated by the activities of histone acetyltransferases
(HATs) and HDACs, which alters chromatin structure and influences activation or repression of gene expression respectively. HDACs keep histones deacetylated and chromatin in a tightly packed state, which leads to a repression of gene expression. In addition, aberrant HDAC activity is associated with the development of cancer. [120].
HDAC inhibitors are currently being investigated as a viable therapeutic option [120, 121], because it is thought that the use of HDAC inhibitors alone or in combination with existing therapies could be an efficient avenue for reversing hormone therapy resistance by re-sensitizing tumors to antiestrogens. In the case of ER, part of the rationale for this comes from evidence that HDAC1 overexpression inhibits ERα transcriptional activity [122] and a retrospective study showed that HDAC6 expression was associated with increased survival of breast cancer patients who had hormone-responsive tumors and underwent treatment with tamoxifen [123]. Some of the different HDACs currently under
40 investigation in clinical trials include vorinostat, panobinostat and valproic acid
[121], though they have only proven to be effective in the case of hematological malignancies and not solid tumors.
Angiogenesis: As tumors progress from hyperplasia to invasive carcinomas, tumor survival is also dependent on the development of new blood vessels or angiogenesis [95]. A major angiogenic factor that has been investigated in this context as a therapeutic target is vascular endothelial growth factor (VEGF).
VEGF is a growth-promoting and survival factor for vascular endothelial cells and it mediates its effects through two known receptor tyrosine kinase (RTK) VEGF receptors (VEGFRs), VEGFR-1 or fms-related tyrosine kinase 1 (Flt-1) and
VEGFR-2 or kinase-insert domain receptor (KDR) [124, 125]. Mice deficient in
VEGF and its cognate receptors die in utero and VEGF is commonly overexpressed in many cancers including breast cancer [125-127]. In preclinical models, breast cancer cells that have increased VEGF expression have higher growth potential and increased metastatic potential in xenograft models [127].
Some studies have shown that E2 has angiogenic potential and mediates its effects via an increase in VEGF expression and other studies have shown that increased VEGF expression attenuates the anti-proliferative activity of anti- hormones [8, 109, 128]. This suggests that E2 induces VEGF and contributes to vascularization in hormone-responsive cancers. Another positive VEGF inducer is hypoxia-inducible factor-1 (HIF-1). HIF-1 is composed of a dimer of HIF-1α and
β. Whereas HIF-1β is constitutively expressed, HIF-1α is rapidly degraded under normal oxygen conditions. Under low oxygen conditions, HIF-1α expression is
41 stabilized, and HIF1-α and β heterodimerize to drive transcription of HIF-1 target genes including VEGF [129]. Estrogens have been shown to regulate HIF-1α expression and there is a positive correlation between ERα and HIF-1α expression in some breast cancers [130]. In a clinical setting, this is relevant because during tumor progression, internal regions within tumors are frequently hypoxic and drive the upregulation of HIF-1 to induce VEGF expression, resulting in enhanced tumor vascularization, breast tumor cell survival and metastasis
[131, 132]. Thus, HIF-1 also represents a potential direct therapeutic target in
VEGF-targeted therapy, although more studies are necessary to validate the benefits of this approach.
Clinical trials with a humanized anti-VEGF monoclonal antibody, bevacizumab (Avastin), in combination with other chemotherapeutic agents has met with considerable success and is currently approved by the FDA as an adjuvant treatment of breast cancer [126, 133]. Clinical trials are also in progress with VEGFR inhibitors [133, 134].
Thus far, I have outlined the characteristics and importance of estrogens and ERs in mammary gland development and breast cancer. To draw more apparent links between all the literature reviewed and this thesis, it is important to introduce the main factor of interest, hexamethylene bisacetamide-indcuble protein 1 (HEXIM1) and expand on our interest in this factor.
42 Hexamethylene-inducible gene 1 (HEXIM1) or Estrogen down-regulated
gene 1 (EDG1)
Identification, Structure and function: HEXIM1 was originally identified as a hexamethylene bisacetamide (HMBA)-inducible gene in vascular smooth muscle cells that suppressed the transcriptional activity of NF-κB [135, 136].
Concurrently, HEXIM1 was identified in our laboratory as an ERα-interacting protein and in other groups as a 7SK small nuclear (sn) RNA-dependent P-TEFb inhibitior [137-139]. 7SK snRNA is an ubiquitously expressed 331-nt snRNA that is conserved amongst vertebrates and identified as a P-TEFb inhibitor [140].
The HEXIM1 cDNA clone has an open reading frame of 1077 bp and is expressed as a 359 amino acid protein in the nucleus of cells [138]. The HEXIM1 protein can be divided into three functional domains: (1) The proline-rich N- terminus that acts as a self-inhibitory domain, (2) a centrally located, basic-rich nuclear localization signal (NLS) that also interacts with 7SK snRNA and (3) the
C-terminus that interacts with and inhibits ERα and P-TEFb activity [62, 138,
141].
The C-terminus can be further subdivided into two coiled-coil regions (CR),
CR1 and CR2 that also mediate HEXIM1 oligomerization [142]. The CR1 region
(amino acids 279-315) is thought to be essential for HEXIM1 binding and inhibiting P-TEFb activity [142] (See Figure I-2 for HEXIM1 structure). The role of
HEXIM1 in transcription has been particularly highlighted in the context of the
Human Immunodeficiency Virus long terminal repeat (HIV-LTR). HEXIM1 has
43
Figure I-2. Structure of HEXIM1. Coiled-coil regions (CR) 1 and 2 are integral to
HEXIM1 inhibition of P-TEFb and ERα activity.
Figure I-2 is adapted from Blazek et al, 2005 Nucleic Acids Res 33:7000 [142].
44 been shown to inhibit the activity of Tat, an essential HIV transactivator via inhibition of P-TEFb activity and recruitment to the HIV-LTR [62, 143]. HEXIM1 has also been shown to interact with and inhibit the transcriptional activity of the glucocorticoid receptor (GR) [144].
HEXIM2, a paralog of HEXIM1, shares some homology (>50% identical) with HEXIM1 beginning at the central NLS and ending at amino acid 343 in the
C-terminus [145]. HEXIM2 can also be induced by HMBA and is expressed as a
286 amino acid protein that also binds to and inhibits P-TEFb activity, although
P-TEFb has been shown to preferentially associate with HEXIM1 [145, 146].
Both HEXIM1 and HEXIM2 form dimers in vivo, and it has been proposed that the dimers function to recruit and inactivate multiple P-TEFb complexes [147,
148].
Deletion of the mouse homolog of HEXIM1, cardiac lineage protein-1
(CLP-1) is embryonic lethal. Analysis of fetuses revealed cardiac hypertrophy to be a major contributing factor to death, accompanied with a decrease in expression of heart and neural crest derivatives expressed 1 (HAND1)—a critical transcription factor involved in cardiac development [149]. In our laboratory, we found that mutation of HEXIM1 in the C-terminus, resulting in expression of
HEXIM1 mutant, HEXIM1 1-312, resulted in significant prenatal lethality due to coronary abnormalities and thin myocardial and ventricular walls that occurred through decrease in VEGF expression [150].
45 HEXIM1 and cancer: In murine leukemia cells, HEXIM1 expression is increased during differentiation [151]. In acute myeloid leukemia (AML), nucleophosmin
(NPM) is a frequently mutated gene, and a recent study linked the expression of
NPM to the regulation of HEXIM1 expression with implications for AML [152]. Our laboratory found that decreased HEXIM1 expression correlates with tumors in human breast cancer when compared to adjacent normal tissue and HEXIM1 expression also correlated with ERα-expressing tumors [138]. We also found that long-term E2 treatment led to a decrease in HEXIM1 expression in breast cancer cells, leading to its initial naming as estrogen down-regulated gene-1 (EDG1)
[138]. Other studies in our laboratory demonstrated that P-TEFb interacts with
ERα via HEXIM1 and this is integral to its inhibitory activity on ERα transcription
[141]. This study was the first to suggest a role for ERα in transcription elongation.
46 STATEMENT OF PURPOSE
The purpose of this dissertation was to two-fold. First, we aimed to characterize the role of HEXIM1 in the regulation of transcriptional elongation during E2/ERα-mediated transcription and provide evidence of the physiologic relevance of this regulation in the mammary gland and breast cancer. This was based on the rationale that HEXIM1 interacts with ERα via cyclin T1 and inhibits
ERα transcriptional activity and thereby inhibits breast cell proliferation [138,
141]. HEXIM1 also inhibits the co-recruitment of ERα and cyclin T1 to the promoter region of pS2, an ERα target gene [141]. The expression of the human
Trefoil factor 1 (TFF1) or pS2 gene is regulated by E2 in MCF-7 breast cancer cells and although there is a positive correlation between pS2 and ER expression in some breast cancers, the function of pS2 in breast cancer is not understood
[153-155]. In gastric mucosal cells, pS2 is secreted in an E2-independent manner and mice lacking pS2 have increased incidence of mucosal carcinomas [156,
157]. However, pS2 has an imperfect ERE sequence and is used as an ER target gene to understand the regulation of ER and associated coregulators [48].
We hypothesized that since HEXIM1 binds to cyclin T1 and inhibits the activity of P-TEFb, this interaction can attenuate E2-mediated ERα transcriptional elongation in cell and animal studies. Using ChIP analyses, we studied the effect of HEXIM1 on the E2-induced recruitment of ERα, P-TEFb and RNAP II to two
ERα target genes, pS2 and cyclin D1 and the relevance of cyclin D1 in the thesis will be expanded on in Chapter II. HEXIM1 expression was also transiently
47 increased or decreased to determine its role in regulating the expression of the
ERα target genes investigated. To investigate the relevance of HEXIM1 regulation of ERα and P-TEFb in physiology, transgenic mice (MMTV/HEXIM1) were also generated in the laboratory that have doxycycline-inducible expression of HEXIM1 in the mammary gland to analyze the effect of increased HEXIM1 expression in the mammary gland.
Our second aim was to characterize the role of HEXIM1 in the regulation of
VEGF in the mammary gland. The rationale for this stemmed from evidence that in breast cancer cells, E2/ERα has been shown to regulate VEGF transcription and we found that the mutation of the HEXIM1 C-terminus (HEXIM1 1-312) led to aberrant VEGF expression that contributed to abnormal vascular development in mice [109, 150]. Therefore, it stood to reason that HEXIM1 could regulate VEGF in the mammary gland, but the mechanism is not known.
The molecular basis for HEXIM1 regulation of VEGF was analyzed by
ChIP assays to determine the effect of HEXIM1 on the recruitment of two positive inducers of VEGF expression, ERα and HIF-1α, to the VEGF promoter. HEXIM1 expression was also increased in breast cancer cells to determine the effect of
HEXIM1 on E2-induced VEGF expression. Mice models generated in our laboratory, MMTV/HEXIM1 and HEXIM1 1-312, were also used to analyze the physiological relevance of the effect of HEXIM1 on VEGF and HIF-1α expression in the mammary gland, and the role of HEXIM1 in tumorigenesis via its effect on angiogenesis.
48 A major obstacle that current therapeutic strategies for breast cancer treatment face is the development of resistance in patients, so a better understanding of the role of novel targets in breast cancer will present other options that can be pursued to possibly enhance patient care. This thesis uncovered novel aspects of the role of HEXIM1 in regulating E2-mediated signaling in transcription elongation and selective HEXIM1 regulation of ERα target genes. Taken together, our studies add more insight into the role of
HEXIM1 in mammary gland and breast cancer and suggest potential for HEXIM1 as a therapeutic target in hormone-responsive breast cancers.
49 CHAPTER II
HEXIM1 regulates 17β-estradiol/Estrogen Receptor-α-mediated expression
of cyclin D1 in mammary cells via modulation of P-TEFb
This work has been published in the Cancer Research journal.
(Ogba, N et al., Cancer Research 2008 September 1; 68(17):7015-7024)
Abstract
Estrogen receptor α (ERα) plays a key role in mammary gland development and is implicated in breast cancer through the transcriptional regulation of genes linked to proliferation and apoptosis. We previously reported that hexamethylene bisacetamide inducible protein 1 (HEXIM1) inhibits the activity of ligand-bound ERα and bridges a functional interaction between ERα and positive transcription elongation factor b (P-TEFb). To examine the consequences of a functional HEXIM1-ERα-P-TEFb interaction in vivo, we generated MMTV/HEXIM1 mice that exhibit mammary epithelial-specific and doxycycline-inducible expression of HEXIM1. Increased HEXIM1 expression in the mammary gland decreased estrogen-driven ductal morphogenesis and inhibited the expression of cyclin D1 and serine 2 phosphorylated RNA polymerase II (S2P RNAP II). In addition, increased HEXIM1 expression in MCF-
7 cells led to a decrease in estrogen-induced cyclin D1 expression, while downregulation of HEXIM1 expression led to an enhancement of estrogen- induced cyclin D1 expression. Studies on the mechanism of HEXIM1 regulation on estrogen action indicated a decrease in estrogen-stimulated recruitment of
50 ERα, P-TEFb and S2P RNAP II to promoter and coding regions of ERα- responsive genes, pS2 and CCND1, with increased HEXIM1 expression in MCF-
7 cells. Notably, increased HEXIM1 expression decreased only estrogen-induced
P-TEFb activity. While there have been previous reports on HEXIM1 inhibition of
P-TEFb activity, our studies add a new dimension by showing that E2/ER is an important regulator of the HEXIM1/P-TEFb functional unit in breast cells.
Together, these studies provide novel insight into the role of HEXIM1 and ERα in mammary epithelial cell function.
Introduction
Mammary gland morphogenesis and development requires input from several genetic and epigenetic pathways regulated by hormones and growth factors including estrogens [11, 13]. Estrogens mediate their actions through estrogen receptors (ERs), ERα, and ERβ, nuclear steroid receptors that classically regulate transcription either by directly binding to estrogen-response elements (EREs) of target genes [12, 20, 76] or indirectly via protein-protein interactions with other transcription factors like SP1 or AP-1 [34]. In both cases, coregulatory proteins are also recruited to the promoter, and together ERs and these factors elicit changes in mRNA and protein levels of ER target genes, and ultimately, a physiological response [12, 34, 76]. Since estrogen signaling controls the balance of growth and apoptosis in normal breast epithelial cells, a disruption of this balance contributes to abnormal cell growth and can lead to tumorigenesis [12, 158]. Therefore, it is important to identify and elucidate the
51 mechanism of action of ERs and their coregulators that give better insight into
ER-mediated transcriptional regulation [159].
In eukaryotic transcription, the elongation stage is highly regulated and important for the generation of full-length mRNA transcripts [56, 57, 160]. One of the positive regulators, positive transcription elongation factor b (P-TEFb) has an essential role in RNA polymerase II (RNAP II) transcription elongation [56, 57]. In many human cell types, the predominant form of P-TEFb consists of cyclin dependent kinase 9 (CDK9) and its regulatory partner, cyclin T1 [160]. It phosphorylates and thereby inhibits the activity of negative elongation factors,
NELF and DSIF (DRB-sensitivity inducing factor) [56]. It also phosphorylates the carboxy-terminal repeat domain (CTD) of the largest subunit of RNAP II [56, 57].
The RNAP II CTD consists of multiple repeats of the heptapeptide sequence,
YSPTSPS, phosphorylated at serine 5 by general transcription factor TFIIH during initiation and at serine 2 by P-TEFb during elongation [56, 57]. These phosphorylation events are crucial for effective transition from an abortive to a productive phase of elongation [62, 160]. P-TEFb is essential for productive HIV-
1 transcriptional elongation and several studies have shown that various transcription factors bind to and recruit P-TEFb to specific promoters stimulating elongation [62, 161].
In previous studies, we identified an ERα-interacting protein, estrogen down-regulated gene-1 (EDG1) (also known as hexamethylene inducible protein
1 (HEXIM1)), and found that it is an inhibitor of ERα transcription and breast cell
52 growth [138]. Additionally, we demonstrated that HEXIM1 expression was lower in human breast tumors when compared to adjacent normal tissue, suggesting a role for HEXIM1 in breast tumorigenesis [138]. Concurrent studies identified
HEXIM1 as a P-TEFb-interacting factor that also inhibits P-TEFb activity [137,
139]. Studies have also shown that HEXIM2, a paralog of HEXIM1, has the same inhibitory effect on P-TEFb [145, 146]. We demonstrated that HEXIM1 modulates a functional interaction between ERα and cyclin T1 in breast epithelial cells, and inhibits the recruitment of ligand-bound ERα (E2/ERα) to the pS2 gene promoter
[141]. We also found that cyclin T1 appeared to be necessary for E2-induction of cyclin D1 protein expression [141]. Cyclin D1 (CCND1) is a D-type cyclin that regulates G1-S cell cycle progression during cell proliferation [162]. CCND1 is also E2/ERα responsive and is thought to play major roles in mammary gland development and breast cancer [13, 115]. However, the contribution of E2/ERα to
HEXIM1 action in breast cells is not well defined. In addition, the precise mechanism by which P-TEFb regulates CCND1 transcription and how this can be linked to mammary gland development and tumorigenesis needs to be further defined.
To address these questions, we developed a transgenic mouse model in our laboratory that is doxycycline-inducible and selectively expresses HEXIM1 in the mammary gland under the control of the mouse mammary tumor virus long terminal repeat (MMTV-LTR) promoter previously described [163]. Using this model, we demonstrate that increased HEXIM1 expression decreased E2- induced CCND1 and serine 2 phosphorylated (S2P) RNAP II expression in the
53 mouse mammary gland. In addition, increasing HEXIM1 expression inhibits E2- induced recruitment of ERα, P-TEFb and S2P RNAP II to ER-responsive genes, pS2 and CCND1 in MCF-7 cells. Finally, we observed that E2 stimulates P-TEFb activity and that HEXIM1 inhibits only E2-induced, and not basal, P-TEFb activity.
These results elucidate the functional consequences of modulating HEXIM1 expression on E2/ERα-driven transcription in the mammary gland and its implications for estrogen-dependent breast cancer.
Materials and Methods
Reagents: 17β-Estradiol (E2) and CDK9 inhibitor, 5,6-dichloro-1-β-D- ribofuranosylbenzimidazole (DRB) were obtained from Sigma Chemical Co. (St.
Louis, MO). Commercially available antibodies were used for immunoprecipitation and Western blot analyses and the HEXIM1 antibody was characterized in previous studies [138, 141].
Transgenic mice (MMTV/HEXIM1) generation: MMTV-rtTA mice were generated as described by Gunther et al [163]. To generate pTET-HEXIM1 mice, a plasmid construct was made by subcloning the coding sequence of human
HEXIM1 gene downstream of the tetracycline-dependent minimal promoter in the pTet-splice transgene construct. After purification, the resulting plasmid was used for pronuclear injection into FVB oocytes (Case Western Reserve University
Transgenic and Targeting Facility). To achieve mammary gland-specific
54 expression of HEXIM1 in a doxycycline-dependent manner, pTET-HEXIM1 mice were crossed with the MTB line, which expressed the reverse tetracycline- dependent transcriptional activator (rtTA) under the control of the mouse mammary tumor virus long terminal repeat (MMTV-LTR). From these matings the bigenic mice, MMTV/HEXIM1, were created. Transgene expression was induced by adding 2 mg/ml doxycycline to the drinking water. Bigenic mice were identified by screening genomic DNA from tail biopsies for the presence of the transgenes using PCR and verified by Western blot analysis. See Table II-1 for list and sequences of primers used.
Whole-mount histology & Immunodetection: Mice were induced with doxycycline at 9 weeks of age, ovariectomized and treated with E2 at 12 weeks.
E2 was administered as daily subcutaneous injections of sesame oil solution containing 1µg of E2. Mammary glands from the MMTV/HEXIM1 mice were collected 25 days after start of E2 treatment for whole-mount staining via the
Carmine-alum technique and Western blot analyses. To generate sections for immunohistochemistry, mammary glands were fixed in 10% formalin, embedded with paraffin, and sectioned (10 µm).
To label proliferating cells, mice were injected with 100 mg/kg body weight
BrdU. Mammary glands were collected 2 h after BrdU injection and embedded in paraffin. Paraffin sections were deparaffinized, rehydrated, incubated in 2 N HCl for 1 hr, treated with 20 ug/ml proteinase K for 10 minutes at room temperature, blocked with 10% normal goat serum, and incubated with a mouse anti-BrdU
55 antibody (PharMingen) for 12-18 hours at 4℃. The antibody was visualized using a biotinylated goat anti-mouse IgG (Vector Laboratories), and the Vector ABC
Elite kit. The sections were lightly counterstained with hematoxylin. Apoptosis was monitored by terminal deoxynucleotidyltransferase-mediated UTP end labeling (TUNEL) staining using the ApopTag® Peroxidase In Situ Apoptosis
Detection Kit (S7100, Chemicon, Temecula, CA).
Immunofluorescent staining was also performed on mammary gland sections from MMTV/HEXIM1 mice (-/+Dox). Antigen retrieval was performed using 10 mM Citrate buffer pH 6.0 and slides were blocked in 3% H202 and a protein blocking solution (10 mg/ml BSA and 1% horse serum) before incubating the sections with the primary antibody, anti-serine 2 and 5 phosphorylated (S2P and S5P) RNA polymerase II (Covance, CA) (1:50) at 4°C overnight. To visualize immunocomplexes, we used Alexa Fluor 594 goat anti-mouse (1:200) (Molecular
Probes). Images were captured using a Leica DMI series (4000 and 6000 B) confocal microscope. The number of S2P- or S5P-postive cells was determined by immunofluorescent labeling and was quantitated from captured images using the Image-Pro Plus software. A minimum of 1000 cells was counted for each lumen structure and a minimum of 2-3 tissue sections per mouse was analyzed.
The number of positive cells is expressed as the percentage of total epithelial cells counted. Three to four animals per treatment group (-/+Dox) were analyzed.
56 Reverse Transcription (RT) PCR Analyses: Human breast epithelial cells,
MCF-7, were maintained as described [141] and were transiently transfected with pCMV-TAG2B or pCMV-TAG2B-HEXIM1 using FuGENE HD reagent (Roche,
IN) according to the manufacturer’s instructions. Forty-eight hours later, cells were treated with ethanol vehicle or 100 nM E2 for 3 hours. Total RNA was extracted using the TRIzol reagent (Invitrogen, CA) and subjected to RT-PCR analyses. Two micrograms of DNase I-treated RNA was used for cDNA synthesis using the MMLV-Reverse Transcriptase kit from Invitrogen according to the manufacturer’s instructions. PCR products were run in 2% agarose gels and visualized by ethidium bromide staining. An eight-bit digital camera captured fluorescence and signal intensities were quantitated using GeneTools software from Syngene (Frederick, MD). Signals from genes of interest were normalized to signals from GAPDH and 36B4 and presented as “relative mRNA levels”. See
Table II-2 for list and sequences of primers used.
Western Analyses: MMTV/HEXIM1 mice were treated as described above and mammary gland tissues were homogenized in 1 ml of Tissue Protein Extraction
Reagent (Pierce, IL) using a polytron homogenizer. MCF-7 cells were treated as described above and total protein was extracted from cells as previously described [141]. Lysates from MCF-7 cells and mammary glands were cleared by centrifugation. The soluble fraction was transferred to a fresh tube and used as protein extract for Western blot analysis as previously described [141].
57 RNA interference: A Pol II promoter-driven miRNA expression vector system
(Invitrogen, CA) was used. To make pcDNA-HEXIM1 miR, miRNA oligos (see
Table II-3 for list and sequences) were annealed and cloned into the pcDNA 6.2
GW/EmGFP vector (Invitrogen) according to the manufacturer’s instructions.
MCF-7 cells were transfected with pcDNA 6.2-GW/EmGFP-miR expression vectors containing either the HEXIM1 miRNA insert or a control LacZ miRNA insert. Following blasticidin selection, cells expressing the highest level of GFP were flow-sorted and expanded. During experiments, cells were treated with ethanol vehicle or 100 nM E2 for 3 hours and harvested as described above for
Western blot analyses.
Chromatin immunoprecipitation (ChIP) assays: MCF-7 cells were plated on
150 mm plates and transfected as described with pCMV-TAG2B or pCMV-
TAG2B-HEXIM1. Forty-eight hours later, cells were treated with ethanol vehicle or 100 nM E2 for 45 and 90 minutes. ChIP assays were carried out as previously described [141]. For hyperphosphorylated RNA polymerase II antibodies (H5 and
H14), immune complexes were collected with 60 µl of Protein G slurry preadsorbed with anti-mouse IgM (M-8644, Sigma). Immunoprecipitated DNA was subjected to PCR, and PCR products were visualized and quantified as described above in “RT-PCR analyses.” Signals from specific immunoprecipitations were normalized to signals from input DNA (10 %) and presented as “fold enrichment” relative to signals from untreated and
58 untransfected sample groups set at “1”. See Table II-4 for list and sequences of
ChIP primers used in this study.
CTD kinase assays: Kinase assays were performed according to previously described protocols with some modifications [143, 164]. The P-TEFb complex was immunoprecipitated from MCF-7 whole cell extracts using 60 µl of Protein A slurry preadsorbed with anti-cyclin T1 or rabbit IgG antibodies (Santa Cruz). The reactions were carried out in lysis buffer [50 mM HEPES pH 7.4, 200 mM NaCl,
10 mM KCl, 1% NP-40, 2 mM EDTA] plus RNasin (40 U/ml) and protease inhibitors for 2 hours at 4°C. After several washes, one half of the immunoprecipitated material was used in Western blot analysis to verify that equal levels of cyclin T1 and HEXIM1 were obtained. The remaining half was further divided into two equal parts and incubated with or without 2 µg of CTD4
peptide, (YSPTSPS)4, in a 25-µl reaction mixture containing 20 mM HEPES-
NaOH pH 7.9, 20 mM Tris-HCl pH 7.9, 7 mM MgCl2, 0.5 mg/ml bovine serum albumin, 30 mM KCl, and 1 µCi of [γ-32P]ATP (3000 Ci/mmol, Amersham
Pharmacia Biotech). For a positive control, 50 µM of CDK9 inhibitor, DRB, was added to some reactions during immunoprecipitations. Reactions were then carried out at 28 °C for 30 min and stopped by adding an equal volume of 2X
SDS protein sample buffer. The phosphorylated CTD4 peptide was visualized by autoradiography after electrophoresis in an 18% SDS-polyacrylamide gel.
Incorporation of [32P] into the CTD peptide was quantified using GeneTools software from Syngene (Frederick, MD).
59 Data analyses: Statistical significance was determined using Student’s t test comparison for unpaired data and was indicated as follows: *, P < 0.05; **, P <
0.005.
Results
Increase in HEXIM1 expression inhibits E2-driven mammary gland development by decreasing cell proliferation and increasing apoptosis
To examine the functional consequences of an interaction between
HEXIM1, ERα and P-TEFb in the mammary gland, we generated a double transgenic mouse model, MMTV/HEXIM1, which inducibly overexpress HEXIM1 in the mammary gland when the mice are treated with doxycycline (+DOX). We first examined the effects of elevated levels of HEXIM1 on E2-driven mammary gland development by having the MMTV/HEXIM1 mice ovariectomized and treated with E2 as described in “Materials and Methods”. In comparing whole mounts of mammary glands from -DOX and +DOX animal groups, we observed decreased ductal branching in the mammary glands of +DOX mice when compared to -DOX mice, as well as compared to single transgenics (MMTV alone) (Figure II-1A, -/+DOX panel insets). We quantified the level of increase of
HEXIM1 expression in the mammary gland as approximately 24% over endogenous levels (p < 0.0124) (See Figure II-2A for Western blot image), so we do not foresee that an overwhelming amount of HEXIM1 is needed to dictate these physiological changes. Since ductal elongation and branching in the
60 mammary gland have been shown to be E2/ERα-dependent [10, 13], these data suggest that HEXIM1 inhibits E2/ERα-driven mammary gland morphogenesis.
Previously, we found that HEXIM1 inhibits ERα transcription [138, 141], so this physiological effect could be due to a dysregulation of ER-responsive genes involved in proliferation and apoptosis. To investigate this, MMTV/HEXIM1 mice were injected with BrdU two hours prior to being sacrificed. BrdU-labeled nuclei in the mammary gland were detected by immunostaining and apoptotic nuclei were stained by TUNEL. Quantitation of positively-labeled mouse mammary epithelial cell nuclei revealed both a decrease in epithelial cell proliferation and an increase in apoptosis (Figure II-1B). Taken together, our data suggest a critical role for HEXIM1 in E2/ERα-driven processes in the mammary gland.
Increased HEXIM1 expression regulates cyclin D1 protein expression levels and Serine 2 phosphorylation of RNA polymerase II in vivo
Our findings that overexpression of HEXIM1 decreased ductal branching and cell proliferation in the mammary gland prompted us to investigate the effect of HEXIM1 overexpression on E2/ERα signaling. As an output, we examined changes in cyclin D1 (CCND1) and c-Myc because both genes are E2/ERα responsive and involved in proliferation integral to mammary gland development and breast cancer [116, 165]. MMTV/HEXIM1 mice were ovariectomized and treated with E2 as described and we found that increased HEXIM1 expression
61 (+DOX) resulted in decreased CCND1 protein expression levels (~67%) in mouse mammary gland cell extracts (Figure II-2A). However, c-Myc expression levels did not significantly change regardless of HEXIM1 expression (Figure II-
3A), suggesting a difference in sensitivity of ERα target genes to HEXIM1.
In addition, since HEXIM1 inhibits P-TEFb activity [62], we examined the effect of increased HEXIM1 expression on P-TEFb activity in the mouse mammary gland by investigating the expression levels of the serine 2 phosphorylated form of RNAP II (S2P RNAP II). In +DOX mice, we observed a decrease in S2P RNAP II levels (~68%) (Figure II-2A). It is important to note here that the antibody used to detect S2P RNAP II recognizes both phosphorylated
(RNAP IIo) and unphosphorylated (RNAP IIa) forms of RNAP II. We also blotted with another antibody that detects only the RNAP IIa form (Hypo RNAP II) and found that there was no observable change between -/+DOX mice groups
(Figure II-2A). Also, HEXIM2, a paralog of HEXIM1, inhibits P-TEFb activity [62], but in the mammary gland, HEXIM1 protein expression levels are significantly higher (Figure II-3B), suggesting a dominant role for HEXIM1 in the mammary gland.
We also examined the expression levels of the serine 5 phosphorylated form of RNAP II (S5P RNAP II), associated with initiation, using immunofluorescent labeling of epithelial nuclei in mammary gland lumen from
MMTV/HEXIM1 mice. We did not observe any changes in the percentage of S5P
62 RNAP II positively-stained nuclei (S5P +ve cells) when we compared -/+DOX animal groups (Figure II-2B). However, in the +DOX animal group, the percentage of S2P RNAP II positively-stained nuclei (S2P +ve cells) within the mammary gland lumen was significantly decreased by 50% (P < 0.05) when compared to the -DOX group (Figure II-2B), verifying our results in the Western blot (Figure II-2A). Given that E2/ERα regulates CCND1 expression [115, 165] and HEXIM1 inhibits P-TEFb activity and associates with ERα [62, 141], the effect of increased HEXIM1 expression levels on CCND1 and S2P RNAP II expression suggest that these changes reflect a complex interaction of multiple pathways that converge at the level of E2/ERα-mediated transcription.
HEXIM1 regulates cyclin D1 and pS2 expression levels in vitro
The effects of increased HEXIM1 expression on CCND1 and S2P RNAP
II expression levels could also be the sum result of the disruption of E2/ERα activity in multiple cell types in the mammary gland. To verify that our observations can be attributed to a more localized event in epithelial cells, we investigated the effects of increased HEXIM1 expression in mammary epithelial
MCF-7 cells. MCF-7 cells were transfected with pCMV-Tag2B-HEXIM1 or control vector and treated with ethanol vehicle or E2 for 3 hours. In control cells, we observed an average 1.5 to 2-fold E2 induction of both CCND1 mRNA and protein levels (Figures II-4A and B); consistent with what has been demonstrated in other studies [115, 159]. As expected, increased HEXIM1 expression diminished E2-induction of CCND1 mRNA and protein expression (Figures II-4A
63 and B, compare lanes 2 and 4). For CCND1 protein expression levels, this was quantified as a 52% decrease in expression by HEXIM1 when normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression (Figure II-4B).
We also investigated the effect of increased HEXIM1 expression on other
ER-responsive genes and found increased HEXIM1 levels decreased E2-induced increases in pS2 mRNA and protein levels (Figures II-4A and B; II-5A). The E2- induced mRNA levels of other genes including c-Myc, Progesterone receptor
(PR) and Cathepsin D remained unchanged with increased HEXIM1 expression
(Figures II-4A and II-5A). In addition, E2-induction of c-Myc protein levels was also unchanged with increased HEXIM1 expression (Figure II-4B, with quantitation in Figure II-5B). These data also suggest that increased HEXIM1 expression may not have a similar effect on all ERα-target genes.
Having observed that increased HEXIM1 expression decreased E2- induced CCND1 and pS2 expression, it stood to reason that HEXIM1 silencing would result in an increase in the induction of both genes by E2 when compared to control cells. HEXIM1 gene expression knockdown (~50%) using miRNA- mediated RNAi resulted in a statistically significant (P < 0.01) enhancement of
E2-induced CCND1 protein expression levels (Figure II-4C). We did not observe a similar effect with pS2 (data not shown) and E2-induced c-Myc protein expression levels remained unchanged (Figure II-4C). Because multiple regulatory proteins are involved in CCND1 regulation [166], we cannot rule out
64 possible actions of other factors in vivo. Also, we cannot rule out the possibility of
HEXIM1 regulation of other key E2 responsive genes involved in cell proliferation and mammary carcinogenesis not studied here. Nonetheless, taken together our data support a novel physiological role for HEXIM1 in E2-driven mammary gland development via regulation of CCND1 levels and serine 2 phosphorylation of
RNAP II.
HEXIM1 inhibits E2-induced recruitment of ERα and cyclin T1 (P-TEFb) to
ERα-responsive genes
Given our observations on the effect of HEXIM1 expression on CCND1 and pS2, it is critical to characterize the molecular events involved in HEXIM1 transcriptional regulation of these ER-responsive genes. We previously reported that HEXIM1 associates with ERα after 90 minutes of E2 stimulation in MCF-7 cells and increased HEXIM1 expression led to a decrease in E2/ERα recruitment to the pS2 promoter [141]. Other reports have shown that P-TEFb associates with the elongating form of RNAP II, S2P RNAP II, which is thought to predominate in the coding regions of genes with increased occupancy towards the 3’ end of genes [63, 167, 168]. Therefore, we hypothesized that increased
HEXIM1 levels would (1.) inhibit the recruitment of ERα to the promoter region of
ER target genes and (2.) inhibit the recruitment of P-TEFb to regions downstream from the promoter at ER-responsive genes, and this would in turn verify the potential regulation of transcriptional elongation of these genes. To investigate this, we carried out chromatin immunoprecipitation (ChIP) assays in
65 MCF-7 cells and examined the effect of E2 on ERα and P-TEFb occupancy at two ERα-responsive genes, pS2 and CCND1.
We found that an increase in HEXIM1 expression in MCF-7 cells correlated with a 2-fold increase in HEXIM1 occupancy at E2-responsive regions within the pS2 and CCND1 [169] promoters (Figure II-6A) that was not significantly affected by E2 treatment. We also found that increased HEXIM1 expression inhibited the recruitment of E2/ERα to the promoter regions of pS2 and CCND1 (Figure II-7B). Now, it is well documented that E2/ERα cycles on and off the promoter of ER-responsive genes and ERα binding is typically diminished after 90 minutes of E2 stimulation [20, 21]. Other studies have also shown that the absolute timing of ERα cycling differs and there can still be significant E2/ERα enrichment at 90 minutes [45, 170]. In our studies, it is clear that E2/ERα binding is less at 90 minutes when compared to 45 minutes (Figure II-7B, compare lanes
2 and 3), and we see a significant decrease at 3 hours (data not shown) indicating E2/ERα is cycling on DNA in our experiments. Regardless of the time point of E2 stimulation, increased HEXIM1 inhibits E2/ERα recruitment to DNA.
For P-TEFb (via cyclin T1), we observed an E2-dependent recruitment pattern that differed slightly at the promoter and coding regions of both pS2 and
CCND1 genes. At the promoters of both pS2 and CCND1, cyclin T1 follows a similar trajectory as ERα in terms of recruitment in control cells, but there is no significant effect on cyclin T1 recruitment with increased HEXIM1 expression
66 (Figure II-7C). However, at the coding regions of both genes, we observed a gradual increase in cyclin T1 recruitment with over time with E2 stimulation and, increased HEXIM1 expression inhibited the recruitment of cyclin T1 (Figure II-
7C). As a further control, we looked at cyclin T1 recruitment to the GAPDH ORF at a region that has been shown to have significant RNAP II enrichment during
GAPDH transcription [171]. We did not observe an E2-dependent recruitment pattern and there were no significant changes in cyclin T1 recruitment with increased HEXIM1 expression (Figure II-6B).
These data suggest that HEXIM1 inhibits ERα transcriptional elongation by inhibiting P-TEFb recruitment, via cyclin T1, to the coding region of some ER- responsive genes. This result is consistent with other studies showing that the recruitment of P-TEFb to promoter-proximal and coding regions stimulates transcriptional elongation [167, 172]. Also, in previous studies, we observed a decrease in the co-recruitment of cyclin T1 with ERα at the pS2 promoter with increased HEXIM1 expression [141], but it appears that the total occupancy of cyclin T1 at the promoter region does not change with increased HEXIM1 expression (Figure II-7C). In this context, our data suggests a dual role for P-
TEFb in ERα transcription. On one hand, it acts as a coactivator for ERα-driven transcription by directly associating with E2/ERα [141], but it also serves its general purpose as a transcription elongation factor, with E2 acting as an enhancer of P-TEFb occupancy in the context of ER-responsive genes.
67 HEXIM1 inhibits E2-induced P-TEFb activity and recruitment of serine 2 phosphorylated RNA polymerase II to coding regions of ER-responsive genes
Since E2 enhanced P-TEFb recruitment to pS2 and CCND1 genes, we investigated if E2 also increased P-TEFb activity as a means of promoting transcriptional elongation of ERα-responsive genes. To do this, we performed kinase assays using endogenous immunoprecipitates of cyclin T1 from MCF-7 cells and examined the kinase activity of P-TEFb with a synthetic peptide substrate, CTD4 (YSPTSPS4). As shown in Figure II-8A, E2 treatment increased
P-TEFb activity in MCF-7 cells (compare lanes 2 and 5). We also observed that increased HEXIM1 expression in MCF-7 cells inhibited an E2-induced increase in
P-TEFb activity (Figure II-8A, compare lanes 2 and 5 to lanes 8 and 11). We confirmed that this inhibition was selective for P-TEFb by adding the CDK9 inhibitor, DRB, to an equal half of the kinase immunoprecipitates (Figure II-8A, lanes 3, 6, 9 and 12) and that the input levels of cyclin T1 and HEXIM1 were evenly loaded (Figure II-9A). Quantitation of 32P incorporation into the CTD4 peptide verified that HEXIM1 significantly abrogates E2-induced P-TEFb activity in MCF-7 cells (Figure II-9B).
We also observed that increased HEXIM1 expression did not inhibit basal
P-TEFb activity in HEXIM1-transfected MCF-7 cells compared to non-transfected cells (Figure II-8A, compare lanes 2 and 8). One reason for this could be attributed to the fact that at basal levels, the P-TEFb complex is thought to occur
68 in two states, 50% free and 50% HEXIM1-7SK RNA bound [62] and in a study by
He et al, gradually decreasing HEXIM1 expression by RNAi did not initially have much effect on this equilibrium because it was the “free” form of HEXIM1 that was being down-regulated, without any effect on the HEXIM1 protein associated with P-TEFb [160]. In our experiments, it is possible that increasing HEXIM1 expression may not be significantly perturbing the P-TEFb equilibrium initially, but increases the availability of free HEXIM1 populations that subsequently diminishes any increases in E2 induced P-TEFb activity.
Because HEXIM1 inhibited the recruitment of cyclin T1 to the coding regions of ER-responsive genes and E2-induced P-TEFb activity, we determined the effect of increased HEXIM1 levels on the recruitment of S2P RNAP II as a mark of the modulation of transcription elongation [56]. At the pS2 promoter, we found that E2 stimulated the enrichment of all forms of RNAP II, without any significant changes when HEXIM1 expression was increased (Figure II-8B). This result differs with what has been observed in some studies, which show low S2P
RNAP II occupancy in comparison to S5P RNAP II at the promoter of transcriptionally active genes [168, 171]. However, it is possible for both S2P and
S5P RNAP II to occupy similar regions on DNA [64], which likely marks the beginning of the transition to elongation, but it is unclear since we did not study proximal upstream or downstream regions to the pS2 promoter. In addition, there was no change in the recruitment of all forms of RNAP II recruitment to the
69 CCND1 promoter in both control and HEXIM1-transfected cells, although it was not as sensitive as the pS2 promoter to E2 stimulation (Figure II-8C).
However, at the pS2 and CCND1 coding regions, we found that increased
HEXIM1 expression inhibited the E2-dependent recruitment of S2P RNAP II, without significant changes in the recruitment of S5P and unphosphorylated
RNAP II forms (Figures II-8B and II-8C). These data indicate that in mammary epithelial cells, HEXIM1 does not affect transcription initiation, since the recruitment patterns of all forms of RNAP II to the promoter-regions of pS2 and
CCND1 were unchanged regardless of HEXIM1 expression levels in the cell.
However, increased HEXIM1 decreases transcription elongation since the recruitment of S2P RNAP II to the coding region was decreased.
Discussion
This study provides novel evidence for a physiological role of HEXIM1/P-
TEFb interaction in attenuating E2/ERα driven transcription in the mammary gland and breast cancer cells. First, we demonstrated that increased HEXIM1 expression in the mammary gland of a transgenic mouse model decreased ductal branching, an E2-driven developmental process, due to changes in proliferation and apoptosis. We correlated these changes with a decrease in
CCND1 and S2P RNAP II expression in vivo and in vitro. We also show that overexpression of HEXIM1 diminished E2-induced recruitment of ERα and cyclin
T1 to the promoter and coding regions, respectively, of ER-responsive genes.
70 Further, we show that E2 enhances the activity of the P-TEFb kinase, CDK9, which is inhibited by increased HEXIM1 expression. Surprisingly, increased
HEXIM1 expression inhibited only E2-induced increases in P-TEFb activity and not basal P-TEFb kinase activity. In our studies, this inhibition of P-TEFb activity translates to a decrease in the recruitment of S2P RNAP II, and not other forms of RNAP II, to the coding regions of ER-responsive genes, pS2 and CCND1.
These findings support a role for P-TEFb and transcription elongation in cell proliferation but more importantly, the data suggests a novel mechanism of action for HEXIM1 that can be recapitulated in vivo and a possible therapeutic role for HEXIM1 in hormone-dependent breast cancer.
Given that the regulation of CCND1 is complex [166], we do not assume that our findings represent a comprehensive explanation regarding E2-regulation of CCND1. Also, other sites within CCND1 contribute to the transcriptional output
[115]. We investigated the recruitment patterns of ERα, P-TEFb and RNAP II to two sites within the gene: an E2-responsive region of the promoter and the coding region. Perhaps, a more extensive analysis of different sites within the genes, pS2 and CCND1, and even other ER-responsive genes will reveal other insight into mechanism of HEXIM1 regulation of these genes. However, we believe that the information gathered from this study was sufficient to demonstrate the regulatory effects of HEXIM1 on ERα and P-TEFb recruitment to pS2 and CCND1, suggesting a role for HEXIM1 and P-TEFb in ERα transcriptional regulation of some, but not all ERα target genes.
71 Based on our previous [141] and current studies, we speculate that E2 enhances an ERα-P-TEFb interaction, and this increases the population of active
P-TEFb at the gene locus of ER-responsive genes, which in turn phosphorylates the CTD of RNAP II. This phosphorylation event positions the gene in an active elongation state with increased S2P RNAP II occupancy at the coding regions, and enhanced recruitment of other forms of RNAP II marking the gene in an active transcription state [63, 64]. However, increased HEXIM1 expression inhibits ERα and P-TEFb enrichment at the promoter and coding regions, respectively, of ER-responsive genes thus, decreasing the population of P-TEFb available to phosphorylate RNAP II to the serine 2 phosphorylated form associated with transcriptional elongation. In addition, we observed that increased HEXIM1 inhibits E2-induced P-TEFb activity and postulated that this was due to an increase in the “free” form of HEXIM1, which serves to diminish any subsequent increases in P-TEFb activity. Taken together, this scenario could represent the mechanism by which HEXIM1 modulates ERα-mediated transcription in the context of some ER-responsive genes (See Figure II-10 for proposed model).
The understanding of general mechanisms that control elongation stem from studies showing that HIV-1 harnesses P-TEFb as a cofactor to promote efficient mRNA transcript synthesis [62]. These and other studies have raised questions about whether P-TEFb acts as a general transcription elongation factor or serves in a gene-selective or context-dependent manner. The P-TEFb
72 complex components, cyclin T1 and CDK9, have not been shown to have sequence-specific DNA binding activity, but transcription factors interact with and recruit P-TEFb to their respective promoter targets [62, 173, 174]. In addition,
DNA microarray analyses of hearts from cyclin T1 transgenic mice indicate selective increases in subsets of genes, rather than a global increase in mRNA expression when compared to non-transgenic mice [175]. Our studies suggest that in breast epithelial cells, P-TEFb can be modulated by E2/ERα and HEXIM1 in the context of some ERα target genes, although identical E2-induced recruitment patterns for P-TEFb to pS2 and CCND1 genes suggest a general transcription elongation mechanism is also involved. In addition, the interaction of
P-TEFb and E2/ERα supports a positive aspect of ERα transcription elongation regulation, but E2/ERα also interacts with negative elongation factor (NELF) and this interaction inhibits ERα-mediated transcription [68].
HEXIM1 has been shown to have P-TEFb-independent action as seen with the glucocorticoid receptor [144]. We have also reported on P-TEFb-independent action of HEXIM1 in cardiovascular development [150]. Given this evidence, it is clear that HEXIM1 can inhibit transcription in a P-TEFb-dependent and P-TEFb- independent manner. Therefore, we cannot assume that the effect of HEXIM1 in the mammary gland is solely on ERα/P-TEFb, as other factors are involved in mammary gland development. However, we were able to demonstrate a specific inhibition pattern that HEXIM1 exerted on E2-induced events at ER-responsive genes, pS2 and CCND1, and increased HEXIM1 levels inhibited E2-induced P-
73 TEFb activity. Thus, based on our data, the HEXIM1 inhibition patterns observed in this study is largely P-TEFb-dependent in both our cell culture and animal models. Several studies also support an emerging role for HEXIM1 as a regulator of cell growth and differentiation [62, 151]. Conversely, deletion of CLP-1, the mouse HEXIM1 gene, leads to pathological cardiac hypertrophy and perinatal death [149].
In this study, a targeted increase in HEXIM1 expression in the mouse mammary gland driven by a mammary epithelial cell promoter (MMTV-LTR), led to a decrease in ductal branching (Figure II-1A), an E2/ERα driven mammary gland developmental process [10]. This observation was attributed to a decrease in proliferation and an increase in apoptosis (Figure II-1B). The decrease in proliferation was linked to a concurrent decrease in CCND1 expression, further demonstrating that HEXIM1 regulates ER-responsive genes in vivo. Future studies will not only aim to address HEXIM1 regulation of other E2/ERα target genes, but also HEXIM1 regulation of other nuclear receptors relevant in mammary cell function and tumorigenesis.
Acknowledgements
We are grateful to the laboratories of Drs. David Schultz, Cheng-Ming
Chiang and Berdis for reagents and technical advice with the ChIP and CTD kinase assays. We would like to Dr. Huayun Deng for his help with some of the
74 animal work and advice on immunohistochemistry. We also thank Dr. Lewis
Chodosh (University of Pennsylvania) for the MTB mice and the pTet-Splice vector. This work was supported by National Institute of Health Grant CA92440 to M.M.M and a Department of Defense Predoctoral Fellowship W81XWH-06-1-
0426 to N.O.
75
Figure II-1. Increased HEXIM1 expression inhibits estrogen-regulated mammary gland morphogenesis due to changes in proliferation and apoptosis.
A. MMTV/HEXIM1 mice were treated as described in Materials and Methods.
Representative whole mounts were obtained from MMTV and MMTV/HEXIM1 mice with Carmine alum stain. Original magnification is X40 for all panels.
B. BrdU-labeled nuclei were detected by immunostaining and apoptotic nuclei
76 were stained by TUNEL. Quantitation of positively-labeled MMEC nuclei from at least 1000 nuclei each from 5 mice per group (-/+DOX) is shown. Original magnification is X40 for all panels.
77
Figure II-2. Increased HEXIM1 expression inhibits E2-induced cyclin D1 expression and serine 2 phosphorylation of RNAP II in mouse mammary gland
A. MMTV/HEXIM1 mice were treated as described in Materials and Methods.
Mammary gland tissue extracts from MMTV/HEXIM1 mice were subjected to
Western blot using indicated antibodies for immunoblotting. Anti-cytokeratin 18 was used as an epithelial cell marker and a loading control. RNAP IIa and IIo
78 indicate the “hypo” and “hyper” phosphorylated forms of RNAP II respectively.
Samples were collected from 3-5 mice per group (-/+Dox) and columns represent quantitation of Western analyses for cyclin D1 and S2P RNAP II; bars, SE; *, P<
0.01.
B. Immunofluorescent detection of S2P and S5P RNAP II. Representative images of mammary gland sections from MMTV/HEXIM1 mice (-/+ Dox) stained as described in Materials and Methods with S2P and S5P RNAP II-specific antibodies. Nuclei were counterstained with DAPI as indicated. Original magnification is X20 for all panels. Quantitation of S2P or S5P positively stained
(S2P+ or S5P+) luminal epithelial cells was performed as described in Materials and Methods. Columns represent percentage (mean + SEM) of total S2P/S5P+ luminal epithelial cells with 3-4 mice per group (-/+ Dox) and a minimum of 1000 cells per animal analyzed; bars, SE; *, P<0.05
79
Figure II-3. Increased HEXIM1 expression does not inhibit c-Myc expression in mouse mammary gland.
A. MMTV/HEXIM1 mice were treated as described in “Materials and Methods” and mammary gland tissue extracts were subjected to Western blot. Samples were collected from 3 mice per group (-/+Dox) and an anti-c-Myc antibody was used for immunoblotting. Columns represent quantitation of Western analyses for c-Myc protein relative to a non-specific band; bars, SE.
B. Western blot analyses of HEXIM1 and HEXIM2 expression in mouse brain, heart, and mammary gland (MG) tissue extracts using anti-HEXIM1 and anti-
HEXIM2 antibodies.
80
Figure II-4. HEXIM1 regulates E2-induced cyclin D1 expression in breast epithelial cells
A. MCF-7 cells were transfected with pCMV-Tag2B-HEXIM1 or empty vector, treated with 100 nM E2 or ethanol vehicle for 3 hours. RNA was extracted and subjected to RT-PCR as described in “Materials and Methods”. RT-PCR results quantitated for changes in cyclin D1 mRNA using GAPDH as a control. Columns,
81 mean of four independent replicates; bars, SE; *, P<0.05
B. Representative Western blot using indicated antibodies for immunoblotting.
Changes in cyclin D1 protein levels were quantitated and normalized to GAPDH.
Columns, mean of three independent replicates; bars, SE; *, P<0.05
C. Representative Western blot indicating miRNA-mediated silencing of HEXIM1 in MCF-7 cells. E2-induced changes in cyclin D1 between LacZ and HEXIM1 miRNA stable cells were quantitated and normalized to GAPDH. Columns, mean of three independent replicates; bars, SE; *, P<0.01
82
Figure II-5. Increased HEXIM1 expression inhibits E2-induced pS2 expression in breast epithelial cells.
A. MCF-7 cells were transfected with pCMV-Tag2B-HEXIM1 or empty vector and treated with 100 nM E2 or ethanol vehicle for 3 hours. RNA was extracted and subjected to RT-PCR as described in “Materials and Methods.” Changes in pS2, c-Myc, PR and Cathepsin D mRNA were quantitated using acidic ribosomal phosphoprotein, 36B4, as a control. Columns, mean of three to five independent replicates; bars, SE.
83 B. Changes in c-Myc protein levels were quantitated and normalized to GAPDH
(See Figure 3B). Columns, mean of three independent replicates; bars, SE.
84
Figure II-6. Increased HEXIM1 expression leads to increase in HEXIM1 occupancy on DNA of ER target genes
A. Increased HEXIM1 expression leads to increased HEXIM1 recruitment to promoter regions of ER target genes, pS2 and CCND1. Samples were prepared from MCF-7 cells as described in Figure 4. Chromatin immunoprecipitation was done with antibodies against HEXIM1. Panels on the left, DNA fragments were analyzed by PCR with primers specific for the promoter-proximal region of pS2 and CCND1 as indicated. Panels on the right, Quantitations of HEXIM1 IP enrichment at pS2 and CCND1 promoters. Columns, mean of two independent replicates; bars, SE.
B. Increased HEXIM1 expression does not inhibit recruitment of cyclin T1 to coding region of GAPDH gene. MCF-7 cells were treated as described in Figure
4. ChIP analysis of cyclin T1 recruitment to GAPDH coding region (ORF) was
85 carried out using primers specific for the region and DNA fragments were analyzed by PCR. Panel is representative of two independent replicates.
86
Figure II-7. Effect of increased HEXIM1 expression on E2-dependent recruitment of ERα and P-TEFb (cyclin T1) to ER-responsive genes.
A. Primers used in ChIP assays are directed at regions indicated for pS2 and
CCND1 genes.
87 B. Increased HEXIM1 expression leads to decrease in E2/ERα recruitment to promoter regions of ER target genes, pS2 and CCND1. Samples were prepared from MCF-7 cells transiently transfected with pCMV-Tag2B-HEXIM1 or empty vector, treated with ethanol vehicle or 100 nM E2 for 45 and 90 minutes.
Chromatin immunoprecipitation was done with antibodies against ERα and rabbit
IgG (as an IP control). Panels on the left, DNA fragments were analyzed by PCR with primers specific for the promoter-proximal region of pS2 and CCND1 as indicated in (A). Panels on the right, Quantitations of ERα IP enrichment at pS2 and CCND1 promoters. Columns, mean of two independent replicates; bars, SE;
*, P<0.05; **, P<0.005.
C. HEXIM1 inhibits E2-dependent recruitment of cyclin T1 to coding regions of pS2 and CCND1. MCF-7 cells were treated as described in (B). ChIP analysis of cyclin T1 recruitment to E2-responsive region of pS2 and CCND1 promoter and coding regions was carried out using primers specific for the regions indicated in
(A). Panels on the left, DNA fragments were analyzed by PCR with primers specific for the regions indicated. Panels on the right, Quantitations of cyclin T1
IP enrichment at pS2 gene. Columns, mean of 2-3 independent replicates; bars,
SE; *, P<0.05
88
Figure II-8. Increased HEXIM1 expression inhibits E2-induced P-TEFb activity and recruitment of serine 2 (hyperphosphorylated) RNA polymerase II to the coding region of ER-responsive genes
A. Increased HEXIM1 expression decreased E2-induced CTD4 peptide phosphorylation. MCF-7 cells were transiently transfected with pCMV-Tag2B-
HEXIM1 or empty vector and treated with ethanol vehicle or 100 nM E2 for 90 minutes. Cell lysates were subjected to immunoprecipitation with antibodies
89 against cyclin T1 and rabbit IgG (as an IP control). The immunoprecipitates were divided into two halves with one half getting 2 µg of the CTD4 peptide added to the reaction (-/+ CTD4). Fifty µM DRB was also added to some immunoprecipitates as a kinase assay control. The kinase reactions were analyzed by SDS-PAGE using autoradiography. Equal volumes of kinase reactions were also analyzed by Western blot to check specificity of anti-cyclin
T1 antibody in immunoprecipitation. Panels are representative of at least four independent experiments.
B. HEXIM1 inhibits E2-dependent recruitment of S2P RNAP II to pS2 ORF. MCF-
7 cells were treated as described in Figure 4 and subjected to ChIP analysis with antibodies against serine 2 phosphorylated (S2P) RNAP II, serine 5 phosphorylated (S5P) RNAP II and the unphosphosphorylated form of RNAP II
(8WG16). The regions of pS2 amplified by PCR are as indicated. Columns, mean of 3-5 independent replicates; bars, SE; **, P<0.005
C. HEXIM1 inhibits E2-dependent recruitment of S2P RNAP II to CCND1 ORF.
MCF-7 cells were treated as described. The regions of CCND1 amplified by PCR are as indicated. Columns, mean of 3-4 independent replicates; bars, SE; *,
P<0.05
90
Figure II-9. Increased HEXIM1 expression inhibits E2-induced P-TEFb activity
A. Five percent of the whole cell lysate was subjected to Western blotting with anti-cyclin T1, -HEXIM1 and -GAPDH antibodies to check for even loading of proteins. The remaining lysate was used in kinase assays as described.
B. 32P-incorporation into the CTD4 peptide (Figure II-8A) was quantified in arbitrary units and plotted as percent (%) phosphorylation, with phosphorylation levels in vehicle-treated cells set at 100%. Columns, mean of four independent replicates; bars, SE; *, P<0.05.
91
Figure II-10. Proposed model for HEXIM1 action on ERα and P-TEFb at ER- responsive genes, pS2 and CCND1, in mammary cells.
92 TABLE II-1: Primer sequences used for transgenic mouse genotyping
93 TABLE II-2: Primers used for reverse transcriptase PCR (RT-PCR) reactions
94 TABLE II-3: miRNA oligonucleotide sequences used for HEXIM1 knockdown experiments
95 TABLE II-4: Primers used in Chromatin immunoprecipitation (ChIP) experiments
96
CHAPTER III
HEXIM1 modulates vascular endothelial growth factor expression and
function in breast cancer cells
This work has been submitted to the Cancer Research journal.
(Ogba, N et al., Cancer Research 2009)
Abstract
Estrogens regulate several factors involved in cancer progression including vascular endothelial growth factor (VEGF), an important mediator of angiogenesis. Recently, we found that mutation of the C-terminus of transcription factor Hexamethylene bisacetamide inducible protein 1 (HEXIM1) in mice leads to abnormalities in cardiovascular development due to aberrant VEGF expression. HEXIM1 regulation of genes has also been shown to be either dependent or independent of its effect on positive transcription elongation factor b (P-TEFb). However, it is not known whether HEXIM1 regulates VEGF in the mammary gland. We demonstrate that HEXIM1 regulates estrogen-induced
VEGF transcription via inhibition of Estrogen Receptor alpha recruitment to the
GC-rich proximal region in the VEGF promoter in a P-TEFb-independent manner in MCF-7 cells. Under hypoxic conditions, HEXIM1 inhibits estrogen-induced protein expression and recruitment of Hypoxia-inducible factor-1 alpha (HIF-1
97 alpha) to the hypoxia response element in the VEGF promoter. In the mouse mammary gland, increased HEXIM1 expression decreased estrogen-driven
VEGF and HIF-1 alpha expression. Conversely, a mutation in the C-terminus of
HEXIM1 (HEXIM11-312) led to increased VEGF and HIF-1 alpha expression and vascularization in mammary glands of heterozygous HEXIM11-312 mice when compared to their wild-type littermates. Additionally, HEXIM11-312 mice have a higher incidence of carcinogen-induced mammary tumors when compared to their wild-type littermates. These tumors have increased vascularization, suggesting an inhibitory role for HEXIM1 during angiogenesis. Our data provide evidence of a novel role for HEXIM1 in cancer progression supported by its anti- angiogenic potential.
Introduction
It is well known that estrogens play a significant role in the etiology and progression of breast cancers [76]. Estrogens mediate their actions through estrogen receptors (ERs), ERα and ERβ, nuclear steroid receptors that classically regulate transcription either directly by binding to estrogen-response elements of target genes or indirectly via protein-protein interactions with other transcription factors [34, 76]. Another factor that is known to play an important role in tumor progression is the vascular endothelial growth factor (VEGF) [125].
VEGF mediates its effects through its receptors, VEGFR1 and VEGFR2 and regulates angiogenesis in both physiological and pathological processes including tumor-associated angiogenesis [125]. VEGF is expressed in many
98 breast tumors and protein and mRNA levels serve as prognostic factors [176].
VEGF also represents a major target for tumor therapy [177].
Several studies have shown that estrogens modulate VEGF expression in breast and uterus tissues and in breast cancer cell lines [109]. The VEGF gene is also known to be estrogen-responsive and have ERα-regulatory components
[178, 179]. In addition, many tumors co-express ERα and VEGF [109, 179].
VEGF expression is also induced by hypoxia [125, 130]. The regulation of VEGF expression by hypoxia occurs due to the stabilization of hypoxia inducible factor-
1 alpha (HIF-1α) protein levels, which interacts with its constitutively expressed binding partner, HIF-1β, and the heterodimer binds the hypoxia-response element in the VEGF promoter to induce its expression [130]. HIF-1α has been shown to be a positive regulator of tumor progression [132] and high levels of
HIF-1α expression occur in ERα-positive and negative breast cancers [130, 180].
Also, estrogens have been shown to induce the recruitment of HIF-1α and HIF-
1β to the VEGF promoter in the rat uterus [181].
We have shown that increased HEXIM1 expression inhibits breast cell growth and HEXIM1 interacts with ERα and decreases its transcriptional activity
[138, 141]. Studies have also shown that HEXIM1 interacts with the positive transcription elongation factor b (P-TEFb) via cyclin T1 and inhibits its activity
[62, 66]. P-TEFb is a complex of cyclin T1 and cyclin-dependent kinase 9 (CDK9) that phosphorylates the carboxy terminal domain of RNA polymerase II (RNAP II)
99 to promote productive phases of transcription [56]. We found that estrogen enhances P-TEFb activity and increases the recruitment of P-TEFb to some ERα target genes [66]. HEXIM1 regulation of these genes involves inhibiting P-TEFb activity and recruitment to the ERα target genes [66]. In addition, recent work from our laboratory uncovered a novel role for HEXIM1 during heart and vascular development as a regulator of VEGF using a mouse model with a C-terminus mutation in HEXIM1 [150].
Although it is known that estrogens can induce VEGF expression via ERα in breast cancer cells, it is not known whether this regulation is dependent on P-
TEFb. Also, it is not known whether HEXIM1 regulates VEGF expression in the mammary gland and in mammary tumors. In this study, we demonstrate that in breast cells, HEXIM1 regulates VEGF expression via its effect on transcription factors ERα and HIF-1α, suggesting an important role for HEXIM1 in mammary tumorigenesis.
Materials and Methods
Antibodies: Anti-ERα (sc-543), anti-HIF-1α (sc-10790), anti-cyclin T1 (sc-
10750), anti-VEGF (sc-7269) and anti-normal rabbit IgG (sc-2027) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Anti- unphosphorylated RNA polymerase II (8WG16) and anti-serine 2 (H5) phosphorylated RNA polymerase II was purchased from Covance Inc. (Berkeley,
100 CA). Anti-GAPDH was obtained from Chemicon (Temecula, CA) and the
HEXIM1 antibody was characterized in previous studies [138, 141].
Reverse Transcription (RT) PCR Analyses: MCF-7 and MDA-MB-231 cells were obtained from American Type Culture Collection (Manassas, VA) and maintained as previously described [138, 141]. Cells were transiently transfected with pCMV-Tag2B, pCMV-Tag2B-ERα, or pCMV-Tag2B-HEXIM1 using FuGENE
HD transfection reagent (Roche, IN) according to the manufacturer’s instructions.
Total RNA was extracted using the TRIzol reagent (Invitrogen, CA) and 4 micrograms of RNA was reverse transcribed using the MMLV-Reverse
Transcriptase kit from Invitrogen according to the manufacturer’s instructions.
PCR products were run in 2% agarose gels and visualized by ethidium bromide staining. A 12-bit digital camera captured fluorescence and signal intensities were quantified using the Alphaimager software from Alpha innotech (San
Leandro, CA). Signals from genes of interest were normalized to signals from
GAPDH and presented as “fold change.” The following primers were used: VEGF
(forward): 5’-CTT TCT GCT GTC TTG GGT G-3’, VEGF (reverse): 5’-ACT TCG
TGA TGA TTC TGC C-3’, HIF-1α (forward): 5’-TGC TAA TGC CAC CAC TAC C-
3’, HIF-1α (reverse): 5’-TGA CTC CTT TTC CTG CTC TG-3’, GAPDH (forward):
5’-TCC ACT GGC GTC TTC ACC-3’ and GADPH (reverse): 5’-GGC AGA GAT
GAT GAC CCT TTT-3’.
101 Western Analyses: MCF-7 and MDA-MB-231 cells were treated as described and total protein was extracted from cells and used for Western blot analysis as previously described [66].
ELISA assays for VEGF secretion: To detect levels of secreted VEGF from
MCF-7 cells, the medium was replaced with 1 mL per well of fresh medium, and the cells were subjected to hypoxia (1% oxygen) or normoxia (21% oxygen) for
12 hours. Then, the cell supernatants were collected, clarified by centrifugation at
4000 rpm for 5 minutes, and stored at –80°C. The amount of VEGF in the supernatant was determined with the Quantikine VEGF-ELISA kit (R&D Systems,
Inc., MN) according to the manufacturer's instructions.
Chromatin immunoprecipitation (ChIP) assays: MCF-7 and MDA-MB-231 cells were plated on 150 mm plates and transfected as described with pCMV-
Tag2B, pCMV-Tag2B-ERα, or pCMV-Tag2B-HEXIM1. Cells were then treated with ethanol or 100 nM E2 and grown under normoxic or hypoxic conditions as indicated. ChIP assays were carried out as previously described [66]. Signals from specific immunoprecipitations were normalized to signals from input DNA and presented as “fold enrichment” relative to signals from untreated and untransfected sample groups set at “1”. VEGF primers used were: VEGF GC-rich proximal region, (forward): 5’-TTT AAA AGT CGG CTG GTA GC-3’ and
(reverse): 5’-AGG GAT AAA ACC CGG ATC AA-3’ which amplify a 194-bp region of the VEGF promoter proximal to GC-rich/Sp1 binding sites; VEGF
102 promoter negative control region, (forward): 5’-GAG AGA GAC GGG GTC AGA
GA-3’ and (reverse): 5’-CTG TCT GTC TGT CCG TCA GC-3’ which amplify a
163-bp region in the VEGF promoter that do not contain GC-rich sites; VEGF hypoxia-response element, (forward): 5’-ACA GAC GTT CCT TAG TGC TGG-3’ and (reverse): 5'-AGC TGA GAA CGG GAA GCT GTG-3’, which amplify a 262- bp region of the VEGF promoter containing the hypoxia response element.
CTD kinase assays: Briefly, Chinese Hamster Ovary (CHO) cells were transfected with pCMV-Tag2B, pCMV-Tag2B-HEXIM1, or pCMV-Tag2B-
HEXIM11-312. The P-TEFb complex was immunoprecipitated from whole cell extracts using 60 µl of Protein A slurry preadsorbed with anti-cyclin T1 or rabbit
IgG antibodies (Santa Cruz). The immunoprecipitated material was incubated with or without 2 µg of CTD4 peptide, (YSPTSPS)4 and for a positive control, 50
µM of CDK9 inhibitor, DRB, was added to some reactions during immunoprecipitations. Kinase reactions were carried out at 28 °C for 30 min and stopped by adding an equal volume of 2X SDS protein sample buffer. The phosphorylated CTD4 peptide was visualized by autoradiography after electrophoresis in an 18% SDS-polyacrylamide gel. Incorporation of 32P into the
CTD peptide was quantified using GeneTools software from Syngene (Frederick,
MD).
103 Mice Studies: MMTV/HEXIM1 transgenic mice were generated and treated as previously described [66]. HEXIM11-312 knock-in mutant mice were generated as previously described [150]. Wild-type pregnant mice and pregnant mice heterozygous for the HEXIM11-312 mutant allele were allowed to deliver and nurse their litters. Litters were culled to 6 pups per lactating mice. Twenty-one days after delivery, the mammary glands were collected. Mammary glands were also collected from 7 to 9-week old adult mice either heterozygous or homozygous for the HEXIM11-312 mutant allele and their wild-type littermates. All tissues were processed for western blot analyses or immunohistochemistry. To examine the effect of HEXIM11-312 mutant protein on mammary tumorigenesis, wild type mice and mice homozygous for the HEXIM11-312 mutant allele were exposed to two sub-threshold doses of 7,12-dimethylbenz[a]anthracene (DMBA). DMBA (0.5 mg) dissolved in 100 µl of corn oil was delivered in two doses (separated by one week) via oral gavage. Mammary tumors were collected and processed for immunohistochemistry.
Immunohistochemistry: To label proliferating cells, mice were injected with bromodeoxyuridine (BrDU) and sacrificed two hours later. BrDU labeled nuclei were detected and quantified as previously described [150]. To assess vascularization, mammary glands were fixed in 4% paraformaldehyde, embedded in OCT and stored at -80°C. Ten µM sections were dried at room temperature for up to 2 hours and slides were washed in 1X PBS followed by 10 minute incubation in 3% H202. To detect blood vessels, sections were incubated
104 with CD31 or PECAM-1 antibody (BD Biosciences, CA) at a 1:50 dilution at 4°C overnight. Following washes, sections were treated with the anti-rat Ig HRP- conjugated secondary antibody (BD Biosciences) at a 1:200 dilution and specific staining was visualized via a 3,3’-diaminobenzidine (DAB) substrate. Image capture was performed with the QCapture Pro software at 4X magnification using an Olympus Q-color3 camera.
Quantification of CD31 positive staining: The brown signal from CD31 immunostaining was filtered using the same filtering conditions for all of the samples and regions on the tissue sections were marked to outline areas to be quantified. A morphological image processing operation called flood-fill was employed to identify the actual pixels within the image that belonged to the regions of interest. Next, CD31 stained pixels were identified by employing a ratio of red to blue channel intensity. An empirically determined low threshold of 1.1 was used for this ratio in order to segment out PECAM regions. For quantifying, we obtained a count of all pixels above the threshold and divided it by the total number of pixels in the region of interest and the data are presented as a percentage of CD31 staining.
Data analyses: Statistical significance was determined using Student’s t test comparison for unpaired data and was indicated as follows: *, P < 0.05; **, P <
0.005.
105 Results
Increase in HEXIM1 expression inhibits estrogen-induced VEGF expression in breast cancer cells
To determine the effect of HEXIM1 on VEGF expression, MCF-7 breast cancer cells were transfected with empty vector or pCMV-Tag2B-HEXIM1 and treated with ethanol (vehicle) or increasing concentrations of 17-beta estradiol
(E2). There was a significant increase in VEGF mRNA expression in cells treated with 10 nM E2 (Figure III-1A), so we used 10 nM E2 treatments in other expression analyses conducted in this study. Increased HEXIM1 expression inhibited E2-induced increases in VEGF mRNA expression (Figure III-1A) and E2- induced secretion of VEGF protein from MCF-7 cells (Figure III-1B).
From previous studies, we know that HEXIM1 inhibits ligand-bound ERα transcriptional activity in the context of some ERα target genes [66, 141]. In ERα- negative MDA-MB-231 cells, we found no change in VEGF expression in response to estrogen treatment consistent with other reports [182] or in response to increased HEXIM1 expression (Figure III-2A). To determine whether the effect of increased HEXIM1 expression on E2-induced VEGF expression could be attributed to its effect on ERα, we transfected MDA-MB-231 cells with pCMV-
Tag2B-ERα and either empty vector or pCMVTag2B-HEXIM1, and treated cells with vehicle or E2. MDA-MB-231 cells are typically not E2-responsive except in some cases of ERα or ERβ-overexpression [182], but in our hands, we found
106 that VEGF mRNA was slightly, but significantly responsive to E2 in ERα- expressing MDA-MB-231 cells (Figure III-2B) and increased HEXIM1 expression inhibits the E2-induced increase of VEGF mRNA expression in these cells (Figure
III-2B).
Increased HEXIM1 expression inhibits the recruitment of E2/ERα to the
VEGF promoter independent of positive transcription elongation factor b
Previous studies have determined that the VEGF promoter contains estrogen-responsive elements and GC-rich motifs that contribute to E2/ERα- driven VEGF transactivation [178, 179]. To determine the effect of HEXIM1 on
VEGF transcription, we carried out chromatin immunoprecipitation (ChIP) assays to examine changes in the recruitment of ERα to a region in the VEGF promoter proximal to GC-rich/Sp1 binding elements. MCF-7 cells were transfected with empty vector or pCMV-Tag2B-HEXIM1 and the cells were treated with vehicle or
E2. We found that increased HEXIM1 expression led to an increase in HEXIM1 occupancy at the VEGF promoter that did not appear to be E2-dependent (Figure
III-1C). This increased occupancy of HEXIM1 led to a decrease in the recruitment of E2/ERα and RNA polymerase II to the VEGF promoter (Figure III-1C). As a negative control, we examined the recruitment of ERα to a region in the VEGF promoter that does not contain GC-rich sites and did not observe any recruitment to this region (Figure III-1C).
107 Previous studies from our laboratory have determined that HEXIM1 inhibits
E2-driven transcription of some ERα target genes in a P-TEFb-dependent manner [66, 141]. To determine whether P-TEFb is involved in E2/ERα-driven
VEGF transcription, we immunoprecipitated cyclin T1 in ChIP assays and found that cyclin T1 recruitment was not enhanced by E2 treatment (Figure III-1C) and increased HEXIM1 expression did not significantly affect its occupancy at the
VEGF promoter (Figure III-1C). As a control, we also examined the recruitment of cyclin T1 to the negative control region of the VEGF promoter described earlier and found that it was also not recruited to this region (Figure III-1C). Overall, these data suggest that P-TEFb recruitment to the VEGF promoter is not dependent on E2 and likely not involved in HEXIM1 regulation of E2/ERα-driven
VEGF transcription.
Under hypoxia, HEXIM1 inhibition of VEGF expression correlates with a decrease in E2-induced HIF-1α expression
Low oxygen tension is another positive regulator of VEGF expression
[130]. In the context of tumors, hypoxic conditions facilitate the upregulation of
HIF-1α expression and increase in HIF-1α transcriptional activity on its target genes, including VEGF, which enhances the formation of blood vessels and facilitates tumor progression [130, 132]. To determine the effect of HEXIM1 on hypoxia-induced VEGF expression in the presence or absence of E2, we transfected MCF-7 cells with empty vector or pCMV-Tag2B-HEXIM1 and
108 subjected the cells to either normal oxygen (21% O2) or hypoxic (1% O2) conditions. We found that increased HEXIM1 expression inhibited E2-induced increases in VEGF mRNA expression under both normoxic and hypoxic conditions (Figure III-3A). However, under hypoxic conditions alone, HEXIM1 did not inhibit VEGF mRNA expression (Figure III-3A), suggesting that the effect of
HEXIM1 on VEGF expression may involve the modulation of E2 and hypoxia in concert. There was no change in HIF-1α mRNA expression in response to E2 treatment or increased HEXIM1 expression under normoxic and hypoxic conditions (Figure III-3A) suggesting that any effects of HEXIM1 on HIF-1α are probably via protein regulation.
Studies have shown that both estrogens and hypoxia cooperate to regulate
VEGF expression in breast cancer cells [183, 184]. We found that E2 and hypoxia induce a slightly higher fold increase in VEGF mRNA expression when compared to E2 under normoxic conditions that is statistically significant (p <
0.05) (Figure III-3A). Western blot analyses showed that under hypoxia, E2 induced an increase in HIF-1α protein expression that was inhibited with increased HEXIM1 expression in MCF-7 cells (Figure III-3B), suggesting that E2 and HIF-1α may also cooperate in MCF-7 cells to regulate VEGF expression. In
MDA-MB-231 cells, we found that E2 did not enhance HIF-1α protein expression and HEXIM1 did not affect HIF-1α protein expression (Figure III-3B). Thus far, these data suggest that E2/ERα regulates HIF-1α expression to enhance VEGF
109 expression in breast cancer cells and that HEXIM1 modulates ERα-regulated
HIF-1α expression.
HEXIM1 inhibits E2-induced HIF-1α recruitment to the hypoxia-response element in the VEGF promoter
Estrogens have been shown to induce the recruitment of HIF-1α to the
VEGF promoter in the rat uterus [181]. To determine the effect of HEXIM1 on E2 and HIF-1α in the context of VEGF transcription we performed ChIP assays on
MCF-7 cells that were treated with vehicle or E2 and grown under normal oxygen or hypoxic (0.5% O2) conditions. We found that E2 enhanced the recruitment of
HIF-1α to the hypoxia response element (HRE) in the VEGF promoter (Figure III-
4A). In MCF-7 cells treated with hypoxia mimetic, cobalt chloride (CoCl2) [185], we observed similar findings (Figure III-4A).
To determine the effect of HEXIM1 on E2-induced HIF-1α recruitment to the VEGF HRE in the presence or absence of ERα, we carried out ChIP assays with MCF-7 and MDA-MB-231 cells. Under hypoxic conditions, E2 induced the recruitment of HIF-1α to the VEGF HRE in MCF-7 cells (Figure III-4B) as observed in Figure III-4A. Increased HEXIM1 expression in MCF-7 cells under hypoxia resulted in a decrease in E2-induced recruitment of HIF-1α to the VEGF
HRE (Figure III-4B). In MDA-MB-231 cells, hypoxia induces increased HIF-1α
110 recruitment to the VEGF HRE but neither E2 nor HEXIM1 significantly affect its occupancy on DNA (Figure III-4B). We also observed that some HIF-1α was immunoprecipitated under normoxic conditions in MCF-7 and MDA-MB-231 cells
(Figures III-4A and 4B), suggesting that HIF-1α binds to the HRE under normal oxygen conditions and may play a role in regulating VEGF expression, but comparatively, hypoxia significantly enhanced the relative amount of HIF-1α present at the VEGF HRE. Taken together, these data suggest that HEXIM1 regulates VEGF transcription via both ERα- and HIF-1α-dependent mechanisms.
These findings shed a new light on the role of HEXIM1 in breast cancer cells and its possible role in regulating angiogenesis via VEGF in vivo.
HEXIM1 modulates VEGF and HIF-1α expression and angiogenesis in the mouse mammary gland.
To determine whether increased HEXIM1 expression would significantly alter E2-regulated VEGF and HIF-1α expression in the mammary gland, we extracted mammary glands from MMTV/HEXIM1 transgenic mice used in previous studies [66]. These mice inducibly express HEXIM1 in the mammary gland when treated with doxycycline and were ovariectomized and treated with
E2 to monitor changes in gene expression that are modulated by E2. We found that increased HEXIM1 expression significantly decreased VEGF and HIF-1α protein expression in the mammary gland (Figure III-5A).
111 To verify the physiological relevance of HEXIM1 regulation on VEGF, we generated mice expressing a knock-in mutation of HEXIM1 in our laboratory that have been described [150]. HEXIM1 is expressed at full length as a 359 amino acid protein, with the C-terminus containing inhibitory domains for ERα and P-
TEFb [62, 141]. These mice carry an insertional mutation in HEXIM1 that disrupts the C-terminus. While this form of HEXIM1, HEXIM11-312, is still able to interact with P-TEFb [150], it does not have the ability to inhibit ERα as suggested by a previous report from our laboratory [141]. We found that the HEXIM1 C-terminus mutation leads to an increase in VEGF and HIF-1α protein expression in the mammary glands of mice carrying the heterozygous allele (HEXIM1 het) when compared to their wild-type littermates (Figure III-5B). Taken together, these data suggest that HEXIM1 modulates VEGF and HIF-1α expression partly via ERα in the mammary gland.
Since VEGF is a proangiogenic factor, we wanted to determine if the enhanced expression of VEGF in the mammary glands of HEXIM1 het mice corresponded to increased vascularization in the mammary gland. A hallmark of angiogenesis is the presence of the platelet endothelial cell adhesion molecule-1
(PECAM-1) or cluster of differentiation molecule 31 (CD31) on the cell surface of endothelial cells [186]. To do this, we examined any changes in CD31 expression in mammary glands from lactating HEXIM1 het mice and their wild-type lactating littermates using immunohistochemistry. We used mammary glands from lactating mice because increased vascularization and VEGF expression is critical
112 for alveolar development and milk production [187]. We found that there was an increase in CD31 positive staining in the mammary glands of HEXIM1 het mice when compared to their wild-type littermates (Figure III-5C). Taken together, this suggests that HEXIM1 regulates VEGF and HIF-1α expression in mammary cells and this in turn regulates the development of blood vessels in the mammary gland.
In the mammary glands of mice homozygous for the HEXIM1 mutant allele
(HEXIM11-312) we also found that there was an increase in VEGF expression
(Figure III-6A) that does not appear to involve P-TEFb regulation as there was no change in the protein expression of cyclin D1 and the Serine 2 phosphorylated form of RNAP II (Ser2 phosph RNAP II) (Figure III-6A), which is phosphorylated by P-TEFb [56, 66]. To demonstrate that the C-terminus mutation in HEXIM1 did not disrupt its potential to inhibit P-TEFb activity, we carried out in vitro kinase assays to compare the activity of wild-type HEXIM1 to HEXIM11-312 in Chinese hamster ovary (CHO) cells. We found that HEXIM11-312 is able to inhibit P-TEFb activity comparable to wild-type HEXIM1 (Figure III-7A). This suggests that while the effects of the HEXIM1 insertional mutation in these mice can be partly attributed to an attenuation of ERα inhibition by HEXIM1, the observed phenotypic effects are not due to a disruption of its P-TEFb-inhibitory function.
These data support the in vitro data from the breast cancer cells that suggest a
P-TEFb-independent nature for the regulation of VEGF by HEXIM1 in breast cells. To characterize the effect of enhanced VEGF expression in the mammary
113 glands of mice, we looked for any differences in proliferation in the mammary glands of HEXIM11-312 mice and their wild-type littermates and found that there was no significant difference in proliferation, as detected by Bromodeoxyuridine
(BrDU) incorporation (Figure III-7B).
HEXIM1 C-terminus mutation increases incidence of carcinogen-induced mammary tumorigenesis and correlates with increased tumor vascularization
During tumorigenesis, hypoxic environments within the tumor enhance
VEGF secretion and facilitate migration and proliferation of endothelial cells at the tumor site [125, 130]. To determine the effect of the HEXIM1 C-terminus mutant on tumorigenesis, we used a well-known experimental model of carcinogen-induced mammary tumors [188] and treated HEXIM11-312 mice and their wild-type littermates with sub-threshold levels of the carcinogen 7,12- dimethylbenz(a)anthracene (DMBA) via oral gavage. Moreover, our HEXIM1 mutant mice (HEXIM11-312) are in the C57/BL6 background strain that is known to be relatively resistant to carcinogen-induced tumors [189]. We found that the
HEXIM11-312 mice developed mammary tumors at a significantly higher incidence
(p < 0.001) than their wild-type littermates (Figure III-6B).
To determine whether enhanced vascularization of the mammary tumors contributed to the increase in tumor incidence in HEXIM11-312 mice, mammary
114 tumors that developed were excised and processed for immunohistochemistry to detect any changes in vascularization. We found that tumors from HEXIM11-312 mice exhibited increased vascularization, evidenced by an increase in CD31 positive staining, when compared to their wild-type littermates (Figure III-6C).
Taken together, the in vivo data support a novel role for HEXIM1 in mammary gland development and in mammary tumorigenesis by regulating angiogenesis.
Discussion
The present study uncovers a novel role for HEXIM1 in mammary development and cancer progression as a regulator of VEGF transcription. We found that in breast cancer cells, HEXIM1 inhibited E2-induced VEGF transcription via ERα. HEXIM1 also inhibits VEGF transcription via a decrease in
E2-induced HIF-1α protein expression and recruitment to the VEGF promoter under hypoxic conditions. Increased HEXIM1 expression in the mammary gland of MMTV/HEXIM1 transgenic mice leads to a decrease in VEGF and HIF-1α protein expression. Conversely, mutation of the C-terminus of HEXIM1
(HEXIM11-312) in mice led to enhanced VEGF and HIF-1α protein expression and vascularization in the mammary gland. Also, the HEXIM11-312 mice have increased susceptibility to developing carcinogen-induced mammary tumors that exhibit increased vascularization when compared to their wild-type littermates, demonstrating an important role for HEXIM1 during mammary gland development and tumorigenesis (Figure III-8).
115 Studies support the regulation of VEGF expression and angiogenesis in the female reproductive system and in breast cancer by estrogens, but specific mechanisms of the regulation are not always clear [109, 190, 191]. Estrogen- responsive elements have been identified in the VEGF gene [178, 179], and the recruitment of E2/ERα to these regions has also been reported in breast and uterine cells [179, 181]. It has also been reported that direct interaction between the tumor suppressor, BRCA1, and ERα inhibits E2-driven VEGF transcription and secretion in breast cancer cells [192]. In this study, we show that HEXIM1, an ERα-interacting protein and tumor suppressor [138, 141], inhibits VEGF transcription by inhibiting the recruitment of E2/ERα to the VEGF promoter in breast cancer cells. HEXIM1 interacts with and inhibits the activity of the P-TEFb to regulate gene expression [62, 66, 141]. Other studies also support P-TEFb independent functions of HEXIM1 [144, 150]. Notably, in this study we found that both E2 and HEXIM1 did not affect P-TEFb recruitment to the VEGF promoter, suggesting that P-TEFb may not be involved in the regulation of VEGF by
HEXIM1.
Although HEXIM1 inhibited E2-driven VEGF transcription in an ERα- dependent manner, it was likely that HEXIM1 also influenced other positive modulators of VEGF transcription. Hypoxia, a strong inducer of VEGF expression, regulates VEGF transcription through the hypoxia-inducible factor-1 binding the HRE in the VEGF promoter [130, 180]. HIF-1α also plays a role in tumor progression and metastasis [132]. Studies also implicate estrogens as
116 regulators of HIF-1α in uterine cells via the phosphatidylinositol 3-kinase/Akt pathway [181, 193] and since both estrogen and hypoxia are involved in tumor development and progression, it is thought that they cooperate to regulate VEGF expression [183, 184]. We found that hypoxia and E2 induced a slight increase in
VEGF expression compared to E2 alone in MCF-7 cells that was inhibited by
HEXIM1. This correlated with a decrease in the E2-induced HIF-1α expression and recruitment of HIF-1α to the VEGF HRE with increased HEXIM1 expression in MCF-7 cells. Overall, these data support a novel role for HEXIM1 in regulating
VEGF in breast cancer cells during tumor progression.
VEGF also plays an important role in endothelial cell migration and proliferation and the resulting angiogenesis contributes to physiological and pathological processes [177]. In a recent study, we reported a HEXIM1 C- terminus mutation leads to a decrease in VEGF expression in the developing mouse heart [150]. This occurs through the attenuation of the inhibitory effect of
C/EBPα by HEXIM1 on VEGF gene transcription in cardiomyocytes [150].
In our current study, we found that the same C-terminus mutation in
HEXIM1 (HEXIM11-312) led to an increase in VEGF and HIF-1α expression in the mouse mammary gland that correlated with an increase in angiogenesis in the mammary gland when compared to their wild-type littermates through a possible attenuation of ERα inhibition by HEXIM1 [141]. Conversely, increased HEXIM1
117 expression in the mammary gland of MMTV/HEXIM1 mice leads to a decrease in
VEGF and HIF-1α protein expression. This is consistent with our breast epithelial cell studies, which indicate that HEXIM1 inhibits the actions of ERα and HIF-1α, two positive regulators of VEGF gene transcription. In addition, the HEXIM11-312 mice are more susceptible to developing carcinogen-induced mammary gland tumors with enhanced vascularization. The fact that HEXIM1 is displaying different effects on VEGF in cardiomyocytes versus the mammary epithelial cells suggests that there may be tissue-specific factors that associate with HEXIM1 in each tissue to regulate VEGF expression.
VEGF-targeted therapy includes targeting circulating VEGF and VEGF receptor blockade [177]. Since cancer cells typically develop resistance to cancer therapeutic drugs, it is important to identify potential targets with multiple mechanisms of action. In breast cancer cells, HEXIM1 regulates factors that contribute to proliferation [66, 141] and as we show in this study angiogenesis— two critical processes that occur during mammary tumorigenesis. These studies elucidate reasons why HEXIM1 might be a desirable therapeutic target for breast cancer. Future studies will aim to elucidate the specific mechanisms of HEXIM1 regulation of HIF-1α expression.
118 Acknowledgements
We thank Drs. Anthony Berdis and Jay Prendergast for reagents and their help with kinase and ELISA assays. This work was supported by National
Institute of Health grant CA92440 and American Heart Association grant to
M.M.M and a Department of Defense Predoctoral Fellowship W81XWH-06-1-
0426 to N.O.
119
Figure III-1. Increased HEXIM1 expression inhibits E2-induced transcription of VEGF via ERα in a P-TEFb-independent manner in breast cancer cells.
A. MCF-7 cells were transfected with pCMV-Tag2B-HEXIM1 or empty vector and treated with ethanol (vehicle) or 1 or 10 nM 17-beta estradiol (E2) for 4 hours.
Graph shows fold change of VEGF mRNA levels measured by reverse transcriptase PCR (RT-PCR) (n = 2-3 independent experiments); bars = SEM; *,
120 p < 0.05; a = E2-induction relative to vehicle, b = E2-induction with Flag-HEXIM1 relative to control vector.
B. MCF-7 cells were transfected with pCMV-Tag2B-HEXIM1 or empty vector and treated with ethanol or 10 nM E2 for 12 hours. Secreted VEGF protein levels were measured by ELISA (n = 2 independent experiments); bars = SEM; **, p <
0.005; a = E2-induction relative to vehicle, b = E2-induction with Flag-HEXIM1 relative to control vector.
C. MCF-7 cells were transfected with pCMV-Tag2B-HEXIM1 or empty vector and treated with ethanol or 100 nM E2 for 45 minutes. Results show ChIP analyses of lysates immunoprecipitated with ERα, HEXIM1, Cyclin T1, RNA polymerase II
(RNAP II) and rabbit immunoglobulin (IgG) antibodies. PCR amplification of the proximal GC-rich fragment or negative control region in the VEGF promoter was performed and graphs show quantification of PCR products as indicated (n = 2-3 independent experiments); Bars = SEM; *, p < 0.05.
121
Figure III-2. Increased HEXIM1 expression inhibits E2-induced VEGF expression in ERα-expressing MDA-MB-231 cells
A. MDA-MB-231 cells were transfected with pCMV-Tag2B-HEXIM1 or empty vector and treated with ethanol (vehicle) or 1 or 10 nM 17-beta estradiol (E2) for 4 hours. Graph shows fold change of VEGF mRNA levels measured by reverse transcriptase PCR (RT-PCR) (n = 2 independent experiments done); bars =
SEM.
B. MDA-MB-231 cells were transfected with pCMV-Tag2B-ERα, pCMV-Tag2B-
HEXIM1 or empty vector and treated with ethanol or 10 nM E2 for 12 hours.
Western blot analyses confirmed expression of ERα and HEXIM1 in transfected cells. Graph shows fold change of VEGF mRNA levels measured by RT-PCR (n
= 3 independent experiments); bars = SEM; *, p < 0.05; a = E2-induction relative to vehicle, b = E2-induction with Flag-HEXIM1 relative to control vector.
122
Figure III-3. Increased HEXIM1 expression inhibits E2-induced VEGF mRNA expression under hypoxia that correlates with a decrease in E2-induced
HIF-1α expression
A. MCF-7 cells were transfected with pCMV-Tag2B-HEXIM1 or empty vector, treated with ethanol or 10 nM E2 and grown under normal oxygen (21% O2) or hypoxic (1% O2) conditions for 12 hours. Results show fold change of VEGF mRNA levels measured by RT-PCR (n = 3 independent experiments); bars =
SEM; *, p < 0.05; **, p < 0.005; a = E2-induction relative to vehicle, b = E2-
123 induction with Flag-HEXIM1 relative to control vector, c = E2-induction under hypoxia relative to normoxia.
B. MCF-7 and MDA-MB-231 cells were transfected with pCMV-Tag2B-HEXIM1 or empty vector, treated with ethanol or 10 nM E2 and grown under normoxic and hypoxic conditions as indicated for at least 12 hours. Western blot analyses show changes in HIF-1α protein expression and protein expression of HEXIM1 and
GAPDH (loading control). (n = 2-3 independent experiments); bars = SEM; *, p <
0.01.
124
Figure III-4. Increased HEXIM1 expression inhibits E2-induced recruitment of HIF-1α to VEGF Hypoxia Response Element
A. MCF-7 cells were treated with ethanol or 100 nM E2 and subjected to normoxic or hypoxic conditions as indicated or treated with 100 µM cobalt chloride for 6 hours. Results show ChIP analyses of lysates immunoprecipitated with HIF-1α and rabbit immunoglobulin (IgG) antibodies and DNA fragments were analyzed by PCR primers specific for the hypoxic response element (HRE)
125 in the VEGF promoter (n = 2-3 independent experiments); bars = SEM; **, p <
0.005.
B. MCF-7 or MDA-MB-231 cells were transfected with pCMV-Tag2B-HEXIM1 or empty vector, treated with ethanol or 100 nM E2 and subjected to normoxic or hypoxic conditions as indicated for 16 hours. Results show ChIP analyses of HIF-
1α immunoprecipitates and PCR amplification of fragment containing VEGF
HRE. Graphs show quantification of HIF-1α immunoprecipitates (n = 2 independent experiments); bars = SEM; *, p < 0.05.
126
Figure III-5. HEXIM1 modulates VEGF and HIF-1α expression and vascularization in mouse mammary gland.
A. MMTV/HEXIM1 mice were treated as previously described and mammary gland tissue extracts were subjected to Western blot. Antibodies for VEGF, HIF-
1α, and HEXIM1 were used for immunoblotting. Anti-cytokeratin 18 was used as an epithelial cell marker and a loading control. Graph panel shows quantification
127 of VEGF and HIF-1α expression from mice treated with or without doxycycline
(DOX) (n = 3-4 mice per group); Bars = SEM; *, p < 0.05; **, p < 0.005.
B. Western blot analyses of mammary gland extracts from adult wild-type mice
(WT) and mice heterozygous for the HEXIM1 1-312 mutant allele (HEXIM1 het) using VEGF and HIF-1α antibodies. Blots were probed for cytokeratin 18 to normalize for epithelial cell content. Graph shows quantification of VEGF expression from WT and HEXIM1 1-312 heterozygous mice (n = at least 3 mice per group); Bars = SEM; *, p < 0.05.
C. Immunohistochemical detection of CD31 in mammary glands of lactating adult
WT or HEXIM1 het mice. Panel is representative of 4 mice per group and graph shows quantification of %CD31-positive staining; Bars = SEM; **, p < 0.005.
128
Figure III-6. Expression of HEXIM1 C-terminus mutant enhances carcinogen-induced mammary tumorigenesis and correlates with increased vascularization of tumors
A. Mammary gland extracts from adult WT mice and mice homozygous for the
HEXIM1 1-312 mutant allele (HEXIM1 1-312) were subjected to Western blot analyses using antibodies for VEGF, cyclin D1, serine 2 phosphorylated, and hypophosphorylated RNAP II. Blots were probed for cytokeratin 18 to normalize for epithelial cell content. Panel is representative of at least 3 mice per group.
B. The graph describes DMBA-induced tumor incidence in HEXIM1 1-312 mice and their WT littermates assessed by palpitation and histopathological
129 examination of excised tumors. DMBA was administered at 8 weeks of age by oral gavage. The frequency of palpable mammary tumors in HEXIM1 1-312 mice was statistically significant from that of the WT mice (p < 0.001); (n = 12 mice per group).
C. Immunohistochemical detection of CD31 in DMBA-induced mammary tumors excised from adult WT or HEXIM1 1-312 mice. Panel is representative of at least
3 mice per group and graph shows quantification of %CD31-positive staining;
Bars = SEM; *, p < 0.05.
130
Figure III-7. HEXIM1 C-terminus mutant inhibits P-TEFb activity and does not affect proliferation in mammary gland.
A. Chinese Hamster Ovary (CHO) cells were transfected with empty vector, pCMV-Tag2B-HEXIM1 (WT) or pCMV-Tag2B-HEXIM11-312 and the cell lysates were immunoprecipitated with a cyclin T1 antibody (Cyclin T1 IP). The immunoprecipitates were divided into two halves and 50 µM DRB was added to one half as a control. All IPs subjected to in vitro kinase assays using an exogenous substrate, CTD4 peptide. 32P-incorporation into the CTD4 peptide was quantified in arbitrary units and plotted as percent (%) phosphorylation (n = 2 independent experiments); Bars = SEM; * p < 0.05; ** p < 0.005.
B. Adult WT and HEXIM1 1-312 mice were injected with BrdU and sacrificed 2 hours later. BrdU-labeled nuclei were detected by immunostaining and quantified.
131
Figure III-8. Model: HEXIM1 regulates VEGF expression via ERα and HIF-1α to modulate angiogenesis and tumorigenesis.
132 CHAPTER IV
SUMMARY AND FUTURE DIRECTIONS
SUMMARY
HEXIM1 was identified in our laboratory as an ERα-interacting protein that inhibited ERα transcription via a novel interaction with P-TEFb and inhibited breast cell proliferation [138, 141]. HEXIM1 also inhibits P-TEFb activity [62].
A. HEXIM1 inhibits E2-induced P-TEFb activity that drives ERα transcriptional elongation and proliferation in mammary cells
In the current HEXIM1/P-TEFb studies (Chapter II), we uncovered novel aspects of E2/ERα and HEXIM1 function in the context of ERα transcription. We demonstrated that HEXIM1 regulates E2-driven transcription of certain ERα target genes. We also found that E2 enhances the activity of transcription elongation factor, P-TEFb, and that HEXIM1 inhibits E2-induced P-TEFb activity, which translates to an inhibitory effect on P-TEFb and Serine 2 phosphorylated
RNAP II (Ser2 RNAP II) recruitment to the coding regions of ERα target genes.
We were able to demonstrate that this played a role in the expression of ERα target genes, cyclin D1 and pS2 [66]. Other studies support the importance of the recruitment of Ser2 RNAP II to the coding region or 3’ end of genes in proper mRNA processing in mammalian cells [56, 64].
133 To demonstrate a physiological relevance for the interactions between
ERα, HEXIM1 and P-TEFb in the mammary gland, we used a transgenic mouse with targeted HEXIM1 expression in the mammary gland and found that increased HEXIM1 expression led to a decrease in ductal branching, an E2/ERα- driven process [66]. Therefore, based on these studies, we conclude that
HEXIM1 decreases cell proliferation in breast cancer cells and in the mammary gland by inhibiting the transcriptional elongation of some ERα target genes in a
P-TEFb-dependent manner.
B. HEXIM1 regulates E2-induced HIF-1α and VEGF expression and resulting angiogenesis in mammary gland and tumorigenesis
Within the last few years, a lot of evidence has emerged with mechanistic detail concerning the role of functional domains of HEXIM1 and a significant portion of these studies have highlighted the role of HEXIM1 in P-TEFb regulation in the context of HIV transcription, inflammation and cell differentiation
[62]. In addition, it is known that mice deficient in HEXIM1 are embryonic lethal and exhibit cardiac abnormalities [149]. Recent studies from our laboratory discovered a novel function for HEXIM1, where mice that express HEXIM1 with a
C-terminus mutation exhibit vascular abnormalities due to a decrease in VEGF expression [150].
134 In the current HEXIM1/VEGF project described (Chapter III), our studies focused on the role of HEXIM1 in regulating VEGF in the mammary gland, as the function of HEXIM1 and VEGF in this context was unknown.
We found that HEXIM1 regulates E2/ERα-driven VEGF transcription in a P-
TEFb-independent manner via an inhibitory effect on ERα and HIF-1α. The inhibitory effect of HEXIM1 on HIF-1α is E2/ERα-dependent and disrupting the
ERα-inhibitory domain in HEXIM1 (HEXIM1 1-312) leads to enhanced VEGF and
HIF-1α expression in the mouse mammary gland. Because P-TEFb inhibition is unaltered in the HEXIM1 1-312 mice, there is no change in either cyclin D1 expression or an increase in mammary epithelial cell proliferation but we found that there is enhanced endothelial cell proliferation in the mammary gland. In a carcinogenesis model, HEXIM1 1-312 expression in mice leads to enhanced tumorigenesis and vascularization of tumors [194]. Based on these studies, we conclude that in the context of estrogen-driven angiogenesis, HEXIM1 regulates angiogenesis in the mammary gland and during tumorigenesis by inhibiting
VEGF transcription. The inhibition of VEGF transcription by HEXIM1 is via
E2/ERα and HIF-1α in a P-TEFb-independent manner and suggests an important role for HEXIM1/ERα regulation in mammary tumorigenesis.
135 FUTURE DIRECTIONS
Generally, our studies suggest that HEXIM1 regulates E2-driven ERα transcriptional activity by modulating the action of other ERα transcription coregulators. In addition, the regulation of ERα target genes by HEXIM1 has functional consquences in the mammary gland and in processes including proliferation and angiogenesis that contribute to mammary tumorigenesis (See
Figure 4-I for summary model).
Although these studies demonstrate a role for HEXIM1 in the regulation of
E2-driven ERα transcriptional activity, there are still many questions that can be addressed to gain a better understanding of the role of HEXIM1 in the regulation of the activity of ERα and other transcription factors, the role of HEXIM1 in mammary gland development and its role in tumorigenesis. The outlined studies to address these questions include but are not limited to the following:
Further dissect the role of HEXIM1 in the transcriptional regulation of ERα- target genes
Our studies elucidated a role for HEXIM1 in regulating the transcriptional elongation of ERα target genes via P-TEFb [66, 141]. We found that the inhibitory effect of HEXIM1 on E2-induced P-TEFb and Serine 2 phosphorylated
RNAP II (S2P RNAP II) occurred in the coding regions of ER target genes and not the promoter-proximal regions [66]. Previous studies from our laboratory
136
Figure IV-1. Summary model of HEXIM1 mechanism of action in breast cancer.
137 have also shown that the inhibition of ERα transcription by HEXIM1 does not involve the regulation of histone deacetylases (HDACs) because HDAC inhibitor, trichostatin, had no effect on HEXIM1 inhibition of transcriptional activity [141].
While our studies provided evidence for how HEXIM1 regulates ERα target genes via E2-induced P-TEFb and independent of HDACs, we did not address other potential mechanisms of HEXIM1 regulation of ERα transcription that might involve factors or coregulators associated with initiation, elongation and termination.
During well-defined phases of transcription, the assembly and disassembly of complexes including members of the p160 family of coactivators, p300,
CARM-1, SMRT, NCoR, cofactor of BRCA1 (COBRA1) have all been shown to regulate ERα transcriptional activity [21, 68, 195]. COBRA1 is a component of the negative elongation factor (NELF) that has been shown to repress ERα transcription by RNAP II promoter proximal pausing [68].
Using ERα positive (MCF-7, T47D cell lines) or negative (MDA-MB-231 cell line) breast cancer cells, we can perform ChIP and Re-ChIP assays to can determine whether HEXIM1 associates with and regulates the recruitment of these coregulators and RNAP II to the promoter of ERα target genes to influence
ERα transcription. To determine whether HEXIM1 is necessary for the recruitment of any of the coregulators, we can carry out the proposed experiments using breast cancer cell lines that have been stably down regulated for endogenous HEXIM1 expression and examine any changes in the
138 recruitment of the factors of interest in the presence or absence of HEXIM1.
These studies will potentially provide a mechanistic detail of the regulation of
ERα transcription by HEXIM1 and reveal whether the function of HEXIM1 can be localized to the elongation phase of the ERα transcription cycle.
In addition, while our studies show that the inhibition of P-TEFb by
HEXIM1 leads to a decrease in the recruitment of S2P RNAP II to the coding regions of genes, we also found that there was no change in the recruitment of the unphosphorylated and phosphorylated forms of RNAP II to the promoter proximal regions of ER target genes [66].
On one hand, this observation suggests that the effect of HEXIM1 is specific to E2-induced P-TEFb activity. However, we only analyzed one region in the coding regions of the genes investigated, therefore, it is necessary to perform a more extensive ChIP analysis that would examine several regions upstream and downstream of the initiation site to strongly demonstrate a role for HEXIM1 in transcriptional elongation and not other processes including RNAP II promoter pausing, a mechanism of gene regulation that also contributes to the generation of abortive mRNA transcripts or inefficient mRNA processing.
139 Identification of HEXIM1 binding sites on target genes of ERα and other nuclear receptors in breast epithelial cells via ChIP-on-chip arrays
Our studies demonstrated that HEXIM1 could bind to DNA of ERα target genes indirectly via ERα or P-TEFb at ER-responsive sites including ERE, AP-1 and Sp1/GC-rich regions within the promoter and coding regions of these genes
[66, 141, 194]. Other studies have demonstrated that HEXIM1 inhibits the transcriptional activity of other transcription factors including NF-κB and the glucocorticoid receptor (GR) in different cell contexts [136, 144]. Additionally, although many transcription factors including nuclear receptors, androgen receptor (AR), aryl hydrocarbon receptor (AhR) and Peroxisome Proliferation-
Activated Receptor-gamma (PPAR-γ) depend on P-TEFb for transcription [173,
196, 197], some genes in the p53 pathway do not require P-TEFb for their transcriptional activity [64].
To identify HEXIM1 target genes using an unbiased approach, we can carry out ChIP-on-chip experiments. This would involve the immunoprecipitation of chromatin bound by HEXIM1 and after purification of DNA, it can be used as a template for microarray hybridization using a microarray chip made up of promoter sequences from several genes. For instance, the GeneChip human promoter 1.0R array is commercially available from Affymetrix and is made up of over 25,000 human promoters with a 10 kb coverage for each promoter proximal to the start site. Also, we can perform more exhaustive analyses using a genome-wide GeneChip Human Tiling 2.0R array set which contains seven whole genome arrays including the promoter array mentioned above. However, it
140 would extend our analysis to include other genomic regions and expand the relationship of HEXIM1 to other transcription regulatory factors and epigenetic modifications.
Using computational algorithmic and analyses programs, we can eliminate false discovery rates and make high confidence predictions about which of the cis-regulatory sites identified with high HEXIM1 enrichment correlate to sequences likely to contribute to gene expression [198].
A high degree of conservation is thought to suggest evolutionary maintenance and suggest a functional role for cis-regulatory elements in chromatin [199]. To begin to determine whether the HEXIM1 binding sites support functional role, it would be important to compare the sequence of the binding site between genomes of multiple vertebrate species. Also, databases like TRANSFAC which house information on transcription factors and their experimentally proven binding sites and regulated genes can be used to further weed out any false positives and make correlations between HEXIM1 and possible interacting factors [200].
It would then be necessary to conduct extensive smaller scale ChIP and
RT-PCR experiments in the presence or absence of HEXIM1 to validate the functional significance of the HEXIM1 binding sites identified through the ChIP- on-chip arrays on gene expression.
Two major challenges to this experiment will be determining the cell lines to be used since HEXIM1 exhibits gene and cell-context dependent effects [62,
141 201] and data analyses due to the considerable amount of information that will be garnered from the experiments. To approach this, we can narrow our focus at the outset of the experiments to a few principal groups of target genes.
For instance, knowing that we will investigate HEXIM1 binding sites on
ERα target genes, we can use at least two ERα-positive breast cancer cells lines
(MCF-7 or T47D) and some ERα-negative breast cancer cells (MDA-MB-231) in the presence or absence of E2 to determine the effect of E2 on endogenous
HEXIM1 and ERα binding to cis-regulatory regions within the ERα target genes.
Studies using ChIP-on-chip have already been done to identify novel ERα binding sites and it was found that ERα can be recruited anywhere from 10 to 22 kb upstream of the transcription initiation site in ERα target genes using genome- wide ChIP-on-chip arrays [198]. These sites were subsequently validated as transcription enhancer regions necessary for the association of transcription
Forkhead factor, FoxA1, which is required for ERα binding and transcription of some ERα target genes. RNAP II binding sites were also identified and correlated with active gene expression [198]. Therefore, for preliminary analyses, we could use the information gathered from this study to determine whether
HEXIM1 binds already identified cis-regulatory elements in ERα target genes.
Given that HEXIM1 displayed selective regulation of ERα target genes
(Chapter II) and other genes have been shown to be able to bypass the requirement for P-TEFb activity in their transcription [64], it would be important to distinguish which genes do not require P-TEFb activity. Using MCF-7 breast
142 cancer cells that have been treated or not with E2, we can immunoprecipitate both HEXIM1 and P-TEFb using appropriate antibodies, and carry out ChIP-on- chIP experiments as described. We can then determine which binding sites on
ERα target genes are enriched for both cyclin T1 and HEXIM1 (as an indicator of
P-TEFb dependency) and HEXIM1 alone (to suggest possible P-TEFb- independent regulation) in the promoter tiling arrays.
These binding sites can subsequently be validated using ChIP and RT-
PCR experiments to demonstrate their functional significance in regulating gene expression of the ERα target genes in either a P-TEFb-dependent or - independent manner.
Identification of transcriptional regulatory regions within HEXIM1 gene
HEXIM1 is ubiquitously expressed in many mammalian cell types, albeit in varying degrees. In previous studies, we found that HEXIM1 mRNA expression was highest in human endocrine tissues including the uterus, cervix, ovary, testis and prostate, relative to the lung, liver and brain [138]. We also found that E2 decreases HEXIM1 mRNA and protein expression in breast cancer cells [138].
Other studies have found that hexamethylene bisacetamide (HMBA), a bipolar differentiation agent, induces HEXIM1 expression in vascular smooth muscle cells [135]. More recently, HMBA was found to increase HEXIM1 expression in a
P-TEFb-dependent manner [160]. Also, HEXIM1 is degraded via association with p53-ubiquitin E3 ligase, human double minute-2 (HDM2) [202]. However, other
143 than P-TEFb, it is not known what transcriptional mechanisms contribute to how
HEXIM1 expression is regulated.
Since E2 decreases HEXIM1 mRNA and protein expression [138], we can assume that the regulation of HEXIM1 by E2 is not solely due to an effect on protein stability or synthesis. Also, it is known that E2/ERα binding to DNA of ERα target genes can either lead to the activation or repression of gene expression
[198], suggesting that it is possible for ERα to negatively regulate HEXIM1.
To first determine whether ERα is a bona fide trans-acting factor and identify other potential transcription factors in HEXIM1 transcription, we can use the TRANSFAC database to determine if the HEXIM1 promoter contains known cis-regulatory elements for identified transcription factors. Next, it would be necessary to determine whether the transcription factor of interest, for example
ERα, regulates HEXIM1 transcript formation. To do this, we can perform transcript runoff assays using MCF-7 cells in the presence or absence of E2.
Additionally, we can identify cis-regulatory elements in the HEXIM1 promoter necessary for its transcriptional activation by cloning fragments with various deletions into reporter vectors. We will then determine the activity of these promoter reporter vectors in the presence or absence of ERα to determine whether ERα represses HEXIM1 transcription. Alternatively, we could use the
ChIP-Sequencing (ChIP-Seq) approach to identify cis-regulatory regions that
HEXIM1 binds without bias towards any previously identified regions.
144 Once we identify responsive cis-regulatory regions from the reporter and
ChIP-Seq assays, ChIP experiments can then be performed to characterize these regions within the promoter and RT-PCR experiments can be carried out to make any correlations to a functional impact on HEXIM1 expression.
In the event that ERα is shown to be a trans-acting factor in HEXIM1 transcription, we could subsequently carry out extensive analyses in a ChIP-on- chip genome-wide array experiment in the presence or absence of ERα to determine other cis-regulatory elements to which ERα binds in the HEXIM1 gene to influence its expression. Alternatively, we can do preliminary analyses using existing data to identify ERα binding sites in the HEXIM1 gene [198, 199].
Finally, to demonstrate the physiological relevance of any identified factors that regulate HEXIM1 expression, we can use cell and animal models to determine their relevance on HEXIM1 in the regulation of cell proliferation and other processes that contribute to tumorigenesis including angiogenesis and apoptosis.
Role of HEXIM1 in apoptosis
Apoptosis or programmed cell death is an important process that occurs during development and a disruption in the normal apoptotic process contributes to pathological physiology including tumorigenesis. Apoptosis occurs due to various stimuli including growth factor withdrawal, stress and overexpression of proapototic stimuli and classic characteristic features in the apoptotic cell include
145 DNA condensation and fragmentation, plasma membrane blebbing and cytoplasmic shrinkage. Other molecular events associated with apoptosis include the cleavage and activation of caspase enzymes. Activated caspases in turn cleave and inactivate Poly(ADP)-ribose polymerase 1 (PARP-1), which is important in monitoring double stranded DNA breaks [203-206].
Reports implicate E2/ERα as a regulator of anti-apoptotic factor, Bcl-2, and
Bcl-2 expression correlates with ERα in Bcl-2 positive mammary tumors [207,
208]. In addition, E2/ERα regulates VEGF transcription [179, 194], and studies have shown that VEGF transcription regulates the expression of anti-apoptotic proteins, Bcl-2 and A1 in endothelial and breast cancer cells via the PI3K-Akt signaling pathway [125, 209]. Conversely, the withdrawal of E2 from chick oviduct results in increased caspase-1, -3 and -6 expression and activation [204].
HMBA has also been shown to decrease Bcl-2 protein expression and induce apoptosis in myeloma cells [210]. In our studies, we found that increased
HEXIM1 expression in the mouse mammary gland leads to an increase in apoptosis [66]. These data suggest a potential role for HEXIM1 in regulating apoptosis.
To determine whether HEXIM1 regulates E2-induced expression of apoptotic factors, using breast epithelial cell lines, MCF-7 (ERα and β-positive) and MDA-MB-231 (ERα-negative), we will carry out gene expression studies
(RT-PCR, Westerns) to assess the effect of the presence or absence of HEXIM1 on the expression of pro- and anti-apoptotic factors, Bcl-2, PARP-1 and
146 Caspase-1 and -3 in the context of E2/ERα signaling. The activity of caspases-1 and 3 can be determined by monitoring the levels of their cleavage products and the cleavage products of PARP-1. In addition, we can correlate the activity of the apoptotic proteins to the levels of DNA fragmentation associated with apoptosis that occurs using DNA fragmentation assays.
We will also address if this mechanism of HEXIM1 regulation is primarily transcriptional via ChIP assays. Because other studies have shown that E2 regulates Bcl-2 expression, we will be assessing the role of HEXIM1 in regulating the recruitment of ERα to the Bcl-2 gene to determine if HEXIM1 regulates
E2/ERα signaling at the Bcl-2 gene. In addition, other studies have shown that
VEGF induces the expression of Bcl-2 via the PI3K-Akt signaling pathway, so we will also carry out gene expression studies looking at components of this signaling pathway including extracellular signal-related kinases 1/2 (ERK 1/2),
STAT and AKT [211]. Controls will include the use of PI3K/AKT and ERK kinase inhibitors and growth factors that initiate PI3K/AKT and ERK signaling.
It is possible that HEXIM1 will have effects on apoptotic regulatory genes independent of ERα given its interaction with P-TEFb and its gene context dependent effects [62, 66, 201]. Loss of ERβ protein expression is correlated with tumor progression and ERβ has been shown to act as a tumor suppressor and inhibit cell proliferation in breast, ovarian, prostate and colon cancers [29,
212, 213]. In breast cells, ERβ has been reported to inhibit cell proliferation by decreasing cyclin D1, c-myc and cyclin A gene transcription and thereby
147 enhancing p21 and p27 expression, which potentiates G2 cell cycle arrest [214].
Additionally, ERβ has been shown to enhance tamoxifen-induced cell death in breast cancer cells [215].
To determine whether HEXIM1 enhances apoptosis via ERβ, we can first determine whether the presence or absence of either HEXIM1 or ERβ diminishes the ability of either factor to induce apoptosis in cell culture. Other studies can be carried out to determine whether ERβ and HEXIM1 interact using coimmunoprecipitation (CoIP) assays in the presence or absence of selective
ERβ selective agonist, diarylpropionitrile (DPN). DPN has been shown to induce a decrease in cell proliferation via ERβ in breast cells [29]. In addition, while there may not be evidence of a direct or indirect interaction between HEXIM1 and ERβ via CoIP, ChIP assays can also be carried out to investigate whether HEXIM1 regulates ERβ transcriptional activity and can therefore influence the effect of
ERβ on apoptosis.
Several other transcription factors are also implicated in regulating apoptosis including NF-κB and p53 [216, 217]. A previous report demonstrated that HEXIM1 regulates NF-κB in vascular smooth muscle cells [136]. Another report showed that P-TEFb activity is integral for the transcriptional activity of some p53 target genes [64], and thus could be potentially affected by HEXIM1.
148 Further examine the role of HEXIM1 in mammary gland development Our studies showed that increased HEXIM1 expression in the mouse mammary gland led to a decrease in ductal branching, an E2/ERα-driven process
[66]. However, the role of HEXIM1 in other stages of mammary gland development is not known. Other steroid hormones and their cognate receptors like the progesterone receptor (PR) come into play after the initial effects of
E2/ERα have occured to influence tertiary ductal branching during puberty. We know that increased HEXIM1 expression did not decrease E2-induced PR expression in breast cancer cells [66], suggesting selective effects of HEXIM1 on
ERα target genes.
To further determine what the role of HEXIM1 is in mammary gland development, it might be informative to decipher if HEXIM1 expression levels fluctuate during mammary gland development. If HEXIM1 expression levels are found to change during the course of development, it would be important to determine if the hormonal milieu during puberty and pregnancy influence its expression. Involution occurs after lactation stops and is characterized by the collapse of mammary gland structures and degradation of the basement membrane due to apoptosis and phagocytosis. It would be interesting to determine whether HEXIM1 levels are increased during this process, linking
HEXIM1 physiologically during mammary gland development as an apoptotic factor.
We could also use our MMTV/HEXIM1 transgenic mice or mice with a conditional knockout of HEXIM1 in the mammary gland in developmental studies
149 to determine the effect of HEXIM1 expression on tertiary ductal branching and lobulo-alveolar development driven by progesterone and prolactin (PRL) respectively during pregnancy [218, 219].
Role of HEXIM1 in mammary tumorigenesis
In previous studies, we demonstrated that HEXIM1 expression was decreased in human breast cancer relative to HEXIM1 expression levels in adjacent normal tissue [138]. Our studies have shown that HEXIM1 regulates ER target genes that influence breast cell proliferation and the development of blood vessels [66, 138, 141]. In addition, we found that the loss of a functional domain in HEXIM1 leads to an increase in susceptibility to carcinogen-induced tumorigenesis in the mouse mammary gland (Chapter III). These data suggest a role for HEXIM1 in tumorigenesis.
The aromatase transgenic mouse model is commonly used to study E2- dependent carcinogenesis. Overexpression of aromatase in the mouse mammary gland leads to enlargement of ducts with significant hyperplastic lesions which are thought to increase the risk of developing neoplasia and have been shown to increase susceptibility to carcinogens [220, 221].
To determine the role of HEXIM1 in cancer initiation processes, we can mate our MMTV/HEXIM1 mice with aromatase transgenic mice to elucidate whether increased HEXIM1 expression decreases aromatase-induced hyperplasia in the mammary gland. Also, these mice can be treated or not with
150 suboptimal doses of 7,12-dimethylbenz[a]anthracene (DMBA) to determine whether HEXIM1 plays a protective role in susceptibility to DMBA-induced tumorigenesis. Conversely, mice with a conditional knockout of HEXIM1 in the mammary gland in aromatase transgenic mice can be generated to determine whether the loss of HEXIM1 contributes to an enhanced rate of estrogen-induced tumorigenesis.
Although our studies have focused on the effects of HEXIM1 on E2/ERα- associated processes in proliferation, mammary gland development and tumorigenesis, the effect of HEXIM1 in ERα negative cancers remains to be explored. Previous studies from our laboratory showed that a decrease in
HEXIM1 expression led to an increase in cell proliferation in both ERα positive and negative breast cancer cell lines [138], suggesting a broad role for HEXIM1 in regulating cell growth. The HER2 gene is amplified in many cancers that are typically ER negative and HER2 overexpression contributes to tumorigenesis
[222]. In mice, HER2/neu overexpression via the MMTV promoter in the mammary gland enhances the rate of tumor progression [223]. We could mate our MMTV/HEXIM1 mice with the MMTV/neu mice to determine whether increased HEXIM1 expression would decrease the rate of tumor formation.
151 Further elucidate HEXIM1 regulation of HIF-1α and VEGF and its effect on tumorigenesis
Both HIF-1α and VEGF are implicated in tumorigenesis and have been shown to play key roles in metastasis [132, 224, 225]. Increased VEGF and HIF-
1α expression are linked to more aggressive phenotypes in human breast cancer, but there is also a positive correlation between HIF-1α, VEGF and ERα expression in tumors [226]. In mouse models, although HIF-1α is not required for the formation of mammary tumors, it enhances the rate of tumor formation [132].
HIF-1α is also required for VEGF-induced angiogenesis associated with early tumorigenesis [132]. In addition, metastasis associated proteins have been shown to stabilize HIF-1α expression in both ERα positive and negative breast cancer cells [227].
Our studies implicate HEXIM1 in the regulation of E2-induced HIF-1α and
VEGF expression in breast cancer cells and the mouse mammary gland with functional consequences on vascularization and tumorigenesis (Chapter III). In the case of VEGF, the regulation by HEXIM1 appears to be transcriptional, but in the case of HIF-1α, it appears to be either due to a decrease in protein synthesis or stability. Our studies did not demonstrate the potential mechanism by which
HEXIM1 regulates E2-induced HIF-1α expression.
To do so, breast cancer cells that have been induced with hypoxic stimuli in the presence or absence of E2 and HEXIM1 can be treated with either protease or protein synthesis inhibitors. Changes in the rates of HIF-1α protein
152 stability can then be monitored. While we did not observe an interaction between
HEXIM1 and HIF-1α in CoIP experiments, other preliminary studies in our laboratory suggest that increased HEXIM1 expression decreases HIF-1α stability, but further studies are necessary to verify this.
Other studies that can be carried out would aim to define the role of
HEXIM1 during tumor progression as it relates to the expression of HIF-1α and
VEGF given their role in metastasis, but appropriate animal models would be necessary to do this.
Polyomavirus middle T antigen (PyMT) is a membrane attached protein that is a potent oncogene because it induces typrosine kinase activity in factors including the c-src family and PI3K associated with the development of mouse tumors [228, 229]. Mice with mammary gland-directed expression of PyMT
(MMTV/PyMT) develop mammary tumors that go through distinct stages of progression comparable to human breast cancer in terms of morphology and the expression of ERα, PR and ErRB2 [229]. These mice also go through the
“angiogenic switch” process where there is an increase in blood vessel density surrounding tumors and exhibit high levels of pulmonary metastasis [229]. To determine the role of HEXIM1 during metastasis, our MMTV/HEXIM1 mice can be mated with MMTV/PyMT mice and we can then investigate whether an increase in HEXIM1 expression regulates HIF-1α and VEGF expression in the mammary gland as the tumors progress from hyperplasia to invasive ductal carcinomas.
153 CONCLUDING REMARKS
Emerging evidence continues to unravel the clinical significance of selective modulation of nuclear receptors and transcription factors by their cognate ligands or other coregulators [27]. We envision HEXIM1 to be one such coregulator and speculate that given the growth-inhibitory and anti-angiogenic potential of HEXIM1, it can be harnessed as a potential therapeutic strategy in
ERα positive breast cancer, once tools for safe and local up-regulation of its expression become available. The studies conducted during this thesis support ongoing work in the Montano lab involving the development of localized delivery of derivatives of HMBA in the mammary gland to upregulate HEXIM1 expression using biodegradable polymer poly(lactic-co-gylycolic acid) (PLGA) [230].
However, as several genes contribute to ERα positive breast cancer and tumorigenesis, further work is necessary to elucidate the effect of HEXIM1 on other ERα target genes and other novel target genes to move closer to this goal.
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