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Endocrinology |
1 Biocenter Oulu and Research Center for Molecular Endocrinology, WHO Collaborating Centre for Research on Reproductive Health, Oulu; and 2 Department of Pathology, University of Oulu, Oulu, Finland
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(ER-) and ß (ER-ß), progesterone receptor, Ki67, and c-erbB-2 expressions analyzed by immunohistochemical techniques and with the Tumor-Node-Metastasis classification, tumor grade, disease-free interval, and survival of the patients. Signals for 17HSD1 mRNA were detected in 16%, 17HSD2 in 25%, and 17HSD5 in 65% of the breast cancer specimens. No association between the 17HSD1, 17HSD2, and 17HSD5 expressions was detected. A significant association was observed between ER- and ER-ß (P = 0.02; odds ratio, 1.96) expressions. There was also a significant inverse association between ER- and 17HSD1 (P = 0.04; odds ratio, 0.53), as well as ER- and 17HSD5 (P = 0.001; odds ratio, 0.35). Patients with tumors expressing 17HSD1 mRNA or protein had significantly shorter overall and disease-free survival than the other patients (P = 0.0010 and 0.0134, log rank). The expression of 17HSD5 was significantly higher in breast tumor specimens than in normal tissue (P = 0.033; odds ratio, 5.56). The group with 17HSD5 overexpression had a worse prognosis than the other patients (P = 0.0146). ER- also associated with survival (P = 0.045). Cox multivariate analyses showed that 17HSD1 mRNA, tumor size, and ER- had independent prognostic significance. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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17ß-Hydroxysteroid dehydrogenase (17HSD) enzymes are important regulators of the physiologic activities of sex steroids by catalyzing the interconversion between 17-ketosteroids and 17ß-hydroxysteroids (8)
. Thus far, nine isoforms of the 17HSD enzymes have been identified in humans. 17HSD1 is essential for the production of active estradiol from estrone (9)
, whereas 17HSD2 catalyzes the oxidation of estradiol to less active estrone. 17HSD2 also catalyzes the metabolism of androgens (testosterone to androstenedione) and the activation of 20-hydroxyprogesterone to progesterone (10
, 11)
.
Both 17HSD1 and 17HSD2 are expressed in the epithelium of normal breast tissue of premenopausal women (12)
, and oxidative activity seems to be the dominant form in nontumorous cells (12
, 13)
. Breast cancer cell lines have been shown to express 17HSD1, 17HSD2, or both enzymes (14)
. 17HSD5, a member of the aldo-keto reductase superfamily, is a reductive 17HSD present in normal breast (15)
. Recent studies have shown that progesterone and prostaglandin D2 are also substrates of 17HSD type 5 (15, 16, 17)
. In addition, 17HSD5 has some activity as a reductase of 3-keto and 17-keto and as an oxidase of 3- and 17ß-hydroxysteroids (15)
.
In this study, we investigated the expressions of the mRNA and protein for 17HSD1 and the mRNA for the 17HSD2 and 17HSD5 enzymes and correlated those with estrogen receptor (ER)-, ER-ß, progesterone receptor (PR), Ki67, and c-erbB-2 expressions, with Tumor-Node-Metastasis classification and grade of the tumors, and with disease-free interval and survival of the patients to ascertain the role of these enzymes in breast cancer progression.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The Tumor-Node-Metastasis status of 443 samples was known. There were 137 T1, 218 T2, 56 T3, and 32 T4 lesions. A total of 216 cases was axillary node positive, and distant metastases were present in 27 cases. Data on menopausal status were not available because we used archival specimens. However, by using the WHO criteria (19)
, we classified the patients according to their menopausal status. Patient ages 
50 years were considered premenopausal. A total of 36.1% of the patients was premenopausal and 63.9% postmenopausal.
In situ Hybridization.
In situ hybridization reactions were performed according to a standard procedure (20)
. Probes for 17HSDs were prepared from a 376-bp fragment (nucleotides 1–376) of human 17HSD1 cDNA (21)
, a 380-bp fragment (nucleotides 191–570) of human 17HSD2 cDNA (10)
, and a 594-bp fragment (nucleotides 407-1000) of human 17HSD5 cDNA (16)
cloned into pGEM-4Z (Promega, Madison, WI) or pCRII-TOPO (Invitrogen, Carlsbad, CA) plasmids and used as templates for in vitro transcription. The transcription of sense and antisense [-35S]CTP-labeled (1300 Ci/mmol; DuPont NEN, Boston, MA) RNA probes from linearized plasmids was according to the riboprobe in vitro transcription system (Promega), using T7 or SP6 RNA polymerases.
The in situ hybridization results were evaluated semiquantitatively by dividing the signal intensity into four categories: –, no signal present; +, weak signal; ++, moderate signal; and +++, strong signal. The results were evaluated by two independent researchers.
Immunohistochemistry.
The antiserum against human 17HSD1 has been described previously (22)
. The ER-, PR, and c-erbB-2 antibodies were from Novocastra Laboratories (Newcastle upon-Tyne, United Kingdom), the ER-ß antibody from Affinity Bioreagents (Golden, CO), and the Ki67 antibody from Zymed Laboratories (San Francisco, CA). The immunochemical stainings were made according to the manufactures’ instructions.
In the immunoreactivity scores, the labeling index was determined as follows: 0 to 20%, negative; 20 to 40%, slightly positive; 40 to 60%, moderately positive; and >60%, strongly positive. The Ki67 scores of the labeling index were classified as follows: <5%, negative; 5 to 15%, slightly positive; 15 to 30%, moderately positive; and >30%, strongly positive.
Statistical Analyses.
Categorical variables were analyzed by 
2 and Fisher’s exact tests, and odds ratio was also calculated. Comparison of in situ hybridization and immunohistochemistry was made using kappa statistics. Analysis of survival was performed using the Kaplan-Meier method, and differences between survival curves were examined for significance using the log-rank test. Multivariable analyses were performed with the Cox regression model to determine the independent prognostic value of variables. P of <0.05 was considered statistically significant in all cases.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Both 17HSD1 and 17HSD2 mRNAs were detected in normal breast tissue of premenopausal women (Fig. 1)
. The mRNA were localized in the ductal or lobular epithelial cells. No expression of 17HSD1 or 17HSD2 mRNA was observed in the normal tissue specimens from postmenopausal women (data not shown). Fig. 2
shows 17HSD1 and 17HSD2 mRNA expression in malignant breast lesions. Variable expression patterns for 17HSD1 and 17HSD2 mRNA in the epithelial cells of different lesions were observed. Stromal cells were devoid of expression of these enzymes. No significant differences were observed for the 17HSD1 enzyme in malignant tissue between the pre- and postmenopausal groups. In contrast, the number of cases showing signals for 17HSD2 mRNA was higher in premenopausal than postmenopausal patients (P < 0.01; odds ratio, 1.51).
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= 0.571). The number of 17HSD1-positive breast cancer specimens was 20% in the immunohistochemical analysis and 16% in in situ hybridization (Table 1)
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No association was found between 17HSD1, 17HSD2, and 17HSD5 mRNA expression and tumor grade 1 to 2. 17HSD1 mRNA expression did not associate with T (P = 0.37), N (P = 0.42), or M (P = 0.99) status. Similar observations were made for 17HSD2 mRNA and T (P = 0.12), N (P = 0.41), and M (P = 0.72) status. The overexpression of 17HSD5 was associated with N (P = 0.012; odds ratio, 2.86) but not with T or M status.
A significant association was observed between ER- and ER-ß (P = 0.02; odds ratio, 1.96) expression. There was also a significant inverse association between ER- and 17HSD1 (P = 0.04; odds ratio, 0.53), as well as ER- and 17HSD5 (P = 0.001; odds ratio, 0.35) but not with 17HSD2 (P = 0.92). ER-ß did not associate with 17HSD1, 17HSD2, or 17HSD5 mRNA (P = 0.70, P = 0.96 and 0.07) or with the T (P = 0.66) or N (P = 0.13) status of the tumors. However, tumors with low ER-ß expression showed significantly more metastatic growth (P = 0.0006; odds ratio, 6.26). Tumors expressing 17HSD1, 17HSD2, and/or 17HSD5 mRNA did not associate with the expression of Ki67 (P = 0.88, P = 0.24 and 0.69) or with c-erb-b2 status (P = 0.28, P = 0.97 and 0.52).
Our study also revealed that patients with tumors expressing 17HSD1 mRNA had significantly worse survival (P = 0.0010, log rank) and a shorter disease-free interval (P = 0.0134) than all other cases (Fig. 4)
. No such association was found with tumors expressing 17HSD2 mRNA (survival, P = 0.339; disease-free interval, P = 0.37). The group with 17HSD5 overexpression had a worse survival than the groups with lower or no expression (P = 0.0146). Patients with ER-–positive breast cancer had better survival than those without (P = 0.045). Multivariate Cox analysis (forward stepwise regression) was used to determine the possible independent prognostic significance of the following parameters: tumor size, the presence of nodal and distant metastases, grade of the tumor, ER-, ER-ß, PR, 17HSD1, 17HSD2, 17HSD5, Ki67, and c-erb-b2. Because nodal status and presence of metastasis showed association with tumor size, they were not included in the model. According to the analysis, tumor size, 17HSD1, and ER- had independent prognostic value (Table 2)
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study, we showed that mRNA for the 17HSD1 and 17HSD2 enzymes are present in the epithelial cells of normal breast tissues of premenopausal women but not in postmenopausal women, which is in agreement with the previous study of Miettinen et al. (12) , demonstrating constant expression of 17HSD1 and 17HSD2 mRNA in the breast throughout the menstrual cycle. In breast cancer tissues, the enzymes were expressed in a significant portion of specimens from both premenopausal and postmenopausal patients. In benign breast tissue and breast cancer, variable amounts of 17HSD1 and 17HSD2 enzyme protein have been reported (25, 26, 27, 28, 29) . Although we detected 17HSD1 mRNA in about one fifth of our specimens, some other studies reported the presence of 17HSD1 enzyme protein in approximately half of the specimens. Recently, Gunnarsson et al. (29) , using reverse transcription-PCR, detected 17HSD1 in all of their 84 archival breast cancer specimens studied. The reason for the discrepancies is not known, but it may be partially explained by the different methodologies used and by the relatively small patient series in most previous studies. In our series, 25% of cancer specimens were positive for 17HSD2. Suzuki et al. (27) did not detect 17HSD2 enzyme in the specimens by immunohistochemistry but demonstrated both 17HSD1 and 17HSD2 enzyme protein in a fraction of the breast cancer specimens in another study (28) . Gunnarsson et al. (29) , using reverse transcription-PCR, detected 17HSD2 in 31% of specimens. In normal breast epithelium of premenopausal women, 17HSD2 may have a protective role against estrogen influence.
The expression of 17HSD5 in breast tumor specimens was significantly higher than that in normal breast tissue. Taken together, 65% of breast cancer specimens were positive for 17HSD5 expression. 17HSD5 has a broad tissue distribution, and in in vitro assays, it recognizes several different substrates (15
, 17)
. In the prostate, 17HSD5 catalyzes the formation of testosterone and inactivation of dihydrotestosterone (30)
. Human 17HSD5 also has a high 20-HSD activity that inactivates progesterone (31)
. In myeloid leukemia cell lines, this enzyme possesses marked 11-ketoreductase activity, converting prostaglandin D2 to prostaglandin F2 and functions to regulate cell differentiation (17)
. Thus far, little is known about the role of 17HSD5 in breast tissue. In our study, overexpression of 17HSD5 associated with positive nodal status and that the patient group with overexpression had a worse prognosis than other patients. The activities of 17HSD5 suggest that it might be involved in the production of progesterone and sex steroids in the breast.
Accumulation of estradiol in breast cancer tissue has been detected in postmenopausal women and may result from higher aromatase, steroid sulfatase, and reductive 17HSD activity. A recent study (32) showed that the estradiol/estrone ratio and the expression of 17HSD1, but not that of aromatase or sulfatase, are higher in breast cancer tissues of postmenopausal compared with premenopausal patients. This suggests that 17HSD1 is mainly responsible for the accumulation of estradiol in breast cancer tissue.
Breast cancer is a multifactorial disease. A number of biological parameters appear to strongly influence tumor behavior and have been tested as prognostic parameters in patients with breast cancer (33)
. Our study revealed that patients with tumors expressing 17HSD1 mRNA had shorter disease-free and overall survival than the other cases in both ER-positive and -negative patients, which indicates that 17HSD1 mRNA is a prognostic marker in breast cancer progression regardless of the ER status. A previous study showed that a high level of 17HSD1 correlated with an increased risk to develop late relapse of breast cancer (29)
in ER-positive breast cancer patients. In a recent study, Gunnarsson et al. (34)
found amplification of the HSD17B1 gene in 
15% postmenopausal breast cancer patients and concluded that amplification of HSD17B1 might be an indicator of adverse prognosis among ER-positive patients. Our present study using large number of breast cancer patients clearly demonstrates that 17HSD1 is an independent prognostic factor in breast cancer in both pre- and postmenopausal patients.
ER and PR measurements from breast cancer specimens have been routinely used to estimate patient prognosis and select optimal treatment therapies because a majority of ER-positive breast cancers respond to endocrine treatment (e.g., ref. 35
). Estrogens exert their effect through two members of the nuclear receptor superfamily, ER- and ER-ß. It has been shown that ER-–positive breast cancer cells proliferate in response to estradiol (24)
. However, it appears that, in the mammary gland, the proliferating cells are not ones that express ER- because Ki67 is not found in cells expressing ER- (36)
. In the human breast, the function of ER-ß is even less well understood than that of ER-. One reason for this is the presence of variant isoforms, the relative expressions of which have been suggested to change during cancer progression in the breast (37)
. Our study showed that ER- is an independent prognostic factor in breast cancer, which is in line with previous reports (e.g., ref. 38
). Moreover, we found negative correlations between 17HSD1 and ER- and 17HSD5 and ER, respectively.
A significant correlation between ER- and ER-ß status was detected. This is in agreement with the suggested coexpression of ER- and ER-ß in most breast cancers (39
, 40)
. There is evidence that the ER-ß acts as a physiologic regulator of ER- (41)
and thereby decreases the invasion of breast cancer cells. This is in line with our finding showing more metastasis in tumors with low ER-ß.
In normal developing mammary gland, c-erbB-2 has been suggested to have an important role in the regulation of cell growth and differentiation. Data from clinical trials have shown that its overexpression is associated with a poor outcome, including shorter disease-free and overall survival (42) . Overexpression of c-erbB-2 is characterized by a loss of estrogen sensitivity of tumor cells and aggressive tumor growth, leading to the development of resistance against a number of therapeutics (34 , 42) . Gunnarsson et al. (34) detected a correlation between HSD17B1 and ERBB2 gene amplification in breast cancer. No correlation between 17HSDs and c-erbB-2 expression was detected in our study.
In summary, our results demonstrate that 17HSD1 is an independent prognostic factor in breast cancer. Because high 17HSD1 expression is associated with a poor prognosis, it is clear that inhibition of this enzyme could be a beneficial therapy for breast cancer patients selected based on the expression of 17HSD1 in tissue specimens. Patient group with 17HSD5 overexpression had a worse prognosis than the groups with low or no expression of 17HSD5, but 17HSD5 was not an independent prognostic factor in breast cancer. However, overexpression of 17HSD5 was associated with nodal status.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Pirkko T. Vihko, Research Center for Molecular Endocrinology, WHO Collaborating Centre, P. O. Box 5000, FI-90014 University of Oulu, Oulu, Finland. Phone: 358-40-5431734; Fax: 358-8-3155631; E-mail: pvihko{at}whoccr.oulu.fi
Received 2/10/04. Revised 8/ 3/04. Accepted 8/16/04.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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