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Selective inhibition of human 3?-hydroxysteroid dehydrogenase type 1 as a potential treatment for breast cancer.

ABSTRACT: Human 3?-hydroxysteroid dehydrogenase/isomerase type 1 (3?-HSD1) is a critical enzyme in the conversion of DHEA to estradiol in breast tumors and may be a target enzyme for inhibition in the treatment of breast cancer in postmenopausal women. Human 3?-HSD2 participates in the production of cortisol and aldosterone in the human adrenal gland in this population. In our recombinant human breast tumor MCF-7 Tet-off cells that express either 3?-HSD1 or 3?-HSD2, trilostane and epostane inhibit the DHEA-induced proliferation of MCF-7 3?-HSD1 cells with 12-16-fold lower IC(50) values compared to the MCF-7 3?-HSD2 cells. Trilostane and epostane also competitively inhibit purified human 3?-HSD1 with 12-16-fold lower K(i) values compared to the noncompetitive K(i) values measured for human 3?-HSD2. Using our structural model of 3?-HSD1, trilostane was docked in the active site of 3?-HSD1, and Arg195 in 3?-HSD1 or Pro195 in 3?-HSD2 was identified as a potentially critical residue. The R195P-1 mutant of 3?-HSD1 and the P195R-2 mutant of 3?-HSD2 were created, expressed and purified. Kinetic analyses of enzyme inhibition suggest that the high-affinity, competitive inhibition of 3?-HSD1 by trilostane may be related to the presence of Arg195 in 3?-HSD1 versus Pro195 in 3?-HSD2. In addition, His156 in 3?-HSD1 may play a role in the higher affinity of 3?-HSD1 for substrates and inhibitors compared to 3?-HSD2 containing Try156. Structural modeling of the 3?-HSD1 dimer identified a possible interaction between His156 on one subunit and Gln105 on the other. Kinetic analyses of the H156Y-1, Q105M-1 and Q105M-2 support subunit interactions that contribute to the higher affinity of 3?-HSD1 for the inhibitor, epostane, compared to 3?-HSD2. Article from the Special issue on Targeted Inhibitors.

SUBMITTER: Thomas JL

PROVIDER: S-EPMC2999670 | biostudies-literature | 2011 May

REPOSITORIES: biostudies-literature

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Selective inhibition of human 3β-hydroxysteroid dehydrogenase type 1 as a potential treatment for breast cancer.

Thomas James L JL Bucholtz Kevin M KM Kacsoh Balint B

The Journal of steroid biochemistry and molecular biology 20100822 1-2

Human 3β-hydroxysteroid dehydrogenase/isomerase type 1 (3β-HSD1) is a critical enzyme in the conversion of DHEA to estradiol in breast tumors and may be a target enzyme for inhibition in the treatment of breast cancer in postmenopausal women. Human 3β-HSD2 participates in the production of cortisol and aldosterone in the human adrenal gland in this population. In our recombinant human breast tumor MCF-7 Tet-off cells that express either 3β-HSD1 or 3β-HSD2, trilostane and epostane inhibit the DHE ...[more]

PMID: 20736065

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Project description:Human 17beta-hydroxysteroid dehydrogenase type 1 (17?-HSD1) is a steroid-converting enzyme that has long been known to play critical roles in estradiol synthesis and more recently in dihydrotestosterone (DHT) inactivation, showing a dual function that promotes breast cancer cell proliferation. Previously, we reported the first observation of the influence of the enzyme on endogenous estrogen-responsive gene expression. Here, we demonstrate the impact of 17?-HSD1 expression on the breast cancer cell proteome and investigate its role in cell migration.17?-HSD1 was stably transfected in MCF7 cells and the proteome of the generated cells overexpressing 17?-HSD1 (MCF7-17?HSD1 cells) was compared to that of the wild type MCF7 cells. Proteomics study was performed using two-dimensional gel electrophoresis followed by mass spectrometry analysis of differentially expressed protein spots. Reverse transcription quantitative real-time PCR (RT-qPCR) was used to investigate the transcription of individual gene. The effect of 17?-HSD1 on MCF7 cell migration was verified by a wound-healing assay.Proteomic data demonstrate that the expression of more than 59 proteins is modulated following 17?-HSD1 overexpression. 17?-HSD1 regulates the expression of important genes and proteins that are relevant to cell growth control, such as BRCA2 and CDKN1A interacting protein (BCCIP) and proliferating cell nuclear antigen (PCNA) which are down- and upregulated in MCF7-17?HSD1 cells, respectively. RT-qPCR data reveal that 17?-HSD1 increases the mRNA levels of estrogen receptors (ER) alpha and beta by 171 and 120%, respectively, while decreasing that of the androgen receptor by 64%. Interestingly, 17?-HSD1 increases the mRNA transcript (by 3.6 times) and the protein expression of the metastasis suppressor gene nm23-H1 and the expression of the two enzymes are closely correlated. We have further shown that 17?-HSD1 expression is associated with an increase of MCF7 cell migration.In addition to the regulation of important genes, we have demonstrated for the first time that 17?-HSD1 increases breast cancer cell migration, in spite of its positive regulation of the antimetastatic gene NM23. This is also correlated to its stimulation of breast cancer cell growth, further confirming its targeting in ER positive breast cancer. The novel findings in this study suggest several directions for future research on the contribution of 17?-HSD1 to breast cancer progression and related treatment.

Project description:Aldo-keto reductase (AKR) 1C3 catalyzes the NADPH-dependent reduction of Delta(4)-androstene-3,17-dione to yield testosterone, reduction of estrone to yield 17beta-estradiol and reduction of progesterone to yield 20alpha-hydroxyprogesterone. In addition, it functions as a prostaglandin (PG) F synthase and reduces PGH(2) to PGF(2)alpha and PGD(2) to 11beta-PGF(2). Immunohistochemistry showed that AKR1C3 is over-expressed in invasive ductal carcinoma of the breast. Retroviral expression of AKR1C3 in MCF-7 breast carcinoma cells shows that each of the assigned reactions occur in a breast cell microenvironment. Steroid and prostaglandin conversions were monitored by radiochromatography. Prostaglandin conversion was validated by a second method using HPLC coupled to APCI-MRM/MS. The combined effect of the AKR1C3 catalyzed 17- and 20-ketosteroid reductions will be to increase the 17beta-estradiol:progesterone ratio in the breast. In addition, formation of PGF(2) epimers would activate F prostanoid receptors and deprive PPARgamma of its putative anti-proliferative PGJ(2) ligands. Thus, AKR1C3 is a source of proliferative signals and a potential therapeutic target for hormone-dependent and -independent breast cancer. Two strategies for AKR1C3 inhibition based on non-steroidal anti-inflammatory drugs were developed. The first strategy uses the Ullmann coupling reaction to generate N-phenylanthranilate derivatives that inhibit AKR1C enzymes without affecting PGH(2) synthase (PGHS) 1 or PGHS-2. The second strategy exploits the selective inhibition of AKR1C3 by indomethacin, which did not inhibit highly related AKR1C1 or AKR1C2. Using known structure-activity relationships for the inhibition of PGHS-1 and PGHS-2 by indole acetic acids we obtained N-(4-chlorobenzoyl)-melatonin as a specific AKR1C3 inhibitor (K(I)=6.0muM) that does not inhibit PGHS-1, PGHS-2, AKR1C1, or AKR1C2. Both strategies are informed by crystal structures of ternary AKR1C3.NADP(+).NSAID complexes. The identification of NSAID analogs as specific inhibitors of AKR1C3 will help validate its role in the proliferation of breast cancer cells.

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Selective inhibition of human 3?-hydroxysteroid dehydrogenase type 1 as a potential treatment for breast cancer.

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Selective inhibition of human 3β-hydroxysteroid dehydrogenase type 1 as a potential treatment for breast cancer.

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