Estrogen Regulation and Physiopathologic Significance of Alternative Promoters in Breast Cancer

Published OnlineFirst April 20, 2010; DOI: 10.1158/0008-5472.CAN-09-3988 Published OnlineFirst on April 20, 2010 as 10.1158/0008-5472.CAN-09-3988

Tumor and Stem Cell Biology Cancer Research Estrogen Regulation and Physiopathologic Significance of Alternative Promoters in Breast Cancer

Martin Dutertre1, Lise Gratadou1, Etienne Dardenne1, Sophie Germann1, Samaan Samaan1, Rosette Lidereau2, Keltouma Driouch2, Pierre de la Grange3, and Didier Auboeuf1

Abstract Alternative promoters (AP) occur in >30% protein-coding genes and contribute to proteome diversity. How- ever, large-scale analyses of AP regulation are lacking, and little is known about their potential physiopathologic significance. To better understand the transcriptomic effect of estrogens, which play a major role in breast cancer, we analyzed gene and AP regulation by estradiol in MCF7 cells using pan-genomic exon arrays. We thereby identified novel estrogen-regulated genes (ERG) and determined the regulation of AP-encoded transcripts in 150 regulated genes. In <30% cases, APs were regulated in a similar manner by estradiol, whereas in >70% cases, they were regulated differentially. The patterns of AP regulation correlated with the patterns of estrogen receptor α (ERα) and CCCTC-binding factor (CTCF) binding sites at regulated gene loci. Interest- ingly, among genes with differentially regulated (DR) APs, we identified cases where estradiol regulated APs in an opposite manner, sometimes without affecting global gene expression levels. This promoter switch was mediated by the DDX5/DDX17 family of ERα coregulators. Finally, genes with DR promoters were preferentially involved in specific processes (e.g., cell structure and motility, and cell cycle). We show, in particular, that isoforms encoded by the NET1 gene APs, which are inversely regulated by estradiol, play distinct roles in cell adhesion and cell cycle regulation and that their expression is differentially associated with prognosis in ER+ breast cancer. Altogether, this study identifies the patterns of AP regulation in ERGs and shows the contribution of AP-encoded isoforms to the estradiol-regulated transcriptome as well as their physiopathologic significance in breast cancer. Cancer Res; 70(9); 3760–70. ©2010 AACR.

Introduction phenotype (e.g., induction of cell proliferation) and helped identify prognostic markers for breast cancer (5, 9, 10). Estrogen receptors (ER), especially ERα, belong to the nu- It is now established that >30% of human protein-coding clear receptor superfamily of transcription factors and play a genes have alternative promoters (AP), and multiple biologi- major role in breast cancer. Indeed, ∼50% of breast tumors cal functions of APs have been shown (11–14). Firstly, APs are ER positive (ER+) and can be growth inhibited by phar- may allow for gene regulation by multiple signals and in mul- macologic blockade of estrogen. In addition, the ER status is tiple tissues. Secondly, APs may affect the efficiency of trans- one of the most widely used markers of good prognosis in lation by affecting the sequence of 5′-untranslated regions breast cancer (1). Therefore, intensive efforts are currently (UTR). Thirdly, APs may affect open reading frames in mRNAs made to elucidate the mechanisms of estrogen actions on and give rise to protein isoforms with different NH2-terminal breast cancer cells. Many estrogen-regulated genes (ERG) sequences and activities. Therefore, APs contribute a signifi- have been identified mainly through microarray analyses in cant part of the proteome diversity and may have a wide bio- the MCF7 breast cancer cell line (2–8). The identification of logical effect. However, little is known about their regulation; ERGs has helped understand how estrogens affect cell in particular, large-scale analyses of AP regulation are lacking. Indeed, classic 3′-biased expression microarrays and promoter arrays are not designed to interrogate APs. Authors' Affiliations: 1Institut National de la Sante et de la Recherche α Medicale, U590, Centre Léon Bérard, Lyon, France; 2Institut National Recent studies indicated that most ER binding sites in de la Sante et de la Recherche Medicale, U735, Centre René the genome of MCF7 cells are not located within promoter Huguenin, Saint-Cloud, France; and 3GenoSplice Technology, Institut regions but up to >50 kb away (15, 16), opening up wide pos- Universitaire d'Hématologie, Paris, France sibilities for the regulation of APs by estrogens. However, few Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). studies have examined the effect of estrogens on APs. Studies of the Xenopus vitellogenin A1 gene and of the human GREB1 Corresponding Author: Martin Dutertre, Institut National de la Sante et de la Recherche Medicale U590, Centre Léon Bérard, 28 rue Laennec, gene showed that estrogen can stimulate several promoters 69373 Lyon (Cedex 08), France. Phone: 33-4-26-55-67-45; Fax: 33-1- within a target gene (17, 18). More recently, a ChIP-chip 42-38-53-45; E-mail: [email protected]. study using a custom array of APs showed selective regu- doi: 10.1158/0008-5472.CAN-09-3988 lation of RNA polymerase II (Pol II) levels at APs in response ©2010 American Association for Cancer Research. to estrogen (19). However, Pol II levels at promoters do not

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always reflect productive initiation (20, 21), and therefore, probes for further analyses: exonic location according to such an analysis does not permit to determine which iso- cDNAs (13), GC content below 18, and no cross-hybridizing forms are generated in response to estradiol. In the present potential. The strategy used to annotate AP expression and study, we determined the contribution of AP-encoded trans- regulation is described in Supplementary Fig. S2. Briefly, to cripts to the estrogen-regulated transcriptome in MCF7 cells annotate AP expression, AP-associated exons were compared using the Affymetrix Human Exon (HuEx) arrays, which have for probe intensities using a Student's t test. To annotate AP probes for most exons in the genome. regulation, the intensities of AP-associated probes were compared between estrogen-treated and control cells using a Stu- Materials and Methods dent's paired t test. To identify estrogen-regulated APs within nonregulated genes, the gene-normalized intensities of each Cell culture. MCF7 cells were maintained in DMEM with AP-associated exon were compared between estradiol-treated 10% fetal bovine serum. Except for transfections, cells were and control cells using a Student's paired t test (splicing index grown for 3 days in phenol red–free medium supplemented method). Probe data were visualized in their gene context using with 2% charcoal-stripped serum before treatment with 17β- the EASANA visualization module (GenoSplice Technology). estradiol (10 nmol/L; Sigma) or 0.1% ethanol as a control. Chromatin immunoprecipitation. Chromatin immuno- Transient transfections were performed using Lipofectamine precipitation (ChIP) was performed as described previously RNAiMax (Invitrogen) and the indicated small interfering (24) using antibodies against Pol II (CTD4H8), acetylated his- RNAs (siRNA; siGL2, CGUACGCGGAAUACUUCGAdTdT; tone H3 (Upstate), and control mouse immunoglobulins. Im- siL, GACUUCACUUUGAAAAGAAdTdT; siS, CCAUACGA- munoprecipitated DNA was purified using Qiagen columns GUCCUAGAUGUdTdT; siDDX5/17, GGCUAGAUGUGGAA- and analyzed by quantitative PCR. GAUGU). Adherent cells were harvested using trypsin. Tumor samples and survival studies. Biopsies from 74 Adherent and floating cells were counted with a Coulter ER+ primary unilateral breast carcinomas were collected. The counter. Cell cycle analyses were performed by propidium io- patients (mean age, 59.3 y; range, 35–91) received no radiother- dide staining and fluorescence-activated cell sorting analysis. apy or chemotherapy before surgery. Sixty-nine percent of RNA and protein analyses. Total RNA was extracted using tumors were lymph node positive. The median follow-up was Trizol (Invitrogen). RNA treated with DNase I (Ambion) was 8.5 years (range, 1.4–16.2), and 26 patients relapsed at distant reverse transcribed using SuperScript II and random primers sites. Total RNA was analyzed by quantitative reverse tran- (Invitrogen). PCR was performed using GO-Tag (Promega). scription-PCR (RT-PCR) using TBP levels for normalization. Quantitative PCR was performed using Master SYBR Green Associations between transcript levels and metastasis-free I on a LightCycler (Roche) using 18S RNA for normalization. survival (MFS) were determined by the Kaplan-Meier method PCR primers are described in Supplementary Table S1. Pro- using the log-rank test for statistical analysis. tein extracts were prepared in 50 mmol/L Tris (pH 8.0), 0.4 mol/L NaCl, 5 mmol/L EDTA, 1% NP40, 0.2% SDS, and Results 1 mmol/L DTT with protease inhibitors. Following SDS- PAGE, blots were hybridized to anti-NET1 (Abcam) and Identification of novel ERGs using exon arrays. To iden- anti-actin (Sigma) antibodies. tify the APs regulated by estradiol, MCF7 cells were treated Microarray hybridization. RNA was purified on Qiagen with estradiol or vehicle for 6 or 24 hours, and total RNA columns, and RNA integrity was verified using a Bioanalyzer. was analyzed with HuEx arrays. HuEx arrays contain probes Total RNA (1 μg) was processed with the GeneChip WT targeting >1 million of exons from well-annotated or compu- Sense Target Labeling kit and hybridized to GeneChip Hu- tationally predicted genes. In this study, we focused on genes man Exon 1.0 ST arrays (Affymetrix), following the manufac- with known cDNAs. For each gene, the average intensity of turer's instructions. Stained arrays were scanned using the exonic probes was calculated in each sample from four inde- GeneChip Scanner 3000-7G, and quality assessment was per- pendent experiments. Using cutoffs of 1.5 for fold change and formed using Expression Console (Affymetrix). For data ana- 0.05 for P value, 983 genes were regulated by estradiol at 6 lyses, probe intensities were normalized using the quantile and/or 24 hours (Supplementary Fig. S1; gene lists are given normalization method, and background correction was made in Supplementary Tables S2–S4). Of 983 genes, 637 (65%) had using antigenomic probes (22). been identified in a large compilation of previous studies Array data analysis at the gene level. Using the Array- (2–8), which showed the reliability of the method we used Assist software (Stratagene), the average intensity of exonic to identify ERGs (Fig. 1A, Known). However, 346 genes probes was calculated for each gene in each sample, and the (35%) had not been identified as ERGs in the previous studies estradiol and control groups were compared using a Stu- (Fig. 1A, New). Using a cutoff of 2 for fold change, there were dent's paired t test (fold > 1.5, P < 0.05). A compilation of still 61 genes (25%) that had not been previously found to be 3,047 genes regulated by ER ligands in various cell lines regulated by estradiol. We validated (five of five tested) by was retrieved from previous studies (2–8). Gene functions quantitative RT-PCR the regulation of several novel ERGs were analyzed using the Panther software (23). (Supplementary Fig. S1). Similar functions than previously re- Array data analysis at the exon level. Exon-level analyses ported, including cell cycle, were enriched in the 983 ERGs of array data were performed using the EASANA system identified (Supplementary Fig. S1). Altogether, these data in- (GenoSplice Technology). Several criteria were used to select dicated that HuEx arrays allowed to reliably identify ERGs.

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analysis indicated that both promoters E2 and E3 were upregulated by estradiol (Fig. 2A, item 2), in agreement with RT- PCR analysis (Fig. 2A, item 3) and with a previous study (18). Likewise, APs from the RBBP8, TPD52L1, SCL7A5, STC2, FKBP5, and SLC22A5 genes were similarly activated by estradiol (Supplementary Fig. S3). We also identified APs that were simultaneously repressed by estradiol as illustrated with the TGFB3, BMF, LMO7, NPHP3, and MYO1B genes (Sup- plementary Fig. S3). A total of 43 genes containing CR promoters were identified (Supplementary Table S5), and 100% (11 of 11) were validated by RT-PCR (Supplementary Fig. S3). The second category corresponds to APs that were differentially expressed or regulated (DE/DR) in response to estradiol. For example, both EASANA and RT-PCR analyses of the GREB1 gene indicated that promoter E1 was lowly expressed compared with the other two promoters (Fig. 2A; data not shown), as previously reported (18). Similar cases of DE APs were found in 92 genes (Supplementary Table S5), and 85% (11 of 13) were validated by RT-PCR (Supplementary Fig. S4). More interestingly, we identified genes where coex- pressed APs were DR by estradiol (i.e., they were regulated in a selective manner). Figure 2B illustrates the case of the UNG gene, which contains two promoters (26). Both EASANA and RT-PCR analyses indicated that the E1 promoter was upregulated by estradiol, whereas the E2 promoter was not regulated (Fig. 2B). Globally, 29 cases of genes with DR APs were identified (Supplementary Table S5), and 100% (5 of 5) were validated by RT-PCR, namely, the UNG, EFHD1, GRB7, DSCR1, and MCM7 genes (Supplementary Fig. S5; see below). Globally, of 301 ERGs with APs, we could determine the expression regulation of APs for 150 (50%) genes (Supple- mentary Table S5). Of these, 43 genes (29%) contained APs that were similarly regulated by estradiol (CR genes), and 118 genes (79%) contained APs that were DE/DR in response to estradiol (DE/DR genes); among DE/DR genes, there were 92 DE and 29 DR genes (Fig. 1C). It should be noted that 19 genes containing more than two promoters were classified in Figure 1. Identification of ERGs and AP regulation patterns using exon two categories. The global validation rate by RT-PCR was arrays. A, identification of ERGs in MCF7 cells using exon arrays. Number of previously known and novel ERGs. B, number of promoters in ERGs. 93% (27 of 29). The detailed annotation of AP expression C, patterns of AP expression and regulation in ERGs (see text). and regulation in ERGs is given in Supplementary Table S5. Patterns of AP regulation in ERGs correlate with patterns of ERα and CTCF binding sites. To determine whether the Determination of AP regulation by estradiol. Among the distinct patterns of AP regulation in ERGs may be explained 983 ERGs, 301 (31%) contain annotated APs in the FAST DB by distinct patterns of ERα binding sites at gene loci, we fo- database (Fig. 1B). To identify the APs regulated by estradiol cused on the 12 CR and 9 DR genes validated by RT-PCR in ERGs, we then analyzed the array data at the exon level. throughout this study (Supplementary Table S6). We used For this, we used the EASANA web interface that allows to ERα ChIP-chip data previously obtained using genome-wide display probes corresponding to alternative exons, particu- tiling arrays, which showed ERα binding site enrichment larly exons associated with APs, as defined by FAST DB within 50 kb of ERGs (15, 27). We also analyzed CTCF bind- (see below; refs. 13, 25). Using the strategy described in Sup- ing sites, which are insulators that were recently mapped ge- plementary Fig. S2, we defined two broad categories of AP nome-wide and shown to isolate genes from the action of regulation by estradiol. distant regulatory sites, including ERα binding sites (28–30). The first category corresponds to APs with similar expres- As expected from previous studies (15, 16, 27), only 6 (4 CR sion regulation in response to estradiol and that we defined and 2 DR) of 21 genes analyzed had an ERα binding site as coregulated (CR) APs. To illustrate this category, Fig. 2A within 5 kb of an AP (Supplementary Table S6). However, represents the analysis of the GREB1 gene, which is con- 10 genes (6 CR and 4 DR) had an ERα binding site and an trolled by three different promoters (18), corresponding to AP located in the same CTCF block (region between two exons E1, E2, and E3 in FAST DB (Fig. 2A, item 1). EASANA consecutive CTCF binding sites; Supplementary Table S6).

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Figure 2. Two main patterns of AP regulation by estradiol. A, coregulation of APs in the GREB1 gene. B, differential regulation of APs in the UNG gene. Item 1, gene representation in EASANA; red arrows indicate promoters. Item 2, HuEx array data analysis in EASANA. The exonic positions and intensities of the probes are represented (some exons have no probes). The height of bars represents the mean intensity (in log2) of a single probe in the presence of estradiol. The horizontal yellow line represents background levels. Bar colors indicate estradiol effect on probe intensity (green, decrease; red, increase; black, no change). Item 3, RT-PCR results and transcript schematics (boxes, exons; arrows, transcription start sites; stars, translation initiation sites; stop signs, translation stop sites). GREB1 transcripts starting in exon 1 are poorly expressed in MCF7 cells and are therefore not shown. C, schematics of typical patterns of ERα and CTCF binding sites in CR and DR genes. Regulated and nonregulated promoters are represented with plain and crossed arrows, respectively; ERα binding sites with circles; and CTCF binding sites with rectangles. In brackets, number of cases (from 21 genes analyzed). D, proportions of CR and DR cases where APs are located in the same CTCF block (clustered APs) or in different CTCF blocks (insulated APs).

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These data suggest that a large proportion of both CR and isoforms. This switch was confirmed by competitive amplifi- DR genes are direct ER target genes. In the majority of these cation of both isoforms by RT-PCR (Fig. 3B, bottom). genes, either CR or DR, both APs were located within 50 kb The opposite effects of estradiol on AP expression, some- of an ERα binding site (Supplementary Fig. S6A). However, times without changes in global gene expression levels, set a whereas in CR genes all APs were located in the same CTCF novel paradigm of qualitative gene regulation by hormone. block as an ERα binding site (Fig. 2C, CR), in DR genes only Further supporting the significance of this finding, the oppo- one (regulated) promoter was located in the same CTCF site effects of estradiol on NET1 isoform expression were ob- block as an ERα binding site (Fig. 2C, DR; Supplementary served at different time points and in a second breast cancer Fig. S6B; Supplementary Table S6). Thus, in genes with cell line (Supplementary Fig. S7). Moreover, the differential ERα binding sites, the distinct patterns of AP regulation effect of estradiol on NET1 APs was specific, as progesterone (CR versus DR) may be explained by distinct patterns of that stimulates NET1 expression in T47D cells (31) strongly ERα location relative to APs and CTCF binding sites. As all increased the expression of both isoforms (Supplementary ERGs do not contain ERα binding sites, we then considered Fig. S7). This result also indicated that the E4 promoter can the location of CTCF binding sites in the 21 genes analyzed be activated at the same time as the E1 promoter. As an ERα relative to APs only. In 85% CR cases, APs were located in binding site was mapped 8 kb downstream of the NET1 gene thesameCTCFblock(“clustered APs”), whereas in 67% (15), it might be a direct transcriptional target gene of estro- DR cases APs were separated by a CTCF binding site (“insu- gen. To determine whether the inverse regulation of NET1 lated APs”; Fig. 2D). The binding of CTCF between APs was isoforms by estradiol was indeed a transcriptional effect, validated by ChIP in MCF7 cells (Supplementary Fig. S6C). we analyzed the effect of estradiol on Pol II levels on NET1 Thus, distinct patterns of AP regulation by estradiol were as- promoters using ChIP assay. Pol II levels increased over the sociated with different patterns of ERα and CTCF binding E1 promoter while decreasing over the E4 promoter in sites at gene loci. response to estradiol (Fig. 4A). These data indicate that estra- Some genes are essentially regulated in a qualitative diol may have opposite effects on the transcriptional activity manner by estradiol. Further analyzing DR genes, we of promoters within the same target gene. AP-selective effects observed that estradiol can regulate the expression of two of estradiol on Pol II or pre-mRNA levels were also observed APs in an opposite manner. Indeed, EASANA analysis of the for other genes (Supplementary Fig. S8). MCM7 gene predicted that the E1 promoter was upregulated Our finding that both upregulation and downregulation by estradiol, like the global gene level, whereas the E2 promot- of promoters may occur within the same gene in response er was downregulated (Fig. 3A). Quantitative RT-PCR analysis to estradiol suggested that these processes might be medi- confirmed that the E1 promoter was strongly upregulated by ated in part by common factors. Although little is known estradiol (∼6-fold), whereas the E2 promoter was downregu- about the mechanisms of gene downregulation by estradiol, lated (∼2-fold; Fig. 3A). several coactivators recruited by ERα to target genes, in- Our finding that AP-encoded transcripts may be selectively cluding the DDX5/DDX17 family of proteins (also called or inversely regulated by estradiol within a gene (Figs. 2B and p68/p72), were recently shown to repress transcription in 3A; Supplementary Fig. S5) raised the possibility that estradi- specific promoter contexts (32–35). To test whether these ol might regulate APs within genes that, as a whole, are not coregulators might play a role in the inverse regulation of significantly regulated. Such DR genes would have been APs by estrogen, MCF7 cells were transfected with a siRNA missed in our first analysis of gene regulation by estradiol, efficiently targeting both DDX5 and DDX17 (siDDX5/17; where all the probes of a given gene were averaged. We there- Supplementary Fig. S9). DDX5/DDX17 depletion nearly abo- fore generated an algorithm to search for estradiolregulated lished the estradiol-mediated effect on the relative expression APs within genes that are not globally regulated. A stringent levels of AP-encoded isoforms of the NET1 gene (Fig. 4B), re- bioinformatic analysis, followed by RT-PCR validation, iden- pressing the estradiol-stimulated isoform while inducing the tified this pattern of regulation in the LMBR1, TPD52, estradiol-repressed isoform (Fig. 4C). Similar observations NUMA1,andNET1 genes (Fig. 3B; Supplementary Fig. S5). were made for the MCM7 gene (Supplementary Fig. S9). Con- In the NET1 gene, two promoters are located upstream of sistently, on DDX5/DDX17 depletion, Pol II levels and histone exons E1 (producing the long form) and E4 (producing the H3 acetylation were decreased at the NET1 P1 promoter short form; Fig. 3B). Quantitative RT-PCR analysis confirmed while increasing at the P4 promoter (Fig. 4D). Altogether, the prediction that the E1 promoter was upregulated by es- these data show the ability of estradiol to switch the expres- tradiol (∼3.5-fold), whereas the E4 promoter was slightly sion of promoters within target genes, and identify the downregulated (Fig. 3B). Remarkably, the whole NET1 gene DDX5/DDX17 coregulators as potential mediators of this expression, as measured by summarizing all the exonic promoter-switching effect. probes, was not significantly affected by estradiol (−1.2-fold). Global effect of APs on transcriptome regulation by This prediction was confirmed by quantitative RT-PCR anal- estradiol. Taking into account that APs occur in 31% ERGs ysis with primers located in the 3′ part of the gene measuring and that their AP expression/regulation status could be deter- both isoforms (Fig. 3B, Total). The fact that NET1 isoforms mined in 50% cases, our data (Fig. 1C) imply that CR and DE/ were regulated in an opposite manner by estradiol, whereas DR promoters occur in 9% and 24% ERGs, respectively. APs overall NET1 mRNA level was not affected, suggested that often encode distinct protein isoforms (11, 12), and we found hormone induced a switch in the expression of AP-encoded this to be the case in 76% ERGs with APs (Supplementary

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Figure 3. Opposite regulation of APs by estradiol in the MCM7 (A) and NET1 (B) genes. Gene representation (item 1) and probe data analysis in EASANA (item 2) as in Fig. 2. RT-PCR analyses (items 3 and 4). Amplicons are indicated. Transcripts are schematized.

Fig. S10). Thus, ∼23% ERGs are predicted to encode protein tions of DE/DR and CR genes. In comparison with the whole isoforms through APs, thereby contributing to proteome reg- genome, DE/DR genes were enriched in several functions, in- ulation by estrogen. In particular, the coregulation of APs cluding cell cycle, DNA metabolism, cell structure and motil- with distinct open reading frames potentially increases the ity, and related molecular functions (Supplementary Fig. S10). number of regulated protein isoforms by estradiol. Mean- These functions were also enriched in ERGs as a whole but while, the differential regulation of APs may serve to regulate not in CR genes (Supplementary Figs. S1 and S10). Related protein isoforms in a selective manner. Consistently, AP- functions were also enriched in DE/DR genes when directly specific open reading frames were more frequent in DR genes compared with CR genes (Supplementary Fig. S10). Altoge- than in CR genes (88% versus 69%; P = 0.03; Supplementary ther, these data suggest that the ability of estradiol to regulate Fig. S10). To further assess the potential contribution of specific AP-encoded isoforms may be preferentially used to APs to biological responses to estrogen, we analyzed the func- regulate gene subsets involved in specific functions.

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was recently proposed (37), which might be relevant to the well-known mitogenic effect of estrogen on ER+ breast cancer cells. To investigate the functions of NET1 isoforms in MCF7 cells, we developed siRNAs selectively targeting either the long (siL) or the short (siS) NET1 isoform (Fig. 5A). Several days after transfection, adherent and floating cells were counted separately to determine the potential role of NET1 isoforms in cell adhesion; in addition, cell cycle analysis was performed on adherent cells to determine the potential effect of NET1 isoforms on cell proliferation. When compared with transfection with a control siRNA targeting luciferase (siGL2), transfection with siL and siS led to ∼30% and 75% decreases in the number of adherent cells, respectively (Supplementary Fig. S11). As expected, the proportion of floating cells was markedly increased by siS and only slightly affected by siL (Fig. 5B). These effects did not seem to be due to increases in cell death (Supplementary Fig. S11) and agree with the established role of the NET1 short isoform in stress fiber formation and cell adhesion (36). Conversely, cell cycle analysis indicated that siL decreased by 33% the proportion of cells in S phase relative to cells in G1, whereas siS had no significant effect (Fig. 5C). These data suggest a selective role for the NET1 long isoform in promoting the G1-S transition of the cell cycle in MCF7 cells. The differential effect of NET1 isoforms on cell growth and adhesion was confirmed using an indepen- dent set of siRNAs and was not due to an interference with ERα (Supplementary Fig. S12). Finally, the depletion of the Figure 4. Mechanism of AP regulation in the NET1 gene. A, effects of NET1 long isoform decreased estrogen-stimulated MCF7 cell estradiol on Pol II levels over the P1 and P4 promoters of NET1,as growth to levels observed in the absence of estrogen (Fig. 5D). determined by ChIP assay. B to D, effects of siDDX5/17 on NET1 isoform Altogether, these data indicate that NET1 isoforms play differ- levels (B and C) and on Pol II and acetylated histone H3 levels on NET1 promoters (D) in estradiol-treated MCF7 cells. siGL2, siRNA targeting ential roles in the regulation of cell adhesion and prolifera- luciferase. tion. Remarkably, estradiol selectively induces the isoform of NET1 that supports cell proliferation. Physiopathologic significance of promoter-encoded isoforms. Differential functions of AP-encoded isoforms. We then Because estrogen and ER play important roles in breast can- assessed the potential role of DR isoforms in breast cancer cer cell proliferation, we next examined the expression of cells, focusing on NET1, a RhoA-specific guanyl nucleotide AP-encoded isoforms that are inversely regulated by estradiol exchange factor. NET1 APs encode long (594 amino acids) in a collection of 74 ER+ breast tumors. Higher levels of the and short (542 amino acids) protein isoforms that have dis- estrogen-stimulated long isoform of NET1 were associated tinct properties when transfected into cells (36). However, with shorter MFS, whereas higher levels of the short isoform little is known about the endogenous expression of these were not (Fig. 6A). In fact, when normalized to total NET1 protein isoforms. We therefore analyzed their expression in mRNA levels, higher levels of the short isoform were associ- MCF7 cells. Using an antibody directed against the COOH ated with longer MFS (Supplementary Fig. S13). As expected terminus of NET1, both the long and short forms could be from these data, the combination of high levels of the long detected; the identity of the isoforms was confirmed using form and low levels of the short form was associated with isoform-selective siRNAs (Fig. 5A). As expected from analyses reduced MFS (Supplementary Fig. S13). In contrast, total at the RNA level (Fig. 3B), only the long protein isoform was NET1 mRNA levels were not associated with MFS (Fig. 6A). upregulated by estradiol (Fig. 5A). These data indicated that These data show the value of measuring individual isoforms whereas both protein isoforms encoded by the NET1 gene to identify prognostic markers. Likewise, in the case of promoters were expressed in MCF7 cells, estradiol treatment MCM7, higher levels of the estrogen-stimulated P1 isoform, selectively increased the NET1 long protein isoform levels, but not of the estradiol-repressed P2 isoform, were associa- owing to the selective upregulation of the E1 promoter. ted with shorter MFS (Fig. 6B). In fact, higher levels of the P2 Previous studies of NET1 isoforms showed that transfec- isoform tended to be associated with a delay in the occur- tion of the short form (also called NET1A), but not of the long rence of death or metastasis, and the combination of high form, induces the formation of stress fibers, which are actin P1 and low P2 expression levels was associated with shorter fibers mediating cell adhesion to substrate (36). On the other MFS (Fig. 6B). Thus, for both the NET1 and MCM7 genes, hand, the potential functions of the long NET1 isoform are higher expression levels of the estrogen-stimulated isoform currently unknown. In addition, a role for NET1 in cell growth were selectively associated with shorter MFS in ER+ breast

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cancer. These data suggested the physiopathologic signifi- The resulting large data sets (Supplementary Tables S2–S5) cance of estrogen-regulated APs. In the case of NET1, estra- significantly increase the knowledge of the estrogen-regulated diol selectively induces the isoform of NET1 that supports transcriptome in MCF7 breast cancer cells. In particular, the cell proliferation (Fig. 3 and 5), in line with the adverse prog- annotation of AP expression regulation in ERGs will help un- nostic value of this isoform in ER+ breast cancer. Also in line derstand the biological consequences of gene regulations by with these data, the combined overexpression of DDX5 and estradiol because APs most often are associated with alterna- DDX17 tended to be associated with shorter MFS in ER+ tive open reading frames, and in many cases, the resulting breast cancer (Supplementary Fig. S13). protein isoforms have differential activities (Supplementary Table S5; see below). In addition, AP-encoded transcripts have Discussion distinct 5′-UTRs that may affect translation efficiency (11, 12). The identification of the regulated AP-encoded transcripts Although >30% of human genes have APs (11, 14), little is within ERGs will also help understand the mechanisms of known about AP regulation by transcriptional stimuli and gene regulation by estrogen. Our data show that APs in ERGs about their physiopathologic significance. In this study, using can be regulated either similarly (29% of cases) or differen- pan-genomic exon arrays, we determined the patterns of AP tially (79% of cases) by estradiol (some genes with more than regulation by estrogen in ERGs and showed their significance two promoters have both patterns). Both regulation patterns in breast cancer. seem to apply to direct ERGs according to the presence of The identification of many novel ERGs in this study was ERα binding sites in the gene vicinity. Coregulation of APs likely due in part to the higher number and whole-exon dis- by estrogen was previously shown in the case of the GREB1 tribution of probes in exon arrays when compared with gene (18), which we confirmed and extended to various other classic 3′-biased expression microarrays (22). In addition, genes. Differential AP regulation by estrogen is also sup- the ability to look at the regulation of individual promoters ported by AP-selective effects on Pol II and pre-mRNA levels allowed us to identify ERGs whose overall expression levels (Fig. 4A; Supplementary Fig. S8; ref. 19). Importantly, our were not affected. This also allowed us to determine the ex- data indicate that differential AP regulation may occur pression and regulation of AP-encoded transcripts in ERGs. not only through selective effects but also through opposite

Figure 5. Biological effect of differential AP regulation. A, Western blot analysis of NET1 protein isoform expression in MCF7 cells treated with estradiol or transfected with siRNAs targeting luciferase (siGL2), NET1 exons 2 to 3 (siL), or NET1 exon 4 (siS). B to D, effects of NET1 isoform-specific siRNAs (siL and siS) on MCF7 cell adhesion (B), cell cycle progression (C; *, P < 0.05), and estrogen-stimulated cell growth (D).

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Figure 6. Prognostic value of AP-encoded isoforms. Patients with ER+ breast tumors were analyzed for MFS as a function of NET1 (A) and MCM7 (B) transcript levels in tumors (Kaplan-Meier curves). For each indicated transcript, tumors were divided into high and low expressors. The P values of intergroup comparisons are indicated.

effects on AP expression. Indeed, this study identifies a novel specific promoter contexts (32, 34, 35, 39). Their dual role type of ERGs that are regulated by estradiol in a qualitative in promoter activation and repression might rely on their manner, sometimes in the absence of changes in global gene ability to interact with both coactivators (e.g., the histone expression level (e.g., NET1 and NUMA1 genes; Fig. 3; Supple- acetylase CBP) and corepressors (e.g., the histone deacetylase mentary Fig. S5). These data also indicate that ERα associa- HDAC1; refs. 32–35), thereby leading to either increased or tion in the vicinity of a gene may affect its expression not decreased acetylation of histones (Fig. 4D). Our data also only quantitatively but also qualitatively. This might explain, support the recent finding that ER coactivators may be in- in part, why many ERα binding sites in the genome are not volved in promoter downregulation by estrogen (40, 41). This associated with changes in the expression level of neighbor- is an important observation because a large proportion of ing genes (16). ERGs and APs are downregulated by estrogen (this study We identified several factors that seem to determine, in and refs. 5, 15). part, the pattern of AP regulation by estradiol. Firstly, in Interestingly, ERGs with APs DE/DR in response to estra- the case of genes with ERα binding sites located in the close diol were enriched in specific functions relevant to the effects vicinity of promoters, the differential regulation of APs cor- of estrogen on breast cancer cells (e.g., cell cycle; Supplemen- related with their differential association with ERα binding tary Fig. S10). Moreover, our data suggest that the differential sites (Fig. 2C). Secondly, in the case of genes with no “local” regulation of APs by estradiol has physiopathologic signifi- ERα binding sites, our data suggest that promoter selectivity cance. Although very few instances of promoter-encoded iso- may be due to the occurrence of CTCF binding sites between forms associated with prognosis have been described overall APs (Fig. 2C and D). In support of this hypothesis, CTCF (42, 43), this study identifies promoter-encoded isoforms of binding sites were recently involved as insulators of distant NET1 and MCM7 as differentially associated with poor prog- ERα binding sites and in the tissue-specific regulation of APs nosis in ER+ breast cancer. Such markers may be useful in the (29, 30, 38). Thirdly, we identified the DDX5/DDX17 family of understanding and management of the subset of ER+ breast proteins as mediators of promoter switches induced by estra- cancer that has poor prognosis (9, 10). Both NET1 and MCM7 diol (Fig. 4). These proteins are ERα interactants and coac- are potentially involved in oncogenesis. A partial NET1 cDNA tivators that were also shown to repress transcription in was cloned in a screen for oncogenes, and both isoforms of

3768 Cancer Res; 70(9) May 1, 2010 Cancer Research

Alternative Promoter Regulation by ER in Breast Cancer

NET1 interact with the tumor suppressor DLG1 (44). Al- in breast cancer. More generally, we propose that AP profil- though neither isoform of NET1 transformed NIH 3T3 cells ing will help improve the analysis of gene regulation and the (36), NET1 isoforms have differential activities that might un- identification of prognostic markers in cancer. derlie their differential association with prognosis. Indeed, our data suggest a selective involvement of the NET1 long Disclosure of Potential Conflicts of Interest isoform in cell proliferation, whereas the short isoform promotes stress fiber formation and cell adhesion (this study No potential conflicts of interest were disclosed. and ref. 36). These differential activities may be due to differential protein localization; indeed, whereas the long isoform Grant Support is located in nuclei, the short isoform can also be detected in the cytoplasm (36). MCM7 plays a major role in DNA repli- Institut National de la Sante et de la Recherche Medicale AVENIR, Ligue cation (45). The MCM7 promoters have the potential to give Nationale Contre le Cancer (Comité de Paris), Agence Nationale de la Recher- rise to protein isoforms with different NH -terminal se- che, Institut National du Cancer, Groupement Entreprises Françaises Lutte 2 Contre Cancer, and European Union FP6 (EURASNET). M. Dutertre was sup- quences, but there are no data available about these iso- ported by Ligue Nationale Contre le Cancer and Institut National de la Sante et forms. The NET1 and MCM7 genes might be prototypes of de la Recherche Medicale. L. Gratadou was supported by Agence Nationale de la genes with AP-encoded isoforms that are differentially in- Recherche. S. Germann was supported by Association pour la Recherche sur le + Cancer. volved in ER breast cancer. Finally, other genes whose APs The costs of publication of this article were defrayed in part by the payment are DR by estradiol are thought to play roles in cancer (Sup- of page charges. This article must therefore be hereby marked advertisement in plementary Fig. S5). Altogether, this study shows the contri- accordance with 18 U.S.C. Section 1734 solely to indicate this fact. bution of AP-encoded isoforms to the estrogen-regulated Received 10/29/2009; revised 02/17/2010; accepted 02/22/2010; published transcriptome, as well as their physiopathologic significance OnlineFirst 04/20/2010.

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3770 Cancer Res; 70(9) May 1, 2010 Cancer Research

Estrogen Regulation and Physiopathologic Significance of Alternative Promoters in Breast Cancer

Martin Dutertre, Lise Gratadou, Etienne Dardenne, et al.

Cancer Res Published OnlineFirst April 20, 2010.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-09-3988

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