Characterizing the role of GREB1 in regulation of breast cancer
proliferation
Dissertation
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of
Philosophy in the Graduate School of The Ohio State University
By
Corinne Nicole Haines
Graduate Program in Molecular, Cellular, and Developmental Biology
The Ohio State University
2019
Thesis Committee
Craig J. Burd PhD., Advisor
Sharon L. Amacher, PhD.
Anne M. Strohecker, PhD.
Philip N. Tsichlis, PhD.
1
Copyrighted by
Corinne Nicole Haines
2019
2
Abstract
Breast cancer is the most frequently diagnosed malignancy in women.
The vast majority (>70%) of breast cancer patients are diagnosed with the estrogen receptor-positive subtype. These cancers express the transcription factor estrogen receptor α (ER) and depend on its activation and subsequent regulation of estrogen-responsive genes for survival and proliferation. Patients diagnosed with ER-positive breast cancer are typically given endocrine therapies that target the activity of the ER. Although endocrine therapies are initially effective, patients invariably develop resistance to currently available treatments, underscoring the need to better characterize the mechanism by which ER-target genes drive proliferation in order to develop new and innovative therapies.
One gene target of ER, growth regulation by estrogen in breast cancer 1
(GREB1), is associated with proliferation of ER-positive breast cancer cells. The
GREB1 gene encodes three distinct protein isoforms: GREB1a, GREB1b, and
GREB1c. Unfortunately, few studies have investigated the mechanism by which
GREB1 regulates proliferation and no studies have investigated the isoform specific molecular functions of GREB1. Here, I investigate the role of the GREB1 isoforms in modulation of ER activity and proliferation. I show that all three isoforms interact with ER through their homologous amino terminus. Analysis of
ii isoform-specific regulation of ER activity reveals that none of the GREB1 isoforms are potent regulators of ER activity. Interestingly, exogenous expression of GREB1a, but not GREB1b, was associated with increased expression of some
ER-target genes and this function was independent of ER activity. While these data demonstrate diverging roles for GREB1a and GREB1b in modulation of gene expression, exogenous expression of either GREB1a or GREB1b resulted in similar patterns of growth repression in breast cancer cell lines, independent of
ER expression. Analysis of GREB1 isoform expression in cell lines and patient samples reveals that while GREB1a is the predominant isoform, there appears to be an increase in the expression of both GREB1b and GREB1c in malignant patient samples compared to normal patient samples. Taken together, these data suggest that GREB1a has an isoform-specific function in transcriptional regulation while all three isoforms share an ER-independent function to modulate proliferation.
Despite the clear relationship between GREB1 expression and proliferation of breast cancer cells, the explicit mechanism by which GREB1 regulates this process remains unclear. As GREB1a is the predominant isoform in cell lines and patient samples, I focused further investigation on this isoform.
Our studies indicate that knockdown of GREB1 results in growth arrest and exogenous expression of GREB1 induces cellular senescence, suggesting a dynamic role for GREB1 in the modulation of proliferation. Analysis of signaling pathways known to regulate both senescence and proliferation revealed that
iii
GREB1 is able to modulate signaling through the PI3K/Akt/mTOR pathway. I show that GREB1 acts through the canonical PI3K pathway to regulate PIP3 levels and activation of Akt and downstream effectors. Importantly, growth suppression of estrogen-dependent breast cancer cells by GREB1 knockdown is rescued by expression of constitutively activated Akt. Together, these data demonstrate a novel mechanism by which GREB1 modulates signaling through the PI3K/Akt/mTOR pathway to regulate proliferation of breast cancer. These findings are critical to better understanding how ER-target genes drive proliferation of breast cancer and developing innovative therapies that act downstream of ER-signaling.
iv
Dedication
This work is dedicated to my mother, Kimberley Haines, and my brother, Kel
Haines, for their unconditional love and support.
v
Acknowledgments
I am forever grateful for the numerous individuals that have supported and encouraged me throughout my graduate career.
I am beyond appreciative of my graduate advisor, Dr. Craig Burd. When I decided to come to OSU I had a clear idea of the type of mentor and lab environment I was looking for: approachable, hands-on, engaging, motivating, and fun. I am thankful to say I found all of those characteristics in my mentor,
Craig, and the Burd lab. Craig, your unwavering support and guidance have allowed me to become a better scientist, writer, and presenter. I have learned more in the past 5 years in your lab than I could have ever imagined. Thank you for your resourcefulness and your willingness to support my ideas.
Thank you to my committee members, Drs. Sharon Amacher, Anne
Strohecker, Denis Guttridge, and Philip Tsichlis for your continued support and advice. Your input and guidance helped to move my project forward and enabled me to become a better scientist. To Dr. Christin Burd, thank you for all of your advice, care, and especially your tough love.
My graduate career would not have been the same without the companionship of my fellow graduate students: Clarissa Wormsbaecher, Becky
Hennessey, Kyle LaPak, Brandon Murphy, and Xiangang Guan. To Ali Shapiro,
vi
Alina Murphy, Makanko Komara, Evi Zhang, and Tirzah Weiss, thank you for all of your help over the years.
Finally, thank you to my family and friends for helping to make graduate school bearable. To my mom, Kimberley Haines, there are no words to express my immense gratitude and appreciation for everything you have done and continue to do for me that has allowed me to get to this point in my life. You are truly my inspiration and the one that has made all of this possible. Thank you. To my brother, Kel Haines, thank you for always being there to cheer me up and for encouraging my love of science. To my “work dad,” Bill Neville, thank you for your unwavering care and support, advice, and for being the best Pelotonia riding partner. To Garrett Strawser, thank you for being the one to make me laugh and smile at the end of everyday- even when nothing in lab worked and I’m hangry.
You guys have been the best cheerleaders ever and I couldn’t have done it without you. Finally, thank you to my cat, Ethel, for always welcoming me home with loud meows and warm cuddles.
vii
Vita
2006-2010……..Grove City High School, Grove City, OH
2010-2014……..B.S. Biology, Denison University, Granville, OH
2014-2019……..Graduate Research Associate, Graduate Teaching Associate,
and Pelotonia Fellow, The Ohio State University, Columbus, OH
Publications
Haines CN, Braunreiter KM, Mo XM, Burd CJ. (2018). “GREB1 isoforms regulate proliferation independent of ERalpha co-regulator activities in breast cancer.”
Endocrine-related cancer.
Fields of Study
Major Field: Molecular, Cellular, and Developmental Biology
Research Area: Cancer Biology
viii
Table of Contents
Abstract ...... ii Dedication ...... v Acknowledgments ...... vi Vita ...... viii Table of Contents ...... ix List of Tables ...... xiii List of Figures ...... xiv Chapter 1 : Introduction ...... 1 1.1 Breast Cancer Incidence, Risk Factors, and Diagnosis ...... 1 1.2 Estrogen and Estrogen Receptors ...... 2 1.2.1 The Estrogen Receptors ...... 3 1.2.2 Structure of the Estrogen Receptors ...... 4 1.2.3Estrogen Receptor Action ...... 4 1.2.4 Role of the Estrogen Receptor in Mammary Gland Development and Breast Cancer ...... 9 1.3 Breast Cancer Treatment and Resistance ...... 12 1.3.1 Endocrine Therapy ...... 13 1.3.2 Other Targeted Therapies and Immunotherapy ...... 15 1.3.3 Resistance to Endocrine Therapies ...... 15 1.4 Signal Transduction Pathways in Breast Cancer ...... 17 1.4.1 Receptor Tyrosine Kinases (RTKs) and Breast Cancer ...... 17 1.4.2 PI3K/Akt/mTOR Pathway is Frequently Altered in Breast Cancer ...... 18 1.5 Growth Regulation by Estrogen in Breast Cancer 1 (GREB1) ...... 21 1.5.1 Identification and Structure ...... 21 1.5.2 Hormone-Dependent Regulation of GREB1 Expression ...... 22 1.5.3 Expression and Localization of GREB1 ...... 25
ix
1.5.4 Function and Clinical Relevance of GREB1 in Breast Cancer ...... 26 1.5.5 Function and Clinical Relevance of GREB1 in Other Hormone- Dependent Diseases ...... 30 1.6 Statement of Research Purpose ...... 32 Chapter 2 : GREB1 isoforms regulate proliferation independent of ERα co- regulator activities in breast cancer ...... 34 2.1 Abstract ...... 34 2.2 Introduction ...... 35 2.3 Materials and Methods ...... 37 2.3.1 Cell Lines and Reagents ...... 37 2.3.2 Plasmids ...... 38 2.3.3 Immunoblot Analysis and Antibodies ...... 38 2.3.4 Adenovirus ...... 39 2.3.5 Immunoprecipitation ...... 40 2.3.6 Gene Expression Analysis ...... 40 2.3.7 GREB1 Isoform Expression ...... 42 2.3.8 Reporter Assay ...... 43 2.3.9 MTT Assays ...... 44 2.3.10 EdU Incorporation Assay ...... 44 2.3.11 Cell Viability Assay ...... 44 2.4 Results ...... 45 2.4.1 The amino terminus of GREB1 isoforms interact with ERα ...... 45 2.4.2 GREB1 isoforms are not robust regulators of ERα activity ...... 47 2.4.3 Elevated expression of GREB1a and GREB1b differentially regulate ERα target genes ...... 50 2.4.4 GREB1 expression is not sufficient to drive hormone-independent growth...... 55 2.4.5 GREB1a and GREB1b modulate proliferation of breast cancer cells independent of ERα status ...... 57 2.4.6 GREB1b and GREB1c expression is increased in primary and metastatic patient tumor samples ...... 60 2.5 Discussion ...... 64 2.5.1 GREB1 isoforms are not potent regulators of ERα activity ...... 65 2.5.2 GREB1 regulates proliferation through ER independent activities ... 66 x
Chapter 3 : GREB1 regulates proliferation of estrogen receptor positive breast cancer through modulation of PI3K/Akt/mTOR signaling ...... 69 3.1 Abstract ...... 69 3.2 Introduction ...... 70 3.3 Materials and methods ...... 73 3.3.1 Cell lines and reagents ...... 73 3.3.2 Plasmids ...... 73 3.3.3 Immunoblot analysis and antibodies ...... 74 3.3.4 Adenovirus ...... 74 3.3.5 Alamar blue assay ...... 74 3.3.6 SA-β-gal staining ...... 75 3.3.7 Conditioned media assay ...... 75 3.3.8 Co-culture assay ...... 75 3.3.9 Immunofluorescence microscopy ...... 76 3.4 Results ...... 77 3.4.1 GREB1 initiates cellular senescence ...... 77 3.4.2 Exogenous GREB1 expression induces hyperactivation of the PI3K/Akt/mTOR pathway...... 78 3.4.3.GREB1-induced hyperactivation of Akt is PI3K-dependent ...... 81 3.4.4 GREB1 activates Akt through intracellular mechanisms ...... 85 3.4.5 Exogenous GREB1 promotes recruitment of Akt to the plasma membrane ...... 86 3.4.6 GREB1 regulates breast cancer proliferation through activation of the PI3K/Akt/mTOR pathway...... 93 3.5 Discussion ...... 96 3.5.1 GREB1-induced activation of Akt is dependent on PI3K ...... 96 3.5.2 GREB1 regulates proliferation of ER+ breast cancer cells through modulation of Akt activity ...... 98 3.5.3 GREB1 and endocrine resistance ...... 99 Chapter 4 : Conclusions and Future Directions ...... 101 4.1 GREB1 is not a potent regulator of ER activity ...... 102 4.2 GREB1 regulates proliferation of breast cancer cell lines through fine- tuning of PI3K/Akt/mTOR signaling ...... 103 4.3 GREB1 and clathrin-mediated endocytosis ...... 105 xi
4.4 Molecular Function of GREB1 ...... 109 4.5 GREB1 and clinical determinants of breast cancer progression and pathway activation ...... 112 References…………………………………………………………………………....113
xii
List of Tables
Table 2.1 SYBR Real-Time PCR Primers ...... 41 Table 2.2 Taqman Real-Time PCR Primers and Probes ...... 43 Table 4.1 GREB1 rapid immunoprecipitation and mass spectrometry of endogenous protein (RIME) reveals interaction of GREB1 with endocytic proteins...... 107
xiii
List of Figures
Figure 1.1 Mechanisms of ligand-dependent and ligand-independent ER activity...... 6 Figure 1.2 Human GREB1 encodes for three protein isoforms...... 22 Figure 2.1 GREB1a protein aggregates at high temperatures...... 39 Figure 2.2 The amino terminus of GREB1 isoforms interact with ERα...... 46 Figure 2.3 GREB1 isoforms are not robust regulators of ERα activity...... 49 Figure 2.4 GREB1 knockdown does not affect ERα activity...... 50 Figure 2.5 GREB1 isoform expression varies between breast cancer cell lines. 52 Figure 2.6 GREB1a and GREB1b differentially regulate ERα target genes...... 53 Figure 2.7 GREB1 preferentially binds ERα in the absence of ligand and in the cytoplasm...... 55 Figure 2.8 GREB1 knockdown reduces proliferation ...... 56 Figure 2.9 GREB1 expression is not sufficient to drive hormone-independent growth...... 57 Figure 2.10 Elevated GREB1a and GREB1b expression reduces proliferation of breast cancer cells independent of ERα status...... 59 Figure 2.11 Elevated GREB1a and GREB1b expression reduces proliferation of breast cancer cells...... 60 Figure 2.12 Proportion of GREB1b and GREB1c expression increases during formation of primary tumor...... 62 Figure 2.13 Expression of GREB1b and GREB1c is higher in malignant tissue. 63 Figure 3.1 Exogenous GREB1 initiates cellular senescence...... 78 Figure 3.2 GREB1 modulates PI3K/Akt pathway signaling...... 80 Figure 3.3 GREB1-induced hyperactivation of Akt is PI3K-dependent...... 84 Figure 3.4 GREB1 activates Akt through intracellular mechanisms...... 86 Figure 3.5 Exogenous GREB1 promotes recruitment of Akt to the plasma membrane...... 88 Figure 3.6 Endogenous GREB1 re-localizes to the cytoplasm under growth- stimulatory conditions...... 92 Figure 3.7 GREB1 regulates breast cancer proliferation through activation of the PI3K/Akt/mTOR pathway ...... 95 Figure 4.1 Receptor tyrosine kinase trafficking through CME...... 107 Figure 4.2 Stimulation of clathrin-mediated endocytosis (CME) causes GREB1 to re-localize from the nucleus to the cytoplasm...... 108 Figure 4.3 GREB1 co-localizes with proteins involved in clathrin-mediated endocytosis (CME)...... 109 xiv
Figure 4.4 GREB1 fragment design...... 111 Figure 4.5 GREB1 localization differs among tumor grades...... 113
xv
Chapter 1 : Introduction
1.1 Breast Cancer Incidence, Risk Factors, and Diagnosis
Breast cancer is the consequence of genetic aberrations within the cells that line the milk-producing ducts within the mammary gland. The uncontrolled proliferation of these cells results in the formation of a solid tumor within the mammary gland [1]. Breast cancer is by far the most frequently diagnosed malignancy in humans [2]. In the United States, it is expected that over 268,000 individuals will be diagnosed with breast cancer and more than 41,000 individuals will succumb to their disease in 2019 alone [2]. Worldwide, breast cancer incidence is projected to continue rising despite efforts to prevent the disease [2].
For most countries, the rise in breast cancer incidence is unsurprising due to an increase in the number of women with breast cancer risk factors. Risk factors can be broken up into non-modifiable and modifiable risks. For breast cancer, non-modifiable risks include gender, race, older age, family history of breast cancer, genetic mutations (ex. BRCA1/2), higher breast density, radiation treatment, younger age of menarche, and older age of menopause [3, 4].
Modifiable risk factors include fewer pregnancies, shorter time or no time spent
1 breastfeeding, obesity, inactivity, alcohol consumption, smoking, contraceptive use, and hormone replacement therapy [3, 4].
Using these risk factors, several models and scoring systems have been created to estimate a woman’s risk of developing breast cancer over time [3, 4].
This information is then used to implement preventative measures such as lifestyle and behavior changes, chemoprevention, and/or surgery for high-risk patients [3, 4]. In addition to preventative measures, physicians use these risk factors to determine if and when patients will receive mammogram screening and/or genetic screening [3, 4]. Mammogram screening, which uses imaging to detect breast density, is the most frequently used breast cancer diagnostic method [5]. Following an irregular mammogram, patients typically receive a breast tissue biopsy to test for malignancy and to determine prognosis and therapy regimen if cancer is found [5].
1.2 Estrogen and Estrogen Receptors
1.2.1 Estrogen Biosynthesis and Role in Development and Physiology
The majority of risk factors linked to breast cancer are associated with lifetime exposure to estrogens. Estrogen is a steroid hormone produced from the enzymatic conversion of androgens (adrostenedione and testosterone) by aromatase in subcutaneous fat, muscle, bone, brain, ovarian, and the breast tissue [6-9]. The most abundant and most potent form of estrogen in humans is
17β-estradiol (estradiol) [1, 7-9]. In premenopausal women, the vast majority of estrogen is produced by the ovaries to support the development of sexual
2 characteristics, regulation of ovulation, and preparation of tissues for reproduction [7, 9]. In both men and women, local synthesis of estrogen plays a key role in the development and maintenance of the cardiovascular, musculoskeletal, immune, and central nervous systems [1, 9].
1.2.1 The Estrogen Receptors
The estrogen receptor (ER) exists in two isoforms, ER and ER, each encoded in separate genes (ESR1 and ESR2, respectively) on different chromosomes [1, 9, 10]. Multiple variants exist for each isoform although the biological functionality of the different variants is unclear [1, 10]. The two isoforms, ER and ER, share a high degree of homology with the exception of their amino terminal domains [1, 10]. Additionally, ER and ER have similar affinities for their primary ligand, estradiol and bind the same estrogen response elements within the DNA [1, 10]. As the estrogen receptors belong to the family of proteins referred to as nuclear receptors, these proteins are primarily localized within the nucleus of their indicated tissues, although some membraneous localization of the ERs has been observed [1, 11-13]. Interestingly, when expressed simultaneously the biological function of these proteins appears to oppose one another at some DNA binding sites (ex. Cyclin D1), suggesting the proliferative response to estradiol through these receptors is dependent on a balance between ER and ER [1, 14-16].
3
1.2.2 Structure of the Estrogen Receptors
The ER belongs to the steroid hormone nuclear receptor family of proteins, which share a general protein structure encompassing a DNA-binding domain (DBD), a ligand-binding domain (LBD), and activation function domains
(AF) [1, 17]. The DBD is centrally located in the protein structure of the ER, plays a role in receptor dimerization, and acts to recognize and bind to specific DNA sequences [1, 10]. The LBD is located on the carboxyl-terminus of the ER and acts to bind ligands to allow for receptor dimerization and translocation to the nucleus [1, 10]. The LBD itself is generous in size allowing the ER to bind a wide range of compounds [1]. The AF domains, AF-1 and AF-2, are located on the amino terminus and the carboxyl-terminus, respectively, and both act to recruit co-regulators to DNA-bound ER [1]. The AF-1 domain functions independent of ligand binding and is involved in orchestrating protein-protein interactions and transcriptional activation [10]. The AF-2 domain is located within the LBD and contains helix 12, which when bound to ligand is stabilized in a conformation that exposes a shallow hydrophobic site allowing for optimal binding to the LxxLL motif of co-activators [1]. Binding of co-activators to ER on gene promoters allows for transcription of target genes [1].
1.2.3 Estrogen Receptor Action
Evidence suggests that the ER can regulate biological processes through multiple distinct mechanisms that are both ligand-dependent and ligand- independent [1]. In the presence of ligand, the ER can affect biological processes
4 through rapid, non-genomic mechanisms or through both direct and tethered genomic mechanisms (Fig. 1.1)[1]. In the absence of ligand, growth factor signaling can activate ER to induce changes in gene expression (Fig. 1.1)[1].
5
Figure 1.1 Mechanisms of ligand-dependent and ligand-independent ER activity. The canonical mechanism of ER activity involves liganded ER binding directly to estrogen response elements (EREs) within the genome to regulate transcription. Alternatively, liganded ER can interact with other transcription factors and regulate transcription through indirect DNA binding. Liganded ER, either at the membrane or within the cytoplasm, can act through signaling proteins (SP) to activate various signaling cascades, increase nitric oxide (NO) production, or induce changes in ion flux. Independent of ligand, the ER can be phosphorylated by kinases activated by various growth factors (GFs). Phosphorylated ER can then bind DNA and regulate gene transcription. Figure adapted from [1].
6
1.2.3.1 Rapid, Non-Genomic Effects
Some results of estrogen stimulation occur too rapidly to be mediated by transcription of RNA followed by protein synthesis, leading to the discovery of rapid, non-genomic effects of estrogen and ER (Fig. 1.1) [1, 12, 13, 18].
Specifically, activation of ER by estrogen binding has been associated with activation of signaling cascades and increases in ion flux (Fig. 1.1) [1, 18].
Liganded ER has been shown to either directly or indirectly activate the IGF-1 receptor [19], the Her2/neu receptor [20], and the EGF receptor [21], which lead to downstream signaling through the MAPK and PI3K/Akt/mTOR pathways (Fig.
1.1) [1, 22]. Additionally, liganded ER has been shown to regulate calcium flux
[23] and production of cAMP by adenylate cyclase [21, 24]. Although these rapid, non-genomic effects of ER have been studied extensively, it remains unclear if these effects are a result of a membrane-bound ER variant or the classical ER [1,
12, 18]. As the ER contains no transmembrane domains, localization of the classical ER to the plasma membrane may be a result of palmitoylation or myristoylation [1, 12]. However, a member of the 7-transmembrane G protein- coupled receptor family, GPR30, has been shown to bind to and respond to estrogen in a large variety of cell types [25, 26]. Now widely considered as an estrogen receptor, GPR30 is known to function in a mechanism clearly distinct from that of ERα and ERβ [25, 26]. Several studies have now shown that GPR30 plays a critical role in activating the MAPK and PI3K/Akt/mTOR pathways in response to estrogen [25, 26]. Despite these reports, the extranuclear function of
7
ER remains relatively understudied, but strongly suggests that the biological outcomes of estrogen exposure are the result of synergy between cytoplasmic estrogen signaling cascades and estrogen-induced genomic regulation.
1.2.3.2 Genomic Effects
The canonical mechanism of estrogen signaling is through regulation of
ER-target genes [1]. Upon binding of estrogen to the ER, chaperone proteins release the ER and allow for dimerization of liganded-ER and translocation to the nucleus [8]. Within the nucleus, the ER can bind to estrogen response elements
(EREs) within the promoters of target genes (direct mechanism) or can bind to other transcription factors or chromatin remodelers (Ex. Fos/Jun, NFkB,
SWI/SNF) that interact directly with the DNA (tethered mechanism) (Fig. 1.1) [1,
8, 12]. Binding of an agonist to the ER exposes hydrophobic surfaces on the receptor that allows for recruitment of transcriptional co-factors and chromatin remodelers [1]. The interaction of ER with transcriptional co-factors is essential for proper estrogen signaling as these proteins typically exist in complexes, have various enzymatic functions, and act to recruit general transcriptional machinery to the ER [9, 17]. Whether ER acts as a transcriptional activator or repressor depends on which co-factors are expressed and interact with the ER [10]. Co- factors can act as either co-activators to enable the transcription of estrogen- responsive genes (Ex. SRC family, CBP/p300, TRAP/DRIP) or as co-repressors to inhibit the expression of estrogen-responsive genes (Ex. N-CoR, SMRT,
HDAC2) [9, 10]. Thus, the overall expression of estrogen-responsive genes
8 depends heavily on which co-regulators are expressed and available when the cell is exposed to hormone [10, 27].
1.2.3.3 Ligand-Independent Effects
In the absence of agonist, the ER can also be activated by phosphorylation at Ser118 primarily through growth factor-activated kinases such as ERK1/2, PKA, and PKC [1, 10, 28]. The phosphorylation and activation of ER in the absence of ligand enables transcription of ER-target genes (Fig. 1.1) [1,
10, 28]. Thus, it is believed that this method of ER activation is responsible for the hormone-independent growth of some breast tumors [29, 30].
1.2.4 Role of the Estrogen Receptor in Mammary Gland Development and Breast Cancer
Mammary gland development is unique to mammals and is responsible for providing milk and nutrients to the young. Although both males and females begin with similar rudimentary mammary glands during embryogenesis, the bulk of mammary gland development occurs during female puberty when high levels of estrogen are beginning to be produced by the ovaries [9, 31]. Additional growth and involution of the mammary gland, as well as expansion and regression of ductal branching, is associated with the menstrual cycle, pregnancy, and lactation when levels of circulating hormones fluctuate [9, 31].
The mammary gland is made of many specialized cell types including epithelial cells, adipocytes, fibroblasts, and vascular and immune cells [9, 31]. The ductal structure in the adult breast is primarily composed of epithelial cells and
9 fibroblasts [9]. The epithelial cells line the ducts and lobules and are primed to produce milk whereas the fibroblasts act to send communicative signals to epithelial cells to promote survival, proliferation, and morphogenesis [9, 31]. The estrogen receptor is primarily expressed within the epithelial cells of the developed breast, and thus, is thought to be the primary site of estrogenic action
[9, 32]. Interestingly, immunohistochemical analysis revealed that only about 15-
25% of the epithelial cells in the adult breast are ER+ and are largely non- mitogenic [9, 33]. Instead, ER+ epithelial cells in the normal, resting breast are thought to support the proliferation of surrounding, ER- cells [9].
Although the explicit path to breast cancer development is unclear, data from animal models suggests that many types of breast cancer begin with hyperproliferation of the epithelial cells lining the ducts followed by a pre- neoplastic phase known as ductal carcinoma in situ (DCIS) [9]. Invasive breast carcinoma occurs when the mass breaches the basement membrane and invades the surrounding tissue [9, 34]. Once the mass leaves the local site, metastatic breast cancers typically disseminate to the lymph nodes in the armpit or to the lungs, liver, bone or brain [34]. Unfortunately, even if the primary tumor is removed, micro-metastases may remain within the body and metastatic disease may be detected months or years after initial treatment for breast cancer
[34]. It is estimated that as many as 30% of women diagnosed with early-stage breast cancer will develop metastatic disease, however, predicting which patients have a higher risk of recurrence remains difficult and varies greatly from
10 individual to individual and depends heavily on the biology and grade of the tumor at initial diagnosis [34].
Breast cancer, like most malignancies, is a very heterogeneous disease and clinical behaviors vary significantly across the patient population [35].
However, patients are typically grouped into one of four intrinsic breast cancer subtypes: luminal A, luminal B, human epidermal growth factor receptor 2- positive (HER2+), or triple negative/basal-like [36]. Luminal A breast cancers are classified based on the expression of either or both ERα (henceforth referred to as ER in the context of breast cancer) and progesterone receptor (PR), lack of
HER2 expression, and low levels of Ki-67 staining, a marker for proliferation [36].
Patients diagnosed with the luminal A subtype typically have a low grade tumor and have a better prognosis than other subtypes [36]. Luminal B breast cancers are further divided based on the expression of HER2 [36]. Luminal B HER2- breast cancers are categorized based on the expression of either or both the ER and the PR, lack of HER2 expression, and high Ki-67 staining [36]. Luminal B
HER2+ breast cancers express either or both the ER and the PR and have overexpressed or amplified HER2 [36]. HER2+ breast cancers are characterized only by the overexpression of amplification of HER2 and a lack of either ER or
PR expression. Triple negative/basal-like breast cancers lack the expression of
ER, PR, and HER2 [36]. Classification of tumors into these defined subtypes aids in guiding therapy recommendations for the patient [36].
11
The vast majority of breast cancer patients are diagnosed with ER+ tumors that express estrogen receptor α [8, 37-39]. In stark contrast to the epithelial cells of the normal, resting breast, ER+ breast cancer cells rely heavily on estrogen and the ER for cell growth, proliferation, and survival [9]. The proliferation of ER+ breast cancer cells is supported by higher local levels of estrogen driven by the expression of aromatase within the fibroblasts of the tumor microenvironment [8, 40]. Although the explicit mechanism by which ER drives proliferation of cancer cells remains unclear, it is hypothesized that ER simply drives the expression of genes that regulate the cell cycle transition and proliferation, such as cyclin D1 and c-myc [8, 41-44]. Unfortunately, as many as
50% of women diagnosed with ER+ breast cancer will fail currently available therapies [39], highlighting the need to better understand the mechanism by which ER-target genes drive growth of breast cancer cells.
1.3 Breast Cancer Treatment and Resistance
Treatment of breast cancer patients is heavily dictated by the molecular subtype associated with their diagnosis [36]. Patients diagnosed with the Luminal
A subtype are typically given only endocrine therapy [36]. Endocrine therapy also makes up a portion of the treatment regimen for patients diagnosed with the luminal B subtype, who typically receive chemotherapy in addition to endocrine therapy [36]. For patients diagnosed with HER2+ breast cancer, chemotherapy with the addition of trastuzumab, which targets HER2 specifically, remains the mainstay of therapeutic treatment [36]. Chemotherapy is the only currently
12 approved therapy for patients diagnosed with triple-negative breast cancer [36].
For all breast cancer patients, surgery to remove a portion of the breast (breast- conserving surgery) or the entire breast (mastectomy) along with the tumor remains an important treatment modality regardless of subtype [45]. In addition to surgery, radiation therapy is typically used to reduce local recurrence of residual microscopic disease [45].
1.3.1 Endocrine Therapy
Endocrine therapy can be broken down into three major categories based on the mechanism by which they act: selective ER modulators (SERMs), selective ER downregulators (SERDs), and aromatase inhibitors (AIs).
SERMs are non-steroidal compounds that act as ligands for the ER and have mixed agonist/antagonist profiles depending on their target tissue [46]. The majority of SERMs are used in an adjuvant setting in which the patient also receives surgery and sometimes chemotherapy [46]. The most common SERM, tamoxifen, remains the mainstay of ER+ breast cancer treatment [9, 46].
Tamoxifen, similar to endogenous estrogens, requires metabolic activation to form its active metabolite, 4-hydroxytamoxifen (4-OHT) [46]. 4-OHT disrupts estrogen signaling by competitive binding to the ER, leading to conformational changes to the co-factor binding site [1]. In the breast, interaction of 4-OHT with the ER reduces ER-target gene expression [1]. However, tamoxifen acts as an
ER agonist in the uterus and bone, inducing expression of ER-target genes [1].
Adjuvant tamoxifen treatment in ER+ breast cancer patients for 5 years resulted
13 in a 51% decrease in breast cancer recurrence and a 28% decrease in mortality that was sustained after the 5 years of treatment [9]. Toremifene and raloxifene act in a similar manner to tamoxifen and are associated with similar efficacy and safety in breast cancer patients [46]. However, neither toremifene nor raloxifene induce uterine growth as is frequently seen in patients treated with tamoxifen
[46].
SERDs, act by competitively binding to the ER and causing degradation via the ubiquitin-proteasome pathway [8]. The first SERD approved for use in patients was fulvestrant in 2002 [8]. Fulvestrant has been shown to be more potent than tamoxifen in vitro and does not induce the partial agonist properties associated with SERMs [8]. However, due to the limitations in bioavailability, fulvestrant has to be given as a 500mg/month intramuscular injection, prompting the current search for an orally bioavailable SERD [47, 48].
AIs act to reduce local estrogen biosynthesis, therefore restricting intratumoral estrogen levels [8]. Although tamoxifen is the mainstay of endocrine therapies, studies suggest that AIs are typically more effective than tamoxifen in the adjuvant setting with reduced relapse [49, 50]. AIs exist as both reversible and irreversible inhibitors. Reversible AIs include letrozole and anastrozole which are non-steroidal and competitively bind to aromatase [8]. Exemestane is a steroidal AI that irreversibly binds to aromatase leading to its inactivation [8]. AIs are typically used in post-menopausal women and are effective in the adjuvant and post-metastatic setting [49].
14
1.3.2 Other Targeted Therapies and Immunotherapy
For patients whose breast cancers do not express hormone receptors or whose cancer does not respond to endocrine therapies, chemotherapy remains the mainstay of therapeutic treatment [36]. However, targeted therapies are emerging as promising treatment options. As mentioned previously, HER2+ patients are typically treated with chemotherapy in addition to trastuzumab, an antibody targeting the HER2 protein that is overexpressed in these tumors [36].
In addition to trastuzumab, there are a number of drugs and antibodies that have emerged targeting the activity of the HER2 protein and are currently being explored as potential treatment options [51]. For patients with hormone receptor- positive, HER2- tumors, CDK4/6 inhibitors (palbociclib, ribociclib, and abemaciclib) that act to inhibit cell cycle progression have recently been approved by the Federal Drug Administration (FDA) for treatment with aromatase inhibitors as a first-line therapy [51]. In patients with BRCA1/2 mutations, PARP inhibitors (olaparib and talazoparib) that act to impair DNA damage repair have been shown to be particularly efficacious and are now approved by the FDA [51].
Although there are currently no FDA-approved immunotherapies for breast cancer, clinical trials are actively investigating the use of immune checkpoint inhibitors in breast cancer treatment [51].
1.3.3 Resistance to Endocrine Therapies
Although endocrine therapies are the cornerstone of treatment for ER+ breast cancer, resistance to endocrine therapy is a major barrier to successful
15 clinical management in a large number of patients [8, 52]. Resistance to endocrine therapy can present in the neoadjuvant setting and within the clinical progression of the primary disease (innate resistance) or may present as a relapse or cancer recurrence during or after adjuvant endocrine therapy
(acquired resistance) [8]. For patients with early stage, ER+ breast cancer, the five year recurrence on endocrine therapy is ~10-15% [53]. However, it is estimated that 30-50% of early stage, ER+ breast cancer patients have innate resistance to endocrine therapies [54-56]. The vast majority of patients diagnosed with advanced or metastatic ER+ breast cancer develop resistance to endocrine therapy within 2-3 years [57, 58]. Thus, there is a critical need to better understand the explicit mechanisms behind both innate and acquired resistance to endocrine therapies in order to improve treatment options for a large patient population.
Several lines of evidence indicate that the expression and activity of the
ER is maintained in tumors that are resistant to endocrine therapy, suggesting the ER has simply found a way around the antagonist to promote cell growth [9,
59]. Mutations within the ER that lead to constitutive activity (Y537N) and ligand- independent activity (D538G) have been reported, however, they are detectable at very low levels in the patient population (<1%) and the clinical relevance in terms of response and resistance to endocrine treatment remains unclear [9].
Post-translational modification of the ER, namely phosphorylation at Ser118,
Ser167, and Thr311, has been associated with ligand-independent activation of
16 the ER and may account for the continued activity of the ER in the presence of antagonist [9]. Phosphorylation and activation of the ER has been shown to result from the activation of the MAPK/ERK, PI3K/Akt, and p38 MAPK pathways in response to epidermal growth factor (EGF), insulin-like growth factors (IGF), and stress/cytokines, respectively [8]. Overexpression of receptor tyrosine kinases (RTKs) that activate these pathways and somatic mutations within key players involved in these pathways have been reported in breast cancers that are resistant to endocrine therapies [8, 9]. Additionally, activation of the
PI3K/Akt/mTOR pathway has been associated with both innate and acquired resistance in breast cancer patients [60-62]. Thus, it is likely that endocrine resistance is the result of crosstalk between the ER and complex signal transduction pathways.
1.4 Signal Transduction Pathways in Breast Cancer
1.4.1 Receptor Tyrosine Kinases (RTKs) and Breast Cancer
Receptor tyrosine kinases (RTKs) are single-pass membrane receptors that receive environmental cues and activate various signaling cascades [63]. In the absence of ligand, the RTK is held in an inactive state by autoinhibitory mechanisms [63]. Binding of ligand results in dimerization and activation of the
RTK, which recruits the Src homology 2 (SH2) or phosphotyrosine-binding (PTB) domains of effector proteins [63]. Docked effector proteins can then phosphorylate associated proteins to further transduce the environmental signal
[63]. Following activation, the RTK is sent to the lysosome for degradation,
17 recycled back to the plasma membrane, or translocated to other cellular compartments [64-66]. RTKs act to regulate numerous signaling cascades including the MAPK, PI3K/Akt, and JAK/STAT pathways, which are responsible for differentiation, metabolism, cell survival, and proliferation [63, 67].
Several RTKs including the epidermal growth factor receptors (EGFRs), vascular endothelial growth factor receptors (VEGFRs), platelet-derived growth factor receptors (PDGFRs), insulin-like growth factor receptors (IGFRs), and fibroblast growth factor receptors (FGFRs) are overexpressed in many different types of cancer, including breast cancer [63]. Members of the EGFR family are particularly associated with breast cancer. EGFR (ERBB1) is overexpressed in
15-30% of breast cancer patients and is associated with poor outcome, particularly in triple negative breast cancer [67]. HER2 (ERBB2), which does not bind ligands directly but associates with ligand-bound EGFR or ERBB3, is overexpressed or amplified in 20-30% of breast cancer patients and is also associated with poor outcome [67]. Overexpression of these RTKs is associated with increased cancer stemness, angiogenesis, proliferation, and metastasis [63].
1.4.2 PI3K/Akt/mTOR Pathway is Frequently Altered in Breast Cancer
The phosphoinositide 3-kinase (PI3K) enzyme exists as a dimer consisting of a regulatory subunit (p85α/β, p55α/γ, or 950α) and a catalytic subunit
(p110α/β) [60, 68, 69]. The PI3K enzyme is activated when the SH2 domains of the regulatory subunit are recruited to the phosphotyrosine residues in activated
RTKs or G-protein-coupled receptors (GPCRs) at the membrane [60, 68, 69].
18
This recruitment causes a conformational change within the regulatory subunit, releasing the autoinhibitory contacts within the dimer, leading to activation of the
PI3K enzyme [60, 68, 69]. The PI3K enzyme then phosphorylates phosphatidylinositol-3,4-biphosphate (PIP2) to generate phosphatidylinositol-3,4- triphosphate (PIP3) [60, 68, 69]. The tumor suppressors phosphatase and tensin homolog (PTEN) and inositol polyphosphate-4-phosphatase, type II, B (INPP4B) terminate PI3K signaling by removing the 3’-phosphate from PIP3 converting it back to PIP2 [60, 68, 69]. The accumulation of PIP3 recruits proteins containing pleckstrin homology (PH) domains, such as PDK1 and Akt, to the plasma membrane [60, 68, 69]. Recruitment of PDK1 and Akt to the plasma membrane results in the phosphorylation of Akt by PDK1 at Thr308 within the activation loop
[70]. Although this phosphorylation event is necessary and sufficient for many downstream events, phosphorylation of Akt in the hydrophobic motif at Ser473 by mTOR complex 2 (mTORC2) stabilizes Akt and allows for maximal activity [70].
Once activated, Akt acts to phosphorylate numerous downstream effectors that regulate processes such as proliferation, metabolism, apoptosis, cell-cycle arrest, and differentiation [68, 70].
The PI3K/Akt/mTOR pathway is the most frequently altered pathway in breast cancer [60]. Mutations of numerous players within this pathway have been documented in breast cancer including p110α, p110β, p85α, AKT1, AKT2, PDK1, and PTEN [60]. Further, activation of the PI3K/Akt/mTOR pathway has been associated with resistance to endocrine therapies [60]. In MCF7 cells,
19 overexpression of a constitutively active form of Akt conferred resistance to tamoxifen [71]. Similarly, knockdown of PTEN, which results in hyperactivation of
Akt, conferred resistance to both tamoxifen and fulvestrant in MCF7 cells [55].
Data from long-term endocrine-treated cell lines suggests that hyperactivation of the PI3K/Akt/mTOR pathway allows the cells to grow in the absence of hormone as suppression of this pathway inhibits proliferation [61]. These in vitro studies are corroborated by patient data that indicates patients with a protein “signature” of PI3K activation had a worse outcome after adjuvant endocrine therapy compared to patients lacking this signature [61]. Further, patients given endocrine therapy that had activated Akt have been shown to be more prone to relapse with distant metastases [72]. Studies such as these have led to the development of PI3K/Akt/mTOR-targeted treatments. Buparlisib, a pan-PI3K inhibitor, along with fulvestrant resulted in a near-complete tumor regression in a
MCF7 xenograft model [73]. In the BELLE-3 clinical trial, patients with ER+ breast cancer that have progressed on AIs were given fulvestrant plus either buparlisib or placebo [74]. Patients that received buparlisib had a significant improvement in progression-free survival (>2-fold) compared to those that received fulvestrant alone [74]. Although the safety profile of buparlisib plus fulvestrant did not meet the standards necessary for further development in this setting, it did provide evidence that a PI3K inhibitor in combination with endocrine therapy is superior to endocrine therapy alone in patients with ER+, HER2-, advanced breast cancer [74]. Current studies are focused on identifying
20
PI3K/Akt/mTOR inhibitor and endocrine therapy combinations that will overcome endocrine resistance and identifying biomarkers to refine sensitive patient populations [75].
1.5 Growth Regulation by Estrogen in Breast Cancer 1 (GREB1)
1.5.1 Identification and Structure
The KIAA0575 gene was first reported in a study predicting the coding sequences associated with cDNA libraries from the human brain [76]. This gene was mapped to human chromosome 2p25.1 and is comprised of approximately
106 kilobases encoding 38 exons and 40 introns [44, 77]. In 2000, Ghosh et al rediscovered the KIAA0575 gene in a suppression subtractive hybridization screen for estrogen regulated genes in MCF7 ER+ breast cancer cells and re- named the gene growth regulation by estrogen of breast cancer 1, or GREB1
[44].
The GREB1 gene encodes three distinct isoforms: GREB1a, GREB1b, and GREB1c [44, 77-79]. Each transcript contains a unique 5’ untranslated region (UTR) but splices to a conserved exon encoding the translational start site and resulting in a homologous amino terminus (Fig. 1.2) [44, 77-79]. GREB1a encodes the full-length isoform with 38 exons, whereas GREB1b and GREB1c terminate after exons 10 and 9, respectively (Fig. 1.22) [44, 77-79]. The truncated isoforms, GREB1b (457 amino acids) and GREB1c (409 amino acids), are homologous with the first 449 and 386 amino acids, respectively, of the full- length isoform, GREB1a (1949 amino acids) [78]. None of the isoforms of
21
GREB1 have any homology to another protein and all isoforms lack any clearly defined functional domains [79]. Further, the majority of published research on the function of GREB1 has failed to differentiate between the three known isoforms, complicating the search for the molecular function of this protein.
Figure 1.2 Human GREB1 encodes for three protein isoforms. Schematic depicting the three splice variants of GREB1: GREB1a (ENST00000381486.6), GREB1b (ENST00000381483.6), and GREB1c (ENST00000263834.9). The three splice variants have unique promoters and 5’ untranslated regions. All isoforms share a homologous amino terminus (patterned exons) as depicted above.
1.5.2 Hormone-Dependent Regulation of GREB1 Expression
In a search for estrogen-induced genes that regulate proliferation of ER+ breast cancer, Ghosh and colleagues initially identified GREB1 as one of only
22 three genes that were consistently upregulated by estrogen in three ER+ breast cancer cell lines (MCF7, T47D, and BT474) [44]. Of these three genes, GREB1 was the only gene to be suppressed by both tamoxifen (ER antagonist in the breast) and fulvestrant (ER downregulator), suggesting GREB1 expression is directly influenced by ER activity [44]. This hypothesis was supported by cyclohexamide experiments indicating that GREB1 expression is not dependent on protein translation, suggesting GREB1 is a direct target of ER activity [44].
Since then, GREB1 has widely been used as a read-out of ER activity and numerous studies have shown that GREB1 expression is induced nearly 25-fold by estrogen in human breast cancer, ovarian cancer, and endometrial cancer cell lines [44, 80-83]. Further, studies have shown that GREB1 expression in breast tumors directly correlates to circulating estrogen levels in pre-menopausal patients [84-86]. All of the data suggesting GREB1 is a direct target of ER is compounded by the fact that expression of GREB1 is highly correlated with ER positivity in breast cancer patient samples and cell lines [44, 79, 80, 87, 88].
As mentioned previously, one mechanism by which ER regulates transcription of target genes in through direct binding to estrogen response elements (EREs) [89]. The GREB1 promoter contains three distal EREs located at 1.6, 9.5, and 21.5 kb upstream of the transcriptional start site [89, 90].
Luciferase assays in MCF7 breast cancer cell lines confirmed that all three EREs are functional and act synergistically to regulate the expression of GREB1 [89,
90]. Histone acetylation centered on the GREB1 EREs was observed in
23 response to estrogen, suggesting accessibility of the promoter for transcription initiation [89, 90]. Binding of ER, ER co-activator, SRC3, and RNA Pol II was confirmed via chromatin immunoprecipitation (ChIP) at all there ERE sites within the promoter of GREB1 [89, 90]. Further, treatment of MCF7 cells induced physical interaction of all three EREs along with the transcriptional start site of
GREB1 via chromatin looping as detected by chromatin conformation capture assays [90]. Taken together, these data clearly demonstrate that ER acts directly on the GREB1 promoter to regulate transcription of the GREB1 gene.
Expression of GREB1 appears to also be regulated through estrogen- induced epigenetic mechanisms. Data suggests that estrogen stimulation of
MCF7 and T47D breast cancer cell lines reduces the expression of miR-26a/b by nearly 50% [91]. MicroRNAs (miRs) are small, non-coding RNAs that act to fine- tune gene expression by promoting mRNA degradation and inhibiting translation
[91]. Expression of miR-26a/b along with a luciferase reporter controlled by the
3’UTR of GREB1 resulted in reduced reporter activity, suggesting that GREB1 is targeted by miR-26a/b through its 3’UTR [91]. Previous studies have suggested that expression of miR-26a/b is controlled by c-myc, a known gene target of ER
[91-93]. Knockdown of c-myc resulted in decreased expression of miR-26a/b and increased the expression of GREB1 [91]. Additionally, research in a tamoxifen- resistant model of breast cancer has revealed that the histone methyltransferase,
EZH2, acts to regulate expression of GREB1 [94]. Specifically, the authors found that methylation of H3K27 within the promoter of GREB1 is partially dependent
24 on EZH2 expression in tamoxifen-resistant MCF7 cells, resulting in increased expression of GREB1 when EZH2 is knocked down [94]. However, the role of
EZH2 in regulation of GREB1 expression in tamoxifen responsive cells remains unclear as EZH2 expression itself is partly regulated by estrogen signaling [95].
In addition to estrogen, expression of GREB1 has been shown to be induced by other steroidal hormones such as androgen and progesterone. In
LNCaP, androgen receptor-positive (AR+) prostate cancer cells, expression of
GREB1 was shown to increase in response to androgen treatment in a dose- dependent manner [96]. Expression of GREB1 was inhibited by treatment with anti-androgens bicalutamide, flutamide, nilutamide, and cyproterone acetate [96], suggesting the GREB1 gene is regulated by AR in prostate cancer cells. This was confirmed by ChIP assays indicating that AR binds to the androgen response element (ARE) within the GREB1 promoter [96]. Although the data for progesterone-induced regulation of GREB1 expression is not as robust as that for androgen and estrogen, data from human endometrial stromal cells suggests that GREB1 expression increases upon treatment with progesterone and is decreased when the progesterone receptor is knocked out in these cells [97].
These data indicate that GREB1 expression is broadly regulated by hormone levels and may play a role in other hormone-dependent malignancies.
1.5.3 Expression and Localization of GREB1
As GREB1 expression is primarily dictated by hormones, it is predominately expressed in hormone-responsive tissues. In humans, GREB1
25 has been reported in the brain [76], breast [44, 87, 88], ovary [83], prostate [96], endometrium [97], and uterus [97, 98]. Homologous genes exist in the mouse and zebrafish. In the mouse, GREB1 expression has been detected in the liver, musculoskeletal system, nervous system, reproductive tract, urinary system, and visual system [99]. In zebrafish, GREB1 is found in the pituitary gland, brain, liver, and gonads [100].
As the GREB1 protein has no explicit functional domains, some clue to its molecular function may come from its intracellular localization. Unfortunately, no studies have investigated the intracellular localization of GREB1 in mouse and zebrafish. In humans, GREB1 appears to be expressed primarily in the nucleus as detected by immunohistochemistry in breast cancer patient samples and cell lines [87, 88]. However, in human endometrial tissue sections GREB1 is predominately expressed in the cytoplasm [101]. Thus, further studies investigating the intracellular localization of GREB1 are necessary in order to aid in determining the molecular function of this protein.
1.5.4 Function and Clinical Relevance of GREB1 in Breast Cancer
Considering that expression of GREB1 appears to be hormone- dependent, it comes as no surprise that expression of GREB1 is highly correlated to expression of the ER in breast cancer cell lines and patient samples
[44, 80, 87, 88]. In breast cancer cell lines, high levels of GREB1 mRNA and protein expression have been shown to be restricted to those expressing the ER
[80, 88]. In breast cancer patient samples, expression of GREB1 mRNA has
26 been shown to be nearly 3.5-fold higher in ER+ tumors when compared to ER- tumors [44]. In the validation of a novel GREB1 monoclonal antibody, one group showed that in a tissue microarray (TMA) of 192 patient samples nearly 28% of all patients and 40% of ER+ tumors expressed GREB1 [88]. Similar findings were later published using the same GREB1 monoclonal antibody and immunohistochemistry from a cohort of 415 patients in which 46% of all patients expressed GREB1 protein [87]. Of the GREB1-positive staining cohort, 96.3% of the tumors were ER+ [87]. Together, these data suggest a strong correlation between GREB1 expression and ER-positivity in breast tumors.
Although little is known about the molecular function of the GREB1 protein, expression of GREB1 appears to be critical for proliferation of ER+ breast cancer cells. The first data to show the necessity of GREB1 for proliferation was performed in MCF7 breast cancer cells transfected with siRNA targeted to a non-specific control or to GREB1and grown in the presence or absence of estradiol [80]. When GREB1 was knocked down in the presence of estradiol, proliferation of the MCF7 cells was reduced to hormone-deprived levels, suggesting GREB1 is imperative for hormone-dependent growth [80].
These findings were later confirmed by a separate group. Using a soft agar assay, it was shown that MCF7 cells transfected with siRNA targeted to GREB1 formed significantly fewer colonies than those transfected with control siRNA, suggesting GREB1 is required for anchorage-independent growth of ER+ breast cancer cells [87]. However, despite these findings indicating a critical role for
27
GREB1 in the proliferation of ER+ breast cancer cells, the mechanism by which
GREB1 regulates proliferation remains unknown.
Some indication of the molecular function of GREB1 came from a study investigating the interactome of the ER in the presence of agonist or antagonist in breast cancer cell lines. Following the treatment of MCF7 cells with either agonist (estradiol) or antagonist (tamoxifen), the ER was purified using rapid immunoprecipitation followed by mass spectrometry of endogenous proteins
(RIME) to identify interacting proteins [87]. These data revealed that GREB1 was the most estrogen-induced ER-interacting protein [87]. Although direct binding of
ER and GREB1 was not confirmed using co-immunoprecipitation, the authors showed using ChIP-sequencing that nearly all of the chromatin binding sites of
GREB1 were shared with the ER and many were shared with ER co-factors,
CBP and p300 [87]. In order to determine the functional role of GREB1, the authors used siRNA to knockdown GREB1 and analyze the expression of estrogen-responsive genes [87]. Following transfection of MCF7 cells with siRNA targeted to GREB1 or a non-specific control and stimulation with estradiol, expression of estrogen-responsive genes was analyzed via microarray analysis
[87]. According to their analysis, knockdown of GREB1 resulted in the loss of differential expression of over 50% of estrogen-responsive genes, suggesting that GREB1 is a key co-regulator of ER activity in MCF7 cells [87]. Following knockdown of GREB1, binding of ER and ER co-factors, CBP and p300, at target gene promoters was analyzed via ChIP analysis [87]. The data showed that
28 binding of CBP and p300, but not ER, was significantly decreased at all tested regions when GREB1 was knocked down, suggesting GREB1 may stabilize the interaction of ER co-factors with ER on target gene promoters [87]. However, analysis of the published microarray data [87] revealed that the siRNA used to target GREB1 may have had an off-target effect, resulting in the loss of nearly
40% of ER expression when GREB1 was knocked down. Thus, it remains unclear if the loss of GREB1 or the loss of ER expression is responsible the dysregulation of estrogen-responsive genes and the loss of CBP and p300 binding at target gene promoters in this study and further studies are necessary to clarify the role of GREB1 in regulation of ER activity in breast cancer.
Although few studies have investigated the correlation between GREB1 expression and clinical determinants of breast cancer, those that are published suggest that GREB1 expression is associated with better prognostic outcome in
ER+ breast cancer patients. Analysis of GREB1 expression via immunohistochemistry in a cohort of 338 patients revealed that higher expression of GREB1 was associated with increased survival [87]. The same study analyzed the expression of GREB1 in both parental and tamoxifen-resistant MCF7 cells and found that expression of GREB1 was lost in the cells that were resistant to tamoxifen [87], suggesting a role for GREB1 expression in sensitivity to endocrine therapy. These findings align with those published in a report of a 76- gene signature, including GREB1, which was associated with prolonged disease- free survival in tamoxifen treated patients [102]. Further, expression of GREB1
29 mRNA was analyzed in two individual cohorts of 136 and 255 patients each which included only patients that had received adjuvant tamoxifen monotherapy
[94, 103, 104]. The individual cohorts were then stratified into a top 50% with high GREB1 expression and a low 50% with low GREB1 expression [94]. In both cohorts, the top 50% with high GREB1 expression had prolonged disease-free survival compared to the lower 50% [94]. Additionally, the two patient cohorts were stratified based on the sensitivity to tamoxifen treatment and GREB1 expression was analyzed [94]. This analysis revealed that GREB1 expression was significantly reduced in tamoxifen-resistant patients compared to tamoxifen- sensitive patients, suggesting that higher GREB1 expression is both associated with sensitivity to endocrine therapy and better prognosis [94]. Patients who respond to endocrine therapies have better prognosis than those with hormone- refractory tumors, thus, the association of GREB1 with both better prognosis and sensitivity to endocrine therapy is unsurprising. Despite these encouraging correlations, the role of GREB1 in mediating sensitivity to endocrine therapy remains unclear.
1.5.5 Function and Clinical Relevance of GREB1 in Other Hormone-Dependent
Diseases
Although the vast majority of research on GREB1 has been performed in breast cancer, expression of GREB1 has been noted in other hormone- responsive diseases including ovarian cancer, prostate cancer, and endometriosis.
30
In ovarian cancer, exposure to external estrogens plays a key role in the development of tumors within the epithelium of the ovary [83]. The ER is expressed in 61-79% of ovarian cancers and both pre-clinical and clinical data suggest that the ER plays a critical role for growth of ovarian cancer [105, 106].
In ovarian cancer cell lines, GREB1 has been shown to be a highly estrogen- induced gene and is correlated to ER-positivity [83]. Knockdown of GREB1 reduced proliferation of ovarian cancer cell lines in vitro and in xenograft models
[83]. Further, expression of GREB1 was shown to be much higher in ovarian cancer cell lines compared to early-passage, normal ovarian surface epithelial cells [83]. Together, these data suggest a critical role for GREB1 in the proliferation of ER+ ovarian cancer cells, although the mechanism by which
GREB1 mediates proliferation remains unclear.
Prostate cancer is the most frequently diagnosed malignancy in men [2]. Androgenic hormones and their receptor, the androgen receptor (AR), play a critical role in the development and progression of prostate cancer [96].
Expression of GREB1 has been reported in benign prostatic hypertrophy, localized prostate cancer, and hormone-refractory prostate cancer cell line models [96]. In prostate cancer cell lines, expression of GREB1 has been shown to be regulated by the AR and increase in an androgen dose-dependent manner
[96]. Similarly to breast and ovarian cancer cell lines, knockdown of GREB1 results in significantly decreased androgen-induced proliferation of prostate cancer cell lines, indicating a compelling role for GREB1 in prostate cancer as
31 well [96]. However, few studies have investigated the role of GREB1 in prostate cancer and further studies are necessary to determine the clinical importance of
GREB1 in this malignancy.
The endometrium is the inner epithelial layer of the uterus that is surrounded by the muscular layer of myometrium [107]. The endometrium typically goes through cyclical periods of proliferation in response to circulating hormones, primarily estrogens [107]. Endometriosis is a commonly-diagnosed disorder in women in which endometrial tissue establishes and proliferates outside of the uterus, such as on the pelvic peritoneum and ovaries, and can increase a woman’s risk of developing ovarian and endometrial cancers [78, 101,
107]. Similar to normal endometrial tissue, proliferation of endometriotic tissue is regulated by estrogen and the ER [101]. Analysis of endometrial tissue from patients with and without endometriosis revealed that expression of GREB1 is significantly higher in patients diagnosed with endometriosis [101]. Further, analysis of normal endometrial tissue and endometriotic lesions within the same patient revealed that GREB1 expression was increased in the endometriotic lesions [101]. Although no proliferation-based assays were performed, these data suggest that GREB1 may play a role in the establishment and maintenance of ectopic endometrial tissue.
1.6 Statement of Research Purpose
Expression of GREB1 has been shown to be highly correlated to ER- positivity in breast cancer patients and necessary for the proliferation of ER+
32 breast cancer cells, making GREB1 an attractive therapeutic target [80, 87].
However, high levels of GREB1 expression have also been shown to be associated with better prognosis for ER+ breast cancer patients, creating a paradox in regards to GREB1 expression [87]. Accumulating evidence suggests a role for GREB1 in sensitivity to endocrine therapies [87, 94, 102], which may explain this contradiction in part. However, few studies have examined the explicit mechanism by which GREB1 regulates proliferation of breast cancer cells and no studies have investigated the function of the distinct GREB1 isoforms in breast cancer. Therefore, the role of the three GREB1 isoforms in regulation of
ER activity and proliferation of breast cancer cell lines will be studied in Chapter
2. In Chapter 3, a potential mechanism by which GREB1 regulates proliferation of breast cancer cell lines through modulation of signaling cascades will be investigated. Future directions investigating the clinical and functional significance of cytoplasmic GREB1 will be investigated in Chapter 4.
33
Chapter 2 : GREB1 isoforms regulate proliferation independent of ERα co- regulator activities in breast cancer
2.1 Abstract
Activation of the transcription factor estrogen receptor α (ERα) and the subsequent regulation of estrogen-responsive genes play a crucial role in the development and progression of the majority of breast cancers. One gene target of ERα, growth regulation by estrogen in breast cancer 1 (GREB1), is associated with proliferation and regulation of ERα activity in estrogen-responsive breast cancer cells. The GREB1 gene encodes three distinct isoforms: GREB1a,
GREB1b, and GREB1c, whose molecular functions are largely unknown. Here, I investigate the role of these isoforms in regulation of ERα activity and proliferation. Interaction between GREB1 and ERα was mapped to the amino terminus shared by all GREB1 variants. Analysis of isoform specific regulation of
ERα activity suggests none of the GREB1 isoforms possess potent co-regulator activity. Exogenous expression of GREB1a resulted in elevated expression of some ER-target genes, independent of ERα activity. Despite this slight specificity of GREB1a for gene regulation, exogenous expression of either GREB1a or
GREB1b resulted in decreased proliferation in both ER-positive and ER-negative breast carcinoma cell lines, demonstrating an ER-independent function of
GREB1. Interestingly, I show an increase in the expression of GREB1b and 34
GREB1c mRNA in malignant breast tissue compared to normal patient samples, suggesting a selective preference for these isoforms during malignant transformation. Together, these data suggests GREB1a has an isoform specific function as a transcriptional regulator while all isoforms share an ER-independent activity that regulates proliferation.
2.2 Introduction
The majority (~70%) of breast cancers rely on estrogen receptor (ER) activity for cell growth and survival [8, 9, 41]. Once activated by estrogens, ER translocates to the nucleus where it dimerizes on estrogen response elements
(EREs) to regulate the transcription of target genes [1]. Two ER isoforms exist (α and β), each encoded by distinct genes [1]. In estrogen-dependent breast cancer, the activation of ERα ultimately leads to proliferation [8]. ER-positive breast cancers are treated with endocrine therapies that disrupt the activity of
ERα [1]. Unfortunately, patients develop resistance to endocrine therapies through re-activation of ERα, allowing for estrogen-responsive gene expression and progression of the disease [1, 8, 9, 39, 41]. Therefore, a better understanding of how gene targets of ERα allow for increased proliferation of breast cancer cells is needed in order to develop new and innovative therapies.
One primary gene target of ERα, growth regulation by estrogen in breast cancer 1 (GREB1), is of particular interest in the modulation of proliferation in
ER-positive breast cancer. Initially identified as a gene upregulated by estrogen and repressed by an ERα antagonist in ER-positive breast cancer cells, GREB1
35 expression is controlled by three distal EREs [44, 89, 90]. The expression of
GREB1 is highly correlated to ER-positivity in breast cancer cell lines and patient tumor samples, suggesting a functional role for this protein in ER-positive breast cancer [44, 80, 87, 88]. Loss of GREB1 expression in the estrogen-dependent
MCF7 breast cancer cell line reduces proliferation and anchorage-independent growth, suggesting GREB1 is essential for hormone-dependent proliferation in
ER-positive breast cancer cells [80, 87].
A previous study suggested that GREB1 may act as a co-factor for ERα, interacting preferentially with agonist-bound ERα [87]. Chromatin immunoprecipitation experiments demonstrated that over 95% GREB1 chromatin binding sites overlapped with ERα chromatin binding sites [87]. These data suggest that GREB1 has the potential to regulate the activity of ERα through modulation of co-factor binding.
Despite evidence suggesting an important functional role of GREB1 in breast cancer, no study has differentiated between the three distinct protein isoforms encoded by the GREB1 gene: GREB1a, GREB1b, and GREB1c [44]. Each transcript is estrogen-dependent and contains a unique 5’ untranslated region, which splices to a conserved exon that encodes the translational start site [44].
Thus, the amino terminus of the three protein isoforms is identical. The transcripts for GREB1b and GREB1c differ from GREB1a due to alternative splicing after exon 10 and exon 9, respectively [44]. In each instance, the alternative exon encodes a stop codon resulting in truncated versions of the full-
36 length isoform, GREB1a (Figure 1.2). Despite the alternative splicing events,
GREB1b and GREB1c contain only 8 and 23 unique amino acids, respectively
(Figure 1.2). None of the GREB1 isoforms have any homology to other proteins or known functional domains that may suggest their molecular function and the
GREB1b and GREB1c isoforms have never been investigated.
Here, I set out to better characterize the contribution of the three different
GREB1isoforms to the modulation of ERα activity and proliferation in breast cancer cell lines. To this end, I mapped the binding of GREB1 to ERα protein.
Despite the interaction of all GREB1 isoforms with ERα protein, none of the
GREB1 isoforms potently regulate ERα transcriptional activity. Further, I have shown that both GREB1a and GREB1b have the ability to regulate proliferation of breast cancer cell lines independent of ERα expression. These data suggest that GREB1 has additional molecular functions beyond acting as a transcriptional co-regulator of ERα.
2.3 Materials and Methods
2.3.1 Cell Lines and Reagents
HEK-293AD, HEK-293T, MCF7, T47D, MDA-MB-231, and MDA-MB-468 cells were validated using Short Tandem Repeat analysis by the Genomics Core in the Research Technology Support Facility (Michigan State University, East
Lansing, MI 48824). Cell lines were maintained in DMEM supplemented with phenol red (Gibco by Life Technologies), 5% (vol/vol) fetal bovine serum (FBS;
Sigma), 1% (vol/vol) penicillin-streptomycin (Corning), and 2 mM L-glutamine
37
(HyClone). For hormone-free conditions, cells were cultured in phenol-red-free
DMEM (Gibco by Life Technologies) supplemented with 5% (vol/vol) charcoal- dextran-treated FBS (CDT, Sigma), 1% (vol/vol) penicillin-streptomycin, and 2 mM L-glutamine. Cells were treated with either vehicle control (ethanol) or 10 nM estradiol (E2; Sigma) for the indicated time.
2.3.2 Plasmids
pcDNA-ERα, H2B-GFP, 3XERE-Luciferase, and PS2-Luciferase have been previously described [108-111]. GREB1a, GREB1b, and GREB1c cDNA were amplified from MCF7 reverse transcribed RNA and cloned into pJET 2.1 vector (Thermo). GREB1a, GREB1b, and GREB1c inserts were removed from pJet2.1 vectors by restriction digestion and inserted into pcDNA 3.1 vector
(Thermo) with a 3XFLAG coding sequence in front of the multiple cloning site. pcDNA 3XFLAG-GREB1 (1-500), (492-992), (984-1477), and (1469-1949) were generated by PCR amplification of the specific fragments from pcDNA 3XFLAG-
GREB1a and inserted into pcDNA 3XFLAG via Gibson cloning (NEB). GIPZ
Lentiviral non-specific shRNA (# RHS4346) and GREB1-targeted shRNA plasmids (V2LHS_139192 and V3LHS_372339) were purchased from Open
Biosystems. CMV-Renilla luciferase reporter construct was purchased from
Promega.
2.3.3 Immunoblot Analysis and Antibodies
Cells were lysed in Buffer E (10 mM Tris-HCl, pH8.0, 60 mM NaCl, 1 mM
EDTA, 0.3% IGEPAL) with added protease inhibitors (Sigma, P8340). Lysates
38 were incubated with Laemmli buffer at 37°C for 30 minutes as incubation of cell lysates at higher than 55°C causes GREB1a to aggregate (Figure 2.1). Lysates were subjected to SDS-PAGE and immunoblots visualized using Licor Odyssey as previously described [112]. Immunoblots were probed with the following antibodies: GREB1 (Abcam; ab72999), FLAG (Sigma; F1804), ERα (GeneTex;
GTX62423), ERα (Santa Cruz; SC-8005), and β-actin (Cell Signaling; 3700).
Figure 2.1 GREB1a protein aggregates at high temperatures. MCF7 whole cell lysate was incubated with Laemmli buffer at increasing temperatures for 30 minutes. Following SDS-PAGE electrophoresis, the gel was transferred to PVDF membrane without removing the stacking gel. The expected GREB1a band at 216 kDa can be detected via immunoblot with GREB1 specific antibodies when the sample is incubated at 37°C and 55°C, however aggregates begin to form in the stacking gel when the sample is heated at 55°C or above.
2.3.4 Adenovirus
GREB1a and GREB1b were moved from 3XFLAG plasmids to a pshuttle-
IRES GFP 3XFLAG plasmid (Agilent). Shuttle vectors were recombined with pAdeasy using BJ5183-AD1 bacteria (Agilent). Adenovirus was produced and
39 amplified in HEK-293AD cells (Agilent) then purified by CsCl gradient. Ad5-CMV- eGFP adenovirus (Baylor College of Medicine Vector Development Labs,
Houston, TX 77030) was used as a control.
2.3.5 Immunoprecipitation
Endogenous GREB1 was immunoprecipitated from MCF7 cells. Individual isoforms or fragments were immunoprecipitated from HEK-293AD cells transfected with H2BGFP, ERα, and indicated GREB1 plasmids using PEI transfection reagent (Polysciences, Inc.). Cells were lysed in Buffer E and immunoprecipitation was performed with anti-FLAG M2 Magnetic Beads (Sigma,
M8823) or GREB1 antibody with PureProteome Protein A magnetic beads
(Abcam; ab72999 and EMD Millipore). Beads were washed in 250 mM NaCl
Buffer E, incubated with Laemmli buffer at 37°C for 30 minutes, and immunoblot analysis performed as described.
2.3.6 Gene Expression Analysis
Total RNA was harvested using RiboZol (Amresco) and reverse transcribed using the RevertAid RT Kit (Thermo). Relative gene expression was analyzed via real-time PCR using SYBR Green (BioRad) and the indicated primers (Table 2.1). Data are relative to RPL13a, normalized to control, and are depicted as mean ± SE. Data represent a minimum of three biological replicates and statistical significance was determined using either a one-way ANOVA with post-hoc Tukey’s HSD test (knock-down experiment) or two-way ANOVA with a post-hoc Tukey’s HSD test (estrogen-induced gene expression experiments).
40
Table 2.1 SYBR Real-Time PCR Primers
41
2.3.7 GREB1 Isoform Expression
Tissue samples were provided by the “Total Cancer Care Protocol: A
Lifetime Partnership with Patients of the James/OSUCCC”, IRB protocol
2013H0197 (The James Comprehensive Cancer Center, The Ohio State
University, Columbus, OH 43240). RNA was harvested using RiboZol (Amresco) and was converted to cDNA using the Revertaid RT Kit (Thermo). Absolute mRNA copy number was determined from a standard curve generated using
Taqman probes for GREB1a, GREB1b, and GREB1c (Table 2.2). The Kruskal-
Wallis multiple comparison method was used to determine statistical significance.
Cancer RNA-seq Nexus [113] was used to determine mRNA levels of
GREB1a, GREB1b, and GREB1c from a publically available data set of transcript expression from breast cancer patients [114].
42
Table 2.2 Taqman Real-Time PCR Primers and Probes
2.3.8 Reporter Assay
HEK-293AD cells were cultured in hormone-depleted media and transfected with CMV-Renilla, PS2-Luciferase or 3XERE-Luciferase, pcDNA-
ERα, and pcDNA 3XFLAG (EV), pcDNA 3XFLAG-GREB1a, or pcDNA 3XFLAG-
GREB1b. The following day, cells were treated with 10 nM estradiol (E2) or vehicle control (ethanol) for 48 hours. Lysates were harvested and analyzed using the Dual-Luciferase kit (Promega). Data represents three biological replicates and statistical significance was determined using a two-way ANOVA with a post-hoc Tukey’s HSD test.
43
2.3.9 MTT Assays
Cells transduced with GFP, GREB1a, or GREB1b adenovirus were treated with 5 mg/ml MTT solution (Sigma) at 37⁰C for 3 hours. Formazan was solubilized in 4 mM HCl, 0.1% IGEPAL in isopropanol and absorbance was measured at 570 nm. Data are depicted as mean absorbance normalized to day
0 ± SD for each condition from 3 biological replicates. Statistical significance for
MTT assays was determined using either a one-way ANOVA with post-hoc
Tukey’s HSD test (shRNA experiments), two-way ANOVA with a post-hoc
Tukey’s HSD test (exogenous expression in presence and absence of hormone), or a two-tailed Student’s t-test (exogenous expression in cell line panels).
2.3.10 EdU Incorporation Assay
MCF7 cells were transduced with GFP, GREB1a, or GREB1b adenovirus.
Proliferation was measured after 6 hours of EdU labeling using the Click-iT EdU
Imaging Kit (Invitrogen) following the manufacturers protocol. Images of EdU stained cells were blindly quantified and statistical significance was determined using a one-way ANOVA with a post-hoc Tukey’s HSD test.
2.3.11 Cell Viability Assay
MCF7 cells were transduced with GFP, GREB1a, or GREB1b adenovirus.
After 72 hours, the cells were harvested and stained with a LIVE/Dead Fixable
Dead Cell Stain Kit (Invitrogen). Cells were analyzed on a LSR II flow cytometer
(BD Biosciences). Unstained MCF7 cells and stained, heat-treated MCF7 cells
44 served as controls. Statistical significance was determined using one-way
ANOVA with a post-hoc Tukey’s HSD test.
2.4 Results
2.4.1 The amino terminus of GREB1 isoforms interact with ERα
To confirm previous studies that have indicated that GREB1 interacts with
ERα [87], I performed immunoprecipitations from MCF7 breast carcinoma whole cell lysate. As previously reported [87], immunoblot analysis revealed an interaction between GREB1a and ERα (Fig. 2.2A). To map this interaction, plasmids expressing roughly 500 amino acid fragments of GREB1a tagged with
3XFLAG (Fig. 2.2B) were transfected with ERα plasmid into HEK-293AD cells and immunoprecipitated from whole cell lysate with anti-FLAG magnetic beads.
Immunoblot analysis showed Fragment #1 (60 kDa) immunoprecipitated with
ERα, while no interaction was observed with Fragment #2 (56 kDa), which contains the LxxLL motif known to bind nuclear receptors, Fragment #3 (62 kDa), or Fragment #4 (56 kDa) (Fig. 2.2C). These data suggest that the amino terminus of GREB1a interacts with ERα.
The interaction between ERα and the amino terminus of GREB1 was particularly interesting as this region is shared by all three isoforms of GREB1
(Fig. 1.2). To determine if all three isoforms of GREB1 are able to bind to ERα each isoform or empty vector was co-transfected with ERα plasmid into HEK-
293AD cells. Using FLAG antibody, each isoform was immunoprecipitated from whole cell lysate and subjected to immunoblot analysis. While ERα was not
45 pulled-down with the empty vector control (EV), 3XFLAG-GREB1a (228 kDa),
3XFLAG-GREB1b (56 kDa), and 3XFLAG-GREB1c (49 kDa) were all able to pull down ERα (Fig. 2.2D). These data indicate that all three protein isoforms of
GREB1 are able to interact with ERα.
Figure 2.2 The amino terminus of GREB1 isoforms interact with ERα. A, Endogenous GREB1 was immunoprecipitated from MCF7 whole cell lysate and purified complexes were subjected to immunoblot analysis with GREB1 and ERα antibodies. B, 3xFLAG-tagged fragments of GREB1a were generated according to the schematic. C, Plasmids expressing the individual fragments were co-transfected with ERα into HEK-293AD cells and immunoprecipitated from whole cell lysate using anti-FLAG magnetic beads. Immunoblot analysis of input and FLAG IP lysates was performed with FLAG and ERα antibodies. D, 3xFLAG-tagged GREB1a, GREB1b, GREB1c, or empty vector (EV) was co- transfected with ERα into HEK-293AD cells. Immunoprecipitation with anti-FLAG magnetic beads was followed by immunoblot analysis of input and FLAG IP lysates with FLAG and ERα antibodies.
46
2.4.2 GREB1 isoforms are not robust regulators of ERα activity
Previous studies have suggested that GREB1 expression is an essential co-regulator of ERα activity [87]. Our binding data (Fig. 2.2) suggests the potential for all three GREB1 isoforms to regulate the transcriptional activity of
ERα. To determine the contribution of the GREB1 isoforms to ERα activity, I utilized shRNA-mediated knockdown of GREB1 with constructs targeting either all isoforms (shRNA #1), or only GREB1a (shRNA #2; Fig. 2.3A). The homology between GREB1b and GREB1c makes targeting these specific isoforms impossible. Knockdown of GREB1a in MCF7 cells was confirmed via immunoblot analysis. Compared to non-specific shRNA control, expression of GREB1a protein was reduced by 88% with shRNA #1 and by 79% with shRNA #2 when normalized to the loading control, beta actin (Fig. 2.3B). Knockdown of GREB1b and GREB1c protein was unable to be confirmed as endogenous levels of these isoforms are undetectable via immunoblot.
Expression of control genes and knockdown of GREB1 isoforms was assessed via real-time PCR. There was no change in the expression of either
ERα or HPRT (control) when either shRNA #1 or shRNA #2 was used compared to control shRNA (Fig. 2.3C).When using the shRNA targeted to all isoforms of
GREB1 (shRNA #1), there was a significant reduction in the expression of
GREB1a and a non-significant decrease in GREB1b and GREB1c (Fig. 2.3C).
The shRNA specifically targeted to GREB1a (shRNA #2) resulted in a significant decrease in the expression of only GREB1a (Fig. 2.3C). The effect of GREB1
47 knockdown on ERα activity was assessed via real-time PCR at gene targets previously identified to be regulated by GREB1 [87]. Knockdown of GREB1 using shRNA #1 resulted in a slight, but non-significant, decrease in the expression of
C-MYC, CASP7, CAV1, and EGR3 (Fig. 2.3D). Knockdown of GREB1a using shRNA#2 resulted in a slight, but significant, decrease in the expression of CAV1
(Fig. 2.3D). However, some ER-target genes (PS2, PR, and SDF1 (i.e. CXCL12)) were unaffected by knockdown with either shRNA (Fig. 2.3D). Further, knockdown of GREB1 had no impact on ERα activity at these same genes when cells were hormone-starved and stimulated with estrogen (Fig. 2.4). These data suggest that while GREB1a is predominantly responsible for the modest co- activator function at some genes (CAV1), it does not significantly regulate most
ER-target genes.
48
Figure 2.3 GREB1 isoforms are not robust regulators of ERα activity. MCF7 cells were transduced with a non-specific shRNA (shNS) or one of the two shRNAs targeting GREB1. A, Diagram showing the relative targeting location of the shRNAs used to knockdown all isoforms of GREB1 (shRNA #1) or GREB1a specifically (shRNA #2). B, Immunoblot depicting the relative expression of GREB1a in MCF7 cells treated with shRNA. Densitometry analysis of GREB1 normalized to beta actin, relative to shNS. C-D, Real-time PCR was used to assess the relative expression of control genes (C) and known ERα target genes (D). Results are displayed as expression relative to RPL13a and presented as mean ± SE; n=5.; *p ≤ 0.05.
49
Figure 2.4 GREB1 knockdown does not affect ERα activity. MCF7 cells were transduced with a control shRNA (shNS), shRNA targeting all GREB1 isoforms (shRNA #1), or only GREB1a only (shRNA #2) in hormone- depleted media. The cells were then treated with ethanol (-E2) or 10 nM estradiol (+E2) for 48 hours. A, Immunoblot depicting the knockdown of GREB1 protein. B, ER-target gene expression was measured via real-time PCR. Results are displayed as expression relative to RPL13a mean + SE; n=4. Statistical significance was tested by two-way ANOVA with post-hoc Tukey’s HSD test. There was no statistical difference in the expression of ER-target genes when GREB1 was knocked-down.
2.4.3 Elevated expression of GREB1a and GREB1b differentially regulate ERα target genes
In order to better assess the co-regulatory potential of specific GREB1 isoforms I expressed exogenous GREB1a or GREB1b in MCF7 cells. As the amino acid sequence of GREB1b is extremely similar to that of GREB1c and is more abundant (Fig. 2.5), I focused only on the elevated expression of GREB1b.
MCF7 cells were transduced with adenovirus expressing GREB1a, GREB1b, or
GFP control. Relative expression of the GREB1 isoforms compared to GFP
50 control was determined using immunoblot analysis 24 hours post-infection (Fig.
2.6A). Transduced cells were treated with 10 nM estradiol (+E2) or vehicle control (ethanol, -E2) for 2 hours to measure early estrogen response genes.
Transcript expression of estrogen-responsive genes previously shown to be regulated by GREB1 (EGR3, PR, PS2 and SDF1) [87] was measured using real- time PCR. Treatment of GFP-transduced MCF7 cells with estradiol resulted in the expected increase in expression of estrogen-responsive genes, EGR3, PR,
PS2, and SDF1 (Fig. 2.6B). As this analysis is performed after only 2 hours of estradiol stimulation, accumulation of some target genes was not robust. When
GREB1a was expressed at elevated levels in the absence of estradiol, there was an increase in the basal expression levels of two estrogen-responsive genes,
PS2 and SDF1, compared to GFP-expressing and GREB1b-expressing cells
(Fig. 2.6B, -E2). Following estradiol treatment, cells that expressed GREB1a at elevated levels had significantly higher induction of the same estrogen- responsive genes, PS2 and SDF1, compared to GFP-expressing and GREB1b- expressing cells (Fig. 2.6B). Exogenous GREB1b expression had no effect on the expression of estrogen-responsive genes in the absence or presence of estradiol in comparison to GFP-expressing cells (Fig. 2.6B). These data suggest that GREB1a and GREB1b differentially regulate the expression of some estrogen-responsive genes, with only GREB1a potentiating their expression.
51
Figure 2.5 GREB1 isoform expression varies between breast cancer cell lines. Absolute mRNA copy number was determined from a standard curve generated using Taqman probes for A, GREB1a, B, GREB1b, and C, GREB1c. D, Graph depicting the percentage of total GREB1 expression of each isoform.
52
Figure 2.6 GREB1a and GREB1b differentially regulate ERα target genes. A, MCF7 cells were transduced with GFP, GREB1a, or GREB1b adenovirus. Immunoblot depicting overexpression of GREB1a or GREB1b in comparison to control, GFP-transduced MCF7 cells. B, MCF7 cells were transduced with GFP, GREB1a, or GREB1b adenovirus then starved of hormone for 72 hours and treated with ethanol (-E2) or 10nM estradiol (+E2) for 2 hours. Real-time PCR was used to measure relative levels of known ERα target genes. Results are displayed as expression relative to RPL13a mean + SE; n=3. C, The indicated reporters were transfected with 3xFLAG-tagged GREB1a, GREB1b, or empty vector (EV), and ERα, into HEK 293 cells and luciferase activity was measured after 48 hours with either ethanol(-E2) or 10 nM estradiol (+E2).. Results are displayed as firefly luciferase activity relative to Renilla luciferase activity+ SE; n=3. *p ≤ 0.05, **p≤ 0.01, ***p≤ 0.001.
As exogenous GREB1a expression increased the expression of some ER- target genes in the absence of ligand, I investigated whether GREB1 was able to 53 interact with ERα under these conditions. I found that GREB1a preferentially interacts with ERα in the absence of ligand and that this interaction occurs primarily in the cytoplasm (Fig. 2.7), suggesting that GREB1a may potentiate ligand-independent ER activity. Thus, luciferase assays were used to test receptor activity in a cell line with no endogenous expression of ERα or GREB1.
Luciferase reporter assays using PS2 and 3X- ERE promoters were performed in
HEK-293AD cells with exogenous expression of either empty vector (EV),
GREB1a, or GREB1b. Expression of neither GREB1a nor GREB1b significantly impacted the activity of ERα on either the PS2- or 3XERE-Luciferase reporters in the presence or absence of hormone compared to empty vector control (Fig.
2.6C). These data confirm that neither GREB1a nor GREB1b are potent co- activators of ERα at target genes and indicate that GREB1a can potentiate transcription of some ER-target genes independent of the receptor.
54
Figure 2.7 GREB1 preferentially binds ERα in the absence of ligand and in the cytoplasm. A, HEK-293AD cells were co-transfected with 4.5μg of 3xFLAG-tagged GREB1a and 1.5μg ERα, starved of hormone for 72 hours, and treated with vehicle control (EtOh), 10 nM estradiol (E2), or 100 nM 4-hydroxytamoxifen for 1 hour. Cells were then harvested and GREB1a was immunoprecipitated from whole cell lysate using anti-FLAG magnetic beads. Immunoblot analysis of input and FLAG IP lysates was performed with FLAG and ERα antibodies. GREB1 preferentially interacted with ERα in the absence of either estradiol or tamoxifen. B, MCF7 cells were lysed in Buffer E with added protease inhibitors as described. Whole cell lysate was then layered over a sucrose pad (100 mg/mL sucrose, Buffer E, 1mM DTT, and protease inhibitors) and centrifuged at 2500 rpm at 4°C for 15 minutes to pellet nuclei. The supernatant was set aside as the cytoplasmic fraction. Nuclei were washed twice and lysed via sonication in Buffer E with added protease inhibitors. Immunoprecipitation of endogenous GREB1 and immunoblot analysis were performed as described. The vast majority of GREB1 and ERα was co-immunoprecipitated from the cytoplasmic fraction.
2.4.4 GREB1 expression is not sufficient to drive hormone-independent growth
GREB1 has long been implicated in the regulation of proliferation. While knockdown experiments have demonstrated the requirement of GREB1 for estrogen-dependent proliferation (Fig. 2.8) [80, 87], here I tested if GREB1a or
GREB1b expression was sufficient to drive proliferation under hormone-deprived conditions. As GREB1b encompasses the majority of GREB1c, only ectopic expression of GREB1a and GREB1b was investigated. MCF7 cells were
55 transduced with adenovirus expressing GREB1a, GREB1b, or GFP and immunoblot analysis confirmed elevated expression in transduced cells (Fig.
2.9A). As expected, GREB1a protein expression decreased in cells cultured in
CDT media (Fig. 2.9A). CDT media inhibited the proliferation of uninfected and
GFP transduced MCF7 cells compared to MCF7 cells grown in FBS media, demonstrating the hormone-dependence of these ER-positive breast cancer cells
(Fig. 2.9B). Elevated expression of either GREB1a or GREB1b was unable to promote hormone-independent proliferation (Fig. 2.9B). These data suggest that while GREB1 has been shown to be necessary for proliferation (Fig. 2.8) [80, 87], exogenous expression of either GREB1a or GREB1b is insufficient to drive hormone-independent proliferation.
Figure 2.8 GREB1 knockdown reduces proliferation MCF7 cells were transduced with a control shRNA (shNS), shRNA targeting all GREB1 isoforms (shRNA #1), or only GREB1a only (shRNA #2) and plated for MTT proliferation assay. Data are plotted as mean absorbance normalized to MTT Day 0 ± SD; n=3 for each cell line. Statistical significance was determined by one-way ANOVA with post-hoc Tukey’s HSD test; *p ≤ 0.05, *** p ≤ 0.001, **** p ≤ 0.0001. 56
Figure 2.9 GREB1 expression is not sufficient to drive hormone- independent growth. MCF7 cells were grown in full-serum media (FBS) or hormone-depleted media (CDT) and transduced with GFP, GREB1a, or GREB1b adenovirus. A, Immunoblot depicts GREB1 expression under each condition. B, Cell proliferation under the above conditions was measured via MTT assay. Data are plotted as mean absorbance normalized to MTT Day 0 ± SD; n=3. *p ≤ 0.05, **p≤ 0.01.
2.4.5 GREB1a and GREB1b modulate proliferation of breast cancer cells independent of ERα status
Although elevated expression of GREB1 isoforms did not induce hormone-independent proliferation, these isoforms could still contribute a growth advantage in the presence of estrogen. Thus, I tested the ability of exogenous
GREB1a and GREB1b to enhance proliferation of ER-positive breast cancer cell lines (MCF7, T47D, and ZR751) and ER-negative breast cancer cell lines (MDA-
MB-231 and MDA-MB-468), which do not express high levels of endogenous
GREB1 (Fig. 2.5). Immunoblot analysis with GREB1 antibody confirmed the elevated expression of GREB1a or GREB1b in all cell lines following transduction with adenovirus (Fig. 2.10A and Fig. 2.11A). Proliferation was measured over the
57 course of 5 days in all cell lines using MTT assays. Interestingly, elevated expression of GREB1a and GREB1b significantly impaired the proliferation of all three ER-positive cell lines and both ER-negative cell lines (Fig. 2.10B and Fig.
2.11B).
In order to determine if exogenous GREB1 expression affected cell viability, MCF7 cells were stained with LIVE/DEAD viability dye and analyzed by flow cytometry 72 hours post-transduction with GFP, GREB1a, or GREB1b adenovirus. No toxicity was associated with exogenous GREB1a expression and only a slight, but significant, decrease in cell viability was associated with exogenous GREB1b expression (Fig. 2.10C).
To confirm that GREB1 was driving changes in proliferation rather than metabolism, as measured by the MTT assay, proliferation of MCF7 cells was measured by EdU incorporation following transduction with GFP, GREB1a, or
GREB1b adenovirus. There was a significant decrease in the incorporation of
EdU into cells transduced with GREB1a and GREB1b in comparison to GFP- transduced cells (Fig. 2.10D). Together, these data indicate that elevated expression of GREB1 inhibits proliferation of breast cancer cell lines independent of ERα status. This activity is conserved between both GREB1a and GREB1b isoforms.
58
Figure 2.10 Elevated GREB1a and GREB1b expression reduces proliferation of breast cancer cells independent of ERα status. ER-positive (MCF7) and ER-negative (MDA-MB-468) cells were transduced with adenovirus expressing GFP, GREB1a, or GREB1b. A, Immunoblot showing elevated expression of GREB1a and GREB1b in transduced cells compared to GFP treated MCF7 cells. B, Proliferation of MCF7 and MDA-MB-468 cells after transduction with GREB1a, GREB1b, or GFP adenovirus was measured by MTT assay. Data are plotted as mean absorbance normalized to MTT Day 0 ± SD; n=3 for each cell line. C, MCF7 cells were transduced with GREB1a, GREB1b, or GFP adenovirus. After 72 hours, cells were stained with LIVE/DEAD Fixable Dead Cell Stain and analyzed by flow cytometry. Data are depicted as live cells as a mean percent total cells ± SE; n=3. D, MCF7 cells were transduced with GREB1a, GREB1b or GFP adenovirus. After 72 hours, cells were treated with EdU and fixed after 6 hours. Data are displayed as mean percent of EdU-positive cells ± SE; n=3. *p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.
59
Figure 2.11 Elevated GREB1a and GREB1b expression reduces proliferation of breast cancer cells. ER-positive (T47D and ZR751) and ER-negative (MDA-MB-231) cells were transduced with GREB1a, GREB1b, or GFP adenovirus. A, Immunoblot depicting the relative elevated expression of GREB1a compared to GFP- transduced cells. B, Proliferation of GREB1a- and GFP-transduced cells was measured using MTT assay. Data are plotted as mean absorbance normalized to MTT Day 0 ± SD; n=3 for each cell line. C, Immunoblot depicting the relative elevated expression of GREB1b compared to GFP-transduced cells. D, Proliferation of GREB1b- and GFP-transduced cells was measured using MTT assay. Data are plotted as mean absorbance normalized to MTT Day 0 ± SD; n=3 for each cell line. Statistical significance was determined by one-way ANOVA with post-hoc Tukey’s HSD test; *p ≤ 0.05.
2.4.6 GREB1b and GREB1c expression is increased in primary and metastatic patient tumor samples
Based on the finding that the GREB1 isoforms have different effects on gene expression (Fig. 2.6), I examined isoform expression in human breast 60 cancer cell lines and breast cancer patient samples using absolute quantification of mRNA copy number. As expected, and in line with immunoblot data (Fig. 2.10 and Fig. 2.11), GREB1a was the predominant form in ER-positive cell lines (Fig.
2.5) and patient samples (Fig. 2.12). High levels of GREB1 expression were limited to ER-positive breast cancer cell lines T47D, MCF7, and ZR751 in which
GREB1a represented at least 90% of all GREB1 transcripts (Fig. 2.5). Similarly,
ER-positive cancer samples had higher levels of GREB1 expression than ER- negative breast cancer samples indicative of the increased ER expression in cancer (Fig. 2.12A-C). Expression of all GREB1 isoforms increased from normal to primary disease (Fig. 2.12A-C). Remarkably, the proportion of GREB1b and
GREB1c mRNA increased significantly from normal to primary and metastatic tissue in matched patient samples, although GREB1a was still the most highly expressed isoform (Fig. 2.13). I calculated the proportion of GREB1 isoform expression using a larger, publically available data set of transcript expression in normal and malignant breast tissue [113, 114]. This data showed a similar trend in which the proportion of GREB1b and GREB1c mRNA was significantly higher in tumor samples compared to normal breast tissue. Together, these data suggest that increased expression of all GREB1 isoforms is associated with both
ER-positivity and primary or metastatic disease state.
61
Figure 2.12 Proportion of GREB1b and GREB1c expression increases during formation of primary tumor. Absolute mRNA copy number was determined from a standard curve generated using Taqman probes for A, GREB1a, B, GREB1b, and C, GREB1c in human normal, primary, and metastatic breast tissue. Each bar represents an individual patient sample. D, Graph depicting the percentage of total GREB1 expression of each isoform. The proportion of GREB1b and GREB1c to total GREB1 increases from normal to primary and metastatic disease (p ≤ 0.05).
62
Figure 2.13 Expression of GREB1b and GREB1c is higher in malignant tissue. A, Absolute mRNA copy number was determined from a standard curve generated using Taqman probes for GREB1a, GREB1b, and GREB1c in patient matched normal and tumor tissue samples. The percentage of total GREB1 expression of each isoform was compared between normal and primary or metastatic tissue from each patient using the Kruskal-Wallis multiple comparison method. The percent of GREB1b and GREB1c expression is significantly higher in primary tumor tissue in four of the five matched patient samples (p≤0.05). B, GREB1a, GREB1b, and GREB1c transcript expression was generated from a publically available dataset of normal and tumor tissue from breast cancer patients [15, 16]. The percentage of total GREB1 expression of each isoform was compared between normal and tumor samples using differential expression analysis [16]. Expression of GREB1b and GREB1c is significantly higher in tumor tissue than in normal tissue (p≤0.005). 63
2.5 Discussion
Many efforts have been made to identify ER-regulated genes that control proliferation and GREB1 was identified as one such gene [44]. Despite routinely being used as a molecular marker for ER activity, and nearly two decades of research, the molecular functions of this protein remain unclear beyond its reported ERα co-regulatory activity. Further, published studies have failed to differentiate between the three known protein isoforms encoded by the GREB1 gene, making it unclear which isoform may be involved in regulation of ERα activity and proliferation of breast cancer. Here, I demonstrate that all three isoforms have the ability to interact with ERα (Fig. 2.2), although none are potent regulators of ERα activity (Fig. 2.3 and Fig. 2.4). I further show that only GREB1a has the ability to enhance expression of ERα target genes (Fig. 2.6). Our data demonstrate the surprising ability of exogenously expressed GREB1a and
GREB1b to repress proliferation of breast cancer cell lines, independent of ERα expression (Fig. 2.10 and Fig. 2.11). In an effort to determine the significance of
GREB1 isoform expression, I quantified the transcript abundance of GREB1a, -b, and -c. All three isoforms were detected in a panel of breast cancer cell lines
(Fig. 2.5) and patient normal and tumor samples (Fig. 2.12 and Fig. 2.13). While
GREB1a is universally the most expressed transcript, breast cancer progression is associated with increased expression of the GREB1b and GREB1c isoforms
(Fig. 2.13). These findings underscore the notion that not all isoforms of GREB1 have conserved functional roles in breast cancer.
64
2.5.1 GREB1 isoforms are not potent regulators of ERα activity
Previous studies have suggested that GREB1 may interact with ER to regulate the receptor’s activity through modulation of co-factor binding [87]. I demonstrate that ER interacts with the amino terminus of all three isoforms of
GREB1 (Fig. 2.2) independent of the LxxLL motif within GREB1a (Fig. 2.2C).
However, the ability of these isoforms to enhance ERα activity was disparate.
Elevated expression of only GREB1a was able to enhance the expression of
ERα target genes (Fig. 2.6B). Notably, this pronounced effect on the expression of known ER target genes was evident both in the presence and absence of hormone (Fig. 2.6B). Previous reports suggest that GREB1 interaction with ER is ligand inducible [87]; however our data show that GREB1 preferentially interacts with ERα in the absence of ligand and in the cytoplasm (Fig. 2.7). The increase in basal expression of some ER-target genes seems to account for most of the transcriptional changes at these targets and appears to be receptor- independent. In contrast to GREB1a, elevated expression of GREB1b had no effect on ER target gene expression (Fig. 2.6B). While GREB1c was not tested, the protein isoform differs from GREB1b by the absence of exon 10 in the coding region and would not be expected to have additional functions than those seen in
GREB1b.
Surprisingly, the overall effect of GREB1 on ER target gene expression was modest at best. In fact, knockdown of all GREB1 isoforms resulted in only a 3-
30% decrease in the expression of target genes in the presence of endogenous 65 hormone (Fig. 2.3D) and knockdown of GREB1 isoforms had no effect on estrogen-induced expression of these target genes (Fig. 2.4). Previous studies using microarray analysis following knockdown of GREB1 with siRNA had demonstrated a 26-91% change in these same genes [87]. However, the previous microarray study also showed a 43% decrease in ER gene expression upon knockdown of GREB1 (6 hour time point) [87], while our data did not demonstrate a similar decrease in receptor levels (Fig.2.3C). Thus, the impact of
GREB1 knockdown on the expression of estrogen-responsive genes in the previous publication may be a result of reduced ER expression that may be dependent upon the methodology.
2.5.2 GREB1 regulates proliferation through ER independent activities
Previous studies have established GREB1 as a critical regulator of proliferation in breast cancer [80, 87], prostate cancer [96], and ovarian cancer
[83]. However, it was unclear which of the individual isoforms of GREB1 are responsible for this proliferative phenotype.
Specifically, one study found that knockdown of GREB1 in the presence or absence of hormone reduces proliferation of MCF7 breast cancer cell lines to that of estrogen-depleted conditions of control cells suggesting the necessity of
GREB1 for hormone-induced proliferation [80]. Thus, I hypothesized that elevated expression of GREB1 may circumvent the requirement of hormone for the proliferation of MCF7 breast cancer cells, presumably through activation of estrogen responsive genes. I show that exogenous GREB1a expression can 66 increase target gene expression even under estrogen-depleted conditions (Fig.
2.6B). However, I found that elevated expression of either of the predominant
GREB1 isoforms, GREB1a or GREB1b, did not induce hormone-independent growth of MCF7 breast cancer cells (Fig. 2.9). These data suggest that the modest role of GREB1 in the modulation of ER-target genes (Fig. 2.3) is not sufficient to drive proliferation of breast cancer cells in hormone-free conditions.
Surprisingly, elevated expression of GREB1a or GREB1b significantly reduced the proliferation of all ER-positive breast cancer cell lines even in hormone-containing media (Fig. 2.10 and Fig. 2.11), suggesting that the role of
GREB1 in regulating proliferation is not solely growth-promoting. The proliferation of ER-negative breast cancer cell lines was also severely inhibited by exogenous expression of GREB1a and GREB1b (Fig. 2.10 and Fig. 2.11), suggesting
GREB1 is able to modulate proliferation independent of ERα expression and activity. These data demonstrate that GREB1 has alternative functions beyond being a nuclear receptor co-regulator. This growth inhibitory role of GREB1 may explain why higher GREB1 expression in prostate cancer patients and hypomethylation of the GREB1 promoter in ovarian cancer patients correlates with better outcome [115]. I postulate that there is a homeostatic level of GREB1 that is growth-promoting while reduced or elevated expression results in growth inhibition in breast cancer cells.
Interestingly, previous analysis in tamoxifen-resistant MCF7 cells suggested that therapy resistance can be attributed to a loss of GREB1 co-
67 activator function [87]. While exogenous restoration of GREB1 caused a decrease in proliferation in the presence of tamoxifen, our data suggests this decrease may not be caused by restoration of tamoxifen sensitivity but rather an overexpression of GREB1. As I demonstrate herein, exogenous GREB1 expression causes a decrease in proliferation of most breast cancer cell lines regardless of ERα status and activity (Fig. 2.10 and Fig. 2.11). Thus, this previously published data supports the notion that an optimal level of GREB1 expression is necessary for proliferation of breast cancer cells.
While there remains a need for further research to be done in order to determine the discrete molecular function of the GREB1 isoforms, the data herein suggests a function of GREB1 isoforms outside of the canonical hormone receptor transcriptional network.
68
Chapter 3 : GREB1 regulates proliferation of estrogen receptor positive breast cancer through modulation of PI3K/Akt/mTOR signaling
3.1 Abstract
Over 70% of breast cancers express the estrogen receptor (ER) and depend on ER activity for survival and proliferation. While hormone therapies that target receptor activity are initially effective, patients invariably develop resistance highlighting the need for new and innovative therapies. While the mechanism by which ER regulates proliferation is poorly understood, one gene target of ER, growth regulation by estrogen in breast cancer 1 (GREB1), has been implicated in this process. Despite the relationship between GREB1 and estrogen-induced proliferation of breast cancer cells, the mechanism by which
GREB1 regulates proliferation has been largely uncharacterized. I show that knockdown of GREB1 results in growth arrest and that exogenous GREB1 expression halts proliferation and induces senescence, suggesting an optimal level of GREB1 expression necessary for proliferation of breast cancer cell lines.
Under both of these conditions, GREB1 is able to regulate signaling through the
PI3K/Akt/mTOR pathway, which has been implicated in the survival and proliferation of many different types of malignant cells, including breast cancer. I show that GREB1 is acting intrinsically through PI3K to regulate Akt activity.
Critically, growth suppression of estrogen-dependent breast cancer cells by 69
GREB1 knockdown is rescued by expression of constitutively activated Akt.
Together, these data identify a novel mechanism by which GREB1 regulates breast cancer proliferation through Akt activation and provides a mechanistic link between estrogen signaling and the PI3K pathway.
3.2 Introduction
Breast cancer is the most frequently diagnosed malignancy in women [116].
Over 70% of breast cancer patients are diagnosed with the estrogen receptor- positive (ER+) subtype, which is characterized by the expression of the transcription factor ER and dependence on ER activity for tumor cell growth and survival [8, 9, 37-39]. Patients diagnosed with the ER+ subtype of breast cancer are typically prescribed endocrine therapies that target ER activity [8, 9, 39].
However, resistance to endocrine therapies invariably occurs, leading to re- activation of the ER, expression of ER-target genes, and ultimately patient relapse [8, 9, 39]. Treatment options for patients that are resistant to endocrine therapies are limited, highlighting the need for innovative therapies that target downstream of ER [8, 9, 39]. Unfortunately, the development of these new therapies has been stymied by a lack in understanding of the mechanism by which ER-target genes drive proliferation of ER+ breast cancer.
Crosstalk between the ER and the PI3K/Akt/mTOR pathway has been implicated in ER+ breast cancer progression and resistance to endocrine therapies [55, 60, 61, 71]. The PIK3CA gene, which encodes the catalytic subunit of the PI3K enzyme, is the most commonly mutated gene in ER+ breast cancer
70 patients with over 40% incidence [117]. This mutation is speculated to be a causal event in breast cancer progression, suggesting there is some need to upregulate this pathway in the development of ER+ breast cancer [117]. PI3K and Akt have been shown to phosphorylate and activate ER at Ser167, resulting in upregulation of estrogen responsive genes [71]. Conversely, studies have indicated the ER can interact with PI3K to regulate its kinase activity in an estrogen-dependent manner [118]. Further, inhibition of the PI3K/Akt/mTOR pathway increases ER expression and sensitivity of breast cancer cells to tamoxifen treatment, suggesting that activation of this pathway is associated with resistance to endocrine therapies through downregulation of ER [119]. Together, these data indicate interdependence between ER and PI3K/Akt/mTOR signaling, however, the molecular basis and clinical relevance for this cooperation remains unclear.
The PI3K/Akt/mTOR pathway is activated downstream of receptor tyrosine kinases (RTKs) in response to environmental cues such as growth factors and cytokines [68-70]. Activation of PI3K results in the conversion of phosphatidylinositol-3,4-biphosphate (PIP2) to phosphatidylinositol-3,4- triphosphate (PIP3) [68-70].The tumor suppressor, phosphatase tensin homolog
(PTEN) opposes the conversion of PIP2 to PIP3, effectively terminating
PI3K/Akt/mTOR signaling [68-70]. Generation of PIP3 recruits phosphoinositide- dependent kinase 1 (PDK1) and Akt to the plasma membrane [68-70].
Phosphorylation of Akt at Thr308 by PDK1 and Ser473 by mTORC2 results in
71 the maximal activation of this kinase, further transducing the environmental signal [68-70].
Herein, I show that the ER gene target, growth regulation by estrogen in breast cancer 1 (GREB1), is a regulator of the PI3K/Akt/mTOR pathway, linking
ER activation to this critical signaling pathway. GREB1 was identified as gene whose expression is highly correlated to ER-positivity in breast cancer cell lines and patient samples [44, 80, 87, 88]. Previous studies have shown that knockdown of GREB1 results in significantly reduced proliferation and colony formation of ER+ breast cancer cell lines indicating a required role for GREB1 in regulation of estrogen-dependent proliferation [79, 80, 87]. However, it appears that an optimal level of GREB1 expression is necessary for proliferation of breast cancer cell lines as exogenous expression of GREB1 inhibits growth [79].
Interestingly, growth repression by exogenous expression of GREB1 was also observed in ER-negative cell lines, indicating the ability of GREB1 to regulate proliferation of breast cancer cells independent of ER activity [79]. Despite the clear association of GREB1 and proliferation of ER+ breast cancer, the molecular function of the protein and the mechanism by which it regulates proliferation remain largely unknown. In this study, I characterize a novel mechanism by which GREB1 regulates proliferation of ER+ breast cancer cell lines through activation of Akt.
72
3.3 Materials and methods
3.3.1 Cell lines and reagents
MCF7, T47D, ZR751, HCC1500, MDA-MB-231, HEK-293AD, and HEK-
293T cells were validated using Short Tandem Repeat analysis by the Genomics
Core in the Research Technology Support Facility (Michigan State University,
East Lansing, MI 48824). Cells were maintained as previously described [79]. For experiments with EGF stimulation, cells were cultured in serum-free media for 16 hours before being stimulated with 1 ng/mL recombinant human EGF (Thermo) for the indicated time. The inhibitors GDC-0941, GSK2334470, and MK-2206 were obtained from Cayman Chemicals and were used at indicated concentrations for 24 hours prior to harvest of the cells.
3.3.2 Plasmids
3XFLAG-GREB1, 3XFLAG PCDNA, H2BGFP, and pcDNA3-FLAG PTEN have been described previously [79, 109, 120]. GIPZ lentiviral non-specific shRNA (# RHS4346) and lentiviral GREB1-targeted shRNA plasmids
(V2LHS_139192 and V3LHS_372339) were obtained from Open Biosystems.
MISSION shRNA constructs targeted to PIK3Ca (TRCN0000196 582,
TRCN0000195 203, TRCN0000010 406), PTEN (TRCN0000002 745,
TRCN0000002 747, TRCN0000002 749), and PDK1 (TRCN0000001 476,
TRCN0000039 778, TRCN0000039 782, TRCN0000010 413) were purchased from Sigma Aldrich. Myristoylated (Myr) AKT1 from pBabe-Puro-Myr-Flag-AKT1
73
[121] was cloned into a pLenti-hygro backbone to create pLenti hygro Myr FLAG
AKT1 (CA AKT) using standard Gibson cloning (NEB).
3.3.3 Immunoblot analysis and antibodies
Cell lysates were prepared, subjected to immunoblot analysis, and visualized on a LI-COR Odyssey system as previously described [79, 112].
Immunoblots were probed with the following antibodies: GREB1 (abcam; ab72999), β-actin (Cell Signaling Technologies (CST); 3700), phospho-p38
(CST; 9211S), p38 (CST; 9212), phospho-MEK1/2 (CST; 9121), MEK1/2 (CST;
9122), phospho-ERK1/2 (CST; 4370); ERK1/2 (CST; 4695), phospho-MKK3/6
(CST; 12280), phospho-MSK1 (CST; 9595), phospho-ATF2 (CST; 5112), phospho-HSP27 (CST; 9709), phospho-MAPKAPK2 (CST; 3007), phospho-
PTEN (CST; 9551), PTEN (CST; 9556), phospho-PDK1 (CST; 3438), PDK1
(CST; 5662), phospho-Akt Thr308 (CST; 13038), phospho-Akt Ser473 (CST;
9271S), Akt (CST; 2920S), phospho-GSK3β (CST; 5558), mTOR (CST; 2983),
Rictor (CST; 2114), Raptor (CST; 2280), and p110α (CST; 4249T).
3.3.4 Adenovirus
GREB1 adenovirus was purified as previously described [79]. Ad5-CMV- eGFP adenovirus (Baylor College of Medicine Vector Development Labs,
Houston, TX 77030) was used as a control.
3.3.5 Alamar blue assay
Cells were treated with 0.04 g/L resazurin sodium salt in phosphate buffered saline (PBS) at 37°C for 1 hour. Fluorescence was measured using a
74
540/35 excitation filter and a 590/20 emission filter. Data are depicted as mean fluorescence normalized to day 0 ± SD for each condition from 3 biological replicates. Statistical significance for alamar blue assays was determined using either a two-tailed Student’s t-test (exogenous GREB1 expression) or a one-way
ANOVA with post-hoc Tukey’s HSD test (shRNA experiments).
3.3.6 SA-β-gal staining
Cells transduced with GFP or GREB1 adenovirus were plated on poly-L- lysine coated coverslips. Cells were fixed and stained for SA-β-gal activity using the Senescence β-Galactosidase Staining Kit (CST #9860) as previously described [122].
3.3.7 Conditioned media assay
MCF7 cells were transduced with either GFP or GREB1 adenovirus. After
24 hours, transduced cells were washed in PBS and fresh media added. The following day, media was collected from the transduced cells and centrifuged at
500 x g for 5 minutes to pellet any cellular debris. Target cells were washed twice with PBS and conditioned media added. After 24 hours, all cells were harvested by scraping in cold PBS containing 10 nM calyculin A (Cell Signaling).
Immunoblot analysis was performed as described above.
3.3.8 Co-culture assay
MCF7 cells were transduced with adenovirus expressing either GFP or
GREB1 (both adenovirus vectors express GFP). The following day, transduced cells were cultured at a 1:1 ratio with un-transduced MCF7 cells. Cells were
75 harvested after 24 hours by trypsinization and washed twice with cold PBS containing 10 nM calyculin A (Cell Signaling). Cells were sorted from both the adGFP and adGREB1 co-cultures using a Becton Dickinson FACSAria II cell sorter into GFP-positive (transduced) and GFP-negative (un-transduced) populations. Cell lysates were prepared from the sorted populations and immunoblot analysis was performed as described above.
3.3.9 Immunofluorescence microscopy
Cells were plated on poly-L-lysine-coated coverslips. Following treatment, cells were fixed in 4% methanol-free formaldehyde (Thermo) diluted in PBS for
15 minutes at room temperature. Cells were then washed three times in PBS before permeabilization with 0.5% saponin, 1% BSA PBS solution at room temperature for 15 minutes. The cells were labeled with the indicated primary antibodies for 2 hours at room temperature in a humidified chamber. Coverslips were then washed three times in PBS before incubation with secondary antibodies (Alexa Fluor 555 goat anti-mouse and Alexa Fluor 555 goat anti- rabbit; Invitrogen) at room temperature for 1 hour in a humidified chamber, protected from light. Coverslips were mounted on microscope slides with
VECTASHIELD Hard Set Mounting Medium with DAPI (Vector Laboratories).
Images were obtained using a spinning disk confocal microscope (Ultra-VIEW
VoX CSU-X1 system; Perkin Elmer) and analyzed using Velocity (Perkin Elmer).
76
3.4 Results
3.4.1 GREB1 initiates cellular senescence
Our previous work has suggested that an optimal level of GREB1 expression is necessary for proliferation of breast cancer cell lines, independent of ER status. This work showed that both GREB1 knockdown and exogenous expression of GREB1 results in growth arrest [79]. I previously reported that exogenous expression of GREB1 did not induce apoptosis [79], thus I investigated the ability of GREB1 overexpression to induce cellular senescence.
Two ER+ breast cancer cell lines, MCF7 and ZR751 cells, were transduced with adenovirus expressing either GFP or GREB1. As senescence is the result of long-term exposure to stress, cells were fixed and stained for SA-β-galactosidase activity, a marker of cellular senescence [122, 123], following 7 days of exogenous GREB1 expression. Compared to GFP control cells, cells overexpressing GREB1 had a large, flattened morphology and characteristic blue staining associated with SA-β-galactosidase activity (Fig. 3.1). This data suggest that exogenous GREB1 expression is able to induce cellular senescence to inhibit proliferation of breast cancer cell lines.
77
Figure 3.1 Exogenous GREB1 initiates cellular senescence. MCF7 or ZR751 cells were transduced with adenovirus expressing GFP or GREB1. Cells were fixed and stained for SA-β- galactosidase activity 7 days post-transduction.
3.4.2 Exogenous GREB1 expression induces hyperactivation of the PI3K/Akt/mTOR pathway
In order to delineate the mechanism by which GREB1 regulates proliferation I chose to focus our attention on two signaling pathways thought to play a critical role in regulating both senescent and proliferative phenotypes: the p38 MAPK pathway (Fig. 3.2A) and the PI3K/Akt/mTOR pathway (Fig. 3.2B)
[124-126]. MCF7 cells were transduced with adenovirus expressing GFP or
GREB1 and after 24 hours, cell lysates were analyzed by immunoblot for activation of various nodes in the p38 MAPK pathway and the PI3K/Akt/mTOR pathway. Following 24 hours, visualization of GFP in both GFP-transduced and
GREB1-transduced cells confirming the expression of our adenoviral vectors.
Our data indicate that exogenous GREB1 expression induces an increase in the activation and phosphorylation of p38 and its downstream effector, ATF2, however, the activation and/or expression of upstream regulators of p38 78
(MEK1/2, ERK1/2, MKK3/6) and other downstream effectors (MSK1,
MAPKAPK2, and HSP27) were largely unaffected (Fig. 3.2C). Analysis of the
PI3K/Akt/mTOR pathway revealed hyperactivation of Akt, as well as increased phosphorylation of GSK3β, a downstream effector of Akt, when GREB1 was overexpressed in MCF7 cells (Fig. 3.2D).
As upstream and downstream effectors of the p38 MAPK pathway were largely unaffected by GREB1 expression, I focused the remainder of our study on the PI3K/Akt/mTOR pathway. To confirm the ability of endogenous GREB1 to regulate signaling through the PI3K/Akt/mTOR pathway, MCF7 cells were transduced with lentivirus expressing non-specific shRNA (shNS) or one of two shRNAs targeting GREB1 (shGREB1 #1 or shGREB1 #2) and activation of nodes within this pathway were analyzed via immunoblot. Knockdown of GREB1 resulted in reduced activation of Akt (Fig. 3.2E) and reduced phosphorylation
GSK3β (Fig. 3.2E)
79
Figure 3.2 GREB1 modulates PI3K/Akt pathway signaling. (Figure 3.2 caption continued on page 81) 80
(Figure 3.2 caption continued from page 80) A, Diagram depicting the p38 MAPK signaling cascade. B, Simplified diagram depicting the PI3K/Akt/mTOR signaling cascade. C, MCF7 cells were transduced with adenovirus expressing GFP or GREB1. Cell lysates were harvested 24 hours post-transduction and subjected to SDS-PAGE followed by immunoblot analysis for indicated proteins in the p38 MAPK pathway or D, the PI3K/Akt pathway. E, Lysate from MCF7cells transduced with lentivirus expressing non-specific shRNA (shNS) or one of two shRNAs targeted to GREB1 (shGREB1 #1 or shGREB1 #2) were analyzed via immunoblot for activation proteins in the PI3K/Akt pathway. Densitometry analysis was performed and ratio of indicated phosphorylated protein to beta actin is graphed next to immunoblot.
3.4.3 GREB1-induced hyperactivation of Akt is PI3K-dependent
Akt requires phosphorylation at two sites, Thr308 and Ser473, by PDK1 and mTORC2 respectively, for maximal activation [70, 127, 128]. Both phosphorylation events are dependent on PI3K and occur downstream of PI3K conversion of PIP2 to PIP3 [70, 128]. Thus, I sought to determine if GREB1 was acting to regulate Akt activation upstream or downstream of PI3K. To this end, I selectively targeted various nodes within the PI3K/Akt/mTOR pathway for inhibition or knockdown and assessed the ability of exogenous GREB1 to induce hyperactivation of Akt.
MCF7 cells were simultaneously treated with inhibitors to PI3K
(GDC0941), PDK1 (GSK2334470), or AKT (MK2206) (Fig. 3.3A) and transduced with adenovirus expressing GFP or GREB1. After 24 hours, cell lysates were harvested and activation of Akt at Thr308 and Ser473 were evaluated by immunoblot analysis. The expected hyperactivation of Akt was observed in
81
DMSO-treated cells that were transduced with GREB1 adenovirus (Fig. 3.3B).
PI3K inhibition by GDC-0941 demonstrated a marked decrease in basal Akt activation in the control GFP-transduced cells (Fig. 3.3B). A decrease in GREB- mediated activation was also observed, particularly at Ser473 (Fig. 3.3B).
Inhibition of PDK1 by GSK2334470 resulted in the expected decrease in activation of Akt at Thr308, the site phosphorylated by PDK1, while Ser473 was unaffected, in GFP-transduced cells (Fig. 3.3B). Inhibition of PDK1 did not affect
GREB1-induced hyperactivation of Akt at either site (Fig. 3.3B). However, when
Akt activation by PDK1 and mTOR was inhibited by MK2206, activation of Akt was drastically reduced in both GFP- and GREB1-transduced cells (Fig. 3.3B).
Together, these data suggest that exogenous GREB1-induced hyperactivation of
Akt occurs through canonical PI3K pathway signaling.
To confirm this result, MCF7 cells were transduced with lentivirus expressing shRNA targeted to a non-specific control (NS), PIK3Ca, PTEN, or
PDK1. Following selection, cells were transduced with adenovirus expressing
GFP or GREB1. After 24 hours, cell lysates were harvested and analyzed via immunoblot. Control cells transduced with non-specific shRNA demonstrated the expected increase in phosphorylation of Akt (Thr308 and Ser473) and GSK3β when GREB1 was exogenously expressed (Fig. 3.3C). Knockdown of PIK3Ca expression reduced Akt activation in both control and GREB1-expressing cells
(Fig. 3.3C). GREB1-induced hyper-phosphorylation of GSK3β was also reduced when PIK3Ca was knocked-down (Fig. 3.3C). Knockdown of PTEN enhanced
82
GREB1-induced hyperactivation of Akt at Thr308, suggesting the mechanism of
GREB1 action is not through phosphatase inhibition (Fig. 3.3C). In contrast to our pharmacological approach (Fig. 3.3B), GREB1-induced hyperactivation of Akt at
Thr308 was completely blocked by knockdown of PDK1, the primary kinase for this site [70, 127-129](Fig. 3.3C). As expected, PDK1 knockdown had no effect on GREB1-induced hyperactivation of Akt at Ser473, as this is not the primary kinase for this site [70, 127, 128] (Fig. 3.3C). Knockdown of PDK1 diminished
GREB1-induced phosphorylation of GSK3β (Fig. 3.3C). These data further demonstrate that GREB1-induced hyperactivation of Akt is dependent on signaling through the canonical PI3K pathway.
83
Figure 3.3 GREB1-induced hyperactivation of Akt is PI3K-dependent. A, Schematic of the PI3K/Akt/mTOR pathway and inhibitor targets. B, MCF7 cells were transduced with adenovirus expressing GFP or GREB1 and treated with denoted inhibitors at indicated concentrations simultaneously. Cell lysates were harvested 24 hours post-transduction and immunoblot analysis was performed with the indicated antibodies. Representative image of three replicates is shown. C, MCF7 cells were transduced with lentivirus targeted to a nonspecific control (shNS), PIK3Ca, PTEN, or PDK1. Following selection, cells were transduced with GFP or GREB1 adenovirus. Cell lysates were harvested 24 hours post adenovirus transduction. Following SDS-PAGE, immunoblot analysis was performed with indicated antibodies. Representative images of three replicates is shown
84
3.4.4 GREB1 activates Akt through intracellular mechanisms
Canonical activation of PI3K and Akt occurs through activation of receptor tyrosine kinases (RTK) or G-protein-coupled receptors (GPCR) by external stimuli [70, 130]. I first sought to determine if GREB1-mediated Akt regulation is dependent upon induction and secretion of a signaling molecule that activates the PI3K/Akt/mTOR pathway. To this end, MCF7 cells were transduced with adenovirus expressing GFP or GREB1. Media from transduced cells was then transferred to un-transduced MCF7 cells. After 24 hours, cell lysates were harvested and activation of Akt was analyzed by immunoblot. As expected, exogenous expression of GREB1 induced hyperactivation of Akt at Ser473 when compared to GFP-transduced cells (Fig, 3.4A). However, conditioned media from neither GFP-transduced cells nor GREB1-transduced cells was able to induce hyperactivation of Akt in un-transduced cells (Fig. 3.4A).
Alternatively, exogenous GREB1 may induce the expression of a membrane-bound signaling molecule that could activate the PI3K/Akt/mTOR pathway. To investigate this possibility, MCF7 cells were transduced with adenovirus expressing GFP or GREB1 and then co-cultured at a 1:1 ratio with un-transduced MCF7 cells. After 24 hours, the cells were harvested and GFP- positive, adenovirus-transduced cells (GFP or GREB1), were sorted from GFP- negative, un-transduced cells. All populations were then analyzed for activation of Akt by immunoblot. Both GFP-transduced cells and cells co-cultured with the
GFP-transduced cells had similar levels of Akt activation (Fig. 3.4B). Exogenous
85 expression of GREB1 induced the expected hyperactivation of Akt at Ser473 within the transduced cells; however, the un-transduced co-culture cells did not demonstrate Akt hyperactivation (Fig. 3.4B).These data demonstrate that GREB1 regulates Akt activation through a cell-intrinsic mechanism.
Figure 3.4 GREB1 activates Akt through intracellular mechanisms. A, Conditioned media from MCF7 cells transduced with GFP or GREB1 adenovirus was added to un-transduced cells. Cell lysates from transduced cells (GFP or GREB1) and un-transduced cells cultured in conditioned media (GFP CM or GREB1 CM) were harvested 24 hours later. Lysates were subjected to SDS-PAGE and immunoblot performed for indicated proteins. B, MCF7 cells were transduced with adenovirus expressing GFP or GREB1. Transduced cells were then cultured with un-transduced cells at a 1:1 ratio for 24 hours. Cells were harvested and sorted for GFP. Cell lysates were analyzed via immunoblot for expression of the indicated proteins.
3.4.5 Exogenous GREB1 promotes recruitment of Akt to the plasma membrane
Akt is typically activated via recruitment to the plasma membrane by interaction with PIP3, however, it is believed that there are other pools of 86 activated Akt on endomembrane surfaces and within the nucleus [70]. To determine the localization of activated Akt induced by exogenous GREB1 expression, MCF7 cells were transduced with either GFP or GREB1 adenovirus.
Following transduction, the cells were serum-starved for 16 hours to reduce basal Akt activation before stimulation of the pathway with EGF. Cells were then fixed and stained with DAPI and the indicated Akt antibodies. As both adenoviral vectors expressed GFP, I focused our imaging on transduced cell populations expressing moderate levels of GFP and excluding high- and low-GFP expressing cells. In serum-starved, GFP-transduced cells, staining for activated Akt was minimal and staining for total Akt resulted in diffuse staining throughout the cytoplasm (Fig. 3.5A-C). In contrast, cells transduced with GREB1 adenovirus under serum-starved conditions had distinct staining for activated and total Akt, primarily localized to the plasma membrane (Fig. 3.5A-C). When stimulated with
EGF for 5 minutes, both GFP- and GREB1-transduced cells stained for activated
Akt and both activated and total Akt were found at the plasma membrane (Fig.
3.5A-C). Activation of Akt and focal localization of total Akt at the plasma membrane was noticeably stronger in GREB1-transduced cells when compared to GFP-transduced cells in the presence of EGF (Fig. 3.5A-C). These data further suggest that GREB1 may act through PI3K to increase PIP3 levels and
Akt relocalization to the plasma membrane.
87
Figure 3.5 Exogenous GREB1 promotes recruitment of Akt to the plasma membrane. (Figure 3.5 continued on page 89)
88
(Figure 3.5 continued from page 88 and continued to page 90)
89
(Figure 3.5 continued from page 88 and 89)
MCF7 cells were transduced with adenovirus expressing GFP or GREB1. The cells were then cultured in serum-free media for 16 hours before being stimulated with 1 ng/mL EGF for 0 or 5 minutes. Cells were fixed and stained for DAPI, A, p- Akt (Ser473), B, p-Akt (Thr308), or C, total Akt. Immunofluorescence microscopy was used to visualize the activation and localization of Akt.
90
Previous studies have suggested that GREB1 is primarily localized to the nucleus in patient samples and breast cancer cell lines [87, 88], thus, it remained unclear how GREB1 was able to regulate signaling through a primarily cytoplasmic pathway. Interestingly, I discovered that under serum-starved conditions endogenous GREB1 is diffuse throughout the cytoplasm and nucleus in MCF7 cells, but upon stimulation with EGF, the vast majority of GREB1 rapidly re-localizes to the cytoplasm (Fig. 3.6A-B). As this is contradictory to previously published reports, I performed nuclear/cytoplasmic fractionation to verify cytoplasmic expression of GREB1. Under normal growth conditions (i.e. media containing FBS), GREB1 is primarily located within the cytoplasm of MCF7 cells
(Fig. 3.6C-D).
91
Figure 3.6 Endogenous GREB1 re-localizes to the cytoplasm under growth- stimulatory conditions. A, MCF7 cells were serum starved for 4 hours and stimulated with 1 ng/mL EGF for 0, 5, or 15 minutes. Cells were fixed and stained for DAPI and endogenous GREB1. Immunofluorescence microscopy was used to visualize GREB1 localization. B, Ten fields were blindly quantified for cytoplasmic and nuclear staining of GREB1. Graph depicts percent of cells with nuclear GREB1 staining. Quantification performed by Alexander Young and Nanditha Ravichandran. C, Cytoplasmic and nuclear fractions were extracted from MCF7 whole cell lysate grown under normal growth conditions using high-speed centrifugation. Fractionated cell lysates were subjected to SDS-PAGE and analyzed via immunoblot for indicated proteins. D, MCF7 cells cultured in full serum media were fixed and stained for DAPI and endogenous GREB1. Immunofluorescence microscopy was used to visualize GREB1 localization under normal growth conditions. Staining and microscopy performed by Karen Wernke.
92
3.4.6 GREB1 regulates breast cancer proliferation through activation of the PI3K/Akt/mTOR pathway
Mutation and hyperactivation of key nodes within the PI3K/Akt/mTOR pathway are frequently found in ER+ breast tumors [60, 75, 131]. Thus, I sought to determine if constitutively activated PI3K/Akt/mTOR signaling can bypass the necessity of GREB1 expression for proliferation. I made use of T47D cells which are ER+ and GREB1-expressing, but harbor a PI3KCAH1047L mutation rendering this pathway constitutively active and unresponsive to typical PI3K/Akt/mTOR- activating stimuli, including GREB1 exogenous expression (Fig. 3.7A). T47D cells were transduced with shRNA targeting a nonspecific control (shNS) or shRNA targeting GREB1 (shGREB1 #1 or shGREB1 #2). Immunoblot analysis confirmed knockdown of GREB1, as well as hyperactivation of Akt in T47D cells
(Fig. 3.7B). Proliferation of these cells was then monitored via alamar blue assay.
GREB1 knockdown had no effect on the proliferation of T47D cells (Fig. 3.7C).
The proliferation of ER+ and GREB1-expressing MCF7 cells has previously been shown to be dependent on expression of GREB1 [79, 80, 87].
While MCF7 cells also harbor a mutation in PIK3CAE545K, which is thought to be an activating mutation [132], I have confirmed that these cells are still responsive to typical PI3K/Akt/mTOR-activating stimuli (e.g. EGF and fetal bovine serum).
Thus, I sought to determine if constitutively activated Akt (myristoylated-Akt) would rescue proliferation in GREB1-depleted MCF7 cells. MCF7 cells were transduced with empty vector lentivirus (EV) or lentivirus expressing constitutively activated Akt (CA AKT) in combination with lentivirus expressing 93 shRNA targeted to a non-specific control (shNS) or to GREB1 (shGREB1 #1 or shGREB1 #2). The knockdown of GREB1, and presumably decrease in Akt signaling, significantly impaired the growth of MCF7 cells co-transduced with empty vector lentivirus (Fig. 3.7D). Expression of constitutively active Akt restores the proliferation caused by GREB1 knockdown to that of control transduced cells (Fig. 3.7E). Together, these data demonstrate that the primary mechanism by which GREB1 drives estrogen-dependent proliferation is through modulation of Akt activity.
94
Figure 3.7 GREB1 regulates breast cancer proliferation through activation of the PI3K/Akt/mTOR pathway A, A panel of ER+ breast cancer cell lines were transduced with adenovirus expressing GFP or GREB1. Cell lysates were harvested 24 hours post- transduction and analyzed via immunoblot for indicated proteins. T47D cells were transduced with lentivirus expressing non-specific shRNA or shRNA targeted to GREB1 (shGREB1 #1 or shGREB1 #2). B, Immunoblot depicting the expression of indicated proteins. C, Proliferation was measured via alamar blue assay. Data are plotted as mean fluorescence normalized to Day 0 ± SD; n=3. D, MCF7 cells were transduced with lentivirus expressing empty vector (EV) and either non- specific shRNA or shRNA targeted to GREB1 (shGREB1 #1 or shGREB1 #2). Proliferation was measured via almar blue assay. Data are plotted as mean fluorescence normalized to Day 0 ± SD; n=3. E, MCF7 cells were transduced with lentivirus expressing myristoylated Akt (CA AKT) and either non-specific shRNA or shRNA targeted to GREB1 (shGREB1 #1 or shGREB1 #2). Proliferation was measured via almar blue assay. Data are plotted as mean fluorescence normalized to Day 0 ± SD; n=3. *p ≤ 0.05, ****p ≤ 0.0001. 95
3.5 Discussion
Despite extensive research on hormone signaling in breast cancer, the explicit mechanism by which ER drives proliferation remains largely undefined. In order to delineate this mechanism, concerted efforts have been made to identify
ER-target genes involved in estrogen-induced proliferation of breast cancer cells.
Several of these studies have identified GREB1 as a gene that is critically involved in the regulation of estrogen-stimulated proliferation of breast cancer cell lines [44, 80, 87]. Previous studies have suggested that GREB1 regulates proliferation through modulation of ER activity [87]. However, our findings show that GREB1 is not a potent regulator of ER activity and has the ability to affect the proliferation of breast cancer cell lines independent of ER expression and action [79]. Here, I suggest a novel mechanism by which GREB1 regulates proliferation through fine-tuning of PI3K/Akt/mTOR signaling.
3.5.1 GREB1-induced activation of Akt is dependent on PI3K
Expression of GREB1 is necessary for proliferation of ER+ breast cancer cell lines [79, 80, 87]. However, our data also show that exogenous expression of
GREB1 induces growth arrest and cellular senescence (Fig. 3.1), suggesting the ability of GREB1 to regulate both proliferative and anti-proliferative pathways. In order to elucidate the mechanism by which GREB1 controls proliferation, I sought to determine how expression of GREB1 influences signaling through pathways shown to be highly involved in growth regulation of cancer cells, namely the MAPK and PI3K/Akt/mTOR pathways [69, 124-126]. Exogenous 96 expression of GREB1 induced dramatic hyperactivation of Akt through the PI3K pathway while the MAPK pathway was largely unaffected (Fig. 3.2A-D). Further, knockdown of GREB1 reduced activation of Akt and phosphorylation of downstream effector, GSK3β (Fig. 3.2E). Taken together, these data are the first to suggest the ability of GREB1 to regulate mitogenic signaling cascades.
Disruption of the PI3K/Akt/mTOR pathway using pharmaceutical and genetic approaches resulted in decreased GREB1-induced activation of Akt (Fig.
3.3), suggesting the ability of GREB1 to directly influence signaling through the canonical PI3K/Akt/mTOR pathway. Interestingly, knockdown and disruption of the key Akt-activators (PI3K and PDK1), reduced the effect of exogenous
GREB1 expression of activation of Akt (Fig. 3.3), suggesting that GREB1 influences this pathway upstream of PI3K. Further, I show that GREB1 must be acting to regulate the PI3K/Akt/mTOR pathway intrinsically within the cell as exogenous expression did not induce expression of a paracrine signaling molecule that would affect the activation of the PI3K/Akt/mTOR pathway in neighboring cells (Fig. 3.4). Instead, our data indicate an accumulation of total and activated Akt at the plasma membrane upon exogenous expression of
GREB1 (Fig. 3.5). Although the explicit mechanism by which GREB1 facilitates the recruitment of activated Akt to the plasma membrane is still unclear, these data are the first to identify a cytoplasmic function for the purportedly nuclear protein [87, 88, 94].
97
3.5.2 GREB1 regulates proliferation of ER+ breast cancer cells through modulation of Akt activity
Several studies have indicated complex crosstalk between ER signaling and PI3K/Akt/mTOR pathway activation and implications for this crosstalk in resistance to endocrine therapy in breast cancer patients [28, 60, 61, 71, 133,
134]. However, no studies have described a comprehensive connection between activation of these signaling pathways and proliferation of breast cancer cells.
The difficulty to assess this connection is compounded by the fact that the vast majority of available breast cancer cell lines contain mutations involved in the
PI3K/Akt/mTOR pathway [135]. Although most ER+ breast cancer cell lines contain mutations within this pathway, the specific mutations have distinctly different effects on the activation of the PI3K/Akt/mTOR pathway. Specifically, breast cancer cell lines harboring PIK3CAH1047R mutations (ex. T47D) have significantly higher intrinsic PI3K activity compared to breast cancer cell lines harboring PIK3CAE545Kmutations (ex. MCF7) or PTENL108R mutations (ex. ZR751) which have subtle effects on activation of the PI3K/Akt/mTOR pathway [117,
136]. Cell lines that harbor mutations with elevated intrinsic PI3K activity, such as
T47D cells, are unable to be stimulated further by exogenous GREB1 expression
(Fig. 3.7A). Using this to our advantage, I assessed the necessity of GREB1- induced PI3K/Akt/mTOR activation on proliferation of ER+ breast cancer cell lines. I show that in T47D cells, which harbor a constitutively active
PI3K/Akt/mTOR pathway, GREB1 is no longer required for proliferation (Fig.
3.7B-C). However, in MCF7 cells, which harbor a PIK3CA mutation but still 98 respond to pathway-activating stimuli, GREB1 is still required but knockdown of
GREB1 can be rescued by constitutively active Akt (Fig. 3.7D-E). Taken together, these data provide strong evidence that GREB1 acts to regulate proliferation of ER+ breast cancer cell lines through fine-tuning of
PI3K/Akt/mTOR activity.
3.5.3 GREB1 and endocrine resistance
Despite the clear association between GREB1 and proliferation of breast cancer cells, expression of GREB1 has been correlated to better prognosis in
ER+ breast cancer patients [87, 94]. In a study that included only patients that received adjuvant tamoxifen monotherapy, higher GREB1 expression correlated with both prolonged disease-free survival and sensitivity to tamoxifen treatment
[94]. Similarly, in an in vitro model of tamoxifen resistance, MCF7 cells that were resistant to tamoxifen treatment had significantly less GREB1 expression compared to the parental line, suggesting GREB1 expression is lost in hormone- refractory breast cancer cells [87]. Despite these findings, the role of GREB1 in mediating sensitivity to hormone therapy remains unclear. Here, I show that proliferation of breast cancer cells with constitutively active PI3K/Akt/mTOR signaling no longer require GREB1 expression (Fig. 3.7). Constitutive activation of the PI3K/Akt/mTOR pathway is frequently associated with resistance to endocrine therapies and is the basis for numerous clinical trials investigating
PI3K/Akt/mTOR pathway inhibitors in endocrine-resistant patient populations [55,
60, 61, 71]. In patients with hormone-refractory disease with hyperactivation of
99 the PI3K/Akt/mTOR pathway, the pressure to express GREB1 is lost. Thus, decreased GREB1 expression in advanced disease may be the result of constitutive PI3K/Akt/mTOR activity rather than a cause of therapeutic bypass.
These findings warrant further research into the use of GREB1 as a clinical biomarker for treatment selection.
100
Chapter 4 : Conclusions and Future Directions
Breast cancer is the most frequently diagnosed malignancy in women with over
268,000 new cases expected in the United States in 2019 alone [2]. The vast majority of these women will be diagnosed with the estrogen receptor (ER) positive subtype of breast cancer [8, 9]. These tumors are characterized by the expression of the ER and the requirement for the activity of the ER at target genes for proliferation and survival [8, 9]. Patients diagnosed with ER-positive breast cancer are typically given endocrine therapies that target the activity of the
ER [8, 9]. However, patients invariably develop resistance to endocrine therapies. Hormone-refractory tumors commonly still express the ER and rely on its activity for tumor growth [8, 9]. Thus, there is a critical need to identify new therapeutic targets downstream of ER activity. One gene target of ER, growth regulation by estrogen in breast cancer 1 (GREB1), is required for proliferation of
ER-positive breast cancer cell lines and has been touted as both a potential clinical biomarker and therapeutic target [78, 80, 87]. However, understanding the potential of GREB1 as both a biomarker and therapeutic target has been stymied by a lack of a known molecular function and explicit mechanism by which it regulates proliferation.
101
4.1 GREB1 is not a potent regulator of ER activity
A previous study identified GREB1 as the most estrogen-induced ER interactor and suggested that GREB1 was a critical co-regulator of the ER [87].
However, the clarity of these published results was clouded by a lack of specific
GREB1 knockdown and a lack of differentiation between the known isoforms of
GREB1. The GREB1 gene encodes three distinct protein isoforms: GREB1a,
GREB1b, and GREB1c [44]. The GREB1a isoform is the full-length protein and
GREB1b and GREB1c are truncated splice variants [44]. In Chapter 2, I investigated the molecular functions of these isoforms and their role in regulation of ER activity. I show that all isoforms of GREB1 interact with the ER through their homologous amino terminus (Fig. 2.2), although isoform-specific knockdown reveals that no isoforms are potent regulators of ER activity (Fig. 2.3 and Fig.
2.4). Exogenous expression of GREB1a, but not GREB1b, resulted in elevated basal expression of ER target genes, however, this effect was independent of ER activity (Fig. 2.6). Thus, I investigated the conditions under which GREB1a interacted with the ER. Interestingly, I found that GREB1a preferentially interacted with ER in the cytoplasm (Fig. 2.7), bolstering the notion that GREB1a is not a potent transcriptional co-regulator of ER. However, the molecular basis for the interaction between ER and GREB1a in the cytoplasm remains unclear.
As cytoplasmic ER has been reported to regulate signaling cascades [22], future research should investigate the ability of GREB1 to mitigate these rapid, non- genomic effects of the ER.
102
4.2 GREB1 regulates proliferation of breast cancer cell lines through fine- tuning of PI3K/Akt/mTOR signaling
Previous studies, as well as our own, have shown that GREB1 expression is required for the proliferation of ER-positive breast cancer cell lines (Fig. 2.8)
[80, 87]. In Chapter 2 I show that exogenous GREB1 is insufficient to induce hormone-independent growth of breast cancer cell lines (Fig. 2.9). Further, I show that exogenous expression of GREB1 impairs the growth of both ER- positive and ER-negative cell lines in the presence of hormone (Fig. 2.10-2.11), suggesting the ability of GREB1 to modulate proliferation is independent of ER activity.
In Chapter 3 I investigate the ER-independent mechanism by which
GREB1 regulates proliferation. I show that exogenous expression of GREB1 induces cellular senescence (Fig. 3.1) and hyperactivation of the PI3K/Akt/mTOR pathway (Fig. 3.2). Our data indicate that exogenous GREB1 acts cell- intrinsically (Fig. 3.4) through the canonical PI3K/Akt/mTOR pathway to induce hyperactivation of Akt (Fig. 3.3) at the plasma membrane (Fig. 3.5). Further, I show that knockdown of GREB1 reduces activation of Akt and phosphorylation of
GSK3β (Fig. 3.2), suggesting endogenous GREB1 has the ability to fine-tune signaling through the PI3K/Akt/mTOR pathway. Critically, growth inhibition by
GREB1 knockdown can be rescued by expression of constitutively active Akt
(Fig. 3.7), suggesting that the primary mechanism by which GREB1 induces estrogen-dependent growth is through modulation of PI3K/Akt/mTOR signaling.
These findings are the first to provide a comprehensive connection between 103
PI3K/Akt/mTOR signaling, ER signaling, and proliferation of breast cancer cells.
However, the explicit mechanism by which GREB1 is able to regulate
PI3K/Akt/mTOR activity remains unclear. As GREB1 contains no well-defined functional domains, it is possible that GREB1 acts as a molecular scaffold at the plasma membrane, aiding in the recruitment of effector molecules and signal transduction. Specifically, my studies indicate PI3K is necessary for GREB1- induced activation of Akt (Fig. 3.3) and exogenous expression of GREB1 induces accumulation of activated Akt at the plasma membrane. Thus, I hypothesize that
GREB1 acts as a molecular scaffold to induce the activation of PI3K by RTKs, thus aiding in the accumulation of PIP3 at the plasma membrane, resulting in activation of downstream effectors such as Akt.
Mutation and hyperactivation of the PI3K/Akt/mTOR pathway is frequently associated with endocrine resistance in ER+ breast cancer patients [60, 75, 131].
Interestingly, a few studies have suggested that GREB1 loss is responsible for endocrine-resistance in MCF7 breast cancer cells and have shown that low
GREB1 expression is associated with worse prognosis in ER+ breast cancer patients [87, 94]. Interestingly, my results indicate that GREB1 expression is no longer required for proliferation in ER+ breast cancer cells that express hyperactive Akt (Fig. 3.7). I hypothesize that ER+ breast cancer cells that have developed resistance to endocrine therapies as a result of mutation and hyperactivation of the PI3K/Akt/mTOR pathway would no longer require GREB1 expression. In endocrine-sensitive, ER+ breast tumors, the ER drives expression
104 of GREB1 to upregulate signaling through the PI3K/Akt/mTOR pathway and drive proliferation. However, I hypothesize that in endocrine-resistant, ER+ breast tumors, the PI3K/Akt/mTOR pathway is mutated and hyperactivated, bypassing the requirement for ER-driven GREB1 expression. Thus, loss of GREB1 expression would be a result, not a driver, of endocrine resistance. Further studies analyzing the connection between GREB1 expression, activation and mutation of the PI3K/Akt/mTOR pathway, and endocrine resistance are necessary to test this hypothesis.
4.3 GREB1 and clathrin-mediated endocytosis
Preliminary GREB1 rapid immunoprecipitation mass spectrometry of endogenous protein (RIME) experiments performed by our collaborator, Jason
Carroll, identified many proteins involved in clathrin-mediated endocytosis (CME) as GREB1 interactors (Table 4.1). CME is a highly conserved vesicular transport process that regulates the internalization of receptors involved in rapid signal transduction pathways (Fig. 4.1) [137]. Many of the identified GREB1-interactors are involved specifically in the early stages of clathrin-coated vesicle formation and early endosome formation (Table 4.1) [138-141]. Our preliminary studies show that upon stimulation of CME, GREB1 re-localizes from an even distribution between the nucleus and cytoplasm to being found only in the cytoplasm (Fig.
4.2A). This rapid re-localization of GREB1 is inhibited by a small-molecule inhibitor of CME (Fig. 4.2B) suggesting that stimulation of CME is the cue for
GREB1 to move to the cytosol. Further, I show that endogenous GREB1 co-
105 localizes with markers of CME, clathrin heavy chain (CHC) and Rab5 (Early
Endosome-GFP) following stimulation of CME (Fig. 4.3). Taken together, these data provide compelling evidence that GREB1 is involved in CME.
Interestingly, emerging evidence suggests a role for PI3K in actin and membrane reorganization during early stages of CME [142-144]. Further, several studies have suggested that the clathrin-coated pits generated during CME act as microdomains within the cell that are imperative for certain signaling events, including EGF-stimulated Akt activation [144]. As our preliminary data suggests that GREB1 localizes and interacts with proteins involved in CME, future work should be dedicated to investigating the role GREB1 may be playing in CME and whether this function of GREB1 is required for GREB1-induced PI3K/Akt/mTOR signaling in breast cancer cells.
106
Table 4.1 GREB1 rapid immunoprecipitation and mass spectrometry of endogenous protein (RIME) reveals interaction of GREB1 with endocytic proteins.
Figure 4.1 Receptor tyrosine kinase trafficking through CME. Some receptor tyrosine kinases (RTKs), such as EGFR, are internalized by CME. Upon activation, a clathrin-coated pit forms at the plasma membrane and the receptor is then routed to the early endosome and can be recycled back to the membrane by the endocytic recycling compartment or degraded in the lysosome.
107
Figure 4.2 Stimulation of clathrin-mediated endocytosis (CME) causes GREB1 to re-localize from the nucleus to the cytoplasm. A, MCF7 cells were serum starved and then stimulated with ligands known to induce CME, EGF and TGFβ, or with a ligand that does not induce CME, TNFα. Cells were fixed following treatment with ligand at t=0, 5, and 15 minutes. Immunofluorescence microscopy using DAPI and GREB1 antibody was used to determine the localization of GREB1. B, MCF7 cells were serum-starved and then pre-treated with dynasore, a small molecule inhibitor of CME. The cells were fixed following treatment with EGF (1 ng/ml) for 0, 5, and 30 minutes. Immunofluorescence microscopy using DAPI and GREB1-specific antibody was used to determine the localization of GREB1.
108
Figure 4.3 GREB1 co-localizes with proteins involved in clathrin-mediated endocytosis (CME). A, MCF7 cells were serum-starved and stimulated with EGF (1 ng/mL) prior to fixation. Immunofluorescence microscopy was used to visualize localization of clathrin heavy chain (CHC, GFP) and GREB1 (red). B, MCF7 cells were labelled with CellLight® Early Endosome-GFP, BacMam 2.0 (Thermo) overnight, serum- starved, and stimulated with EGF (1 ng/mL) prior to fixation. Cells were stained with DAPI and GREB1 antibody (red). Immunofluorescence microscopy was used to visualize localization of early endosomes and GREB1. Cells were randomly selected and representative images are shown.
4.4 Molecular Function of GREB1
While I have made strides in identifying cellular processes of which GREB1 is involved, the molecular function of GREB1 remains elusive. Identification of a discrete molecular function of GREB1 has been made particularly challenging due to the lack of homology to other proteins and lack of well-characterized functional domains. In order to determine which portions of GREB1 are functionally relevant Dr. Craig Burd and I have generated 3XFLAG plasmids expressing different fragments of GREB1 (Fig. 4.4). Using these expression 109 plasmids I can use various analytical methods such as immunoblot, co- immunoprecipitation, and immunofluorescence microscopy to identify critical portions of GREB1 for its role in the processes outlined above.
I am particularly interested in understanding which domains of GREB1 are necessary for the activation of the PI3K/Akt/mTOR signaling cascade. To this end, I will individually express the fragments outlined in Fig. 4.4 in MCF7 cells and test for activation and localization of Akt via immunoblot and immunofluorescence microscopy. Once necessary domains are identified, I can perform immunoprecipitation followed by immunoblot or mass spectrometry in
MCF7 cells expressing GREB1 containing the necessary domains and expressing GREB1 that lacks the necessary domains. These studies will aid in determining the mechanism by which GREB1 supports activation of the
PI3K/Akt/mTOR pathway.
Additionally, GREB1 contains multiple putative nuclear localization and nuclear export signals throughout the amino acid sequence. The fragments outlined in Fig. 4.4 could also be used to identify portions of the GREB1 that are necessary and sufficient for the subcellular localization of GREB1. I will express the individual fragments in MCF7 cells, stain the cells with DAPI and FLAG antibody, and analyze the localization of the fragments by immunofluorescence microscopy. These experiments will be performed under normal growth conditions (full serum), serum-starvation, and growth stimulation (serum- starvation followed by 5 minutes EGF treatment). These results can then be
110 compared to those in Fig. 3.6 and Fig. 4.2 to determine the domains of GREB1 necessary for cytoplasmic and nuclear localization.
Figure 4.4 GREB1 fragment design. Diagram depicting the fragments of GREB1 and relative localization of the putative nuclear receptor binding site (LxxLL), putative proline-rich domain (PR), and putative transmembrane domain (TD).
To further characterize the role of GREB1 in the processes outlined above, I have generated plasmids expressing GREB1 fused to a promiscuous biotin ligase enzyme (miniTurboID), which will initiate covalent tagging of endogenous proteins [145]. Biotinylated proteins can then be harvested using streptavidin-coated beads and analyzed by mass spectrometry to identify novel
GREB1-interacting proteins and proximal. Although previous GREB1 RIME experiments were performed from nuclear lysates, presumably cytoplasmic proteins were identified (Table 4.1), suggesting the fractionation was not clean.
111
The GREB1 miniTurboID experiments will be not require fractionation and I expect that data generated from these experiments will provide a more complete picture of the GREB1 interactome. Co-immunoprecipitation and co-localization experiments can be used to confirm candidate interactors. Future studies will investigate the role of GREB1 in mediating the function of interacting proteins in order to develop a better understanding of the molecular function of GREB1.
4.5 GREB1 and clinical determinants of breast cancer progression and pathway activation
Previous studies have suggested that expression of GREB1 in breast cancer patients is associated with better prognosis and sensitivity to endocrine therapies [87, 94]. However, the role GREB1 plays in response to endocrine therapy remains unclear. Our data demonstrate a dynamic role for GREB1 in regulation of proliferation and modulation of PI3K/Akt/mTOR signaling, a pathway frequently associated with sensitivity to endocrine therapy [60, 61]. Further, I have shown that higher cytoplasmic GREB1 localization is associated with more aggressive, late stage disease in prostate cancer tissue samples (Fig. 4.5), suggesting a correlation between localization of GREB1 and disease progression. Taken together, I believe that these findings warrant further research to establish the connection between GREB1 expression and localization, activation of the PI3K/Akt/mTOR pathway, and clinical determinants of breast cancer progression.
112
Figure 4.5 GREB1 localization differs among tumor grades. A, Human prostate cancer tissue samples were analyzed for GREB1 expression using immunohistochemistry. Representative images display differences in GREB1 localization with strong cytoplasmic staining (top) and strong nuclear staining (bottom). B, Expression of GREB1 in the cytoplasm was quantified (H- score) and plotted based on Gleason score of the tumor. Each marker represents a separate field within a tumor sample. C, Intensity of GREB1 staining was quantified in the epithelial cytoplasmic and nuclear compartments using InForm software. Data are displayed as the ratio of nuclear staining intensity to cytoplasmic staining intensity based on tumor grade with pT2a being low-grade, confined only to one side of the prostate, and pT4 being high-grade, spreading to nearby tissues. Experiments in Figure 4.5 were performed by Alina Murphy.
Future studies will investigate correlations between both GREB1 expression/localization and activation of Akt in breast cancer patient samples.
These correlations could then be extrapolated to include available patient data
113 such as stage at diagnosis, histological grade, nodal status at diagnosis, ER status, recurrence, survival, and treatment response. In addition to these correlative studies, I will investigate the role of GREB1 in development of endocrine resistance in vitro. Using cell line models of tamoxifen resistance, I will evaluate the sufficiency and necessity of GREB1 for sensitivity to endocrine therapies and investigate if GREB1-mediated PI3K/Akt/mTOR signaling plays a role in development of tamoxifen resistance. The association of the
PI3K/Akt/mTOR pathway with endocrine resistance is the basis for numerous clinical trials investigating pathway inhibitors in endocrine-resistant patient populations [75]. However, identifying sensitive populations remains a major obstacle for current clinical trials [75]. Findings from this proposed future work will aid in determining the potential use of GREB1 as a clinical biomarker for treatment selection for the growing number of women diagnosed with breast cancer.
114
References
1. Heldring, N., et al., Estrogen receptors: how do they signal and what are their targets. Physiol Rev, 2007. 87(3): p. 905-31.
2. Siegel, R.L., K.D. Miller, and A. Jemal, Cancer statistics, 2019. CA Cancer J Clin, 2019. 69(1): p. 7-34.
3. Rahib, L., et al., Projecting cancer incidence and deaths to 2030: the unexpected burden of thyroid, liver, and pancreas cancers in the United States. Cancer Res, 2014. 74(11): p. 2913-21.
4. Winters, S., et al., Breast Cancer Epidemiology, Prevention, and Screening. Prog Mol Biol Transl Sci, 2017. 151: p. 1-32.
5. Nunez, C., Blood-based protein biomarkers in breast cancer. Clin Chim Acta, 2018. 490: p. 113-127.
6. Wang, Y., et al., Gene-expression profiles to predict distant metastasis of lymph- node-negative primary breast cancer. Lancet, 2005. 365(9460): p. 671-9.
7. Nelson, L.R. and S.E. Bulun, Estrogen production and action. J Am Acad Dermatol, 2001. 45(3 Suppl): p. S116-24.
8. Dixon, J.M., Endocrine Resistance in Breast Cancer. New Journal of Science, 2014. 2014: p. 1-27.
9. Ali, S. and R.C. Coombes, Endocrine-responsive breast cancer and strategies for combating resistance. Nat Rev Cancer, 2002. 2(2): p. 101-12.
10. Nilsson, S., et al., Mechanisms of estrogen action. Physiol Rev, 2001. 81(4): p. 1535-65.
11. Soltysik, K. and P. Czekaj, Membrane estrogen receptors - is it an alternative way of estrogen action? J Physiol Pharmacol, 2013. 64(2): p. 129-42.
12. Bjornstrom, L. and M. Sjoberg, Mechanisms of estrogen receptor signaling: convergence of genomic and nongenomic actions on target genes. Mol Endocrinol, 2005. 19(4): p. 833-42.
115
13. Acconcia, F. and M. Marino, The Effects of 17beta-estradiol in Cancer are Mediated by Estrogen Receptor Signaling at the Plasma Membrane. Front Physiol, 2011. 2: p. 30.
14. Matthews, J., et al., Estrogen receptor (ER) beta modulates ERalpha-mediated transcriptional activation by altering the recruitment of c-Fos and c-Jun to estrogen-responsive promoters. Mol Endocrinol, 2006. 20(3): p. 534-43.
15. Matthews, J. and J.A. Gustafsson, Estrogen signaling: a subtle balance between ER alpha and ER beta. Mol Interv, 2003. 3(5): p. 281-92.
16. Liu, M.M., et al., Opposing action of estrogen receptors alpha and beta on cyclin D1 gene expression. J Biol Chem, 2002. 277(27): p. 24353-60.
17. Aranda, A. and A. Pascual, Nuclear hormone receptors and gene expression. Physiol Rev, 2001. 81(3): p. 1269-304.
18. Simoncini, T., et al., Genomic and non-genomic effects of estrogens on endothelial cells. Steroids, 2004. 69(8-9): p. 537-42.
19. Kahlert, S., et al., Estrogen receptor alpha rapidly activates the IGF-1 receptor pathway. J Biol Chem, 2000. 275(24): p. 18447-53.
20. Wong, C.W., et al., Estrogen receptor-interacting protein that modulates its nongenomic activity-crosstalk with Src/Erk phosphorylation cascade. Proc Natl Acad Sci U S A, 2002. 99(23): p. 14783-8.
21. Razandi, M., et al., Proximal events in signaling by plasma membrane estrogen receptors. J Biol Chem, 2003. 278(4): p. 2701-12.
22. Migliaccio, A., et al., Tyrosine kinase/p21ras/MAP-kinase pathway activation by estradiol-receptor complex in MCF-7 cells. Embo j, 1996. 15(6): p. 1292-300.
23. Improta-Brears, T., et al., Estrogen-induced activation of mitogen-activated protein kinase requires mobilization of intracellular calcium. Proc Natl Acad Sci U S A, 1999. 96(8): p. 4686-91.
24. Aronica, S.M., W.L. Kraus, and B.S. Katzenellenbogen, Estrogen action via the cAMP signaling pathway: stimulation of adenylate cyclase and cAMP-regulated gene transcription. Proc Natl Acad Sci U S A, 1994. 91(18): p. 8517-21.
25. Maggiolini, M. and D. Picard, The unfolding stories of GPR30, a new membrane- bound estrogen receptor. J Endocrinol, 2010. 204(2): p. 105-14.
26. Prossnitz, E.R., J.B. Arterburn, and L.A. Sklar, GPR30: A G protein-coupled receptor for estrogen. Mol Cell Endocrinol, 2007. 265-266: p. 138-42.
116
27. Welboren, W.J., et al., Genomic actions of estrogen receptor alpha: what are the targets and how are they regulated? Endocr Relat Cancer, 2009. 16(4): p. 1073- 89.
28. Kato, S., et al., Activation of the estrogen receptor through phosphorylation by mitogen-activated protein kinase. Science, 1995. 270(5241): p. 1491-4.
29. Shim, W.S., et al., Estradiol hypersensitivity and mitogen-activated protein kinase expression in long-term estrogen deprived human breast cancer cells in vivo. Endocrinology, 2000. 141(1): p. 396-405.
30. Coutts, A.S. and L.C. Murphy, Elevated mitogen-activated protein kinase activity in estrogen-nonresponsive human breast cancer cells. Cancer Res, 1998. 58(18): p. 4071-4.
31. Inman, J.L., et al., Mammary gland development: cell fate specification, stem cells and the microenvironment. Development, 2015. 142(6): p. 1028-42.
32. Boyd, M., R.H. Hildebrandt, and S.A. Bartow, Expression of the estrogen receptor gene in developing and adult human breast. Breast Cancer Res Treat, 1996. 37(3): p. 243-51.
33. Ricketts, D., et al., Estrogen and progesterone receptors in the normal female breast. Cancer Res, 1991. 51(7): p. 1817-22.
34. Feng, Y., et al., Breast cancer development and progression: Risk factors, cancer stem cells, signaling pathways, genomics, and molecular pathogenesis. Genes & diseases, 2018. 5(2): p. 77-106.
35. Provenzano, E., G.A. Ulaner, and S.F. Chin, Molecular Classification of Breast Cancer. PET Clin, 2018. 13(3): p. 325-338.
36. Goldhirsch, A., et al., Strategies for subtypes--dealing with the diversity of breast cancer: highlights of the St. Gallen International Expert Consensus on the Primary Therapy of Early Breast Cancer 2011. Ann Oncol, 2011. 22(8): p. 1736- 47.
37. Patani, N. and L.A. Martin, Understanding response and resistance to oestrogen deprivation in ER-positive breast cancer. Mol Cell Endocrinol, 2014. 382(1): p. 683-694.
38. Libson, S. and M. Lippman, A review of clinical aspects of breast cancer. Int Rev Psychiatry, 2014. 26(1): p. 4-15.
39. Clarke, R., J.J. Tyson, and J.M. Dixon, Endocrine resistance in breast cancer--An overview and update. Mol Cell Endocrinol, 2015. 418 Pt 3: p. 220-34.
117
40. Miller, W.R. and A.P. Forrest, Oestradiol synthesis by a human breast carcinoma. Lancet, 1974. 2(7885): p. 866-8.
41. Tryfonidis, K., et al., Endocrine treatment in breast cancer: Cure, resistance and beyond. Cancer Treat Rev, 2016. 50: p. 68-81.
42. Hui, R., et al., Constitutive overexpression of cyclin D1 but not cyclin E confers acute resistance to antiestrogens in T-47D breast cancer cells. Cancer Res, 2002. 62(23): p. 6916-23.
43. Dubik, D. and R.P. Shiu, Mechanism of estrogen activation of c-myc oncogene expression. Oncogene, 1992. 7(8): p. 1587-94.
44. Ghosh, M.G., D.A. Thompson, and R.J. Weigel, PDZK1 and GREB1 are estrogen-regulated genes expressed in hormone-responsive breast cancer. Cancer Res, 2000. 60(22): p. 6367-75.
45. Tsoutsou, P.G., et al., Emerging Opportunities of Radiotherapy Combined With Immunotherapy in the Era of Breast Cancer Heterogeneity. Front Oncol, 2018. 8: p. 609.
46. Martinkovich, S., et al., Selective estrogen receptor modulators: tissue specificity and clinical utility. Clin Interv Aging, 2014. 9: p. 1437-52.
47. Nathan, M.R. and P. Schmid, A Review of Fulvestrant in Breast Cancer. Oncol Ther, 2017. 5(1): p. 17-29.
48. Patel, H.K. and T. Bihani, Selective estrogen receptor modulators (SERMs) and selective estrogen receptor degraders (SERDs) in cancer treatment. Pharmacol Ther, 2018. 186: p. 1-24.
49. Chumsri, S., et al., Aromatase, aromatase inhibitors, and breast cancer. J Steroid Biochem Mol Biol, 2011. 125(1-2): p. 13-22.
50. Smith, I.E. and M. Dowsett, Aromatase inhibitors in breast cancer. N Engl J Med, 2003. 348(24): p. 2431-42.
51. Waks, A.G. and E.P. Winer, Breast Cancer Treatment: A Review. Jama, 2019. 321(3): p. 288-300.
52. Larionov, A.A. and W.R. Miller, Challenges in defining predictive markers for response to endocrine therapy in breast cancer. Future Oncol, 2009. 5(9): p. 1415-28.
53. Dowsett, M., et al., Meta-analysis of breast cancer outcomes in adjuvant trials of aromatase inhibitors versus tamoxifen. J Clin Oncol, 2010. 28(3): p. 509-18.
118
54. Colleoni, M. and E. Montagna, Neoadjuvant therapy for ER-positive breast cancers. Ann Oncol, 2012. 23 Suppl 10: p. x243-8.
55. Miller, W.R., et al., Gene expression profiles differentiating between breast cancers clinically responsive or resistant to letrozole. J Clin Oncol, 2009. 27(9): p. 1382-7.
56. Mustacchi, G., et al., Neo-adjuvant exemestane in elderly patients with breast cancer: a phase II, multicentre, open-label, Italian study. Ann Oncol, 2009. 20(4): p. 655-9.
57. Hasson, S.P., et al., Endocrine resistance in breast cancer: focus on the phosphatidylinositol 3-kinase/akt/mammalian target of rapamycin signaling pathway. Breast Care (Basel), 2013. 8(4): p. 248-55.
58. Ring, A. and M. Dowsett, Mechanisms of tamoxifen resistance. Endocr Relat Cancer, 2004. 11(4): p. 643-58.
59. Kuukasjarvi, T., et al., Loss of estrogen receptor in recurrent breast cancer is associated with poor response to endocrine therapy. J Clin Oncol, 1996. 14(9): p. 2584-9.
60. Miller, T.W., J.M. Balko, and C.L. Arteaga, Phosphatidylinositol 3-kinase and antiestrogen resistance in breast cancer. J Clin Oncol, 2011. 29(33): p. 4452-61.
61. Miller, T.W., et al., Hyperactivation of phosphatidylinositol-3 kinase promotes escape from hormone dependence in estrogen receptor-positive human breast cancer. J Clin Invest, 2010. 120(7): p. 2406-13.
62. Zardavas, D., D. Fumagalli, and S. Loi, Phosphatidylinositol 3- kinase/AKT/mammalian target of rapamycin pathway inhibition: a breakthrough in the management of luminal (ER+/HER2-) breast cancers? Curr Opin Oncol, 2012. 24(6): p. 623-34.
63. Butti, R., et al., Receptor tyrosine kinases (RTKs) in breast cancer: signaling, therapeutic implications and challenges. Mol Cancer, 2018. 17(1): p. 34.
64. Lee, H.H., Y.N. Wang, and M.C. Hung, Non-canonical signaling mode of the epidermal growth factor receptor family. Am J Cancer Res, 2015. 5(10): p. 2944- 58.
65. von Zastrow, M. and A. Sorkin, Signaling on the endocytic pathway. Curr Opin Cell Biol, 2007. 19(4): p. 436-45.
66. Waterman, H. and Y. Yarden, Molecular mechanisms underlying endocytosis and sorting of ErbB receptor tyrosine kinases. FEBS Lett, 2001. 490(3): p. 142- 52.
119
67. Hsu, J.L. and M.C. Hung, The role of HER2, EGFR, and other receptor tyrosine kinases in breast cancer. Cancer Metastasis Rev, 2016. 35(4): p. 575-588.
68. Fruman, D.A., et al., The PI3K Pathway in Human Disease. Cell, 2017. 170(4): p. 605-635.
69. Fruman, D.A. and C. Rommel, PI3K and Cancer: Lessons, Challenges and Opportunities. Nat Rev Drug Discov, 2014. 13(2): p. 140-56.
70. Manning, B.D. and A. Toker, AKT/PKB Signaling: Navigating the Network. Cell, 2017. 169(3): p. 381-405.
71. Campbell, R.A., et al., Phosphatidylinositol 3-kinase/AKT-mediated activation of estrogen receptor alpha: a new model for anti-estrogen resistance. J Biol Chem, 2001. 276(13): p. 9817-24.
72. Perez-Tenorio, G. and O. Stal, Activation of AKT/PKB in breast cancer predicts a worse outcome among endocrine treated patients. Br J Cancer, 2002. 86(4): p. 540-5.
73. Miller, T.W., et al., ERalpha-dependent E2F transcription can mediate resistance to estrogen deprivation in human breast cancer. Cancer Discov, 2011. 1(4): p. 338-51.
74. Di Leo, A., et al., Buparlisib plus fulvestrant in postmenopausal women with hormone-receptor-positive, HER2-negative, advanced breast cancer progressing on or after mTOR inhibition (BELLE-3): a randomised, double-blind, placebo- controlled, phase 3 trial. Lancet Oncol, 2018. 19(1): p. 87-100.
75. Keegan, N.M., et al., PI3K inhibition to overcome endocrine resistance in breast cancer. Expert Opin Investig Drugs, 2018. 27(1): p. 1-15.
76. Nagase, T., et al., Prediction of the coding sequences of unidentified human genes. XII. The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro. DNA Res, 1998. 5(6): p. 355-64.
77. Cheng, M., S. Michalski, and R. Kommagani, Role for Growth Regulation by Estrogen in Breast Cancer 1 (GREB1) in Hormone-Dependent Cancers. Int J Mol Sci, 2018. 19(9).
78. Hodgkinson, K.M. and B.C. Vanderhyden, Consideration of GREB1 as a potential therapeutic target for hormone-responsive or endocrine-resistant cancers. Expert Opin Ther Targets, 2014. 18(9): p. 1065-76.
79. Haines, C.N., et al., GREB1 isoforms regulate proliferation independent of ERalpha co-regulator activities in breast cancer. Endocr Relat Cancer, 2018. 25(7): p. 735-746.
120
80. Rae, J.M., et al., GREB 1 is a critical regulator of hormone dependent breast cancer growth. Breast Cancer Res Treat, 2005. 92(2): p. 141-9.
81. Purcell, D.J., et al., Novel CARM1-Interacting Protein, DZIP3, Is a Transcriptional Coactivator of Estrogen Receptor-alpha. Mol Endocrinol, 2015. 29(12): p. 1708- 19.
82. McDermott, M.S., et al., Inhibition of CDK8 mediator kinase suppresses estrogen dependent transcription and the growth of estrogen receptor positive breast cancer. Oncotarget, 2017. 8(8): p. 12558-12575.
83. Laviolette, L.A., et al., 17beta-estradiol upregulates GREB1 and accelerates ovarian tumor progression in vivo. Int J Cancer, 2014. 135(5): p. 1072-84.
84. Haynes, B.P., et al., Expression of key oestrogen-regulated genes differs substantially across the menstrual cycle in oestrogen receptor-positive primary breast cancer. Breast Cancer Res Treat, 2013. 138(1): p. 157-65.
85. Haakensen, V.D., et al., Serum estradiol levels associated with specific gene expression patterns in normal breast tissue and in breast carcinomas. BMC Cancer, 2011. 11: p. 332.
86. Dunbier, A.K., et al., Relationship between plasma estradiol levels and estrogen- responsive gene expression in estrogen receptor-positive breast cancer in postmenopausal women. J Clin Oncol, 2010. 28(7): p. 1161-7.
87. Mohammed, H., et al., Endogenous purification reveals GREB1 as a key estrogen receptor regulatory factor. Cell Rep, 2013. 3(2): p. 342-9.
88. Hnatyszyn, H.J., et al., Correlation of GREB1 mRNA with protein expression in breast cancer: validation of a novel GREB1 monoclonal antibody. Breast Cancer Res Treat, 2010. 122(2): p. 371-80.
89. Sun, J., Z. Nawaz, and J.M. Slingerland, Long-range activation of GREB1 by estrogen receptor via three distal consensus estrogen-responsive elements in breast cancer cells. Mol Endocrinol, 2007. 21(11): p. 2651-62.
90. Deschenes, J., et al., Regulation of GREB1 transcription by estrogen receptor alpha through a multipartite enhancer spread over 20 kb of upstream flanking sequences. J Biol Chem, 2007. 282(24): p. 17335-9.
91. Tan, S., et al., Identification of miR-26 as a key mediator of estrogen stimulated cell proliferation by targeting CHD1, GREB1 and KPNA2. Breast Cancer Res, 2014. 16(2): p. R40.
92. Chang, T.C., et al., Widespread microRNA repression by Myc contributes to tumorigenesis. Nat Genet, 2008. 40(1): p. 43-50.
121
93. Kota, J., et al., Therapeutic microRNA delivery suppresses tumorigenesis in a murine liver cancer model. Cell, 2009. 137(6): p. 1005-17.
94. Wu, Y., et al., Tamoxifen Resistance in Breast Cancer Is Regulated by the EZH2- ERalpha-GREB1 Transcriptional Axis. Cancer Res, 2018. 78(3): p. 671-684.
95. Bhan, A., et al., Histone methyltransferase EZH2 is transcriptionally induced by estradiol as well as estrogenic endocrine disruptors bisphenol-A and diethylstilbestrol. J Mol Biol, 2014. 426(20): p. 3426-41.
96. Rae, J.M., et al., GREB1 is a novel androgen-regulated gene required for prostate cancer growth. Prostate, 2006. 66(8): p. 886-94.
97. Camden, A.J., et al., Growth regulation by estrogen in breast cancer 1 (GREB1) is a novel progesterone-responsive gene required for human endometrial stromal decidualization. Mol Hum Reprod, 2017. 23(9): p. 646-653.
98. Brunetti, M., et al., RNA-sequencing identifies novel GREB1-NCOA2 fusion gene in a uterine sarcoma with the chromosomal translocation t(2;8)(p25;q13). Genes Chromosomes Cancer, 2018. 57(4): p. 176-181.
99. Finger, J.H., et al., The mouse Gene Expression Database (GXD): 2017 update. Nucleic Acids Res, 2017. 45(D1): p. D730-d736.
100. Li, S.Z., et al., greb1 regulates convergent extension movement and pituitary development in zebrafish. Gene, 2017. 627: p. 176-187.
101. Pellegrini, C., et al., The expression of estrogen receptors as well as GREB1, c- MYC, and cyclin D1, estrogen-regulated genes implicated in proliferation, is increased in peritoneal endometriosis. Fertil Steril, 2012. 98(5): p. 1200-8.
102. Lippman, M.E., J.M. Rae, and A.M. Chinnaiyan, An expression signature of estrogen-regulated genes predicts disease-free survival in tamoxifen-treated patients better than progesterone receptor status. Trans Am Clin Climatol Assoc, 2008. 119: p. 77-90; discussion 90-2.
103. Loi, S., et al., Predicting prognosis using molecular profiling in estrogen receptor- positive breast cancer treated with tamoxifen. BMC Genomics, 2008. 9: p. 239.
104. Zhang, Y., et al., The 76-gene signature defines high-risk patients that benefit from adjuvant tamoxifen therapy. Breast Cancer Res Treat, 2009. 116(2): p. 303- 9.
105. Langdon, S.P., et al., Endocrine therapy in epithelial ovarian cancer. Expert Rev Anticancer Ther, 2017. 17(2): p. 109-117.
122
106. Slotman, B.J. and B.R. Rao, Ovarian cancer (review). Etiology, diagnosis, prognosis, surgery, radiotherapy, chemotherapy and endocrine therapy. Anticancer Res, 1988. 8(3): p. 417-34.
107. Gibson, D.A., et al., Endometrial Intracrinology: Oestrogens, Androgens and Endometrial Disorders. Int J Mol Sci, 2018. 19(10).
108. Hall, J.M. and K.S. Korach, Analysis of the molecular mechanisms of human estrogen receptors alpha and beta reveals differential specificity in target promoter regulation by xenoestrogens. J Biol Chem, 2002. 277(46): p. 44455-61.
109. Burd, C.J., et al., Cyclin D1 binding to the androgen receptor (AR) NH2-terminal domain inhibits activation function 2 association and reveals dual roles for AR corepression. Mol Endocrinol, 2005. 19(3): p. 607-20.
110. Norris, J.D., et al., Identification of a third autonomous activation domain within the human estrogen receptor. Mol Endocrinol, 1997. 11(6): p. 747-54.
111. Zacharewski, T.R., et al., Antiestrogenic effect of 2,3,7,8-tetrachlorodibenzo-p- dioxin on 17 beta-estradiol-induced pS2 expression. Cancer Res, 1994. 54(10): p. 2707-13.
112. Patterson, A.R., et al., Sustained reprogramming of the estrogen response after chronic exposure to endocrine disruptors. Mol Endocrinol, 2015. 29(3): p. 384-95.
113. Li, J.R., et al., Cancer RNA-Seq Nexus: a database of phenotype-specific transcriptome profiling in cancer cells. Nucleic Acids Res, 2016. 44(D1): p. D944- 51.
114. Varley, K.E., et al., Recurrent read-through fusion transcripts in breast cancer. Breast Cancer Res Treat, 2014. 146(2): p. 287-97.
115. Bauerschlag, D.O., et al., Progression-free survival in ovarian cancer is reflected in epigenetic DNA methylation profiles. Oncology, 2011. 80(1-2): p. 12-20.
116. Siegel, R.L., K.D. Miller, and A. Jemal, Cancer statistics, 2018. CA Cancer J Clin, 2018. 68(1): p. 7-30.
117. Ellis, M.J. and C.M. Perou, The genomic landscape of breast cancer as a therapeutic roadmap. Cancer Discov, 2013. 3(1): p. 27-34.
118. Simoncini, T., et al., Interaction of oestrogen receptor with the regulatory subunit of phosphatidylinositol-3-OH kinase. Nature, 2000. 407(6803): p. 538-41.
119. Creighton, C.J., et al., Proteomic and transcriptomic profiling reveals a link between the PI3K pathway and lower estrogen-receptor (ER) levels and activity in ER+ breast cancer. Breast Cancer Res, 2010. 12(3): p. R40.
123
120. Lee, M.S., et al., PI3K/AKT activation induces PTEN ubiquitination and destabilization accelerating tumourigenesis. Nat Commun, 2015. 6: p. 7769.
121. Boehm, J.S., et al., Integrative genomic approaches identify IKBKE as a breast cancer oncogene. Cell, 2007. 129(6): p. 1065-79.
122. Guan, X., et al., Stromal Senescence By Prolonged CDK4/6 Inhibition Potentiates Tumor Growth. Mol Cancer Res, 2017. 15(3): p. 237-249.
123. Debacq-Chainiaux, F., et al., Protocols to detect senescence-associated beta- galactosidase (SA-betagal) activity, a biomarker of senescent cells in culture and in vivo. Nat Protoc, 2009. 4(12): p. 1798-806.
124. Xu, Y., et al., Emerging roles of the p38 MAPK and PI3K/AKT/mTOR pathways in oncogene-induced senescence. Trends Biochem Sci, 2014. 39(6): p. 268-76.
125. Freund, A., C.K. Patil, and J. Campisi, p38MAPK is a novel DNA damage response-independent regulator of the senescence-associated secretory phenotype. EMBO J, 2011. 30(8): p. 1536-48.
126. Courtois-Cox, S., S.L. Jones, and K. Cichowski, Many roads lead to oncogene- induced senescence. Oncogene, 2008. 27(20): p. 2801-9.
127. Du, K. and P.N. Tsichlis, Regulation of the Akt kinase by interacting proteins. Oncogene, 2005. 24(50): p. 7401-9.
128. Bellacosa, A., et al., Akt activation by growth factors is a multiple-step process: the role of the PH domain. Oncogene, 1998. 17(3): p. 313-25.
129. Bellacosa, A., et al., Activation of AKT kinases in cancer: implications for therapeutic targeting. Adv Cancer Res, 2005. 94: p. 29-86.
130. Liu, P., et al., Targeting the phosphoinositide 3-kinase pathway in cancer. Nature reviews. Drug discovery, 2009. 8(8): p. 627-644.
131. Ciruelos Gil, E.M., Targeting the PI3K/AKT/mTOR pathway in estrogen receptor- positive breast cancer. Cancer Treat Rev, 2014. 40(7): p. 862-71.
132. Bosch, A., et al., PI3K inhibition results in enhanced estrogen receptor function and dependence in hormone receptor-positive breast cancer. Sci Transl Med, 2015. 7(283): p. 283ra51.
133. Bhat-Nakshatri, P., et al., AKT alters genome-wide estrogen receptor alpha binding and impacts estrogen signaling in breast cancer. Mol Cell Biol, 2008. 28(24): p. 7487-503.
124
134. Chen, D., et al., Phosphorylation of human estrogen receptor alpha at serine 118 by two distinct signal transduction pathways revealed by phosphorylation-specific antisera. Oncogene, 2002. 21(32): p. 4921-31.
135. Smith, S.E., et al., Molecular characterization of breast cancer cell lines through multiple omic approaches. Breast Cancer Res, 2017. 19(1): p. 65.
136. Jamieson, S., et al., A drug targeting only p110alpha can block phosphoinositide 3-kinase signalling and tumour growth in certain cell types. Biochem J, 2011. 438(1): p. 53-62.
137. Takei, K. and V. Haucke, Clathrin-mediated endocytosis: membrane factors pull the trigger. Trends Cell Biol, 2001. 11(9): p. 385-91.
138. McMahon, H.T. and E. Boucrot, Molecular mechanism and physiological functions of clathrin-mediated endocytosis. Nat Rev Mol Cell Biol, 2011. 12(8): p. 517-33.
139. Le Roy, C. and J.L. Wrana, Clathrin- and non-clathrin-mediated endocytic regulation of cell signalling. Nat Rev Mol Cell Biol, 2005. 6(2): p. 112-26.
140. Scita, G. and P.P. Di Fiore, The endocytic matrix. Nature, 2010. 463(7280): p. 464-73.
141. Grant, B.D. and J.G. Donaldson, Pathways and mechanisms of endocytic recycling. Nat Rev Mol Cell Biol, 2009. 10(9): p. 597-608.
142. Basquin, C., et al., The signalling factor PI3K is a specific regulator of the clathrin-independent dynamin-dependent endocytosis of IL-2 receptors. J Cell Sci, 2013. 126(Pt 5): p. 1099-108.
143. Basquin, C. and N. Sauvonnet, Phosphoinositide 3-kinase at the crossroad between endocytosis and signaling of cytokine receptors. Commun Integr Biol, 2013. 6(4): p. e24243.
144. Lucarelli, S., R.C. Delos Santos, and C.N. Antonescu, Measurement of Epidermal Growth Factor Receptor-Derived Signals Within Plasma Membrane Clathrin Structures. Methods Mol Biol, 2017. 1652: p. 191-225.
145. Branon, T.C., et al., Efficient proximity labeling in living cells and organisms with TurboID. Nat Biotechnol, 2018. 36(9): p. 880-887.
125