Profiling Estrogen-Regulated Gene Expression Changes in Normal And

Proﬁling estrogen-regulated gene expression changes in normal and malignant human ovarian surface epithelial cells

Viqar Syed1, Xiang Zhang1, Kin-Mang Lau1,4, Robert Cheng1, Kasturi Mukherjee1 and Shuk-Mei Ho*,1,2,3

1Department of Surgery, University of Massachusetts Medical School, Worcester, MA, USA; 2Department of Cell Biology, University of Massachusetts Medical School, Worcester, MA, USA; 3Department of Physiology, University of Massachusetts Medical School, Worcester, MA, USA

Estrogens regulate normal ovarian surface epithelium been identified due to their differential responses to E2 (OSE) cell functions but also affect epithelial ovarian between the two cell types. These findings may explain the cancer (OCa) development. Little is known about how paradoxical roles of estrogens in regulating normal and estrogens play such opposing roles. Transcriptional malignant OSE cell functions. profiling using a cDNA microarray containing 2400 Oncogene (2005) 24, 8128–8143. doi:10.1038/sj.onc.1208959 named genes identified 155 genes whose expression was published online 22 August 2005 altered by estradiol-17b (E2) in three immortalized normal human ovarian surface epithelial (HOSE) cell Keywords: erythrocyte adducin a subunit clone; 5.1 lines and 315 genes whose expression was affected by the RNA-binding protein S1; nuclear protein SkiP; Rap-2; hormone in three established OCa (OVCA) cell lines. All plexin A3; Sex gene but 19 of the genes in these two sets were different. Among the 19 overlapping genes, five were found to show discordant responses between HOSE and OVCA cell lines. The five genes are those that encode clone 5.1 RNA- Introduction binding protein (RNPS1), erythrocyte adducin a subunit (ADD1), plexin A3 (PLXNA3 or the SEX gene), nuclear Ovarian cancers (OCas), in particular those arising from protein SkiP (SKIIP), and Rap-2 (rap-2). RNPS1, the surface epithelium of the ovary, are the deadliest of ADD1, rap-2, and SKIIP were upregulated by E2 in the gynecologic cancers, with an annual incidence close HOSE cells but downregulated by estrogen in OVCA to 22 220 cases and a death of 16 210 cases (AACR, cells, whereas PLXNA3 showed the reverse pattern of 2005). Several theories have been advanced regarding regulation. The estrogen effects was observed within 6– the etiology of epithelial OCa (EOCa), including an 18 h of treatment. In silicon analyses revealed presence of imbalance in cell growth and apoptosis brought about estrogen response elements in the proximal promoters of by an alteration in the ratio of estrogen to progestin, all five genes. RNPS1, ADD1, and PLXNA3 were which is well supported by epidemiological data (Riman underexpressed in OVCA cell lines compared to HOSE et al., 2004). A decreased incidence of EOCa is observed cell lines, while the opposite was true for rap-2 and SKIIP. among ever users of oral contraceptives during their Functional studies showed that RNPS1 and ADD1 reproductive years. In contrast, among postmenopausal exerted multiple antitumor actions in OVCA cells, while women, the use of hormone replacement therapies PLXNA3 only inhibited cell invasiveness. In contrast, rap- (HRTs) has been shown to increase the risk of EOCa 2 was found to cause significant oncogenic effects in in some but not all population-based studies (Garg OVCA cells, while SKIIP promotes only anchorage- et al., 1998; Riman et al., 2002; Sit et al., 2002; Bakken independent growth. In sum, gene profiling data reveal et al., 2004). Furthermore, ever users of estrogen that (1) E2 exerts different actions on HOSE cells than on replacement therapy (ERT) are more at risk than ever OVCA cells by affecting two distinct transcriptomes with users of HRT formulations supplemented with progestin few overlapping genes and (2) among the overlapping (Riman et al., 2002). Data from a recently completed, genes, a set of putative oncogenes/tumor suppressors have large-scale randomized controlled trial, the Women’s Health Initiative, corroborate observations in population-based studies (Anderson et al., 2003). *Correspondence: S-M Ho, Department of Surgery, Room 504, The best-described action of estrogens related to OCa Lazare Research Building, University of Massachusetts Medical is stimulation of cell proliferation by physiological School, 364 Plantation Street, Worcester, MA 01605, USA; concentrations (nanomolar) of the hormones in OCa E-mail: [email protected] cells (Nash et al., 1989; Langdon et al., 1990; Galtier- 4Current address: Department of Anatomical and Cellular Pathology, Prince of Wales Hospital, Shatin, New Territories, Hong Kong, China Dereure et al., 1992; Syed et al., 2001) and in normal Received 4 May 2005; revised 21 June 2005; accepted 21 June 2005; ovarian surface epithelial (OSE) cells (Bai et al., 2000; published online 22 August 2005 Syed et al., 2001). Chronic increases in cell replication Estrogen-regulated genes expression in ovarian cancer cells V Syed et al 8129 have been linked to genomic instability (Stopper et al., that may be involved in the observed connection 2003; Gorringe et al., 2005), which could be a causative between estrogen exposure and ovarian carcinogenesis factor of tumor initiation and progression. Conversely, in women. antiestrogens (such as tamoxifen and ICI 182,780), which are effective in inhibiting OCa cell proliferation in cultures (Langdon et al., 1990), colony formation in soft Results agar (Runge et al., 1986a, b), and in vivo growth (Langdon et al., 1993, 1994), may afford protection Identification of genes whose expression was regulated by against OCa. The action of estrogen is believed to be E2 in OVCA cell lines in a manner opposite to that mediated by the two estrogen receptors (ERs), ER-a and observed in HOSE cell lines ER-b, which through differential regulation of gene transcription may exert opposite actions on OCa cell Transcriptional profiling was employed to identify genes growth and survival (Bardin et al., 2004). Specific genes whose expression was altered after 5 days of E2 have been identified to be regulated by estrogens in OCa treatment in all three HOSE cell lines or in all three cells; these include ezrin, a member of the membrane– OVCA cell lines based on t-statistics, as described before cytoskeleton-linking proteins, which controls cell moti- (Lau et al., 2003; Ho et al., 2003). A total of 155 genes lity and invasion (Song et al., 2005); cyclin D1, a were identified as differentially expressed in the HOSE regulator of cell cycle progression (Bardin et al., 2004); cell lines and 315 genes in the OVCA cell lines after E2 the growth stimulatory cytokine stromal cell-derived treatment (Figure 1a). Among the 155 genes affected by factor 1 (SDF-1) (Hall and Korach, 2003); transforming the E2 treatment in HOSE cells, 96 (62%) were growth factor-a (TGF-a) (Simpson et al., 1998); and upregulated while 59 (38%) were downregulated. In procathepsin D (Galtier-Dereure et al., 1992). The contrast, of the 315 genes whose expression was altered translational products of these and other estrogen- by the E2 treatment in OVCA cell lines, only 72 (23%) regulated genes may play important roles in the were upregulated and 243 (77%) were downregulated. development and/or progression of OCa. Gene ontogeny analyses revealed that the E2-regulated It is now recognized that aberrant transcription of a genes fall into 16 broadly classified functional categories multitude of genes is involved in the pathogenesis of any (Figure 1b). They include categories of genes that human disease, including OCa. For this reason, tradi- encode receptors, transport proteins, binding proteins, tional studies that relied upon the investigation of a few tumor suppressors, and oncogenic molecules, as well as candidate genes at a time may not be the most efficient those involved in signal transduction, protein modifica- approach with which to identify novel key molecular tion, apoptosis, protein biosynthesis, transcription regulators governing the process. With the advent of processing, cell cycle progression, differentiation, and global transcriptional profiling, microarrays that con- cell adhesion. tain thousands of probes are now used as discovery Comparisons were then made to identify genes whose platforms for large-scale identification of gene expres- regulation by estrogen was reversed in OVCA cell lines sion changes associated with a specific physiological when compared to HOSE cell lines. Among the 19 genes state or response to a stimulus. Transcriptional profiling whose expression was altered by E2 in all HOSE and has been used to identify transcriptomes associated with OVCA cell lines, 13 genes were found to be regulated in EOCa (Hough et al., 2001; Lee et al., 2004) or different the same manner by E2 in both HOSE and OVCA cell types of EOCas (Welsh et al., 2001; Lancaster et al., lines; five were identified as genes upregulated by the 2004), as well as those that predict clinical outcomes hormone treatment, and eight as downregulated (Lassus et al., 2003; Santin et al., 2004), responses to (Table 1). The remaining six genes exhibited discordance chemotherapy, or drug resistance (Sakamoto et al., with regard to their responses to the E2 treatment 2001; Sugimura et al., 2004). However, this approach between HOSE and OVCA cell lines. Of the six has not been utilized to identify genes affected by differentially regulated genes, five were upregulated by estrogens in normal or malignant OSE cells. E2 in HOSE cell cultures and downregulated by the In this study, a microarray containing 2400 known hormone in OVCA cell cultures, while the sixth gene human genes was used to identify those whose expres- (Sex gene or plexin A3) exhibited an opposite pattern of sion was regulated by 17b-estradiol (E2) in three dysregulation (Table 1). A literature search of PubMed immortalized, nontumorigenic human OSE (HOSE) cell on April 8, 2005 found no reports linking any one of lines and three OCa (OVCA) cell lines. Comparison of these genes to estrogen or to OCa. The E2-regulated these two sets of genes revealed only 19 whose genes in HOSE and OVCA cell lines were subjected to a expression is regulated by E2 in HOSE and OVCA cell web-based service for pathway-mapping analysis. A list lines. Among them, five genes were found to be of best-matching pathways for HOSE and OVCA cells is regulated by E2 in HOSE and OVCA cell lines, but in illustrated in Table 2. The genes affected by E2 in both opposite directions. Functional studies demonstrated cell types are implicated in vital cellular functions. that they possess antitumorigenic or oncogenic proper- Based on the basal expression levels of identified ties to varying degrees. Taken together, these findings genes in HOSE and OVCA cell lines, we have led us to postulate that malignant transformation of categorized them into two groups for data presentation: human OSE cells causes dysregulation of a set of E2- (1) genes encoding clone 5.1 RNA-binding protein regulated tumor suppressors/oncogenes, a mechanism (RNPS1), erythrocyte adducin a subunit (ADD1), and

Oncogene Estrogen-regulated genes expression in ovarian cancer cells V Syed et al 8130 a HOSE OVCA 1 1 Phosphorylation/ 2 Phosphorylation/ 2 Dephosphorylation Transcription 15 Dephosphorylation Transcription 7% Regulation 16 4 Metabolite/Ion 4% Regulation 15 11% Others Development/ Transport 16 14% Metabolite/Ion 3 19% Differentiation 14 2% Others Transport Apoptosis 15% 3% Cell 3 4 7% 1% Communication Apoptosis Development/ 5 1% 14 3% Differentiation Cell Communication Protein Biosynthesis/ 13 3% Common genes 6% Modification/ Signal Transduction 13 6 5 Trafficking 12 9% Signal Transduction Cell Cycle 6% Carbohydrate Protein 7% 6% Biosynthesis/ 12 Metabolism 6 7 11 Modification/ Carbohydrate 5% Cell Cycle Cell Adhesion/Cell Cell Proliferation Trafficking Metabolism 6% Structure 6% 5% 10 7 18% 9 8 9% 11 DNA 7 7 Cell Adhesion/Cell 10 Oncogene Cell Motility Cell Proliferation Metabolism/Repair Tumor Suppressor Cell Motility Structure DNA 3% 3% 4% 5% 1% 2% 6% Metabolism/Repair 3% 155 19 315

b HOSE OVCA 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

50 20 40 10 30 0 20 -10 10 -20

Number of genes -30 Number of genes 0 -10 -40 -20 -50

Figure 1 Estrogen-regulated gene expression in HOSE and OVCA cells. (a) The pie diagram depicts gene distribution based on functional categories in HOSE and OVCA cell lines. (b) Number of genes upregulated (solid bars) and downregulated (open bars) in each category are shown by histograms

Table 1 Estrogen-regulated genes in HOSE and OVCA cells Acc. no. Genes HOSE s.d. OVCA s.d. HOSE OVCA Average Average P-value P-value

A-20,18 X58141 mRNA for erythrocyte adducin 1.264 0.105 0.570 0.024 0.025 0.001 alpha subunit D-16, 19 M55040 Acetylcholinesterase (ACHE) 0.597 0.232 0.567 0.060 0.048 0.003 A-17, 10 AF035582 CASK 0.767 0.081 0.631 0.059 0.019 0.004 A-22, 17 M83653 Cytoplasmic phosphotyrosyl 1.645 0.368 0.512 0.105 0.047 0.008 protein phosphatase A-4, 24 U94592 Uncoupling protein homolog 1.805 0.113 1.740 0.193 0.003 0.011 (UCPH) B-14, 20 L76703 Protein phosphatase 2A 0.549 0.189 0.573 0.115 0.027 0.012 B56-epsilon (PP2A) B-24, 7 X87689 mRNA for putative p64 CLCP 1.433 0.198 1.648 0.199 0.032 0.015 protein A-15, 2 U51432 Nuclear protein Skip 1.261 0.136 0.817 0.062 0.040 0.018 D-22, 17 X07743 mRNA for pleckstrin (P47) 0.626 0.076 0.502 0.181 0.007 0.021 D-17, 24 X87852 mRNA for SEX gene 0.701 0.154 1.168 0.061 0.039 0.021 D-6, 16 Y07759 mRNA for myosin heavy chain 12 0.764 0.036 0.636 0.158 0.004 0.029 D-7, 5 L10386 Transglutaminase E3 (TGASE3) 0.720 0.069 0.584 0.182 0.010 0.029 A-19, 17 L37368 (clone E5.1) RNA-binding protein 1.806 0.195 0.564 0.193 0.009 0.030 C-16, 7 U09559 Rch1 (RCH1) 2.044 0.076 1.293 0.135 0.001 0.032 C-3, 16 M18112 poly(ADP-ribose) polymerase 1.268 0.149 1.281 0.130 0.045 0.032 A-11, 24 X12534 rap2 mRNA for ras-related protein 1.188 0.032 0.670 0.165 0.005 0.037 A-4, 20 U10248 Ribosomal protein L29 (humrpl29) 1.704 0.189 1.410 0.215 0.012 0.040 D-8, 1 X64838 mRNA for restin 0.717 0.121 0.523 0.258 0.028 0.043 D-21, 11 AB004903 mRNA for STAT induced STAT 0.737 0.099 0.927 0.040 0.022 0.044 inhibitor-2

Column 1 shows position of gene on the microarray. Column 2 represents the accession number of gene. Column 3 shows the name of the gene. Column 4 represents the average fold increase/decrease expression of E2-regulated genes in three normal human ovarian surface epithelial (HOSE) cell lines. Column 5 shows the the standard deviation of three HOSE lines. Columns 6 and 7 show the average fold change and standard deviation of three OVCA lines, respectively. Columns 8 and 9 indicate P-values for HOSE and OVCA cells, respectively

Oncogene Estrogen-regulated genes expression in ovarian cancer cells V Syed et al 8131 Table 2 Pathways affected by E2 treatment of HOSE and OVCA treatment (Figure 2). Cotreatment of cell cultures with cells E2 and ICI reversed the action of the estrogen, whereas HOSE the antiestrogen alone exerted no direct effects. Phosphatidylinositol signaling system 2.3% (21) Purine metabolism 1.4% (13) Ribosome 1.3% (12) RNPS1, ADD1, PLXNA3, rap-2, and SKIIP transcripts Integrin-mediated cell adhesion 1.2% (11) are induced in OVCA cell lines by E2 in a time-dependent Glycerolipid metabolism 1.1% (10) manner; changes were observed within 6–18 h of exposure Arginine and proline metabolism 1.0% (9) Cell cycle 1.0% (9) To study the time-dependent induction of RNPS1, Alzheimer’s disease 0.9% (8) ADD1, PLXNA3, rap-2, and SKIIP, OVCA cells Inositol phosphate metabolism 0.9% (8) Oxidative phosphorylation 0.9% (8) (OVCA 420 and OVCA 429) were cultured and treated with E2 (10À6 M) or vehicle for 6, 18, and 36 h. At the OVCA termination of culture, cells were collected and RNA Phosphatidylinositol signaling system 2.7% (14) was prepared. The decrease in rap-2 transcript was first Glycolysis/Gluconeogenesis 1.7% (9) detected in RNA of OVCA 429 from 6 h of treatment Integrin-mediated celladhesion 1.7% (9) Glycerolipid metabolism 1.5% (8) with E2, whereas RNPS1, ADD1, and SKIIP transcripts Purine metabolism 1.5% (8) were significantly decreased and PLXNA3 transcript Arginine and proline metabolism 1.2% (6) increased after 18 h of E2 treatment (Figure 3). The Oxidative phosphorylation 1.2% (6) changes in mRNA expression for all five genes persisted Cell cycle 1.0% (5) Fatty acid metabolism 1.0% (5) at the 36 h time point. No change was observed in Pentose phosphate pathway 1.0% (5) vehicle-treated OVCA cells at any time point.

Pathway-mapping analyses of all the genes regulated by estrogen in HOSE and OVCA cells by using the Database for Annotation, Effects of forced expression of RNPS1, ADD1, and Visualization and Integrated Discovery (DAVID) web based program. PLXNA3 in OVCA cell lines The table shows names of signaling pathways and in parentheses the number of genes in that category Having established that RNPS1, ADD1, and PLXNA3 were underexpressed in OVCA cell lines when compared to HOSE cell lines (Figure 2a), alterations in tumorigenicity following forced expression of RNPS1, ADD1, plexin A3 (PLXNA3 or the SEX gene) and (2) genes or PLXNA3 in OVCA cell lines were assessed as changes coding cytoplasmic phosphotyrosyl protein phosphatase in cell growth in culture, colony formation efficacy in (or acid phosphatase 1; ACP1), nuclear protein SkiP (or soft agar, and cell invasiveness through Matrigel. SKI interacting protein; SKIIP), and rap-2 (Rap Overexpression of a targeted gene was accomplished GTPase homolog; rap-2). Post hoc analyses by real-time via transient transfection of an expression vector reverse transcription–PCR (RT–PCR) confirmed dis- containing the gene into individual OVCA cell lines. cordant regulation of this set of genes by E2 between Successful induction of higher levels of expression was HOSE and OVCA cell lines, as revealed by the evident from increased gene transcripts in the trans- microarray analyses (Figure 2). fected cultures (Figure 4a). Overexpression of RNPS1 or ADD1 in OVCA cell lines significantly reduced cell growth (Figure 4b), colony formation efficacy in soft Expression levels of RNPS1, ADD1, PLXNA3, rap-2, agar (Figure 5), and cell invasion through Matrigel and SKIIP in HOSE and OVCA cell lines and their regulation by estrogen (5 days of treatment) (Figure 6). In contrast, overexpression of PLXNA3 in OVCA cells failed to alter cell growth (Figure 4b) and Lower levels of RNPS1, ADD1, and PLXNA3 tran- colony formation efficacy in soft agar (Figure 5). It scripts were found in OVCA cell lines compared to induced only a reduction in cell invasiveness through HOSE cell lines (Figure 2). E2 increased RNPS1 and Matrigel (Figure 6). No change in tumor phenotypes ADD1 mRNA levels in HOSE cell lines but reduced was observed in OVCA cells transfected with empty their levels in OVCA cell lines. In contrast, PLXNA3 vectors. expression was downreguated by E2 in HOSE cell lines and upregulated in OVCA cell lines (Figure 2). The Effects of inhibited rap-2 and SKIIP expression in OVCA effects of E2 (5-day treatment) on the expression of these cell lines genes were hormone specific, since treatment of HOSE or OVCA cells with ICI, a pure ER antagonist, Transcript levels of rap-2 and SKIIP were found to be abolished the E2-induced changes in all cases. Exposure higher in OVCA cell lines than in HOSE cell lines of cell lines to ICI alone induced no effect (Figure 2). (Figure 2a). Reduction in expression of these genes was Basal levels of rap-2, SKIIP,andACP1 expression achieved by transfection of gene-specific antisense were higher in OVCA cell lines compared to HOSE cell oligodeoxynucleotides (ODNs) at two concentrations lines (Figure 2). Significant elevations in rap-2, SKIIP, (2.5 and 5.0 mM) into individual OVCA cell lines. and ACP1 mRNA levels were observed in HOSE cell Significant loss of transcript expression was observed lines after exposure to E2, while the levels of these in transfected OVCA cell cultures (Figure 7a). Reduc- transcripts were reduced in OVCA cell lines by E2 tion in rap-2 expression was associated with inhibition

Oncogene Estrogen-regulated genes expression in ovarian cancer cells V Syed et al 8132

Figure 2 Real-time RT–PCR analysis of mRNA for human RNA-binding protein (RNPS1), erythrocyte adducin a subunit (ADD1), SEX gene (PLXNA3), rap-2, human nuclear protein SKIIP, and phosphotyrosyl protein phosphatase (ACP1) transcripts in total RNA samples from normal immortalized and malignant HOSE cell lines. Three normal immortalized (HOSE 642, HOSE 6-8, and HOSE 12- 12), and malignant OSE cell lines (OVCA 420, OVCA 429, and OVCA 432) were treated with estrogen (10À6 M) for 5 days to isolate total RNA. Real-time RT–PCRs were performed as described in Materials and methods. (a) Gray and open columns represent the relative abundance of RNPS1, ADD1, PLXNA3, rap-2, SKIIP, and ACP1 in individual, immortalized, untreated HOSE and OCa cell lines, respectively. (b and c) The relative abundance of three genes in E2-treated HOSE and OVCA cell lines, respectively. Open columns represent untreated cells, black columns represent E2-treated cells, hatched columns represent cells treated with E2 and ICI, and gray columns represent ICI-treated cells. Two independent RT–PCRs were performed with total RNA of HOSE and OVCA cell lines. Levels of expression in control were set to 1. Three independent cDNA syntheses and three PCRs were run from each RNA preparation. The means of the replicated measurements were calculated and are shown. The data are shown as the means7s.d. Statistically signiﬁcant differences in the transcript levels in estrogen-treated cell lines and those observed in untreated cell cultures at Po0.05 are shown

in cell growth (Figure 7b), colony formation in soft agar lines, elicited only a reduction in colony formation (Figure 8), and cell invasiveness through Matrigel efﬁcacy (Figure 8); it did not affect cell growth (Figure 9) in OVCA cell cultures transfected with rap- (Figure 7b) or cell invasiveness through Matrigel 2 antisense ODNs. These changes were not observed in (Figure 9) in OVCA cell lines exhibiting lowered OVCA cell cultures transfected with the mismatched expression of the SKIIP transcript. Mismatched ODN- ODN control. In contrast, inhibition of SKIIP expres- transfected controls did not alter gene expression or sion, via antisense ODN transfection to OVCA cell tumor phenotypes.

Oncogene Estrogen-regulated genes expression in ovarian cancer cells V Syed et al 8133 estrogen-regulated genes were analysed for the presence of cis-regulatory elements or transcriptional factor (TF)- binding sites that might mediate estrogen action, either directly or through tethering. Results (Table 3) showed that the promoters of all five genes contain estrogen response elements (EREs) or consensus sequence for estrogen receptor-related-a (ERR-a). In addition, TF- binding sites for activator protein-1 (AP-1), specificity protein (Sp-1), and nuclear factor-kB (NF-kB) are commonly found in these genes. In this regard, TF- binding sites for NF-kB and stimulating protein-1 (SP1) are present in the promoters of all five genes whereas AP1 sites were detected in the promoter of three genes.

Discussion

Using transcriptional profiling and stringent data mining criteria, we here identified five genes (RNPS1, ADD1, rap-2, SKIIP, and PLXNA3) whose expression was regulated by E2 in HOSE and OVCA cell lines in opposite directions. These genes have not been reported to be regulated by the hormone in previous studies. Specifically, RNPS1, ADD1, rap-2, and SKIIP are upregulated by E2 in HOSE cells but downregulated by estrogen in OVCA cells, whereas PLXNA3 shows the reverse pattern of regulation by the hormone. RNPS1, ADD1, and PLXNA3 were found to be underexpressed in OVCA cell lines compared to HOSE cell lines. Functional studies showed that RNPS1 and ADD1 exert multiple antitumor actions in OVCA cells, while PLXNA3 inhibits only cell invasiveness. In contrast, rap-2 and SKIIP were found to be overexpressed in OVCA cell lines when compared to HOSE cell lines. Experiments involving antisense-mediated downregulation of rap-2 demonstrated that this gene exerts a broad spectrum of oncogenic effects in OVCA cells, whereas SKIIP promotes only anchorage-independent growth in these cancer cells. Based on these findings, we identify a set of novel E2-regulated genes with tumor suppressive or oncogenic characteristics whose biological roles may have significant relevance to ovarian carcinogenesis and Figure 3 Time-dependent E2-induced expression of RNPS1, implications for the relationship between ERT/HRT ADD1, PLXNA3, rap-2, and SKIIP in OVCA cell lines. OVCA usage and risk of EOCa. cells were treated with E2 (10À6 M) or vehicle for 6, 18, and 36 h. The time-course studies were conducted twice on separate To our knowledge, we are the first to report occasions. The results are the means of two independent experi- expression of RNPS1 in human OSE cells and regula- ments. Total RNAs were extracted and analysed for the expression tion by E2. We observed significant tumor suppressor of RNPS1, ADD1, PLXNA3, rap-2, and SKIIP. Two independent activity of this gene when it is overexpressed in OCa RT–PCRs were performed with total RNA of two OVCA cell lines. Relative mRNA levels were expressed as percentage changes after cells. RNPS1 encodes a protein that functions as a cDNA normalized to those of GAP-DH cDNA. The data are splicing coactivator in the processing of pre-mRNA to shown as the means7s.d. Statistically significant differences in the mRNA (Badolato et al., 1995; Mayeda et al., 1999). It transcript levels in estrogen-treated cell lines and those observed in also plays a role in mRNA nuclear export and mRNA untreated cell cultures at Po0.05 are shown integrity surveillance (Kim et al., 2001; Lykke-Andersen et al., 2001). Since proper processing of pre-mRNA and Analysis of promoters of ADD1, RNPS1, SKIIP, Rap-2, maintenance of postsplicing surveillance are crucial to and PLXNA3 genes with altered expression after normal cell function, the loss of RNPS1 expression may estrogen exposure disrupt the composition and/or stability of numerous cellular mRNAs, which in turn contribute to tumor Using the Matinspector program (Quandt et al., 1995), growth and progression. Our findings are in agreement the proximal promoter regions of the five putative with the frequent association between splicing dysregu-

Oncogene Estrogen-regulated genes expression in ovarian cancer cells V Syed et al 8134

Figure 4 Effect of human RNA-binding protein (RNPS1), erythrocyte adducin a subunit (ADD1), and SEX gene (PLXNA3) overexpression on cell growth in a panel of OCa cell lines. Three OCa cell lines were transfected using Lipofectamine Plus with 1.0 and 0.5 mg of either RNPS1, ADD1,orPLXNA3 plasmids or their respective empty vectors (1.0 mg). Control cells were treated with Lipofectamine Plus alone. (a) Expression of RNPS1, ADD1,orPLXNA3 in transfected OCa cell lines. Total RNAs were extracted and subjected to RT–PCR analysis. The primers used for RNPS1 mRNA were 50-AGC TCT TCC TCA GCA TCC AG-30 (forward) and 50-CAC TTT GGT GGG CTT AGG AG-30 (reverse); for ADD1, 50-GGA ACA AGA TCC GAG AGC AG-30 (forward) 50-ACG GTC TGT GAG TGT GGT GA-30 (reverse), and plexin A3 mRNA 50-GCT GAG AGG ATC GTG TGT GA-30 (forward) and 50- CCT GAG ATG GTA AGC CGT GT-30 (reverse). The primers of GAPDH were 50-CATCACCATCTTCCAGGAGC-30 (forward) and 50-GGATGATGTTCTGGAGAGCC-30 (reverse). The number of PCR cycles used for GAPDH was 25 and for other cDNAs it was 30. All other conditions were the same for PCRs: initial denaturation for 6 min at 941C, cycle denaturation for 1 min at 941C, annealing for 1 min at 601C, and extension at 721C for 1 min. (b) Cell growth was quantiﬁed by MTS. The value shown is the percentage growth compared with that of untreated control cells (100%). The data shown represent the mean (7s.d.) of three independent experiments. Statistically signiﬁcant decreases in gene expression/cell growth compared to that seen in control cells are indicated by *Po0.05

lation and malignancy (Faustino and Cooper, 2003). maintenance and remodeling of cell membrane skeleton They are also in concordance with a recent study that (Matsuoka et al., 2000). Phosphorylation by protein demonstrates loss of expression of nmt55/p54nrb protein, kinase C or Rho-associated kinase inhibits the activity which also has a role in pre-mRNA splicing, in of adducin, which in turn induces cytoskeleton remodel- ERnegative breast cancers (Pavao et al., 2001). ing and promotes cell motility. Although adducin has Like RNPS1, the expression of ADD1 was reported been best studied in erythrocytes concerning cell shape and characterized for the ﬁrst time, in OSE cells. ADD1 and anemia (Birkenmeier and Barker, 2004), recent encodes a cytoskeleton protein, the adducin a subunit investigations have reported its expression in a variety of that serves as an obligatory partner for adducin b and g. tissues including brain, muscle, and epithelia (Bennett Adducin a/b and a/g heterodimers oligomerize to form a and Baines, 2001). A limited number of studies have adducin tetramer complex that mediates interaction shown changes in adducin levels and/or phosphoryla- between spectrin and actin, which is essential for the tion during cancer development and the effect on the

Oncogene Estrogen-regulated genes expression in ovarian cancer cells V Syed et al 8135

Figure 6 Effect of human RNA-binding protein (RNPS1), erythrocyte adducin a subunit (ADD1), and SEX gene (PLXNA3) overexpression on cell invasion in a panel of OCa cell lines. Three OCa cell lines were transfected using Lipofectamine Plus with 1.0 mg of either RNPS1, ADD1,orPLXNA3 plasmid or their respective empty vectors (1.0 mg). Control cells were treated with Lipofectamine Plus alone. Number of cells invading Matrigel were 7 Figure 5 Effect of human RNA-binding protein (RNPS1), counted. The data shown represent the mean ( s.d.) of two erythrocyte adducin a subunit (ADD1), and SEX gene (PLXNA3) independent experiments. Statistically significant changes in overexpression on soft agar in a panel of OCa cell lines. Three number of cells, as compared to those seen in control cells, are ovarian cancer cell lines were transfected using Lipofectamine Plus indicated by *Po0.05 with 1.0 mg of either RNPS1, ADD1,orPLXNA3 plasmid or their respective empty vectors (1.0 mg). Control cells were treated with Lipofectamine Plus alone. Colonies were counted after 5 weeks. The data shown represent the mean (7s.d.) of two independent experiments. Statistically significant changes in number of colonies, (Fowler et al., 1998). In the present study, lower levels of as compared to those seen in control cells, are indicated by ADD1 mRNA were observed in OVCA cells than in *Po0.05 HOSE cells. E2 was shown to upregulate ADD1 expression in HOSE cell lines but downregulate its expression in OVCA cells. Since OSE is involved in repeated follicular ruptures and repairs of the follicle motility and metastatic potentials of cancer cells. wall during reproductive age, a great demand for Specifically, adducin a and b transcripts were found to cytoskeleton remodeling and expression of various be upregulated in basal cell carcinoma and squamous adducin subunits are expected in OSE cells. It is also cell carcinoma when compared with levels in normal not surprising to find ADD1 expression under E2 keratinocytes (Bowen et al., 2003). In contrast, adducin regulation, since primary and immortalized OSE cells g was found to be downregulated in migrating glioma can convert estrone to E2 (Okamura et al., 2003) and cells (Mariani et al., 2001). In renal carcinomas, total OSE cells are often trapped in the estrogen-rich stroma adducin levels were decreased, but Ser-660-phosphory- as inclusion cysts (Katabuchi and Okamura, 2003). Our lated adducin levels were increased, possibly due to findings that overexpression of ADD1 in OVCA cells is enhanced protein kinase C d activity in these tumors associated with reduced cell proliferation, soft agar

Oncogene Estrogen-regulated genes expression in ovarian cancer cells V Syed et al 8136

Figure 7 Effect of human rap-2 and SKIIP antisense oligonucleotides and their mismatch oligonucleotides on cell growth in a panel of OCa cell lines. Three OCa cell lines were transfected using Lipofectamine Plus with 5.0 or 2.5 mM of either rap-2 and SKIIP or respective mismatch (5.0 mM). Control cells were treated with Lipofectamine Plus alone. (a) rap-2 and SKIIP mRNA expression. Total RNAs were extracted from antisense-treated cells and subjected to RT–PCR analysis. For amplification of rap-2 50-GGG CTT CAT CCT CGT CTA CA-30 (forward) and 50-ACT CTT CAG CAA GGG CTC TG-30 (reverse) primers and for SKIPP 50-TTT TGG AGA TGG AGG TGC TT-30 (forward) and 50-TCC TTT GGA ACC AGG TCA GT-30 (reverse) primers were used. The GAPDH primers were same as in Figure 5. The number of PCR cycles used for GAPDH and SKIIP was 25 and for rap-2 cDNAs it was 30. The PCR conditions were initial denaturation for 6 min at 941C, cycle denaturation for 1 min at 941C, annealing for 1 min at 601C, and extension at 721C for 1 min. (b) Cell growth was quantified by MTS assay. The value shown is the percentage growth compared with that of untreated control cells (100%). The data shown represent the mean (7s.d.) of three independent experiments. Statistically significant decreases in gene expression/cell growth compared to those seen in control cells are indicated by *Po0.05

colony formation, and invasion suggest a tumor ity, as well as tumor-promoting actions. Specifically, suppressor function of adducin in OCa cells. Although SEX, through interaction with neuropilins, serves as a still unclear, such a tumor suppressive function could be receptor for semaphorin-3F and possibly for semaphor- mediated via promotion of cell differentiation and in-3A (Cheng et al., 2001). Semaphorins 3B and 3F have suppression of mobility in the cancer cells. potent tumor suppressor activities for small cell lung The third gene that is underexpressed in OVCA cells carcinoma (Roche and Drabkin, 2001), an OCa cell line when compared with HOSE cells is PLXNA3 or SEX HEY (Tse et al., 2002), and a mouse fibrosacroma cell gene. It has been mapped to the chromosome region line A9 (Xiang et al., 2002). In the present study, Xq28 and encodes a transmembrane protein named PLXNA3 was found to be expressed at significant levels SEX (Maestrini et al., 1996). The SEX belongs to the in HOSE cell lines and downregulated by E2. These plexin family of proteins, which share a highly conserved findings suggest participation of SEX, in the environ- cytoplasmic sex-plexin (SP) domain believed to trans- ment of declining estrogens after ovulation, in the repair duce intracellular responses (Tamagnone and Comoglio, of the ruptured wall of the developing corpus luteum by 2000). It is now known that plexins, either alone or guiding OSE cell migration. Furthermore, we observed through interaction with neuropilins, serve as receptors lower levels of PLXNA3 expression in OVCA cell lines for semaphorins (Tamagnone and Comoglio, 2000), a and demonstrated suppression of cell invasion when this superfamily of over 30 signaling molecules involved in a gene is forced to express. This latter finding is consistent broad spectrum of normal and aberrant cell functions with the postulate that PLXNA3 suppresses metastases (Neufeld et al., 2005). These include axon guidance for OCa. To our knowledge, this is the first demonstra- during nervous system development, regulation of cell tion of expression of PLXNA3 in normal and malignant migration, antitumorigenic and antiangiogenesis activ- OSE cells and its regulation by E2.

Oncogene Estrogen-regulated genes expression in ovarian cancer cells V Syed et al 8137

Figure 9 Effect of human rap-2 and SKIIP antisense oligonucleo- Figure 8 Effect of human rap-2 and SKIIP antisense oligonucleotides and their mismatch oligonucleotides on cell invasion in a tides and their mismatch oligonucleotides on colony formation of panel of OCa cell lines. Three OCa cell lines were transfected using OVCA cells. Three OCa cell lines were transfected using Lipofectamine Plus with 5.0 mM of either rap-2 and SKIIP or Lipofectamine Plus with 5.0 mM of either rap-2 or SKIIP antisense respective mismatch (5.0 mM). Control cells were treated with oligonucleotides or respective mismatch (5.0 mM). Control cells Lipofectamine Plus alone. Numbers of cell migrating through were treated with Lipofectamine Plus alone. Transfected OVCA Matrigel were counted. The data shown represent the mean (7s.d.) cell lines were plated on soft agar. Every third day, rap-2 and of two independent experiments. Statistically significant changes in SKIIP antisense oligonucleotides and their mismatch oligonucleo- cell invasion, as compared to those seen in control cells, are tides were added to the plates. Colonies were counted after 5 weeks. indicated by *Po0.05 The value shown is the percentage compared with that of untreated control cells (100%). The data shown represent the mean (7s.d.) of two independent experiments. Statistically significant changes in colonies, as compared to those seen in control cells, are indicated by *Po0.05 effectors through regions that are highly discriminative in terms of effector recognition. The effector recognition region of Rap-2 differs from those of Ras and Rap-1 by a single amino acid (amino acid 39 in Rap-2 is Phe, The rap-2 belongs to the Ras family of small GTP- while in Ras and Rap-1 it is Ser) and therefore may binding proteins (Campbell et al., 1998). The protein confer to Rap-2 specific signaling roles distinct from was first purified from a cytosolic fraction of platelets as those of Rap-1 and other Ras proteins. It has been a GTP-binding protein of 25 kDa (Nagata et al., 1989) reported that Rap-2 activation is important for cell and the gene subsequently cloned with its close relative spreading, actin polymerization, enhancement of B-cell rap-1 from a cDNA library (Lerosey et al., 1991). It is adhesion, promotion of integrin activation, and phos- now known that rap-2 encodes a protein of 183 amino phorylation of Pyk2, a tyrosine kinase involved in acids with high identity with Rap-1 and K-ras p21 (60 regulating cell morphology and migration (Jimenez and 46%, respectively) (Lerosey et al., 1991; Janoueix- et al., 1991; Lerosey et al., 1991; McLeod et al., 2004). Lerosey et al., 1992). The Ras family proteins regulate Rap-1, on the other hand, has been reported to trigger many cellular processes including proliferation, differ- similar but not identical actions, including activation of entiation, and apoptosis (Campbell et al., 1998), serving integrins in T cells and macrophages (Caron et al., 2000; as molecular switches by cycling between the GTP- Katagiri et al., 2000; Reedquist et al., 2000) and bound active and the GDP-bound inactive form. The inhibition of cell adhesion to extracellular matrix GTP-bound forms interact physically with downstream proteins (Tsukamoto et al., 1999). In the present study,

Oncogene Estrogen-regulated genes expression in ovarian cancer cells V Syed et al 8138 Table 3 Promoter analysis of estrogen regulated genes by matin- Table 3 (continued ) spector Gene Concenses Position to Strand Gene Concenses Position to Strand sequences for ATG sequences for ATG transcription transcription factors factors SP1 À2157 (+) ADD1 AP1 À2857 (+) SP1 À2085 (+) Acc. no. X58141 AP1 À2857 (À) SP1 À1961 (À) ER À590 (À) SP1 À1893 (+) ER À149 (+) SP1 À1009 (À) NF-kB À866 (À) SP1 À1002 (À) NF-kB À808 (À) SP1 À1582 (+) The promoter regions of the five genes differentially regulated by SP1 À685 (À) estrogen in normal (HOSE) and malignant (OVCA) ovarian surface epithelial cells were analysed for the presence of ERE, ER, estrogen- RNPS1 AP negative related receptor (ERR), and other TFs that modulate the estrogen Acc. no. L37368 ER À3985 (À) response such as AP1, SP1, or NF-kB using the Matinspector ER À1573 (+) program. Their position to ATG and the strand on which they are c-Rel À4008 (À) present are also indicated. ER: estrogen receptor; ERR, estrogen NF-kB À3639 (À) receptor related; ERE, estrogen respose element; AP1, activator NF-kB À3627 (+) protein 1; SP1, stimulating protein 1; NF-kB, NF-kappaB NF-kB À3495 (+) NF-kB À263 (+) NF-kB À3602 (À) NF-kB À532 (+) NF-kB (p50) À3370 (À) NF-kB (p50) À3369 (+) we observed overexpression of rap-2 in OVCA cells SP1 À4802 (À) SP1 À4787 (+) when compared to HOSE cells. Antisense ODN- SP1 À3791 (+) mediated downregulation of rap-2 expression reduced SP1 À3760 (+) cell growth, colony formation in soft agar, and SP1 À3254 (+) invasiveness in OVCA cell lines, thus suggesting that SP1 À3250 (+) SP1 À3115 (À) rap-2 has potent oncogenic actions in these cell lines. These findings are in marked contrast to our previous SKIIP AP1 À1107 (À) observations for rap-1, which is expressed in HOSE and Acc. no. U 51432 AP1 À1362 (À) OVCA cells and regulated by the follicular stimulating ER À1739 (+) hormone (Ho et al., 2003). Antisense ODN-mediated c-Rel À2579 (À) c-Rel À245 (À) downregulation of rap-1 in OVCA cells induced no c-Rel À48 (À) effects on OVCA cell growth, tumorigenicity, and NF-kB À278 (À) invasiveness (Ho et al., 2003). The differential actions NF-kB À428 (+) of rap-1 and rap-2 in OVCA cells are likely cell-type NF-kB (p50) À1505 (À) SP1 À957 (À) specific, since Jimenez et al. (1991) reported that rap-1 SP1 À42 (À) exerts antagonistic effects on Ras-induced transformation, whereas rap-2 exhibited no such effects. Rap-2 AP1 negative The last gene that was upregulated in OVCA cells and Acc. no. X 12534 ERR À2303 (À) downregulated in HOSE cells by E2 is SKIIP, which ERR À2095 (À) c-Rel À1776 (À) encodes the nuclear Ski interacting protein (SkiP). SkiP c-Rel À1183 (+) is the human homolog of Drosophila melanogaster SP1 À1062 (+) nuclear protein Bx42 (Folk et al., 1996; Dahl et al., SP1 À1041 (+) 1998). Recent evidence suggests that SkiP, like its SP1 À470 (+) SP1 À443 (À) Drosophila counterpart, plays an important role in SP1 À424 (À) transcriptional regulation and is widely expressed in SP1 À344 (+) many tissues. It was first described as a nuclear protein SP1 À315 (+) that interacted with the oncoprotein v-Ski to mediate its SP1 À310 (+) transforming activities (Dahl et al., 1998). Recent SP1 À95 (+) SP1 À90 (+) studies demonstrate that SkiP interacts with many SP1 À31 (+) transcriptional regulators to modulate transcription. Some of the SkiP partners recently identified include PLXNA3 AP1 À2660 (À) NotchIC (Zhou et al., 2000), Smad proteins (Leong Acc. no. X 87852 ERE À120 (+) NF-kB À2889 (À) et al., 2001), retinoblastoma tumor suppressor (Pratha- NF-kB À2083 (À) pam et al., 2002), vitamin D receptor (VDR) (Zhang NF-kB À792 (+) et al., 2001), N-CoR/SMRT, and p300 (Leong et al., NF-kB (p50) À1977 (À) 2004). Increased SKIIP expression has been found to NF-kB (p50) À1394 (À) NF-kB (p50) À1393 (+) enhance gene expression mediated by vitamin D, retinoic acid, estrogen, and glucocorticoid (Baudino

Oncogene Estrogen-regulated genes expression in ovarian cancer cells V Syed et al 8139 et al., 1998). Furthermore, SkiP has been identified as Since the gene profiling was conducted after cell novel binding partner of E7, the major transforming cultures were treated for 5 days with E2, the likelihood protein of human papillomavirus (Prathapam et al., that some of the genes are directly regulated by 2002). This interaction leads to inhibition of the estrogens while others are indirectly modulated by the transcriptional activation activity of SkiP and may hormone exists. However, with regard to the five promote the transforming activities of E7. Findings putative E2-regulated genes selected for functional from this study demonstrate overexpression of SKIIP in studies, we strongly believed that they are regulated by OVCA cell lines and abrogation of anchorage-indepen- estrogen in a direct manner. First, the expression for all dent growth in these OCa cells when SKIIP expression five genes was found to be altered within the first 6–18 h was abrogated by an antisense ODN. These findings are of estrogen exposure. Secondly, in silicon analyses of consistent with the notion that SKIIP possesses their proximal promoters revealed presence of putative oncogenic properties in OCa cells. EREs or ERR-a sites in all five genes. The other most Finally, from a more global perspective, we recognize commonly noted TF-binding sites are NF-kB and SP1, from our data that OVCA cells differ markedly from and both have been shown to mediate estrogen action HOSE cells in their response to E2. Almost twice as via ER tethering (Chadwick et al., 2005; DeNardo et al., many genes were identified as regulated by estrogen in 2005; Ghisletti et al., 2005; Kim et al., 2005). At present, the OVCA cell lines (315 genes) than in the HOSE cell it is difficult to speculate how estrogen could upregulate lines (155 genes). Importantly, E2 primarily down- a gene in HOSE cells but downregulate its expression in regulated expression of the affected genes in OVCA cell OVCA cells. Alternative utilization of cis-regulatory lines (77%) but upregulated the affected genes in HOSE elements or TF-binding sites may be a mechanism. It is cell lines (62%). An even more striking finding is that possible that the neoplastic transformation process only 19 genes were found to be regulated by E2 in both alters levels of various TFs and hence affects regulation HOSE and OVCA cells; five of them were regulated in via an direct mechanism such as transactivation via a opposite directions. A logical explanation is that ERE versus and indirect mechanism through tethering neoplastic transformation significantly changes the to a different TF. The functional outcomes of these two phenotype of the OSE cells and affects their response processes might yield opposite direction of regulation. to this hormone. Under normal conditions, OSE cells In summary, we have demonstrated successful usage are exposed to fluctuating levels of estrogens in the of transcriptional profiling, in combination with func- follicular fluid during the reproductive years. Unless the tional studies, in the discovery of novel, putative tumor OSE is trapped as inclusion cysts, it is not chronically suppressors or oncogenes for OCa that are under E2 exposed to high levels of estrogens. After menopause, regulation. Further investigations of the roles of these the OSE is not likely to be stimulated by high levels of genes may result in a better understanding of ovarian estrogens, except in ERT/HRT users. In contrast, it is carcinogenesis and its connection to the risk presented well documented that OCas produce their own estro- by ERT/HRT usage. gens, sometimes at rather high levels (Heinonen et al., 1982; Taube et al., 2002). We (Lau et al., 1999) and others (Ho, 2003) have previously reported expression Materials and methods of ER-a and -b in OSE and OCa cells, although the expression of ER-a has been shown to undergo marked Cell lines and culture conditions reduction in OCa cells when compared to OSE cells. The HOSE cell lines (HOSE 642, HOSE 6–8, and HOSE Collectively, these differences may help explain the 12–12) were derived from scrapings of normal/benign ovaries differential responses of these two cell types to estrogen from women with noncancer gynecologic indications and stimulation at the transcriptional level. In other words, immortalized with human papillomavirus E6 and E7 (Tsao the OSE’s response to E2 is intimately linked to the et al., 1995). The OVCA cell lines (OVCA 429, OVCA 420, and normal physiology of follicular growth and ovulation, OVCA 432) were established from patients with late-stage whereas OCa cells are under persistent stimulation by serous OCa (Bast et al., 1981). In our previous publication, we estrogens formed de novo in the carcinomas. It is logical described our use of these cell lines, including the phenotypes to speculate that estrogens regulate very different and culture conditions (Lau et al., 1999; Syed and Ho, 2003; Syed et al., 2001, 2002, 2005). functions in normal cells than in malignant OSE cells. Of interest, many of the genes affected by E2 in both types of cells are involved in vital cellular functions Treatment of HOSE and OVCA cell cultures (Table 2) such as phosphoinositide 3 kinase signaling, The HOSE and OVCA cell lines were seeded at 105 cells per glycolysis/gluconeogenesis, integrin-mediated cell adhe- T-25 flask (Falcon, Becton Dickinson Labware, Bedford, MA, sion, glycerolipid metabolism, purine metabolism, ami- USA; culture area ¼ 25 cm2), allowed to attach for 48 h in no-acid metabolism, oxidative phosphorylation, cell medium with fetal bovine serum (FBS), and then switched to cycle, fatty acid metabolism, and pentose phosphate medium with charcoal-stripped (CS)-FBS. Cell cultures were subsequently treated daily for 5 days with and without 10À6 M metabolism. However, due to the limited number of of E2 (Sigma). For post hoc confirmation experiments, HOSE probes on the microarrays (2400), we were unable to and OVCA cell lines were treated with E2 in the absence and discern specific gene ontology pathways that are unique presence of 10À5 M ICI 182,780 or fulvestrant (ICI), a gift from to the nontumorigenic or the malignant cell lines after Dr B M Vose (Zeneca Pharmaceuticals, Cheshire, UK). To pathway-mapping analyses. study the time-dependent expression of candidate genes,

Oncogene Estrogen-regulated genes expression in ovarian cancer cells V Syed et al 8140 OVCA cells were treated with 10À6 M E2 for 6, 18, and 36 h verse). For amplification of cDNA of glyceraldehyde 3- before RNA extraction. phosphate dehydrogenase (GAPDH), the forward primer was 50-ATGTTCGTCATGGGTGTGAA-30 and the reverse RNA isolation, probe preparation and transcriptome comparison primer was 50-GTCTTCTGGGTGGCAGTGAT-30. For PCR reaction, 2.5 ml of cDNA derived from reverse transcription of Total RNA was extracted, subjected to quality check, and used an RNA sample was amplified in a 25 ml reaction mixture in transcriptional profiling with MICROMAXt microarrays containing 12.5 ml of SYBER Green Master Mix and 0.375 ml (Perkin Elmer, CT), which contain 2400 known human genes, of 10 mM each primer and 9.25 ml of water. as previously described (Syed et al., 2005). The Tyramide All samples were amplified in triplicate. Two-step PCR was Signal Amplification (TSA) Labeling and Detection system carried out. After initial denaturation of the samples at 951C was used to enhance sensitivity, allowing probe coverage to for 4 min, 40 cycles of 951C for 30 s, and 601C for 1 min were reach or exceed 80% (Lau et al., 2003). The transcriptome in performed for the generation of all amplimers. A melting curve each E2-treated HOSE or OVCA cell line was compared to was run after PCR by increasing temperature from 55 to 951C that of its untreated counterpart on a microarray. (0.51C increments). A single peak was obtained for targets, supporting the specificity of the reaction. PCRs for all Signal normalization, data processing, and mining of gene of templates were performed in triplicate in a 96-well plate interest format, along with positive and negative controls. Relative Signal normalization, data processing, and gene mining were expression of the real-time RT–PCR products was determined by the WWCt method. The data are represented as conducted according to our previously published reports (Lau 7 et al., 2003; Syed et al., 2005). Briefly, the fluorescent intensity means s.d. of each spot was calculated by local median background subtraction. Dye bias and experimental and systematic Construction of the clone 5.1 and adducin expression plasmids variations were removed as described before (Yang et al., 2002; Lau et al., 2003). Log ratios were centered on zero to Total RNA from a prostate cancer cell line (DU-145) isolated achieve within-slide normalization and across-slide compar- with the TRI reagent (Sigma, St Louis, MO, USA) was reverse isons. Global medium intensity was used for normalization transcribed with random hexamers using the GeneAmp RNA because it offers the most stability in terms of estimating PCR kit (Applied Biosystems, Foster City, CA, USA). cDNAs spatially-dependent and intensity-dependent trends in the log corresponding to the coding sequence of clone 5.1 RNA- ratios (Yang et al., 2002). Differentially expressed genes binding protein S1 (RNPS1, GenBank accession number associated with treatment with E2 were then identified by NM_006711.2) and erythrocyte adducin a subunit (ADD1, computing the t-values using the two-sample Welch t-statistics GenBank accession number X87852.1) were amplified with the method (Callow et al., 2000) and the P-values for each gene for Platinum Taq DNA polymerase high-fidelity kit (Invitrogen, the three HOSE cell lines and separately for the three OVCA Carlsbad, CA, USA) using specific forward and reverse cell lines. Genes with an absolute t-value greater than 2 and a primers with BamHI and XbaI recognition sites added to their 50 ends, respectively. The forward primer B1-RNF (AGG ATC P-value o0.05 were identified as differentially expressed due to E2 treatment. To conduct pathway-mapping analyses, all CGA TTT ATC AGG AGT GAA AAA GAA GA) and GeneChip entries with a minimum of two-fold absolute reverse primer RNR-Xba (TTC TAG ATT ATC GGG AGG changes were submitted to the Database for Annotation, AGT TGG AGC T) were used to amplify RNPS1 CDS, while Visualization and Integrated Discovery (DAVID) (Dennis forward primer B1-ADF (AGG ATC CAA TGG TGA TTC et al., 2003). Mapped pathways from different transcriptomes TCG TGC TGC GGT) and reverse primer ADR-Xba (TTC were compared visually. TAG ATC AGG AGT CAC TCT TCT TCT TGC T) were used to amplify erythrocyte adducing-a subunit CDS. The PCR conditions comprised an initiation denaturation at 941C Reverse transcription and quantitative real-time PCR for 2 min followed by 35 cycles of 941C for 20 s, 601C for 30 s, The estrogen-regulated gene expression pattern was confirmed and 681C for 2 min 45 s The last cycle was followed by by real-time PCR. The quantitative real-time PCR reaction additional extension incubation of 7 min at 721C. The PCR was run in iCycler from Bio-Rad (Bio-Rad Laboratories, products were gel-purified by the QIAquick gel extraction kit Hercules, CA, USA) using SybrGreen PCR master mix (Qiagen, Valencia, CA, USA) and TA cloned into pGEM-T (Applied Biosystems, Foster City, CA, USA). The primers easy vector (Promega, Madison, WI, USA) for sequencing. used for PCR were designed based on the following mRNA The inserts with the expected sequence were subcloned into sequences from the GenBank: (1) the erythrocyte adducin a pcDNA4/HisMax C at BamHI and XbaI sites and re- subunit mRNA (accession # X58141): 50-TGTCCATGATT sequenced before large-scale preparation of the plasmid with CTGCAAAGC-30 (forward) 50-GCTGCTGGGTAGACATT the EndoFree plasmid Maxi kit (Qiagen). TGG-30 (reverse); (2) specific cytoplasmic phosphotyrosyl protein phosphatase mRNA (accession # M83653): 50-GCT A Transfection of OVCA cells with expression vectors or antisense CC AAG TCC GTG CTG TT-30 (forward) and 50-GAA TGC ODNs CGT GCC TCT TCA T-30 (reverse); (3) nuclear protein SkiP mRNA (accession # U51432): 50-GACAGCATGACCGGAA The OVCA cell lines (1 Â 105 cells) were transfected with the TCTT-30 (forward) and 50-CCATACCCTTGGATTGGTTG- pSV--galactosidase vector (Promega, Madison, WI, USA) at 30 (reverse); (4) the SEX gene or plexin A3 mRNA (accession # 1 mg/ml and the expression vector containing a transgene or its X87852): 50-CAGCGAGTGCCAGTTTGTAA-30 (forward) and respective empty control vectors at 0.5 and 1 mg/ml using 50-CATTGATGATGCTCCAGGTG-30 (reverse); (5) clone 5.1 LipofectAMINE PLUS (Life Technologies Inc., Rockville, RNA-binding protein mRNA (accession # L37368): 50-GGA MD, USA) in serum-free medium for 3 h at 371C, followed by GGAGAAACGTGGAGTAGG-30 (forward) and 50-GTCCA incubation with complete medium for 24 h. Some of the ACAGCCAGAGAGAGG-30 (reverse); (6) the rap-2 mRNA transfected cells were stained for X-Gal to estimate transfec- (accession # X12534): 50-GAAGTATCGTCCAGCGAAGG tion efficiency. Transfection efficiencies for OVCA 420, OVCA -30 (forward) and 50-CATGGGTCATCTTTGTCAGG-30 (re- 429, and OVCA 432 approximated 74, 67, and 68%,

Oncogene Estrogen-regulated genes expression in ovarian cancer cells V Syed et al 8141 respectively. Antisense ODNs (18-mers) phosphorothioate membrane filter of 8 mM pore size (Costar, Cambridge, MA, against the human SKIIP and rap-2 mRNAs consisted of the USA) coated with Matrigel (Collaborative Research Inc., MA, antisense sequences of 50-CATCTTCTACCGCTTCTT-30 USA). The lower chamber was filled with 10% HI-FBS (AS-SKIIP)and50-CATCGTCCCTCCGCGGCC-30 (AS-rap-2), containing complete medium. At 24 h after seeding, cell respectively. The sequences of the mismatched controls for AS- invasiveness was determined according to the number of cells SKIIP and AS-rap-2 were 50-GATCATCAACCGCTACAA-30 that had migrated to the reverse side of the filter (Ho et al., (AS MM-SKIIP) and 50-GATCGTCGCTGCGCGGGG-30 2003; Syed et al., 2005). (AS MM-rap-2), respectively. The protocols used for transfection of antisense and mismatched ODNs into OVCA cells for cell proliferation, colony formation, and cell invasion assays Promoter analysis by matinspector were as described previously (Syed et al., 2001, 2002; Ho et al., The promoter regions of those genes having altered expression 2003). following estrogen exposure based on microarray analysis were analysed for the presence of putative ERE/ER-binding sites. MTS assay Most of the promoter regions analysed had been previously characterized experimentally; however, in some cases, experi- OVCA cells transfected with the expression vector containing a mentally determined promoters were not available, and 3 kb of transgene or with an antisense ODN were seeded in 96-well the 50 regions of the genes of interest was analysed. TF-binding plates, and numbers of cells in cultures were assessed by the site analyses for ERE/ER were performed using the Matin- MTS assay as described before (Syed et al., 2001; Ho et al., spector web-based search algorithm available from Genomatix 2003). Relative cell growth was expressed as fold increases/ Software (Quandt et al., 1995). The search algorithm is decreases over cells transfected with the empty vector or with a described in detail at http://www.Genomatix.de. mistmatched ODN control.

Soft agar colony formation assay Statistical analyses OVCA cells were transfected with an expression vector Statistical analyses were carried out using ANOVA, followed containing a transgene or an antisense ODN as previously by Tukey’s post hoc test. Values are presented as mean7s.d. described (Syed et al., 2005). After 16–18 h, 10 000 cells were and are considered signiﬁcant at Po0.05. resuspended in 0.35% soft agar and layered on top of 1 ml of 0.6% solidiﬁed agar in a 35 mm dish. Soft agar plates seeded with cells transfected with an antisense ODN were treated with Acknowledgements antisense every third day. The soft agar colonies were allowed We thank Dr Kun-Liang Guan at the Department of to grow at 371C for 21 days. Colonies were counted under an Biological Chemistry, University of Michigan for providing inverted microscope after 21 days for 2 weeks at weekly the Sex gene expression vector and Dr Samuel C Mok at the intervals. Laboratory of Gynecologic Oncology, Department of Ob- stetrics, Gynecology, and Reproductive Biology, Brigham and Women’s Hospital, Boston for providing the HOSE and Cell invasion assay OVCA cell lines. We thank the editorial staff of Brigham and OVCA cells transfected with an expression vector containing a Women’s Hospital in Boston for their excellent editorial transgene or an antisense ODN were added to the upper service. This study was supported, in part, by NIH Grants chamber (400 000 cells/well) of a prehydrated polycarbonate CA091250 (to VS) and CA94221 (to SMH).

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