Analysis of 17β-hydroxysteroid dehydrogenase types 5, 7, and 12 genetic sequence variants in breast cancer cases from French Canadian Families with high risk of breast and ovarian cancer

A family history and estrogen exposure are well-known risk factors for breast cancer. Members of the 17β-hydroxysteroid dehydrogenase family are responsible for important steps in the metabolism of androgens and estrogens in peripheral tissues, including the mammary gland. The crucial biological function of 17β-HSDs renders these genes good candidates for being involved in breast cancer etiology. This study screened for mutations in HSD17B7 and HSD17B12 genes, which encode enzymes involved in estradiol biosynthesis and in AKR1C3, which codes for 17β-HSD type 5 enzyme involved in androgen and progesterone metabolism, to assess whether high penetrance allelic variants in these genes could be involved in breast cancer susceptibility. Mutation screening of 50 breast cancer cases from non-BRCA1/2 high-risk French Canadian families failed to identify germline likely high-risk mutations in HSD17B7, HSD17B12 and AKR1C3 genes. However, 107 sequence variants were identified, including seven missense variants. Assessment of the impact of missense variants on enzymatic activity of the corresponding enzymes revealed no difference in catalytic properties between variants of 17β-HSD types 7 and 12 and wild-type enzymes, while variants p.Glu77Gly and p.Lys183Arg in 17β-HSD type 5 showed a slightly decreased activity. Finally, a haplotype-based approach was used to determine tagging SNPs providing valuable information for studies investigating associations of common variants in these genes with breast cancer risk.     

Studies on the 5α-androstane-3β, 17β-diol-6α-hydroxylase and on the 5α-androstane-3β, 17β-diol-7α-hydroxylase in anterior hypophysis of male rats.

In anterior pituitaries from male rats, it appeared that 5α-androstane-3β, 17β-diol was quickly metabolized into 5α-androstane-3β,6α-17β-triol and 5α-androstane-3β,7α, 17β-triol by action of 6α- and 7α-hydroxylases. Hydroxysteroid hydroxylases were located in endoplasmic reticulum and were dependent on NADPH⁺. Their optimum pH was 8.0, optima temperature, 37°C, and their apparent Km was 2.7 μM. Hydroxylative reactions were not reversible and not modified by gonadectomy. Hydroxylation seemed an efficient control of the pituitary level of 5α-andros-tane-3β, 17β-diol.     

Two 17β-hydroxysteroid dehydrogenases (17HSDs) of estradiol biosynthesis: 17HSD type 1 and type 7

Two 17β-hydroxysteroid dehydrogenases (17HSDs), type 1 and type 7, are enzymes of estradiol biosynthesis, in addition to which rodent type 1 enzymes are also able to catalyze androgens. Both of the 17HSDs are abundantly expressed in ovaries, the type 1 enzyme in granulosa cells and type 7 in luteinized cells. The expression of 17HSD7, which has also been described as a prolactin receptor-associated protein (PRAP), is particularly up-regulated in corpus luteum during the second half of rodent pregnancy. A moderate or slight signal for mouse 17HSD7/PRAP mRNA has also been demonstrated in samples of placenta and mammary gland, for example. Human, but not rodent, 17HSD1 is expressed in placenta, breast epithelium and endometrium in addition to ovaries. A cell-specific enhancer, silencer and promoter in the hHSD17B1 gene participate in the regulation of type 1 enzyme expression. The enhancer consists of several subunits, including a retinoic acid response element, the silencer has a binding motif for GATA factors, and the proximal promoter contains adjacent and competing AP-2 and Sp binding sites.     

Role of 17β-hydroxysteroid dehydrogenase type 1 in endocrine and intracrine estradiol biosynthesis

Enzymes with 17β-hydroxysteroid dehydrogenase (17β-HSD) activity catalyse reactions between the low-active female sex steroid, estrone, and the more potent estradiol, for example. 17β-HSD activity is essential for glandular (endocrine) sex hormone biosynthesis, but it is also present in several extra-gonadal tissues. Hence, 17β-HSD enzymes also take part in local (intracrine) estradiol production in the target tissues of estrogen action. Four distinct 17β-HSD isozymes have been characterized so far, and the data strongly suggests that different 17β-HSD isozymes have distinct roles in endocrine and intracrine metabolism of sex steroids. Current data suggest that 17β-HSD type 1 is the principal isoenzyme involved in glandular estradiol production both in humans and rodents. During ovarian follicular development and luteinization, rat 17β-HSD type 1 is regulated by gonadotropins, and the effects of gonadotropins are modulated by steroid hormones and paracrine growth factors. Human 17β-HSD type 1 favors the reduction reaction, thereby converting estrone to estradiol both in vitro and in cultured cells. Hence, the enzymatic properties of the enzyme are also in line with its suggested role in estradiol biosynthesis. Interestingly, 17β-HSD type 1 is also expressed in certain target tissues of estrogen action such as normal and malignant human breast and endometrium. Hence, 17β-HSD type 1 could be one of the factors leading to a relatively high tissue/plasma ratio of estradiol in breast cancer tissues of postmenopausal women. We conclude that 17β-HSD type 1 has a central role in regulating the circulating estradiol concentration as well as its local production in estrogen target cells.     

Parallel solid-phase synthesis of 3β-peptido-3α-hydroxy-5α-androstan-17-one derivatives for inhibition of type 3 17β-hydroxysteroid dehydrogenase

Type 3 17β-hydroxysteroid dehydrogenase (17β-HSD), a key steroidogenic enzyme, transforms 4-androstene-3,17-dione (Δ⁴-dione) into testosterone. In order to produce potential inhibitors, we performed solid-phase synthesis of model libraries of 3β-peptido-3α-hydroxy-5α-androstan-17-ones with 1, 2, or 3 levels of molecular diversity, obtaining good overall yields (23–58%) and a high average purity (86%, without any purification steps) using the Leznoff's acetal linker. The libraries were rapidly synthesized in a parallel format and the generated compounds were tested as inhibitors of type 3 17β-HSD. Potent inhibitors were identified from these model libraries, especially six members of the level 3 library having at least one phenyl group. One of them, the 3β-(N-heptanoyl- -phenylalanine- -leucine-aminomethyl)-3α-hydroxy-5α-androstan-17-one (42) inhibited the enzyme with an IC₅₀ value of 227 nM, which is twice as potent as the natural substrate Δ⁴-dione when used itself as an inhibitor. Using the proliferation of androgen-sensitive (AR⁺) Shionogi cells as model of androgenicity, the compound 42 induced only a slight proliferation at 1 μM (less than previously reported type 3 17β-HSD inhibitors) and, interestingly, no proliferation at 0.1 μM.     

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.     

Laminin 5 Promotes Activation and Apoptosis of the T Cells Expressing α3β1 Integrin

By introducing an α3 gene-containing plasmid into a human T cell line Jurkat, we prepared the T cells, which express a high level of the α3β1 integrin, to assess the role of laminin 5 in the skin immune system. The α3β1-expressing T cells adhered to laminin 5 and exhibited spreading. These adhered T cells showed a significant tyrosine phosphorylation of intracellular proteins including p59^fynupon T-cell receptor (TCR) stimulation. Six hours after cross-linking TCR, these cells on laminin 5 secreted a three times higher level of IL-2 than those on a BSA-coated plate. Twenty hours after the stimulation, 48% of the α3β1-expressing T cells on laminin 5 caused apoptosis. The protein level of cyclin D3 and E decreased, while that of p53 increased in these T cells. These data suggest that laminin 5 may play at least two regulatory roles for T cell functions: augmentation of IL-2 production by antigen-stimulated T cells and induction of apoptosis in these T cells.     

17α-Ethyl-5β-estrane-3α,17β-diol, a biological marker for the abuse of norethandrolone and ethylestrenol in slaughter cattle

The metabolism of the illegal growth promoter ethylestrenol (EES) was evaluated in bovine liver cells and subcellular fractions of bovine liver preparations. Incubations with bovine microsomal preparations revealed that EES is extensively biotransformed into norethandrolone (NE), another illegal growth promoter. Furthermore, incubations of monolayer cultures of hepatocytes with NE indicated that NE itself is rapidly reduced to 17α-ethyl-5β-estrane-3α,17β-diol (EED). In vivo tests confirmed that, after administration of either EES or NE, EED is excreted as a major metabolite. Therefore, it was concluded that, both in urine and faeces samples, EED can be used as a biological marker for the illegal use of EES and/or NE. Moreover, by monitoring EED in urine or faeces samples, the detection period after NE administration is significantly prolonged. These findings were further confirmed by three cases of norethandrolone abuse in a routine screening program for forbidden growth promoters.     

Differential estrogenic 17β-hydroxysteroid dehydrogenase activity and type 12 17β-hydroxysteroid dehydrogenase expression levels in preadipocytes and differentiated adipocytes

Estradiol (E2) is produced locally in adipose tissue and could play an important role in fat distribution and accumulation, especially in women. It is well recognized that aromatase is expressed in adipose tissue; however the identity of its estrogenic 17β-hydroxysteroid dehydrogenase (17β-HSD) partner is not identified. To gain a better knowledge about the enzyme responsible for the conversion of estrone into estradiol, we determined the activity and expression levels of known estrogenic 17β-HSDs, namely types 1, 7 and 12 17β-HSD in preadipocytes before and after differentiation into mature adipocytes using an adipogenic media. Estrogenic 17β-HSD activity was assessed using [¹⁴C]-labelled estrone, while mRNA expression levels of types 1, 7 and 12 17β-HSD were quantified using real-time PCR and protein expression levels of type 12 17β-HSD was determined using immunoblot analysis. The data indicate that there is a low conversion of E1 into E2 in preadipocytes; however this activity is increased 5-fold (p < 0.0001) in differentiated adipocytes. The increased estrogenic 17β-HSD activity is consistent with the increase in protein expression levels of 17β-HSD12.     

Estradiol-17β and 2-hydroxyestradiol-17β-induced differential production of prostaglandins by cells dispersed from human intrauterine tissues at parturition

Prostaglandin (PGE, 6-keto PGF_1α) output by cells dispersed from human amnion and decidua in the presence of increasing levels (0–5000 ng/ml) of estradiol-17β (E₂) or 2-hydroxyestradiol-17β (2-OH E₂) was studied in relation to parturition. Tissues were obtained from women at term either before (CS) or after (SL) spontaneous labor and vaginal delivery. In the absence of estrogens, the output of both PGs from amnion increased significantly with labor. No significant increase in decidua PG output occurred with labor. Neither estrogen influenced CS amnion PG output. However, both E₂ and 2-OH E₂ stimulated SL amnion PGE output (2-OH E₂>E₂) while having no affect on 6-keto PGF_1α output. Only the highest dose of 2-OH E₂ stimulated PGE output in CS decidua, but both estrogens significantly inhibited 6-keto PGF_1α output in this tissue. In SL decidua only 2-OH E₂ significantly stimulated PGE, and neither estrogen affected 6-keto PGF_1α output. These results might suggest that estrogens modulate PG biosynthesis at the level of endoperoxide to primary PG conversion.     

17 beta-hydroxysteroid dehydrogenases and cancers

17β-Hydroxysteroid dehydrogenases (17HSDs) catalyze the interconversions between active 17β-hydroxysteroids and less-active 17-ketosteroids thereby affecting the availability of biologically active estrogens and androgens in a variety of tissues. The enzymes have different enzymatic properties and characteristic cell-specific expression patterns, suggesting differential physiological functions for the enzymes. Epidemiological and endocrine evidence indicate that estrogens play a key role in the etiology of breast cancer while androgens are involved in mechanisms controlling the growth of prostatic cells, both normal and malignant. Recently, we have developed, using LNCaP prostate cancer cell lines, a cell model to study the progression of prostate cancer. In the model LNCaP cells are transformed in culture condition to more aggressive cells, able to grow in suspension cultures. Our results suggest that substantial changes in androgen and estrogen metabolism occur in the cells during the process. These changes lead to increased production of active estrogens during transformation of the cells. Data from studies of breast cell lines and tissues suggest that the oxidative 17HSD type 2 may predominate in human non-malignant breast epithelial cells, while the reductive 17HSD type 1 activity prevails in malignant cells. Deprivation of an estrogen response by using specific 17HSD type 1 inhibitors is a tempting approach to treat estrogen-dependent breast cancer. Our recent studies demonstrate that in addition to sex hormone target tissues, estrogens may be important in the development of cancer in some other tissues previously not considered as estrogen target tissues such as colon. Our data show that the abundant expression of 17HSD type 2 present in normal colonic mucosa is significantly decreased during colon cancer development.     

The interaction of estrogen receptor α and caveolin-3 regulates connexin43 phosphorylation in metabolic inhibition-treated rat cardiomyocytes

Caveolin-3, the major caveolin isoform in cardiomyocytes, plays an important role in the rapid signaling pathways initiated by stimulation of the membrane-associated molecules. To examine the role of caveolin-3 in regulating estrogen receptor α in cardiomyocytes, we investigate whether the membrane estrogen receptor α associates with caveolin-3 and whether this association is linked to the 17β-estradiol-mediated signals. In control cardiomyocytes, following discontinuous sucrose gradient centrifugation, caveolin-3 was found predominantly in the lipid raft buoyant fractions, whereas it was distributed to both the buoyant and non-lipid raft heavy fractions following metabolic inhibition treatment. Confocal microscopy showed that estrogen receptor α co-localized with caveolin-3 on the plasma membrane of neonatal and adult rat cardiomyocytes. This membrane labeling of estrogen receptor α was not seen following treatment with the cholesterol-depleting agent methyl-β-cyclodextrin (5 mM), whereas metabolic inhibition had little effect on the membrane distribution of estrogen receptor α. Metabolic inhibition induced tyrosine phosphorylation of caveolin-3 and decreased its association with estrogen receptor α, both effects being mediated via a Src activation mechanism, since they were inhibited by the selective tyrosine kinase inhibitor PP2. Metabolic inhibition also induced tyrosine phosphorylation of connexin43 and increased its association with c-Src, both effects being prevented by 17β-estradiol (200 nM). The effect of 17β-estradiol on metabolic inhibition-induced tyrosine phosphorylation of connexin43 was inhibited by the specific estrogen receptor antagonist ICI182780. These data identify cardiac caveolin-3 as juxtamembrane scaffolding for estrogen receptor α docking at caveolae, which provide a unique compartment for conveying 17β-estradiol-elicited, rapid signaling to regulate connexin43 phosphorylation during ischemia.     

Protein kinase D1 regulates ERα‐positive breast cancer cell growth response to 17β‐estradiol and contributes to poor prognosis in patients

About 70% of human breast cancers express and are dependent for growth on estrogen receptor α (ERα), and therefore are sensitive to antiestrogen therapies. However, progression to an advanced, more aggressive phenotype is associated with acquisition of resistance to antiestrogens and/or invasive potential. In this study, we highlight the role of the serine/threonine‐protein kinase D1 (PKD1) in ERα‐positive breast cancers. Growth of ERα‐positive MCF‐7 and MDA‐MB‐415 human breast cancer cells was assayed in adherent or anchorage‐independent conditions in cells overexpressing or depleted for PKD1. PKD1 induces cell growth through both an ERα‐dependent manner, by increasing ERα expression and cell sensitivity to 17β‐estradiol, and an ERα‐independent manner, by reducing cell dependence to estrogens and conferring partial resistance to antiestrogen ICI 182,780. PKD1 knockdown in MDA‐MB‐415 cells strongly reduced estrogen‐dependent and independent invasion. Quantification of PKD1 mRNA levels in 38 cancerous and non‐cancerous breast cell lines and in 152 ERα‐positive breast tumours from patients treated with adjuvant tamoxifen showed an association between PKD1 and ERα expression in 76.3% (29/38) of the breast cell lines tested and a strong correlation between PKD1 expression and invasiveness (P < 0.0001). In tamoxifen‐treated patients, tumours with high PKD1 mRNA levels (n = 77, 50.66%) were significantly associated with less metastasis‐free survival than tumours with low PKD1 mRNA expression (n = 75, 49.34%; P = 0.031). Moreover, PKD1 mRNA levels are strongly positively associated with EGFR and vimentin levels (P < 0.0000001). Thus, our study defines PKD1 as a novel attractive prognostic factor and a potential therapeutic target in breast cancer.     

15β-hydroxysteroids (Part IV). Steroids of the human perinatal period: The synthesis of 3α,15β,17α-trihydroxy-5α-pregnan-20-one and its A/B-ring configurational isomers

In recent years several 15β-hydroxysteroids have emerged pathognomonic of adrenal disorders in human neonates of which 3α,15β,17α-trihydroxy-5β-pregnan-20-one (2) was the first to be identified in the urine of newborn infants affected with congenital adrenal hyperplasia. In this investigation we report the synthesis of the three remaining 3ξ,5ξ-isomers, namely 3α,15β,17α-trihydroxy-5α-pregnan-20-one (3), 3β,15β,17α-trihydroxy-5α-pregnan-20-one (7) and 3β,15β,17α-trihydroxy-5β-pregnan-20-one (8) for their definitive identification in pathological conditions in human neonates. 3β,15β-Diacetoxy-17α-hydroxy-5-pregnen-20-one (11), a product of chemical synthesis was converted to the isomeric 3 and 7, while conversion of 15β,17α-dihydroxy-4-pregnen-3,20-dione (4), a product of microbiological transformation, resulted in the preparation of 8. In brief, selective acetate hydrolysis of 11 gave 15β-acetoxy-3β,17α-dihydroxy-5-pregnen-20-one (12) which on catalytic hydrogenation gave 15β-acetoxy-3β,17α-dihydroxy-5α-pregnan-20-one (13) a common intermediate for the synthesis of the 3β(and α),5α-isomers. Hydrolysis of the 15β-acetate gave 7, whereas oxidation with pyridinium chlorochromate gave 15β-acetoxy-17α-hydroxy-5α-pregnan-3,20-dione (14) which on reduction with -Selectride and hydrolysis of the 15β-acetate gave 3. Finally, hydrogenation of 4 gave 15β,17α-dihydroxy-5β-pregnan-3,20-dione (10) which on reduction with -Selectride gave 8.     

Disposition of 17β-trenbolone in humans

The urinary excretion and metabolic pattern of 17β-trenbolone, a synthetic anabolic steroid hormone used as a growth promotor for beef cattle in several countries, has been studied in a human subject. For the separation of the metabolites of 17β-trenbolone, a reversed-phase high-performance liquid chromatographic method was established. The method was tested with metabolites obtained from incubation of 17β-trenbolone with rat liver microsomes. Fifteen metabolites could be well separated in one run by using a concave acetonitrile—water—methanol gradient. After ingestion of the tracer-labelled hormone at a dose of 0.04 mg/kg body weight 54% of the administered radioactivity was found in the urine after 26 h and 63% after 72 h. Of the urinary material 54% was present as glucuronides, which contained mostly 17α-trenbolone, 17β-trenbolone and trendione. At least five other polar metabolites, presumably hydroxylated products, were found in smaller amounts, mostly in the unconjugated and sulphated fractions. Thus, the disposition of 17β-trenbolone in humans differs significantly from that in rats, which may have a bearing on the toxicological evaluation of the hormone.     

Progestins and danazol effect on cell-to-cell adhesion, and E-cadherin and α- and β-catenin mRNA expressions

The first step of invasion and metastasis is the detachment of cancer cells in the primary tumor, which is mainly controlled by the function in the adherens junction, consisting of E-cadherin associated proteins (E-cadherin, α- and β-catenins, vinculin, α-actinin, and actin). The cell-to-cell aggregation activity and the expressions of E-cadherin, and α- and β-catenin mRNAs in Ishikawa cells of well-differentiated endometrial cancer were significantly suppressed by estrogen. These suppressions were reversed by progesterone, medroxyprogesterone acetate (MPA) and danazol. Proteins in the adherens junction appeared to be expressed intact and to be functional in Ishikawa cells. Persistent estrogen predominant milieu might contribute to the detachment of well-differentiated endometrial cancer cells, leading to spreading of those cells, while progestins and danazol protect estrogen-induced spreading of those cells.     

Synthesis and biological activity of 17α-alkynylamide derivatives of estradiol

Seven estradiol (E₂) derivatives with an alkynylamide side chain at the 17α position were synthesized starting from ethynylestradiol (EE₂). The main chemical step was the coupling reaction of the acetylide ion of EE₂ with carbon dioxide, glutaric anhydride or bromoalkyl ortho ester. The synthesis of these compounds is fast (3–6 steps according to the compound) and is easily achieved with good yield. Five compounds with different side chain lenghts were evaluated for uterotrophic and antiuterotrophic activity in the CD-1 mouse. None of the tested compounds shows estrogenic activity in this sensitive in vitro system. At low doses (1 and 3 μg), a 14–57% inhibition of E₂-induced uterine growth was observed while no additional inhibition was observed at the 10, 20 and 30 μg doses. In human breast carcinoma cells in culture, all compounds show estrogenic activity at high concentrations while only compound 39 (N-buty,N-methyl-8-[3′,17′β-dihydroxy estra-1′,3′,5′(10′)-trien-17′α-yl]-7-octynamide) possesses antiproliferative or antiestrogenic effects. No significant correlation could be demonstrated between alkynylamide side chain length and estrogenic or antiestrogenic activity. Among the compounds tested, the derivative of EE₂ possessing a five-methylene (CH₂) side chain (compound 39) possesses the best antiestrogenic activity (44 ± 7% in the CD-1 mouse uterus assay at the 3μg dose and 57 ± 4% at 0.1 nM in human ZR-75-1 cancer cells in culture).