Expression of 17beta-hydroxysteroid dehydrogenase type 2 and type 5 in breast cancer and adjacent non-malignant tissue: a correlation to clinicopathological parameters

Estrogens play an important role in the development and progression of breast cancer. 17β-Hydroxysteroid dehydrogenase (17β-HSD) type 2 and type 5 are involved in sex steroid metabolism. 17β-HSD type 2 converts estradiol to estrone while 17β-HSD type 5 converts androstenedione to testosterone. Using immunocytochemistry, we have studied the expression of 17β-HSD type 2 and type 5 in 50 specimens of breast carcinoma and adjacent non-malignant tissues. The results were correlated with the estrogen receptor α (ERα) and β (ERβ), progesterone receptor A (PRA) and B (PRB), androgen receptor and CDC47 and with the tumor stage, tumor size, nodal status and menopausal status. 17β-HSD type 2 was expressed in 20% and 17β-HSD type 5 in 56% of breast cancer specimens. In adjacent normal tissues, both enzymes were highly expressed in almost all the patients. No significant association could be found between the expression of 17β-HSD type 2 and 17β-HSD type 5 and between the expression of each enzyme and the clinicopathological parameters studied. The decrease in 17β-HSD type 2 and 17β-HSD type 5 expressions in breast cancer may play a predominant role in the development and/or progression of the cancer by modifying the intratumoral levels of estrogens and androgens.     

Structure–function relationships in 3α-hydroxysteroid dehydrogenases: a comparison of the rat and human isoforms

3α-Hydroxysteroid dehydrogenases (3α-HSDs) inactivate steroid hormones in the liver, regulate 5α-dihydrotestosterone (5α-DHT) levels in the prostate, and form the neurosteroid, allopregnanolone in the CNS. Four human 3α-HSD isoforms exist and correspond to AKR1C1–AKR1C4 of the aldo-keto reductase (AKR) superfamily. Unlike the related rat 3α-HSD (AKR1C9) which is positional and stereospecific, the human enzymes display varying ratios of 3-, 17-, and 20-ketosteroid reductase activity as well as 3α-, 17β-, and 20α-hydroxysteroid oxidase activity. Their k_cat values are 50–100-fold lower than that observed for AKR1C9. Based on their product profiles and discrete tissue localization, the human enzymes may regulate the levels of active androgens, estrogens, and progestins in target tissues. The X-ray crystal structures of AKR1C9 and AKR1C2 (human type 3 3α-HSD, bile acid binding protein and peripheral 3α-HSD) reveal that the AKR1C2 structure can bind steroids backwards (D-ring in the A-ring position) and upside down (β-face inverted) relative to the position of a 3-ketosteroid in AKR1C9 and this may account for its functional plasticity. Stopped-flow studies on both enzymes indicate that the conformational changes associated with binding cofactor (the first ligand) are slow; they are similar in both enzymes but are not rate-determining. Instead the low k_cat seen in AKR1C2 (50-fold less than AKR1C9) may be due to substrate “wobble” at the plastic active site.     

Mapping of the HSD17B2 gene encoding type II 17β-hydroxysteroid dehydrogenase close to D16S422 on chromosome 16q24.1–q24.2

The enzymes of the 17β-hydroxysteroid dehydrogenase (17β-HSD) gene family are responsible for a key step in the formation and degradation of androgens and estrogens: catalyzing the interconversion of 17-ketosteroids and their active 17β-hydroxysteroid counterparts. The structure of human type II 17β-HSD cDNA was recently reported. This enzyme catalyzes the interconversion of Δ⁴-androstenedione and testosterone, androstanedione and dihydrotestosterone, and estrone and 17β-estradiol, whereas type I 17β-HSD catalyzes exclusively the interconversion of estrogens. To locate the HSD17B2 gene, the novel dinucleotide CA repeat sequence found 571 bp downstream from the end of exon 1 was genotyped into eight CEPH reference families by PCR. Two-point linkage analysis was performed between the latter polymorphism and the 2066 microsatellite markers of Généthon. The maximal pairwise lod score (Z_max = 33.3) with a maximal recombination fraction (θ_max) of 0.008 was obtained with the marker D16S422 located on 16q24.1–q24.2. To define further the localization of the HSD17B2 gene, we constructed a high-resolution genetic map of the region flanking the polymorphic HSD17B2 gene including eight Généthon markers. The order of the HSD17B2 gene and markers is qter-D16S516 — D16S504 — D16S507 — D16S505 — D16S511 — [HSD17B2—D16S422]—D16S520—D16S413—tel.     

Genetic variation in the HSD17B1 gene and risk of prostate cancer

Steroid hormones are believed to play an important role in prostate carcinogenesis, but epidemiological evidence linking prostate cancer and steroid hormone genes has been inconclusive, in part due to small sample sizes or incomplete characterization of genetic variation at the locus of interest. Here we report on the results of a comprehensive study of the association between HSD17B1 and prostate cancer by the Breast and Prostate Cancer Cohort Consortium, a large collaborative study. HSD17B1 encodes 17β-hydroxysteroid dehydrogenase 1, an enzyme that converts dihydroepiandrosterone to the testosterone precursor Δ5-androsterone-3β,17β-diol and converts estrone to estradiol. The Breast and Prostate Cancer Cohort Consortium researchers systematically characterized variation in HSD17B1 by targeted resequencing and dense genotyping; selected haplotype-tagging single nucleotide polymorphisms (htSNPs) that efficiently predict common variants in U.S. and European whites, Latinos, Japanese Americans, and Native Hawaiians; and genotyped these htSNPs in 8,290 prostate cancer cases and 9,367 study-, age-, and ethnicity-matched controls. We found no evidence that HSD17B1 htSNPs (including the nonsynonymous coding SNP S312G) or htSNP haplotypes were associated with risk of prostate cancer or tumor stage in the pooled multiethnic sample or in U.S. and European whites. Analyses stratified by age, body mass index, and family history of disease found no subgroup-specific associations between these HSD17B1 htSNPs and prostate cancer. We found significant evidence of heterogeneity in associations between HSD17B1 haplotypes and prostate cancer across ethnicity: one haplotype had a significant (p < 0.002) inverse association with risk of prostate cancer in Latinos and Japanese Americans but showed no evidence of association in African Americans, Native Hawaiians, or whites. However, the smaller numbers of Latinos and Japanese Americans in this study makes these subgroup analyses less reliable. These results suggest that the germline variants in HSD17B1 characterized by these htSNPs do not substantially influence the risk of prostate cancer in U.S. and European whites.     

Selective inhibition of 17β-hydroxysteroid dehydrogenase type 1 (17βHSD1) reduces estrogen responsive cell growth of T47-D breast cancer cells

The most potent estrogen estradiol (E2) plays a pivotal role in the initiation and progression of estrogen dependent diseases. 17β-Hydroxysteroid dehydrogenase type 1 (17βHSD1) catalyses the NADPH-dependent E2-formation from estrone (E1). It is often overexpressed in breast cancer and endometriosis. For this reason, inhibition of 17βHSD1 is a promising strategy for the treatment of these diseases. In the present paper, we investigate the estrogen responsive cell growth of T47-D breast cancer cells, the intracellular inhibitory activity of non-steroidal 17βHSD1-inhibitors and their effects on estrogen dependent cell growth in vitro. At equal concentrations the estrogens E1 and E2 induced the same extent of growth stimulation indicating fast intracellular conversion of E1 into E2. Application of inhibitors selectively prevented stimulation of proliferation evoked by E1-treatment whereas E2-mediated stimulation was not affected. Furthermore, intracellular E2-formation from E1 was significantly inhibited with IC₅₀-values in the nanomolar range. In conclusion, our findings strongly support suitability of non-steroidal 17βHSD1-inhibitors for the treatment of estrogen dependent diseases.     

Cloning, Expression, and Physical Mapping of the 3β-Hydroxysteroid Dehydrogenase Gene Cluster (HSD3BP1–HSD3BP5) in Human

Seven members of the human 3β-hydroxysteroid dehydrogenase (3β-HSD) gene family (HGMW-approved symbols HSD3BP1–HSD3BP5) have been cloned and physically mapped. HSD3B1 and 2 express 3β-HSD enzymes; HSD3Bψ1–5 are unprocessed pseudogenes that are closely related to HSD3B1 and 2 but contain no corresponding open reading frames. mRNA is expressed from ψ4 and ψ5 in several tissues, but with altered splice sites that disrupt reading frames. A 0.5-Mb contig of 3 yeast artificial chromosome and 32 bacterial artificial chromosome genomic clones contained no additional members of the gene family. The seven genes and pseudogenes mapped within 230 kb in the order HSD3Bψ5–ψ4–ψ3–HSD3B1–ψ1–ψ2–HSD3B2. HSD3B1 and 2 are in direct repeat, 100 kb apart. Six HSD3B2 mutations involve substitutions that are present in several of the pseudogenes. In four cases, mutations arose in CpG sites that are conserved within the gene cluster. The tendency for CpG sites to mutate by transition provides an adequate explanation for these HSD3B2 mutations, which are unlikely to be due to recombination or conversion within the gene family.     

Intracrinology of estrogens and androgens in breast carcinoma

Intratumoral metabolism and synthesis of biologically active steroids such as estradiol and 5-dihydrotestosterone as a result of interactions of various enzymes are considered to play very important roles in the pathogenesis and development of hormone-dependent breast carcinoma. Among these enzymes involved in estrogen metabolism, intratumoral aromatase play an important role in converting androgens to estrogens in situ from serum and serving as the source of estrogens, especially in postmenopausal patients with breast carcinoma. However, other enzymes such as 17β-hydroxysteroid dehydrogenase (17β-HSD) isozymes, estrogen sulfatase (STS), and estrogen sulfotransferase, which contribute to in situ availability of biologically active estrogens, also play pivotal roles in this intratumoral estrogen production above. Androgen action on human breast carcinoma has not been well-studied but are considered important not only in hormonal regulation but also other biological features of carcinoma cells. Intracrine mechanisms also play important roles in androgen actions on human breast carcinoma cells. Among the enzymes involved in biologically active androgen metabolism and/or synthesis, both 17β-hydroxysteroid dehydrogenase type 5 (17βHSD5; conversion from circulating androstenedione to testosterone) and 5-reductase (5Red; reduction of testosterone to DHT (5-dihydrotestosterone) were expressed in breast carcinoma tissues, and in situ production of DHT has been proposed in human breast cancer tissues. However, intracrine mechanisms of androgens as well as their biological or clinical significance in the patients with breast cancer have not been fully elucidated in contrast to those in estrogens.     

Aldo-keto reductases AKR1C1, AKR1C2 and AKR1C3 may enhance progesterone metabolism in ovarian endometriosis

Endometriosis is a very common disease that is characterized by increased formation of estradiol and disturbed progesterone action. This latter is usually explained by a lack of progesterone receptor B (PR-B) expression, while the role of pre-receptor metabolism of progesterone is not yet fully understood. In normal endometrium, progesterone is metabolized by reductive 20α-hydroxysteroid dehydrogenases (20α-HSDs), 3α/β-HSDs and 5α/β-reductases. The aldo-keto reductases 1C1 and 1C3 (AKR1C1 and AKR1C3) are the major reductive 20α-HSDs, while the oxidative reaction is catalyzed by 17β-HSD type 2 (HSD17B2). Also, 3α-HSD and 3β-HSD activities have been associated with the AKR1C isozymes. Additionally, 5α-reductase types 1 and 2 (SRD5A1, SRD5A2) and 5β-reductase (AKR1D1) are responsible for the formation of 5α- and 5β-reduced pregnanes. In this study, we examined the expression of PR-AB and the progesterone metabolizing enzymes in 31 specimens of ovarian endometriosis and 28 specimens of normal endometrium. Real-time PCR analysis revealed significantly decreased mRNA levels of PR-AB, HSD17B2 and SRD5A2, significantly increased mRNA levels of AKR1C1, AKR1C2, AKR1C3 and SRD5A1, and negligible mRNA levels of AKR1D1. Immunohistochemistry staining of endometriotic tissue compared to control endometrium showed significantly lower PR-B levels in epithelial cells and no significant differences in stromal cells, there were no significant differences in the expression of AKR1C3 and significantly higher AKR1C2 levels were seen only in stromal cells. Our expression analysis data at the mRNA level and partially at the cellular level thus suggest enhanced metabolism of progesterone by SRD5A1 and the 20α-HSD and 3α/β-HSD activities of AKR1C1, AKR1C2 and AKR1C3.     

Characterization of rat and mouse NAD+-dependent 3alpha/17beta/20alpha-hydroxysteroid dehydrogenases and identification of substrate specificity determinants by site-directed mutagenesis

In this study, we characterized rat and mouse aldo-keto reductases (AKR1C16 and AKR1C13, respectively) with 92% sequence identity. The recombinant enzymes oxidized non-steroidal alcohols using NAD⁺ as the preferred coenzyme, and showed low 3α/17β/20α-hydroxysteroid dehydrogenase (HSD) activities. The substrate specificity differs from that of rat NAD⁺-dependent 3α-HSD (AKR1C17) that shares 95% sequence identity with AKR1C16. To elucidate the residues determining the substrate specificity of the enzymes, we performed site-directed mutagenesis of Tyr24, Asp128 and Phe129 of AKR1C16 with the corresponding residues (Ser, Tyr and Leu, respectively) of AKR1C17. The double mutation (Asp128/Tyr-Phe129/Leu) had few effects on the substrate specificity, while the Tyr24/Ser mutant showed only 3α-HSD activity, and the triple mutation of the three residues produced an enzyme that had almost the same properties as AKR1C17. The importance of the residue 24 for substrate recognition was verified by the mutagenesis of Ser24/Tyr of AKR1C17 which resulted in a decrease in 3α-HSD activity and appearance of 17β- and 20α-HSD activities. AKR1C16 is also 92% identical with rat NAD⁺-dependent 17β-HSD (AKR1C24), which possesses Tyr24. The replacement of Asp128, Phe129 and Ser137 of AKR1C16 with the corresponding residues (Glu, Ser and Phe, respectively) of AKR1C24 increased the catalytic efficiency for 17β- and 20α-hydroxysteroids.     

Angiotensin II Triggers Expression of the Adrenal Gland Zona Glomerulosa-Specific 3β-Hydroxysteroid Dehydrogenase Isoenzyme through De Novo Protein Synthesis of the Orphan Nuclear Receptors NGFIB and NURR1

The 3β-hydroxysteroid dehydrogenase (3β-HSD) is an enzyme crucial for steroid synthesis. Two different 3β-HSD isoforms exist in humans. Classically, HSD3B2 was considered the principal isoform present in the adrenal. However, we recently showed that the alternative isoform, HSD3B1, is expressed specifically within the adrenal zona glomerulosa (ZG), where aldosterone is produced, raising the question of why this isozyme needs to be expressed in this cell type. Here we show that in both human and mouse, expression of the ZG isoform 3β-HSD is rapidly induced upon angiotensin II (AngII) stimulation. AngII is the key peptide hormone regulating the capacity of aldosterone synthesis. Using the human adrenocortical H295R cells as a model system, we show that the ZG isoform HSD3B1 differs from HSD3B2 in the ability to respond to AngII. Mechanistically, the induction of HSD3B1 involves de novo protein synthesis of the nuclear orphan receptors NGFIB and NURR1. The HSD3B1 promoter contains a functional NGFIB/NURR1-responsive element to which these proteins bind in response to AngII. Knockdown of these proteins and overexpression of a dominant negative NGFIB both reduce the AngII responsiveness of HSD3B1. Thus, the AngII-NGFIB/NURR1 pathway controls HSD3B1. Our work reveals HSD3B1 as a new regulatory target of AngII.     

Expression of splice variants of 1α-hydroxylase in mcf-7 breast cancer cells

1,25-Dihydroxyvitamin D₃ (calcitriol) is the most active natural metabolite of Vitamin D₃. It has strong antiproliferative and differentiating effects on various cell types including breast cancer cells. 25-Hydroxyvitamin D₃-1α-hydroxylase (1α-hydroxylase, CYP27B1) is one of the key enzymes in the formation of calcitriol. It has been found in breast cancer cells suggesting an autocrine regulation of formation of calcitriol in these cells. Alternative splicing of the encoding genes for this enzyme can possibly play a role in regulating the enzyme level and can explain tissue specific variations of 1α-hydroxylase activity. Splice variants containing intron 1 may encode for truncated proteins with deletion of protein domains which are essential for its enzymatic activity. In order to obtain more information on the abundance of 1α-hydroxylase splice variants, we performed a highly specific nested touchdown PCR in MCF-7 cells. The full-length sequence of 1α-hydroxylase and two different splice variants of this enzyme containing intron 1 were isolated. By Western blot technique we then confirmed the protein products of the full-length enzyme and its splice variants. We hypothesize that that the expression of splice variants can lead to a quantitatively lower expression of the mRNA of the full-length enzyme. The abundance of less active 1α-hydroxylase protein variants can alter the local synthesis of calcitriol in the cells and may explain variations of enzymatic activity in different cells and tissues.     

Specific estradiol biosynthetic pathway in choriocarcinoma (JEG-3) cell line

Estradiol (E2) plays a crucial role in all reproduction processes. In the placenta, it is well recognized that E2 is synthesized from fetal dehydroepiandrosterone sulfate (DHEAS). However, there is some controversy about the biosynthetic pathway involved, some authors suggest that E2 is produced by aromatization of testosterone (T), while others suggest that E2 is produced by the conversion of estrone (E1) into E2 by type 1 17β-HSD, subsequent to the aromatization of 4-androstenedione (4-dione) into E1. In the present report, using the precursor [¹⁴C]DHEA, inhibitors of steroidogenic enzymes (chemical inhibitors and siRNA) and a choriocarcinoma (JEG-3) cell line that expresses all the enzymes necessary to transform DHEA into E2, we could determine the sequential steps and the specific steroidogenic enzymes involved in the transformation of DHEA into E2. Quantification of mRNA expression levels using real-time PCR, strongly suggests that type 1 3β-hydroxysteroid dehydrogenase (3β-HSD1), aromatase and type 1 17β-HSD (17β-HSD1) that are highly expressed in JEG-3 cells are the enzymes responsible for the transformation of DHEA into E2. Analysis of the intermediates produced in the absence and presence of 3β-HSD, aromatase and 17β-HSD1 inhibitors permits to determine the following sequential steps: DHEA is transformed into 4-dione by 3β-HSD1, then 4-dione is aromatized into E1 by aromatase and E1 is finally transformed into E2 by 17β-HSD1. Our data are clearly in favor of the pathway in which the step of aromatization precedes the step of reduction by 17β-HSD.     

3D-structure of human estrogenic 17β-HSD1: binding with various steroids

Human estrogenic dehydrogenase (17β-HSD1) catalyses the last step in the biosynthesis of the active estrogens that stimulate the proliferation of breast cancer cells. While the primary substrate for the enzyme is estrone, the enzyme has some activity for the non-estrogenic substrates. To better understand the structure–function relationships of 17β-HSD1 and to provide a better ground for the design of inhibitors, we have determined the crystal structures of 17β-HSD1 in complex with different steroids.The structure of the complex of estradiol with the enzyme determined previously (Azzi et al., Nature Structural Biology 3, 665–668) showed that the narrow active site was highly complementary to the substrate. The substrate specificity is due to a combination of hydrogen bonding and hydrophobic interactions between the steroid and the enzyme binding pocket. We have now determined structures of 17β-HSD1 in complex with dihydrotestosterone and 20α-OH-progesterone. In the case of the C19 androgen, several residues within the enzyme active site make some small adjustments to accommodate the increased bulk of the substrate. In addition, the C19 steroids bind in a slightly different position from estradiol with shifts in positions of up to 1.4 Å. The altered binding position avoids unfavorable steric interactions between Leu 149 and the C19 methyl group (Han et al., unpublished). The known kinetic parameters for these substrates can be rationalized in light of the structures presented. These results give evidence for the structural basis of steroid recognition by 17β-HSD1 and throw light on the design of new inhibitors for this pivotal steroid enzyme.     

Tibolone and its delta-4, 7α-methyl norethisterone metabolite are reversible inhibitors of human aromatase

Tibolone is used for the treatment of climacteric symptoms and osteoporosis in menopausal women. After ingestion, it is rapidly converted to a number of metabolites including 3α- and 3β-hydroxy derivatives and the delta-4, 7α-methylnorethisterone (7α-MeNET) metabolite, which is rapidly cleared from circulation. Tibolone and some of its metabolites act in a tissue-selective manner to inhibit steroid sulphatase (STS) and 17β-hydroxysteroid dehydrogenase Type 1 (17β-HSD1) activities but also stimulate steroid sulphotransferase and 17β-HSD2 activities. In the present study we have examined whether the ability of tibolone and its 7α-MeNET metabolites to regulate the activities of enzymes involved in oestrogen formation or inactivation extends to another key enzyme involved in oestrogen synthesis, the aromatase, which converts androstenedione to oestrone. Using JEG-3 choriocarcinoma cells, which have a high level of aromatase activity, tibolone and 7α-MeNET, but not the 3α- or 3β-hydroxy metabolites, were found to inhibit aromatase activity in intact cells and also lysates prepared from these cells (up to 61% inhibition at 10 μM). An investigation into the nature of aromatase inhibition by these compounds revealed that they inhibit aromatase activity by a reversible mechanism. Tibolone and 7α-MeNET also inhibited aromatase activity in MCF-7 breast cancer cells, which have a much lower level of aromatase activity than JEG-3 cells. It is concluded that, in addition to inhibiting STS and 17β-HSD1, tibolone and 7α-MeNET may exert some of their tissue-selective effects in regulating oestrogen synthesis by also inhibiting aromatase activity.     

Androgen-induced human breast cancer cell proliferation is mediated by discrete mechanisms in estrogen receptor-α-positive and -negative breast cancer cells

Androgens have important physiological effects in women. Not only are they the precursor hormones for estrogen biosynthesis in the ovaries and extragonadal tissues, but also act directly via androgen receptors (ARs) throughout the body. Studies of the role of androgens on breast cancer development are controversial and the mechanisms involved are not fully understood. In this report we demonstrate that a non-aromatizable androgen metabolite, dihydrotestosterone (DHT), stimulated cell proliferation in vitro of both estrogen receptor-α (ER-α)-positive MCF-7 cells and ER-α-negative MDA-MB-231 human breast cancer cells. A contribution of ER to the proliferative effect of DHT in MCF-7 cells was supported by actions of small interfering RNA (siRNA) ER-α transfection and of the specific inhibitor of ER, ICI 182,780 to block DHT-induced proliferation. A contribution of the possible conversion of DHT to androstane-3α, 17β-diol was not excluded in these MCF-7 cell studies. In MDA-MB-231 cells, a novel mechanism was implicated, in that anti-integrin αvβ3 or an Arg-Gly-Asp (RGD) peptide targeted at a small molecule binding domain of the integrin eliminated the DHT effect on cell proliferation. Anti-integrin αvβ3 did not affect DHT action on MCF-7 cells. A contribution from classical androgen receptor to the DHT effect in each cell line was excluded. A proliferative DHT signal is transduced in both ER-α-positive and ER-α-negative breast cancer cells, but by discrete mechanisms.