This item is only available as the following downloads:
1 THE INTERPLAY OF GENOMIC AND NON GENOMIC ESTROGEN RECEPTOR SIGNALING PATHWAYS IN MEDIATING CELLULAR REPSONSES TO XENOESTROGENS BY LEY CODY SMITH A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULF ILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2013
2 2013 Ley Cody Smith
3 To my family
4 ACKNOWLEDGEMENTS I thank my advisor, Dr. Tara Sabo Attwood, for always being helpful and never he sitating to answer any question Tara is highly motivated, personable, and truly loves to teach. I consider myself very fortunate to have her as a mentor. H er expertise in molecular toxicolog y has been an invaluable resource th roughout the progression of my m It is unusual to find an adviser who is as invested in her which is t he main reason that motivated me to continue my project into a PhD under her direction, and I greatly look forward to the oppo rtunity. I would also like to thank Dr. Nancy Denslow. The evolution of my project toward understanding cellular signaling networks would not have been possible without Dr. tha t Dr. Denslow is o ne of the most caring professor s with whom I have ever had the pleasure of working and I cannot express how satisfying it is to have an adviser so excited about your research project. Dr. Paul Cooke was also an invaluable addition to m y committee due to his extensive knowledge of the field of endocr ine disruption Most importantly Dr. Cooke was always very friendly and approachable for which I am extremely grateful. I would also like to thank Dr. Barber who gave me the opportunity to work in his lab while pursuing my undergraduate degree. The experience motivated my decision to pursue a graduate degree in toxicological sciences. I am grateful for his mentorship in the beginning of my career. Beyond my advisers, I would like to th ank everyone in the Center for Environmental and Human Toxicology at the University of Florida. In particular I want to thank Erica Anderson who taught me everything she knew about molecular biology
5 based scientific techniques while shadowing her in Dr. B I would not be where I am today without the solid foundation in scientific research that she provided. I also want to thank Dr. Noel Takeuchi and Nick Doperalski who were always there to help me and who made working in the Barber lab fun. Further, I want to thank Ca ndice Harley who never hesitated to lend a helping hand and on who m I c ould always rely for good advic e whenever I had a problem. I also want to thank Dr. Joe Bisesi and Dr. Gustavo Dominguez. Without Gustavo, the Sabo Attwood lab would have fallen apart a long time ago. More importantly he was a great friend and was always good for a laugh. Joe has also been an excellent office mate and is also very helpful in the lab I also want to acknowledge Dr. Roxanna Weil, Georgia Hinkley, Joh n Munson, Roxanne Werner, and Kevin Kroll for never hesitating to help me whenever I had a problem. I want to thank Dr. P. Lee Ferguson for running my protein samples on the mass spectrometer and Sharon Norton for training me in how to use the qPCR machine Most importantly, I want to thank my family who has always supported me and I know would be proud of me no matter what I did I thank my dad for always reminding that I can do whatever I want to do as long as I set my mind to it. I thank my mom for alwa ys reminding me to have fun and especially for going out of her way by requesting the Gainesville region for work so that she could visit me every couple of months. The reminder of home was always welcomed and refreshing. I thank my brother for always bei ng there for me. I know it is not because we are twins and he has no choice, but because he really cares. I also thank my sister for always looking up to me and reminding me to try my best so that I can set the best possible example I thank my
6 grandparen ts, Granny and John, for unconditiona lly supporting me and loving me. I feel very fortunate to have such a close relationship with them I also w ant to acknowledge my grandparents who could not be there Nana and Papa. I miss them every day and am sad that they could not be there to see me beg in my career I will always cherish the time we spent together. Beyond those mentioned above, I thank all of my friends I consider myself incredibly fortunate to have met so many amazing people thr oughout high school and college. I hope to continue all of these relationships far into the future.
7 TABLE OF CONTENTS page ACKNOWLEDGEMENTS ................................ ................................ ............................... 4 LIST OF TABLES ................................ ................................ ................................ ............ 9 LIST OF FIGURES ................................ ................................ ................................ ........ 10 LIST OF ABBREVIATIONS ................................ ................................ ........................... 11 ABSTRACT ................................ ................................ ................................ ................... 12 CHAPTER 1 LITERATURE REV IEW ................................ ................................ .......................... 14 Endocrine Disruption ................................ ................................ ............................... 14 G Protein Coupled Estrogen Receptor (G PER ) ................................ ...................... 15 GPER Ligands ................................ ................................ ................................ ........ 16 GPER Mediated Transcriptional Modulation ................................ ........................... 18 Cell Type Specific GPER Mediated Responses ................................ ..................... 18 Dose Dependent GPER Mediated Cellular Proliferation ................................ ........ 19 Potential for Receptor Crosstalk ................................ ................................ ............. 20 GPER and Xenoestrogens ................................ ................................ ...................... 21 Cell Line ................................ ................................ ................................ .................. 21 Research Objectives ................................ ................................ ............................... 22 2 GPER SPECIFIC AGONIST G 1 INHIBITS ESR MEDIATED CELLUAR RESPONSES TO XENOESTROGENS ................................ ................................ .. 24 Introduction ................................ ................................ ................................ ............. 24 Material s and Methods ................................ ................................ ............................ 27 Chemicals ................................ ................................ ................................ ......... 27 Cell Culture ................................ ................................ ................................ ....... 27 Proliferation Assa ys ................................ ................................ .......................... 28 Reporter Gene Activity Assays ................................ ................................ ......... 28 Quantitative Real Time PCR ................................ ................................ ............ 29 Protein Isolation ................................ ................................ ................................ 30 Phosphoproteomic Analysis ................................ ................................ ............. 30 Western Blots for NDRG2 ................................ ................................ ................ 31 Statistical Analysis ................................ ................................ ............................ 31 Results ................................ ................................ ................................ .................... 32 G 1 Suppresses Nuclear ER driven Cell Proliferation ................................ ...... 32 G 1 Inhibits Xenoestrogen Induced Cellular Proliferation ................................ 32 G 1 Suppresses ER Mediated Transcriptional Activation ................................ 33
8 G 1 Mediated Suppression of Xenoestrogen Driven Transcriptional Activation is Dose Dependent ................................ ................................ ....... 34 G 1 Does Not Affect pS2 Gene Expression ................................ ...................... 35 Xenoestrogen Induced pS2 Expression is not Sensitive to G 1 ....................... 35 G 1 and E 2 Exhibit Differential Phosphorylation Patterns ................................ 36 NDRG2 is a Plausible Novel Target of G 1 Mediated Signaling ....................... 37 NDRG2 mRNA Expression is Downregulated by ESR Activation .................... 38 Discussion ................................ ................................ ................................ .............. 38 3 GENERAL CONCLUSIONS ................................ ................................ ................... 60 LIST OF REFERENCES ................................ ................................ ............................... 62 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 69
9 LIST OF TABLES page 2 1 Peptides differentially phosphorylated by G 1 and sorted into bio logical ................................ .............................. 57
10 LIST OF FIGURES page 1 1 Signaling pathways activated by GPER.. ................................ ........................... 23 2 1 Co exposure to 1 M G 1 suppresses nuclear ER driven proliferation of MCF 7 cells. ................................ ................................ ................................ ....... 47 2 2 Co exposure to 1 M G 1 suppresses xenoestrogen induced pr oliferation of MCF 7 cells. ................................ ................................ ................................ ....... 48 2 3 Co exposure to 1 M G 1 suppresses ESR mediated activation at an ERE. .... 49 2 4 GPER mediated s uppression of ERE activation is dose dependent. ................. 50 2 5 pS2 expression is responsive to nuclear ER agonists but not G 1. .................... 51 2 6 X enoestrogen induced pS2 expression is not responsive to G 1. ...................... 52 2 7 Distribution of the number of phosphorylated proteins identified in MCF 7 cells exposed to E 2 or G 1 compared to control cel ls.. ................................ ....... 53 2 8 Phosphorylated peptides organized by biological process. ............................... 54 2 9 Pathway showing relationship between proteins phosp horylated specifically by G 1 in MCF 7 cells. ................................ ................................ ..................... 55 2 10 Phosphorylation of NDRG2.. ................................ ................................ .............. 55 2.11 Nuclear ERs suppress the expression of NDRG2 mRNA. ................................ .. 56
11 LIST OF ABBREVIATIONS BPA Bisphenol A DPN ESR2 Specific Agonist E 2 Endogenous Estradiol ERE Estrogen Response Element ESR Nuclear Estrogen Receptor s G 1 GPER Specific Agonist G 15 GPER Specific Antagonist GPER G Protein Coupled Estrogen Receptor MCF 7 ESR positive, GPER positive human breast cancer cell line NDRG2 N Myc Downstream Regulated Gene 2 PPT ESR1 Specific Agonist
12 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for Master of Science THE INTERPLAY OF GENO MIC AND NON GENOMIC ESTROGEN RECEPTOR SIGNALING PATHWAYS IN MEDIATING CELLULAR RESPONSES TO XENOESTROGENS By Ley Cody Smith May 2013 Chair: Tara Sabo Attwood Major: Veterinary Medical Sciences Estrogen can exert cellular effects through both nuclear ( ESR1 and ESR2) and membrane bound receptors (GPER). It is unclear if these receptors act independently role in proliferation, activation of reporter genes and protein ph osphorylation in breast cancer cells (MCF 7), we employed selective agonists for ESR1 (PPT), ESR2 (DPN) and GPER (G 1). We also determined the impact of the xenoest rogens BPA and genistein estradiol (E 2 ), PPT, DPN, BPA, and genistein each independently enhanced cell proliferation and activation of a n estrogen response element (ERE) driven reporter gene when compared to control cells, whereas G 1 had no significant impact. However, G 1 inhibited E 2 PPT DPN BPA, and genistein induced proliferation and ERE activation at doses greater than 500 nM. Furthermore, inhibition of ESR activity was not receptor isotype specific and preliminary studies using the GPER antagonist (G 15) suggests these observations are GPER independent. As membrane receptors initiate a cascade of phosphorylation even ts, we performed a global phospho proteom i c analysis on cells exposed to E 2 or G 1. Of the
13 238 phosphorylated p roteins identified only 10 sites were similarly phosphorylated between E 2 and G 1 whereas 31 and 13 sites were specifically modified by each ligand, phosphorylated by G 1 as pu tative proteins that modulate the inhibition of E 2 and xenoestrogen mediated ESR activity and cell proliferation. Taken together, these results reveal a novel role for G 1 in interfering with nuclear receptor activity driven by E 2 and xenoestrogens.
14 CH APTER 1 LITERATURE REVIEW Endocrine Disruption Endocrine disruptors are environmental contaminants that can interfere with normal hormonal signaling by disrupting the tightly regulated synthesis, structure or function of hormones and contribute to adverse effects in human and wildlife population s ( Colborn et al. 1993 ; Kavlock et al. 1996 ) The idea that exogenous chemicals could bind to hormone receptors and cause adverse effects was motivated by the observation of impaired reproductive performance in sheep grazing on red clover pastures in Australia ( Morley et al. 1964 ) This concept was translated to humans when it was noticed that a rare form of vaginal cancer occurred in daughters of women who had been administered a synthetic estrogen, Diethylstilbestrol (DES ), during pregnancy to prevent miscarriages ( Herbst et al. 1970 ; Herbst et al. 1999 ) The unfortunate consequences of DES exposure an d subsequent research solidified the both naturally occurring and synthetic chemical s may disrupt the normal functions of the endocrine system and its hormones in organisms ( Patisaul and Adewale, 2009 ) Since then, the most widely studied endocrine disruptors are those that interfere with signaling by the endogenous estrogen, e stradiol (E 2 ) which is necessitat ed by the drastic increase in number of chemicals identified with estrogenic activity in the past few decade s ( Thomas and Dong, 2006 ) These x enoestrogens can be naturally occurring, such as the soy based phytoestrogens (i.e. g enistein ), or are anthropogenic and encompass a diverse array of chemicals that are ubiquitously used in pesticides, plastics, and pharmaceuticals. Many of these compounds are suspect ed to contribute to a myriad of disorders including
15 cardiovascular disease, hypertension, metabolic diseases, immune di sorders, and reproductive cancers ( Deroo and Korach, 2006 ) The classical mechanism of action of xenoestrogens is well established to be mediated by ligand activated transcription fa ctors known as the nuclear estrogen receptors (ESR1 and ESR2). Once engaged by ligand the ESRs adopt a conformational change that promotes dimerization and recruitment of various co regulatory proteins to the transcriptional complex ( McKenna and O'Malley, 2001 ) This ESR receptosome then interacts with cis acting estrogen response elements in the promoter s of estrogen responsive genes by either direct DNA binding to classical estrogen response elements (ERE) or through alternate elements (AP 1, Sp 1) via tethering mechanisms thereby initiating aberrant gene expression ( Hall et al. 2001 ) GPER Recently, a putative membrane bound estrogen receptor G protein coupled estrogen receptor (GPER) has been implicated in mediating the rapid, non genomic effects of E 2 by influencing cell adhesion, migration, survival, and proliferation ( Revankar et al. 2005c ) The GPER is a member of the seven transmembrane domain G protein coupled receptor (GPCR) superfamily which represents the largest class of signaling molecules in the human genome ( Venter et al. 2001 ) Studies to date support a number of theories regarding a functional role for GPER and not all studies are in agreement. For example, the subcellular location of GPER is debated as multiple studies have indicated expression at eit her the plasma membrane or endoplasmic reticulum ( Revankar et al. 2005b ; Thomas et al. 2005a ; Filardo et al. 2007 ) and still others suggest the GPER cycles between these two cellular compartments ( Cheng et al. 2011 ) Initially, GPER was linked to E 2 signaling when it was found to be required for
16 E 2 induced rapid activation of t he mitogen activated protein kinase (MAPK), extracellular signal regulated kinase 1 and 2 (ERK 1/ 2, p42/44) ( Filardo et al. 2000 ) Activation of ERK 1/ subunit protein complex dependent pertussis toxin sensitive signaling mechanism that requires both Src and Shc ( Filardo et al. 2000 ) which supports a G PCR initiated mechanism opposed to a nuclear E S R dependent mechanism that was suggested by previous studies ( Improta Brears et al. 1999 ; Pedram et al. 2006 ; Madak Erdogan et al. 2008 ) Since this initial observation, a number of studies collectively support a proposed GPER mediated signaling pathw ay that is depicted in Figure 1.1. This diagram shows that once activated by various ligands (E 2 xenoestrogens, t amoxifen G 1), GPER phosphorylates Shc which results in matrix metalloproteinase (MMP) mediated cleavage and release of heparin bound e pider mal g rowth f actor (proHB EGF) from the cell surface EGF transactiva tes the e pidermal g rowth f actor r eceptor (EGFR ) which ultimately leads to activation of MAPK ( Filardo et al. 2000 ) and phosph oinositide 3 kinase (PI3K) ( Revankar et al. 2005b ; Ariazi et al. 2010 ) which play a role in influencing cell g rowth through modulation of downstream genes (c fos, cyclins) Nuclear ESR s may also feed into this pathway. Aside from this proposed model E 2 activated GPER stimulates adenyl cyclase and intracellular cAMP production to promote the activation of Protei n Kinase A (PKA) and subsequent suppression of EGF induced ERK 1/ 2. This forms a regulatory loop that returns GPER induced signaling back to baseline levels ( Filardo et al. 2002 ) GPER Ligands Significant overlap exists in the ligand specificity of the ESRs and GPER as multiple compounds in addition to the natural ligand E 2 such as the ESR antagonists ICI 182,780 and tamoxifen (TAM) also display binding affinities for GPER ( Hall et al.
17 2001 ; Korach et al. 2003 ; Revankar et al. 2005c ) These ESR antagonists surprisingly act as GPER agonists suggesting crosstalk may occu r between nuclear and membrane receptor pathways that may even lead to opposing control of cellular effects. E fforts to investigate the consequences of select activation of the GPER have been made possible with the development of a specific receptor agon ist. Through a combination of virtual and bioactivity screenings aimed at sifting through vast numbers of molecules with similar structure to E 2 a substituted dihydroquinoline was discovered to competitively inhibit binding of E 2 to GPER and is known as G 1 ( Bologa et al. 2006 ) Binding assays yielded high affinity for GPER ( K i of 11 nM) compared to E 2 ( K i of 5.7 6.6 nM ) whereas negligible bind ing of G 1 to the ESRs up to 1 0 uM was observed ( Bologa et al. 2006 ) Experiments to assay the intracellular effects of G 1 revealed that GPER activation by this compound caused increases in intrac ellular calcium mobilization, accumulation of PIP3 indicative of PI3K activation and cell migration which were all consistent with E 2 induced GPER activation ( Bologa et al. 2006 ) thereby satisf ying the requirement for a high affinity, selective GPER agonist. The same method was then utilized to search for a GPER selective antagonist. Results revealed a compound related in structure to G 1 tha t binds GPER and prevents both E 2 and G 1 mediated mobilization of intracellular calcium and PI3K activation in breast cancer cells lacking ESRs ( Dennis et al. 2009 ) In murine epithelial uterine cells G 15 was able to block E 2 induced proliferation ( Dennis et al. 2009 ) and has also been shown to block G 1 induced ERK 1/ 2 activation and E SR1 phosphorylation at serine 118 ( Lucki and Sewer, 2011 ) However, these results are complicated by the ability of G 15 to weakly bind and activate ESRs at high doses (1 0 M) that were employed in
18 these studies ( Dennis et al. 2011 ) Overall, d ev elopment of these GPER specific ligands can be valuable tools for answering questions aimed at teasing apart non genomic and genomic E 2 and xenoestrogen mediated cellular responses. GPER Mediated Transcriptional Modulation Before the identification of sel ective ligands, the mechanisms by which GPER modulates kinase activity had been documented but little was known about the downstream transcriptional effects as a consequence of activation. A few recent studies that have employed the selective agonist G 1 and a series of select kinase inhibitors show GPER up regulate s oncogene, c fos in an EGFR dependent manner via rapid ERK 1/ 2 phosphorylation in breast cancer cell lines. Studies using a series of ESR positive (MCF 7) and ESR negative (SKBR3) cell lines confirmed this effect was GPER mediated ( Maggiolini e t al. 2004 ) In addition, GPER has been found to up regulate expression of connective tissue growth factor (CTGF) and Bcl 2 by E 2 ( Kanda and Watanabe, 2003 ; Pandey et al. 2009 ) Othe r genes that are down regulated by G 1 include those involved in regulating the cell cycle (c yclins, p21) ( Lubig et al. 2012 ) Cell Type Specific GPER Mediated Responses Studi es to elucidate the cellular impacts of GPER signaling pathways have yielded complex and in some cases, controversial results It has been proposed that GPER activation leading to proliferative effects depends on the cell type and corresponding ESR expre ssion profile. For example, in SKBR3 cells which express GPER but not ESRs, exposure to either 100 nM E 2 or 100 nM G 1 increase d proliferation which suggests that GPER signaling is sufficient to stimulate cell growth independent of the ESRs ( Albanito et al. 2007 ; Albanito et al. 2008 ) The increased
19 proliferation in SKBR3 cells coincides with up regulation the CTGF gene suggesting that modulation of this mRNA may influence proliferation in the absence of ESRs ( Pandey et al. 2009 ) Likewise, GPER knockdown inhibits the E 2 induced proliferative effects in SKRB3 cells. However, other studies have indicated that GPER activation by G 1 (100 n M 1 ( Lubig et al. 2012 ) Further, GPER knockdown potentiates E 2 induced proliferation of breast cancer cells that express GPER and ESRs (MCF 7) suggesting an inhibitory role ( Ariazi et al. 2010 ) These results collectively indicate that the ESR expression profile may influence the pro or anti proliferative properties of GPER activation and suggest a compensatory mechanism may exist between the ESRs and GPER. Dose Depend ent GPER Mediated Cellular Proliferation The proliferative effects of GPER signaling are also dependent on dose of ligand. Low dose (10 nM) exposure to G 1 has been shown to stimulate proliferation of MCF 7 cells through up regulation of acid ceramidase ( ASAH1) ( Lucki and Sewer, 2011 ) while a high dose (1 M) inhibits E 2 induced proliferation of MCF 7 cells ( Ariazi et al. 2010 ) Collectively, these results indicate that activation of GPER with high doses of G 1 has an inhibitory effect on proliferation whereas low dose exposure has a stimula tory effect. Attempts to explain this phenomenon have yielded a current hypothesis that suggests G 1 signals through a pathway independent of the GPER at high doses. This notion is supported by the failure of pretreatment of cells with G 15 to rescue the effects of G 1 at high doses, namely inhibition of bre ast cancer cell growth ( Wang et al. 2012 ) Furthermore, knockdown of GPER using siRNA strategies also failed to rescue G 1 mediated effects. Finally, it has been suggested that the membrane bound ER splice
20 36 may mediate the rapid, non genomic effects of E 2 and G 1 at high doses ( Kang et al. 2010 ) Potential for Receptor Crosstalk GPER and ESRs may participate in crosstalk at some level as both receptors regulate distinct and overlapping transcriptional profiles ( Notas et al. 201 1 ) As previously mentioned, many cellular responses to E 2 and the GPER selective agonist, G 1, are dependent on the ESR (1/2) expression profile which suggests the presence of certain receptor types influences the downstream responses that are cell typ e specific As evidence for convergent signaling pathways between ESR and GPER E 2 and TAM induced activation of ERK 1/ 2 via GPER enhances E SR1 transcriptional activity by phosphorylating serine 118 within the transcriptional activation function I (AF I ) domain ( Kato et al. 1995 ) In addition, both the ESR 1 and GPER are able to stimulate PI3K which results in the accumulation of PIP3 ( Revankar et al. 2005b ) It has also been suggested that extranuclear and n uclear signaling inputs converge on similar target genes For example, GPER and ESR knockdown experiments revealed a requirement for both receptors in maximal induction of the c fos gene This study concluded that both ESRs and GPER signal through overlap ping signal transduction pathway s in ovarian cancer cell s ( Albanito et al. 2007 ) In support of divergent signaling pathways, knockdown of GP ER in breast cancer cells lacking ESRs is sufficient to block the E 2 and G 1 induced growth stimulation and c fos induction suggesting ESR s are not required for these effects ( Albanito et al. 2007 ) Further, G 1 had no effect on the expression of the ESR target gene, progesterone rece ptor ( Albanito et al. 2007 ) Most striking however, is the ability of the ESR antagonis t ICI 182,780 and partial antagonist TAM to act as GPER agonists
21 and activate ERK 1/ 2 by GPER mediated transactivation of EGFR ( Filardo et al. 2000 ; Revankar et al. 2005b ) GPER and Xenoestrogens The GPER represents a potential target for interference by endocrine disrupting chemicals as it has been shown that se veral xenoestrogens are capable of binding to this receptor with low affinity up regulating adenyl cyclase activity and consequently increasing intracellular levels of cAMP ( Thomas and Dong, 2006 ) The soy based phytoestrogen g enistein and the plasticizer b isphenol A (BP A) bind to GPER with relative binding affinities of 13.41 % and 2.83 % compared to E 2 (100%) respectively ( Thomas and Dong, 2006 ) and have both been shown to activate ERK 1/ 2 signaling via a GPER dependent mechanism ( Maggiolini et al. 2004 ; Dong et al. 2011 ) Exposure to BPA has been linked to impaired reproductive performance ( et al. 2005 ; Sugiura Ogasawara et al. 2005 ; Rasier et al. 2006 ) and Genistein exposure has been shown to promote proliferation of estrogen dependent breast and thyroid cancer cells ( Allred et al. 2001 ; Vivacqua et al. 2006 ) which necessitates the need to elucidate the complex molecular mechanisms of action of EDCs. Cell Line MCF 7 human breast cancer cells are an ideal model to study the interaction between the ESRs and the GPER as they are known to express each r eceptor (ESR1, ESR2, GPER) and are very well characterized with respect to E 2 action. Further, the effects of Genistein and BPA exposure on MCF 7 proliferation are well known thus rendering them an appropriate system in which to study the effect of GPER and ESR interplay on established cellular endpoints in response to xenoestrogens ( Hsieh et al. 1998 ; Pupo et al. 2012 )
22 Research Objectives The interplay of GPER and ESR activation and its estrogen/xenoestrogen dependence in controlling downstream c ellular response s is currently unknown. Studies aimed at elucidating the mechanisms of xenoestrogen action have been primarily limited to probes of their interaction with ESRs. Current knowledge suggests that the GPER may represent an alternate and highly relevant (e.g. fast and sensitive) mechanism by which environmental contaminants exert their effects on biological systems. Based on these notions the objectives of this research are two fold; (1) to elucidate the impact of low ver sus high doses of G 1 activated GPER on ESR mediated function and cellular responses ; (2) to identify downstream targets phosphorylated by G 1 as a means to uncover novel pathways modulated both dependently and independently of GPER and potentially xenoestrogens. To perform these studies we employ ed a series of cell based assays coupled with phosphoproteome analysis. Overall, these studies provide a groundwork for elucidating novel mechanisms of xenoestrogen actions.
23 Figure 1 1. Signaling p athways a ctivated by GPER. E 2 xenoestrogens, and G 1 are able to bind to GPER, shown here localized on the endoplasmic reticulum, and transactivate EGFR through matrix metalloproteinase mediated cleavage and release of proHP EGF. EGFR activation leads to multiple downstream signaling e vents including activation of MAPKs and PI3K which results in the expression of transcription factors ( c fos ) which ultimate leads to cell cycle progression. Adapted from Prossnitz, E. R. and M. Maggiolin i (2009). Molecular and cellular endocrinololgy 308 ( 1): 32 38.
24 CHAPTER 2 GPER SPECIFIC AGONIST G 1 INHIBITS E S R MEDIATED CEL LUAR RESPONSES TO XENOESTROGENS Introduction Endocrine disruptors are environmental contaminants that can interfere with normal hormonal signaling by either disrupting the tightly r egulated synthesis, structure or function of hormones thereby causing adverse effects in human and wildlife population s ( Colborn et al. 1993 ; Kavlock et al. 1996 ) The most widely studied endocrine disruptors are those that interfere with signaling by the endogenous estrogen, e stradiol (E 2 ) which is necessitated by the drastic increase in number of chemicals identified with estrogenic activity in the past decade ( Thomas and Dong, 2006 ) Xenoestrogens can be naturally occurring, such as the soy based phytoestrogens (i. e. Genistein ), or they can be anthropogenic and include a diverse array of chemicals that are ubiquitously used in pesticides, plastics, and pharmaceuticals. Many of these compounds are suspect ed to contribute to a myriad of disorders including reproductiv e abnormalities, cardiovascular disease, hypertension, metabolic diseases, immune disorders, and reproductive cancers ( Deroo and Korach, 2006 ) A classical mode of action of xenoestr ogens is well established to be mediated by ligand activated transcription factors known as the nuclear estrogen receptors (ESR1 and ESR2). Once engaged by ligand the ESRs adopt a conformational change that promotes dimerization and recruitment of variou s co regulatory proteins to the transcriptional complex ( McKenna and O'Malley, 2001 ) This ESR recepto some then interacts with cis acting estrogen response elements in the promoters of estrogen responsive genes by either direct DNA binding to classical estrogen response elements
25 (ERE) or through alternate elements (AP 1, Sp 1) via tethering mechanisms the reby initiating aberrant gene expression ( Hall et al. 2001 ) Xenoestrogens such as bisphenol A ( BPA ) and Genistein have been shown to weakly bind and modulate ESR activity. The consequences of these actions are vast and are cell and tissue type specific. In breast cancer cells (MCF 7) activation of ESRs by E 2 and xenoestrogens is a major driver of proliferation. Recently, a putative membrane bound estrogen receptor, G protein coupled estrogen receptor (GPER), has been implicated in mediating the rapid, non genomic effects of E 2 ( Revankar et al. 2005c ) The GPER is a member of the seven transmembrane domain, G protein coupled receptor superfamily and is required for E 2 induced rapid res ponses such as activation of ERK 1/ 2, mobilization of intracellular calcium stores, and stimulation of intracellular cAMP production ( Revankar et al. 2005b ; Prossnitz et al. 2008 ) The role of GPER in mediating cell responses to E 2 is a source of controversy as the subcellular location has been extensively debated and has been suggested to signal from both the endoplasmic reticulum and plasma membr anes ( Revankar et al. 2005c ; Thomas et al. 2005a ; Filardo e t al. 2007 ) while other studies have indicated that it shuttles between these two compartments ( Cheng et al. 2011 ) The fact that xenoestrogens have been shown to have weak affinity for GPER suggest s modulation of this receptor may represent a potential novel mechanism wh ereby EDCs exert biological effects. Evaluating ESR and GPER specific actions is complicated due to significant overlap in ligand specificity. Multiple well established ESR ligands, such as E 2 ICI 182,780 and tamoxifen (TAM) display similar binding affin ities ( Hall et al. 2001 ; Korach
26 et al. 2003 ; Revankar et al. 2 005c ) for both receptors, the latter compounds act ing as antagonists for ESR while agonizing the GPER. This has led to the idea that ESR and GPER likely engage in crosstalk, and may even modulate compensatory or opposing cell signaling pathways as it ha s been shown that GPER is able to mediate proliferative effects of E 2 in breast cancer cells lacking ESRs ( Pandey et al. 2009 ) Sorting cellular signaling networks specifically modulated by GPER and ESRs required identification of GPER specific agonists and antagonists. A combination of virtual and bioactivity screenings revealed a substituted dihydroquinoline termed G 1, that is a selective GPER agonist ( Bologa et al. 2006 ) Availability of this ligand has allow ed for studies aimed at investigating a GPER specific role in non genomic biological responses to E 2 However, a number of studies that have employed G 1, primarily in cell based experiments, have produced mixed resu lts For example, in assessing proliferation, some studies show G 1 enhances cell growth while others find it inhibits proliferation in breast cancer cells both expressing and lacking ESRs ( Albanito et al. 2007 ; Albanito et al. 2008 ; Ariazi et al. 2010 ; Lucki and Se wer, 2011 ; Lubig et al. 2012 ) Interestingly, it has been suggested that G 1 may signal independently of GPER at high doses ( Wang et al. 2012 ) Based on the current body of knowledge, the goals of this research we re (1) to determine the impact of GPER activation on breast cancer cell growth and ESR activity in response to E 2 and the xenoestrogens BPA and Genisten and (2) to identify novel pathways modulated by E 2 and G 1 using a global phosphoproteomic approach. While we originally set out to look at GPER activation by G 1 and xenoestrogens, our results defined a role for G 1 in suppressing E 2 and xenoestrogen driven cell ular proliferation
27 and ESR activity. We further show that this inhibition of ESRs i s not isot ype specific and likely occurs independently of GPER. Finally, through phosphoproteomic s analysis we present plausible protein targets, such as NDRG2, that are selectively phosphorylated by G 1 and may contribute to the observed inhibition of cell growth and ESR activity. These results are significant in that they suggest that high dose s of G 1 may act through pathways independent of GPER to modulate ESR driven cell growth by E 2 and the xenoestrogens BPA and Genistein Materials and Methods Chemicals All chemicals used in the studies were dissolved in DMSO and included ; estradiol (Sigma A ldric h, St. Louis, MO ), bisphenol A (BPA, supplied by NIEHS), Genistein (Acros Organics Morris Plains, NJ ) ( propyl pyrazole triol ( PPT Tocris Ellisville, MO ), diarylpropionitrile (DPN, Tocris) () 1 [(3a R *,4 S *,9b S *) 4 (6 Bromo 1,3 benzodioxol 5 yl) 3a,4,5,9b tetrahydro 3 H cyclopenta[ c ]quinolin 8 yl] eth anone ( G 1 Santa Cruz Biotechnology Santa Cruz, CA ), and (3a S *,4 R *,9b R *) 4 (6 Bromo 1,3 benzodioxol 5 yl) 3a,4,5,9b 3 H cyclopenta[ c ] quinolone ( G 15 Tocris). Cell Culture Human breast cancer cells (MCF 7, purchased from American Type Tissue Collection) w ere cultured in Minimum Essential Medium (MEM) without phenol red (Cellgro 17 305 CV, Manassas, VA) supplemented with 10% heat inactivated fetal bovine serum (FBS), 2 mM L glutamine, Penstrep, 0.1 mM non essential amino acids, 1 mM sodium pyruvate, and 1.5 g/L sodium bicarbonate. Cells were maintained at 37C in humidified 5% CO 2 atmosphere. All exposures were performed in phenol red free MEM with 10% charcoal stripped FBS and the supplements listed above.
28 Proliferation Assays MCF 7 cells were seeded at a concentration of 4 X 10 3 cells per well of a 96 well Tissue Culture Treated M icroplate (Costar Corning Incorporated) in 100ul cell culture medium. The cells were allowed to adhere overnight then were switched to cell culture medium containing 10% charco a l stripped FBS for 24 hours. Cells were then exposed vehicle control for 24, 48, 72 and 96 hours. Proliferation was measured by modified MTT assay (Promega Corp., WI, USA). Br iefly, proliferation was measured every 24 hours by incubating cells in 15 l Dye Solution for 4 hours followed by addition of 100L Solubilization/Stop buffer. After 1 hour, absorbance measurements (570 nm) were acquired on a BioTek Synergy H1 plate reade r. Exposure media was changed every 48 hours throughout the time course. Reporter Gene Activity Assays MCF 7 cells were seeded at a concentration of 2 X 10 5 cells per well of a 24 well T issue C ulture T reated P lates (Multiwell TM Falcon) in 1.0 ml of compl ete cell culture medium. The cells were allowed to adhere overnight then were switched to 10% charcoal stripped FBS containing medium without Penstrep. Cells were transiently transfected with 500 ng reporter plasmid containing a 2X estrogen response elem ent (ERE) upstream of the luciferase gene and 50 ng Renilla pRL TK using Lipofectamine 2000 (Invitrogen). After 24 hours, cells were exposed to chemicals dissolved in DMSO for 24 hours. Cells were washed with 500 l 1X Phosphate Buffered Saline without M agnesium and Calcium (Corning cellgro, Manassas, VA) and collected in 100 l 1X Passive Lysis Buffer (Promega). To facilitate lysis, cells were rocked on a shaking platform at 50 rpm for 20 minutes at room temperature. Cell lysates were collected and
29 pla ced in 1.5 ml microcentrifuge tubes and centrifuged at 12,000 x g at 4C. Thereafter, luciferase activity was measured using the Dual Luciferase Reporter Kit (Promega Corp., WI, USA) on a BioTek Synergy H1 plate reader. To each well of a 96 well LUMITRAC 200 white immunology plate (USA Scientific), 20 l of each lysate was added followed by 50 l of the Firefly Luciferase Reagent. The activity in each well was read, followed by the addition of 50 l of Stop & Glo Substrate. Firefly luciferase luminescence was normalized to Renilla luminescence and reported as either fold change over control or p ercent maximal response. Quantitative Real Time PCR MCF 7 cells were seeded at a concentration of 2 X 10 5 cells per well of a 24 well T issue C ulture T reated P late ( Multiwell TM Falcon) in 1.0 ml of complete cell culture medium. The cells were allowed to adhere overnight then were switched to complete cell culture medium containing 10% charcoal stripped FBS. After 24 hours, cells were exposed to chemicals dissolved in DMSO or vehicle control for 24 hours. Cells were with the optional on column DNase treatment (Qiagen). Thereafter, total RNA was quantified on a Biotek Syngery H1 plate reader and 1 g was reverse transcribed per Promega). TaqMan probes specific to TFF1/pS2 (Hs00170216_m1), NDRG2 (Hs01045114_g1), FOS (Hs04194186_s1), and the housekeeping gen e, GAPDH (Hs02758991_g1) were purchased from Applied Biosystems (Carlsbad, CA). Amplification was performed in duplicate in TaqMan Universal PCR Master Mix (Applied Biosystems) using the standard run time on the 7500 Fast Real Time PCR System
30 (Applied Bio systems). Target gene expression was normalized to GAPDH and relative quantitation was calculated using the Delta Delta CT method. Protein Isolation MCF 7 cells were seeded at a concentration of 3 X 10 5 cells per ml on 60 mm suspension culture plates (Mult iwell TM Falcon) coated with Bactogelatin and in 5 ml complete phenol red free MEM. The cells were allowed to adhere overnight then switched to MEM with 10% charcoal stripped FBS. After 24 hours, cells were exposed to chemicals dissolved in DMSO or vehic le control for 30 minutes. Cells were then washed three times with 2 ml ice cold PBS and harvested in 100 ul ice cold whole cell lysis buffer (20 mM Tris pH 7.6, 1% Triton X 100, 137 mM NaCl, 0.1 mM EDTA, 1X EDTA Free Protease Inhibitor Tablet (Pierce 88 661)). Lysates were passed through a 26 gauge needle and incubated on ice for 30 minutes, then centrifuged at 14,000 g for 15 min at 4C. Supernatant was collected and quantified by the Bradford Protein Assay (BioRad). For Alkaline Phosphatase treatment 80 g protein was incubated with 80 units (1 unit/ g protein) of Calf Intestinal Alkaline Phosphatase (Promega) at 37C for 2 hours. Phosphoproteomic Analysis MCF 7 cells were exposed to 10 nM E 2 1 M G 1 or vehicle control for 30 minutes and total prot ein was isolated from MCF 7 cells as previously described. Protein phosphorylation was assessed using a quantitative, label free approach at Duke University within the Proteomics Core Facility ( Soderblom et al. 2011 ) Briefly, protein extracts were digested with trypsin and subjected t o phospho peptide enrichment using TiO 2 packed spin columns. After elution, phospho peptides were identified using DIA MS E HPLC MS/MS with the addition of phosphorylation as a considered modification on
31 serine, tyrosine, and threonine residues. Proteins were identified using MASCOT, and phosphorylation sites were mapped via a combination of automated database searching and de novo (manual) sequencing. Analysis of the phosphoproteome was performed using Scaffold ( Searl e, 2010 ) and pathway analysis was performed using Pathway Studio Version 9.0 (Elsevier). Western Blots for NDRG2 Total protein was isolated as previously described and 80 g protein was diluted in Laemm li Sample buffer and separated on 16.5% acrylamide Mini PROTEAN Tris Tricine Precast Gels (BioRad). Protein was transferred to Nitrobind Cast 0.45 micron Pure Nitrocellulose membrane (GE Water & Process Technologies) at 35 volts overnight at 4C in Towbin buffer ( 25 mM Tris, 192 mM glycine, 20% (v/v) met hanol (pH 8.3) ) using a wet transfer apparatus (BioRad). Thereafter, the blot was blocked for one hour in 5% Blotto and then probed with rabbit antibody specific to human NDRG2 (Novus Biologicals, Littleton, CO). The blot was probed overnight with primar y antibody at 4C at a dilution of 1:300 in Tris buffered saline with Tween 20 (pH 7.6) After washing, the blot was incubated with the secondary antibody, ECL TM anti rabbit IgG, Horseradish Peroxidase Linked (GE Healthcare, UK) at a dilution of 1:5000 fo r 1 hour at room temperature then incubated with NOVEX ECL HRP Chemiluminescent Substrate Reagent kit (Invitrogen, Carlsbad, CA) for 1 minute at room temperature. The blot was imaged using the ChemiDoc XRS (BioRad). Statistical Analysis Data obtained fr om the gene reporter and qRT PCR assays were analyzed by one hoc multiple comparisons to
32 determine statistical differences between treatments. Differences were considered significant with a p value < 0.05. Res ults G 1 Suppresses Nuclear ER driven Cell Proliferation To explore the effect of GPER activation by the selective agonist, G 1, ( Bologa et al. 20 06 ) on the proliferation of breast cancer cells we utilized a modified MMT assay MCF 7 cells were exposed to 10 nM E 2 alone and in combination with 1 M G 1 and proliferation was measured every 24 hours for either 72 or 96 hours total. As expected, E 2 increased cellular proliferation compared to the untreated control group over a peri od of 96 hours (Figure 2.1 ). Interestingly, exposure to 10 nM E 2 in the presence of 1 M G 1 abolished proliferation which was undistinguishable from the control cells Of note, individual exposures to low (10 nM) and high (1 M) doses of G 1 individually had no effect on proliferation. In order to determine the ER isoform specificity of the inhibitory effect of G 1 on MCF 7 proliferation, cells were exposed to 100 nM PPT ( ESR1 specific agonist), or 100 nM DPN (ESR2 specific agonist) individually and in combination with 1 M G 1. While exposure of MCF 7 cells to both 100 nM PPT and 100nM DPN increased proliferation over the untreated control group, co exposure to 1 M G 1 decreased proliferation to the level of control cells for both treatments (Figure 2.1). G 1 Inhibits Xenoestrogen Induced Cellular Proliferation To determine whether G 1 could also suppress xenoestrogen driven cellular proliferation, MCF 7 cells were exposed to 10 M BPA or 1 M Genistein individually and in combination with a low or high dose of G 1 (10 nM or 1 M, respectively). Cell proliferation was assessed by mo dified MTT assay every 24 hours for 96 hours. In
33 agreement with previous reports ( Hsieh et al. 1998 ; Schafer et al. 1999 ; Nakaya et al. 2007 ) exposure to BPA and Genistein increased proliferation of MCF 7 cells compared to the untreated control group over the course of 96 hours (Figure 2.2). Similar to results obtained with E 2 co exposure with the high dose of G 1 (1 M) decreased BPA and Genistein driven proliferation to the level of the untreated control group over the course of 96 hours (Figure 2.2). This inhibition was not observed when the low dose (10 nM) of G 1 was used. G 1 Suppresses ER Mediated Transcriptional Activation To further examine a plausible mechanism for the G 1 mediated inhibition of E 2 driven cell proliferation, we examined the ability of G 1 to modulate E S R activity by utilizing a 2X ERE driven luciferase reporter gene assay. In addition to E 2 E S R isoform specific agonists (PPT, DPN) were used individually and in combination with G 1 to also examine effects on ESR isoform specific activity We also employed the GPER a nta gonist G 15 i n these studies. MCF 7 cells were transfected with a 2XERE Luciferase reporter gene and exposed to E 2 or the ESR isoform specific agonists individually and in combination with 1 M G 1 for 24 hours. Data from these experiments revealed that individual exp osures to 10 nM E 2 100 nM DPN and 100 nM PPT significantly increased ERE activation 12, 10 and 14 fold, respectively, over the untreated control group (Figure 2. 3 ). These results indicated that both ESRs activated transcription via 2XERE interaction in o ur model cell line. E xposing MCF 7 cells to 1 M G 1 did not have any effect on ERE activation. In agreement with results from the proliferation assays, exposure to 10 nM E 2 100 nM DPN or 100 nM PPT in combination with 1 M G 1 significantly decreased E RE activation compared to individual exposures with 10 nM E 2 100 nM DPN and 100 nM PPT (Figure 2. 3 ). Surprisingly attempts to
34 rescue this inhibition by G 1 in E 2 and DPN exposed cells by pretreating cells with 20 M G 15 were unsuccessful G 1 Mediated Suppression of Xenoestrogen Driven Transcriptional Activation is Dose Dependent After establishing that a high dose of G 1 inhibited ESR mediated transcriptional activation at an ERE, we next sought to determine whether the inhibition was dose dependent. For these experiments, MCF 7 cells were transfected with a 2XERE Luciferase reporter gene and exposed to 10 nM E 2 individually and in combination with increasing concentrations of G 1 (10 nM, 100 nM, 500 nM or 1 M) for 24 hours. Results revealed a dose d ependent decrease in ERE activation with a downward trend in ERE activation starting at 500 nM G 1 that resulted in a significant reduction ( 60% ) for the highest dose of G 1 tested (1 M) compared to cells exposed only to 10 nM E 2 (Figure 2. 4 ). Because 1 M G 1 also inhibited xenoestrogen induced proliferation, we next examined the potential inhibitory effects of low and high doses of G 1 on xenoestrogen induced ERE activat ion. For these experiments, EC 50 values for BPA and Genistein were calculated based on a dose response assessment of ERE activity using the pharmacology function in SigmaPlot (Systat Software, San Jose, CA). The calculated EC 50 values for BPA and Genistein were 640 nM and 284 nM, respectively. Transfected MCF 7 cells were exposed to th ese concentrations individually and in combination with increasing concentrations of G 1 (10 nM, 100 nM, 500 nM, or 1 M) for 24 hours. Similar to results observed with E 2 low doses of G 1 (10 nM 100 nM) had no effect on Genistein induced ERE activatio n whereas co exposure to 500 nM G 1 and 1 M G 1 resulted in a 40% reduction in ERE activation compared to cel ls exposed to Genistein individually (Figure 2. 4 ). Contrary to results observed in the E 2 and
35 Genistein co exposure experiments, a downward trend in BPA (640 nM) induced activation began at doses as low as 10 nM G 1 with the greatest reduction of BPA driven ERE activation occurring at 1 M G 1 (40% of individual exposure) Additional experiments with more doses are required to delineate if signifi cant differences exist in the inhibitory dose response between BPA and Genistein G 1 Does Not Affect pS2 Gene Expression Next, we sought to examine the effect of G 1 on transcription of the well described E 2 responsive gene pS2 (TFF1). In order to veri fy that this was a good candidate gene for co exposure experiments, we first examined pS2 responsiveness to an array of G 1 doses. For these experiments, MCF 7 cells were exposed to E 2 (10 nM), DPN (100 nM), PPT (100 nM) or various doses of G 1 (10 nM, 10 0 nM, 500 nM, 1 M) for 24 hours and pS2 expression was measured by qRT PCR. Results revealed that exposure to 10 nM E 2 100 nM DPN, and 100 nM PPT increased pS2 gene expression 12, 16, and 2 fold, respectively (Figure 2. 5 ). Exposure to any of the doses of G 1 (10 nM 1 M) did not have any effect on pS2 expression (Figure 2. 5 ). Xenoestrogen Induced pS2 Expression is not Sensitive to G 1 After validating pS2 as an acceptable gene to study G 1 mediated inhibition of ESR driven pS2 expression, we next so ught to determine the effect of co exposure to low and high dose G 1 on E 2 BPA, and Genistein induced pS2 expression. For these studies MCF 7 cells were exposed to 10 nM E 2 10 M BPA, or 1 M Genistein individually and in combination with 10 nM G 1 or 1 M G 1 for 24 hours and pS2 expression was measured by qRT PCR. Results revealed a trend of increased expression after exposure to (xen o)estrogens individually and a trend of pS2 expression potentiation after co exposure to 10 nM G 1 and 1 M G 1 (Figure 2. 6 ). These results
36 revealed that E 2 and xeno estrogen induced pS2 expression is not sensitive to the inhibitory effects of G 1. G 1 and E 2 Exhibit Differential Phosphorylation Patterns In order to identify alternate pathways of G 1 mediated inhibition of ESR function, we utilized global phosphoproteomic strategies. MCF 7 cells were exposed to vehicle control, 10 nM E 2 or 1 M G 1 and total protein was isolated after 30 minutes in order to identify early targets of G 1 and E 2 induced non genomic signalin g pathways. Total protein was digested with trypsin and enriched for phosphorylated peptides using a TiO 2 based spin method ( Soderblom et al. 2011 ) Phospho peptides were identified using LC MS/MS strategies and mat ched to proteins using MASCOT. Results revealed 238 proteins that were phosphorylated on specific serine, threonine, or tyrosine residues These identified peptides were organized into a Venn diagram based on treatment group (Figure 2. 7 ). Overall, 13 and 37 phospho peptides were differentially phosphorylated by G 1 and E 2 r espectively while 10 peptides revealed phosphorylation by both compounds but at specific sites The proteins among the 238 identified were further grouped according to associated biological processes using the Gene Ontology (GO) Consortium (Figure 2. 8 ). A large number of proteins were assigned to the categories of development, localization and response to stimulus. Interestingly, of those differentially phosphorylated by G 1, a number were sorted into the biological process of reproduction and these 27 pr oteins are listed in Table 2. 1 A number of distinc t phosphorylation trends are evident from the data For example, a number of proteins were phosphorylated only by G 1 (NDRG2, RS6, SRPK1, WAPL) whereas others were dephosphorylated by G 1 (JIP4, PEBP1, UB R4, UNG, BAG6, IF4G1, RLA2) compared
37 to control and E 2 treatments Other trends included proteins that were phosphorylated only by E 2 or showed shared phosphorylation patterns by E 2 and G 1. Robust pathway analysis (Pathway Studio, Elsevier) of proteins di fferentially phosphorylated by G 1 revealed N myc downstream regulated gene 2 (NDRG2) and s numerous other proteins in particular kinases, that have been established to be ac tivated by G 1 (i.e. MAPK AKT) ( Bologa et al. 2006 ) (Figure 2. 9 ). While GPER thetical pathway, modulation of these proteins by G 1 may occur independently of the GPER. NDRG2 is a Plausible Novel Target of G 1 Mediated Signaling In order to validate the results of the phosphoproteomic analysis we attempted to examine differential p hosphorylation of NDRG2 on Serine 332 by E 2 and G 1 by Western blot analysis T he addition of phosphates alter s the migration pattern of the protein in a polyacrylamide gel which can be visualized as a higher molecular weight band compared to the native un phosphorylated form. Results from the Western blot show ed the presence of NDRG2 as a series of bands which likely indicate multiple differentially phosphorylated sites on the protein (Figure 2. 10 ). It is difficult to discern whether there were any noticea ble differences between treatments as the banding, particularly for E 2 and G 1, occurred in a diffuse pattern. However, evidence that these bands represented phosphorylated forms of the protein was supported by the presence of discrete and intense lower mo lecular weight bands when protein fractions were treated with alkaline phosphatase.
38 NDRG2 mRNA Expression is Downregulated by ESR Activation In order to characterize the transcriptional regulation of NDRG2, we examined the effect of E 2 and G 1 treatment o n NDRG2 mRNA expression by qRT PCR. For this experiment, MCF 7 cells were exposed to 10 nM E 2 1 M G 1, 10 M G 15, 100 nM PPT, or 100 nM DPN for 24 hours. Results revealed that NDRG2 expression was not affected by 1 M G 1 while exposure to 10 nM E 2 1 00 nM PPT, and 100 nM DPN significantly decreased NDRG2 expression compared to control cells Surprisingly, a trend of decreased NDRG2 expression was observed in cells treated with G 15 Discussion The biochemistry and molecular biology of nuclear ESRs has been a focus of study for decades, but a newly discovered ( Revankar et al. 2005a ; Thomas et al. 2005b ) putative membrane receptor for estrogens, GPER ( Valdes and Weeks, 2009 ) has been found to contribute to rapid non genomic actions of E 2 However, the precise role of GPER in modulating E 2 signaling and activity is controversial (rev iewed in Langer et al.) ( Langer et al. 2010 ) and its function as an estrogen receptor is heavily debated. It is possible that GPER and ESRs are capable of signal ing through both convergent and divergent pathways which complicates attempts to fully understand their role in E 2 signaling and downstream consequences. In addition, the clear findings that xenoestrogens, such as BPA and Genistein bind and modulate both ESR and GPER activity highlights the need to more thoroughly examine genomic and non genomic driven cellular processes as an alternate and highly relevant mode of endocrine disruption in the context of environmental exposures. We began investigations to a ssess the role of GPER in mediating E 2 and xenoestrogen driven cellular responses in MCF 7 cells. We also wanted to specifically
39 determine the role of each ESR isoform (ESR1, ESR2) in these endpoints. Based on results from our proliferation studies we can draw a number of conclusions. First G 1 inhibits E 2 driven proliferation but only at high doses > 500 nM This is in agreement with other reports that show G 1 (100 nM 1 M) inh ibits proliferation of breast ( MDA MB 231) and ovarian granulosa (KGN) cancer cells ( Ariazi et al. 2010 ; Wang et al. 2012 ) Recent studies have suggested this inhibitory effect occurs independ ently of GPER as G 15, the GPER antagonist, and siRNA to knock down GPER expression, both failed to rescue G 1 inhibited proliferation of ovarian cells A few other studies have shown similar effects by G 1 on proliferation in cells that do not express GPE R. ( Wang et a l. 2012 ) These studies, including ours, all utilized G 1 at relatively high doses (500 nM 1 M), suggesting the observed reduction in cellular proliferation has a dose threshold. While some reports have indicated that low doses of G 1 (10 nM 100 nM) stimulate the proliferation of breast cancer cells ( Lucki and Sewer, 2011 ) our results contradict this ob servation as 10 nM G 1 did not stimulate growth compared to the untreated control group. The reasons for these discordant observations are not clear but could be a result of variable cell lines and experimental conditions. For example, in some studies, pr oliferation was observed in cells exposed to G 1 that expressed GPER but not ESRs (SKBR3) ( Albanito et al. 2007 ; Albanito et al. 2008 ; Pandey et al. 2009 ) which implies GPER function largely relies on the ESR expression profile of the cell. However, other studies have indicated that GPER activation by G 1 inhi bits proliferation in the same cell line ( Lubig et al. 2012 ) Disparate results have also been observed in cells that express both types of receptor s ( GPER and ESRs ) For examp le, one study
40 using MCF 7 cells showed that a low dose exposure to G 1 increased proliferation (10 nM) ( Lucki and Sewer, 2011 ) while another study reported a high dose (1 M) was inhibitory ( Ariazi et al. 2010 ) It should be noted that in the former study these cells were manipulated to overexpress proteins of interest (i.e. ASAH1) which may change the proliferative capacity of G 1 ( Lucki and Sewer, 2011 ) Future experiments include examining the specific role of GPER in mediating the inhibitory effects of high d oses of G 1 on xenoestrogen induced MCF 7 proliferation by utilizing GPER blocking experiments (utilizing G 15 and siRNA) as it has been reported that signaling by high doses of G 1 (500 nM 1 M) may occur independently of GPER ( Wang et al. 2012 ) A second conclusion that emerged from the proliferation studies is the novel observation that the inhibitory effect of G 1 is not ESR isotype specific. Proliferation induced by both ESR specific agonists PPT (E SR1 specific agonist) and DPN (E SR 2 specific agonist) was inhibi ted in the presence of 1 M G 1 revealing that the mechanism of inhibition was not specific to either receptor subtype. Although further studies are required to elucidate the specific mechanisms of action of G 1, a few studies suggest that a sustained incr ease in cytosolic Ca 2+ concentrations that is dependent on GPER but independent of E 2 ( Ariazi et al. 2010 ) Furthermore, these studies revealed that G 1 blocks cell cycle progression and modulates the expression of relevant genes (p53, p21, cyclin D1). Whether these effects contribute to our obse rved results remains to be determined. Third, G 1 (at 1 M) inhibited proliferation stimulated by BPA and Genistein Because previous reports indicated that the environmentally relevant xenoestrogens BP A and Genistein show binding affinities for the ESRs ( Kuiper et al. 1997 ) and more
41 recently GPER with weak affinity ( Thomas and Dong, 2006 ) and are able to stimulate proliferation of MCF 7 cells ( Hsieh et al. 199 8 ; Schafer et al. 1999 ; Recchia et al. 2004 ; Nakaya et al. 2007 ) we examined the ability of G 1 to inhibit xenoestrogen induced cellular proliferation. High doses of BPA and Gen (10 M and 1 M, respectively) were used because they are known to significantly increase proliferation of MCF 7 cells at these concentrations By utilizing co exposure exp eriments, we showed that while 1 M G 1 does in fact inhibit proliferation stimulated by BPA and Genistein 10 nM G 1 was insufficient (Figure 2.2). Because these results paralleled those observed in the co exposure experiments utilizing the ESR agonists, they suggest that BPA and Genistein largely promote proliferation of MCF 7 cells through a genomic mechanism of ESR action and provide additional support for the existence of divergent signaling pathways activated by a high dose of G 1 in which non genomi c signaling opposes genomic actions of the ESRs. As proliferation of MCF 7 cells by E 2 is known to be controlled via nuclear ESRs, we performed reporter gene assays to measure the ability of these receptors to transcriptional ly activat e an ERE luciferase gene Our results revealed that G 1 inhibited ERE activation by E 2 PPT and DPN which paralleled results from the proliferation assays (Figure 2. 3 ). These results suggest that G 1 is influencing ESR activity in a non isoform specific manner. While the spe cific mechanism of action is not known, these results suggest that a number of mechanisms are involved which may be at the level of DNA interaction through modulation of ESR transcriptional co regulatory proteins ( Hall and McDonnell, 2005 ) increased ESR degradation ( Nawaz et al. 1999 ) or inducing ESR aggregation in the nucleus ( Hung, 2004 ) Interestingly, attempts to block G 1
42 mediated inhibition of ESR driven ERE activation by pretreatment with G 15 failed to rescue these effects (Figure 2. 3 ) which suggests that transcriptional inhibition by 1 M G 1 may oc cur independently of GPER. This observation, while not supported by all studies, has been recently corroborated by another group ( Wang et al. 2012 ) Our data however should be interpreted with caution as G 15 has been shown to weakly bind nuclear ESRs at the doses u sed in our study ( Dennis et al. 2011 ) Future studies to employ knockdown strategies, similar to those observed by Wang et al., ( Wang et al. 2012 ) are planned to more carefully address the role of GPER in the observed effects. With the exception of BPA where a trend in decreased activation was seen at low doses, G 1 only inhibited ERE ac tivity at doses > 500 nM (Figure 2. 4 ). These results coincide with our observation that a low dose (10 nM) of G 1 had no effect on ligand induced proliferation and correlated with inhibition of cell growth at doses above 500 nM Of note, low doses of G 1 (10 nM 100 nM) also did not inhibit ERE activation driven by lower doses of E 2 (10 pM 100 pM) which refutes the possibility that the in ability of low doses of G 1 to inhibit ESR function is due to saturation of the ESRs (data not shown). These resul ts lend additional support to the notion that high doses of G 1 may signal independently of GPER to inhibit ESR function by estrogenic ligands. While reporter assays allow for examining ESR activity of a specific ERE, promoters of E 2 responsive endogenous genes are much more complex and are typically regulated by more than just a consensus response element. To that end, we examined the capability of G 1 to inhibit the expression of a known ESR driven gene. The pS2 (TFF1) gene is well established to be ind ucible by E 2 via ESRs in MCF 7 cells ( Jakowlew et al. 1984 ) and therefore was chosen as a candidate gene with which to
43 study the effects of low and high doses of G 1. Our results substantiated previous results of pS2 induction by E 2 and select xenoestr ogens ( Jakowlew et al. 1984 ; Recchia et al. 2004 ) and revealed that expression is not modulated by G 1 alone (Figure 2. 5 ). Interestingly, results revealed that E 2 and xenoestroge n induced pS2 expression is not sensitive to low or high doses of G 1 i n co exposure conditions (Figure 2. 6 ). Although this may suggest that G 1 mediated inhibition of proliferation occurs farther upstream of transcriptional activation, it is more likely due to the complexity of the pS2 promoter. It has been shown that the pS2 promoter contains only a half ERE and is regulated by additional transcription factors such as AP 1 ( Barkhem et al. 2002 ) Taken together, our results indicate that the inhibition of ESR activity is not at the level of degrading the ESRs but interfering with interactions of the receptors at select response elements (ERE). Further studies are required to define these mechanisms. Only a handful of studies have begun to investigate the precise mecha nisms involved in G 1 mediated inhibition of proliferation. To date, these investigations have primarily focused on a few select kinases (MAPK, etc ) and downstream genes involved in cell cycle progression (cyclins etc ) ( Lubig et al. 2012 ) To our knowledge, no studies have assessed the global proteome to begin to identify alternate pathways that are modulated by G 1. The propagation of signaling pathways largely relies on reve rsible post translation modifications such as phosphorylation ( Metodiev and Alldridge, 2008 ) and as such, global phosphoproteomic strategies are emerging as a powerful tool to decipher complex signaling networks ( Harsha and Pandey, 2010 ; Sudhir et al. 2011 ) Although it is well established that G 1 activ ates various signaling cascades including the PI3K pathway through modulation of the GPER ( Bologa et al. 2006 ) our results
44 and those of others have indicated that the inhibitory effects of G 1 at high doses may occur either independently of GPER ( Wang et al. 2012 ) or modulate alternate pathways driven in some yet unknown way by GPER. As an initial attempt to identify novel targets of G 1 mediated signaling in order to uncover pathways by which G 1 inhibits ESR functions we performed a global phosphoproteome analysis on cells exposed to E 2 or G 1 for 30 minutes Results of these studies revealed 238 peptides in total that were phosphorylated on specific se rine, threonine, and tyrosine residues. A number of the peptides that were differentially phosphorylated by G 1 compared to controls are involved in reproduction as determined by GO analysis (Figure 2. 8 ) and are annotated in greater detail in Table 2.1. Ou r observation that a high dose of G 1 and E 2 exhibited differential phosphorylation patterns (Figure 2. 7 ) is intriguing because it substantiates the notion that G 1 may signal independently of GPER at high doses as previous studies have indicated that low doses of G 1 activates kinases analogous to E 2 ( Kato et al. 1995 ; Revankar et al. 2005c ; Albanito et al. 2007 ) Of particular interest is the G 1 specific phosphorylation of the candidate tumor suppressor protein NDRG2 which was one of many differentially phosphorylated proteins that were associated with reprod uction and cell growth. NDRG2 has been shown to play a role in cell growth inhibition of colon cancer cells when phosphorylated at the particular residue (S332) identified in our phosphoproteomic analysis ( Kim et al. 2009 ) Neither the control nor E 2 treatments resulte d in S332 phosphorylation. We attempted to validate the G 1 specific phosphorylation of NDRG2 by Western blot and results support variable phosphorylation patterns between E 2 and G 1 however ; we were unable to discern the precise phosphorylation status o f S332 using this method
45 (Figure 2.10) A number of reports reveal numerous phosphorylation sites on NDRG2 that have been linked with various functions. For example, phosphorylation of S332 inhibits the insulin induced phosphorylation of T348 resulting in inhibition of muscle growth and metabolism of muscle cells ( Burchfield et al. 2004 ) These studies highlight the complexity and multiple phosphorylation sites that are important in governing its function. We have recently obtained a plasmid encoding NDRG2 (kindly supplied by Dr. Lim, Sookmyung Women's University, Seoul Korea) which has bee n mutated at S332 (to alanine) rendering it incapable of being phosphorylated. This tool will allow us to perform additional studies to address the role of S332 in G 1 inhibitory effects on cell proliferation. Regardless, the results of our phosphoproteomic analysis provided a useful first pass screening of the phosphoproteome and have laid a framework on which to build future experiments to address G 1 modulated cell signaling pathways Based on reports that the NDRG2 promoter contains a response eleme nt shown to interact with ESR2 and its expression is modulated by E 2 in several cancer lines through promoter methylation, mutation, and genomic deletion ( Liu et al. 2007 ) we performed experiments to assess the impact of ESR specific ligands and G 1 on NDRG2 mRNA levels. Here we report that E 2 PPT and DPN decrease NDRG2 expression at 24 hours post exposure whereas G 1 had no effect compared to control cells (Figure 2.11) These results suggest that while ESRs decrease NDRG2 expression thereby allowing cell growth to occur, G 1 does not alter e xpression levels, but may phosphorylate the protein at select residues such as S332 which may contribute to cell cycle inhibition. While NDRG2 has been shown to act as an inhibitor of cell proliferation in othe r cells ( Kim et al. 2009 ) one study showed that ESR2 increas es transcriptional
46 activation of the NDRG2 promoter using a reporter assay after 12 hours in MCF 7 cells ( Li et al. 2011 ) Although we observed an opposite effect, it is difficult to directly compare results as the published study only assessed a portion of the NDRG2 promoter. It is also possible that differences are the result of periodicity and further time points should be evaluated. Additional studies to discern the impact of xenoestrogens and the role of GPER on NDRG2 phosphorylation are planned. Collectively, results of the present work indicate that dose s of the GPER agonist G 1 > 500 nM inhibit E 2 and xenoestr ogen induced, ESR mediated proliferation of human breast cancer cells possibly by interference with ESR mediated transcriptional activation These data support the possibility that G 1 may be acting independent ly of GPER through selective phosphorylation and activation of the tumor suppressor protein NDRG2 ( Lubig et al. 2012 ) The se collective results are significant because they highlight a novel role for G 1 in inhibiting E 2 and xenoestrogen dependent proliferation. While these data do not necessarily support a role for xenoestrogens in activating GPER, elucidating pathways that inhibit their effects (through ESRs) have enormous implications for adjuvant therap ies in the treat ment of estrogen receptor positive breast cancers
47 Figure 2 1. Co exposure to 1 M G 1 suppresses nuclear ER d riven p roliferation of MCF 7 c ells. Cells were seeded in 96 well cell culture plates and exposed to chemicals dissolved in DMSO for 72 or 96 hours. Proliferation was determined by a modified MTT assay and graphed as 570 nm absorbance over time. A) Exposure to 10 nM E2, 10 nM G 1 or 1 M G 1. B) Exposure to 10 nM E 2 in combination with 1 M G 1. C) Exposure to DPN (ESR2 agonist) with and without 1 M G 1. D) Exposure to the PPT (ESR1 agonist) with and without 1 M G 1.
48 Figure 2 2. Co e xposure to 1 M G 1 s uppresses x enoestrogen i nduced p roliferation of MCF 7 c ells. Cells were seeded in 96 well cell culture plates and exposed to chemicals dissolv ed in DMSO for 96 hours. Proliferation was determined by a modified MTT assay and graphed a s 570 nm absorbance over time. A) Exposure to 10 M BPA with and without 10 nM G 1 or 1 M G 1. B) Exposure to 1 M genistein with and without 10 nM G 1 or 1 M G 1.
49 Figure 2 3 Co e xposure to 1 M G 1 s uppresses ESR m ediated a ctivation at an ERE. MCF 7 cells were transfected with a 2XERE Luciferase reporter construct for 24 hours and exposed to chemicals dissolved in DMSO for 24 hours thereafter. A) Exposure to 10 nM E 2 1 M G 1 and 20 M G 15. B) Exposure to 10 nM E 2 and co exposures with 1 M G 1 and 20 M G 15. C) Exposure to 100 nM DPN and co exposures with 1 M G 1 and 20 M G 15. D) Exposure to 100 nM PPT and co exposures with 1 M G 1. Results are repres ented as the mean SEM of three experiments. Letters denote significance at p < 0.05.
50 Figure 2 4 GPER m ediated s uppression of ERE a ctivation is d ose d ependent. MCF 7 cells were transfected with 2XERE Luciferase reporter gene and exposed to chemic als dissol ved in DMSO for 24 hours. Bars r epresent percent of maximal activity (RLU) for E 2 BPA, or genistein A) Exposure to 10 nM E 2 with inc reasing concentrations of G 1. B) Exposure to 640 nM BPA with inc reasing concentrations of G 1. C) Exposure to 2 84 nM genistein with increasing concentrations of G 1. Results are represented as mean SD. Letters are significant at p < 0.05. Note; r esults presented in panel 3 represent data from one experiment so statistics could not be performed.
51 Figure 2 5 pS 2 e xpression is r esponsive to n uclear ER a gonists but not G 1. MCF 7 cells were exposed to 10 nM E 2 100 nM DPN, 100 nM PPT, G 1 (10 nM, 100 nM, 500 nM, 1 M) for 24 hours. Results are represented as mean SD of one experiment.
52 Figure 2 6 Xenoestro gen i nduced pS2 e xpression is n ot r esponsive to G 1. MCF 7 cells were exposed to chemicals d issolved in DMSO for 24 hours. A) Exposure to 10 nM E 2 or 1 M G 1. B) Exposure to 10 Results are represented as mean SD of one experiment.
53 Figure 2 7. Distribution of the n umber of p hosphorylated p roteins i dentified in MCF 7 c ells e xposed to E 2 or G 1 c ompared to c ontrol c ells. The V enn diagram depicts the distribution of peptides phosphorylated for each treatment after exposure for 30 minutes. Representative peptides are shown for specific, relevant proteins identified within discrete treatments, with phosphorylation sites noted as bold underli ned pS or pT in each sequence.
54 Figure 2 8 Phosphorylated p eptides organized by b iological p rocess. Pie chart showing the representative biological processes determined by GO analysis of the 238 proteins phosphorylated by E 2 and/or G 1 a fter 30 minutes of exposure.
55 Figure 2 9 Pathway s howing r elationship between p roteins p hosphorylated s pecifically by G 1 in MCF 7 c ells. The larger central circular entities (NDRG2 and SRPK1) are phosphorylated proteins identified in the phosphor proteomic play a role in G 1 signaling through GPER. The shapes represent the following; diamonds growth factors/peptides; semi circles kinases; circles proteins; others r eceptors and transcription factors. Lines represent various relationships between entities including post translational modifications, expression, and general relationships. Figure 2 10 Phosphorylation of NDRG2. MCF 7 cells were exposed to DMSO, 10 nM E 2 or 1 M G 1 for 30 minutes and total protein was separated on Tris Tricine polyacrylamide gel and probled with anti NDRG2. Lanes 2, 4, and 6 represent protein fractions treated with alkaline phosphatase (AP)
56 Figure 2. 11 Nuclear ERs s uppress the e x pression of NDRG2 mRNA. MCF 7 cells were exposed to 10 nM E 2 100 nM PPT, 100 nM DPN, 1 M G 1, or 10 M G 15. Bars represent mean SD of two experiments Letters are significant at p < 0.05.
57 Table 2 1 Peptides differentially phosphorylated by G 1 and s Protein ID Name Phosphorylation by Treatment Phos pho Site(s) Sequence Protein Probability (%) MASCOT ID Score Control E2 G1 NDRG2 NMYC downstream regulated gene 2 1 S332 (R)TA S LTSAASVDG NR(S) 88% 28.6 RS6 40S ribosomal protein S6 1 S235, S236 (R)RL SS LR(A) 88% 25.0 SRPK1 Serine/threonine protein kinase 1 S51 (R)GSAPHSESDLPEQEEEILG S DDDEQEDPNDYcK(G) 88% 32.3 WAPL Wings apart like protein 1,2 S77, S221 S226 (K)RPE S PSEI S PIKGSVR(T); (K)VEEESTGDPFGFD S DDESLP VSSK(N) 100% 31.6 JIP4 C Jun amino terminal kinase interacting protein 4 1,2 1,2 S730, S733 (R)SA S QS S LDKLDQELKEQQK( E) 70 100% 32.1 PEBP1 Phosphatidylethanolamine binding protein 1 1 1 S52 (K)NRPT S ISWDGLDSGK(L) 89 90% 30.3 UBR4 E3 ubiquitin protein ligase 1 1 S2719 (R)HVTLPS S PR(S) 68 76% 26.3 UNG Uracil DNA glycosylase 1 1,2,3, 4 S23, T60, S63, S64 (R)HAP S PEPAVQGTGVAGVPEE SGDAAAIPAK(K); (K)KAPAGQEEPG T PP SS PLSA E QLDR(I) 85 100% 32.8 BAG6 Large proline rich protein 1,2 1,2 1 S113, S1117 (R)APPQTHLPSGASSGTGSASA THGGG S PPGTR(G); (R)LQEDPNY S PQRFPNAQR(A) 74 100% 33.2
58 IF4G1 Eukaryotic translation initiation factor 4 gamma 1 1,2,3,4 1,2,3, 4 1,2 S118 5, S1187. T205, T207 (R) S F S KEVEER(S); (R)TAS T P T PPQTGGGLEPQANG ETPQVAVIVRPDDR(S) 88 100% 34.1 RLA2 60S acidic ribosomal protein P2 1,2 1,2 2 S102, S105 (K)DEKKEE S EE S DDDMGFGLFD ( ) 89 100% 29.5 AGFG1 Afr GAP domain 1 1 S181 (K)GTPSQ S PVV GR 95% 27.4 NPM Nucleophosmin 1 1 S125 (K)cGSGPVHISGqHLVAVEEDAE S EDEEEEDVK(L) 90 100% 33.6 PACS1 Phosphofurin acidic cluster sorting protein 1 1,2,3 2,3 1,2,3 S495, S529, S531 (K)TDLQGSA S PSKVEGVHTPR( Q); (R)TNS S D S ERSPDLGHSTQIPR( K) 89 1 00% 31.6 PARN Poly(A) specific ribonuclease P 1,2 3,4 1,2 S572, S583, T589, T594 (R)NLSP S QEEAGLEDGV S GEIS D T ELEQ T DScAEPLSEGR(K) 88 89% 34.1 PSA3 Proteasome subunit alpha type 3 1,2 1 1,2 S243, S250 (K)YAKE S LKEEDE S DDDNM( ) 89 100% 28.5 SCRIB Scribble 1 S 1316, S1319 (K)mAESPcSPSGQQPP S PP S PD ELPANVK(Q) 89% 32.7 UBP2L Ubiquitin associated protein 2 like 4 1,2,3, 4 4 S454, S462, S604, S609 (K)SPAVATSTAAPPPPS S PLPSK (S); (K)ST S APQMSPGSSDNQSSSP QPAQQK(L); (R)RYP S SISS S PQKDLTQAK(N) 88 100% 30.6 L MNA Prelamin A/C 1 1 S22 (R)SGAQASSTPL S PTR(I) 100% 28.6
59 RD23A UV excision repair protein RAD23 homolog A 1 1 S128 (R)EDKSPSEE S APTTSPESVSG SVPSSGSSGR(E) 88 89% 33.4 TRI25 E3 ubiquitin/ISG15 ligase 1 S100 (R)ASAP S PNAQVAcDHcLK(E) 100% 32.1 AKT1 Serine/Threonine protein kinase 1,2 1,2 1,2 S124, S129 (R)SG S PSDN S GAEEMEVSLAKP K(H) 88 90% 31.5 CTNA1 Catenin alpha 1 1 1 1 S641 (R)TPEELDD S DFETEDFDVR(S) 88 90% 29.4 HMCS1 Hydroxymethylglutaryl CoA synthase, cytop lasmic 1 1 1 S516 (R)LPATAAEPEAAVI S nGEH( ) 88 90% 30.6 HNRPK Heterogeneous nuclear ribonucleoprotein K 1 1 1 S284 (R)DYDDM S PR(R) 76 100% 25.0 IMA2 Importin subunit alpha 2 1 1 1 S62 (R)NVSSFPDDAT S PLQENR(N) 88 90% 30.3 NACA Nascent polypeptide associated complex subunit alpha 1 1 1 S166 (K)VQGEAVSNIQENTQTPTVQE E S EEEEVDETGVEVK(D) 88 90% 34.9 XRN2 5' 3' exoribonuclease 2 1,2 1,2 1,2 S499, S501 (R)KAED S D S EPEPEDNVR(L) 88 90% 26.7
60 CHAPTER 3 GENERAL CONCLUSIO NS The initial goal of this research was to decipher the potential interplay between genomic and non genomic signaling pathways initiated by the GPER and ESRs in response to xenoestrogens. Uncovering the complex cellular responses to xenoestrogen exposure offers valuable information regarding their contribution to disease. However our investigations le d us in a different direction as it was revealed that the putative GPER specific agonist (G 1) is able to inhibit ESR function initiated by E 2 and xenoestro gens and that these mechanisms of inhibition may occur independently of GPER. These results reveled for the first time, a novel role for G 1 in modulating the actions of E 2 and xenoestrogens that may have value in the development of therapeutics in the tre atment of endocrine disease. This could be of particular importance in the treatment of estrogen receptor positive breast cancer which proliferates in response to ESR agonists. In the US, approximately 1 in 8 women will develop breast cancer in her lifeti me and ESR positive breast cancers are the most common type constituting 70% of diagnoses. Remarkably, 30% of women who develop ESR positive breast cancer are or become endocrine resistant and do not respond to antihormone therapeutics such as the select ive estrogen receptor modulator (SERM), tamoxifen ( Garca Becerra et al. 2012 ) These staggering statistics necessitate the identification of a more comprehensive treatment to mitigate ESR mediated proliferation. Although it is still unknown whether exposure to xenoestrogens can impact cancer risk, experimental evidence is mounting. For example, dietary exposure to Genistein is of particular concern in estrogen responsive breast cancers as the
61 estrogenic propertie s of Genistein are largely determined by the levels of circulating E 2 and ESR expression profile. In the absence of circulating E 2, Genistein has been found to increase the growth of ESR positive breast cancer cells ( Chen et al. 2003 ) In light of this, G 1 may prove to be a beneficial therapeutic in the treatment of postmenopausal women diagnosed with estrogen respo nsive breast cancer. Further, BPA has been shown to increase proliferation of breast cancer cells ., ( Schafer et al. 1999 ; Recchia et al. 2004 ) and even decrease the efficacy of chemotherapeutic drugs by antagonizin g their cytotoxic properties independent of ESRs in vitro ( LaPensee et al. 2009 ) Based on our results, G 1 may present a potential alternative to chemotherapeutics. The fact that G 1 mediated inhibitory pathways of ESR function may occur independently of GPER require s uncovering novel targets of G 1 in order to construct a plausible cell ular signaling network to determine the mechanism by which G 1 functions at high doses. Elucidating the complex mechanism of G 1 action may further substantiate its use as an adjuvant therapeutic in the treatment of xenoestrogen mediated disease particular est rogen responsive breast cancer.
62 LIST OF REFERENCES Albanito, L., Madeo, A., Lappano, R., Vivacqua, A., Rago, V., Carpino, A., Oprea, T.I., Prossnitz E.R., Musti, A.M., Ando, S., 2007. G protein coupled receptor 30 estradiol and selective GPR30 ligand G 1 in ovarian cancer cells. Cancer Research 67 1859 1866. Albanito, L., Sisci, D. Aquila, S., Brunelli, E., Vivacqua, A., Madeo, A., Lappano, R., Pandey, D.P., Picard, D., Mauro, L., 2008. Epidermal growth factor induces G protein coupled receptor 30 expression in estrogen receptor negative breast cancer cells. Endocrinology 149 3799 3808. Allred, C.D., Allred, K.F., Ju, Y.H., Virant, S.M., Helferich, W.G., 2001. Soy diets containing varying amounts of genistein stimulate growth of estrogen dependent (MCF 7) tumors in a dose dependent manner. Cancer Research 61 5045 5050. Ariazi, E .A., Brailoiu, E., Yerrum, S., Shupp, H.A., Slifker, M.J., Cunliffe, H.E., Black, M.A., Donato, A.L., Arterburn, J.B., Oprea, T.I., Prossnitz, E.R., Dun, N.J., Jordan, V.C., 2010. The G Protein Coupled Receptor GPR30 Inhibits Proliferation of Estrogen Rece ptor Positive Breast Cancer Cells. Cancer Research 70 1184 1194. Barkhem, T., Haldosn, L. A., Gustafsson, J. ., Nilsson, S., 2002. pS2 Gene expression in HepG2 cells: complex regulation through crosstalk between the responsive element, and the activator protein 1 response element. Mole cular pharmacology 61 1273 1283. Bologa, C.G., Revankar, C.M., Young, S.M., Edwards, B.S., Arterburn, J.B., Kiselyov, A.S., Parker, M.A., Tkachenko, S.E., Savchuck, N.P., Sklar, L.A., 2006. Virtual and biomolecular screening converge on a selective agoni st for GPR30. Nature chemical biology 2 207 212. Burchfield, J.G., Lennard, A.J., Narasimhan, S., Hughes, W.E., Wasinger, V.C., Corthals, G.L., Okuda, T., Kondoh, H., Biden, T.J., Schmitz Peiffer, C., 2004. Akt mediates insulin stimulated phosphorylation of Ndrg2. Journal of Biological Chemistry 279 18623 18632. Chen, W. F., Huang, M. H., Tzang, C. H., Yang, M., Wong, M. S., 2003. Inhibitory actions of genistein in human breast cancer (MCF 7) cells. Biochimica et Biophysica Acta (BBA) Molecular Basis of Disease 1638 187 196. Cheng, S. B., Graeber, C.T., Quinn, J.A., Filardo, E.J., 2011. Retrograde transport of the transmembrane estrogen receptor, G protein coupled receptor 30 (GPR30/GPER) from the plasma membrane towards the nucleus. Steroids 76 892 896.
63 Colborn, T., vom Saal, F.S., Soto, A.M., 1993. Developmental effects of endocrine disrupting chemicals in wildlife and humans. Environmental health perspectives 101 378. Dennis, M.K., Burai, R., Ramesh, C., Petrie, W.K., Alcon, S.N., Nayak, T.K., B ologa, C.G., Leitao, A., Brailoiu, E., Deliu, E., 2009. In vivo effects of a GPR30 antagonist. Nature chemical biology 5 421 427. Dennis, M.K., Field, A.S., Burai, R., Ramesh, C., Petrie, W.K., Bologa, C.G., Oprea, T.I., Yamaguchi, Y., Hayashi, S.I., Skl ar, L.A., 2011. Identification of a GPER/GPR30 antagonist with improved estrogen receptor counterselectivity. The Journal of steroid biochemistry and molecular biology 127 358 366. Deroo, B.J., Korach, K.S., 2006. Estrogen receptors and human disease. Jo urnal of Clinical Investigation 116 561. Dong, S., Terasaka, S., Kiyama, R., 2011. Bisphenol A induces a rapid activation of Erk1/2 through GPR30 in human breast cancer cells. Environmental pollution 159 212 218. Filardo, E., Quinn, J., Pang, Y., Graeb er, C., Shaw, S., Dong, J., Thomas, P., 2007. Activation of the novel estrogen receptor G protein coupled receptor 30 (GPR30) at the plasma membrane. Endocrinology 148 3236 3245. Filardo, E.J., Quinn, J.A., Bland, K.I., Frackelton, A.R., 2000. Estrogen i nduced activation of Erk 1 and Erk 2 requires the G protein coupled receptor homolog, GPR30, and occurs via trans activation of the epidermal growth factor receptor through release of HB EGF. Molecular Endocrinology 14 1649 1660. Filardo, E.J., Quinn, J. A., Frackelton, A.R., Bland, K.I., 2002. Estrogen Action Via the G Protein Coupled Receptor, GPR30: Stimulation of Adenylyl Cyclase and cAMP Mediated Attenuation of the Epidermal Growth Factor Receptor to MAPK Signaling Axis. Molecular Endocrinology 16 70 84. Garca Becerra, R., Santos, N., Daz, L., Camacho, J., 2012. Mechanisms of Resistance to Endocrine Therapy in Breast Cancer: Focus on Signaling Pathways, miRNAs and Genetically Based Resistance. International Journal of Molecular Sciences 14 108 145 Hall, J.M., Couse, J.F., Korach, K.S., 2001. The multifaceted mechanisms of estradiol and estrogen receptor signaling. Journal of Biological Chemistry 276 36869 36872. Hall, J.M., McDonnell, D.P., 2005. Coregulators in nuclear estrogen receptor action : from concept to therapeutic targeting. Molecular interventions 5 343.
64 Harsha, H., Pandey, A., 2010. Phosphoproteomics in cancer. Molecular oncology 4 482 495. Herbst, A., Green Jr, T., Ulfelder, H., 1970. Primary carcinoma of the vagina. An analysis o f 68 cases. American journal of obstetrics and gynecology 106 210. Herbst, A.L., Ulfelder, H., Poskanzer, D.C., Longo, L., 1999. Adenocarcinoma of the vagina. Association of maternal stilbestrol therapy with tumor appearance in young women. 1971. America n journal of obstetrics and gynecology 181 1574. Hsieh, C.Y., Santell, R.C., Haslam, S.Z., Helferich, W.G., 1998. Estrogenic effects of genistein on the growth of estrogen receptor positive human breast cancer (MCF 7) cells in vitro and in vivo. Cancer R esearch 58 3833 3838. Hung, H., 2004. Inhibition of estrogen receptor alpha expression and function in MCF 7 cells by kaempferol. Journal of cellular physiology 198 197 208. Improta Brears, T., Whorton, A.R., Codazzi, F., York, J.D., Meyer, T., McDonne ll, D.P., 1999. Estrogen induced activation of mitogen activated protein kinase requires mobilization of intracellular calcium. Proceedings of the National Academy of Sciences 96 4686 4691. Jakowlew, S.B., Breathnach, R., Jeltsch, J. M., Masiakowski, P., Chambon, P., 1984. Sequence of the pS2 mRNA induced by estrogen in the human breast cancer cell line MCF 7. Nucleic acids research 12 2861 2878. Estradiol inhibits oxidative stress induced apoptosis in keratinocytes by promoting Bcl 2 expression. Journal of investigative dermatology 121 1500 1509. Kang, L., Zhang, X., Xie, Y., Tu, Y., Wang, D., Liu, Z., Wang, Z. Y., 2010. Involvement of estrogen receptor variant ER signaling. Molecular Endocrinology 24 709 721. Kato, S., Endoh, H., Masuhiro, Y., Kitamoto, T., Uchiyama, S., Sasaki, H., Masushige, S., Gotoh, Y., Nishida, E., Kawashima, H., 1995. Activation of the estrogen receptor through phosphorylation by mitogen activated protein kinase. Science (New York, NY) 270 1491. Kavlock, R.J., Daston, G.P., DeRosa, C., Fenner Crisp, P., Gray, L.E., Kaattari, S., Lucier, G., Luster M., Mac, M.J., Maczka, C., 1996. Research needs for the risk assessment of health and environmental effects of endocrine disruptors: a report of the US EPA sponsored workshop. Environmental health perspectives 104 715.
65 Kim, Y.J., Yoon, S.Y., Kim, J. T. Choi, S.C., Lim, J. S., Kim, J.H., Song, E.Y., Lee, H.G., Choi, I., Kim, J.W., 2009. NDRG2 suppresses cell proliferation through down regulation of AP 1 activity in human colon carcinoma cells. International Journal of Cancer 124 7 15. Korach, K.S., Em men, J., Walker, V.R., Hewitt, S.C., Yates, M., Hall, J.M., Swope, D.L., Harrell, J.C., Couse, J.F., 2003. Update on animal models developed for analyses of estrogen receptor biological activity. The Journal of steroid biochemistry and molecular biology 86 387 391. Kuiper, G.G., Carlsson, B., Grandien, K., Enmark, E., Hggblad, J., Nilsson, S., Gustafsson, J. ., 1997. Comparison of the ligand binding specificity and 138 863 870. Langer, G., Bader, B., Meoli, L., Isensee, J., Delbeck, M., Noppinger, P.R., Otto, C., 2010. A critical review of fundamental controversies in the field of GPR30 research. Steroids 75 603 610. LaPensee, E.W., Tuttle, T.R., Fox, S.R., Ben Jonathan, N., 2009. Bi sphenol A at low nanomolar doses confers chemoresistance in estrogen receptor positive and negative breast cancer cells. Environmental health perspectives 117 175. Li, Y., Yang, J., Li, S., Zhang, J., Zheng, J., Hou, W., Zhao, H., Guo, Y., Liu, X., Dou K., 2011. N myc downstream regulated gene 2, a novel estrogen targeted gene, is involved in the regulation of Na+/K+ ATPase. Journal of Biological Chemistry 286 32289 32299. Liu, N., Wang, L., Liu, X., Yang, Q., Zhang, J., Zhang, W., Wu, Y., Shen, L., Zhang, Y., Yang, A., 2007. Promoter methylation, mutation, and genomic deletion are involved in the decreased< i> NDRG 2 expression levels in several cancer cell lines. Biochemical and biophysical research communications 358 164 169. Lubig, J., Lattr ich, C., Springwald, A., Hring, J., Schler, S., Ortmann, O., Treeck, O., 2012. Effects of a Combined Treatment With GPR30 Agonist G 1 and Herceptin on Growth and Gene Expression of Human Breast Cancer Cell Lines. Cancer Investigation 30 372 379. Lucki, N.C., Sewer, M.B., 2011. Genistein stimulates MCF 7 breast cancer cell growth by inducing acid ceramidase (ASAH1) gene expression. Journal of Biological Chemistry 286 19399 19409. Madak Erdogan, Z., Kieser, K.J., Kim, S.H., Komm, B., Katzenellenbogen, J .A., Katzenellenbogen, B.S., 2008. Nuclear and extranuclear pathway inputs in the regulation of global gene expression by estrogen receptors. Molecular Endocrinology 22 2116 2127.
66 Maggiolini, M., Vivacqua, A., Fasanella, G., Recchia, A.G., Sisci, D., Pezz i, V. Montanaro, D., Musti, A.M., Picard, D., And, S., 2004. The G protein coupled receptor GPR30 mediates c fos up estradiol and phytoestrogens in breast cancer cells. Journal of Biological Chemistry 279 27008 27016. McKenna, N., O'Malley B.W., 2001. Nuclear Receptors, Coregulators, Ligands, and Selective Receptor Modulators. Annals of the New York Academy of Sciences 949 3 5. Metodiev, M., Alldridge, L., 2008. Phosphoproteomics: A possible route to novel biomarkers of breast cancer. Pr oteomics Clinical Applications 2 181 194. hormone production by porcine ovarian granulosa cells caused by bisphenol A and bisphenol A dimethacrylate. Molecular and cellular endocrinology 244 57 62. Morley, F., Axelsen, A., Bennett, D., 1964. Effects of grazing red clover (Trifolium pratense L.) during the joining season on ewe fertility. ASAP, pp. Nakaya, M., Onda, H., Sasaki, K., Yukiyoshi, A., Tachibana, H., Yamada, K., 2007. Effect of royal jelly on bispheno l A induced proliferation of human breast cancer cells. Bioscience, biotechnology, and biochemistry 71 253 255. dependent degradation of the human estrogen receptor. Pro ceedings of the National Academy of Sciences 96 1858 1862. Notas, G., Kampa, M., Pelekanou, V., Castanas, E., 2011. Interplay of estrogen receptors and GPR30 for the regulation of early membrane initiated transcriptional effects: A pharmacological approa ch. Steroids. Pandey, D.P., Lappano, R., Albanito, L., Madeo, A., Maggiolini, M., Picard, D., 2009. Estrogenic GPR30 signalling induces proliferation and migration of breast cancer cells through CTGF. The EMBO journal 28 523 532. Patisaul, H.B., Adewale H.B., 2009. Long term effects of environmental endocrine disruptors on reproductive physiology and behavior. Frontiers in behavioral neuroscience 3 Pedram, A., Razandi, M., Levin, E.R., 2006. Nature of functional estrogen receptors at the plasma membra ne. Molecular Endocrinology 20 1996 2009.
67 Prossnitz, E.R., Arterburn, J.B., Smith, H.O., Oprea, T.I., Sklar, L.A., Hathaway, H.J., 2008. Estrogen signaling through the transmembrane G protein coupled receptor GPR30. Annu. Rev. Physiol. 70 165 190. Pupo M., Pisano, A., Lappano, R., Santolla, M.F., De Francesco, E.M., Abonante, S., Rosano, C., Maggiolini, M., 2012. Bisphenol A Induces Gene Expression Changes and Proliferative Effects through GPER in Breast Cancer Cells and Cancer Associated Fibroblasts. Environmental health perspectives 120 1177. Rasier, G., Toppari, J., Parent, A.S., Bourguignon, J.P., 2006. Female sexual maturation and reproduction after prepubertal exposure to estrogens and endocrine disrupting chemicals: a review of rodent and human data. Molecular and cellular endocrinology 254 187 201. Recchia, A., Vivacqua, A., Gabriele, S., Carpino, A., Fasanella, G., Rago, V., Bonofiglio, D., Maggiolini, M., 2004. Xenoestrogens and the induction of proliferative effects in breast cancer cells via direct and contaminants 21 134 144. Revankar, C.M., Cimino, D.F., Sklar, L.A., Arterburn, J.B., Prossnitz, E.R., 2005a. A transmembrane intracellular estrogen receptor mediates rapid cell signaling. Science 307 1625 1630. Revankar, C.M., Cimino, D.F., Sklar, L.A., Arterburn, J.B., Prossnitz, E.R., 2005b. A transmembrane intracellular estrogen receptor mediates rapid cell signaling. Science Signalling 307 1625. Revankar, C.M., Cimino, D.F., Sklar, L.A., Arter burn, J.B., Prossnitz, E.R., 2005c. A Transmembrane Intracellular Estrogen Receptor Mediates Rapid Cell Signaling. Science 307 1625 1630. Schafer, T.E., Lapp, C.A., Hanes, C.M., Lewis, J.B., Wataha, J.C., Schuster, G.S., 1999. Estrogenicity of bisphenol A and bisphenol A dimethacrylate in vitro. Journal of biomedical materials research 45 192 197. Searle, B.C., 2010. Scaffold: A bioinformatic tool for validating MS/MS based proteomic studies. Proteomics 10 1265 1269. Soderblom, E.J., Philipp, M., Thom pson, J.W., Caron, M.G., Moseley, M.A., 2011. Quantitative label free phosphoproteomics strategy for multifaceted experimental designs. Analytical chemistry 83 3758. Sudhir, P. R., Hsu, C. L., Wang, M. J., Wang, Y. T., Chen, Y. J., Sung, T. Y., Hsu, W. L ., Yang, U. C., Chen, J. Y., 2011. Phosphoproteomics identifies oncogenic Ras signaling targets and their involvement in lung adenocarcinomas. PloS one 6 e20199.
68 Sugiura Ogasawara, M., Ozaki, Y., Sonta, S., Makino, T., Suzumori, K., 2005. Exposure to bisp henol A is associated with recurrent miscarriage. Human reproduction 20 2325 2329. Thomas, P., Dong, J., 2006. Binding and activation of the seven transmembrane estrogen receptor GPR30 by environmental estrogens: a potential novel mechanism of endocrine disruption. The Journal of steroid biochemistry and molecular biology 102 175 179. Thomas, P., Pang, Y., Filardo, E., Dong, J., 2005a. Identity of an estrogen membrane receptor coupled to a G protein in human breast cancer cells. Endocrinology 146 624 6 32. Thomas, P., Pang, Y., Filardo, E.J., Dong, J., 2005b. Identity of an estrogen membrane receptor coupled to a G protein in human breast cancer cells. Endocrinology 146 624 632. Valdes, J.J., Weeks, O.I., 2009. Lithium: a potential estrogen signaling modulator. J Appl Biomed 7 175 188. Venter, J.C., Adams, M.D., Myers, E.W., Li, P.W., Mural, R.J., Sutton, G.G., Smith, H.O., Yandell, M., Evans, C.A., Holt, R.A., 2001. The sequence of the human genome. Science Signalling 291 1304. Vivacqua, A., Bonof iglio, D., Albanito, L., Madeo, A., Rago, V., Carpino, A., Musti, A.M., Estradiol, genistein, and 4 hydroxytamoxifen induce the proliferation of thyroid cancer cells through the G protein coupled receptor GPR 30. Molecular pharmacology 70 1414 1423. Wang, C., Lv, X., Jiang, C., Davis, J.S., 2012. The putative G protein coupled estrogen receptor agonist G 1 suppresses proliferation of ovarian and breast cancer cells in a GPER independent manner. American Journ al of Translational Research 4 390
69 BIOGRAPHICAL SKETCH Ley Cody Smith was born on August 1, 1988 in Orlando, FL After graduating from Willia m R. Boone High School in 2006, he enrolled at the University of Flori da where he received his Bachelor of Science in integrative biology in 2010. Mr. Smith then worked as a research associate in the lab of Dr. David Barber in the Center for Environmental and Human Toxicology for Thereafter, h e enrolled in the University of Florida graduate school in August 2011 and received his Master of Science in the spring of 2013 under the direction of Dr. Tara Sabo Attwood. He was awarded a Graduate School Fellowship form the University of Fl orida in the spring of 2013 and will begin his Doctor of Philosophy program studying interdisciplinary toxicology in the fall of 2013 also under the direction of Dr. Tara Sabo Attwood.