Tissue-specific transcripts of human steroid sulfatase are under control of estrogen signaling pathways in breast carcinoma

Journal of Steroid Biochemistry & Molecular Biology 105 (2007) 76–84

Tissue-specific transcripts of human steroid sulfatase are under control of estrogen signaling pathways in breast carcinoma Tetiana Zaichuk ∗ , David Ivancic, Denise Scholtens, Carol Schiller, Seema A. Khan ∗∗ Department of Surgery, Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Feinberg School of Medicine, Chicago, IL 60611, United States Received 5 September 2006; accepted 18 December 2006

Abstract Steroid sulfatase (STS) increases the pool of precursors of biologically active steroids, thereby playing an important role in breast cancer development. Mechanisms that control STS expression remain poorly understood. In present study we investigated alterations in the 5 region of STS gene to gain insight into the mechanism(s) that regulates its expression in mammary epithelial cells. We found that at least four alternatively spliced transcripts of STS gene can be produced from at least four different leader exons. Distinct expression patterns of the STS variants were observed in human tissues. Expression profiles of estrogen receptor ␣ (ER␣)-positive and ER␣-negative breast carcinomas showed that these two categories of tumors and their adjacent benign tissues display remarkably different expression of STS isoforms. Coexpression of STS isoforms with ER isotypes suggests their cell-type specific coregulation. In addition, we identified ER␣ as essential regulator of STS transcription and provide evidence of direct estradiol-dependent binding of ER␣ to multiple STS cis-regulatory regions in vivo. Our results indicate that STS isoforms are under control of estrogen signaling pathways and their differential expression may play a significant role in breast cancer biology. © 2007 Elsevier Ltd. All rights reserved. Keywords: Steroid sulfatase; Steroid biosynthesis; Estrogen receptor; Breast cancer

1. Introduction Local estrogen production is important in the development of breast malignancies [1–4]. Hydrolysis of alkyl and aryl 3␤hydroxysteroid sulfates (e.g., dehydroepiandrosterone sulfate DHEAS and estrone sulfate E1S, respectively) is catalyzed by steroid sulfatase (EC 3.1.6.2, STS, also known as arylsulfatase C) to form unconjugated DHEA and E1 which can ∗ Corresponding author at: Northwestern University, Feinberg School of Medicine, Department of Surgery, Robert H. Lurie Medical Research Center 4-220, 303 E. Superior str., Chicago, IL 60611, United States. Tel.: +1 312 503 2109; fax: +1 312 503 0095. ∗∗ Corresponding author at: Lynn Sage Breast Center, Department of Surgery, Feinberg School of Medicine of Northwestern University, 675 North St Clair Street, Galter 13-174, Chicago, IL 60611, United States. Tel.: +1 312 695 0288; fax: +1 312 695 4956. E-mail addresses: [email protected] (T. Zaichuk), [email protected] (S.A. Khan).

0960-0760/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.jsbmb.2006.12.101

be converted to potent estrogens [5]. Elevated DHEAS levels stimulate growth of cultured breast cancer cells [6] and high STS expression is associated with increased risk of breast cancer recurrence [7–9]. The adverse prognostic impact of STS expression is confined to estrogen receptor (ER) positive tumors and appears to affect both pre- and post-menopausal women [8,10]. Recently, a non-steroidal STS inhibitor, 667 COUMATE, has entered a Phase 1 trial in postmenopausal women with breast cancer [11,12]. Human placental STS cDNA [13] corresponds to 583 amino acids with an N-terminal signal peptide [14]. However, experimental evidence suggest the existence of tissuespecific STS isozyme(s) with different kinetic parameters for DHEAS and E1S [15–17]. Several studies demonstrate that STS enzyme activity and mRNA level are concordant [7,8,18], however the role of transcription factors, cis-acting elements, and the cellular context in STS regulation remain obscure. The promoter region and 5 upstream regulatory ele-

T. Zaichuk et al. / Journal of Steroid Biochemistry & Molecular Biology 105 (2007) 76–84

ments of the STS gene have been characterized in human placental cells where STS activity is high [19]. This promoter exhibits little basal activity and shows tissue-specificity, suggesting that additional regulatory elements are required to achieve high STS expression [20]. Our incomplete knowledge of STS gene regulation at the transcriptional level may be due to the fact that so far only the placental transcript has been studied. The paucity of experimental data for STS gene regulation in other tissues together with our interest in the involvement of the STS gene in breast carcinoma lead us to investigate whether there may be 5 -variability in STS transcripts. We found that the STS gene, like most of human genes [21], expresses mRNA variants as result of multiple splicing and alternative promoters. Four STS transcripts that differ at their first exons were identified in MCF-7 mammary epithelial cells. The alternative use of these leader exons is, likely, the key step in the regulation of mature STS mRNA level, which is the part of molecular mechanism conferring tissue-specific regulation of STS expression. To understand the role of STS variants in breast cancer, we explored their expression patterns in normal tissue, cancer samples and matched adjacent tissue, and have examined the associations of these with the ER␣ status of the primary tumor. In addition, we identified ER␣ as the mediator of the estradiol (E2)-dependent increase of STS transcription in MCF-7 mammary epithelial cells. Our results indicate that the changes in the balance between different STS isoforms are likely to be important to our understanding of mechanisms of breast cancer initiation and progression.

2. Materials and methods 2.1. Cell culture and reagents MCF-7 cells obtained from American Type Culture Collection (Manassas, VA) were maintained following manufacture’s instructions. Three days before cell exposure to 10−8 M E2 (Sigma, St. Louis, MO) or/and 10−6 M ICI 182,780 (Tocris Cookson, Ellisville, MI.) the medium was replaced by phenol red-free MEM containing 5% steroidstripped serum (JR Scientific Inc., Woodland, CA). Cells were pre-treated with vehicle (DMSO) or 10−6 M MG132 from Sigma for 1 h followed by treatment with E2. Antibodies used in Western blot analysis performed as described previously [32], were: ER␣ (NeoMarkers/Lab Vision, Fremont, CA, Ab-15) and ␤-actin (Sigma, AC-15). Oligonucleotides were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA). 2.2. Tissue specimens Grossly dissected tissue fragments from the 21 primary breast carcinomas and paired adjacent nonneoplastic tissues were provided by Northwestern University Breast

77

SPORE tissue bank following the Institutional Review Board approval. The age of patients ranged from 36 to 80 years, with a mean of 49.2 years. ER␣ measurement were made as part of the routine pathological assessment of tumors by immunohistochemistry (IHC) and scored using a cut-off value of 10% of positive tumor nuclei staining. Four specimens of normal breast tissue were obtained from cosmetic breast reduction surgery and one specimen of normal breast tissue (Ambion Inc., Austin, TX) originated from autopsy. Total RNA from breast tissues specimens was extracted using Trizol (Invitrogen Life Technologies, Carlsbad, CA) and subjected to a single round of linear RNA amplification with a starting amount of 100 ng RNA (Full SpectrumTM , Complete Transcriptome RNA Amplification Kit, System Bioscience, CA). 2.3. RNA ligase-mediated 5 rapid amplification of cDNA ends (5 -RLM-RACE) and sequence analysis Reverse transcription of total RNA (10 ␮g) and secondstrand synthesis were performed using a FirstChoice RLM-RACE kit (Ambion, Austin, TX). In this system only full-length, capped mRNA is reverse transcribed. To assure this DNA-free RNA template was treated with calf intestinal phosphatase (CIP) to remove 5 -phosphates from rRNA, tRNA and fragmented mRNA. The cap structure was then removed by treatment with the tobacco acid pyrophosphatase (TAP) to ligate a synthetic RNA adapter oligonucleotide to the mRNA 5 end with T4 RNA ligase. A random-primed reverse transcription reaction was then carried out to make a cDNA copy of the treated RNA and after RNase treatment the cDNA was size selected by column chromatography. A 0.5 ng cDNA aliquot was used as a template for PCR amplification with an antisense primer annealing to the STS exon 3 (5 -AACCGGTCGATATTGGGAGT-3 ) and a primer containing the synthetic RNA adapter sequence. Then, a nested PCR was applied to the first PCR products using 5 RACE inner primer and STS-specific primer from exon 2 (5 -AGGAGGAAAGGGATCTTCATC-3 ). The final PCR products were analyzed on a 2% agarose gel and visualized by ethidium bromide staining. Gel-purified products were cloned into the pGEM-T vector (Promega, Madison, WI) for DNA sequencing. These sequences are available at GeneBank under accession numbers DQ851171–DQ851173. 2.4. Database search and secondary structure prediction Nucleotide sequence information was subjected to the basic local alignment search tool (BLAST) homology search against the Human Genome resource database of the National Center for Biotechnology Information (NCBI) [22]. Comparing the genomic sequences to the corresponding cDNA sequences identified 5 splice sites. The contexts of alternative ATG start codons were analyzed using software tool for prediction of translation initiation sites [23], and by comparison to the Kozak consensus sequence [24]. The secretory

78

T. Zaichuk et al. / Journal of Steroid Biochemistry & Molecular Biology 105 (2007) 76–84

signal peptides were predicted by Signal IP method [25]. The secondary structure for each STS mRNA was predicted by the mFOLD program based on the Zuker algorithm [26]. The 5 flanking regions of the newly identified transcripts were scanned by TESS (http://www.cbil.upenn.edu/tess/) and Dragon ERE Finder version 3 [27] programs. 2.5. Absolute quantification of STS isoforms and total transcripts by quantitative reverse transcription PCR STS expression profiling was performed using the First Choice Human Total RNA Survey panel (Ambion, Austin, TX). 1 ␮g of total RNA was converted into the cDNA template using a reverse transcription kit (Applied Biosystems, Foster City, CA). Real-time PCR was done on an ABI Prism 7900 Detection System using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA). PCR primers were designed to amplify sequences specific to each isoform with forward primers in alternative exons (see Supplementary materials) and reverse primers in exon 2. 5 CAGTTCCCCAACAACAGGAT-3 and 5 -GGAAAGGGATCTTCATCTTCCTT-3 primers yielded a 63-bp product for 1a transcript; 5 -GTTGCTGAACCTGCACACAGTCAT3 (from exon 1b) and 5 -TTGTTCCCATAGCACCCAGGATCT-3 amplified 217 bp product; 5 -AAGAACCTGGCTCAAGGCTGTGAA-3 and 5 -TTGTTCCCATAGCACCCAGGATCT-3 yielded a 231 bp product for 1c transcript. Primers from exons 5 and 6 for estimation of total amount of STS (5 -GATCATTCAGCAGCCCATGT-3 and 5 -GAGGTAGGACAAGACAAGCAGG-3 ) amplified 115 bp product. No primer was used for exon 0b since this exon is always linked to exon 1b. Product specificity was confirmed initially by sequencing and routinely by melting curve analysis. Gene expression assays were used for ER␣, ER␤ (Hs00174860 m1 and Hs00230957 m1; Applied Biosystem, Foster City, CA) and GAPDH (Invitrogen, Carlsbad, CA) analysis. The standard curves for absolute quantification were generated using 10–107 dilutions of cDNA or plasmid templates. The PCR efficiencies of the target genes and the endogenous reference gene were comparable. 2.6. Chromatin immunoprecipitation (ChIP) ChIP was performed as previously described [28] using the ChIP kit (Upstate Biotechnology, Lake Placid, NY) and ER␣ antibodies (Ab-10 mAb TE 111.5D11, NeoMarkers, Fremont, CA) or control IgG (Santa Cruz Biotechnology, Inc., CA). The following sets of primers were used to amplify immunoprecipitated DNA: 5 -TGATGCAACCTGGCTCTGCAGTTA-3 and 5 -CCAGTGACATGGGCCATAGAGAATGA-3 (1a region I); 5 -TTCACGCCATTCTCCTGCCTCA-3 and 5 -CAAGGGAGAGTGATCAGGATGCTT-3 (1a region II); 5 -ACTCTGGCAGCTTTCTCTATGTC3 and 5 -TCATTCCACAAGGGAAGAGAAGAC-3 (1a region III); 5 -AATTTACAGTCACCCTCCCAGGCT-3 and 5 -AGATCATAGCTCACTGCAGCCTCA-3 (1a control);

5 -TCGCCCAATCAATACAGACTGCAC-3 and 5 -AGGAGACCACGAGTGTTAGCAAGA-3 (1b region IV); 5 AAGACTTCTCCCATTGCCGTCTGT-3 and 5 -TTGCAGAGCTGACTGTATCCTCCA-3 (1b control). To avoid non-specific PCR products, database screening was performed with BLAST search program for every pair of primers used. 2.7. Statistical analysis STS expression levels under E2 treatment were compared with the respective expression levels without drug treatment using statistical analysis by Student’s t-test with GraphPad Prism software version 4. Statistical evaluation of STS expression in human breast specimens was done using the Wilcoxon signed rank test for paired comparisons and the Wilcoxon rank sum test for unpaired comparisons. Nonparametric Spearman correlation coefficients were used to evaluate correlations between ERs and STS cDNA levels. Nominal p-values less than 0.05 were considered statistically significant.

3. Results 3.1. Additional isoforms of human STS cDNA with different 5 -terminal regions To determine the capped full-length RNA sequences at the 5 end of the STS transcripts, a 5 -RLM-RACE on RNA from mammary epithelial cell line MCF-7 was performed. This approach generated four major DNA fragments with respective sizes of 0.18, 0.21, 0.27 and 0.35 kb (Fig. 1A). Sequencing of the PCR-purified products was used to define their transcription start sites. We found that all sequences retrieved by 5 -RLM-RACE approach map to chromosome X upstream of STS exon 2 and contained consensus splice junctions at their 3 ends (Suppl.). These products can be classified into four types. The 0.35 kb product contains exon 1 previously identified in the cells of placental origin [13]. We name this exon 1a to distinguish it from other alternative 5 STS sequences (Fig. 1B, Suppl.). Two additional products (0.27 and 0.21 kb) have an identical 131 bp sequences corresponding to exon 1b recently identified in adipose tissue [29]. The 0.27-kb product contains an additional sequence, identical to adipose-specific exon 0b [29], however, it is 14 bp shorter due to differences in transcription start sites. A third 0.18-kb product has a 101 bp sequence, derived from the STS first intron and is named here as exon 1c. These data indicate that the genomic region of STS has total length of 318 kb and contains both variable and constant regions. Alternative leader exons 1a and 1b are located approximately 30 and 62 kb upstream of exon 2, respectively. Distal exon 0b is mapped 43 kb further upstream from exon 1b and proximal exon 1c is located 953 bp upstream from the constant region. Homology search against the nucleotide sequence

T. Zaichuk et al. / Journal of Steroid Biochemistry & Molecular Biology 105 (2007) 76–84

79

Fig. 1. Alternative initiation of STS transcription. (A) 5 -RLM-RACE analysis of MCF-7 full-length capped STS mRNAs. The sizes of amplified products are indicated. (B) Schematic representation of the exon structure of a portion of the STS gene and the corresponding transcripts. Open boxes indicate the alternative exons encoding STS mRNA isoforms. Grey box indicate exon 2 common for all transcripts. Note, that cDNA for STS transcripts with exon 1c has an open reading frame common for all transcripts.

database of the NCBI [22] indicated that exons 0b and 1b are highly homologous to STS mRNAs of non-human primates (Pan troglodytes XM 520918 and Macaca mulatta XM 001088752), suggesting that a functional role for these transcript has been preserved during evolution. Sequence alignment of the 5 -UTR of the exon 1c with the known STS sequences from other mammalian species showed no significant homology. ATG initiation codon is located within the constant region of 1c transcript in the same open reading frame as the previously described STS isoforms [14,29] (Fig. 1B, Suppl.). The 1c isoform has a sequence consisting of 18 hydrophobic amino acids that specifies a cleavable signal peptide, similarly to the known STS variants.

3.2. Tissue-specific expression of the STS mRNA isoforms To elucidate the physiological significance of alternative STS transcripts, normalized cDNA preparations obtained from various human tissues were analyzed by real time RTPCR (Table 1) with primers designed to distinguish different leader exons. All STS cDNA species are expressed in the majority of human tissues examined. The highest level of total STS gene transcription we identified in the brain (where STS activity is essential for the synthesis of neuroactive steroids [30]) and it mainly consists of the transcripts with exon 1b. We also found relatively high level of the 1b transcripts in the liver, ovary, heart, adipose tissue and muscle (Table 1). The

Table 1 Abundance of alternative STS mRNAs in different human tissues cDNA source

1a cDNA/RNA (amol/mg)

Adipose Bladder Brain Cervix Colon Esophagus Heart Kidney Liver Lung Ovary Placenta Prostate Muscle Small intestine Spleen Testes Thymus Thyroid Trachea

1.2 3.1 7.6 1.2 0.9 1.2 0.6 0.7 0.6 1.8 0.7 2744 1.3 0.95 0.9 0.9 0.9 1.3 0.7 0.7

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.3 0.3 0.1 0.06 0.12 0.6 0.07 0.2 0.04 0.3 0.2 141 0.5 0.33 0.1 0.1 0.03 0.1 0.2 0.1

1b cDNA/RNA (amol/mg) 435 74 6107 244 43 60 498 115 1038 425 906 140 70 426 37 221 274 81 91 351

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

48 7 340 18 3 13 95 16 41 73 41 9 23 10 4 14 34 1.5 26 27

1c cDNA/RNA (amol/mg)

Total STS cDNA/RNA (amol/mg)

0.7 ± 1.0 ± 2.0 ± 1.2 ± 2.0 ± 0.6 ± N/D 2.6 ± 5.7 ± 8.8 ± 5.3 ± 3.5 ± 2.6 ± 0.2 ± 3.0 ± 9.5 ± 1.3 ± 3.9 ± 1.4 ± 3.1 ±

1837 705 12594 1082 395 529 1987 1615 3003 2018 2500 9219 658 1371 384 1054 1150 535 656 1521

0.2 0.2 0.3 0.3 0.7 0.1 0.2 0.1 2.3 1.3 0.1 0.01 0.03 1.1 1.0 0.2 0.9 0.2 0.5

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

17 52 589 79 3.3 17 71 56 376 172 452 44 21 10 28 114 53 5 10 14

cDNA/RNA, amount of quantified cDNA obtained by regression analysis of qRT-PCR and normalized to the amount of starting RNA. STS transcript with exon 1a is expressed at high level in placenta. The main amount of STS mRNA in all other tissues is represented by transcripts containing exon 1b. Transcript with exon 1c is expressed at low level. The highest levels of STS gene transcription are indicated in bold.

80

T. Zaichuk et al. / Journal of Steroid Biochemistry & Molecular Biology 105 (2007) 76–84

a considerable variation among subjects, ranging from 7 to 7466 for total STS, from 3 × 10−3 to 3862 for 1a, from 8 × 10−4 to 12 for 1b and from 10−3 to 113 for 1c (calculated as amol STS/pmol GAPDH) (Fig. 2). Expression of all isoforms strongly correlates with that of total STS. Breast cancers that were ER␣ positive by immunohistochemistry show significantly higher levels of total STS expression than ER␣ negative cancers. (p = 0.0007) (Fig. 2). Interestingly, ER␣ negative tumor samples display significantly lower expression levels of total STS than adjacent benign tissues (p = 0.037) or the normal breast samples (p = 0.0193) (Fig. 2). 1b STS displays a gradually decreased expression across the categories of samples, with the highest expression in normal tissue. The patterns of expression of 1a and 1c transcripts are similar in that the median expression in the cancers is higher than in their matched benign tissues. 1a STS expression in tissue adjacent to ER␣-negative breast cancer is significantly lower than in both normal control breast tissue (p = 0.003) and benign tissue of ER␣-positive patients (p = 0.015) (Fig. 2). In addition, we investigated correlations in expression between STS isoforms and ER isotypes on mRNA levels. In these exploratory analyses, the 1a transcript positively correlates with ER␣ expression in benign breast tissues of patients with ER␣ positive breast carcinoma (rs = 0.794; p = 0.009), but not in the cancer samples themselves (rs = 0.200, p = 0.572) (Table 2). In the same benign tissue adjacent to ER␣ positive breast cancers, there is a positive and significant correlation with the levels of ER␤ RNA and total STS (rs = 0.697; p = 0.029). A strong and significant correlation was also observed between the 1c STS variant and ER␤ expression in ER␣ negative breast cancer samples (rs = 0.746; p = 0.017).

Fig. 2. Patterns of expression of the STS transcripts in the breast tissues. Individual values are presented on a log scale for normalized STS transcripts expression in normal breast, tumors (ER␣-positive and ER␣-negative by IHC) and adjacent benign tissues with marked median values. Horizontal broken lines represent the median values detected in normal breast. Statistically significant differences in STS expression between tissues are shown (*<0.05, **<0.005 and ***<0.001). The Wilcoxon signed rank test for paired comparisons is shown by dotted arrows.

next highest level of total STS expression, consisting of previously described [13] transcript 1a was identified in placental tissue. The 1c transcript is always expressed in quantities that are an order of magnitude lower than 1b transcripts. The highest level of the 1c transcript was observed in the spleen, lung and liver. These results indicate that tissue-specific mechanisms may govern transcriptional regulation of STS and specify the presence of other still unknown STS variants. Variation in the expression of STS isoforms among different tissues suggests that alternative promoters [31] control the expression of STS.

3.4. 17β-Estradiol (E2) regulates STS expression in MCF-7 cells

3.3. Expression of STS mRNAs in the mammary gland: link with ERα status

To understand the molecular basis of the observed differences of STS level in the breast tissues, we estimated the amount of STS transcripts in MCF-7 cells under basal and E2-stimulated conditions. As shown in Fig. 3A, basal transcription of the STS gene in the absence of E2 remains detectable even after 3 days of steroid deprivation. Since

We evaluated the expression of STS variants in 5 normal breast tissues and series of specimens from 21 human breast carcinoma samples paired with adjacent benign breast tissues (Fig. 2). The expression levels of STS variants showed

Table 2 Relation between normalized expression of STS variants and estrogen receptors in breast carcinoma and adjacent tissue

adjacent tissue ER␣-positive (IHC) tumor

tumors adjacent tissue

ER␣-negative (IHC) tumor

tumors

Rs

p

Rs

p

Rs

p

ER␣ ER␤ ER␣ ER␤

0.62 0.70 −0.35 0.61

0.06 0.03 0.33 0.06

0.79 0.20 0.20 0.32

0.009 0.57 0.57 0.39

0.53 0.38 −0.14 0.19

0.12 0.27 0.71 0.59

0.45 0.02 0.04 0.31

0.19 0.96 0.91 0.38

ER␣ ER␤ ER␣ ER␤

−0.32 0.52 −0.04 0.10

0.37 0.13 0.92 0.77

0.58 0.39 0.49 0.64

0.08 0.25 0.15 0.05

0.35 −0.43 −0.27 0.15

0.32 0.22 0.45 0.67

0.50 −0.18 0.52 0.75

0.14 0.63 0.13 0.02

Spearman correlation coefficients and significant p-values (two-tailed) are shown. The significant correlations are indicated in bold.

Rs

p

T. Zaichuk et al. / Journal of Steroid Biochemistry & Molecular Biology 105 (2007) 76–84

81

Fig. 3. (A) Regulation of STS mRNA expression in MCF7 cells. Steroid-depleted MCF-7 cells were treated with E2 (10−8 M) ± MG132 (10−6 M) or ICI 182,780 (10−6 M) for 2 h. STS mRNA levels were assayed by quantitative real time PCR. Data are presented as fold change in STS mRNA levels (normalized to the amount of starting RNA) relative to the vehicle treated cells. Values are expressed as mean ± S.D. of two independent experiments. (B) Effect of MG132 on E2-induced degradation of ER␣. Total cellular extracts prepared from MCF7 cells treated with E2 along or in combination with MG132 were analyzed by Western blotting with a specific anti-ER␣ antibody or an anti-actin antibody for loading control. Note the decrease in ER␣ by E2 and restored ER␣ level in the E2 -stimulated cells exposed to MG132. (C) Temporal ER␣ recruitment to STS cis-regulatory elements analyzed by ChIP. Chromatin was precipitated with indicated antibodies after 1, 2 and 3 h treatment with E2 and the kinetics of the ER␣ association with STS regulatory elements was determined by PCR. The nucleotide position (relative to the transcriptional start sites (+1) of the primer pairs used in the analysis are indicated by double-ended arrows. The locations of the 1a (boxes I-III), 1b (box IV) regions and the control segments (no ERE) are shown. The stars under sequences indicate the nucleotide changes compared with the complete perfect palindromic ERE. Immunoprecipitation with nonspecific IgG served as negative controls. Amplification of input DNA (loading control) is also shown. Note the strong increase of the PCR-amplified DNA fragments in the cells exposed to E2. Similar results were obtained in triple independent experiments.

E2 binding rapidly down-regulates ER␣ [32], we pretreated cells with MG132 proteasomal inhibitor to abolish E2induced ER␣ degradation (Fig. 3B). After 2 h of treatment E2 up-regulates 1a transcript by 1.7-fold, 1b by 1.3-fold and down-regulates the least abundant 1c isoform, whereas MG132 decreases basal expression of all STS isoforms by more than 50%. The combined treatment of MG132 and E2 significantly increases levels of all STS transcripts. Overall, these results demonstrate that blocking the turnover of transcription factors degraded by proteasome decreases basal transcription of the STS gene and increases E2-induced transcription of all STS isoforms. However, it remains to be determined which factors are involved in the inhibition of STS gene transcription by proteasome blockage. To explore a potential role of ER␣ on STS expression we examined the ability of pure antiestrogen to antagonize the E2 effect on STS gene expression. We found that ICI 182,780 exposure causes a down-regulation of the steadystate and E2-induced mRNA levels of all STS variants (Fig. 3A).

3.5. ERα directly regulate transcription of STS isoforms in MCF-7 cells We analyzed the 5 -flanking regions of the STS gene for presence of genomic elements required for E2 response. Estrogen response element-like sequences (ERE) that deviate from the consensus palindromic ERE [33] by 3 bp (region II) and 1 bp (regions III and IV) were located in 5 regions of STS transcripts (Fig. 3C). Computer-assisted analysis also revealed three ERE half-palindromic motifs (region I) in the promoter of the 1a transcript that may contribute to E2induced transactivation [34]. We used ChIP assays to examine ER␣ recruitment to the E2-responsive regions in MCF-7 cells in vivo. Cells were treated for 1, 2 or 3 h with either control vehicle or E2, then protein-DNA complexes were immunoprecipitated with antibodies to ER␣. Unrelated regions (no ERE) were used as a negative control. As shown in Fig. 3C, the EREs in STS cis-regulatory regions interact directly with ER␣ upon E2 addition. While occupancy of regions I and II of the 1a STS variant and region IV of the 1b STS variant is

82

T. Zaichuk et al. / Journal of Steroid Biochemistry & Molecular Biology 105 (2007) 76–84

evident after 1 and 2 h and decline at approximately 3 h of the treatment, recruitment of ER␣ to region III of 1a variant is slower and becomes apparent after 3 h of E2 exposure. This unique timing of different ERE-specific complexes formed with ER␣ probably reflects sequence-dependent recruitment of transcriptionally functional ER␣ complexes responsible for E2 action.

4. Discussion Steroid sulfatase is a membrane-bound microsomal enzyme that hydrolyzes a variety of 3␤-hydroxysteroid sulfates and is expressed in virtually all human tissues where biologically active androgens and estrogens are able to form [11]. To understand the structural organization of STS and to study its regulation, we characterized the 5 heterogeneity of human STS gene. In MCF-7 mammary epithelial cells we found a new, more complex structure of STS cDNA with alternative leader exons. The deduced amino acid sequences of these transcripts specify STS isoforms with diverse cleavable signal peptides. It is well established that heterogeneity in signal peptide sequences modulates the proper folding and localization of proteins to the correct intracellular compartment [35–37]. However, whether STS transcripts indeed encode different proteins and the functional significance of predicted N-terminal differences, awaits further investigations. Alternative leader exons may also provide a mechanism(s) to regulate expression of STS post-transcriptionally, by modifying the translational efficiency of the mRNA. It has long been appreciated that upstream AUGs, open reading frames and secondary structure within 5 -UTR are inhibitors of efficient translation [38]. As determined by the mFOLD program [26], the 1c leader exon is the less structured (initial dG values −26.4 kcal/mol) that may promote, together with the absence of upstream out of frame AUG codons (Supplementary materials), more efficient translation [39] for this transcript. A consequence of STS gene organization is that multiple transcripts are expressed in a tissue-specific manner. All alternative transcripts were found in cDNAs obtained from 20 human tissues. Although the STS variants are expressed at various levels depending on tissue type, the most abundant mRNA species utilize the exon 1b that is highly conservative among primates. This transcript has recently been identified in adipose tissue [29], and our study demonstrated their highest expression in the brain. Of interest, brain-specific and housekeeping genes evolve relatively slowly and are poorly represented among genes associated with disease [40]. The high level of 1a STS mRNA is restricted exclusively to the placenta, where DHEAS, the major steroid released by the fetal adrenal glands is a main source of active estrogens [41]. It is possible that transcription of the least abundant 1c isoform is also activated in a specific spatio-temporal manner or, alternatively, in very scarce cell types. We have noticed, indeed, that 1c transcript is highly expressed in the subset

of ER␣-positive breast tumors and surrounding tissues (see Fig. 2). In breast tissues we found a marked variation in expression levels of all STS transcripts, however, the range of 1b and 1c expression is smaller than that of 1a, suggesting that expression of the 1a transcript is less tightly controlled. When analyzing mRNA expression in relation to ER␣ status of breast cancer samples, we found that the median expression level of all transcripts identified so far is lower in ER␣-negative than in ER␣-positive tumors. ER␣-negative tumors were also significantly lower in total STS expression than normal breast tissues from unaffected women. And, of interest from perspective of the evolution of ER␣negative vs. ER␣-positive breast cancer, benign breast tissue from women with ER␣-negative cancer expressed the 1a transcript at significantly lower levels than normal control breast tissues and benign tissues taken from women with ER␣-positive breast cancer. This indicates, in concordance with previously reported data [42], that STS expression may be important in both autocrine and paracrine regulation of breast cancer cell growth. Of note, the differences in 1a expression seen in benign breast samples may predate the development of breast cancer, and deserve to be investigated as markers of risk for ER␣-positive versus ER␣-negative tumors. Interestingly, in most specimens from unaffected women the 1b STS is the major isoform expressed. In the subset of cancerous, ER␣-positive specimens, the enzymatically more active 1a [29] became predominant and is responsible for the overall increase in the STS expression. We hypothesize that the expression of more active STS isoform(s) may result in dysregulated estrogen formation in such patients and further promote cancer progression. Subsequent analysis with regard to potential interrelationships of STS and ER isotypes revealed that significant positive correlations exist between their mRNA levels, particularly for total STS/ER␤, 1a/ER␣ in tissues adjacent to ER␣-positive tumors and 1c/ER␤ in ER␣-negative breast carcinoma samples. This observation may indicate that the cell type-specific expression of these genes is likely to be coordinately regulated. Moreover, since coexpressed genes tend to encode interacting proteins [43], it is conceivable that cell-type-specific regulation of STS isoforms relies on distinct ER isotypes. In situ hybridization studies using probes designed to anneal specifically to STS leader exons could help to narrow down the specific cell type(s) where coexpression of these transcripts with ER isotypes may be prominent. Expression of STS has been previously demonstrated to be upregulated by 17␤-estradiol in an ER-dependent cell lines [18]. Data presented here indicates that steroid hormonedependent STS regulation can be mediated by ER␣ since the pure antiestrogen ICI 182,780 caused a dramatic reduction of both basal and E2-stimulated expression of all STS mRNA variants in MCF7 cells. Since estrogen induces ER␣ degradation in an autoregulatory feedback loop, we included the

T. Zaichuk et al. / Journal of Steroid Biochemistry & Molecular Biology 105 (2007) 76–84

treatment with proteasomal inhibitor MG132 in our experiments. Consistent with published data [32] we found by immunoblot analysis that pretreatment with MG132 blocked E2-dependent degradation of ER␣ (Fig. 3B). After acute exposure to E2, expression of all STS mRNAs is markedly increased by pretreatment with MG132, while the proteasome blockage alone favors STS downregulation. Multiple regulatory elements other than ER␣ whose activity must be tightly controlled [44] could be differentially regulated by proteasome inhibition and cooperation between ER␣ and other transcription factors involved in the STS regulation by proteasome blockage remain to be determined. A detailed characterization of the dynamics of cell-type specific expression of STS isoforms and STS activity under activating and inhibitory stimulus is the subject of future analysis. To understand the molecular basis of the observed E2-induced STS regulation the E2-responsive regulatory sequences were mapped within the STS locus. We confirmed the functionality of potential ER␣ binding sites within 5 regions of 1a and 1b transcripts by comparing the ability of ER␣ antibodies to precipitate the regulatory sequences associated with ER␣ in MCF-7 cells expressing STS at basal levels and activated by E2. We proved that all ER binding sites including distant from the transcription start sites are involved in transcriptional regulation and demonstrated that STS expression is up-regulated by E2 via increased direct binding of estrogen receptor to these regions. Our temporal studies of ER␣ association showed that complexes involving distinct EREs are formed with different kinetic profiles and affinity, indicating the possible effect of natural variation in ERE sequences and/or cofactor recruitment on ER␣ binding. In conclusion, the findings of the present study suggest that the STS gene exhibits alternative splicing and promoter usage, which are likely to be the basis for tissue-specific regulation. This, in turn can affect the translation efficiency, orchestrating the events leading to the final expression profile of STS. The diversity in the distribution of STS variants in human breast carcinoma presumably reflects specific regulation and function of each of the isoform during tumor progression. Understanding the functional impact of different STS isoforms on breast cancer initiation and progression will provide the means for designing more selective inhibitors able to discriminate between peripheral and intratumoral STS activity.

Acknowledgments We thank Drs. C. Waltenbaugh, A. Chlenski, and A. Levenson for providing critical comments on the manuscript, and Dr. D. Patil and Mr. E. Schilling for technical assistance. This work was supported by the Bluhm Family Program for Breast Cancer Early Detection & Prevention and NIH/NCI P50 CA89018-02.

83

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/ j.jsbmb.2006.12.101.

References [1] R.J. Santen, Determinants of tissue oestradiol levels in human breast cancer, Cancer Surv. 5 (1986) 597–616. [2] J. Russo, M. Hasan Lareef, G. Balogh, S. Guo, I.H. Russo, Estrogen and its metabolites are carcinogenic agents in human breast epithelial cells, J. Steroid Biochem. Mol. Biol. 87 (1) (2003) 1–25. [3] A. Purohit, M.J. Reed, N.C. Morris, G.J. Williams, B.V. Potter, Regulation and inhibition of steroid sulfatase activity in breast cancer, Ann. N.Y. Acad. Sci. 784 (1996) 40–49. [4] J.R. Pasqualini, G.S. Chetrite, Recent insight on the control of enzymes involved in estrogen formation and transformation in human breast cancer, J. Steroid Biochem. Mol. Biol. 93 (2–5) (2005) 221–236. [5] T. Sugawara, S. Fujimoto, The potential function of steroid sulphatase activity in steroid production and steroidogenic acute regulatory protein expression, Biochem. J. 380 (Pt 1) (2004) 153–160. [6] K.E. Calhoun, R.F. Pommier, P. Muller, W.S. Fletcher, S. TothFejel, Dehydroepiandrosterone sulfate causes proliferation of estrogen receptor-positive breast cancer cells despite treatment with fulvestrant, Arch. Surg. 138 (8) (2003) 879–883. [7] T. Utsumi, N. Yoshimura, S. Takeuchi, J. Ando, M. Maruta, K. Maeda, N. Harada, Steroid sulfatase expression is an independent predictor of recurrence in human breast cancer, Cancer Res. 59 (2) (1999) 377–381. [8] Y. Miyoshi, A. Ando, S. Hasegawa, M. Ishitobi, T. Taguchi, Y. Tamaki, S. Noguchi, High expression of steroid sulfatase mRNA predicts poor prognosis in patients with estrogen receptor-positive breast cancer, Clin. Cancer Res. 9 (6) (2003) 2288–2293. [9] T. Suzuki, Y. Miki, T. Nakata, Y. Shiotsu, S. Akinaga, K. Inoue, T. Ishida, M. Kimura, T. Moriya, H. Sasano, Steroid sulfatase and estrogen sulfotransferase in normal human tissue and breast carcinoma, J. Steroid Biochem. Mol. Biol. 86 (3–5) (2003) 449–454. [10] T. Nakata, S. Takashima, Y. Shiotsu, C. Murakata, H. Ishida, S. Akinaga, P.K. Li, H. Sasano, T. Suzuki, T. Saeki, Role of steroid sulfatase in local formation of estrogen in post-menopausal breast cancer patients, J. Steroid Biochem. Mol. Biol. 86 (3–5) (2003) 455–460. [11] M.J. Reed, A. Purohit, L.W. Woo, S.P. Newman, B.V. Potter, Steroid sulfatase: molecular biology, regulation, and inhibition, Endocr. Rev. 26 (2) (2005) 171–202. [12] S.J. Stanway, A. Purohit, L.W. Woo, S. Sufi, D. Vigushin, R. Ward, R.H. Wilson, F.Z. Stanczyk, N. Dobbs, E. Kulinskaya, M. Elliott, B.V. Potter, M.J. Reed, R.C. Coombes, Phase I study of STX 64 (667 Coumate) in breast cancer patients: the first study of a steroid sulfatase inhibitor, Clin. Cancer Res. 12 (2006) 1585–1592. [13] P.H. Yen, E. Allen, B. Marsh, T. Mohandas, N. Wang, R.T. Taggart, L.J. Shapiro, Cloning and expression of steroid sulfatase cDNA and the frequent occurrence of deletions in STS deficiency: implications for X–Y interchange, Cell 49 (4) (1987) 443–454. [14] C. Stein, A. Hille, J. Seidel, S. Rijnbout, A. Waheed, B. Schmidt, H. Geuze, K. von Figura, Cloning and expression of human steroid-sulfatase. Membrane topology, glycosylation, and subcellular distribution in BHK-21 cells, J. Biol. Chem. 264 (23) (1989) 13865–13872. [15] J.H. MacIndoe, The hydrolysis of estrone sulfate and dehydroepiandrosterone sulfate by MCF-7 human breast cancer cells, Endocrinology 123 (3) (1988) 1281–1287. [16] O. Prost, M.O. Turrel, N. Dahan, C. Craveur, G.L. Adessi, Estrone and dehydroepiandrosterone sulfatase activities and plasma estrone sulfate levels in human breast carcinoma, Cancer Res. 44 (2) (1984) 661–664.

84

T. Zaichuk et al. / Journal of Steroid Biochemistry & Molecular Biology 105 (2007) 76–84

[17] O. Prost, G.L. Adessi, Estrone and dehydroepiandrosterone sulfatase activities in normal and pathological human endometrium biopsies, J. Clin. Endocrinol. Metab. 56 (4) (1983) 653–661. [18] T.R. Evans, M.G. Rowlands, Y.A. Luqmani, S.K. Chander, R.C. Coombes, Detection of breast cancer-associated estrone sulfatase in breast cancer biopsies and cell lines using polymerase chain reaction, J. Steroid Biochem. Mol. Biol. 46 (2) (1993) 195–201. [19] Y. Miki, T. Nakata, T. Suzuki, A.D. Darnel, T. Moriya, C. Kaneko, K. Hidaka, Y. Shiotsu, H. Kusaka, H. Sasano, Systemic distribution of steroid sulfatase and estrogen sulfotransferase in human adult and fetal tissues, J. Clin. Endocrinol. Metab. 87 (2002) 5760–5768. [20] X.M. Li, E.S. Alperin, E. Salido, Y. Gong, P. Yen, L.J. Shapiro, Characterization of the promoter region of human steroid sulfatase: a gene which escapes X inactivation, Somat. Cell Mol. Genet. 22 (2) (1996) 105–117. [21] S. Stamm, S. Ben-Ari, I. Rafalska, Y. Tang, Z. Zhang, D. Toiber, T.A. Thanaraj, H. Soreq, Function of alternative splicing, Gene 344 (2005) 1–20. [22] S.F. Altschul, T.L. Madden, A.A. Sch`eaffer, J. Zhang, Z. Zhang, W. Miller, D.J. Lipman, A. Nardi, Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, Nucleic Acids Res. 25 (17) (1997) 3389–3402. [23] A.A. Salamov, T. Nishikawa, M.B. Swindells, Assessing protein coding region integrity in cDNA sequencing projects, Bioinformatics 14 (1998) 384–390. [24] M. Kozak, Possible role of flanking nucleotides in recognition of the AUG initiator codon by eukaryotic ribosomes, Nucleic Acids Res. 9 (1981) 5233–5252. [25] H. Nielsen, S. Brunak, G. von Heijne, Machine learning approaches for the prediction of signal peptides and other protein sorting signals, Protein Eng. 12 (1999) 3–9. [26] M. Zuker, Mfold web server for nucleic acid folding and hybridization prediction, Nucleic Acids Res. (2003) 3406–3415. [27] V.B. Bajic, S.L. Tan, A. Chong, S. Tang, A. Strom, J.A. Gustafsson, C.Y. Lin, E.T. Liu, Dragon ERE Finder version 2: A tool for accurate detection and analysis of estrogen response elements in vertebrate genomes, Nucleic Acids Res. 31 (2003) 3605–3607. [28] J. Matthews, B. Wihlen, J. Thomsen, J.A. Gustafsson, Aryl hydrocarbon receptor-mediated transcription: ligand-dependent recruitment of estrogen receptor alpha to 2,3,7,8-tetrachlorodibenzo-p-dioxin-responsive promoters, Mol. Cell. Biol. 25 (2005) 5317–5328. [29] L.D. Valle, V. Toffolo, A. Nardi, C. Fiore, P. Bernante, R. Di Liddo, P.P. Parnigotto, L. Colombo, Tissue-specific transcriptional initiation and activity of steroid sulfatase complementing dehydroepiandrosterone sulfate uptake and intracrine steroid activations in human adipose tissue, J. Endocrinol. 190 (2006) 129–139. [30] M. Racchi, S. Govoni, S.B. Solerte, C.L. Galli, E. Corsini, Dehydroepiandrosterone and the relationship with aging and memory: a

[31] [32]

[33]

[34]

[35]

[36] [37]

[38] [39] [40]

[41]

[42]

[43]

[44]

possible link with protein kinase C functional machinery, Brain Res. Rev. 37 (2001) 287–293. T.A. Ayoubi, W.J. Van De Ven, Regulation of gene expression by alternative promoters, FASEB J. 10 (1996) 453–460. M. Fan, H. Nakshatri, K.P. Nephew, Inhibiting proteasomal proteolysis sustains estrogen receptor-alpha activation, Mol. Endocrinol. 18 (2004) 2603–2615. F.V. Peale Jr., L.B. Ludwig, S. Zain, R. Hilf, R.A. Bambara, Properties of a high-affinity DNA binding site for estrogen receptor, Proc. Natl. Acad. Sci. U.S.A. 85 (1988) 1038–1042. S. Kato, L. Tora, J. Yamauchi, S. Masushige, M. Bellard, P. Chambon, A far upstream estrogen response element of the ovalbumin gene contains several half-palindromic 5 -TGACC-3 ; motifs acting synergistically, Cell 68 (1992) 731–742. V.R. Lingappa, D.T. Rutkowski, R.S. Hegde, O.S. Andersen, Conformational control through translocational regulation: a new view of secretory and membrane protein folding, Bioessays 24 (8) (2002) 741–748. K.M. Neugebauer, On the importance of being co-transcriptional, J. Cell Sci. 115 (Pt 20) (2002) 3865–3871. D.T. Rutkowski, C.M. Ott, J.R. Polansky, V.R. Lingappa, Signal sequences initiate the pathway of maturation in the endoplasmic reticulum lumen, J. Biol. Chem. 278 (32) (2003) 30365–30372. M. Kozak, An analysis of vertebrate mRNA sequences: intimations of translational control, J. Cell Biol. 115 (1991) 887–903. M. Iacono, F. Mignone, G. Pesole, uAUG and uORFs in human and rodent 5 untranslated mRNAs, Gene 349 (2005) 97–105. E.E. Winter, L. Goodstadt, C.P. Ponting, Elevated rates of protein secretion, evolution, and disease among tissue-specific genes, Genome Res. 14 (2004) 54–61. W.E. Rainey, K.S. Rehman, B.R. Carr, The human fetal adrenal: making adrenal androgens for placental estrogens, Semin. Reprod. Med. 22 (2004) 327–336. G.S. Chetrite, J. Cortes-Prieto, J.C. Philippe, F. Wright, J.R. Pasqualini, Comparison of estrogen concentrations, estrone sulfatase and aromatase activities in normal, and in cancerous, human breast tissues, J. Steroid Biochem. Mol. Biol. 72 (2000) 23–27. H. Ge, Z. Liu, G.M. Church, M. Vidal, Correlation between transcriptome and interactome mapping data from Saccharomyces cerevisiae, Nat. Genet. 29 (2001) 482–486. A. Ciechanover, J.A. DiGiuseppe, B. Bercovich, A. Orian, J.D. Richter, A.L. Schwartz, G.M. Brodeur, Degradation of nuclear oncoproteins by the ubiquitin system in vitro, Proc. Natl. Acad. Sci. U.S.A. 88 (1991) 139–143; J.K. Tobacman, M. Hinkhouse, Z. Khalkhali-Ellis, Steroid sulfatase activity and expression in mammary myoepithelial cells, J. Steroid Biochem. Mol. Biol. 81 (2002) 65–68.