International Journal of Molecular Sciences Article BCAS2 Enhances Carcinogenic Effects of Estrogen Receptor Alpha in Breast Cancer Cells Ángel Salmerón-Hernández 1,2 , María Yamilet Noriega-Reyes 1,2 , Albert Jordan 3 , Noemi Baranda-Avila 1 and Elizabeth Langley 1, * 1 Departamento de Investigación Básica, Instituto Nacional de Cancerología, Av. San Fernando No. 22, Col. Sección XVI, 14080 Mexico City, Mexico; angel.salher@yahoo.com.mx (A.S.-H.); yamilet_noriega@hotmail.com (M.Y.N.-R.); mimi19481@yahoo.com.mx (N.B.-A.) 2 Programa de Doctorado en Ciencias Biomédicas, Universidad Nacional Autónoma de México, 04510 Mexico City, Mexico 3 Institut de Biología Molecular de Barcelona (IBMB-CSIC) Parc Científic de Barcelona, Barcelona, 08028 Cataluña, Spain; ajvbmc@ibmb.csic.es * Correspondence: langleyemx@gmail.com; Tel.: +(55)-5628-0400 (ext. 32075)  Received: 17 January 2019; Accepted: 21 February 2019; Published: 22 February 2019  Abstract: Estrogen receptor alpha (ERα) has an established role in breast cancer biology. Transcriptional activation by ERα is a multistep process modulated by coactivator and corepressor proteins. Breast Cancer Amplified Sequence 2 (BCAS2), is a poorly studied ERα coactivator. In this work, we characterize some of the mechanisms through which this protein increases ERα activity and how this promotes carcinogenic processes in breast cancer cells. Using protein-protein interaction and luciferase assays we show that BCAS2 interacts with ERα both in vitro and in vivo and upregulates transcriptional activation of ERα directly through its N-terminal region (AF-1) and indirectly through its C-terminal (AF-2) region, acting in concert with AF-2 interacting coactivators. Elevated expression of BCAS2 positively affects proliferation, clonogenicity and migration of breast cancer cells and directly activates ERα regulated genes which have been shown to play a role in tumor growth and progression. Finally, we used signal transduction pathway inhibitors to elucidate how BCAS2 is regulated in these cells and observed that BCAS2 is preferentially regulated by the PI3K/AKT signaling pathway. BCAS2 is an AF-1 coactivator of ERα whose overexpression promotes carcinogenic processes, suggesting an important role in the development of estrogen-receptor positive breast cancer. Keywords: estrogen receptor; breast cancer; BCAS2; coactivators 1. Introduction ERα is the primary therapeutic target in breast cancer and is expressed in close to 70% of cases. These tumors can respond to endocrine therapy and be growth inhibited by pharmacologic blockade of estrogen production or ER. The selective ER modulator, Tamoxifen (TAM), is the most widely used hormonal therapy. However, prolonged treatment often derives in endocrine resistance and tumor recurrence [1], through mechanisms which have not been completely elucidated. Some of these mechanisms include ERα loss or mutations, changes in coregulatory proteins, increased cross-talk with other signal transduction pathways resulting in aberrant or ligand independent activation of ER or its coregulators and alternative downstream signaling pathways activating different gene expression programs [2–6]. The transcriptional activity of ERα is dependent on several coregulatory proteins called coactivators and corepressors [7,8]. Coactivators do not usually bind DNA directly but are recruited to target gene promoters through protein-protein interactions with nuclear receptors (NRs). Thus, Ligand binding promotes the formation of multiprotein complexes containing the nuclear Int. J. Mol. Sci. 2019, 20, 966; doi:10.3390/ijms20040966 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2019, 20, 966 2 of 19 receptor (NR), coactivators and basal transcription factors acting in concert to activate transcription [8]. Structural analysis of coactivators has identified a five amino acid (aa) motif, LXXLL (where X is any amino acid), which is sufficient to mediate coregulator binding to the liganded NRs at their AF-2 domain [9]. However, a number of coactivators have recently been discovered that bind to the N-terminus of NRs and activate the AF-1 transcriptional activation function. In general, coactivators increase transcriptional activity through chromatin remodeling, histone acetylation or methylation, as well as recruitment of other coregulators and of the basal transcriptional machinery [10,11]. In contrast, corepressors associate with histone deacetylases to repress transcription and promote a closed chromatin configuration [12]. Besides modulating chromatin structure to activate or repress transcription, coactivators and corepressors can have many other functions including control of splicing and protein degradation through ubiquitination. [13]. Additionally, expression of different coregulators has been implicated in differential tissue and cell type-specific responses to various hormones; however, more research is required to fully understand these mechanisms. Using a yeast two-hybrid assay, we detected BCAS2 as an ERα binding protein, interacting with its N-terminal domain. BCAS2 was previously determined to be a coactivator protein that increases ERα transcriptional activity through its AF-2 domain [14] and has been found to associate with the tumor suppressor p53 protein [15]. In this work, we identified BCAS2 as a protein that interacts with ERα both in vitro and in vivo and regulates the transcriptional activation of ERα through its N-terminal region (AF-1) and indirectly via the C-terminal (AF-2) region. The enhanced expression of BCAS2 in human mammary cancer cell lines increases their proliferation, migration and colony formation. Furthermore, it regulates the expression of genes that have a role in breast cancer tumorigenesis. This suggests that BCAS2 regulates AF-1 activity on the ERα N-terminus and may play a role in regulating estrogen dependent growth in breast cancer. 2. Results 2.1. BCAS2 Interacts Directly with the N-Terminal Region of ERα Using the yeast two-hybrid system to identify proteins that interact with the N-terminal domain of ERα (aa 1-180), we obtained several sequences that encode for proteins that interact with this region, including BCAS2. To verify this interaction and the involvement of the different domains in BCAS2 binding, we performed pull-down assays in vitro using full-length ERα (Full) as well as its N- and C-terminal domains separately, fused to GST (Figure 1A). Assays were carried out in the presence and absence of E2 and interaction was tested with in vitro labeled BCAS2. We observed that BCAS2 interacts with full-length ERα, both in the presence and absence of E2 and that this interaction takes place via the N-terminal domain of ERα and not through its C-terminal domain, even in the presence of ligand (Figure 1B). Additionally, we determined interaction with ERβ and also found that BCAS2 interacts via its N-terminal region (data not shown). This supports our two-hybrid interaction assay but contrasts previous findings where BCAS2 was found to activate ERα only through its C-terminal domain [14]. Coimmunoprecipitation assays in both, transfected monkey kidney COS7 cells and breast cancer MCF7 cells, confirmed interaction between BCAS2 and ERα (Figure 1C,D) in the absence and presence of E2. Additionally, TAM treatment in MCF7 cells did not abrogate BCAS2 binding to ERα. Thus, indicating a fruitful in vivo interaction between these proteins and suggesting that BCAS2 plays a role in directly regulating ERα activity. Int. J. Mol. Sci. 2019, 20, 966 3 of 19 Int. J. Mol. Sci. 2018, 19, x 3 of 19 1. BCAS2 interacts with ERα in vivo and in vitro. (A) Structure of ERα and its N and FigureFigure 1. BCAS2 interacts with ERα in vivo and in vitro. (A) Structure of ERα and its N and C domains C domains used for Glutathione sepharose affinity matrix assays. NTD, amino terminal domain; used for Glutathione sepharose affinity matrix assays. NTD, amino terminal domain; DBD, DNA DBD, DNA binding domain; HR, hinge region; LBD, ligand binding domain. (B) GST pull-down binding domain; assays HR, labeled of biotin hinge region; in vitro LBD, ligand translated binding BCAS2 with domain. GST alone,(B)GST-ERα-Full GST pull-down assays aa (full-length of biotin labeled1-595), in vitro translated GST-ERα-N (aa BCAS2 with GST(aa 1-180) GST-ERα-C alone, GST-ERα-Full 264-595). Western blot(full-length analysis wasaa 1-595), carried outGST-ERα-N using (aa 1-180) GST-ERα-C anti-biotin (aaantibodies. or anti-GST 264-595). Binding Western wasblot analysis assayed in thewas carried presence (+) orout using absence (−)anti-biotin of 100 nM E2.or anti- (C) Coimmunoprecipitation of ERα and BCAS2. COS7 cells were transfected GST antibodies. Binding was assayed in the presence (+) or absence (−) of 100 nM E2. (C) with plasmids expressing ERα and BCAS2 in the presence (+) or absence (−) of 10 nM E2. Immunoprecipitation of whole cell Coimmunoprecipitation of ERα and BCAS2. COS7 cells were transfected with plasmids expressing protein extracts was carried out with antibodies against BCAS2 or ERα and IgG as negative control. ERα and BCAS2 in the presence (+) or absence (−) of 10 nM E2. Immunoprecipitation of whole cell Shown are immunoblots probed with antibodies against both proteins. (D) Coimmunoprecipitation proteinassays extracts was cells in MCF7 carried in theout with of presence antibodies ethanol (OH),against E2 (10BCAS2 or ERα nM) or TAM (100and nM) IgG as negative showing control. interaction Showninare theimmunoblots probed presence or absence with Full of ligand. antibodies blots canagainst be foundboth proteins. (D)Figure in supplementary Coimmunoprecipitation S1. assays in MCF7 cells in the presence of ethanol (OH), E2 (10 nM) or TAM (100 nM) showing interaction 2.2. BCAS2 Enhances ERα Transcriptional Activity in the presence or absence of ligand. Full blots can be found in supplementary Figure S1. It was previously shown that BCAS2 could increase ERα transcriptional activity in CV-1 monkey kidney 2.2. BCAS2 cells [14]. ER Enhances α Transcriptional Thus, we were interested in confirming whether BCAS2 could promote increased Activity ERα transcriptional activity in breast cancer cells. Luciferase reporter gene assays showed that It overexpression was previously ofshown BCAS2 that BCAS2 is able could ERα to enhance increase ERα transcriptional transcriptional activity to anactivity extentin CV-1 or similar monkey kidneygreater cells [14]. Thus, than that we for found were theinterested in confirming SRC-1 coactivator whether in ER positive (MCF7)BCAS2 (Figurecould 2A) or promote increased ER negative (MDA-MB-231) breast cancer cell lines in the presence of E2 (Figure ERα transcriptional activity in breast cancer cells. Luciferase reporter gene assays showed that 2B). Since ERα contains two transcriptional overexpression activation of BCAS2 functions, is able which can to enhance ERαacttranscriptional independently or cooperatively, activity we were to an extent interested similar or greater in determining whether BCAS2 could activate both domains. Thus, transcriptional activity assays than that found for the SRC-1 coactivator in ER positive (MCF7) (Figure 2A) or ER negative (MDA- were performed using the AF-1 and AF-2 domains separately in HEPG2 cells, where it was previously MB-231) breast shown cancer that the cell lines AF-1 domain in the independent can promote presence of E2 (Figureactivity transcriptional 2B). [16]. Since ERαshow Results contains that two transcriptional activation BCAS2 increases both AF-1functions, and AF-2whichmediated can act independently transcriptional activity, in aor dosecooperatively, dependent manner we were interested (Figure 2C,D). This compares with the activity of SRC-1, which also enhances both transcriptionalactivity in determining whether BCAS2 could activate both domains. Thus, transcriptional assays activation were performed using the AF-1 and AF-2 domains separately in HEPG2 cells, where it was functions [17]. previously shown that the AF-1 domain can promote independent transcriptional activity [16]. Results show that BCAS2 increases both AF-1 and AF-2 mediated transcriptional activity, in a dose dependent manner (Figures 2C,D). This compares with the activity of SRC-1, which also enhances both transcriptional activation functions [17]. Int. J. Mol. Sci. 2019, 20, 966 4 of 19 Int. J. Mol. Sci. 2018, 19, x 4 of 19 Figure Figure 2. 2. BCAS2 BCAS2 enhances enhances ERα ERα transcriptional transcriptional activity. activity. Luciferase Luciferase activity activity induced induced by by the the overexpression of BCAS2 in the presence of ethanol (EtOH), 10 nM E2 overexpression of BCAS2 in the presence of ethanol (EtOH), 10 nM E2 or 100 nM TAM (A,B). MCF7or 100 nM TAM (A,B). MCF7 cells cells were were transiently transiently transfected transfected with with anan ERE-luciferase ERE-luciferase reporter reporter vector vector(200 (200ng) ng)and andBCAS2 BCAS2(100 (100ng). ng). Empty Empty pcDNA3.1 pcDNA3.1 vectorvector (EV) (EV) (100 (100 ng) ng) and and SRC-1 SRC-1 (100 (100 ng) ng) were were transfected transfected as as negative negative and and positive positive controls, controls, respectively. respectively. (B) (B)MDA-MB-231 MDA-MB-231cells cellswere weretransfected transfected with withthethe same sameplasmids plasmids as well as well as 50 as ng of ERα. (C,D) BCAS2 enhances AF-1 (aa 1-282) and AF-2 (aa 144-595) transcriptional 50 ng of ERα. (C,D) BCAS2 enhances AF-1 (aa 1-282) and AF-2 (aa 144-595) transcriptional activation activation functions, functions, independently. independently. Each Each plasmid plasmid waswas transfected transfected intointo HEPG2 HEPG2 cellscells along along with with empty empty vector vector (EV) or (EV) concentrations of BCAS2 (50-200 ng), ERE-luc and β-galactosidase or increasing concentrations β-galactosidase in in the the presence presence or absence or absence of of 10 10 nM nM E2.E2. Transfected Transfected SRC-1 SRC-1 coactivator coactivator is is used used as as positive positive control control for transcriptional transcriptional activation. (E) activation. (E) BCAS2 BCAS2 activates activates PRPR transcriptional transcriptional activity. activity. Increasing Increasing concentrations concentrations of of BCAS2 BCAS2 (100 (100 and 200 and 200 ng), ng), as well as β-galactosidase as well vector, were β-galactosidase vector, were transfected transfected into into breast breast cancer cancer T47D T47D cells cells stably stably transfected with transfected with aa luciferase luciferase reporter reporter vector vector forfor PR PR activity activity and and treated treated inin the the absence absence (EtOH) (EtOH) or or presence of 10 nM P4. SRC-1 is used as positive control. Relative luciferase activity presence activity (RLU (RLU LUC/β-gal) LUC/β-gal) is shown is shown as as the mean ±±SE the mean SEofofaa representative representativeassay assaycarried carriedoutout in in triplicate, triplicate, at at least least three three times. times. Statistical analysis is carried out by Student´s t-test comparing activation between Statistical analysis is carried out by Student´s t-test comparing activation between BCAS2 and empty BCAS2 and empty vector (transfected at the same concentration) vector concentration) in in the the presence presence and and absence absenceof E2,**pp<<0.05, ofE2, ** pp << 0.001. 0.05, ** 0.001. 2.3. BCAS2 Has a Positive Effect on the Transcriptional Activity of PR It is known that many coregulators can function on different SHRs. BCAS2 has previously been found to interact with PR in vitro, however, the effect of this interaction was not determined [14]. Int. J. Mol. Sci. 2018, 19, x 5 of 19 Int. J. Mol. Sci. 2019, 20, 966 5 of 19 Therefore, we tested the effect of BCAS2 on the transcriptional activity of PR in a breast cancer cell line (T-47D-MTVL) harboring a PR-dependent luciferase reporter gene [18]. BCAS2 showed a 2.3. BCAS2 positive Has aonPositive effect Effect on the Transcriptional PR transcriptional Activity activity in the of PR of progesterone (P4) (Figure 2E). presence Furthermore, It is known ARthat activity manyascoregulators determinedcan by luciferase function onreporter assays different SHRs.is BCAS2 also increased by BCAS2 has previously in been prostate found toand breast interact cancer with cells PR in (data vitro, not shown however, the and [19]. effect Thisinteraction of this suggests that wasBCAS2 is a coactivator not determined [14]. that can be shared between steroid hormone receptors and activates important growth Therefore, we tested the effect of BCAS2 on the transcriptional activity of PR in a breast cancer cell modulators in breast line cancer cells. (T-47D-MTVL) harboring a PR-dependent luciferase reporter gene [18]. BCAS2 showed a positive effect on PR transcriptional activity in the presence of progesterone (P4) (Figure 2E). Furthermore, 2.4. activity AR BCAS2 Is asRecruited determined to ER Target Gene by luciferase Promoters reporter and assays is Increases Target also increased byGene Expression BCAS2 in prostate and breastIn order to determine whether BCAS2 is a bona fide coactivator, acting on estrogen that cancer cells (data not shown and [19]. This suggests that BCAS2 is a coactivator can be responsive shared between steroid hormone receptors and activates important growth modulators promoters, we carried out ChIP assays to determine whether ERα and BCAS2 could be tethered in breast to cancer cells. the pS2 [20] and WISP2 [21], promoters after different times of induction with E2. We found BCAS2 is recruited to these promoters and that its presence on the promoter begins to show in as little as 30 2.4. BCAS2 Is Recruited to ER Target Gene Promoters and Increases Target Gene Expression min after exposure to E2 and increases over time (Figure 3A,B). Additionally, we used a marker of openIn order to determine chromatin, H3K4me3,whether BCAS2 which also showsis aenrichment bona fide coactivator, acting on after E2 treatment. estrogen Thus, we can responsive conclude promoters, that BCAS2we is acarried out ChIPof true coactivator assays to determine ERα exerting whether its effect ERα directly onand BCAS2 could E2 regulated be tethered promoters to and that the pS2 [20]ofand length WISP2to exposure [21], E2 promoters after different directly influences times of induction with E2. We found BCAS2 is this interaction. recruited to these We then usedpromoters RT-PCR to and that itstranscription observe presence on of thea promoter number ofbegins to showgenes ER regulated in as little as 30cells. in MCF7 min after exposure toofE2BCAS2, Overexpression and increases both inover time (Figure the absence and 3A,B). Additionally, presence wethe of E2, affects used a marker of expression of some open chromatin, H3K4me3, genes containing which response also shows elements for ER enrichment receptors, after E2 treatment. as shown in Figure Thus, 3C–F. we Wecansee conclude an increasethat in BCAS2 mRNA is a true coactivator expression of ERα for the pS2, exertingand C3, IGFBP2 its C-MYC effect directly genes,on E2 regulated suggesting promoters BCAS2 and athat can activate the subset length of genesofdepending exposure toon E2the directly influences promoter context. this interaction. Figure 3. BCAS2 colocalizes with ERα on estrogen regulated promoters upon incubation with E2 and promotes promotes increased increased estrogen-regulated estrogen-regulatedgene geneexpression. expression.(A,B) (A,B)Chromatin Chromatinimmunoprecipitation immunoprecipitationwaswas Int. J. Mol. Sci. 2019, 20, 966 6 of 19 performed in MCF7 cells after different times of exposure to E2 (10 nM) treatment (30–180 min). Statistical analysis was carried out comparing ER or BCAS2 with IgG control using by one-way ANOVA, * p < 0.001. We carried out qRT-PCR of precipitated DNA showing endogenous ERα and BCAS2 localization at promoter regions of pS2 and WISP2 genes. Additionally, Histone 3 methylation (H3k4me3) is shown as a marker for open chromatin. (C–F) MCF7 cells transfected with pcDNA-BCAS2 or empty vector (EV) and incubated in the presence (+) or absence (−) of 10nM E2 for 24h. RTqPCR was used to determine mRNA expression of pS2, C3, insulin-like growth factor binding protein 2 (IGFBP2) and C-MYC and the results were normalized against human GAPDH mRNA amplification. * p < 0.05 compared to the EV group at 24 h in the absence (−) or presence of E2, as indicated in the figure. We then used RT-PCR to observe transcription of a number of ER regulated genes in MCF7 cells. Overexpression of BCAS2, both in the absence and presence of E2, affects the expression of some genes containing response elements for ER receptors, as shown in Figure 3C–F. We see an increase in mRNA expression for the pS2, C3, IGFBP2 and C-MYC genes, suggesting BCAS2 can activate a subset of genes depending on the promoter context. 2.5. BCAS2 Acts Together with Other Coactivators to Enhance ERα Activity In order to delve further into the molecular mechanisms of the BCAS2 coactivator on ERα transcriptional activation, we evaluated whether BCAS2 was capable of affecting the activity of other ERα coactivators preferentially recruited through the AF-2 domain. We tested SRC-1, TIF2 and CBP. All contain LxxLL motifs through which they bind to AF-2. We tested three different cancer cell lines, MCF7, MDA-MB-231 and SK-BR-3 cells. All of our results show that when BCAS2 is combined with any of the three coactivators in the presence of E2, transcriptional activation is enhanced compared to BCAS2 or the coactivator alone (Figure 4A–C). Interestingly, in MCF7 cells (Figure 4A), the combination of BCAS2 with any of the three AF-2 coactivators, also consistently increases transcription in the presence of TAM, which is not observed in the ER− SK-BR-3 (Figure 4C) or in the MDA-MD-231 cell lines (Figure 4B). This suggests that BCAS2 may play a role in TAM resistance in an ER+ cellular environment. Int. J. Mol. Sci. 2019, 20, 966 7 of 19 Int. J. Mol. Sci. 2018, 19, x 7 of 19 Figure Figure 4. BCAS2 4. BCAS2 acts together acts together withwith AF-2AF-2 coactivators coactivators to increase to increase ERα ERα activity. activity. (A) Luciferase (A) Luciferase assays assays werewere carried out with carried pcDNA-BCAS2 out with pcDNA-BCAS2 or empty vector or empty (EV),(EV), vector transfected withwith transfected and without and withoutcoactivators coactivators SRC-1, CBPCBP SRC-1, or TIF2 and treated or TIF2 in the and treated inabsence (EtOH) the absence (EtOH)or presence of 10ofnM or presence 10 of nM E2oforE2100 or nM 100 of nM TAM of TAM in MCF7 cells.cells. in MCF7 Similar assays Similar werewere assays carried out in carried outMDA-MB-231 in MDA-MB-231 (B) and SK-BR-3 (B) and (C) cells SK-BR-3 withwith (C) cells the the addition of transfected addition pSG5-ER of transfected pSG5-ERStatistical analysis Statistical was was analysis carried out using carried one-way out using one-wayANOVAANOVA withwith Bonferroni posttest; Bonferroni * p < *0.01, posttest; ** p <**0.005, p < 0.01, *** p *** p < 0.005, < 0.001. p < 0.001. 2.6. BCAS2 Promotes Cell Proliferation and Colony Formation in Breast Cancer Cells 2.6. BCAS2 Promotes Cell Proliferation and Colony Formation in Breast Cancer Cells In order to determine the effect that BCAS2 may have on breast cancer development, we used In order to determine the effect that BCAS2 may have on breast cancer development, we used MCF7 breast cancer cells to determine its effect on proliferation and clonogenicity. Overexpression MCF7 breast cancer cells to determine its effect on proliferation and clonogenicity. Overexpression of BCAS2 caused a significant increase in estrogen dependent and independent cell proliferation of BCAS2 caused a significant increase in estrogen dependent and independent cell proliferation (Figure 5A). Conversely, BCAS2 knockdown reduces cell viability in the presence of E2 compared to (Figure 5A). Conversely, BCAS2 knockdown reduces cell viability in the presence of E2 compared to cells transfected with control shRNA (shE1) (Figure 5B). cells transfected with control shRNA (shE1) (Figure 5B). We then carried out colony formation assays in MCF7 cells and observed a significant increase in clonogenicity upon BCAS2 overexpression and E2 treatment (Figure 5C). Int. J. Mol. Sci. 2019, 20, 966 8 of 19 Int. J. Mol. Sci. 2018, 19, x 8 of 19 Figure 5.5. BCAS2 enhances breast cancer cancer cell cell growth growth andand colony colony formation. formation. (A) CellCell proliferation proliferation assays assays were werecarried carriedoutoutononMCF7 MCF7cellscellstransfected transfected with BCAS2 with BCAS2 or or emptyemptyvector (EV) vector andand (EV) treated in the treated in absence (EtOH) or presence of E2 for 8 days. Cell viability was assayed every 2 days, the absence (EtOH) or presence of E2 for 8 days. Cell viability was assayed every 2 days, as described as described in materials andand in materials methods. BCAS2 methods. BCAS2overexpression overexpression caused a significant caused increase a significant in proliferation increase both inboth in proliferation the presence and absence in the presence of ligand and absence (** p <(** of ligand 0.001) (B) Cell p < 0.001) (B)viability assaysassays Cell viability were were carried out on carried outMCF7 cells on MCF7 transfected with shBCAS2 cells transfected with shBCAS2or unrelated shE1 in or unrelated shE1theinabsence (EtOH) the absence or presence (EtOH) of E2 of or presence andE2assayed after and assayed 96 h. Inhibition after of BCAS2 caused 96 hours. Inhibition of BCAS2 a significant caused a reduction significantinreduction cell growth in in cellthe presence growth (* ppresence in the < 0.05) of(*E2. p (C) < 0.05) of E2. (C) Colony formation of MCF7 cells transfected with BCAS2 or empty vector. cells Colony formation of MCF7 cells transfected with BCAS2 or empty vector. 1000 transfected 1000 were platedcells transfected per well wereand treated plated withand per well E2 treated or TAMwith every E248 orhTAM for 12 days. every 48Colonies hours forwere thenColonies 12 days. stained and werecounted. Graphical then stained representation and counted. of therepresentation Graphical number of colonies of thecounted numberwith respect to of colonies the number counted with of is shown on right. Data represent three independent assays, * p respect to the number of cells plated is shown on right. Data represent three independent assays, *inp cells plated < 0.05 compared to EV the presence < 0.05 of EtOH compared to EVand ** p presence in the < 0.001 compared of EtOH andto BCAS2 ** p < in the compared 0.001 presence oftoEtOH.BCAS2 in the presence of EtOH. We then carried out colony formation assays in MCF7 cells and observed a significant increase in clonogenicity upon BCAS2 2.7. BCAS2 Promotes overexpression Estradiol Dependent and CellE2 treatment (Figure 5C). Migration 2.7. BCAS2 Promotesthe We analyzed Estradiol role ofDependent BCAS2 inCell Migration cellular migration using wound-healing assays. Our data shows Wegreater analyzedmigration of BCAS2 the role of cells overexpressing BCAS2using in cellular migration as compared to those wound-healing transfected assays. with Our data EV shows after 48 h of E2 treatment (Figure 6). greater migration of cells overexpressing BCAS2 as compared to those transfected with EV after 48 h of E2 treatment (Figure 6). Int. J. Mol. Sci. 2019, 20, 966 9 of 19 Int. J. Mol. Sci. 2018, 19, x 9 of 19 Figure 6. BCAS2 significantly increases cell migration. The effect of BCAS2 overexpression on MCF7 Figure 6. BCAS2 significantly increases cell migration. The effect of BCAS2 overexpression on MCF7 cell migration was determined using a wound healing assay. The wound healing rate of BCAS2 cell migration was determined using a wound healing assay. The wound healing rate of BCAS2 transfected cells was faster than empty vector (EV) in presence of E2. The open wound area at 0h was transfected cells was faster than empty vector (EV) in presence of E2. The open wound area at 0h was regarded as 100%. Values represent mean ± SD of three replicates. * p < 0.05 compared to the EV group regarded at 48 h of as E2 100%. Values treatment. represent 10× mean ± SD of three replicates. * p <0.05 compared to the EV group magnification. at 48 h of E2 treatment. 10× magnification. 2.8. Various Signal Transduction Pathways Are Involved in BCAS2 Activation 2.8. Various Signal Transduction Pathways Are Involved in BCAS2 Activation It is well known that there is cross-talk between MAP kinase and/or PI3K/AKT signal It is wellpathways transduction known that andthereERαisincross-talk betweencells, breast cancer MAPwhichkinasepromotes and/or PI3K/AKT an increase signal in transduction pathways estrogen-mediated and and signaling ERαfavors in breast cancer cells, recruitment which promotes of coactivators, such as an increase SRC-1 in estrogen- and SRC-3 [22,23]. mediated signaling Additionally, activation and favorspathways of these recruitment of coactivators, by HER2, has been highly suchimplicated as SRC-1inand breastSRC-3 cancer[22,23]. and is Additionally, activation of these pathways by HER2, has been highly implicated known to promote endocrine resistance [24,25]. Therefore, we were interested in determining whether in breast cancer and is known these signaltotransduction promote endocrine pathways resistance [24,25]. were capable Therefore,BCAS2 of impacting we were interested activation in determining of ERα. We carried whether out these signal transcriptional transduction activation pathways studies weretwo comparing capable breastofcancer impacting BCAS2 cell lines activation using of ERα.and HER2, MEK-1 We carried PI3K out transcriptional inhibitors. MCF7 is anactivation ERα positive studies comparing cell line with lowtwo HER2 breast cancer cell expression, while lines using is SK-BR-3 HER2, ERα MEK-1 and negative andPI3K inhibitors. MCF7 overexpresses HER2 and is anthus, ERα ERα positive wascell line with low transfected. Our HER2 resultsexpression, show that the while SK- HER2 BR-3 is ERα negative and overexpresses HER2 and thus, ERα was transfected. Our inhibitor, trastuzumab, has little effect on inhibiting ERα activation in our system. However, it causes results show that the HER2abrogation complete inhibitor, of trastuzumab, the BCAS2 induced has little effect in increase ontranscriptional inhibiting ERα activation activity in thein our system. presence of E2 However, (Figure it causes 7A,B). complete The MEK-1 abrogation inhibitor, of thefurther PD98059, BCAS2downstream induced increasein theinMAPK transcriptional signalingactivity pathway, in the presence of E2 (Figure 7A,B). The MEK-1 inhibitor, PD98059, further downstream causes a more marked decrease in activity, inhibiting both ERα activity alone and the BCAS2 induced in the MAPK signaling(Figure increase pathway, 7C).causes a more This effect is marked much more decrease in activity, evident in SK-BR-3 inhibiting both ERα cells, where ERαactivity activityalone and is almost the BCAS2 induced completely abrogated increase (Figure(Figure 7D). This7C).supports This effect is muchfindings previous more evident showing in SK-BR-3 that MEKcells, where activation ERα activity increases is almost [22] ER signaling completely and thus abrogated suggests (Figure that this7D). effectThis supportscaused is probably previous findings showing by inhibiting ER and thatas not MEK activation a direct increases effect on BCAS2.ER signaling [22] and thus suggests that this effect is probably caused by inhibiting Conversely, ER and not asthe blocking a direct PI3K/AKTeffect (Wortmannin) on BCAS2. signal transduction pathway shows little or Conversely, no effect blockingalone on ERα activity the PI3K/AKT but shows (Wortmannin) an important effectsignalontransduction pathway BCAS2 activity, shows inhibiting itslittle or ability nopromote to effect onERαERαtransactivation activity alone but shows (Figure an important effect on BCAS2 activity, inhibiting its ability 7E,F). to promote ERα transactivation (Figure 7E,F). Int.Int. J. Mol. J. Mol.Sci. 2018,20, Sci.2019, 19,966 x 10 of1019of 19 Figure7.7. Signal Figure Signal transduction transductionpathways pathways involved in BCAS2 involved activation. in BCAS2 (A, C and activation. (A, E) MCF7 C and E)cells MCF7werecells transiently were transfected transiently with with transfected an ERE-luciferase reporter an ERE-luciferase vector vector reporter (200 ng) and (200 BCAS2 ng) (100 ng)(100 and BCAS2 in the ng) in absence the absence (EtOH) (EtOH)or presence of 10 of or presence nM10E2nM or 100 nM100 E2 or TAMnMalong TAMwith 50 gwith along of Trastuzumab or 10 mM or 50 g of Trastuzumab of PD98059 or 100 nM of Wortmannin, as indicated, for 48h. Empty vector 10 mM of PD98059 or 100 nM of Wortmannin, as indicated, for 48h. Empty vector (EV) (EV) (100 ng) was used as ng) (100 negative control. (B, D and F) SK-BR-3 cells were transfected and treated in the same was used as negative control. (B, D and F) SK-BR-3 cells were transfected and treated in the same manner as MCF7 cells, except manner as MCF7 thatcells, 50 ngexcept of pSG-ERα that 50were ng ofalso transfected. pSG-ERα Relative were also luciferase transfected. activityluciferase Relative (RLU LUC/β-activity gal) is shown as the mean ± SE of a representative assay carried out (RLU LUC/β-gal) is shown as the mean ± SE of a representative assay carried out in triplicate, at least three times. in triplicate, Representative western blots show HER2 after 48 h of Trastuzumab treatment, ERK and ERK at least three times. Representative western blots show HER2 after 48 h of Trastuzumab treatment, phosphorylation (pERK) after 48 h of Trastuzumab or PD98059 treatment, AKT and AKT ERK and ERK phosphorylation (pERK) after 48 h of Trastuzumab or PD98059 treatment, AKT and AKT phosphorylation (pAKT) after 48 h of Wortmannin treatment, each performed in triplicate or phosphorylation (pAKT) after 48 h of Wortmannin treatment, each performed in triplicate or duplicate (Figure S2). GAPDH is used as control for protein loading. Statistical analysis was carried out using Student’s t-test; ** p < 0.005, *** p < 0.001. Int. J. Mol. Sci. 2019, 20, 966 11 of 19 3. Discussion The identification and characterization of coregulators that modulate ERα activity is a crucial step in understanding the mechanisms that control, not only the expression of estrogen regulated genes but also the initiation and progression of breast cancer. In this study, we have reinforced and enhanced our knowledge about the role of BCAS2 as an ERα coactivator and its regulation in breast cancer cells. BCAS2 had previously been proposed as an ERα coactivator and had been shown to increase its transcriptional activity [14]. However, this group proposed that this activity was carried out solely through the AF-2 transcriptional activation domain. In this study, we confirmed direct interaction with the N-terminal domain of ERα in vitro, without finding interaction via de C-terminal domain (Figure 1B). Additionally, interaction with full-length ERα was observed both in the presence and absence of E2 and TAM (Figure 1C). The fact that there is interaction of BCAS with ERα in the presence of TAM further supports binding to the AF-1 domain, as the conformational change caused to the LBD does not abrogate BCAS2 binding. However, since there have been many sites known to harbor post transcriptional modifications involved in ERα activation in both the hinge and the LBD regions [26], we cannot rule out that some post translational modification is necessary for BCAS2 binding to the AF-2 region and that by using an in vitro GST-pulldown assay we are unable to observe this interaction. Additionally, our construct is lacking a small portion of the hinge region, so this may be a possible binding site for BCAS2. In this respect, there are a number of coactivator proteins that bind the hinge region, such as calmodulin, which promotes ERα dimerization [27]. Luciferase assays, carried out in breast cancer cell lines, confirmed that BCAS2 increases ERα transcriptional activity (Figure 2A,B). However, we also determined that BCAS2 increases transcription through both the AF-1 domain and the AF-2 domain, independently (Figure 2C,D). This is in contrast to data published by Qi et al. [14], who did not observe AF-1 activation by BCAS2 in CV-1 cells. It is likely that this cellular context does not support independent activation through the AF-1 domain alone while in HEPG2 cells both AFs have been found to function independently [16]. Thus, combined with our protein interaction data, this suggests that the effect on the AF-1 domain was carried out through its direct interaction with ERα while the positive effect on the AF-2 domain may require interaction with other coactivators that can interact directly with AF-2. This supports the idea that BCAS2 may be involved in promoting synergism between the AF-1 and AF-2 transactivation functions, as has been suggested for SRC-1 [16]. Thus, we tested the effect of BCAS2 on the activity of three coactivators that preferentially bind to AF-2 through their LxxLL sequences, although it has been shown that they can also bind to the N-terminal domain. We observed that the combination of BCAS2 with SRC-1, TIF2 or CBP increases the transcriptional activity of ERα compared to any of these coactivators alone (Figure 4). This implies that BCAS2 does not compete with these coactivators for receptor binding. This has been observed with other coactivators such as Tip60, which synergizes with TIF2 to increase ERβ transactivation [28]. Moreover, in MCF7 cells, the combination of BCAS2 with any of the coactivators tested, especially TIF2, also increases transcription in the presence of TAM and in some cases in the absence of ligand (Figure 4A). The combination of coactivators may be necessary to promote the agonist activity of TAM through AF-1, even though it abrogates their binding to AF-2. This is not consistently observed in the ER negative cell lines transfected with exogenous ERα (Figure 4C), suggesting that BCAS2 may play a role in TAM resistance in an ER+ cellular environment. Additionally, BCAS2 has been shown to be overexpressed in TAM resistant MCF7 cells and increased expression is associated to shorter relapse-free survival of breast cancer patients [29]. In order to ensure that BCAS2 is a bona fide coactivator, we demonstrated its existence on ER target promoter DNA and show that target gene expression is increased by BCAS2, at least for pS2, IGFBP2 and C3, both in the presence and absence of ligand (Figure 3). It is well known that the activity or expression of many ER coactivators increases during breast carcinogenesis and progression. However, the role that BCAS2 may play in cancer has been poorly elucidated. A number of studies have shown that it is amplified or overexpressed in breast tumors [29–31] and have suggested that this could influence the development of endocrine resistance, Int. J. Mol. Sci. 2019, 20, 966 12 of 19 as has been observed for other coactivators [32–35]. Additionally, it has been found to be overexpressed in prostate cancer and to regulate the androgen receptor by increasing its mRNA expression and stabilizing AR protein [19]. Thus, we would expect that overexpression of BCAS2 can impact the physiologic environment of estrogen dependent breast cancer cells and be involved in carcinogenic processes. We studied the effect of overexpressing BCAS2 on cellular proliferation, clonogenicity and cell migration (Figures 5 and 6). In all cases we observed that overexpression of BCAS2 enhances all of these processes. Additionally, this is also observed in the absence of ligand. This supports a role for BCAS2 in the progression of breast cancer, both before and after endocrine resistance sets in. Studies on other coactivators, such as SRC-1 have also shown that they have a role in increasing cell migration and proliferation in breast cancer [36,37], as well as increasing the expression of Twist and promoting metastasis [38]. The coactivators, CBP and p300, have also been shown to have the capacity to promote cell migration by increasing expression of metalloproteinases 2 and 9 [39]. Thus, these coactivators have been proposed as markers for breast cancer aggressiveness and malignancy. Our data showing that BCAS2 can synergize with these coactivators, as well as promote carcinogenic processes, shows it has important implications in breast cancer. Further studies need to be carried out to determine whether BCAS2 binds directly to other coactivators and to define its target interaction sequence on ERα. Additionally, assays that block these interactions, for example by using synthetic peptides [40], would greatly help to dissect its functions in ER transactivation, metastatic processes and endocrine resistance. In this regard, BCAS2 expression has also been shown to be upregulated by Estrogen Related Receptor β (ERRβ and both ERα and ERβ in the absence of E2, however addition of ligand inhibited its expression [31]. This work also showed that increased BCAS2 led to expression of MMP7 and cleavage of E-cadherin, supporting a role for BCAS2 in epithelial-mesenchymal transition. Conversely, BCAS2 seems to be involved in downregulating β-catenin resulting in a decrease of the pro-apoptotic protein FST. However, the inhibition of β-catenin observed after BCAS2 overexpression also derives in cyclin D1 downregulation, suggesting that proliferation may be inhibited when ERRβ is present. It has been previously demonstrated that there is cross-talk between ERα and various signal transduction pathways, including HER2, which is capable of activating both the MAPK and PI3K/AKT pathways [41]. HER2 overexpression has been observed in 20-30% of patients with breast cancer and has been implicated in TAM resistance, both acquired and de novo [42,43]. Additionally, various clinical studies have also indicated that HER2 overexpression is associated with a lower response to TAM treatment. Moreover, ectopic expression of HER2 in MCF7 cells is sufficient to confer resistance to TAM in these cells [44]. In our results, we show that inhibition of the MAPK and PI3K/AKT result in inhibition of ERα activity. However, the targets seem to be different. On one hand, blocking MEK-1 activity almost completely abrogates ERα activity (Figure 7C,D). On the other hand, inhibition of PI3K only inhibits the increase in activity promoted by BCAS2, without strongly affecting intrinsic ERα activity (Figure 7E,F). This highlights the importance of these signal transduction pathways in ERα regulation and the progression of estrogen-regulated breast cancer. The reduction in BCAS2 activity can probably be explained by a reduction in phosphorylation of both the receptor, as previously reported [25,45] and BCAS2. However, the direct effect on BCAS2 must still be determined. 4. Materials and Methods 4.1. Yeast Two-Hybrid Screen A yeast two-hybrid assay was carried out using the Matchmaker two-hybrid system 3 kit (Clontech Laboratories, Mountain View, CA, USA), following manufacturer’s instructions and as previously described [46]. Briefly, the N-terminal domain of ERα, amino acids (aa) 1-180, was used as bait to screen a human recurrent prostate carcinoma CWR-R1 cell line cDNA library. Positive clones were selected at high stringency in media lacking histidine, leucine, tryptophan and adenine, supplemented with 5 mM 3-Aminotriazole (3-AT) and X–Gal, to detect blue colonies. The resulting colonies were Int. J. Mol. Sci. 2019, 20, 966 13 of 19 assayed for β-galactosidase activity. cDNAs from galactosidase positive yeast colonies were sequenced and analyzed using Basic Local Alignment Search Tool (BLAST) analysis. Full-length BCAS2 was recovered from the pACTII plasmid. 4.2. Cell Culture All cell lines were obtained from ATCC (authenticated using STRS analysis) and tested for mycoplasma. Cell lines were cultured in media as suggested by ATCC, supplemented 5–10% fetal bovine serum (FBS) and antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin) and incubated at 37 ◦ C with 5% CO2 in a humidified atmosphere, for maintenance. The cell lines Hep-G2 and COS-7 were cultured in Eagle´s Minimum Essential Medium (EMEM) and Dulbecco’s Modified Eagle Medium (DMEM). Human breast cancer cell lines: MCF7, MDA-MB-231 and SK-BR-3, were cultured in EMEM with 0.01 mg/mL human recombinant insulin, Leibovitz’s L-15 Medium and McCoy’s 5a Medium, respectively. T-47D 3.17 (transfected to carry one stably integrated copy of a luciferase reporter gene driven by the MMTV promoter) [18] were cultured in RPMI 1640 with 0.2 units/mL bovine insulin. All media were supplemented with 5–10% fetal bovine serum (FBS) and antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin) incubated at 37 ◦ C with 5% CO2 in a humidified atmosphere, for maintenance. For studies with hormone treatments, phenol red-free media was used with charcoal-dextran treated serum (sFBS) to reduce the presence of hormones. 4.3. Plasmids Full-length BCAS2, as obtained in the yeast two-hybrid screen, was amplified from pACTII-BCAS2 and cloned into pcDNA3.1/His (Invitrogen, Carlsbad, CA, USA) to make pcDNA-BCAS2. All glutathione-S-transferase (GST) fusion constructs were generated in a pDEST15 vector, as previously described [42]. Estrogen and progesterone receptor vectors (pSG5-ERα and pSG5-PR) were donated by Dr. Pierre Chambon (INSERM, France). ER activation domain expression vectors, pERα-AF1 (aa 1-283) and pERα-AF2 (aa 144-595), as well as 3X-ERE-Luc were provided by Dr. Donald McDonnell (Duke University, USA) [17]. pSG5-SRC-1, pSG5-CBP and pSG5-TIF2 were obtained from Dr. Edwin Milgrom (INSERM, France). 4.4. Glutathione-S-Transferase (GST) Pull-Down Assays GST fusion proteins: pGST-ERα Full (aa 1-595), pGST-ERα N (aa 1-180), pGST-ERα LBD (aa 264-595) were expressed in E. coli strain BL21, induced with 0.2% L-arabinose and purified with glutathione-Sepharose beads, according to manufacturer’s instructions (GE Healthcare). Biotin labeling and in vitro translation of BCAS2 was performed using the TNT® T7 Quick Coupled Transcription/Translation System (Promega, Madison, WI, USA). 30 µL of bound GST fusion proteins was incubated with 10 µL of BCAS2 and incubated for 2 h in 500 µL of sonication buffer (20 mM Tris-HCl, pH 8, 100 mM NaCl, 0.7 mM EDTA, 0.05% Nonidet P-40, with protease inhibitors) in the presence or absence of 100 nM estradiol (E2). Beads were washed 5 times and eluted by boiling in 50 µL of 2× Laemmli loading buffer. Proteins were separated by SDS-PAGE followed by immunoblot using antibodies against GST (Santa Cruz Biotechnology, Dallas, TX, USA) and biotin (Promega, Madison, WI, USA) and chemiluminescent detection (GE Healthcare, Chicago, IL, USA). 4.5. Coimmunoprecipitation COS-7 cells (1.5 × 106 cells per 10 cm dish) were transfected with 3µg pSG5-ERα and 3 µg pcDNA-BCAS2 using Lipofectamine 2000 (Invitrogen) for 5 h. Cells were then treated for 48 h in the absence (ethanol) or presence of 10 nM E2 and then harvested in lysis buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 1 mM DTT). Cell debris was precipitated by centrifugation and the supernatant was incubated with protein A/G-Sepharose (Merck KGaA, Darmstadt, Germany) at 4 ◦ C for 30 min to remove nonspecific interactions. The pre-cleared lysate was precipitated with antibodies against ERα (Santa Cruz Biotechnology) or BCAS2 (Abcam, Cambridge, UK) at 4 ◦ C for Int. J. Mol. Sci. 2019, 20, 966 14 of 19 2 h and incubated overnight with protein A/G-Sepharose. After washing 4 times, the precipitates were eluted in loading buffer, separated by SDS-PAGE and transferred to a nitrocellulose membrane. Immunoblot was performed with anti-ERα and anti-BCAS2. Coimmunoprecipitation in MCF7 cells was carried out in the same manner, except incubations were carried out with EtOH, 10 nM E2 and 100 nM TAM. 4.6. Transient Transfection and Luciferase Assays To determine the transcriptional activity effect of BCAS2 on SHRs, 7 × 104 MCF7, T-47D 3.17 (T-47D-MTVL), SK-BR-3, MDA-MB-231 or Hep-G2 cells were seeded in 24-well plates and transfected with 200 ng of 3xERE-Luc or MMTV-Luc, 50 ng of receptor expressing plasmid, as required, 50 to 300 ng of the appropriate coregulator expression plasmid or empty vector (EV) and 10 ng of pCMV-β-galactosidase (Promega), as transfection control, for 6 h, using Lipofectamine 2000 (Invitrogen) as indicated by the manufacturer. Additionally, these experiments were performed using constructs expressing only the ERα AF-1 or ERα AF-2 in Hep-G2 cells, to determine whether the activity of BCAS2 requires one or both transcriptional activation sites. Cells were incubated for 48 h in the presence or absence of ligand. Ligands used for ERα were E2 or TAM and progesterone (P4) was used to activate the PR. To determine the effect of BCAS2 on the transcriptional activity of other ERα coactivators, 7 × 104 MCF7, MDA-MB-231 and SK-BR-3 cells were transfected, as previously described, along with 50ng of SRC-1, CBP or TIF2. Finally, to determine the impact of HER2, PI3K and MEK-1 signaling pathways in BCAS2 activation, MCF7 and SK-BR-3 cells were transfected, as previously described, in presence of ethanol (EtOH), E2 or TAM along with Trastuzumab, PD98059 or Wortmannin for 48 h. Cell extracts were assayed for luciferase and β-galactosidase activities using a GloMax® 96 microplate Luminometer (Promega). At least three independent transfections were performed in triplicate for each trial. Results are shown as relative light units (RLU) calculated as a ratio of luciferase and β-galactosidase activities. 4.7. Chromatin Immunoprecipitation (ChIP) ChIP assays were performed as previously described [46]. Briefly, 3 × 106 MCF7 cells were grown in 10 cm dishes in phenol red-free medium with 2% sFBS for 2 days before treatment with vehicle (EtOH) or 10 nM E2 for 30 min – 2 h, as indicated. Cells were crosslinked and harvested in cold PBS and protease inhibitor cocktail (Roche) plus 1mM DTT. The cell button was processed using a ChIP Kit (Upstate) following the manufacturer’s instructions. Immunoprecipitation was performed overnight at 4 ◦ C with the addition of 3 g normal rabbit IgG, rabbit anti-ERα (Santa Cruz Biotechnology), mouse monoclonal anti-BCAS2 (Abcam) or methyl histone H3 antibody (Upstate Biotechnology, Lake Placid, NY, USA). Real-time PCR was carried out to amplify promoter regions from the pS2 (TFF1) [20] and WISP2 [21] genes. Oligonucleotides used were: pS2: fwd 50 -CCATGTTGGCCAGGCTAGTC-30 , rev 50 -ACAACAGTGGCTCACGGGCT-30 ; WISP2: fwd 50 -TGTTGTGCCTCCAGCTCCTG-30 , rev 50 -GGTTTCTGGCAGGCAGATT-30 . 4.8. Real-Time Quantitative PCR Real-time PCR was carried out using the SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s protocol, using a LightCycler 480 (Roche Indianapolis, IN, USA). 4.9. Expression of Endogenous Genes To monitor endogenous expression of estrogen-responsive genes, we used MCF7 cells transfected, as described above, with pcDNA-BCAS2 and treated with E2 for 24 h. The effect of overexpression of BCAS2 on the endogenous expression of E2-dependent genes was determined by RTqPCR. Total RNA was isolated using TRIzol (Invitrogen), following manufacturer’s instructions. We measured mRNA levels of pS2, C3, IGFBP2 and c-Myc. The cDNA was Int. J. Mol. Sci. 2019, 20, 966 15 of 19 obtained from 100 ng of total RNA using SuperScript Vilo cDNA synthesis (Invitrogen, Carlsbad, CA, USA). Glyceraldehyde-3-phosphate dehydrogenase (human GAPDH) mRNA amplification was used for assay normalization and relative units are reported. Oligonucleotides used were: pS2 fwd 50 -CACCATGGAGAACAAGGTGA-30 , rev 50 -TGACACCAGGAAAACCACAA-30 ; C3: fwd 50 -ACCAGCAGACCGTAACCATC-30 , rev 5´-GCAGCCTTGACTTCCACTTC-30 ; IGFBP2: fwd 50 -CCTCAAGTCGGGTATGAAGG-30 , rev 50 -ACCTGGTCCAGTTCCTGTTG-30 ; c-MYC: fwd 50 -AGAGAAGCTGGCCTCCTACC-30 , rev 50 -CGTCGAGGAGAGCAGAGAAT-30 ; GAPDH: fwd 50 -CCTCAACGACCACTTTGTCA-3’; rev: 5’-CCCTGTTGCTGTAGCCAAAT-3’. The data shown were performed three times in triplicate. 4.10. shRNA Transfection Lentivirus stocks were produced in HEK 293T cells by transfection with pLKO.1-BCAS2 siRNA constructs or control siRNA (shRNAs; Sigma Aldrich), packaging plasmid (pCMV R8.91) and envelope plasmid (pVSVG) using Calcium phosphate. Viruses were harvested and concentrated as previously described [42] 3 × 105 MCF7 cells were infected by spin inoculation system [47] centrifuging the virus particles with the cells at 1200 rpm for 2h at room temperature. Cells infected with vectors expressing shRNA-BCAS2 or shRNA-E1 (negative control) pLKO.1 and were selected with 2 g/mL of puromycin (Sigma-Aldrich) 24 h after infection. 4.11. Cell Viability Assays Cell viability was assessed using a tetrazolium salt WST-1 assay (Quick Cell Proliferation, BioVision, Milpitas CA, USA). MCF7 cells (5 × 105 ) were seeded in 6-well plates and transfected with 3 µg pcDNA-BCAS2 or EV, as described previously and left for 48 h. Cells were then seeded in 96-well plates and treated in the absence (EtOH) or presence of E2 and incubated for up to 8 days. Cell viability measurements were taken every 48 h. After incubation, 10 µL of 5 mg/mL WST was added to the cells and the reaction was incubated for 3 h at 37 ◦ C. The assay was read at an absorbance of 450 nm using a microplate reader (BioTek Synergy H1 Hybrid Multi-Mode Microplate Reader) and survival of cells was calculated by dividing the absorbance of the transfected cells by that of the non-transfected. All tests were performed in quadruplicate. Additionally, cell viability was assayed in the same manner after transfecting BCAS2 shRNA constructs, as described previously. Cells were seeded in 96 well plates and allowed to grow for 96 h before determining cell viability. All tests were performed in quadruplicate. 4.12. Colony Formation and Wound Healing Assays To perform clonogenicity assays, MCF7 cells were BCAS2 or EV transfected and 1000 cells/well were seeded in 6-well plates. Treatments were carried out with E2, TAM or vehicle (EtOH) for 12 days. Cells were then dyed with crystal violet 0.05% and the number of colonies was counted. Colony numbers were assessed visually and colonies containing >50 normal-appearing cells were counted. Pictures were taken using a digital camera. Number of colonies is reported as % of colonies (PE), where PE = (# colonies formed/# cells plated) × 100 [48]. Experiments were performed three times. To assess cell motility, wound healing assays were carried out. MCF7 cells were BCAS2 or EV transfected and 500,000 cells/well were plated in 6-well plates. Once cells were 90% confluent, the surface was scratched with a 200 µL tip and cells were treated with E2 for 48 h. Experiments were carried out in the presence of a proliferation inhibitor, AraC (20nM), for 48 h. Pictures were taken every 24 h for 48 h and the % of open wound was calculated. 4.13. Statistical Analysis All experiments were repeated at least three times and data are expressed as the mean ± standard deviation. Statistical analysis of the proliferation results was carried out by two-way ANOVA followed Int. J. Mol. Sci. 2019, 20, 966 16 of 19 by Bonferroni post tests using Graph Pad Prism 6.0 for Mac. Statistical analysis for luciferase assays was carried out using Student´s t-test or one-way ANOVA followed by Dunnet port test. 5. Conclusions We have shown that BCAS2 is a bona fide coactivator of ERα in breast cancer cell lines, being recruited to target gene promoters and increasing expression of a subset of E2 regulated genes. BCAS2 functions primarily through its interaction with AF-1 and collaborates with AF-2 coactivators, having a role in both independent and synergistic activity of these two transcriptional activation functions. Additionally, BCAS2 is involved in promoting various processes of carcinogenesis both in the presence and absence of estradiol. This suggests a role for BCAS2 overexpression in promoting ERα activity in a reduced estrogen environment, as is found during hormonal treatment for breast cancer. Finally, BCAS2 is regulated by the PI3K/AKT signaling pathway. However, the mechanisms involved in this regulation must be studied further. Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/4/ 966/s1. Supplementary Figure S1. Coimmunoprecipitation assays in MCF7 cells in the presence of ethanol (OH), E2 (10nM) or TAM (100nM) showing interaction in the presence or absence of ligand. Input refers to pre-clear protein extracts. Supplementary Figure S2. Raw western blot data studies using inhibitors of signal transduction pathways. (A) Three independent assays showing HER2 expression in SK-BR-3 cells in the presence or absence of the HER 2 inhibitor trastuzumab. (B) Two independent assays showing AKT and pAKT expression in MCF7 cells in the presence and absence of Trastuzumab and AKT activation inhibitor wortmannin. (C) Two independent assays showing ERK and pERK expression in MCF7 cells in the presence or absence of MEK inhibitor PD98059. Author Contributions: A.S.-H. and M.Y.N.-R. carried out the experimental procedures. A.J. supervised the R.T.-PCR assays and those involving progesterone receptor. A.S.H., N.B.-A. and E.L. analyzed the data and wrote the article. E.L. conceived the study and was in charge of overall direction and planning. All authors read and approved the final manuscript. Funding: This work was supported by funding from the Instituto Nacional de Cancerología and grants from Consejo Nacional de Ciencia y Tecnología (CONACyT) numbers 33831-M and 182189 and from Programa de Apoyo a Proyectos de Investigación e Inovación Tecnológica, Dirección General de Asuntos del Personal Academico, UNAM. ASH and MYNR were PhD students in the Programa de Doctorado en Ciencias Biomédicas, Universidad Nacional Autónoma de México and funded by scholarships from CONACyT. The funders had no role in the study design, data collection, analysis or preparation of this manuscript. Acknowledgments: We thank Instituto Nacional de Cancerología for financial support. Conflicts of Interest: The authors declare no conflict of interest. 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