BEX2 Is Overexpressed in a Subset of Primary Breast Cancers and Mediates Nerve Growth Factor/Nuclear Factor-KB Inhibition of Apoptosis in Breast Cancer Cell Lines

Research Article

Ali Naderi,1 Andrew E. Teschendorff,1 Juergen Beigel,1 Massimiliano Cariati,1 Ian O. Ellis,2 James D. Brenton,1 and Carlos Caldas1

1Cancer Genomics Program, Department of Oncology, University of Cambridge, Hutchison/Medical Research Council Research Center, Cambridge, United Kingdom and 2Department of Histopathology, Nottingham City Hospital NHS Trust and University of Nottingham, Nottingham, United Kingdom

Abstract might reflect different cells of origin (4), and importantly, iden- + We have identified a novel subtype of estrogen receptor (ER)- tifying different subgroups of ER tumors may have clinical implications: within tamoxifen-treated tumors, those that are positive breast cancers with improved outcome after tamox- À ER+/progesterone receptor–negative (PR ) and express HER-1 and ifen treatment and characterized by overexpression of the + + BEX2. BEX2 BEX1 HER-2 have worse clinical outcomes than ER /PR tumors (5). gene and its homologue have highly + correlated expression and are part of a cluster enriched for The heterogeneity of ER breast cancer is the result of complex ER response and apoptosis genes. BEX2 expression is induced interactions between estrogen response and growth factor receptor after estradiol (E2) treatment with a peakat 3 h, suggesting signaling pathways, including the insulin-like growth factor and epidermal growth factor families (6). The nerve growth factor BEX2 is an estrogen-regulated gene. BEX2 belongs to a family of genes, including BEX1, NGFRAP1 (alias BEX3), BEXL1 (alias (NGF) pathway has also been implicated in the survival and BEX4 NGFRAP1L1 BEX5 proliferation of breast cancer cells (7). NGF treatment of breast ), and (alias ). Both BEX1 and n n NGFRAP1 interact with p75NTR and modulate nerve growth cancer cells activates nuclear factor- B (NF- B), resulting in factor (NGF) signaling through nuclear factor-KB (NF-KB) to apoptosis inhibition after treatment with a ceramide homologue, regulate cell cycle, apoptosis, and differentiation in neural C2 (8, 9). In breast cancer cells, the effects of NGF on apoptosis and tissues. In breast cancer cells, NGF inhibits C2-induced proliferation are mediated through different receptors: p75NTR apoptosis through binding of p75NTR and NF-KB activation. and p140TrkA, respectively (8, 9). BEX2 In this study, using expression microarray analysis of 135 Here, we show that expression is necessary and + K primary breast tumors, we identified a subset of ER breast cancer sufficient for the NGF-mediated inhibition (through NF- B BEX2 BEX1. BEX1 activation) of C2-induced apoptosis. We also show that BEX2 with overexpression of and was first identified in modulates apoptosis of breast cancer cells in response to E2 blastocytes using differential display analysis, and subsequent M database homology searches identified other family members (50 nmol/L) and tamoxifen (5 and 10 mol/L). Furthermore, BEX2, NGFRAP1 BEX3 BEXL1 NGFRAP1L1 BEX2 [ ( ), , and ], all mapping to overexpression enhances the antiproliferative effect of NGFRAP1 BEX3 tamoxifen at pharmacologic dose (1 Mmol/L). These data Xq22.1-23 (10, 11). ( ) encodes NADE, which BEX2 K interacts with the death domain of p75NTRand mediates suggest that a NGF/ /NF- B pathway is involved in BEX1 regulating apoptosis in breast cancer cells and in modulating apoptosis in neural cells in response to NGF (12). also encodes a small adaptor-like protein that interacts with p75NTR response to tamoxifen in primary tumors. [Cancer Res n 2007;67(14):6725–36] and inhibits NF- B activation in PC12 cells to regulate cell cycle arrest (13). BEX1 and BEX2 have been reported to be silenced by promoter methylation in malignant gliomas (14). We therefore Introduction investigated whether BEX2 and BEX1 could also be involved in The heterogeneity of breast cancer poses a significant challenge NGF signaling in breast cancer cells. in the diagnosis, prognostication, and treatment of the disease. Indeed, using expression analysis, breast cancers can be sub- Materials and Methods classified into at least six subtypes: luminal-like subgroups A, B, and C; basal-like; normal breast-like; and HER-2–like (1–3). Expression Microarray Experiments Different subclasses of estrogen receptor–positive (ER+) tumors The samples used were from a cohort of 135 frozen breast tumors collected at Nottingham City Hospital NHS Trust between 1986 and 1992. The institution research ethics committee approved this study. Total RNA extraction, RNA amplification, and indirect labeling were carried out as described before (15–17). Oligonucleotide microarrays containing 22,575 Note: Supplementary data for this article are available at Cancer Research Online features were used (Agilent Human 1A 60-mer Oligo Microarray, Agilent (http://cancerres.aacrjournals.org/). Current address for A.E. Teschendorff, J. Beigel, J.D. Brenton, and C. Caldas: Cancer Technologies). Hybridization, scanning, feature extraction, and data Research UK Cambridge Research Institute, Li Ka Shing Centre, Robinson Way, normalization were done as previously published (15). Cambridge CB2 0RE, United Kingdom. Requests for reprints: Carlos Caldas or Ali Naderi, Cancer Genomics Program, Microarray Data Analysis Department of Oncology, University of Cambridge, Hutchison/MRC Research Center, A total of 307 slides was compared for correlations between dye reversal Hills Road, Cambridge CB2 2XZ, United Kingdom. Phone: 44-1223-404420; Fax: 44- pairs using Spotfire DecisionSite 8.0, and for each biological specimen, only 1223-331753; E-mail: [email protected] or [email protected]. I2007 American Association for Cancer Research. those genes with a positive correlation were selected for further analysis. We doi:10.1158/0008-5472.CAN-06-4394 further removed those genes with >20% missing data across all samples and www.aacrjournals.org 6725 Cancer Res 2007; 67: (14). July 15, 2007

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2007 American Association for Cancer Research. Cancer Research this resulted in a matrix of 1,203 genes. Missing data points were imputed Western blotting with anti-FLAG antibody (Sigma) at 1:500 dilution of by the k-means nearest neighbor method as described before (18). primary antibody were used to confirm overexpression. Microarray data are deposited in ArrayExpress database (accession number C2/NGF experiments. Cells were grown for 24 h and then incubated E-UCON-1). with C2 and NGF for the apoptotic assays described above. Independent component analysis (ICA; ref. 19) was implemented using Tamoxifen treatment. MCF-7 cells were grown on coverslips. Tamoxifen the Rpackage mlica. 3 Supervised analysis by t test/ANOVA and similarity (Sigma) treatment was carried out at 1, 5, and 10 Amol/L concentrations in ranking were done by Spotfire DecisionSite 8.0. Significance analysis of serum-free medium for 24 h and untreated cells were used as controls. To + microarrays (SAM)4 and prediction analysis of microarrays (PAM)5 were assess the effect of BEX2 overexpression, either BEX2 or empty vector was done following the instructions of the software. The significance analysis of transfected in four replicates followed by tamoxifen treatment at 5 and Pearson’s correlations using Monte-Carlo simulations was done applying 10 Amol/L. Apoptosis was measured using Hoechst and Annexin V-FITC the Rstatistical language. 6 Analysis of the BEX expression signature staining. Annexin V-FITC assay was done using Annexin V-FITC function was done using Ingenuity Pathways Analysis software (Ingenuity fluorescence microscopy kit (BD Biosciences) following the manufacturer’s Systems). Biostatistical analysis was done with Statistical Package for the instructions. Social Sciences version 12.0.1 (SPSS). Single sample prediction (SSP) BEX2 classifier was derived as described before (20). Experiments with Gene Knockdown Cells Generation of BEX2 knockdown cells. BEX2 knockdown (BEX2-KD) in Real-Time PCR Analysis on Tumors MCF-7 and MDA-MB-231 cell lines was carried using SMARTpool small Reverse transcription-PCR (RT-PCR) to assess the expression levels of interfering RNA (siRNA; four oligonucleotides) reagents following the BEX1 and BEX2 on tumor samples was done using gene-specific Taqman manufacturer’s instructions (Dharmacon, Inc.). Cells transfected with assays (Applied Biosystems). Housekeeping genes HPRT1 and RPLP0 were siCONTROL Non-Targeting siRNA (Dharmacon) and grown under the same used as controls. Experimental procedures were done following the conditions were used as controls. All siRNA silencing experiments were manufacturer’s instruction (Applied Biosystems). Relative gene expressions done in four replicates. were calculated as described before (21). C2/NGF experiments. After transfection, MCF-7 and MDA-MB-231 cells (knockdown and controls) were incubated at 37jC for 24 h followed by Analysis of Apoptosis with C2 and NGF Treatments treatments with C2 and NGF as above. Cell culture and apoptotic assays with MCF-7 and MDA-MB-231 cells Estradiol treatment. MCF-7 cells were treated in the following four were done as described in refs. 8, 9. Briefly, cells were grown and treated in groups for 48 h: (a) serum starvation, (b) serum starvation and 17-h- a À À the following groups (in triplicate): ( ) control group (C2 /NGF ), no estradiol at 50 nmol/L, (c) serum starvation in BEX2-KD cells, and (d) b À + h treatment; ( )C2 /NGF , treatment with 200 ng/mL -NGF (R&D Systems); serum starvation in BEX2-silenced cells and 17-h-estradiol at 50 nmol/L. c + À A ( )C2/NGF , treatment with ceramide analogue C2 (Sigma) at 20 mol/L; After 48 h of incubation, cells were harvested, fixed in 4% formaldehyde, and d + + h and ( )C2/NGF , treatments with both -NGF and C2. Apoptosis was then stained with Hoechst to score apoptosis (percentage). scored after staining with Hoechst 33258 (Sigma) as described in refs. 8, 9. Tamoxifen treatment. MCF-7 cells (controls and knockdown) were A RT-PCR Analysis of BEX Expression in Cell Lines with treated with NGF and tamoxifen (5 and 10 mol/L). Apoptosis was assessed after 24 h using Annexin V-FITC assay as described above. Different Treatments Expression levels for BEX1 and BEX2 genes were measured using gene- Assessment of Proliferation in Tamoxifen-Treated Cells BEX BEX specific Taqman assays. relative expression is equal to expression MCF-7 and MDA-MB-231 cells were grown in 96-well plates to 50% in the treated group/average BEX expression in the control group. confluence. Both cell lines were then transfected and treated in the NGF/C2 treatments. The MCF-7 cell line was treated with C2 and NGF following groups: (a) siCONTROL transfection followed by NGF and (b) in four groups as described above. After 24 h of incubation, cells were BEX2-KD followed by NGF. MCF-7 cells were further studied in the harvested. Experiments were carried out in four replicates. following groups: (a) empty vector transfection followed by tamoxifen at Estradiol treatment. MCF-7 cells grown in medium containing phenol 1 Amol/L and (b) BEX2 transfection followed by tamoxifen at 1 Amol/L. red–free DMEM (Invitrogen) and 10% charcoal/dextran-treated serum siCONTROL or empty vector–transfected cells without any treatment were (HyClone) were incubated with 17-h-estradiol (Sigma) at 1 nmol/L used as controls. Proliferation was measured 24 and 72 h after the concentration. Cells were harvested at 0, 1, 3, 6, and 12 h for RT-PCR treatments using Vybrant 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo- measurement of BEX1 and BEX2 expression. Four replicate experiments lium bromide (MTT) proliferation assay kit (Invitrogen) following the were carried out for each treatment condition. manufacturer’s instructions. All experiments were done in eight biological K NF- B inhibition. MCF-7 cells were serum deprived for 24 h and then replicates. treated separately with 18 Amol/L SN50 NF-nB inhibitor peptide (Tebu-Bio) in the presence or absence of C2/NGF. Untreated MCF-7 cells and NF-KB Activity Assays treatment with C2/NGF were used as controls. After another 6, 12, and p50 NF-KB. MCF-7 cells were cultured in the following five groups: (a) 18 h of incubation, cells were harvested. untreated cells (control), (b) C2/NGF treatment (overnight), (c) BEX2 silencing followed by C2/NGF treatment, (d) transfection with empty vector Experiments with BEX2-Transfected Cells (control), and (e) transfection with BEX2 vector. Nuclear extraction was BEX2 transfection. The BEX2 construct in pDream2.1/LIC expression carried out using Nuclear Extraction kit (Panomics, Inc.), and NF-nB DNA- vector (cytomegalovirus promoter and a Flag tag) was obtained from binding activity was measured by ELISA in 10 Ag of starting nuclear extract GenScript Corp. Transfection of MCF-7 and MDA-MB-231 cells was carried (TransBinding NF-nB Assay kit, Panomics). The assays were done in four out in four replicates using ExGen 500 reagent (Fermentas Life Sciences) as biological replicates following the manufacturer’s instructions. The binding instructed by the manufacturer. Cells were cotransfected with 1.2 Agof of p50 NF-nB to DNA was measured for each treatment group, and ratios green fluorescent protein (GFP)-carrying vector with either 1.6 Ag of the + were calculated relative to the untreated control for C2/NGF and silencing empty or BEX2-carrying pDream2.1/LIC vector (BEX2 ). RT-PCR and experiments or empty vector control for the overexpression experiments. Phosphorylated p65 NF-KB. MCF-7 cells were cultured in 96-well plates in five groups: (a) untreated cells (control), (b) transfection with siCONTROL siRNA followed by NGF treatment (overnight), (c) BEX2 d 3 silencing followed by NGF treatment, ( ) transfection with empty vector http://cran.us.r-project.org e BEX2 4 http://www-stat.stanford.edu/~tibs/SAM/ (control), and ( )transfectionwith vector. The amounts of 5 http://www-stat.stanford.edu/~tibs/PAM/ phosphorylated p65 and total p65 NF-nB proteins were measured using 6 http://www.R-project.org ELISA (SuperArray CASE NF-nB p65 S468 kit, Tebu-Bio). Assays were done

Cancer Res 2007; 67: (14). July 15, 2007 6726 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2007 American Association for Cancer Research. BEX2 Is Overexpressed in a Subset of Breast Cancers in eight biological replicates following the manufacturer’s instructions. For overexpressers were luminal subtype A, 30% (n = 6) were luminal each experimental group, the ratio of phosphorylated p65/total p65 was subtype B, and 10% (n = 2) were normal subtype. obtained and relative ratios were calculated as treatment group/control Projection of the sample expression values along the BEX mode À group. showed significant differential activation between ER+ and ER À samples (P =6.4Â 10 9; Supplementary Fig. S3). Thus, we Results concluded that this ICA mode and the genes highly activated in BEX1 and BEX2 are classifiers of ER+ breast cancer. this mode, including the BEX genes, are associated with ERstatus. Expression microarray analysis revealed that BEX2 was the gene We verified that the BEX ICA mode and its association with P with the highest frequency of significant log2 ratios ( < 0.05) estrogen response were robust under repeated runs of the across the cohort: >98% of the samples showed differential algorithm as well as under variations in the number of inferred expression relative to the common reference pool (Supplementary modes (data not shown). Analysis of publicly available data sets Fig. S1). This indicated that the expression of BEX2 varied also showed a majority of the genes in this mode to be differentially À significantly across most samples. Cancer outlier profile analysis expressed in ER+ versus ER breast cancers (data not shown).7 + (COPA), a methodology recently proposed to identify biologically A distinct BEX cluster of genes in breast cancer. Having relevant genes (22), showed that BEX2 and its close homologue, established the significance of the expression of BEX genes in BEX1, ranked among the top genes (Fig. 1A). The mutual presence breast cancer using both COPA and ICA, we asked whether there of these two genes in COPA analysis was striking and led us to were genes significantly correlating with BEX1 and BEX2 (hereafter + further investigate their expression profiles. designated ‘‘BEX cluster’’). To investigate this, we used Pearson’s BEX2 expression relative to the common reference pool correlation coefficients and tested significance of the coefficients separated the breast tumors in two significantly different (P <1 by comparison with null distributions obtained using Monte-Carlo À Â 10 5;Fig.1B) groups with overexpression (n = 20) and under- simulation. The gene correlations were computed to the average expression (n = 115) of the gene. BEX1 expression also varied expression profile of BEX1 and BEX2 because both genes were À significantly between the two groups (P <1Â 10 5), reflecting highly correlated (>0.9). The correlation analysis and Monte-Carlo the strong correlation (Pearson’s correlation coefficient = 0.94) simulation identified 35 genes (Fig. 2A) with significant correla- + in expression patterns of both genes (Fig. 1C). Most interestingly, tions to the expression of BEX1 and BEX2 (BEX cluster), with all samples with BEX overexpression were ER+ (P <0.001; absolute correlation values ranging from approximately 0.25 to 0.4 Fig. 1C). (P < 0.0025, q < 0.06). There were nine of these genes also present in + To validate the expression microarray results, RT-PCR was used to the BEX ICA mode. To validate the BEX cluster, a similar analysis confirm the expression levels of BEX1 and BEX2 in a subset of was done in an independent data set (29). BEX1, together with 23 samples from the original cohort. The correlation coefficients other genes from the cluster, was present in the external data set. between RT-PCR results and expression microarray ratios were 0.87 Sixteen of the overlapping genes (f70%) had significant correla- (BEX2; n = 40) and 0.6 (BEX1; n = 40; P < 0.001; Supplementary tions with BEX1 (P < 0.04; Fig. 2B) and showed the same direction À Fig. S2). BEX1 expression was very low in BEX samples, explaining of expression changes. Considering the differences in methodology + À the wide difference in log ratios between BEX and BEX groups in and variation in the platform used, this overlap supports the + the microarray data. existence of a distinct BEX cluster. BEX1 and BEX2 genes are associated with an estrogen Supervised analysis defines a developmental/apoptosis/cell response expression mode. To gain insight into the possible proliferation BEX expression signature in breast cancer. To À biological role and function of the BEX genes, we applied ICA. identify genes that could differentiate between tumors expressing + ICA decomposes the expression matrix into a set of expression high levels of BEX genes (BEX ; n = 20) from the remainder of À modes, which have been shown to provide better representations tumors (BEX ; n = 115), the samples were divided in two groups BEX2 BEX+ BEXÀ of underlying biological processes or pathways than other based on the expression ratios of ( log2 >0; dimensional reduction methods, such as principal component log2 < 0). This subdivision was validated using an unsupervised analysis (PCA; refs. 19, 23). The explanation for this is that ICA k-means algorithm (k = 2; data not shown). infers expression modes that are as mutually statistically The first supervised analysis used a class prediction algorithm independent as possible. As such modes provide closer approx- based on the nearest shrunken centroid method5 (30). This imations to the underlying biologically relevant processes that algorithm generated an optimal classifier consisting of 37 genes gave rise to the data in the first place. In other words, ICA with a correct classification rate of 85% (Fig. 2C; Supplementary + approximates the expression matrix as a linear superposition of Fig. S4). Notably, 60% of the genes from the BEX cluster were also independent biological processes that are inferred using the members of the optimal classifier. criterion of statistical independence through a nonlinear decorre- To validate these results, further supervised analysis using lation step. In contrast, PCA only does a linear decorrelation and t test/ANOVA and SAM were conducted (Supplementary Excel so does not decompose the expression matrix into the Files S1 and S2). The results showed that approximately 2/3 of the + independent biological processes that generated the data. Our genes overlap among all supervised methods and with the BEX ICA analysis identified an expression mode with strong represen- cluster. tation of BEX1 and BEX2 (Fig. 1D; Supplementary Table S1). The expression patterns in comparison with BEX2 of the 68 BEX1 showed the highest weight in this mode, and BEX2 was genes from the ANOVA-derived signature were analyzed using represented as one of the top 20 genes. Known estrogen response similarity ranking of correlation coefficients (Supplementary Fig. S5; genes, such as GATA3, TFF1, LIV-1, and AREG (24, 25), as well as genes previously shown to be expressed in breast cancers, such as LACRT (26), DCD (27), and BMP7 (28), were also highly activated. Not surprisingly, using the SSP classifier (20), 60% (n = 12) of BEX 7 http://www.oncomine.org/main/index.jsp www.aacrjournals.org 6727 Cancer Res 2007; 67: (14). July 15, 2007

À Supplementary Excel File S3) within the expression matrix of 1,203 absence of NGF. Despite being ER , MDA-MB-231 is also rescued genes filtered for analysis (see Materials and Methods). BEX1 from C2-induced apoptosis by NGF (9). ranked closest to BEX2 with 0.9 similarity and the other 31 with Both cell lines were cotransfected with a BEX2-FLAG expression positive correlation ranked ordered 3 to 33 of 1,203. Likewise, the vector and a GFP expression vector to assess transfection 36 genes with negative correlation with BEX genes ranked the most efficiency. As a negative control, similar experiments used distant (e.g., C10orf81 ranked 1,203 and TNFSF7 ranked 1,201 with cotransfection with an empty vector and GFP. Overexpression of À0.28 and À0.27 similarities, respectively). BEX2 in transfected cells was confirmed using RT-PCR and These findings show that there is a distinct expression signature Western blot with anti-FLAG antibody because none of the + À differentiating BEX and BEX samples, which is reproducible commercially available BEX2 antibodies worked in our hands using different supervised methods. (Fig. 3B). BEX2 overexpression significantly reduced the C2- To gain further insights into the potential functional significance induced apoptosis in both cell lines (P = 0.0001; Fig. 3C; of the BEX expression signature, the Ingenuity Pathways Analysis Supplementary Fig. S7). These data show that BEX2 overexpression software was used. The stability of results was tested by analyzing rescues cells from C2-induced apoptosis in two different breast signatures obtained using SAM, ANOVA, and PAM. Significant cancer cell lines and is sufficient to produce an antiapoptotic effect associations (P < 0.001) were detected between the BEX signature similar to that observed with NGF treatment. and development, apoptosis, and cell proliferation functions Knockdown of BEX2 inhibits the NGF antiapoptotic re- (Supplementary Table S2). sponse in breast cancer cells. We next asked whether the BEX2 expression in MCF-7 breast cancer cells increases with expression of BEX2 is necessary for the NGF antiapoptotic effect. estrogen treatment. Given that BEX1 and BEX2 expression are siRNA was used to knock down BEX2 in MCF-7 and MDA-MB-231 significantly higher in a subset of ER+ breast cancers, we cell lines, and in parallel experiments, nontargeted siRNA (controls) investigated the effect of estrogen on the expression of both genes was used as a control. RT-PCR confirmed that BEX2 transcript in the ER+ cell line MCF-7. MCF-7 cells were treated with estradiol levels were suppressed by f90% in BEX2 siRNA-treated cells (E2) at 1 nmol/L over 12 h. Cells were harvested at 1, 3, 6, and compared with controls (Fig. 3B). BEX2-KD significantly inhibited 12 h time points, and BEX1 and BEX2 expression ratios relative to the antiapoptotic effect of NGF after C2 treatment (P = 0.001) in baseline untreated cells were measured using RT-PCR (Fig. 2D). both cell lines (Fig. 3D) compared with controls. These results BEX2 expression was induced at 60 min, peaked at 3 h (4.1 F 0.2), show that BEX2 expression is required for NGF-mediated and returned to baseline by 12 h. In contrast, E2 did not have any antiapoptotic response in two different breast cancer cell lines. effect on the expression of BEX1, which was not expressed at BEX2 modulates the apoptotic responses of MCF-7 cells to baseline in MCF-7 cells (data not shown). These findings indicate estrogen and tamoxifen. Ceramide is a physiologic mediator of that BEX2 expression increases in response to E2 treatment in the programmed cell death. Although C2 ‘‘mimics’’ the proapoptotic breast cancer cell line MCF-7. activity of cytokines known to interact with the ERresponse, the BEX2 expression in MCF-7 breast cancer cells increases with direct relevance of the ‘‘NGF/BEX2 pathway’’ to apoptosis in C2-induced apoptosis and with NGF treatment. The expression response to estrogen modulation was not addressed in the data suggested a potential link between BEX2 and BEX1 with ER experiments described above. Indeed, tamoxifen treatment in response and apoptosis. We therefore asked whether the expression addition to its antiproliferative effects induces apoptosis in breast of BEX genes in the MCF-7 ER+ breast cancer cell line is modulated cancer cells (31), and E2 has an antiapoptotic activity in breast in response to C2 and NGF using RT-PCR (Fig. 3A). MCF-7 cells cancer cell lines in response to various stressors (32, 33). We were previously shown to express p75NTR(9). therefore treated MCF-7 cells with tamoxifen or E2, using doses Apoptosis in MCF-7 cells were analyzed in four groups as previously shown to modulate apoptosis, and compared responses described in Materials and Methods. We confirmed the previously with and without manipulation of BEX2 expression. published results: no significant change compared with controls Treatment of MCF-7 cells with tamoxifen at 5 and 10 Amol/L À + À in the C2 /NGF+ group, gross apoptosis in the C2 /NGF group, resulted in significant apoptosis (P < 0.01), which was not seen at + + and rescue from apoptosis in the C2 /NGF group (Supplemen- 1 Amol/L (Fig. 4A and B; Supplementary Fig. S8). Apoptosis was tary Fig. S6). significantly reduced by both NGF treatment and BEX2 over- The BEX2 expression ratios relative to control cells were as expression (P < 0.03; Fig. 4A and B). Treatment with the NF-nB follows: (a) 2.62 F 0.44 in response to NGF, (b)4.4F 0.80 in response inhibitor SN50 and BEX2-KD removed the protective effects of to C2, and (c)8.9F 1.61 in response to C2 + NGF treatments BEX2 overexpression and NGF, respectively (P < 0.01; Fig. 4A and (significantly higher than other groups; P < 0.001; Fig. 3A). BEX1 B). These data indicate that activation of the NGF/BEX2 pathway expression was not detected in MCF-7 either at baseline or in inhibits tamoxifen-induced apoptosis and this effect of BEX2 is response to the treatments (data not shown). mediated through NF-nB. These data show that, in MCF-7 cells, BEX2 expression increases MCF-7 cells subjected to serum starvation (48 h) and treated slightly with NGF stimulation, increases significantly with C2 with E2 (50 nmol/L) showed a significant reduction of apoptosis treatment (which induces apoptosis), and increases further with compared with untreated cells (P = 0.01; Fig. 4C). In contrast, in rescue from C2-induced apoptosis by NGF treatment. In contrast, BEX2-KD MCF-7 cells, E2 was not as effective as an antiapoptotic BEX1 was not expressed either at baseline or with any of the agent (Fig. 4C). These results show that BEX2 is necessary for the treatments used. We therefore manipulated BEX2 expression levels E2 antiapoptotic activity in serum-starved MCF-7 cells. in breast cancer cells and analyzed its effects on apoptosis. BEX2 modulation of cell proliferation. Having shown that BEX2 overexpression rescues breast cancer cells from C2- BEX2 plays a role in modulating apoptosis in breast cancer cells in induced apoptosis mimicking NGF treatment. We asked response to NGF and tamoxifen, we next analyzed whether it could whether BEX2 overexpression could rescue breast cancer cells also play a role in modulating cell proliferation using the MTT (MCF-7 and MDA-MB-231) from C2-induced apoptosis in the assay.

Cancer Res 2007; 67: (14). July 15, 2007 6728 www.aacrjournals.org

Figure 1. Expression pattern of BEX genes in primary breast cancers and ICA of breast cancer expression data reveals a BEX mode. A, results of COPA showing top 10 ranked genes. Range of COPA scores for the top 10 genes in each centile and scores of BEX genes. B, correlation of BEX2 log ratios between dye reversal experiments [sample/reference (LogS/R) and reference/sample (LogR/S) are log10 based]. P value is calculated using t test/ANOVA. C, heat map showing the expression of BEX1 and BEX2 across the 135 samples (log10 ratio scales: À1.5 to 1.5). Columns, ER status of the samples. D, graphic showing relative weights of genes in BEX ICA mode. Bold italic, BEX1, BEX2, and known estrogen response genes.

www.aacrjournals.org 6729 Cancer Res 2007; 67: (14). July 15, 2007

Figure 2. BEX cluster and expression signature in breast cancer. A, bar plot of BEX cluster 35 genes identified by correlation analysis. Each column identifies a gene and the direction of the column and its height reflect the nature of the correlation (P and q values of Pearson’s correlation and Monte-Carlo simulation). B, bar plot of overlapping BEX cluster genes with significant correlations to BEX1 (P < 0.04) in van ’t Veer et al.’s study. C, centroid of BEX expression signature by PAM. Weights for the up-regulated and down-regulated genes. Red, genes that overlap with ICA BEX mode. D, graphic of BEX2 relative expression ratios in response to E2 (at 1 nmol/L) in MCF-7 cell line. *, P < 0.01, compared with untreated cells using Mann-Whitney U test.

Cancer Res 2007; 67: (14). July 15, 2007 6730 www.aacrjournals.org

As previously described (34), NGF significantly (P <0.01) modulates the antiproliferative activity of tamoxifen in the MCF-7 increased cell proliferation after 1 and 3 days of treatment. This breast cancer cell line but this effect seems to depend on the was not significantly altered by BEX2-KD (P > 0.3; Fig. 4D). In duration of tamoxifen exposure. addition, BEX2-KD and BEX2 overexpression had no measura- BEX2 mediates NF-nB activation in the NGF antiapoptotic ble effect in the proliferation of these two breast cancer cell lines pathway. The antiapoptotic effect of NGF in breast cancer cell (P > 0.3; data not shown). These data indicate that BEX2 is not lines treated with C2 is mediated through the activation of NF-nB involved in NGF-mediated proliferation. (9). To determine whether BEX2 is directly involved in this pathway MCF-7 cells treated with tamoxifen at 1 Amol/L (pharmacologic in MCF-7 cells, we measured the binding of p50 NF-nB to DNA and dose) for 1 or 3 days showed significantly impaired proliferation the phosphorylation of p65 NF-nB using ELISA assays. NGF (Fig. 4D). BEX2 overexpression further reduced proliferation rate in treatment had no effect on p50 NF-nB binding (Fig. 5A) and cells tamoxifen treated for 24 h (P = 0.01; Fig. 4D) but not in cells increased ratio of phosphorylated p65 NF-nB/total p65 NF-nB tamoxifen treated for 72 h (P > 0.3). These data indicate that BEX2 (P < 0.01; Fig. 5B). BEX2 overexpression significantly increased both

Figure 3. BEX2 and regulation of apoptosis. A, BEX2 relative expression ratios by RT-PCR in MCF-7 cell line. Columns, BEX2 expression ratios. C2À/NGF+, NGF treatment alone; C2+/NGFÀ, C2 treatment alone; C2+/NGF+, C2 and NGF treatments. *, P < 0.001, compared with untreated cells using Mann-Whitney U test. B, RT-PCR and Western blot to confirm BEX2 expression in MCF-7 and MDA-MB-231 cells. For RT-PCR experiments, transfection with BEX2 vector (left) and knockdown using siRNA (right) are shown. Four replicate experiments were done for each condition and ÀDDCT measurements are shown for each set of experiments. Western blot was done using BEX2-transfected MCF-7 (MCF-BEX2) and MDA-MB-231 (MDA-BEX2) cells. Empty vector control cells (MDA-EV and MCF-EV). Anti-FLAG antibody (Sigma) at 1:500 dilution was used (BEX2 construct carries a FLAG tag at the 5¶-end). C, apoptosis in MCF-7 and MDA-MB-231 cells overexpressing BEX2. Columns, percentage of apoptosis. EV, cells transfected with empty vector; BEX2+, cells transfected with BEX2 vector; MDA, MDA-MB-231. D, apoptosis in MCF-7 and MDA-MB-231 cells with BEX2-KD. CT, control siRNA. www.aacrjournals.org 6731 Cancer Res 2007; 67: (14). July 15, 2007

Figure 4. BEX2 modulation of tamoxifen/E2 induced apoptosis and tamoxifen antiproliferative effect. A, apoptosis of MCF-7 in response to tamoxifen using Hoechst staining. TAM, tamoxifen at 10 Amol/L for 24 h; NGF, at 200 ng/mL for 24 h; BEX2 (+), BEX2 transfection. *, P < 0.01 for NGF and TAM versus TAM, BEX2 transfection and TAM versus empty vector and TAM, and BEX2 transfection and TAM + SN50 versus empty vector and TAM. Columns, mean; bars, SE. B, apoptosis of MCF-7 in response to tamoxifen using Annexin V-FITC assay. TAM, tamoxifen at 0, 1, 5, and 10 Amol/L concentrations for 24 h; NGF, at 200 ng/mL. *, P <0.03for TAM versus TAM-5 and BEX2 transfection; **, P < 0.01 for all other experimental conditions. All P values are calculated using Mann-Whitney U test. C, apoptosis of MCF-7 in response to serum starvation and E2. Percentage of apoptotic MCF-7 cells is shown in each treatment group. SS, serum starvation for 48 h; E2, at 50 nmol/L; *, P = 0.01 for E2 treatment versus no treatment. D, effect of BEX2 on proliferation changes mediated by NGF and tamoxifen using MTT assay. BEX2 transfection, BEX2 overexpression; TAM, tamoxifen at 1 Amol/L; MCF, MCF-7. Incubation was done for 1 and 3 d. DOD measured as absorbance difference at 520 nm between the treatment groups and untreated controls. *, P value is for TAM versus TAM/BEX2 overexpression after 1-d incubation using Mann-Whitney U test. p50 NF-nB DNA binding and ratio of phosphorylated p65 NF-nB/ dramatically reduced p50 NF-nB binding (1 F 0.18; P = 0.01; total p65 NF-nB(P = 0.01; Fig. 5A and B). Treatment with C2/NGF Fig. 5A) after C2/NGF treatment. The rise in the ratio of also significantly increased p50 NF-nB DNA binding (P < 0.01; phosphorylated p65 NF-nB/total p65 NF-nB after NGF treatment Fig. 5A), but BEX2-KD, which reverts apoptosis rescue (see Fig. 3D), is also significantly reduced in BEX2-KD cells (P = 0.03; Fig. 5B).

Cancer Res 2007; 67: (14). July 15, 2007 6732 www.aacrjournals.org

These data show that BEX2 expression is both necessary and sufficient for NGF-mediated NF-nB activation. Notably, total p65 protein level by ELISA (and REL-A/p65 gene by RT-PCR) did not significantly change after either NGF treatment or BEX2 overexpression (data not shown). This indicates that the effect of NGF/BEX2 on p65 NF-nB is mediated through an increase in phosphorylation, not a change of protein/gene expression. BEX2 expression is increased by NF-KB inactivation. The ELISA experiments showed that NF-nB activation in C2/NGF treated cells is extremely sensitive to and dependent on BEX2 expression. This suggested that modulation of BEX2 expression is a regulator of the pathway. We therefore analyzed whether BEX2 expression levels change when NF-nB is prevented from relocating to the nucleus despite stimulation of the pathway. MCF-7 cells were treated with SN50, a cell-permeable polypeptide that inhibits translocation of the NF-nB active complex into the nucleus, and this inhibited the antiapoptotic effect of NGF in C2-exposed cells (data not shown). SN50 treatment alone did not change BEX2 expression (Fig. 5C). Overnight treatment with C2/NGF/SN50 for 18 h led to a 21-fold F 3.3 increase (P < 0.001; Fig. 5C)inBEX2 expression. Similar results were obtained after 6 and 12 h of SN50 treatment: relative expressions of 18 F 2.5–fold and 20 F 3–fold, respectively (data not shown). This shows that, upon simultaneous activation of the pathway with NGF/C2 treatment and inhibition of NF-nB DNA binding using SN50, there is a significant further increase of BEX2 expression compared with activation of the pathway alone. Increased BEX2 expression was not observed with other proapoptotic stimuli, such as tamoxifen treatment (10 Amol/L) or serum starvation (data not shown), indicating that the effect of NGF/NF-nB modulation (e.g., with C2 and SN50) on BEX2 expression is not the result of a nonspecific apoptotic response. + + BEX2 /ER breast cancers have a better response to tamoxifen therapy. Having established that in our cohort all BEX+ cases were ER+ breast cancers, we asked whether there was any influence of BEX2 status on outcome among patients treated with tamoxifen therapy. We first divided the cases (n = 34) into BEX2 overexpression or BEX2 underexpression using one of two a criteria: ( )log2 ratios normalized to the median value on the microarrays, showing at least 2-fold expression difference (n = 24), and (b) RT-PCR of samples with <2-fold expression difference by microarray analysis (n = 10), showing DCT<À1SDorDCT > +1 SD of the mean (n = 6). Using these criteria, a total of 30 cases was available for survival analysis and this showed a significantly better (P = 0.03; Fig. 6A; Supplementary Table S3) disease-free interval in

Figure 5. BEX2 and NF-nB activation. A, measurement of p50 NF-nB DNA binding in MCF-7 cells using ELISA. Fold p50 NF-nB DNA binding is compared with untreated cells (C2/NGF and silencing experiments) or empty vector–transfected cells (transfection experiment). C2, 10 Amol/L; NGF, 200 ng/mL. *, P = 0.01 for C2/NGF versus BEX2-KD and C2/NGF and for BEX2 overexpression versus empty vector transfection. B, measurement of phosphorylated p65 NF-nB in MCF-7 cells using ELISA. Relative ratio for phosphorylated p65 NF-nB/total p65 NF-nB is compared with untreated cells (NGF and silencing experiments) or empty vector–transfected cells (transfection experiment). *, P = 0.03 for NGF versus BEX2-KD and NGF; **, P = 0.01 for BEX2 overexpression versus empty vector transfection. C, BEX2 fold expression by RT-PCR in MCF-7 cells treated with C2+/NGF+ and inhibition of NF-nB with SN50 treatment. *, P < 0.001 for C2+/NGF+/SN50+ versus C2+/NGF+. Expression ratios are relative to the average BEX2 expression in untreated MCF-7 cells. All P values are calculated using Mann-Whitney U test. Columns, mean; bars, SE. www.aacrjournals.org 6733 Cancer Res 2007; 67: (14). July 15, 2007

Figure 6. Outcome with tamoxifen therapy and proposed NGF/BEX2/NF-nB apoptotic pathway. A, Kaplan-Meier disease-free survival curve in tamoxifen-treated cases with high versus low BEX2 expression. Cum Survival, cumulative survival; DFI, disease-free interval in months; N, number of events/number of cases in each group. B, schematic presentation of NGF/BEX2/ NF-nB pathway. +, stimulatory effect; À, inhibitory effect; dashed line, hypothetical feedbackloop.

patients with BEX2 overexpression [n = 16; 95% confidence interval NGFRAP1 encodes NADE, which interacts with p75NTR(12, 42–44). (95% CI), 121–161 months] versus those with BEX2 under- More recently, BEX1 was also shown to interact with p75NTRand expression (n = 14; 95% CI, 63–123 months). the data from this group suggest that NGF uses BEX1 to regulate cell cycle arrest (13). The results from these published experiments Discussion showing that BEX1 and NGFRAP1 encode small adaptor proteins The evidence for the involvement of BEX1 and BEX2 genes in that interact with p75NTRconstitute a link between these genes, human cancer has emerged with the report that both genes are NGF signaling, and regulation of apoptosis and differentiation in silenced by promoter methylation in human gliomas (14). BEXL1, neural cells. This provided us with a clue about a possible BEX another BEX family member, is epigenetically silenced in ovarian mechanism for involvement of the gene family in breast cancer (35). The data presented here reveal overexpression of BEX1 cancer in light of the published data showing that NGF inhibits the n and BEX2 in a subset of ER+ breast cancers. We could not detect apoptotic response to C2 in cell lines through p75NTRand NF- B. any correlation in our breast cancers between BEX2 methylation C2 is a ceramide analogue that induces massive apoptosis of breast BEX2 and expression (data not shown). We also reviewed array cancer cells (45, 46). For these studies, we analyzed because BEX1 comparative genomic hybridization data available for the tumors is not expressed in the breast cancer cell lines used. Our BEX2 with BEX overexpression and found no alterations at the genomic experiments show that is necessary for the antiapoptotic D DNA level (data not shown). BEX2 and BEX1 were also highlighted function of NGF in C2-treated breast cancer cells (Fig. 3 ) and BEX2 by outlier analysis (COPA), which recently was used to identify overexpression produces an antiapoptotic effect similar to C significant cancer genes (22). We noted that BEX1 was also that observed with NGF treatment (Fig. 3 ). Interestingly, in À significantly overexpressed in ER+ versus ER tumors in two neuronal cells, ceramide is generated as a second messenger of publicly available data sets, and interestingly, in both data sets sphingomyelin cycle activation by NGF, downstream of p75NTR BEX1 ranks among the top genes in COPA analysis7 (36, 37). ICA, a (47). This probably explains the moderate rise in BEX2 expression completely unsupervised algorithm, identified a mode that we observed in breast cancer cells treated with C2 alone (Fig. 3A). included BEX1 and BEX2. This mode correlated strongly with BEX2-KD inhibited and BEX2 overexpression enhanced NF-nB À ER+ status (P =6.4Â 10 9) and included several well-known activation (both p50 and p65 components; Fig. 5A and B), estrogen response genes. The correlation of BEX1 and BEX2 with indicating that BEX2 is in the NGF/NF-nB pathway upstream of estrogen response genes was further supported using supervised NF-nB in the modulation of apoptosis. In contrast, BEX2 seems not analysis. One of these genes, GATA3, is a transcription factor with to be involved in NGF-mediated proliferation (Fig. 4D), which is known correlation to ERexpression (24). Other estrogen response not surprising because this effect is mediated through a separate genes included LIV-1, TFF1, and AREG (24, 38, 39). Interestingly, receptor (p140TrkA) and downstream pathway (mitogen-activated several BEX-associated genes (GATA3, LIV-1, TFF1, SCUBE2, and protein kinase) in breast cancer cells (8, 9). KIAA0882) are present in the luminal subtype A cluster, which is Our data indicate that BEX2 is also important for apoptosis in associated with estrogen response (2), and indeed, 60% of BEX response to ERmodulation by E2 and tamoxifen by modulating overexpressers were luminal A. The pattern of BEX2 increased NF-nB. NF-nB activity has been shown to play a role in hormone expression in response to E2 treatment (Fig. 2D) is compatible with independence and resistance to tamoxifen and raloxifene in breast that of other estrogen early up-regulated genes (40). cancer (48–50). NF-nB is also activated in anti–estrogen (Fulves- BEX2 interacts with LMO2 and this interaction may regulate the trant)-resistant breast cancer cells, and this resistance can be transcriptional activity of LMO2 through its binding to NSCL2 (41). reversed using NF-nB inhibitors (51). Our experiments with MCF-7

Cancer Res 2007; 67: (14). July 15, 2007 6734 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2007 American Association for Cancer Research. BEX2 Is Overexpressed in a Subset of Breast Cancers cells provide further evidence for this role and suggest direct BEX2 leading to BEX2 overexpression, which in turn enhances the involvement: NF-nB activation by NGF treatment or BEX2 over- tamoxifen antiproliferative effect. expression resulted in reduced tamoxifen-induced apoptosis and In summary, overexpression of BEX genes is a novel classifier of the protective effect of BEX2 overexpression was removed with ER+ breast tumors, and potentially, BEX2 overexpression could NF-nB inhibition (Fig. 4A and B). serve as a marker to identify tumors with better response to We propose a possible explanation to reconcile the cell line tamoxifen therapy. We propose a model where BEX2 is part of both results with the observed better prognosis of tamoxifen-treated the NGF/NF-nB and estrogen response pathways, which regulate patients who were also BEX2 overexpressers. We consistently apoptosis in breast cancer cells (Fig. 6B). In addition, BEX2 observed higher BEX2 expression on simultaneous activation of the cooperates in inhibiting proliferation of breast cancer cells treated pathway (with NGF/C2) and inhibition of NF-nB DNA binding with tamoxifen at pharmacologic dose. These findings suggest the (with SN50) compared with pathway activation alone (Fig. 5C). This tantalizing possibility of the NGF/BEX2/NF-nB pathway being a shows that NF-nB DNA binding is upstream of BEX2 expression, therapeutic target in breast cancer. possibly creating a feedback loop. Others have shown that nuclear extracts from ER+ tumors with better response to tamoxifen have much lower NF-nB DNA-binding activity and ERstimulation results in direct inhibition of NF-nB DNA binding (52–54). We also Acknowledgments observed that BEX2 overexpression enhanced the antiproliferative Received 11/29/2006; revised 4/30/2007; accepted 5/11/2007. effect of tamoxifen after 24 h of treatment (Fig. 4D). It is therefore Grant support: Cancer Research UK. BEX2+ The costs of publication of this article were defrayed in part by the payment of page conceivable that the better prognosis in tamoxifen-treated charges. This article must therefore be hereby marked advertisement in accordance patients is the combined result of relative NF-nB inactivation with 18 U.S.C. Section 1734 solely to indicate this fact.

References neurotrophin receptor, links neurotrophin signaling to expression and regulation of bone morphogenetic the cell cycle. EMBO J 2006;25:1219–30. protein 7 in breast cancer. Int J Oncol 2003;23:89–95. 1. Sorlie T, Perou CM, Tibshirani R, et al. Gene 14. Foltz G, Ryu GY, Yoon JG, et al. Genome-wide analysis 29. van ’t Veer LJ, Dai H, van de Vijver MJ, et al. Gene expression patterns of breast carcinomas distinguish of epigenetic silencing identifies BEX1 and BEX2 as expression profiling predicts clinical outcome of breast tumor subclasses with clinical implications. Proc Natl candidate tumor suppressor genes in malignant glioma. cancer. Nature 2002;415:530–6. Acad Sci U S A 2001;98:10869–74. Cancer Res 2006;66:6665–74. 30. TibshiraniR,HastieT,NarasimhanB,ChuG. 2. Sorlie T, Tibshirani R, Parker J, et al. Repeated 15. Naderi A, Teschendorff AE, Barbosa-Morais NL, et al. Diagnosis of multiple cancer types by shrunken observation of breast tumor subtypes in independent A gene-expression signature to predict survival in breast centroids of gene expression. Proc Natl Acad Sci U S A gene expression data sets. Proc Natl Acad Sci U S A cancer across independent data sets. Oncogene 2007;26: 2002;99:6567–72. 2003;100:8418–23. 1507–16. 31. Zhang GJ, Kimijima I, Onda M, et al. Tamoxifen- 3. Sotiriou C, Neo SY, McShane LM, et al. Breast cancer 16. Naderi A, Ahmed AA, Barbosa-Morais NL, Aparicio S, induced apoptosis in breast cancer cells relates to classification and prognosis based on gene expression Brenton JD, Caldas C. Expression microarray reproduc- down-regulation of bcl-2, but not bax and bcl-X(L), profiles from a population-based study. Proc Natl Acad ibility is improved by optimising purification steps in without alteration of p53 protein levels. Clin Cancer Res Sci U S A 2003;100:10393–8. RNA amplification and labelling. BMC Genomics 2004;5:9. 1999;5:2971–7. 4. Dontu G, El-Ashry D, Wicha MS. Breast cancer, stem/ 17. Naderi A, Ahmed AA, Wang Y, Brenton JD, Caldas C. 32. Razandi M, Pedram A, Levin ER. Plasma membrane progenitor cells and the estrogen receptor. Trends Optimal amounts of fluorescent dye improve expression estrogen receptors signal to antiapoptosis in breast Endocrinol Metab 2004;15:193–7. results in tumor specimens. Mol Biotechnol 2005;30: cancer. Mol Endocrinol 2000;14:1434–1447. 5. Arpino G, Weiss H, Lee AV, et al. Estrogen receptor- 151–4. 33. Fernando RI, Wimalasena J. Estradiol abrogates positive, progesterone receptor-negative breast cancer: 18. Troyanskaya O, Cantor M, Sherlock G, et al. Missing apoptosis in breast cancer cells through inactivation association with growth factor receptor expression and value estimation methods for DNA microarrays. Bio- of BAD: Ras-dependent nongenomic pathways requiring tamoxifen resistance. J Natl Cancer Inst 2005;97:1254–61. informatics 2001;17:520–5. signaling through ERK and Akt. Mol Biol Cell 2004;15: 6. Nicholson RI, Gee JM. Oestrogen and growth factor 19. Liebermeister W. Linear modes of gene expression 3266–84. cross-talk and endocrine insensitivity and acquired determined by independent component analysis. Bio- 34. Chiarenza A, Lazarovici P, Lempereur L, Cantarella G, resistance in breast cancer. Br J Cancer 2000;82:501–13. informatics 2002;18:51–60. Bianchi A, Bernardini R. Tamoxifen inhibits nerve 7. Dolle L, Adriaenssens E, El Yazidi-Belkoura I, Le 20. Hu Z, Fan C, Oh DS, et al. The molecular portraits of growth factor-induced proliferation of the human breast Bourhis X, Nurcombe V, Hondermarck H. Nerve growth breast tumors are conserved across microarray plat- cancerous cell line MCF-7. Cancer Res 2001;61:3002–8. factor receptors and signaling in breast cancer. Curr forms. BMC Genomics 2006;7:96. 35. Chien J, Staub J, Avula R, et al. Epigenetic silencing of Cancer Drug Targets 2004;4:463–70. 21. Ma XJ, Wang Z, Ryan PD, et al. A two-gene expression TCEAL7 (Bex4) in ovarian cancer. Oncogene 2005;24: 8. El Yazidi-Belkoura I, Adriaenssens E, Dolle L, Descamps ratio predicts clinical outcome in breast cancer patients 5089–100. S, Hondermarck H. Tumor necrosis factor receptor- treated with tamoxifen. Cancer Cell 2004;5:607–16. 36. van de Vijver MJ, He YD, van ’t Veer L, et al. A gene- associated death domain protein is involved in the 22. Tomlins SA, Rhodes DR, Perner S, et al. Recurrent expression signature as a predictor of survival in breast neurotrophin receptor-mediated antiapoptotic activity fusion of TMPRSS2 and ETS transcription factor genes cancer. N Engl J Med 2002;347:1999–2009. of nerve growth factor in breast cancer cells. J Biol Chem in prostate cancer. Science 2005;310:644–8. 37. Wang Y, Klijin JG, Zhang Y, et al. Gene-expression 2003;278:16952–6. 23. Lee SI, Batzoglou S. Application of independent profiles to predict distant metastasis of lymph-node- 9. Descamps S, Toillon RA, Adriaenssens E, et al. Nerve component analysis to microarrays. Genome Biol 2003;4: negative primary breast cancer. Lancet 2005;365:671–9. growth factor stimulates proliferation and survival of R76. 38. Taylor KM, Morgan HE, Johnson A, Hadley LJ, human breast cancer cells through two distinct 24. Lacroix M, Leclercq G. About GATA3, HNF3A, and Nicholson RI. Structure-function analysis of LIV-1, the signaling pathways. J Biol Chem 2001;276:17864–70. XBP1, three genes co-expressed with the oestrogen breast cancer-associated protein that belongs to a new 10. Brown AL, Kay GF. Bex1, a gene with increased receptor-a gene (ESR1) in breast cancer. Mol Cell subfamily of zinc transporters. Biochem J 2003;375:51–9. expression in parthenogenetic embryos, is a member of Endocrinol 2004;219:1–7. 39. Martinez-Lacaci I, Saceda M, Plowman GD, et al. a novel gene family on the mouse X chromosome. Hum 25. Vendrell JA, Magnino F, Danis E, et al. Estrogen Estrogen and phorbol esters regulate amphiregulin Mol Genet 1999;8:611–9. regulation in human breast cancer cells of new expression by two separate mechanisms in human 11. Alvarez E, Zhou W, Witta SE, Freed CR. Character- downstream gene targets involved in estrogen metab- breast cancer cell lines. Endocrinology 1995;136:3983–92. ization of the Bex gene family in humans, mice, and rats. olism, cell proliferation and cell transformation. J Mol 40. Carroll JS, Meyer CA, Song J, et al. Genome-wide Gene 2005;357:18–28. Endocrinol 2004;32:397–414. analysis of estrogen receptor binding sites. Nat Genet 12. Mukai J, Hachiya T, Shoji-Hoshino S, et al. NADE, 26. Weigelt B, Bosma AJ, van ’t Veer LJ. Expression of a 2006;38:1289–97. a p75NTR-associated cell death executor, is involved novel lacrimal gland gene lacritin in human breast 41. Han C, Liu H, Liu J, et al. Human Bex2 interacts with in signal transduction mediated by the common tissues. J Cancer Res Clin Oncol 2003;129:735–6. LMO2 and regulates the transcriptional activity of a neurotrophin receptor p75NTR. J Biol Chem 2000;275: 27. Porter D, Weremowicz S, Chin K, et al. A neural novel DNA-binding complex. Nucleic Acids Res 2005;33: 17566–70. survival factor is a candidate oncogene in breast cancer. 6555–65. 13. Vilar M, Murillo-Carretero M, Mira H, Magnusson K, Proc Natl Acad Sci U S A 2003;100:10931–6. 42. Mukai J, Shoji S, Kimura MT, et al. Structure-function Besset V, Ibanez CF. Bex1, a novel interactor of the p75 28. Schwalbe M, Sanger J, Eggers R, et al. Differential analysis of NADE: identification of regions that mediate www.aacrjournals.org 6735 Cancer Res 2007; 67: (14). July 15, 2007

nerve growth factor-induced apoptosis. J Biol Chem associated with activation of cytosolic phospholipase factor nB inhibitor parthenolide restores ICI 182,780 2002;277:13973–82. A2. Cell Death Differ 1999;6:890–901. (Faslodex; fulvestrant)-induced apoptosis in antiestro- 43. Mukai J, Suvant P, Sato TA. Nerve growth factor- 47. Dobrowsky RT, Werner MH, Castellino AM, Chao MV, gen-resistant breast cancer cells. Mol Cancer Ther 2005; dependent regulation of NADE-induced apoptosis. Hannun YA. Activation of the sphingomyelin cycle 4:33–41. Vitam Horm 2003;66:385–402. through the low-affinity neurotrophin receptor. Science 52. Kalaitzidis D, Ok J, Sulak L, Starczynowski DT, 44. Park JA, Lee JY, Sato TA, Koh JY. Co-induction of 1994;265:1596–9. Gilmore TD. Characterization of a human REL-estrogen p75NTRand p75NTR-associateddeath executor in 48. deGraffenried LA, Chandrasekar B, Friedrichs WE, receptor fusion protein with a reverse conditional neurons after zinc exposure in cortical culture or et al. NF-nB inhibition markedly enhances sensitivity of transforming activity in chicken spleen cells. Oncogene transient ischemia in the rat. J Neurosci 2000;20: resistant breast cancer tumor cells to tamoxifen. Ann 2004;23:7580–7. 9096–103. Oncol 2004;15:885–90. 53. Hsu SM, Chen YC, Jiang MC. 17h-estradiol inhibits 45. Gill ZP, Perks CM, Newcomb PV, Holly JM. Insulin- 49. Pratt MA, Bishop TE, White D, et al. Estrogen tumor necrosis factor-a-induced nuclear factor-nB like growth factor-binding protein (IGFBP-3) predis- withdrawal-induced NF-nB activity and bcl-3 expression activation by increasing nuclear factor-nB p105 level poses breast cancer cells to programmed cell death in a in breast cancer cells: roles in growth and hormone in MCF-7 breast cancer cells. Biochem Biophys Res non-IGF-dependent manner. J Biol Chem 1997;272: independence. Mol Cell Biol 2003;23:6887–900. Commun 2000;279:47–52. 25602–7. 50. Liu H, Lee ES, Gajdos C, et al. Apoptotic action of 54. Zhou Y, Eppenberger-Castori S, Marx C, et al. 46. Pirianov G, Danielsson C, Carlberg C, James SY, 17h-estradiol in raloxifene-resistant MCF-7 cells in vitro Activation of nuclear factor-nB (NFnB) identifies a Colston KW. Potentiation by vitamin D analogs of TNFa and in vivo. J Natl Cancer Inst 2003;95:1586–97. high-risk subset of hormone-dependent breast cancers. and ceramide-induced apoptosis in MCF-7 cells is 51. Riggins RB, Zwart A, Nehra R, Clarke R. The nuclear Int J Biochem Cell Biol 2005;37:1130–44.

Cancer Res 2007; 67: (14). July 15, 2007 6736 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2007 American Association for Cancer Research. BEX2 Is Overexpressed in a Subset of Primary Breast Cancers and Mediates Nerve Growth Factor/Nuclear Factor- κB Inhibition of Apoptosis in Breast Cancer Cell Lines

Ali Naderi, Andrew E. Teschendorff, Juergen Beigel, et al.

Cancer Res 2007;67:6725-6736.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/67/14/6725

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2007/07/19/67.14.6725.DC1

Cited articles This article cites 54 articles, 20 of which you can access for free at: http://cancerres.aacrjournals.org/content/67/14/6725.full#ref-list-1

Citing articles This article has been cited by 8 HighWire-hosted articles. Access the articles at: http://cancerres.aacrjournals.org/content/67/14/6725.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/67/14/6725. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.