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Department of Biomedicine and Surgery, Division of Oncology, Linköping University, S-581 85 Linköping, Sweden
(Requests for offprints should be addressed to A Jansson; Email: agnja{at}ibk.liu.se)
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Abstract |
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 Top Abstract IntroductionMaterial and methodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterial and methodsResultsDiscussionReferences |
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and ERß. Oestrogen binding to ER induces cell proliferation whereas binding to ERß inhibits proliferation (Paruthiyil et al. 2004, Strom et al. 2004). Various studies have shown decreased expression of ERß in tumour versus normal tissue in the breast (Shaw et al. 2002, Roger et al. 2001). The primary source of oestrogen in premenopausal women is the ovary but, after menopause, oestrogen biosynthesis in peripheral tissue has a major role. Oestrone sulphatase and aromatase are important enzymes for the local synthesis of oestradiol. An enzyme group that affects the availability of active oestrogens is the 17ß-hydroxysteroid dehydrogenase (17HSD) family (Pasqualini 2004). It is well known that 17HSD activity is responsible for the balance between oestrone and oestradiol in breast tissue. The 17HSD family has 13 known members and several of them have been identified to be of importance in different hormone dependent tissues and tumours. There are multiple family members expressed in breast tissue (Speirs et al. 1998, Peltoketo et al. 1999, Luu-Thee et al. 2005) even though 17HSD type 1 and 2 seem to be the principal 17HSD enzymes involved in breast cancer thus far (Labrie et al. 1997, Miettinen et al. 1999). 17HSD type 1 catalyses reduction of oestrone to oestradiol with NADP(H) as a cofactor, and 17HSD type 2 catalyses oxidation from oestradiol to oestrone with NAD(H) as a cofactor (Vihko et al. 2001).
17HSD type 2 is the dominant form in normal epithelium of the breast, and protects the epithelial cells to balance the amount of oestrone against oestradiol (Speirs et al. 1998). In breast cancer, 17HSD type 1 and type 2 have been mostly investigated and high expression of 17HSD type 1 and low expression of 17HSD type 2 are associated with decreased survival in ER positive breast cancer (Gunnarsson et al. 2001, 2005, Oduwole et al. 2004). The question whether 17HSD type 1 or type 2 is of greatest importance in tumour development is still not clear. Oxidative 17HSD type 2 activity leading to the conversion of oestradiol to oestrone has been detected to be 50 times more predominant than the reductive 17HSD type 1 activity (Vihko et al. 2001). However, Miettinen et al.(1996) suggested that, in cultured cells, 17HSD type 1 has a higher catalytic efficacy than the 17HSD type 2 enzyme.
To address this question, the aim of this study was to investigate how the loss of 17HSD type 2 expression in non-tumour breast epithelial cells and gain of 17HSD type 2 expression in breast cancer-derived cell lines affect oestradiol conversion and proliferation rate. We further investigated how this was influenced by the endogenous expression of 17HSD type 1, ER and ERß in the examined cell lines.
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Material and methods |
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TopAbstractIntroductionMaterial and methodsResultsDiscussionReferences |
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T47D and MCF7 breast cancer epithelial cells (American Type Culture Collection, Manassas, VA, USA) were cultured in phenol-red free Opti-Mem I (Invitrogen, Carlsbad, CA, USA), supplemented with 4% fetal bovine serum (FBS; Invitrogen) and incubated at 37 °C, 5% CO2. In all experiments, charcoal/dextran-treated serum (CTS; HyClone, UT, USA) was used to control the levels of oestradiol. The experiments with T47D were performed between passages 100 and 104 and MCF7 between passages 151 and 157.
The non-tumour breast epithelial cells MCF10A (American Type Culture Collection) and Human Mammal Epithelial Cells (HMEC; Cambrex, Walkersville, MD, USA) were cultured in serum free Mammary Epithelial Growth Medium (MEGM; Cambrex) supplemented with 100 ng/ml cholera toxin (Sigma-Aldrich), and incubated at 37 °C, 5% CO2. The experiments with MCF10A were performed between passages 99 and 101 and HMEC between passages 9 and 11.
Cloning and transient transfection of 17HSD type 2
Synthesis of cDNA from human mammary gland RNA was performed using the 1st strand cDNA Synthesis kit with random hexameres (Roche Diagnostics Corporation, Indianapolis, IN, USA). To generate double-stranded 17HSD type 2 cDNA, a pair of primers 5'GAAGTTATCAGTCGACTGAAGGTGCAGCAA-GTCAC 3' and 5'ATGGTCTAGAAAGCTTAT-TGCCTAGGTGGCCTTTTT 3' containing SalI and HindIII cleavage sites were used in a PCR with human mammary gland cDNA. The amplified PCR fragment was cloned into pDNR-Dual Donor Vector (BD Biosciences Clontech, Mountain View, CA) with BD In-Fusion Enzyme. The 17HSD type 2 gene was subcloned to pLP-IRES2-EGFP Acceptor vector (BD Biosciences Clontech) using Cre recombinase. The plasmids containing the 17HSD type 2 cDNA were sequenced with MegaBACE 500 DNA analysis Systems (Amersham Biosciences) sequencing equipment according to the manufacturer’s protocol.
For transfection with 17HSD type 2, 30 000/well T47D cells and 60 000/well MCF7 cells were seeded in 12 well plates in 1 ml/well in culture medium. Twenty-four hours after seeding, the cells were transfected by FuGENE 6 transfection (Roche Diagnostics Corporation) by adding in each well 0.04 ml containing 0.4 µg plasmid, 1.2 µl FuGENE 6 transfection reagent and Opti-Mem I (Invitrogen). Twenty-four hours after transfection, the cells were washed with PBS and incubated in culture medium before harvest. Cells transfected with an empty vector were used as control.
siRNA
Two 19-nucleotide specific sequences were selected in the coding region of HSD17B2 to generate 21-nucleotide sense and anti-sense strands. The sequences were submitted to BLAST (Basic Local Alignment Search Tool; http://www.ncbi.nlm.nih.gov/BLAST) to ensure the specificity of the siRNA to the targeted sequence. The specific target sequence for the two HSD17B2 siRNA are as follows: 5'AAAGAUGC UUACAGCAAGGUU3'; 5'AAGGCGCUUACUUGU GGAUCU3'. For transfection of the siRNA duplexes, 44 000 cells/well of MCF10A and HMEC were seeded in 12 well plates in 1 ml/well in culture medium. Twenty four hours after seeding, the cells were transfected by Lipofectamine 2000 (Invitrogen) by adding in each well 0.2 ml containing 0.5 µg siRNA, 2 µl Lipofectamine 2000 and Opti-Mem I (Invitrogen). Twenty four hours after transfection, the cells were washed with PBS and incubated in culture medium before harvest. One duplex, which does not recognise any sequence in the human genome, was used as negative control and one duplex against ß-actin (Qiagen) was used as positive control in all experiments.
Real-time PCR
RNA was extracted from the siRNA-treated HMEC and MCF10A cells and controls with unspecific siRNA, and 17HSD type 2-transfected T47D and MCF7 cells and controls with an empty vector, 24, 48 and 72 h after siRNA treatment or transfection using SV Total RNA Isolation System (Promega). The RNA was stored at –70 °C. Synthesis of cDNA from total RNA was performed using the 1st strand cDNA Synthesis kit with random hexameres (Roche Diagnostics Corporation). mRNA expression of 17HSD type 1 and 17HSD type 2 was analysed according to Gunnarsson et al.(2001). Further, mRNA levels of 17HSD type 1, 17HSD type 2, ER and ERß were analysed in untreated HMEC, MCF10A, T47D and MCF7 cells. ER and ERß were analysed employing Assay on demand no Hs00174860 and no Hs00230957 respectively (Applied Biosystems, Warrington, UK) according to the manufacturer’s description. ß-Actin was used as endogenous reference gene except for in the positive controls of the siRNA when phosphogly-cerate kinase was used. The assays were purchased from Applied Biosystems. Standard curves for all analysed genes were run on each plate, using serial diluted cDNA to normalise the runs. The data obtained from ß-actin were used to normalise the sample variation in the amount of input cDNA. All samples were run as triplicates and in all experiments samples without template were used as control. The relative quantification of 17HSD type 1, 17HSD type 2, ER and ERß were performed according to the manufacturer’s description (Protocol P/N 4303859, Perkin Elmer, Foster City, CA, USA).
Measurement of oestradiol levels
The concentration of oestradiol in the medium was measured in 17HSD type 2 siRNA-transfected and control HMEC and MCF10A cells, and 17HSD type 2 transfected and control T47D and MCF7 cells. The medium was changed to a medium containing 10–9 M oestradiol at 48 and 72 h after siRNA treatment or transfection and thereafter incubated at 37 °C with 5% CO2 for 1, 4, 8 and 24 h, and finally frozen in liquid nitrogen. The concentration of oestradiol was measured using electrochemiluminescence immunoassay (ELICA) employing the Elecsys Estradiol II reagent kit (Roche Diagnostics Corporation) analysed with Roche Elecys 1010/2010.
S-Phase measurement with flow cytometry
The proportions of cells in S phase were analysed using DNA flow cytometry according to Vindelöv et al.(1983). The S-phase fraction was analysed in 17HSD type 2 siRNA-transfected and control HMEC and MCF10A cells, and in 17HSD type 2-transfected and control T47D and MCF7 cells, with or without treatment with 10–9 M oestradiol in the medium. The medium was changed at 24 and 48 h after transfection and the cells were harvested at 48 and 72 h after transfection. Ten thousand cells were analysed in each sample using a Becton Dickinson FACSCalibur (Becton Dickinson, San José, CA, USA) and ModFit LT software (Verity Software House Inc., Topsham, ME, USA).
Statistical evaluation
Student’s t-test was used for a comparison between the groups. The results are expressed as ± S.E. of the mean values obtained from three replicates repeated three times. All P-values are two sided, and P < 0.05 was considered to be statistically significant. All the procedures were comprised in the statistical package Statistica 6.0 (StatSoft Scandinavia AB, Uppsala Sweden).
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Results |
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and ERß
Relative quantities of mRNA expression to the endogenous reference of 17HSD type 1, 17HSD type 2, ER and ERß were analysed in untreated HMEC, MCF10A, T47D and MCF7 cells. MCF10A and T47D cells expressed high levels of 17HSD type 1 (Fig. 1A
); MCF10A expressed high levels of 17HSD type 2 and HMEC and T47D intermediate levels (Fig 1B); MCF7 expressed high levels of ER, T47D intermediate levels and HMEC low levels of ER (Fig. 1C); HMEC and T47D expressed high levels of ERß (Fig 1D).
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MCF7 and T47D were transfected with pLP-IRES2-EGFP-HSD17B2 and a control vector, and MCF10A and HMEC were transfected with 17HSD type 2 siRNA and a control duplex. The mRNA expression was analysed at 24, 48 and 72 h after transfection. The levels of 17HSD type 2 increased significantly compared to the controls in MCF7 and T47D, and decreased significantly compared to the controls in MCF10A and HMEC (Table 1). The differences between the groups were greatest at 48 h after treatment. The levels of ß-actin in the positive control samples decreased by 60%, 75% and 55% after 24, 48 and 72 h respectively.
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The concentration of oestradiol levels was measured in samples treated with 10–9 M oestradiol, 48 and 72 h after 17HSD type 2 siRNA transfection or 17HSD type 2 transfection and thereafter incubated at 1, 4, 8 and 24 h. The concentration of oestradiol decreased significantly more in the control compared to the 17HSD type 2 siRNA-transfected HMEC cells after 48 (Fig. 2A) and 72 h (Fig. 2B). There were no differences in oestradiol concentration in the medium from the MCF10A cells neither at 48 (Fig. 2C) nor at 72 h (Fig. 2D). The concentration of oestradiol decreased significantly in 17HSD type 2-transfected MCF7 cells compared with the control after 48 (Fig. 2E) and 72 h (Fig 2F). There were no differences in oestradiol concentration in the medium from the T47D cells at 48 (Fig 2G) or at 72 h (Fig 2H).
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Characterisation of proliferative responses
The S-phase fraction significantly decreased in HMEC cells transfected with 17HSD type 2 siRNA and treated with oestradiol, compared with the control, 48 h after siRNA transfection. This was not detected in the samples that were not treated with oestradiol. There were no significant changes after 72 h (Fig. 3A). The MCF10A cells did not show any significant difference in S-phase fraction between any of the groups at 48 or 72 h (Fig. 3B).
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). There were no differences between any of the groups in the T47D cells at 48 or at 72 h (Fig. 3D). |
Discussion |
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The oxidative pathway (oestradiol to oestrone) has been found to be predominant in normal breast epithelial cells (Speirs et al. 1998). In the non-tumour breast epithelial cells employed in this study, HMEC expressed low endogenous amounts of both 17HSD type 1 and 17HSD type 2, even though the expression of 17HSD type 1 was higher than 17HSD type 2. In the medium from the control cells of HMEC, the oestradiol concentration decreased rapidly in the first 8 h of incubation. When the 17HSD type 2 expression was decreased by siRNA, HMEC showed significantly decreased expression of oestradiol in the controls compared to the treated samples. The MCF10A expressed high endogenous amounts of both 17HSD type 1 and 17HSD type 2, and the expression of 17HSD type 2 was higher than 17HSD type 1. In the medium from the control cells of MCF10A, the oestradiol concentration decreased quickly, but the oestradiol concentration did not differ in the controls compared to the treated samples. The steep decrease in oestradiol levels in the control samples from HMEC contradicts the results from the other cell lines. However, the expression of 17HSD type 1 is low in HMEC. There may also be other members of the 17HSD family that are expressed in HMEC that work in the same direction as 17HSD type 2 and convert oestradiol to oestrone, such as 17HSD type 13. The steep decrease in oestradiol level was detected in the first 8 h of incubation in both the control and treated HMEC and MCF10A cells, but not in MCF7 and T47D. This may be a result of oestrogen sulphotransferase (EST) expression in the non-tumour breast epithelial cells that sulphonates the oestradiol. Suzuki et al.(2003) showed that there is high expression of EST in normal human mammary epithelial cells. Steroid sulphatase (STS) hydrolyses oestrone sulphate to oestrone and in MCF7 and T47D Chetrite et al. (1999a) found the activity of both EST and STS. The STS activity in the tumour cells examined may neutralise the EST activity. Ishibashi et al. (2005) found that oestradiol concentrations in human thymoma were inversely correlated to EST and positively correlated with STS and 17HSD type 1. It is likely that the same pattern can be detected in breast cancer and that 17HSD type 2 can be correlated to high expression of EST. The balance of 17HSD enzymes may be more important in breast cancer cells with both high EST and STS, than in the cells with the same expression pattern as in the thymoma, both in vitro and in vivo.
There were significant differences in the medium from the siRNA treated HMEC cells, but not from the MCF10A cells. This may be a result of more efficient siRNA transfection in the HMEC cells, but also that MCF10A has a higher endogenous expression of 17HSD type 1 than HMEC. Our results suggest that 17HSD type 1 is more dominant than 17HSD type 2, and that there must be excessive amounts of 17HSD type 2 to inhibit the effect of 17HSD type 1. Taken together, the results on oestradiol levels in the medium from MCF7, T47D, HMEC and MCF10A show that 17HSD type 2 is important to maintain oestrone–oestradiol balance in the cells and that factors, such as other 17HSD members can interfere with this balance. Even though changes of oestradiol levels in the medium are moderate following transfection, the influence of 17HSD type 1 and 17HSD type 2 on proliferation of ER positive tumours in vivo may have larger effects in the long term for the patients. Progestin is administrated in contraceptives and, to prevent climacteric symptoms, has been shown to interfere with the regulation of enzymes involved in oestrogen formation, including 17HSD type 1 and 17HSD type 2 expression. In T47D cells, progestin induces 17HSD type 1 expression (Poutanen et al. 1990) and breast tumours from postmenopausal women who received lynestrenol showed higher 17HSD type 1 expression than tumours from untreated patients (Fournier et al. 1985). In contrast, medrogestrone significantly decreases 17HSD type 1 activity in both MCF7 and T47D cells in a dose-dependent manner (Chetrite et al. 1999b) and in the endometrium of oestrogen-dependent benign disease, progestin induced 17HSD type 2 expression (Kitawaki et al. 2000). It would be interesting to investigate how the use of progestins correlates with the expression pattern of 17HSDs in breast cancer patients.
Previous in vitro studies investigating the role of 17HSD type 2 have investigated the conversion of sex steroids (Miettinen et al. 1996, Härkönen et al. 2003, Liu et al. 2005) without studying the effect on proliferation. We found that the fraction of cells in S-phase in the 17HSD type 2-transfected MCF7, as well as in the 17HSD type 2 siRNA-transfected HMEC cells significantly decreased compared to the controls. No differences were detected in the T47D cells and the MCF10A cells. The level of ER is measured in breast cancer for the stratification of treatment, and about 70% of breast cancers show high expression. MCF7 and T47D are cell lines with high ER expression and can be used as models for tumours with high ER expression. Our findings indicate that tumours with high ER expression, high 17HSD type 1 expression and low 17HSD type 2 expression are exposed to high amounts of oestradiol and have a high proliferation rate. These results are in line with our previous findings on ER positive breast cancer when high expression of 17HSD type 1 and low expression of 17HSD type 2 were associated with decreased survival (Gunnarsson et al. 2001, 2005). The ratio of ER/ERß mRNA tends to increase in tumours versus normal tissue, and prompts the mitogenic effect of oestradiol (Roger et al. 2001, Shaw et al. 2002). In the MCF7 cells, the ratio of ER/ERß was higher than in the T47D cells. The oestradiol levels decreased between 48 and 72 h after transfection in the MCF7 cells and, as a result, we found a decrease in S-phase fraction. During this time course, only small effects on the proliferation affected by these differences were possible to detect, even though during a longer time period such decreases may be of importance. The oestradiol levels did not decrease in the T47D cells after transfection. The T47D cells express 5 times more ERß than ER and therefore a slight decrease in proliferation might have been expected in both the transfected cells and the controls. The proliferation in the HMEC cells decreased after siRNA transfection compared to the control, even though the oestradiol levels increased. The HMEC cells express high levels of ERß and hardly detectable levels of ER and exposure of oestradiol might lead to a decrease in S-phase fraction. However, there were no differences in S-phase fraction in the control cells treated with or without oestradiol, which there should have been if ERß was the target of importance. Therefore, other mechanisms may be involved. The MCF10A cells showed no changes in oestradiol after siRNA transfection and are totally negative for both ER and ERß, and no differences in S-phase fraction were detected as expected.
In summary, this article examines the importance of 17HSD type 2 in breast cancer-derived cell lines and in non-tumour breast epithelial cells in relation to endogenous expression of 17HSD type 1, ER and ERß. We found that high or low expression levels of 17HSD type 2 affected the oestradiol concentration significantly; however, the response was dependent on the endogenous expression of 17HSD type 1. The proliferative changes induced by changes in oestradiol concentration were followed by ER and ERß expression. Expression of 17HSD type 1 seems to be dominant over 17HSD type 2. Therefore, it may be important to investigate a ratio between 17HSD type 1 and 17HSD type 2 instead of investigating them individually.
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Acknowledgements |
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Funding |
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This work was funded by the Swedish Cancer Society. There is no conflict of interest that would prejudice impartiality.
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References |
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