Expression of nuclear receptor coregulators in ovarian stromal and epithelial tumours

Molecular and Cellular Endocrinology 229 (2005) 149–160

Expression of nuclear receptor coregulators in ovarian stromal and epithelial tumours S. Hussein-Fikreta,b , P.J. Fullera,b,∗ b

a Prince Henry’s Institute of Medical Research, PO Box 5152, Clayton, Vic. 3168, Australia Monash University Department of Medicine, Monash Medical Centre, Clayton, Vic. 3168, Australia

Received 27 April 2004; accepted 11 August 2004

Abstract Granulosa cell tumours of the ovary (GCT) exhibit high expression of estrogen receptor ␤ (ER␤). A role for estrogen receptors in these tumours may depend on altered co-activator expression. This study examines the expression of the co-activators SRC-1a/e, SRC-2, SRC-3, SRA, and the corepressors NCoR and SMRT in GCT, epithelial ovarian tumours and normal ovary. No significant difference in the expression of SRC-1, SRC-2, SRC-3 or NCoR and SMRT was found. In particular, there was no correlation of co-activator expression with ER␤ expression. There was a significant upregulation in the expression of the novel RNA co-activator SRA in the serous tumours compared with the other tumour types and normal ovary. The findings suggest that ER␤ may require co-activators, other than members of the SRC family for the modulation of transcription in GCT. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Granulosa cell tumours (GCT); Amplified in breast cancer 1 (AIB1); Nuclear corepressor (NCoR); Silencing mediator of the retinoid and thyroid hormone receptor (SMRT); Estrogen receptor (ER)

1. Introduction Ovarian cancer is the fourth leading cause of cancer deaths in women and has the highest mortality rate of all gynaecological malignancies (Riman et al., 1998). The ovarian surface epithelium is the site of origin of 80–90% of ovarian malignancies (Amsterdam and Selvaraj, 1997; Riman et al., 1998). The majority of epithelial ovarian tumours are serous and mucinous cystadenocarcinomas (Russell and Bannantyne, 1989). Sex cord stromal tumours of the ovary represent approximately 10% of all ovarian malignancies; granulosa cell tumours (GCT) account for the majority of these tumours (Amsterdam and Selvaraj, 1997). The steroid hormone estrogen stimulates the proliferation of the granulosa cells of the ovary, and recent studies indicate a role for estrogen in the pathogenesis of ovarian cancer (Richards, 1994; Amsterdam and Selvaraj, 1997; Clinton ∗

Corresponding author. Tel.: +61 3 9594 4379; fax: +61 3 9594 6125. E-mail address: [email protected] (P.J. Fuller).

0303-7207/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2004.08.005

and Hua, 1997; Riman et al., 1998). Furthermore, ovarian surface epithelial cells show a marked proliferative response to estrogen and develop a morphology similar to human epithelial ovarian tumours (Bai et al., 2000). The mitogenic effects of estrogens are mediated through two estrogen receptors, ER␣ and ER␤, which belong to the superfamily of nuclear receptors that function as liganddependent transcription factors. ER␣ is expressed in normal epithelial cells of the ovary and in as many as 60–70% of primary epithelial ovarian tumours (Lau et al., 1999; Havrilesky et al., 2001; Brandenberger et al., 1998; Lindgren et al., 2001; Pujol et al., 1998). Whilst Chu et al. (2000) found low ER␣ expression across the epithelial serous and mucinous tumours and also GCT, ER␤ expression is predominantly and abundantly expressed in GCT, with moderate levels in mucinous tumours and very low levels in serous tumours. The high levels of ER␤ in GCT are not only consistent with the normal phenotype of preovulatory granulosa cells but may be of pathogenetic significance, since the majority of these tumours synthesise estrogens (Amsterdam and

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Selvaraj, 1997; Fuller et al., 2002). Although the presence of ER in ovarian tumours may confer estrogen-responsiveness, endocrine therapy such as antiestrogen therapy has limited success in the treatment of ovarian tumours (Rao and Slotman, 1991). Whilst the levels of ER␣ and ER␤ are important in defining the nature of response to endocrine manipulation in steroid-dependent tissues, the other important factors are the expression patterns of co-activator and corepressor molecules (Glass and Rosenfeld, 2000). Nuclear receptors regulate gene transcription through interactions with co-activator and corepressor proteins. The well-characterised steroid receptor co-activator (SRC) family, also known as the p160 family of co-activators, is potentially an important determinant of the ovarian response to estrogens. Currently, three homologous co-activators, SRC-1, SRC-2/TIF-2/GRIP-1 and SRC-3/AIB1/ACTR/ RAC3/TRAM-1/pCIP, have been identified, which are encoded by separate genes (Glass and Rosenfeld, 2000). All three members of the SRC family interact with nuclear receptors in a ligand-dependent manner and enhance nuclear receptor transcriptional activity. Unlike co-activators, the corepressor molecules, NCoR (nuclear receptor corepressor) and SMRT (silencing mediator of the retinoid and thyroid receptor), function to stabilize the repressed state of unliganded nuclear receptors (although not the steroid receptors) and antagonist-bound nuclear receptors (Glass and Rosenfeld, 2000). Co-activators have been implicated in the neoplastic process on the basis of the observation that the co-activator gene amplified in breast cancer 1 (AIB1) or SRC-3 was amplified and overexpressed in 9.5% of primary breast tumours, as well as being overexpressed in 58% of breast tumours lacking SRC-3 gene amplification (Anzick et al., 1997). A subsequent study found SRC-3 amplification in 7.4% of ovarian tumours. Altered expression levels of other coregulator genes have been reported in breast cancer tissues, including increased levels of the co-activator SRC-2 and the novel steroid receptor RNA activator, SRA (Lanz et al., 1999), as well as decreased levels of the corepressor NCoR (Kurebayashi et al., 2000; Murphy et al., 2000; Lanz et al., 2003). These observations further support the hypothesis that altered coregulator expression may provide a selective growth advantage for hormone-dependent tumours. On the basis of these observations we hypothesised that increased co-activator and/or decreased corepressor expression may occur during ovarian tumourigenesis. The aim of the present study is to examine the gene expression profiles of the SRC family members (human SRC-1 isoforms, SRC-1a and SRC-1e, SRC-2 and SRC-3), the wildtype and deleted isoforms of SRA, and the corepressors NCoR and SMRT, in a panel of ovarian epithelial (serous and mucinous) and stromal (GCT) tumours compared to normal ovary. In addition, we sought to contrast the coregulator gene expression profiles to the expression profiles of ER␣ and ER␤ in the ovarian tumours (Chu et al., 2000). Co-activator gene expression has not previously been examined in GCT, the predominant site

of ER␤ expression. Despite the high ER␤ expression in the GCT relative to normal ovary, in this study we did not find increased levels of the co-activator genes examined. However, we found high SRA expression in the serous tumours, which have previously been shown to express low levels of both ER␣ and ER␤.

2. Materials and methods 2.1. Isolation of RNA from tissue specimens Ovarian granulosa cell tumours (n = 5), mucinous cystadenocarcinomas (n = 6) and serous cystadenocarcinomas (n = 6) were obtained in a study of serum inhibin levels in ovarian tumours (Healy et al., 1993). The tumours were consecutive tumours from which adequate tissue was available for RNA extraction. Some of these tissues have been examined in previous studies for the expression of various genes (Fuller et al., 1999; Chu et al., 2000, 2002). Normal ovarian tissue was obtained from six premenopausal women who had undergone elective hysterectomy with oophorectomy for a range of conditions not associated with ovarian malignancy (Table 1). Normal human uterine endometrium (day 14) and human placenta (first trimester) tissues were obtained as positive control samples. Clinical details for the ovarian tumour are presented in Table 1. RNA was isolated using the guanidine thiocynate/cesium chloride method as described previously (Fuller et al., 1999). 2.2. RT-PCR amplification One microgram of total RNA was reverse transcribed for 90 min at 42 ◦ C in a total volume of 20 ␮l using AMV reverse transcriptase (Roche Molecular Biochemicals, Mannheim, Germany). First strand synthesis for SRC-1, SRC-1a/e, SRC2, SRC-3, SRA, the deleted isoform of SRA (SRA-del), NCoR, SMRT, ER␣, ER␤ and ␤2 -microglobulin (␤2 m) was performed using 30 pmol of oligo dT. The oligonucleotide primers and conditions for ER␣ and ER␤ have previously been described (Chu et al., 2000). The gene-specific oligonucleotide primers for the coregulator genes and ␤2 m (Table 2) were designed to the coding regions from published sequences (Voegel et al., 1996; Anzick et al., 1997; Kalkhoven et al., 1998; Wang et al., 1998; Lanz et al., 1999; Park et al., 1999; Gussow et al., 1987) with OLIGO Primer Analysis software version 5.0 (Natural Biosciences, North Plymouth, Minnesota). SRC-1 primers spanning the exon–intron junction at which alternate splicing occurs, were used to detect the two isoforms of human SRC-1, SRC-1a and SRC-1e. The three SRA isoforms identified to date, differ in their 5 and 3 terminal regions but share an identical core sequence from nucleotides 28 to 720 (Lanz et al., 1999). In addition to these wildtype isoforms of SRA, Leygue et al. (1999) identified a deleted isoform of SRA, referred to as SRA-del that has a 203 bp deletion between nucleotide positions 155 and 357

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Table 1 Clinical information of the patients studied Case no.

Age (years)

Menopausal status

Type

Stage

CA125 (U/l)

Serum inhibin (U/l)

Serum FSH (U/l)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

40 43 49 33 50 48 76 86 20 65 78 64 31 48 43 30 85 56 71 71 75 75 68

Pre Pre Pre Pre Pre Pre Post Post Pre Post Post Post Pre Pre Pre Pre Post Post Post Post Post Post Post

Ov Ov Ov Ov Ov Ov MC MC MC MC MC MC GCT GCT GCT GCT GCT SC SC SC SC SC SC

– – – – – – 1 Benign 1 1 1 2 1 Recurrent 1A 1 Recurrent Unstaged 3C 3C 3C 3C 3C

ND ND ND ND ND ND 53 NA 15 104 NA 638 NA NA 13 NA 24 158 412 NA 862 1075 4600

579 <100 202 <100 396 NA 1940 152 580 641 539 1090 2410 <100 230 1438 953 1405 75 75 75 75 207

ND ND ND ND ND ND 26 33.2 0 70.9 8.6 15.4 0 NA 8.1 4.9 NA 31.1 0 27.4 111.4 57.8 56.3

The inhibin normal range in postmenopausal women is <130 U/l. In cycling women, follicular phase levels are usually <800 U/l. The CA125 clinical range is <35 U/l in postmenopausal women. The FSH normal range for premenopausal women in the follicular phase is 2.5–10.2 IU/l. The tumour types are mucinous cystadenocarcinoma (MC), granulosa cell tumour (GCT) or serous cystadenocarcinoma (SC). Normal ovaries (Ov) are from patients who have undergone total abdominal hysterectomy/bilateral salpingo-oophorectomy for the following conditions: endometriosis/adenomyosis (nos. 1, 6), endometrial carcinoma (no. 5), fibroid uterus (no. 3), cervical adenoma (no. 2) and intractable premenstrual tension (no. 4). Note that the mucinous tumours are classified as borderline. In some cases specific biochemical values were not available (NA) or not determined (ND).

of the SRA core region. A set of primer sequences for SRA were designed to the core region between nucleotides 397 and 700 and will amplify a 304 bp product that corresponds to all SRA isoforms (wildtype and deleted) containing the core region. SRA primers for a separate PCR reaction were designed to the core region between nucleotides 35 and 700 and will coamplify a 666 bp product corresponding to the wildtype SRA isoforms and a 463 bp product corresponding to SRA-del isoform. The primer sequences for SMRT were designed to the coding region at the C-terminus, and will amplify three different splice variants of SMRT (unpublished primer sequences for NCoR and SMRT were kindly provided by Dr. Dinny Graham, Denver, Colorado). One microlitre of each RT reaction was amplified in a single stage PCR with 10 pmol of gene-specific primers and 2.5 U Taq DNA ploymerase (Roche Molecular Biochemicals) in a total volume of 50 ␮l. For coamplification of the full length and deleted form of SRA, a final concentration of 1× PCR buffer, 1× Q-solution and 2.5 U Taq DNA polymerase (Qiagen) was added to the PCR reaction mixture. The thermal cycling profile for the co-activator, corepressor and ␤2 m genes consisted of an initial denaturing step at 95 ◦ C for 5 min and subsequent cycles of denaturation, annealing and extension (as shown in Table 2), with a final 72 ◦ C incubation for 5 min. The thermal cycling profile of ER␣ and ER␤ has previously been reported (Chu et al., 2000). Prior to Southern blot analysis, the appropriate number of cycles

was established for each reaction to ensure that the amplification was in the linear phase. The number of cycles required, which ranged between 22 and 26 cycles, is shown for each reaction in Table 2. Each of the samples was analysed on at least three separate occasions for each gene; in all cases the relative levels of expression were reproduced across experiments. PCR amplification of the SRC-2, SRA, SMRT and ␤2 m genes was performed on a OmniGene thermocycler (Thermo Hybaid, UK), SRA-del was amplified on a PCR EXPRESS thermocycler (Thermo Hybaid, UK), while the other coregulator genes in Table 2 were amplified using a Robocycler Gradient 40 thermocycler (Stratagene, San Diego). The products were visualized on a 1.2–2.0% agarose gel, stained with ethidium bromide and photographed under UV transillumination (results not shown). Controls for the RT-PCR were the reaction mixtures described above, but with reverse transcriptase omitted. The identity of the amplicons was confirmed by automated sequencing in the Wellcome Trust Joint Sequencing Facility at Monash Medical Centre. 2.3. Southern blot analysis Southern blot analyses were used for a semiquantitative estimate of the relative levels of gene expression across the various tissue samples (Figs. 1, 2A, 3A, 3C and 4A). For Southern blot analysis using gene-specific 32 P-labeled internal oligonucleotide probes (Table 2), the PCR products

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Table 2 Oligonucleotide primers/probes for RT-PCR and Southern blot analysis SRC-1 Sense Antisense Probe

5 -CCA GAA TAA ACC GCC AGC AGA GC-3 (1289) 5 -ACT TGT CAT GCC AAC GGG AGA GC-3 (1611) (95 ◦ C 1 min, 60 ◦ C 1 min, 72 ◦ C 1 min; 25 cycles) 5 -GGC AAC ATT GGC TAC AAT CTG ACT TCC GGG TGA GC-3 (1404)

SRC-1a/e Sense Antisense Probe

5 -CAG ATG CCA GGA ATG AAC ACT GT-3 (4231) 5 -GGT CCT GAC GTG GGC TTT TGA GT-3 (4529) (95 ◦ C 1 min, 60 ◦ C 1 min, 72 ◦ C 1 min; 22 cycles) 5 -CAG TGG ATG AGA GCT GGT TGC AGT AGA GGC-3 (4324)

SRC-2 Sense Antisense Probe

5 -GCT TCG CTG TCT CTC AAC CAA AG-3 (848) 5 -ACT GAA TGC CAA TCC TTG TCT CA-3 (1164) (95 ◦ C 40 s, 49 ◦ C 40 s, 72 ◦ C 40 s; 25 cycles) 5 -GGT GCT GGT ATC CAG AGA CGT GAT CTT GCC TTG G-3 (1023)

SRC-3 Sense Antisense Probe

5 -ACG CCG CAT TAC TAC AGG AGA AAG A-3 (950) 5 -TTT GTT GCA GGA TTT CGG AAG AGT T-3 (1273) (95 ◦ C 1 min, 58 ◦ C 1 min 15 s, 72 ◦ C 1 min 15 s; 25 cycles) 5 -AAC GAG AAT CGA TAT ACT GGG GTT TCT GCA TGG CC3 (1210)

SRA Sense (full length) Sense (full length + del) Antisense Probe

5 -CTG CAG GAA CAG TGG GCT GGA GG-3 (397) 5 -AGG AAC GCG GCT GGA ACG A-3 (35) 5 -GGT GAG TCT GGG GAA CCG AGG AT-3 (700) core (95 ◦ C 40 s, 62 ◦ C 40 s, 72 ◦ C 40 s; 25 cycles) core + del (95 ◦ C 40 s, 62 ◦ C 40 s, 72 ◦ C 50 s; 27 cycles) 5 -TCT GCT GCG TCC CAC CGG TGG CTT GAA AGC TCT TG-3 (497)

NcoR Sense Antisense Probe

5 -GAG ATC TTT AAT CTG CCA GCA GTT A-3 (6700) 5 -TCA GTC ATC ACT ATC CGA CAG GGT CTC GTA C-3 (7323) (95 ◦ C 1 min, 52 ◦ C 1 min, 72 ◦ C 1 min; 26 cycles) 5 -GTA CAG AGG AGA CTG AAG AG-3 (7069)

SMRT Sense Antisense Probe

5 -TCT TGG GTG GTG GTG AGG ACG GT-3 (6611) 5 -TGC TGG GTC TGC CAG AGA CCT T-3 (7201) (95 ◦ C 40 s, 64 ◦ C 40 s, 72 ◦ C 40 s; 26 cycles) 5 -GGT GTT CAG CTT CTT GTT G-3 (6858)

␤2 m Sense Antisense Probe

5 -TGA ATT GCT ATG TGT CTG GGT-3 (183) 5 -CCT CCA TGA TGC TGC TTA CAT-3 (1046) (95o C 30 s, 55o C 30 s, 72o C, 1 min; 22 cycles) 5 -TGC CTG CCG TGT GAA CCA TGT GAC TTT GTC-3 (349)

The primer/probe sequences were designed from the following GenBank database sequences: HSU90661 (SRC-1), AJ000881 (SRC-1a), AJ000882 (SRC-1e), X97674 (SRC-2), AF012108 (SRC-3) AF092038 (SRA) and M17987 (␤2 m). The primer/probe sequences for NCoR and SMRT were provided by Dr. Dinny Graham. The numbers in parentheses refer to the location of the primer/probe in the nucleotide sequence. The primer/probe nucleotide positions for NCoR and SMRT are from GenBank database sequences AF044209 and AF125672, respectively. Shown beneath the antisense oligonucleotide primer sequences in parentheses are the thermal cycling profiles for each primer pair.

Fig. 1. Southern blot analysis of the RT-PCR products amplified from normal ovary (Ov), mucinous cystadenocarcinomas (MC), granulosa cell tumours (GCT) and serous cystadenocarcinomas (SC) with gene-specific primers for ER␣ and ER␤. The numbered lanes correspond to the case number designated to the normal ovary and ovarian tumour patient samples shown in Table 1 (patient samples #7 and #14 are omitted in this figure). The first lane (M) contains the molecular weight markers. The negative controls (NoRT), in which the reverse transcriptase was omitted from the reaction, are shown in lanes C1–C4.

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Fig. 2. The gene expression profile of the SRC family members in a panel of normal ovary and ovarian tumour samples. (A) Southern blot analysis of the RT-PCR products amplified from normal ovary (Ov), mucinous cystadenocarcinomas (MC), granulosa cell tumours (GCT) and serous cystadenocarcinomas (SC) with gene-specific primers for SRC-1, SRC-1a/e, SRC-2 and SRC-3. The normal ovaries and ovarian tumours numbered 1–23 are in the order shown in Table 1. The first lane (M) contains the molecular weight markers. The positive controls are endometrium (E) and placenta (P). The negative controls (NoRT), as described in Fig. 1, are shown in lanes C1–C4. Expression of the ␤2 -microglobulin (␤2 m) gene, a control for semiquantitative analysis, is shown in the lower panel. (B) The relative mRNA expression levels of SRC-1, SRC-2 and SRC-3 for each ovarian sample are plotted as a scattergram after semiquantitatively measuring the densitometry value of each amplicon and correcting for ␤2 m. Horizontal lines indicate the mean value. Columns represent normal ovaries (Ov), mucinous cystadenocarcinomas (MC), granulosa cell tumours (GCT) and serous cystadenocarcinomas (SC).

described above were transferred to Hybond N+ membranes (Amersham Pharmacia Biotech, Aylesbury, UK) as described previously (Fuller et al., 1999; Chu et al., 2000). Other amplicons were included in each transfer as hybridization controls (results not shown). Radiolabeled membranes were then exposed to X-ray film, which was scanned using a GS-710 Calibrated Imaging Densitometer

(Biorad, California). Semiquantitative analysis was performed by measuring densitometry values for each band and correcting for ␤2 m, using the Quantity One software version 4.0 (Biorad, California). For each sample the relative coregulator mRNA levels, expressed as arbitrary units, were plotted as a scattergram and the mean for each ovarian tissue type was determined (Figs. 2B, 3B and 4B).

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Fig. 3. Gene expression of wildtype SRA and SRA-del in a panel of normal ovaries and ovarian tumours. (A) A representative autoradiograph of a Southern blot of RT-PCR products amplified from normal ovary (Ov), mucinous cystadenocarcinomas (MC), granulosa cell tumours (GCT) and serous cystadenocarcinomas (SC) with primers that detect all SRA isoforms containing the core sequence (three wildtype SRA isoforms and SRA-del isoform) (304 bp amplicon). The samples in each lane follow the same format as described in Fig. 2A. The lower panel is the same representative autoradiograph shown in Fig. 2A for the ␤2 -microglobulin (␤2 m) housekeeping gene, which is used as a control for RNA quality/quantity/loading. (B) Densitometric analysis of each amplicon for SRA has been corrected for ␤2 m and plotted as a scattergram. The horizontal lines indicate the mean value. The symbol ‘*’ indicates that mean SRA expression levels in the serous cystadenocarcinomas (SC) are significantly (P < 0.01) different when compared with mean levels in the normal ovaries (Ov) and the other tumour groups, the mucinous cystadenocarcinomas (MC) and granulosa cell tumours (GCT). (C) Southern blot analysis of RT-PCR products amplified from representative samples of normal ovary (Ov), mucinous cystadenocarcinomas (MC), granulosa cell tumours (GCT) and serous cystadenocarcinomas (SC) with primers that detect the core sequence of the wildtype SRA isoforms (666 bp amplicon) and the deleted isoform of SRA that contains a 203 bp deletion within the SRA core sequence (463 bp amplicon), referred to as SRA-del. The numbered lanes correspond to the case number designated to the patient samples shown in Table 1. The first lane (M) contains the molecular weight markers. The negative controls (NoRT), as described in Fig. 1, are shown in lanes C1–C4.

2.4. Statistical analysis Statistical analysis for each data set was performed using GB-STAT software V6.5 (Dynamic Systems Inc, Silver Spring, Maryland, USA). They were analysed by one-way ANOVA followed by the Dunnetts test comparing each data set with the normal ovary. The correlation between co-activator and corepressor expression levels in the ovarian tumours was analysed by the calculation of the Pearsons correlation coefficient r.

3. Results 3.1. SRC-1 expression in ovarian tumours To establish whether SRC-1 is expressed in a tumourspecific fashion the levels of SRC-1 expression was assessed

by Southern blot analysis of the PCR products using an SRC1 gene-specific hybridisation probe. Consistent with previous studies (Takeshita et al., 1997; Li and Chen, 1998), the results confirm SRC-1 gene expression in the human placenta positive control. An amplicon of the appropriate size and sequence for SRC-1 was detected in the ovarian tumours. SRC1 expression was widespread across all the ovarian samples examined (Fig. 2A). The relative levels of co-activator expression for each tumour are presented in Fig. 2B. The mean SRC-1 levels were similar in both the normal ovary and all three tumour groups examined. The pattern of expression for SRC-1 was serous ≥ normal ovary ≥ GCT ≥ mucinous. SRC-1 expression is homogeneous within each tumour group examined, except for the low levels of SRC-1 expression observed in one GCT (Fig. 2A: patient case number 15), which is also seen for this tumour with SRC-2 and SRC-3 (see below). This variation in SRC-1 expression does not correlate with the stage or age of the patient (Table 1).

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Fig. 4. The gene expression profile of the corepressors NCoR and SMRT in a panel of normal ovaries and ovarian tumours. (A) Southern blot analysis of the RT-PCR products amplified from normal ovary (Ov), mucinous cystadenocarcinomas (MC), granulosa cell tumours (GCT) and serous cystadenocarcinomas (SC) with gene-specific primers for NCoR and SMRT. The samples in each lane follow the same format as described in Fig. 2A. The ␤2 -microglobulin (␤2 m) gene is included as a control for semiquantitative analysis (lower panel). (B) The mRNA expression levels of NCoR and SMRT for all samples in the normal ovary (Ov), mucinous cystadenocarcinoma (MC), granulosa cell tumour (GCT) and serous cystadenocarcinoma (SC) groups were semiquantitated, as described in Fig. 2B, and expressed as relative densitometry units/␤2 m. The horizontal lines represent the mean value in each group.

3.2. SRC-1 isoform expression in ovarian tumours To explore the relative patterns of expression of the two known isoforms of human SRC-1 (Kalkhoven et al., 1998), primers were designed to amplify the region spanning the exon–intron junction at which alternate splicing occurs; the amplicons thus span a region where a 57 bp deletion results in SRC-1a. Southern blot analysis of the PCR products revealed that both of the SRC-1 isoforms were at a similar abundance across all ovarian tissues examined (Fig. 2A). The SRC-1 isoforms were not expressed in a tumour-specific pattern.

size and sequence for SRC-2. The identity of SRC-2 was further confirmed by Southern blot analysis of amplicons amplified for 25 cycles across the panel of tumours, using an SRC-2 gene-specific oligonucleotide probe (Fig. 2A). The pattern of expression for SRC-2 was serous ≥ mucinous ≥ GCT > normal ovary. The expression levels of SRC-2 in the ovary and tumours are lower than the placenta and endometrium positive controls. In contrast to SRC-1, there is considerable variability in SRC-2 expression within each group, and there is a trend towards higher levels in the tumours compared to the normal controls, however this difference is not statistically significant (Fig. 2B).

3.3. SRC-2 expression in ovarian tumours 3.4. SRC-3 expression in ovarian tumours Expression of the second member of the SRC family, SRC-2, was sought in the panel of tumours using SRC-2 gene-specific primers. Expression of SRC-2, like SRC-1, has previously been shown in human placenta (Voegel et al., 1996; Li and Chen, 1998). PCR amplification of total RNA from the placenta yielded an amplicon of the appropriate

To establish whether SRC-3 is expressed in a tumourspecific fashion, the panel of tumours were subjected to PCR for 25 cycles before Southern blotting with an SRC-3 internal oligonucleotide probe (Fig. 2A). SRC-3 expression in the endometrium and placenta positive controls is consistent

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with other studies (Takeshita et al., 1997; Li and Chen, 1998). Expression of SRC-3 was low across all of the ovarian samples examined. The relative pattern of SRC-3 expression was serous > GCT ≥ mucinous > normal ovary (Fig. 2B). A very faint band was again observed in the GCT from patient 13 (Fig. 2A: patient case number 15). The expression profile of SRC-3 is not as widespread as SRC-1 and SRA expression (see below) across the panel of tumours. However, SRC-3 expression is similar to SRC-2 expression, where levels of both co-activator genes show variability in expression across all groups examined and are expressed at low levels when compared to the positive controls (Fig. 2A and B). 3.5. SRA expression in ovarian tumours Amplification by PCR of total RNA from the panel of tumours yielded an amplicon of the appropriate size and sequence for SRA. This amplicon corresponds to all SRA isoforms containing the core region, including the deleted isoform of SRA which was identified by Leygue et al. (1999). Consistent with another study (Lanz et al., 1999), the results confirm SRA expression in the placenta positive control. Southern blot analysis of the PCR products revealed high and widespread expression of SRA across both the tumours and normal ovary controls, with relatively less variation than that observed for the co-activators, SRC-2 and SRC-3 (Fig. 3A). The pattern of relative expression for SRA was serous  GCT ≥ mucinous > normal ovary. In contrast to the other co-activators, the levels of SRA are significantly higher (P < 0.01) in the serous tumours compared to the normal ovary controls and also the other tumour types (P < 0.01), as shown in the scattergrams in Fig. 3B.

from a cohort of breast tumour samples, and the signal corresponding to SRA-del for each sample was measured and expressed as a percentage of the signal corresponding to wildtype SRA. This approach was applied to the current analysis of the ovarian tumours and normal ovary. Although there appears to be a trend for higher expression of SRA-del relative to wildtype SRA in the serous tumours compared to normal ovary and also the mucinous tumours, SRA-del is expressed at very low levels to almost undetectable levels compared to wildtype SRA. 3.7. NCoR expression in ovarian tumours Expression of the corepressor, NCoR, was sought in the panel of ovarian tumours by Southern blot analysis of PCR products amplified with NCoR gene-specific primers. NCoR expression has previously been reported in the placenta (Wang et al., 1998). An amplicon of the appropriate size and sequence for NCoR was detected at low levels in the placenta positive control when compared to the endometrium (Fig. 4A). NCoR levels in the normal ovary controls and tumours are also lower than the levels observed in the endometrium. The relative pattern of expression for NCoR was GCT > serous ≥ normal ovary > mucinous. The scattergram of NCoR shows that there is considerable variability in NCoR expression within each group, and the differences in the levels of NCoR detected in the normal ovary controls and the different tumour groups are not statistically significant (Fig. 4B). An inverse relationship was not observed between NCoR and SRC-1, SRC-2, SRC-3 or SRA expression levels in the ovarian tumours (results not shown).

3.6. SRA-del expression in ovarian tumours

3.8. SMRT expression in ovarian tumours

A second set of gene-specific primers for SRA was designed to encompass most of the core region, to determine the relative expression levels of the SRA-del isoform, with 203 bp deleted from the core sequence, to the levels of the wildtype SRA isoforms. Sequencing confirmed the identity of the 666 bp amplicon as the SRA core region of the wildtype isoforms and the 463 bp amplicon as the deleted form of the SRA core region between nucleotide positions 155 and 357. Southern blot analysis of RT-PCR products identified the 666 bp amplicon in all of the normal ovary and ovarian tumour samples examined in this study (Fig. 3C). Furthermore, the expression pattern of the 666 bp amplicon in the ovarian samples in this PCR (representative samples shown in Fig. 3C) is consistent with the results obtained for the 304 bp amplicon corresponding to all SRA isoforms containing the core region (wildtype and deleted isoforms) (Fig. 3A). The 463 bp fragment corresponding to SRA-del was detected in most of the ovarian samples examined, but at very low abundance relative to full length SRA (Fig. 3C). In the studies by Leygue et al. (1999) and Murphy et al. (2000), the wildtype and deleted isoforms of SRA were coamplified by PCR

To examine the pattern of expression of the three isoforms of SMRT, total RNA from the panel of tumours was subjected to PCR with primers previously designed to amplify the different splice variants identified in the C-terminal region of SMRT. The amplicons span a region where alternative splicing results in a 90 and 135 bp deletion (unpublished primer sequence information provided by Dr. Dinny Graham). Southern blot analysis of the PCR products detected all isoforms of SMRT in the panel of ovarian samples (Fig. 4A). To examine the relative pattern of SMRT expression the densitometric measurements included all isoforms of SMRT; the pattern was serous ≥ GCT > mucinous > normal ovary. There is a trend for SMRT levels to be higher in the tumours, however the differences between the tumour groups and the normal ovary did not reach significance (Fig. 4B). There was variable expression of the SMRT isoforms between tumours in the serous group (Fig. 4A). However, we detected no tumour-specific expression of the SMRT isoforms tested. As with NCoR, an inverse relationship was not observed between SMRT expression and co-activator expression in the ovarian tumours (results not shown).

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4. Discussion Both ER␣- and ER␤-mediated transcriptional activity of estrogen is influenced by coregulatory molecules known as co-activators and corepressors, which enhance or repress transcriptional activity at ER-responsive gene promoters. In the unliganded state, nuclear receptors remain inactive by recruiting corepressor proteins, whereas ligand activation of a nuclear receptor results in corepressor dissociation and the simultaneous recruitment of co-activator proteins. The SRC family of co-activators are principally ligand-dependent activation function-2 (AF-2) interacting co-activators. The AF-2 receptor surface and the co-activator nuclear receptor interaction domains (LXXLL motifs) are important determinants of the specificity of steroid hormone receptor responses, and hence the enhancer activity of these co-activators may differ between ER␣ and ER␤, providing a further level of regulation in estrogen signalling. Co-activator and corepressor proteins recruited to the receptor surface further regulate the activation state of the nuclear receptor through their ability to remodel chromatin structure (Glass and Rosenfeld, 2000). Altered coregulator expression levels, such as overexpression of the co-activators, may lead to increased transcriptional activation of ER with enhanced ERdependent tumour growth. In breast cancer, amplification of the SRC-3 co-activator gene correlated with high expression levels, increased tumour size and ER- and PR-positivity, supporting the role of this co-activator in ER-dependent tumour development and progression (Anzick et al., 1997; Bautista et al., 1998). Furthermore, SRC-3 amplification was observed in ovarian tumours (Bautista et al., 1998; Tanner et al., 2000). Co-activator overexpression appears to be a common theme in breast tumourigenesis, as amplification and overexpression of the PBP/TRAP220 co-activator gene has also been reported in breast tumours (Zhu et al., 1999), while SRC-2 and CREB binding protein (CBP) levels were upregulated in intraductal carcinomas of the breast when compared with normal breast epithelium (Kurebayashi et al., 2000). In contrast, the same study found a decrease in NCoR expression levels during breast tumour progression (Kurebayashi et al., 2000), suggesting that an increase in co-activator expression and/or a decrease in corepressor expression may occur during tumourigenesis. The levels of coregulator gene expression in ovarian cancer have not been fully characterised. SRC-3 gene amplification studies in ovarian cancer have mainly focussed on epithelial tumours primarily of the serous type. Our study is the first to examine the co-activator and corepressor expression profiles in both ovarian serous and mucinous epithelial tumours (n = 12) and the less common GCT (n = 5) and in normal ovary (n = 6). Many studies have shown that the expression of the SRC-1 gene is widespread across tissues and cell lines (Takeshita et al., 1997; Kalkhoven et al., 1998; Li and Chen, 1998). Consistent with these previous observations is the widespread expression of SRC-1 in both the normal ovaries and ovarian tu-

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mours examined. Two isoforms of SRC-1 have been reported in the human, SRC-1a and SRC-1e, where the latter is the more transcriptionally active form (Kalkhoven et al., 1998). Although higher ratios of SRC-1e to SRC-1a have previously been reported in a range of tumour cell lines (Kalkhoven et al., 1998), both isoforms of SRC-1 were expressed in all ovarian tumour tissues at a similar relative abundance. In addition, there was no tumour-specific pattern of isoform expression in this study. We found no differences in either SRC-2 or SRC-3 expression between the normal ovary and ovarian tumours. In contrast to the widespread and uniform expression of SRC-1, the expression of SRC-2 and SRC-3 is low and variable across all ovarian samples examined. SRC-3 gene amplification has been reported to occur in ovarian tumours with a frequency of 7.4% (9/122) (Bautista et al., 1998) or 29% (9/31) (Tanner et al., 2000). In the study by Bautista et al. (1998) the majority of samples examined were epithelial tumours, 84/122 were serous tumours and 11/122 were mucinous tumours. In the study by Tanner et al. (2000), 16/31 tumours were of the serous type and 3/31 were of the mucinous type, however SRC-3 amplification was reported in only 2/16 serous tumours and 0/3 mucinous tumours. Unfortunately neither study analysed SRC-3 expression. Although SRC-3 gene amplification was not sought in the ovarian tumour samples examined in the present study, we found no evidence of increased SRC-3 expression in the serous and mucinous tumours or the GCT. In the studies of SRC-3 in breast cancer not only has the incidence of gene amplification varied considerably between series (Anzick et al., 1997; Glaeser et al., 2001) but evidence of increased expression has been similarly variable (Anzick et al., 1997; Bouras et al., 2001; Glaeser et al., 2001; List et al., 2001). Furthermore, significant SRC-3 overexpression has been observed not only in breast cancer specimens that carry amplification of the SRC-3 gene (Anzick et al., 1997), but also in breast cancer specimens without amplification of this locus (Anzick et al., 1997; Glaeser et al., 2001), suggesting that overexpression also occurs via mechanisms other than amplification. The lack of SRC-3 overexpression in the ovarian tumours examined in this study may reflect the small sample size or it may be that gene amplification is not invariably associated with increased gene expression. Lanz et al. (1999) isolated the novel co-activator SRA and identified three isoforms. SRA has the ability to interact with and enhance PR- and also ER-mediated transactivation as an RNA transcript. It exists in ribonucleoprotein complexes that include SRC-1 and which are recruited by steroid receptors in a ligand-independent manner in vivo (Lanz et al., 1999). Given this close association of SRA with SRC-1, it is not surprising that in this study both SRC-1 and SRA show widespread expression across the normal ovary and ovarian tumours. The upregulation of SRA expression observed in these ovarian tumour samples, in particular the serous tumours, has been reported in breast tumours (Murphy et al., 2000) and also in ovarian tumours (Lanz et al., 2003), however the study of SRA overexpression in the ovarian tumours

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was limited in terms of an overall small sample size with limited consecutive tumour samples from the different tumour groups. SRA has the ability to interact with the AF-1 domain in the N-terminus of nuclear receptors and can confer agonist activity on RU486-bound PR (Lanz et al., 1999). While antiestrogen therapy can inhibit the SRC family members from potentiating AF-2 function it has no effect on AF1-mediated transactivation by steroid receptors. Therefore, altered SRA levels may be important in the pathogenesis of breast and ovarian tumours as well as in predicting the response of these tumours to antiestrogen therapy. We observed a trend for higher levels of SRA-del, when expressed as a percentage of wildtype SRA, in the serous tumours compared to normal ovary and the mucinous tumours. In breast tumours, the expression of SRA-del relative to wildtype SRA was not significantly different between normal breast and breast tumour samples, suggesting that it is not altered during breast tumourigenesis (Murphy et al., 2000). However, two studies reported that the relative expression of SRA-del correlated with higher tumour grades (Leygue et al., 1999; Murphy et al., 2000). SRA-del has been shown to be an exon 3 deleted variant of SRA (Leygue et al., 1999) and SRA mutations deleted of exon 3 sequences have been reported to not co-activate steroid receptors (Lanz et al., 1999). Furthermore, in this study the relevance of SRA-del in the ovarian tumours is less clear since the expression of SRA-del in the ovarian tumours is very low compared to wildtype SRA. The genes encoding both NCoR and SMRT are ubiquitously expressed, however the levels of both factors are variable across various tissues and cell lines (Wang et al., 1998). A study by Havrilesky et al. (2001) found that the levels of NCoR and SMRT varied widely in primary ovarian epithelial cancers expressing these corepressors. Similarly, we found that NCoR and SMRT expression levels varied across all the ovarian samples and there was no obvious pattern in the expression of NCoR or the SMRT splice variants in the normal and tumour tissues examined in this study. We also found that NCoR and SMRT mRNA expression levels were relatively unaltered in the ovarian tumours compared to normal ovary; another study also found no correlation between the presence of NCoR or SMRT proteins with the ER-status of ovarian tumours (Havrilesky et al., 2001), suggesting that these corepressors are unlikely to account for the hormone unresponsiveness observed in most ovarian tumours. We had anticipated that the expression of one or more co-activators would correlate with estrogen receptor expression. GCT express abundant levels of ER␤ (Chu et al., 2000); however, that observation was not paralled by increased levels of any of the coregulator genes examined in this study. It is tempting to speculate that other known or novel coregulators may have a substantial role in modulating ER␤ activity in the GCT and indeed in normal granulosa cells. Furthermore, in the mucinous tumours, low co-activator expression levels are observed in the presence of modest levels of ER␤ expression (Chu et al., 2000).

Although both mucinous and serous tumours are thought to have arisen from the ovarian surface epithelial cells (Amsterdam and Selvaraj, 1997; Riman et al., 1998), the levels of co-activator gene expression are very different being low in the mucinous tumours and high in the serous tumours. There was a marked upregulation of SRA expression in the serous tumours compared to the other tumour types and normal ovary. This may relate to tumour type or simply be an index of malignancy given the panel of serous tumours examined were stage 3 and hence almost invariably high grade whereas stage 1 mucinous tumours are generally low grade (Table 1). Although SRA is upregulated in breast tumours compared to normal breast (Murphy et al., 2000; Lanz et al., 2003), in two separate studies of breast tumours, expression levels of the SRA-del isoform but not the wildtype SRA isoforms positively correlated with higher breast tumour grade (Leygue et al., 1999; Murphy et al., 2000). SRA-del levels did appear to be higher in the serous tumours when compared to the mucinous tumours, however SRA-del was expressed at very low levels relative to wildtype isoforms of SRA. Given the relatively low levels of ER expression in the serous tumours (Chu et al., 2000), the results from this study suggest that SRA could play a role in amplifying the response to ER or the interaction may be with other steroid receptors. Although we found no significant difference in SRC-1, SRC-2, SRC-3, NCoR and SMRT expression between the normal and tumour groups, the expression of these coregulator genes in the ovarian tumours suggests that these factors may be available for interaction with the ER or other nuclear receptors. Whilst the considerable variability in coregulator expression within the normal and tumour groups of the ovary most likely reflects the small sample size, the lack of difference in coregulator expression between normal and tumour tissues may also reflect a limitation of using the normal ovary as a control tissue. Tissue heterogeneity and individual patient variation make a quantitative comparison with the normal ovarian tissues difficult. Furthermore, in this study we have focussed on expression profiles, so any interpretation of the functional significance must be tempered by the recognition that protein levels have not been examined. This caveat does not however apply to SRA, which is not translated. We speculate that the high SRA expression in this cohort of serous tumours is important in their pathogenesis, and suggestive that estrogen receptor-specific antagonists may be less effective in these tumours as SRA overexpression could potentially render an antagonist agonist. Endocrine therapy including anti-estrogen therapy for ovarian cancers has yielded very limited responses; re-evaluation of therapy with regard to receptor status, coregulator profile and tumour type may be warranted.

Acknowledgements We thank Sue Panckridge for assisting in the preparation of this manuscript, and Dr. Dinny Graham for kindly pro-

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viding the primer sequences for NCoR and SMRT. We thank our colleagues Simon Chu and Francine Brennan for their technical guidance, particularly Simon Chu for his assistance with the ER study, and gratefully acknowledge the contribution of our clinical colleagues Henry Burger, Tom Jobling, David Healy, Bruce Ward and Pam Mamers. This work was supported by project grant (#122201) and a Senior Principal Research Fellowship (PJF) from the National Health and Medical Research Council of Australia.

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