Erα Mediates Estrogen-Induced Expression of the Breast Cancer Metastasis Suppressor Gene Brms1

ERΑ MEDIATES ESTROGEN-INDUCED EXPRESSION OF THE BREAST CANCER METASTASIS SUPPRESSOR GENE BRMS1

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Citation Ma, H., & Gollahon, L. (2016). ERα Mediates Estrogen-Induced Expression of the Breast Cancer Metastasis Suppressor Gene BRMS1. International Journal of Molecular Sciences, 17(2), 158. MDPI AG. Retrieved from http://dx.doi.org/10.3390/ijms17020158 Citable Link http://hdl.handle.net/2346/66800 Terms of Use CC-BY 4.0

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International Journal of Molecular Sciences

Article ERα Mediates Estrogen-Induced Expression of the Breast Cancer Metastasis Suppressor Gene BRMS1

Hongtao Ma and Lauren S. Gollahon *

Department of Biological Sciences, Texas Tech University, 2901 Main St. Suite 108, Lubbock, TX 79409, USA; [email protected] * Correspondence: [email protected]; Tel.: +1-806-834-3287; Fax: +1-806-742-2963

Academic Editor: William Chi-shing Cho Received: 16 December 2015; Accepted: 18 January 2016; Published: 26 January 2016

Abstract: Recently, estrogen has been reported as putatively inhibiting cancer cell invasion and motility. This information is in direct contrast to the paradigm of estrogen as a tumor promoter. However, data suggests that the effects of estrogen are modulated by the receptor isoform with which it interacts. In order to gain a clearer understanding of the role of estrogen in potentially suppressing breast cancer metastasis, we investigated the regulation of estrogen and its receptor on the downstream target gene, breast cancer metastasis suppressor 1 (BRMS1) in MCF-7, SKBR3, TTU-1 and MDA-MB-231 breast cancer cells. Our results showed that estrogen increased the transcription and expression of BRMS1 in the ERα positive breast cancer cell line, MCF-7. Additionally, the ERα speciﬁc agonist PPT also induced the transcription and expression of BRMS1. However, the two remaining estrogen receptor (ER) subtype agonists had no effect on BRMS1 expression. In order to further examine the inﬂuence of ERα on BRMS1 expression, ERα expression was knocked down using siRNA (siERα). Western blot analysis showed that siERα reduced estrogen-induced and PPT-induced BRMS1 expression. In summary, this study demonstrates estrogen, via its α receptor, positively regulates the expression of BRMS1, providing new insight into a potential inhibitory effect of estrogen on metastasis suppression.

Keywords: breast cancer; metastasis; estrogen; estrogen receptor; BRMS1

1. Introduction Breast cancer is the leading female cancer in the United States and the leading cause of cancer-associated death in women. One of the risk factors associated with breast cancer development is a woman’s lifetime exposure to estrogen [1,2]. Estrogen performs its function through its estrogen receptor (ER) in target cells [3]. However, prior (and current) studies gauge the effect of estrogen on the ER in general; not delineated based on the different subtypes. Evidence is slowly emerging that demonstrate distinctions in the functions of ER subtypes with respect to cancer [4]. Our study looked speciﬁcally at the effect of the estrogen on the known ER subtypes to determine if there was a correlation with activation of a putative down-stream target involved in metastasis. Two types of estrogen receptors exist. The nuclear estrogen receptor (nER) is a member of the nuclear hormone family of intracellular receptors. nERs exist as two isoforms, α and β. The estrogen G protein coupled receptor GPR30 is a cytoplasmic receptor, which is predominantly associated with the endoplasmic reticulum [5,6]. Upon estrogen binding, several sites along the nER undergo phosphorylation and subsequent conformational changes freeing the receptor from the Hsp90 aggregate. At this point, the activated receptor subunits dimerize and bind to the estrogen response element (ERE) in the promoter regions of target genes in the nucleus [7–9]. In addition, the nER may also regulate gene expression by binding to transcriptional complexes on other promoter sites,

Int. J. Mol. Sci. 2016, 17, 158; doi:10.3390/ijms17020158 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2016, 17, 158 2 of 15 e.g., Sp1 or AP1 [10–15]. In contrast, upon estrogen binding, the membrane-bound estrogen receptor Int. J. Mol. Sci. 2016, 17, 158 2 of 15 GPR30 activates G proteins, which in turn activate adenyl cyclases, Src, and SphK. These kinases mediateSp1 subsequentor AP1 [10–15]. transcriptional In contrast, responsesupon estrogen [16– binding,18]. the membrane-bound estrogen receptor ItGPR30 is well activates known G that proteins, estrogen which promotes in turn activate the proliferation adenyl cyclases, of ER positiveSrc, and SphK. cancer These cells bothkinasesin vivo and inmediate vitro [19 subsequent–25]. In contrast, transcriptional other studiesresponses have [16–18]. reported estrogen-induced inhibition of cancer cell invasion andIt is motilitywell known [23 that,26– estrogen28]. However, promotes the the mechanism proliferation of of this ER inhibitory positive cancer effect cells remains both in unknown. vivo Recentand data in vitro suggest [19–25]. that In the contrast, ER may other mediate studies transcription have reported of estrogen-induced metastasis-associated inhibition genes, of cancer e.g., nm23, cell invasion and motility [23,26–28]. However, the mechanism of this inhibitory effect remains KAI1, KiSS1, and MTA [29–33]. Breast cancer metastasis suppressor 1 (BRMS1), a known metastasis unknown. Recent data suggest that the ER may mediate transcription of metastasis-associated genes, suppressore.g., nm23, gene, KAI1, has KiSS1, been shownand MTA to [29–33]. suppress Breast metastasis cancer metastasis of a variety suppressor of tumor 1 (BRMS1), cell lines, a known including breast,metastasis melanoma, suppressor bladder, gene, ovarian has been and shown lung [to34 supp–38].ress Several metastasis studies of a have variety indicated of tumor a cell possible lines, link betweenincluding the ER breast, and melanoma, BRMS1. One bladder, study, ovarian involving and lung 161 breast[34–38]. carcinoma Several studies specimens, have indicated showed a that the levelpossible of BRMS1link between mRNA the expressionER and BRMS1. was One marginally study, involving higher in161 ER breastα-positive carcinoma group specimens, than in the ERα-negativeshowed that group the [level39]. Anof BRMS1 analysis mRNA of 238 expression tissue samples was marginally by microarray higher showed in ERα-positive a trend forgroup BRMS1 towardsthan a in positive the ERα association-negative group with [39]. the ERAn [analysis40]. Frolova of 238et tissue al. [41 samples] found by that microarray nuclear showed expression a of BRMS1trend was for positively BRMS1 towards correlated a positive with expression association of with ER. ER-negativethe ER [40]. Frolova breast cancers et al. [41] were found signiﬁcantly that morenuclear likely thanexpression ER-positive of BRMS1 cancers was positively to have low correl BRMS1,ated with and expression loss of nuclear of ER. BRMS1ER-negative was associatedbreast cancers were significantly more likely than ER-positive cancers to have low BRMS1, and loss of with ER-negative cancers. Based on this information, we investigated whether BRMS1 expression nuclear BRMS1 was associated with ER-negative cancers. Based on this information, we investigated in breastwhether cancer BRMS1 cells expression was mediated in breast through cancer cells a speciﬁc was mediated estrogen through receptor a specific in response estrogen receptor to estrogen. Our resultsin response demonstrate to estrogen. that Our BRMS1 results expressiondemonstrate isth upregulatedat BRMS1 expression by estrogen is upregulated stimulation by estrogen through its ERα receptor.stimulation through its ERα receptor.

2. Results2. Results

2.1. ER2.1. Subtype ER Subtype Expression Expression in MCF-7,in MCF-7, TTU-1, TTU-1, MDA-MB-231, MDA-MB-231, and and SKBR3 SKBR3 Cell Cell Lines Lines In orderIn order to test to thetest effects the effects of ER of inﬂuence ER influence on BRMS1, on BRMS1, the ERthe subtypeER subtype expression expression in MCF-7,in MCF-7, TTU-1, MDA-MB-231,TTU-1, MDA-MB-231, and SKBR3 and cell SKBR3 lines wascell lines investigated was investigated by Western by Western analysis. analysis. Results Results showed showed that the expressionthat the of expression the ER subtype of thewas ER dependentsubtype was upon depende thent characteristics upon the characteristics of the individual of the cancerindividual cell line (Figurecancer1). MCF-7 cell line cells, (Figure (histopathologically 1). MCF-7 cells, (histopathologically ER+) expressed ER+) all three expressed ER subtypes, all three ER ER subtypes,α, ERβ, and ERα, ERβ, and GPR30. Neither TTU-1 (histopathologically ER−) nor MDA-MB-231 GPR30. Neither TTU-1 (histopathologically ER´) nor MDA-MB-231 (histopathologically ER´) cells (histopathologically ER−) cells expressed ERα. SKBR3 cells (histopathologically ER−) only expressed α ´ expressedGPR30 ER (Figure. SKBR3 1). Based cells (histopathologicallyon protein expression ERlevels,) only MCF-7, expressed TTU-1 and GPR30 SKBR3 (Figure were1 ).further Based on proteinanalyzed. expression levels, MCF-7, TTU-1 and SKBR3 were further analyzed.

Figure 1. Protein expression levels for breast cancer cells demonstrating different ER subtypes. Figure 1. Protein expression levels for breast cancer cells demonstrating different ER subtypes. Western Western analysis was performed against ERα, ERβ, GPR30 on MCF-7, MDA-MB-231, TTU-1, and analysis was performed against ERα, ERβ, GPR30 on MCF-7, MDA-MB-231, TTU-1, and SKBR3 cells SKBR3 cells using 40 µg of total cell lysate. α-tubulin was used as a loading control. Upon detection, usingprotein 40 µg ofwas total quantitated cell lysate. usingα -tubulinImage J. was used as a loading control. Upon detection, protein was quantitated using Image J.

2.2. E2 and the ERα Agonist PPT Induced BRMS1 Expression in MCF-7 Cells 2.2. E and the ERα Agonist PPT Induced BRMS1 Expression in MCF-7 Cells 2 Since DMSO was used as the vehicle to dissolve E2, PPT, DPN and G-1 (for full name details, see abbreviations list) a control experiment was performed with cells treated at the same DMSO Since DMSO was used as the vehicle to dissolve E2, PPT, DPN and G-1 (for full name details, see abbreviations list) a control experiment was performed with cells treated at the same Int. J. Mol. Sci. 2016, 17, 158 3 of 15 Int. J. Mol. Sci. 2016, 17, 158 3 of 15 Int. J. Mol. Sci. 2016, 17, 158 3 of 15 DMSOconcentration concentration used for used chemical for chemical dissolution, dissolution, over the same over time the points. same timeResults points. show insignificant Results show concentration used for chemical dissolution, over the same time points. Results show insignificant insignificanteffects of effectsDMSO of versus DMSO untreatedversus untreated cells (Figure cells (Figure S1). Additionally, S1). Additionally, these these control control experiments experiments effects of DMSO versus untreated cells (Figure S1). Additionally, these control experiments demonstrateddemonstrated no no significant significant changes changes inin BRMS1BRMS1 expression levels levels in in cells cells treated treated with with DMSO DMSO for for all all demonstrated no significant changes in BRMS1 expression levels in cells treated with DMSO for all timetime points points (Figure (Figure S1). S1). Therefore, Therefore, DMSO DMSO 2 h controls2 h controls were were used used to demonstrate to demonstrate background background BRMS1 time points (Figure S1). Therefore, DMSO 2 h controls were used to demonstrate background BRMS1 levels throughout the remainder of the experiment. levelsBRMS1 throughout levels throughout the remainder the remainder of the experiment. of the experiment. ToTo determine determine whether whether BRMS1 BRMS1 expression expression was modulatedwas modulated in response in response to E , cellsto E were2, cells incubated were To determine whether BRMS1 expression was modulated in response2 to E2, cells were incubated with culture media containing 10 nM E2, the endogenous estrogen receptorKd agonist (Kd = withincubated culture mediawith culture containing media 10 containing nM E2, the 10 endogenousnM E2, the endogenous estrogen receptor estrogen agonist receptor ( agonist= 0.1 nM(Kd and= 0.1 nM and 0.4 nM for ERα and ERβ, respectively [42]) for 48 h. It is also a high affinity ligand for the 0.40.1 nM nM for and ER 0.4α and nM ERforβ ER, respectivelyα and ERβ, respectively [42]) for 48 [42]) h. It for is also48 h. a It high is also affinity a high ligandaffinity for ligand the estrogenfor the estrogen membrane receptor GPR30 (Kd = 9.0 nM) [43]. Western analysis demonstrated that 10 nM E2 membraneestrogen receptormembrane GPR30 receptor (Kd GPR30= 9.0 nM) (Kd [=43 9.0]. WesternnM) [43]. analysis Western demonstrated analysis demonstrated that 10 nM that E 210increased nM E2 increased BRMS1 expression prior to 24 h in MCF-7 cells (Figure 2a). Compared to DMSO, the BRMS1increased expression BRMS1 priorexpression to 24 hprior in MCF-7to 24 h cells in MCF-7 (Figure cells2a). (Figure Compared 2a). Compared to DMSO, to the DMSO, expression the expression of BRMS1 was significantly increased at 8, 16, and 24 h (p ≤ 0.05) (Figure 2b). However, ofexpression BRMS1 was of significantlyBRMS1 was significantly increased at increased 8, 16, and at 8, 24 16, h (andp ď 240.05) h (p (Figure ≤ 0.05) 2(Figureb). However, 2b). However, BRMS1 BRMS1 expression did not significantly change in SKBR3 (Figure 3) or TTU-1 (Figure S2) cells in expressionBRMS1 expression did not significantly did not significantly change in SKBR3change (Figure in SKBR33) or (Figure TTU-1 (Figure3) or TTU-1 S2) cells (Figure in response S2) cells to in E 2 response to E2 (10 nM–1 µM) for up to 48 h. These results suggested that E2 positively regulates (10response nM–1 µ M)to forE2 (10 up nM–1 to 48 h.µM) These for resultsup to 48 suggested h. These thatresults E suggestedpositively that regulates E2 positively BRMS1 regulates expression BRMS1 expression via the ERα. 2 viaBRMS1 the ER αexpression. via the ERα.

FigureFigure 2. Western2. Western analysis analysis of of BRMS1 BRMS1 protein proteinexpression expression induced by E E22 inin MCF-7 MCF-7 cells. cells. (a ()a Cells) Cells were were Figure 2. Western analysis of BRMS1 protein expression induced by E2 in MCF-7 cells. (a) Cells were incubatedincubated with with culture culture media media containing containing 1010 nMnM EE22 for 2, 2, 4, 4, 8, 8, 16, 16, 24, 24, and and 48 48 h. h. Control Control cells cells were were incubated with culture media containing 10 nM E2 for 2, 4, 8, 16, 24, and 48 h. Control cells were incubatedincubated with with culture culture media media containing containing 1010 nMnM DMSODMSO for 2 2 h. At At each each time time interval, interval, cells cells were were incubated with culture media containing 10 nM DMSO for 2 h. At each time interval, cells were harvestedharvested and and 20 20µ gµg of of the the total total protein protein waswas loadedloaded for each sample sample to to determine determine BRMS1 BRMS1 expression expression harvested and 20 µg of the total protein was loaded for each sample to determine BRMS1 expression byby Western Western blot. blot.α -tubulinα-tubulin was was used used as as the the loadingloading control.control. Each Each band band present present was was quantified quantiﬁed using using by Western blot. α-tubulin was used as the loading control. Each band present was quantified using Image J and a set area encompassing the BRMS1 band was divided by the same area forα α-tubulin ImageImage J and J and a set a set area area encompassing encompassing the the BRMS1 BRMS band1 band was was divided divided by theby the same same area area for for-tubulin α-tubulin from from the same lane to determine the relative amount of BRMS1 expression. The DMSO value was set thefrom same the lane same to determinelane to determine the relative the relative amount am ofount BRMS1 of BRMS1 expression. expression. The DMSO The DMSO value value was set was to set 1 for to 1 for comparisons; (b) The bar graph summarizes BRMS1 protein normalized to α-tubulin from comparisons;to 1 for comparisons; (b) The bar (b graph) The bar summarizes graph summarizes BRMS1 protein BRMS1 normalizedprotein normalized to α-tubulin to α-tubulin from the from same the same lane in three independent experiments. Error bars were the average of three independent lanethe in same three lane independent in three independent experiments. experiments. Error bars wereError the bars average were the of threeaverage independent of three independent experiments experiments ± SD. * indicates statistical significance at the p < 0.05 level compared to DMSO. ˘ SD.experiments * indicates ± SD. statistical * indicates signiﬁcance statistical at significance the p < 0.05 at levelthe p compared< 0.05 level to compared DMSO. to DMSO.

Figure 3. Cont. FigureFigure 3.3. Cont.

Int. J. Mol. Sci. 2016, 17, 158 4 of 15 Int. J. Mol. Sci. 2016, 17, 158 4 of 15 Int. J. Mol. Sci. 2016, 17, 158 4 of 15

Figure 3. Analysis of BRMS1 protein expression induced by E2 in SKBR3 cells. (a) SKBR3 cells were

FigureincubatedFigure 3. Analysis3. with Analysis culture of of BRMS1 BRMS1 media protein proteicontainingn expression expression 10 nM inducedinduced E2 for 2, by 4, E 8,22 in in16, SKBR3 SKBR3 24, and cells. cells. 48 ( ah. ()a SKBR3)Control SKBR3 cells cellscells were werewere incubatedincubatedincubated withwith with cultureculture culture mediamedia media containing containing 10 10 nM nM DMSO; EE22 for 2, 2, ( b4, 4,) 8, 8,Bar 16, 16, graph 24, 24, and and summarizes 48 48 h. h. Control Control BRMS1 cells cells wereprotein were incubatednormalizedincubated with withto α culture culture-tubulin media media from containing containing the same 10 10lane nMnM from DMSO;DMSO; three ( (b)) Barindependent Bar graph graph summarizes summarizes experiments BRMS1 BRMS1 from protein protein(a); (c)

normalizedSKBR3normalized cells to were αto -tubulinα incubated -tubulin from from with the the sameculture same lane media lane from fromcontaining three three independent independent100 nM E experiments2 or experiments 1 µM E from2 for from4, (a );8, ( cand()a); SKBR3 (16c) h. cellsControlSKBR3 were cells cells incubated werewere incubated with culture withwith media cultureculture containing media media containing containing 100 nM 100 E 2100 ornM 1nM Eµ2ME orDMSO 12 µMfor 4,forE2 8, for 2 and h;4, (8, 16d )and h.Bar Control 16 graph h. cellssummarizesControl were incubatedcells BRMS1 were withincubatedprotein culture normalized with media culture containing to mediaα-tubulin containing 100 from nM DMSO the100 samenM for DMSO 2lane h; (d from )for Bar 2 graphthreeh; (d) independent summarizesBar graph BRMS1experimentssummarizes protein from BRMS1 normalized (c). proteinAt each to normalized timeα-tubulin interval, fromto αcells-tubulin the were same from harvested lane the from same and three lane 20 µg independentfrom of thethree total independent experiments protein was fromloadedexperiments (c ).for At each each from sample time (c). interval, Atto determineeach time cells interval, wereBRMS1 harvested cellsexpression were and harvested by 20 Westernµg ofand the blot.20 totalµg For of protein theall samples,total was protein loaded α-tubulin was for eachwasloaded used sample for as toeachthe determine loading sample tocontrol. BRMS1 determine Each expression BRMS1 band presex bypression Westernent was by blot.quantified Western For allblot. using samples, For Image all samples,α-tubulin J and αgraphed-tubulin was used as was used as the loading control. Each band present was quantified using Image J and graphed as aspreviously the loading described. control. The Each bar band graphs present and was error quantified bars represent using Image the average J and graphed of three asindependent previously previously described. The bar graphs and error bars represent the average of three independent described.experiments The ± barS.D. graphs No statistical and error significance bars represent was theobserved. average of three independent experiments ˘ S.D. Noexperiments statistical significance ± S.D. No statistical was observed. significance was observed. To further validate this observation, cells were exposed to ER agonists to activate specific ER To further validate this observation, cells were exposed to ER agonists to activate specific ER subtypes.subtypes.To further PPT PPT is validate is an an ER ERα thisα selective selective observation, agonist,agonist, cells whereaswhereas were exposedDPNDPN isis anan to ER ER ββ agonistsselective selective toagonist agonist activate [44]. [44]. specific G-1 G-1 is ais ER a subtypes.knownknown agonist agonist PPT isfor for an GPR30 ERGPR30α selective [16]. [16]. ChangesChanges agonist, inin whereasBRMS1 expressionexpression DPN is an werewere ERβ determined selectivedetermined agonist by by immunoblot. immunoblot. [44]. G-1 is aResults knownResults showed agonistshowed that forthat GPR3010 10 nM nM PPT [16PPT]. increased Changesincreased inBRMS1 BRMS1 expressionpression expression for for wereup up to to determined24 24 h hin in MCF-7 MCF-7 by (Figure immunoblot. (Figure 4a). 4a). ResultsComparedCompared showed with with thatDMSO DMSO 10 nMcontrols, controls, PPT increased thethe expressionexpression BRMS1 of expressionBRMS1BRMS1 waswas forsignificantly significantly up to 24 hincreased inincreased MCF-7 at (Figureat16 16h (hp 4≤(a).p ≤ Compared0.05)0.05) (Figure (Figure with 4a). 4a). DMSO However, However, controls, the the ER theERββexpression and and GPR30GPR30 of agonists BRMS1 DPN wasDPN significantlyand and G-1, G-1, respectively, respectively, increased did at did 16 not hnot (changep changeď 0.05) (FigureBRMS1BRMS14 a).expression expression However, in in the these these ER ERβ ERand subtype subtype GPR30 positivepositive agonists cells DPN forfor and upup toG-1,to 48 48 respectively,h. h. Incubation Incubation did of of MCF-7 not MCF-7 change cells cells BRMS1with with expressionDPNDPN and and G-1 in G-1 these at at concentrations ERconcentrations subtype positive rangingranging cells fromfrom for up10 tonM 48 toto h. 11 Incubation µMµM reflectedreflected of MCF-7similar similar cellsresults results with (data DPN(data not andnot G-1shown).shown). at concentrations These These results results ranging confirmed confirmed from ourour 10 nMinitialinitial to 1dataµM reflectedindicatingindicating similar thatthat ER ER resultsαα activation activation (data notinduced induced shown). BRMS1 BRMS1 These resultsexpression.expression. confirmed our initial data indicating that ERα activation induced BRMS1 expression.

Figure 4. Cont. FigureFigure 4.4. Cont.Cont.

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Figure 4. Western blotblot resultsresultsof ofPPT, PPT, DPN DPN and and G-1 G-1 effects effects on on induction induction of of BRMS1 BRMS1 protein protein expression expression in MCF-7in MCF-7 cells. cells. MCF7 MCF7 cells cells were were incubated incubated with with culture culture media media containing containing (a) 10 (a nM) 10 PPT; nM PPT; (b) 10 (b nM) 10 DPN nM orDPN (c) 100or (c nM) 100 G-1 nM for G-1 2, 4, for 8, 16,2, 4, 24 8, and 16, 48 24 h. and Control 48 h. cellsControl were cells incubated were incubated with culture with media culture containing media 10containing nM DMSO 10 (anM, b )DMSO or 100 nM(a, (bc) foror 2100 h. ProteinsnM (c) for were 2 quantitatedh. Proteins aswere previously quantitated described. as previously Graphs anddescribed. error barsGraphs represent and error the averagebars represent of three the independent average of experimentsthree independent˘ SD. *experiments indicates statistical ± SD. * signiﬁcanceindicates statistical at the p significance< 0.05 level comparedat the p < 0.05 to DMSO. level compared to DMSO.

2.3. E 2 andand the the ER ERαα AgonistAgonist PPT PPT Increased Increased BRMS1 BRMS1 Transcription Transcription in MCF-7 Cells The next question investigated was whether the re resultssults observed at the protein level were also evident atat thethe RNARNA level.level. ToTo address address this, this, real-time real-time quantitative quantitative PCR PCR (RT-PCR) (RT-PCR) was was used used to analyze to analyze the effectthe effect of E 2ofand E2 and PPT PPT on BRMS1 on BRMS1 transcription. transcription. Since Since there th areere multiple are multiple splice splice variants variants of BRMS1 of BRMS1 listed inlisted the NCBIin the database,NCBI database, we chose we to chose analyze to analyze the BRMS1 the BRMS1 transcript transcript NM_015399, NM_015399, which was which first was reported first andreported encodes and the encodes most abundant the most transcript abundant [34]. Additionally,transcript [34]. the Additionally, BRMS1 transcript the NM_001024957BRMS1 transcript uses anNM_001024957 alternative splice uses sitean inalternative exon 10. Thus,splice two site pairs in exon of BRMS1 10. Thus, primers, two BRMS1-V1 pairs of BRMS1 and BRMS1-V12 primers, wereBRMS1-V1 analyzed and for BRMS1-V12 transcriptional were activation analyzed of for BRMS1 transcriptional in MCF-7 cells activation following of BRMS1 incubation in MCF-7 with culture cells mediafollowing containing incubation 10 nMwith E 2cultureand PPT. media Optimization containing experiments10 nM E2 and included PPT. Optimization an initial time experiments course of 1,included 2, 4, 8 and an 16initial h (Figure time 5course). Results of 1, showed 2, 4, 8 that and 10 16 nM h E(Figure2 increased 5). Resu BRMS1lts showed expression that up10 tonM 16 E h2 (Figureincreased5a,b). BRMS1 At 8 h,expression the mRNA up level to of16BRMS1 h (Figure was 5a,b). significantly At 8 h, increased the mRNA for bothlevel pairs of BRMS1 of primers. was Theresignificantly was also increased a significant for both increase pairs of BRMS1of primers. transcription There was at 2also h for a BRMS1-V1significant primers.increase Inof contrast,BRMS1 wetranscription observed attenuationat 2 h for BRMS1-V1 in the time primers. to significantly In contrast, increased we observed mRNA levels attenuation of BRMS1 in tothe 4 andtime 8to h forsignificantly the BRMS1-V12 increased primers mRNA (Figure levels5a). of Furthermore,BRMS1 to 4 and the 8 highest h for the transcription BRMS1-V12 peak primers appeared (Figure at 85a). h forFurthermore, both pairs the of primers highest (Figuretranscript5a,b).ion Moreover,peak appeared RT-PCR at 8 results h for both showed pairs that of primers 10 nM PPT (Figure increased 5a,b). BRMS1Moreover, expression RT-PCR upresults to 16 showed h as well. that At 10 2, nM 4, andPPT 8 in h,creased the mRNA BRMS1 level expression of BRMS1 up was to significantly16 h as well. increasedAt 2, 4, and for both8 h, the sets mRNA of primers level (Figure of BRMS15c,d) with was thesignificantly highest transcription increased peakfor both occurring sets of at primers 4 h for both(Figure primers 5c,d) with (Figure the5 c,d).highest transcription peak occurring at 4 h for both primers (Figure 5c,d).

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Figure 5.5. BRMS1 transcription was induced by treatment with E2 or PPT. BRMS1 transcription for BRMS1 V12 primers (a)) andand BRMS1BRMS1 V1V1 primersprimers ((b)) afterafter treatmenttreatment withwith 1010 nMnM EE22 demonstrated peak induction at 8 h;h; BRMS1BRMS1 transcriptiontranscription for BRMS1 V12 primers (c) and BRMS1 V1 primers (d) afterafter treatment withwith 1010 nMnM PPT showed much earlier peak induction between 2 and 4 h. Values Values with error bars are the average of three independent experiment experimentss ±˘ SD.SD. * *indicates indicates statistical statistical significance signiﬁcance at at the the p p< <0.05, 0.05, compared compared to to DMSO. DMSO.

2.4. siRNA Inhibition of ERα Expression Decreased E2 and PPT-Induced BRMS1 Expression

To investigateinvestigate BRMS1BRMS1 expressionexpression inin responseresponse toto EE22 and PPT-inducedPPT-induced activationactivation followingfollowing ERα knockdown, MCF-7 cells cells were were transfected transfected with with 100 100 nM nM siER siERα αforfor 48 48 h followed h followed by by treatment treatment with with 10 10nM nM E2 Efor2 for 24 24h or h or10 10 nM nM PPT PPT for for 16 16 h. h. Results Results are are shown shown in in Figure Figure 6.6. DMSO was used asas control.control. Control cellscells werewere incubated incubated in in the the same same volume volume of transfectionof transfection reagent reagent without without siER siERα andα treatedand treated with Ewith2 or E PPT2 or followingPPT following the same the same conditions conditions described descri above.bed above. Western Western analysis anal showedysis showed that DMSOthat DMSO and SiRNAand SiRNA control control with DMSOwith DMSO did not did affect not ER αaffectexpression ERα expression (Figure6a,b). (Figure Negative 6a,b). control Negative experiments control wereexperiments also performed were also using performed siRNA-A using (Santa siRNA-A Cruz, Santa(Santa Cruz, Cruz, LA, Santa USA) Cruz, to demonstrateLA, USA) to speciﬁcitydemonstrate of siERspecificityα target of effects siERα (Figure target S3).effects In the (Figure presence S3). of In E2 theand presence PPT, ERα ofexpression E2 and PPT, remained ERα unchanged,expression butremained BRMS1 unchanged, expression but increased BRMS1 as expression previously increased observed. as With previously the addition observed. of siER Withα knockdown, the addition both of EsiER2-inducedα knockdown, BRMS1 expressionboth E2-induced and PPT-induced BRMS1 expression BRMS1 and expression PPT-induced was signiﬁcantly BRMS1 expression decreased was by ~40%significantly and 60%, decreased respectively by ~40% (p ď and0.05) 60%, (Figure respectively6a,b). Taken (p ≤ together,0.05) (Figure these 6a,b). results Taken demonstrate together, these that ERresultsα contributed demonstrate to the that changes ERα contributed in BRMS1 to expression the changes induced in BRMS1 by E2 expression. induced by E2.

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FigureFigure 6.6. TheThe effectseffects ofof siERsiERαα onon BRMS1BRMS1 expressionexpression inin MCF-7MCF-7 cellscells afterafter treatmenttreatment withwith EE22 or PPT.

MCF-7MCF-7 cellscells werewere transfectedtransfected withwith siERsiERαα forfor 4848 hh andand thenthen treatedtreated withwith eithereither EE22,, PPT,PPT, oror DMSODMSO forfor 1616 h.h. siControlsiControl cellscells werewere incubatedincubated inin thethe samesame volumevolume ofoftransfection transfection reagentreagent withoutwithout siERsiERαα.. EachEach lanelane waswas loadedloaded withwith 2020 µµgg andand membranesmembranes werewere probedprobed withwith monoclonalmonoclonal antibodiesantibodies againstagainst ERERαα,, BRMS1,BRMS1, oror αα-tubulin-tubulin asas loadingloading control.control. ControlsControls areare untransfecteduntransfected cellscells treatedtreated withwith DMSO,DMSO, PPTPPT oror α EE22.(. (a) Western blotblot demonstratingdemonstrating knockdownknockdown ofof ERERα andand correspondingcorresponding BRMS1BRMS1 proteinprotein expressionexpression

fromfrom MCF-7MCF-7 cellscells treatedtreated withwith 1010 nMnM EE22.. TheThe barbar graphgraph ((rightright panelpanel)) summarizessummarizes BRMS1BRMS1 proteinprotein normalizednormalized totoα -tubulinα-tubulin from from control control and SiERandα SiERfromα the from same the lane same in three lane independent in three experiments;independent (experiments;b) Western blot (b) demonstratingWestern blot demonstrating knockdown ofknockdown ERα and correspondingof ERα and corresponding BRMS1 protein BRMS1 expression protein fromexpression MCF-7 from cells MCF-7 treated withcells 10treated nM PPT.with The 10 barnM graphPPT. The (right bar panel graph) summarizes (right panel BRMS1) summarizes protein normalizedBRMS1 protein to α-tubulin normalized from controlto α-tubulin and SiER fromα from control the same and laneSiER inα threefrom independent the same lane experiments. in three Valuesindependent with error experiments. bars were Values the average with error of three bars independent were the average experiments of three˘ independentSD. * indicates experiments statistical signiﬁcance at the p < 0.05, compared to siControl for E or PPT treatment. ± SD. * indicates statistical significance at the p < 0.05, compared2 to siControl for E2 or PPT treatment.

3.3. Discussion Discussion InIn thisthis study, study, we we investigated investigated whether whether expression expressi ofon theof breastthe breast cancer cancer metastasis metastasis suppressor suppressor gene BRMS1gene BRMS1 changed changed in response in response to estrogen to estrogen exposure. exposure. Additionally, Additionally, we also addressed we also theaddressed question the of whetherquestion aof specific whether ER a subtypespecific ER was subtype responsible was re forsponsible modulating for modulating BRMS1 expression. BRMS1 expression. Here, we report Here, forwe thereport first for time, the thatfirst time, BRMS1 that is BRMS1 a downstream is a downstream target in ERtarget positive in ER breastpositive cancer breast cells cancer and cells that and its expressionthat its expression is modulated is modulated by the ER αbyin the the presenceERα in the of estrogenpresence or of an estrogen ERα agonist. or an Our ERα results agonist. suggest Our thatresults estrogen suggest can that have estrogen a dual effectcan have on the a dual ERα. effect On one on hand, the ER it isα. knownOn one to hand, stimulate it is tumorknown cell to growthstimulate [45 tumor–47]. However, cell growth in this[45–47]. study, However, we demonstrate in this study, that it we may demonstrate also inhibit that tumor it may cell invasionalso inhibit by upregulationtumor cell invasion of the breastby upregula cancertion metastasis of the breast suppressor cancer gene metastasis BRMS1. suppressor gene BRMS1. ByBy treatingtreating breast breast cancer cancer cells cells with with differential differen expressiontial expression of the ERof subtypesthe ER subtypes with E2, wewith observed E2, we thatobserved the regulation that the of Eregulation2 on the metastasis of E2 on suppressor the metastasis gene BRMS1 suppressor induced gene a time-dependent BRMS1 induced increase a intime-dependent the amount of BRMS1increase protein in the producedamount of only BRMS1 in ER proteinα positive produced MCF-7 only cells. in This ER suggestedα positive thatMCF-7 E2, viacells. its Thisα receptor, suggested positively that E regulated2, via its theα receptor, expression positively of BRMS1. regulated In order the to confirmexpression this of finding, BRMS1. three In ERorder subtypes to confirm were this activated finding, using three receptor ER subtypes specific we agonistsre activated to determine using receptor whether specific BRMS1 expressionagonists to wasdetermine changed. whether Again, BRMS1 the expression expression ofBRMS1 was changed. only showed Again, a the time-dependent expression of increaseBRMS1 only in response showed to a time-dependent increase in response to the ERα agonist PPT. In contrast, agonists for ERβ (DPN)

Int. J. Mol. Sci. 2016, 17, 158 8 of 15 the ERα agonist PPT. In contrast, agonists for ERβ (DPN) and GPR30 (G-1) did not change BRMS1 expression in any of the cell lines tested. The effect of this E2-ERα-BRMS1 relationship was further shown to occur at the transcriptional level. RT PCR showed a corresponding increase in BRMS1 mRNA levels with either E2 or PPT treatment. However, this event occurred prior to BRMS1 protein expression, where mRNA increased from 1 to 8 h, but protein levels continued to increase until 16 h (PPT) or 24 h (E2). In eukaryotic cells, time-delayed gene expression is essential for the function of the circadian clock, somite-segmentation clock, and mitosis [48]. Time-delay is mainly associated with the regulation of protein stability by phosphorylation and ubiquitination [49]. The mechanisms involved in this process include proteasome-dependent proteolysis and negative feedback regulation [50]. Interestingly, others have reported a similar estrogen-triggered delay in gene expression as well [51,52]. Recent studies have shown that circadian clock gene expression is maintained in ER+, low metastatic breast cancers, whereas, the loss of circadian clock gene expression is associated with more aggressive breast cancers [53]. More study is warranted on the importance of this phenomenon with respect to BRMS1 activation. To further elucidate the influence of the ERα on the expression of BRMS1, ERα was knocked down by siRNA in MCF-7 cells. Western analysis showed that siERα reduced E2-induced and PPT-induced BRMS1 expression by approximately ~40% and 60%, respectively. These results confirmed the observation that ERα was responsible for the E2-induced BRMS1 expression in MCF-7 cells (summarized in Figure7). What are the potential consequences of these results? Studies have shown that ERα regulates target gene transcription as a transcriptional factor or co-regulator [7,9,10,14]. As a transcriptional factor, ERα binds to the ERE in the promoter region of the target gene [7,9]. To date, the ERE in the BRMS1 promoter has not been identified. Thus, ERα regulation on BRMS1 expression may not be as a transcriptional factor. However, the BRMS1 51 upstream region shows several putative regulatory elements, and one of them is the binding site for the cAMP response element (CRE)-binding protein (CREB) [36]. As a co-regulator, ERα may regulate gene expression by binding to transcriptional complexes on other promoter sites [10]. ERα has been shown to bind c-Jun/ATF proteins, which bind to the CRE of the cyclin D1 promoter, inducing its expression [15]. This suggests that ERα may induce BRMS1 expression by binding transcriptional complexes on the CRE in the promoter region of BRMS1. In addition, the UCSC Genome Browser has predicted a putative AP-1 binding site in the BRMS1 promoter. It has been shown that ERα upregulates gene transcription by binding the AP-1 complex on AP-1 response elements [11,12]. This information, taken together, suggests that ERα may induce BRMS1 expression through binding transcriptional complexes as a co-activator on other promoter sites (e.g., CRE and AP-1) instead of the ERE. Alternatively, BRMS1 may regulate ERα expression. Justification for this rationale is based on reports that ERα transcription was shown to be activated in human breast cancer cells by HDAC inhibition [54]. HDAC1 interacts with ERα in vitro and in vivo, and suppresses ERα transcription activity. This interaction was mediated by the AF-2 and DBD domains of ERα [55]. However, BRMS1 may interact with the mSin3:HDAC complex, including HDAC1 and HDAC2, reducing target gene transcription [56] For review of metastatic suppression of BRMS1 and Sin3 complexes, please see [57]. Lui et al. [58] reported that BRMS1 suppressed RelA/p65 transcriptional activity by promoting binding of HDAC1 to RelA/p65, where it deacetylates lysine K310 on RelA/p65. BRMS1 also negatively regulates uPA gene expression through recruitment of HDAC1 to the NF-κB binding site of the uPA promoter [59]. These findings indicate that BRMS1 could participate in HDAC inhibition on ERα expression. We have presented a putative model for the relationship(s) between ERα and BRMS1 in Figure7. There is still little known about the function of BRMS1, binding partners or its importance in suppressing metastasis. Recently, Hurst and colleagues have elucidated several important aspects of BRMS1 expression and its involvement in cellular adhesion towards suppressing metastasis [57,60,61]. Given the findings of our study, as well as previous studies discussed above, an important question that needs further investigation is whether the inhibitory effect of estrogen on developing metastases Int. J. Mol. Sci. 2016, 17, 158 9 of 15

is countered by its mitogenic effect. In other words, does estrogen promote proliferation at a greater rate than it inhibits metastasis? The study of ER subtypes and their effects is gaining momentum and recent studies address this issue [53]. Moreover, another emergent area involves the role of miRNA regulation of ER subtypes and implications towards metastasis [62], which may translate to a new strategy for assessing diagnosis, treatment and prognosis of patients with ER-positive breast cancer Int.and J. Mol. its associationSci. 2016, 17, 158 with metastasis. 9 of 15

FigureFigure 7. A proposedproposed model model for for the the relationship relationship between between ERα andERα BRMS1. and BRMS1. (a) The ( BRMS1a) The 5BRMS11 upstream 5′ upstreamregion shows region several shows putative several regulatoryputative regulatory elements, elements, one of them one is of the them binding is the site binding for CREB. site ERforα CREB.may regulate ERα may gene regulate expression gene asexpression a co-regulator as a co on-regulator other promoter on other sites. promoter ERα has sites. been ER shownα has to been bind shownc-Jun/ATF to bind proteins, c-Jun/ATF which proteins, bind to which the CRE bind of theto the cyclin CRE D1 of promoter, the cyclin inducing D1 promoter, its expression. inducing This its expression.suggests that This ER αsuggestsmay induce that BRMS1ERα may expression induce byBRMS1 binding expression transcriptional by binding complexes transcriptional on the CRE complexesin the promoter on the regionCRE in of the BRMS1; promoter (b) Itregion has been of BRMS1; shown ( thatb) It ERhasα beenupregulates shown that gene ER transcriptionα upregulates by genebinding transcription the AP-1 complexby binding on the AP-1 AP-1 response complex elements. on AP-1 This response information, elements. taken This together, information, suggests taken that together,ERα may suggests induce BRMS1 that ER expressionα may induce through BRMS1 binding expression transcriptional through complexes binding as atranscriptional co-activator on complexesother promoter as a co-activator sites (e.g., CRE on other and AP-1).promoter sites (e.g., CRE and AP-1).

4. ExperimentalThere is still little Section known about the function of BRMS1, binding partners or its importance in suppressing metastasis. Recently, Hurst and colleagues have elucidated several important aspects of BRMS14.1. Cell expression Culture and Treatmentand its involvement in cellular adhesion towards suppressing metastasis [57,60,61].MCF-7, Given SKBR3 theand findings MDA-MB-231 of our study, breast canceras well cells as wereprevious purchased studies from discussed ATCC above, (Manassas, an importantVA, USA). question TTU-1 isthat a primary, needs further invasive investigation ductal cancer is whether cell line the derived inhibitory in our effect lab [of63 ].estrogen These cellon developinglines were chosenmetastases for analysis is countered based by on its differences mitogeni inc effect. ER expression In other profiles.words, does MCF-7 estrogen and TTU-1 promote cells proliferationwere cultured at ina greater Dulbecco’s rate than Modified it inhibits Eagle’s metastasis? Medium. The SKBR3 study cells of ER were subtypes maintained and their in phenol effects red is gainingfree McCoy’s momentum 5A Medium and recent (Modified). studies MDA-MB-231address this issue cells were[53]. grownMoreover, in Leibovitz’s another emergent L-15 medium. area involvesAll media the were role phenolof miRNA red-free regulation and supplemented of ER subtypes with and 10% implications fetal bovine towards serum metastasis and 50 µg/mL [62], which may translate to a new strategy for assessing diagnosis, treatment and prognosis of patients gentamycin sulfate. Cells were treated with 10 nM–1 µME2, PPT, DPN, and G-1 (see chemical reagents withbelow) ER-positive over 48 h. breast DMSO cancer was usedand its as association the vehicle with control metastasis. at the same concentrations and collected at the same time points to delineate vehicle effects from treatment effects on BRMS1 expression. Control 4. Experimental Section results are in Supplementary Materials (Figure S1). 4.1. Cell Culture and Treatment MCF-7, SKBR3 and MDA-MB-231 breast cancer cells were purchased from ATCC (Manassas, VA, USA). TTU-1 is a primary, invasive ductal cancer cell line derived in our lab [63]. These cell lines were chosen for analysis based on differences in ER expression profiles. MCF-7 and TTU-1 cells were cultured in Dulbecco’s Modified Eagle’s Medium. SKBR3 cells were maintained in phenol red free McCoy’s 5A Medium (Modified). MDA-MB-231 cells were grown in Leibovitz’s L-15 medium. All media were phenol red-free and supplemented with 10% fetal bovine serum and 50 µg/mL

Int. J. Mol. Sci. 2016, 17, 158 10 of 15

4.2. Chemical Reagents and Antibodies ERα-selective agonist (propyl-pyrazole-triol (PPT)), ERβ-selective agonist (diarylpropionitrile (DPN)), and the GPR30 agonist were purchased from Tocris (Bristol, UK), (referred to as G-1) (˘)-1-[(3aR*,4S*,9bS*)-4-(6-Bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quino lin-8-yl]- ethanone. Estradiol (E2) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies were purchased as follows: mouse monoclonal anti-human ERα and anti-human ERβ from GeneTex Inc. (Irvine, CA, USA); rabbit polyclonal anti-human GPR30 from Santa Cruz; mouse monoclonal anti-human BRMS1 from Sigma-Aldrich and Santa Cruz; mouse monoclonal anti-human α-tubulin from Abcam (Cambridge, MA, USA).

4.3. Western Blot Analysis Total protein (20–40 µg/lane) was separated on an 8% or 10% polyacrylamide SDS gel and transferred to a nitrocellulose membrane (Applied Biosystems, Waltham, MA, USA). The membranes were incubated with primary antibodies overnight at 4 ˝C at the following dilutions: ERα 1:1000; ERβ 1:1000; GPR30 1:200; BRMS1 1:300 (Santa Cruz) and 1:1000 (Sigma); α-tubulin 1:10,000. After washing, the membranes were incubated with goat anti-mouse HRP-conjugated secondary antibody (1:5000) or goat anti-rabbit HRP-conjugated secondary antibody (1:5000), for 1 h at room temperature. A chemiluminescent substrate kit (Pierce, Waltham, MA, USA) was used for signal detection on X-ray ﬁlm. Each band present was quantiﬁed using Image J [64], (National Institutes of Health, Bethesda, MD, USA). A set area encompassing the BRMS1 band was divided by the same area for α-tubulin from the same lane to determine the relative amount of BRMS1 expression. The DMSO value was set to 1 for comparison.

4.4. RNA Isolation and Quantitative RT-PCR Total RNA was extracted from cells using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA). Five micrograms of RNA was reverse transcribed to cDNA using a cDNA synthesis kit (Invitrogen, Waltham, MA, USA) according to the manufacturer’s instructions. Reverse transcription products were then ampliﬁed using a SYBR Green Master Mix (Applied Biosystems) for real-time PCR. BRMS1 (NM_015399) was ﬁrst reported and encodes the most abundant transcript [34]. BRMS1 (NM_001024957) uses an alternative splice site in exon 10. Using the SciTool software program, two pairs of BRMS1 primers were designed based on its transcripts, variant 1 and variant 2. The sequence of variant 2 is completely covered by variant 1. One pair of primers (BRMS1 V1) was suitable only to variant 1, whereas the other pair of primers (BRMS1 V12) was suitable to both variant 1 and 2. The primer pair was selected for a Tm at approximately 60 ˝C, amplicon of less than 150 bp, and low primer penalty. Primers sequences compared against the human genome databank using a “Blast” search revealed that all of these three pair primers were unique for Homo sapiens BRMS1 or GAPDH BRMS1-V1 and BRMS1-V12 primer sequences were as follows:

BRMS1-V12: Forward-51-ATTCAGGTGGCAGGGATCTACAAGG-31 BRMS1-V12: Reverse-51-GCAGCAGCTTCTCACTCTCCA-31 BRMS1-V1: Forward-51-CGACATCCTGGAGGACTGGACAGCCA-31 BRMS1-V1: Reverse-51-TGAACAGCAGGGTCAAGGTCCATCCGA-31

The GAPDH primers used for loading control were:

Forward-51-CACCACCATGGAGAAGGCTG-31 Reverse-51-GAGGCATTGCTGATGATCTTGAGG-31

The BRMS1 and GAPDH primers were diluted to a ﬁnal concentration of 10 µM with RNase-free H2O before use. The 2X SYBR Green master mix (containing HotStart DNA Taq polymerase, ultrapure nucleotides, real-time PCR buffer, SYBR Green dye, and the passive reference dye ROX) (Applied Int. J. Mol. Sci. 2016, 17, 158 11 of 15

Biosystems), forward and reverse primers, cDNA template, and H2O were added based on the number of reactions (each 20 µL reaction: 10 µL SYBR Green Mix (2ˆ), 1 µL cDNA (20 ng/1 µL), 2 µL primer pair mix (10 µM each primer), and 7 µL RNase-free H2O) and mixed well. Twenty microliters of reaction solution was added to each well of a 96 well plate. Each reaction included three replicates. Three reactions without reverse transcriptase and three reactions without cDNA templates in each pair of primers were tested for genomic DNA contamination and general contamination. The real time Ct values should be greater than 35 for these negative controls. The Applied Biosystems 7500 real time PCR instrument (Biotech Core Facility, TTU, Lubbock, TX, USA) was used to run the PCR reaction plate. The two-step thermal cycling program was performed as follows: Real time PCR program:

Step 1: 10 min at 95 ˝C (enzyme activation) Step 2: 40 cycles each of 15 s at 95 ˝C (denature) and 1 min at 60 ˝C (anneal/extend).

The PCR program was followed by a dissociation program:

15 s at 95 ˝C, 1 min at 60 ˝C, and 15 s at 95 ˝C.

Fold change was calculated from the ∆∆Ct values and data were presented as relative expression to DMSO treated control cells. In order to determine BRMS1 transcription levels, the ∆∆Ct method was used.

1. Change all Ct values, which were reported as greater than 35 or N/A (undetermined) to 35. Any Ct value equal to 35 was considered a negative call. 2. Calculate the average Ct value of each time interval treatment across three replicates. 3. Calculate the ∆Ct for each time interval treatment in that group

∆Ct “ average Ct ´ average of GAPDH (housekeeping gene) for that group (1)

4. Calculate ∆∆Ct ∆∆Ct “ ∆Ct ´ ∆Ct control (2)

5. Calculate the fold-change

2´∆∆Ct “ 2´∆Ct experiment{2´∆Ct control (3)

If the fold-change is greater than 1, the result is considered as a fold upregulation. If the fold-change is less than 1, the result is reported as a fold down-regulation.

4.5. siRNA Knockdown Twenty-four hours before transfection, 2 ˆ 105 cells were plated into each well of a 6-well plate containing antibiotic-free medium. Using Lipofectamine™ 2000 (Invitrogen), a 25 bp siRNA against ERα (siERα) (Invitrogen) was transfected into MCF-7 cells, at a concentration of 100 nM, according to the manufacturer’s protocol. Control cells were incubated in the same volume of transfection reagents without siERα. Forty-eight hours post-transfection, the cells were treated with E2 for 24 h or PPT for 16 h, or DMSO for 24 h. Concurrently, siRNA-A (Santa Cruz) was used as a negative control to demonstrate that there was no effect on expression due to transfection. Following exposure, whole cell extracts were harvested for Western analysis. Figure S3 demonstrates no change in protein expression for ERα. Additionally, the siERα does not show any changes in ERβ expression by Western analysis.

4.6. Data Analysis All experiments were repeated three to six times. Two-tailed Student’s t tests were used for comparison between experimental and control groups. A value of p ď 0.05 was set as the criterion for Int. J. Mol. Sci. 2016, 17, 158 12 of 15 statistical signiﬁcance. Bar graphs represented the mean ˘ SD for three independent experiments. All statistical analysis was performed using SAS 9.1.

Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/ 17/2/158/s1. Acknowledgments: The authors would like to thank the Department of Biological Sciences, the Graduate School and the Association of Biologists at Texas Tech University for their support of this work. Author Contributions: Hongtao Ma conceived the study, carried out the experiments, and wrote the initial manuscript draft. Lauren S. Gollahon guided Hongtao Ma in designing the experiments, interpreting the data, supporting the research and finalizing the manuscript. Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations BRMS1 breast cancer metastasis suppressor 1 CRE cyclic AMP-response element DPN 2,3-bis(4-hydroxyphenyl)-proprionitrile

E2 estradiol ER estrogen receptor ERE estrogen response element G-1( ˘)-1-[(3aR*,4S*,9bS*)-4-(6-Bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c] quinolin-8-yl]-ethanone HDAC histone deacetylase Hsp heat shock protein mSin3 mammalian suppressor of defective silencing 3 PPT (4,41,4”-(4-propyl-[1H]-pyrazole-1,3,5-triyl) tris-phenol

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