Lau Kin Chong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 149-158). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgements --- p.v / Table of Contents --- p.vi / List of Tables --- p.xii / List of Figures --- p.xiii / List of Appendices --- p.xv / Abbreviations --- p.xvi / Chapter 1 --- Introduction / Chapter 1.1 --- Mucopolysaccharidosis type II as a lysosomal storage disease --- p.1 / Chapter 1.1.1 --- Prevalence of MPS II --- p.2 / Chapter 1.1.2 --- Pathophysiology of MPS II --- p.4 / Chapter 1.1.3 --- Clinical features of MPS II --- p.4 / Chapter 1.1.4 --- Clinical management of MPS II --- p.6 / Chapter 1.1.4.1 --- Diagnostic methods for MPS II --- p.6 / Chapter 1.1.4.2 --- Treatments for MPS II --- p.7 / Chapter 1.2 --- Iduronate-2-sulfatase protein (IDS) --- p.9 / Chapter 1.2.1 --- Role in GAG degradation --- p.9 / Chapter 1.2.2 --- Post-translational modifications --- p.11 / Chapter 1.2.2.1 --- Formylglycine formation --- p.11 / Chapter 1.2.2.2 --- Glycosylation --- p.12 / Chapter 1.2.2.3 --- Proteolysis --- p.12 / Chapter 1.2.3 --- Iduronate-2-sulfatase gene (IDS) --- p.14 / Chapter 1.2.3.1 --- Properties of IDS mutations --- p.15 / Chapter 1.2.3.2 --- Methylation patterns are correlated with transitional mutations --- p.17 / Chapter 1.2.3.3 --- Genotype-phenotype correlations between IDS gene and MPS II --- p.19 / Chapter 1.3 --- In this study --- p.21 / Chapter 1.3.1 --- Mutational analysis --- p.21 / Chapter 1.3.2 --- In vitro expression of mutant IDS --- p.22 / Chapter 1.3.3 --- Maturation of IDS polypeptides --- p.23 / Chapter 2 --- Materials & Methods / Chapter 2.1 --- Mutation screening for MPS II patients --- p.24 / Chapter 2.1.1 --- Patients --- p.24 / Chapter 2.1.2 --- Genomic DNA extraction --- p.24 / Chapter 2.1.2.1 --- Materials --- p.24 / Chapter 2.1.2.2 --- Methods --- p.25 / Chapter 2.1.3 --- IDS exons amplification by Polymerase Chain Reaction (PCR) --- p.26 / Chapter 2.1.3.1 --- Materials --- p.26 / Chapter 2.1.3.1.1 --- PCR --- p.26 / Chapter 2.1.3.1.2 --- Agarose gel electrophoresis --- p.27 / Chapter 2.1.3.1.3 --- PCR fragments purification --- p.29 / Chapter 2.1.3.2 --- Methods --- p.29 / Chapter 2.1.3.2.1 --- Amplifying IDS exons by PCR --- p.29 / Chapter 2.1.3.2.2 --- Purifying PCR fragments --- p.30 / Chapter 2.1.4 --- DNA sequencing for detecting IDS mutations --- p.30 / Chapter 2.1.4.1 --- Materials --- p.30 / Chapter 2.1.4.2 --- Methods --- p.30 / Chapter 2.1.4.2.1 --- Sequencing reaction --- p.30 / Chapter 2.1.4.2.2 --- Purifying sequencing products --- p.31 / Chapter 2.1.4.2.3 --- Analyzing sequencing results --- p.31 / Chapter 2.1.5 --- Fragment restriction endonuclease analysis --- p.31 / Chapter 2.1.5.1 --- Materials --- p.31 / Chapter 2.1.5.2 --- Methods --- p.32 / Chapter 2.2 --- Isolation of IDS cDNA from peripheral blood --- p.34 / Chapter 2.2.1 --- Materials --- p.34 / Chapter 2.2.1.1 --- Total RNA extraction --- p.34 / Chapter 2.2.1.2 --- Reverse-transcriptase PCR (RT-PCR) --- p.35 / Chapter 2.2.1.3 --- PCR for amplifying IDS cDNA --- p.35 / Chapter 2.2.2 --- Methods --- p.37 / Chapter 2.2.2.1 --- Extracting total RNA by QIAamp RNeasy Mini Kit --- p.37 / Chapter 2.2.2.2 --- Converting IDS mRNA into cDNA by RT-PCR --- p.38 / Chapter 2.2.2.3 --- Isolating IDS cDNA by PCR --- p.39 / Chapter 2.2.2.4 --- Isolating firefly luciferase gene by PCR --- p.39 / Chapter 2.3 --- Introducing IDS cDNA into Gateway Cloning System --- p.40 / Chapter 2.3.1 --- Materials --- p.40 / Chapter 2.3.1.1 --- Directional cloning --- p.40 / Chapter 2.3.1.2 --- LB medium/ agar with antibiotics preparation --- p.42 / Chapter 2.3.1.3 --- Plasmids purification from transformed cells --- p.42 / Chapter 2.3.1.4 --- Validation of IDS inserted plasmids --- p.43 / Chapter 2.3.2 --- Methods --- p.43 / Chapter 2.3.2.1 --- TOPO cloning reaction --- p.43 / Chapter 2.3.2.2 --- Transformation --- p.44 / Chapter 2.3.2.3 --- Small-scale plasmids preparation by QIAprep Miniprep Kit --- p.44 / Chapter 2.3.2.4 --- Sequencing the plasmids --- p.45 / Chapter 2.3.2.5 --- QuikChange II XL site-directed mutagenesis --- p.46 / Chapter 2.3.2.5.1 --- Synthesizing mutant strand with desired mutations --- p.46 / Chapter 2.3.2.5.2 --- Digesting parental strand --- p.46 / Chapter 2.3.2.5.3 --- Transformation --- p.47 / Chapter 2.3.2.6 --- Swapping IDS gene from entry clone to expression vectors --- p.47 / Chapter 2.3.2.6.1 --- LR clonase reaction --- p.47 / Chapter 2.3.2.6.2 --- Transformation --- p.48 / Chapter 2.4 --- Introducing IDS cDNA into RTS pIVEX Wheat Germ vector --- p.49 / Chapter 2.4.1 --- Materials --- p.49 / Chapter 2.4.1.1 --- Restriction digestion --- p.49 / Chapter 2.4.1.2 --- Purification of digested products --- p.50 / Chapter 2.4.1.3 --- Ligation of the IDS insert into pIVE´Xؤ1.3_WG --- p.50 / Chapter 2.4.2 --- Methods --- p.50 / Chapter 2.4.2.1 --- Restriction digestion to create sticky ends --- p.50 / Chapter 2.4.2.2 --- Purifying the digested products --- p.51 / Chapter 2.4.2.3 --- Ligating the IDS insert into pIVE´Xؤ1.3_WG --- p.51 / Chapter 2.4.2.4 --- Transformation --- p.51 / Chapter 2.5 --- Transient expression study of IDS constructs --- p.53 / Chapter 2.5.1 --- Materials --- p.53 / Chapter 2.5.2 --- Methods --- p.55 / Chapter 2.5.2.1 --- Cell culturing --- p.55 / Chapter 2.5.2.2 --- Transfecting IDS constructs by lipofection procedures --- p.55 / Chapter 2.5.2.3 --- Harvesting COS-7 cells --- p.56 / Chapter 2.5.2.4 --- Total RNA extraction from transfected COS-7 cells --- p.57 / Chapter 2.5.2.5 --- RT-PCR showing IDS mRNA stability --- p.58 / Chapter 2.5.2.6 --- Endocytosis of expressed IDS products into COS-7 cells --- p.58 / Chapter 2.6 --- Synthesizing IDS by cell-free in vitro expression systems --- p.59 / Chapter 2.6.1 --- Materials --- p.59 / Chapter 2.6.1.1 --- DNA templates for expression --- p.59 / Chapter 2.6.1.2 --- Commercial cell-free expression kits --- p.60 / Chapter 2.6.1.3 --- Supplements --- p.61 / Chapter 2.6.2 --- Methods --- p.64 / Chapter 2.6.2.1 --- Cell-free expression by ExpressWay plus expression system --- p.64 / Chapter 2.6.2.2 --- Cell-free expression by RTS 100 E.coli HY Kit --- p.64 / Chapter 2.6.2.3 --- Cell-free expression by RTS 100 Wheat Germ CECF Kit --- p.64 / Chapter 2.6.2.4 --- Cell-free expression by TnT Coupled Wheat Germ Extract Systems --- p.65 / Chapter 2.6.2.5 --- Cell-free expression by TNT Coupled Reticulocyte Lysate Systems --- p.66 / Chapter 2.7 --- Investigations of IDS protein expression --- p.67 / Chapter 2.7.1 --- Materials --- p.67 / Chapter 2.7.1.1 --- Isolation of Histidine-tagged proteins --- p.67 / Chapter 2.7.1.2 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis/ SDS-PAGE --- p.67 / Chapter 2.7.1.3 --- Fluorometric activity assay for IDS --- p.69 / Chapter 2.7.1.4 --- Luciferase activity assay --- p.72 / Chapter 2.7.2 --- Methods --- p.72 / Chapter 2.7.2.1 --- Isolating His-tagged IDS from cell-free expression products --- p.72 / Chapter 2.7.2.2 --- Protein staining of expression products --- p.73 / Chapter 2.7.2.2.1 --- Preparation of protein separating gel --- p.73 / Chapter 2.7.2.2.2 --- Preparation of proteins for SDS-PAGE --- p.73 / Chapter 2.7.2.2.3 --- SDS-PAGE analysis --- p.73 / Chapter 2.7.2.3 --- Fluorometric enzyme assay for IDS proteins --- p.74 / Chapter 2.7.2.4 --- Luciferase activity assay --- p.75 / Chapter 3 --- Results / Chapter 3.1 --- Mutational analysis of MPS II and carrier detection --- p.76 / Chapter 3.2 --- Investigating IDS mutants by transient expression --- p.86 / Chapter 3.2.1 --- Fluorometric enzyme assay for measuring IDS activity --- p.86 / Chapter 3.2.2 --- Source of IDS gene for transient expression in COS-7 cells --- p.89 / Chapter 3.2.3 --- In vitro expression of IDS and its mutants in COS-7 cells --- p.92 / Chapter 3.2.3.1 --- Analysis of transient expression in terms of IDS activity --- p.92 / Chapter 3.2.3.2 --- Analysis of IDS mRNA stability in COS-7 cells --- p.95 / Chapter 3.2.3.3 --- Analysis of IDS protein stability in COS-7 cells --- p.95 / Chapter 3.3 --- Cell-free in vitro expression for investigating the IDS mutants --- p.98 / Chapter 3.3.1 --- The five cell-free systems involved --- p.98 / Chapter 3.3.2 --- Source of IDS gene for cell-free in vitro expression --- p.98 / Chapter 3.3.3 --- SDS-PAGE analysis of IDS protein stability in cell-free systems --- p.100 / Chapter 3.3.3.1 --- Wheat germ-based cell-free expression system (Roche) --- p.100 / Chapter 3.3.3.2 --- E.coli-based cell-free expression system (Invitrogen) --- p.102 / Chapter 3.3.3.3 --- E.coli-based cell-free expression system (Roche) --- p.102 / Chapter 3.3.4 --- In Vision His-tag In-gel stain for wild-type IDS and its mutant --- p.103 / Chapter 3.3.5 --- Analysis of IDS activity in cell-free expression systems --- p.107 / Chapter 3.3.6 --- Analysis of the cellular uptake of IDS --- p.110 / Chapter 4 --- Discussions / Chapter 4.1 --- Mutational analysis --- p.113 / Chapter 4.1.1 --- Heterogeneity of IDS mutations --- p.113 / Chapter 4.1.2 --- Role of molecular diagnosis for MPS II --- p.113 / Chapter 4.1.3 --- Two novel mutations and one reported mutation were identified --- p.115 / Chapter 4.1.3.1 --- A novel nonsense mutation: Ser369term --- p.115 / Chapter 4.1.3.2 --- A reported nonsense mutation: Gln389term --- p.115 / Chapter 4.1.3.3 --- A novel missense mutation: Leu339Pro --- p.116 / Chapter 4.2 --- Expression studies of the IDS mutants --- p.117 / Chapter 4.2.1 --- Analysis of transient expression in COS-7 cells --- p.117 / Chapter 4.2.1.1 --- Stability of mutant mRNA --- p.119 / Chapter 4.2.1.2 --- IDS catalytic activity --- p.119 / Chapter 4.2.2 --- Analysis of mutant stability by cell-free expression systems --- p.120 / Chapter 4.2.3 --- Structural analysis of amino acids alterations --- p.121 / Chapter 4.2.3.1 --- p.L339P causes conformational change --- p.122 / Chapter 4.2.3.2 --- p.L339R changes overall charge balance --- p.122 / Chapter 4.2.3.3 --- Mutations at Leu339 residue affect substrate binding --- p.123 / Chapter 4.3 --- Analysis of IDS maturation processing --- p.124 / Chapter 4.3.1 --- Active IDS modifications are not completed in lysosomes --- p.124 / Chapter 4.3.2 --- C-terminal proteolysis is essential for active IDS --- p.125 / Chapter 4.3.3 --- Functional role of glycosylation during IDS processing --- p.126 / Chapter 4.4 --- Analysis of cell-free expression systems --- p.128 / Chapter 4.4.1 --- Microbial systems using E.coli cell extracts: insoluble IDS precursors --- p.128 / Chapter 4.4.2 --- Plant system using wheat germ extracts: soluble IDS precursors --- p.129 / Chapter 4.4.3 --- Mammalian system using rabbit reticulocytes extracts: undetectable --- p.129 / Chapter 4.5 --- Role of transfecting IDS constructs --- p.131 / Chapter 4.6 --- Conclusion --- p.132 / Appendices --- p.133 / Electronic-database and computing system --- p.149 / Bibliography --- p.149

The development of reversible and irreversible inhibitors of steroid sulfatase (STS) and protein tyrosine phosphatase 1B (PTP1B) is reported herein. STS belongs to to the aryl sulfatase family of enzymes that have roles in diverse processes such as hormone regulation, cellular degradation, bone and cartilage development, intracellular communication, and signalling pathways. STS catalyzes the desulfation of sulfated steroids which are the storage forms of many steroids such as the female hormone estrone. Its crucial role in the regulation of estrogen levels has made it a therapeutic target for the treatment of estrogen-dependent cancers. Estrone sulfate derivatives bearing 2- and 4-mono- and difluoromethyl substitutions were examined as quinone methide-generating suicide inhibitors of STS with the goal of developing these small molecules as activity-based probes for proteomic profiling of sulfatases. Kinetic studies suggest that inhibition by the monofluoro derivatives is a result of a quinone methide intermediate that reacts with active-site nucleophiles. However, the main inhibition pathway of the 4-difluoromethyl derivative involved an unexpected process in which initially formed quinone methide diffuses from the active site and decomposes to an aldehyde in solution which then acts as a potent, almost irreversible STS inhibitor. This is the first example where this class of inactivator functions by in situ generation of an aldehyde. 6- and 8-mono- and difluoromethyl coumarin derivatives were also examined as quinone methide-generating suicide inhibitors of STS. The 6-monofluoromethyl derivative acted as a classic suicide inhibitor. The partition ratio of this compound was found to be very large indicating that this class of compounds is not likely suitable as an activity-based probe for proteomic profiling of sulfatases. Boronic acids derived from steroid and coumarin platforms were also examined as STS inhibitors with the goal of improving our understanding of substrate binding specificity of STS. Inhibition constants in the high nanomolar to low micromolar range were observed for the steroidal derivatives. The coumarin derivatives were poor inhibitors. These results suggest that the boronic acid moiety must be attached to a platform very closely resembling a natural substrate in order for it to impart a beneficial effect on binding affinity compared to its phenolic analog. The mode of inhibition observed was reversible and kinetic properties corresponding to the mechanism for slow-binding inhibitors were not observed. PTP1B catalyzes the dephosphorylation of phosphotyrosine residues in the insulin receptor kinase and is a key enzyme in the down regulation of insulin signaling. Inhibitors of PTP1B are considered to have potential as therapeutics for treating type II diabetes mellitus. The difluoromethylenesulfonic (DFMS) acid group, one of the best monoanionic phosphotyrosine mimics reported in the literature, was examined as a phosphotyrosine (pTyr) mimic in a non-peptidyl platform for PTP1B inhibition. The DFMS-bearing inhibitor was found to be an approximately 1000-fold poorer inhibitor than its phosphorus analogue. It was also found that the fluorines in the DFMS inhibitor contributed little to inhibitory potency. In addition, [sulfonamido(difluoromethyl)]-phenylalanine (F2Smp) was examined as a neutral pTyr mimic in commonly used hexapeptide and tripeptide platforms. F2Smp was found to be a poor pTyr mimic. These inhibition studies also revealed that the tripeptide platform is not suitable for assessing pTyr mimics for PTP1B inhibition. Taken together, the kinetic data on the inhibition of STS and PTP1B provide valuable information relevant for future design of inhibitors of these two therapeutic targets.

The development of reversible and irreversible inhibitors of steroid sulfatase (STS) and protein tyrosine phosphatase 1B (PTP1B) is reported herein. STS belongs to to the aryl sulfatase family of enzymes that have roles in diverse processes such as hormone regulation, cellular degradation, bone and cartilage development, intracellular communication, and signalling pathways. STS catalyzes the desulfation of sulfated steroids which are the storage forms of many steroids such as the female hormone estrone. Its crucial role in the regulation of estrogen levels has made it a therapeutic target for the treatment of estrogen-dependent cancers. Estrone sulfate derivatives bearing 2- and 4-mono- and difluoromethyl substitutions were examined as quinone methide-generating suicide inhibitors of STS with the goal of developing these small molecules as activity-based probes for proteomic profiling of sulfatases. Kinetic studies suggest that inhibition by the monofluoro derivatives is a result of a quinone methide intermediate that reacts with active-site nucleophiles. However, the main inhibition pathway of the 4-difluoromethyl derivative involved an unexpected process in which initially formed quinone methide diffuses from the active site and decomposes to an aldehyde in solution which then acts as a potent, almost irreversible STS inhibitor. This is the first example where this class of inactivator functions by in situ generation of an aldehyde. 6- and 8-mono- and difluoromethyl coumarin derivatives were also examined as quinone methide-generating suicide inhibitors of STS. The 6-monofluoromethyl derivative acted as a classic suicide inhibitor. The partition ratio of this compound was found to be very large indicating that this class of compounds is not likely suitable as an activity-based probe for proteomic profiling of sulfatases. Boronic acids derived from steroid and coumarin platforms were also examined as STS inhibitors with the goal of improving our understanding of substrate binding specificity of STS. Inhibition constants in the high nanomolar to low micromolar range were observed for the steroidal derivatives. The coumarin derivatives were poor inhibitors. These results suggest that the boronic acid moiety must be attached to a platform very closely resembling a natural substrate in order for it to impart a beneficial effect on binding affinity compared to its phenolic analog. The mode of inhibition observed was reversible and kinetic properties corresponding to the mechanism for slow-binding inhibitors were not observed. PTP1B catalyzes the dephosphorylation of phosphotyrosine residues in the insulin receptor kinase and is a key enzyme in the down regulation of insulin signaling. Inhibitors of PTP1B are considered to have potential as therapeutics for treating type II diabetes mellitus. The difluoromethylenesulfonic (DFMS) acid group, one of the best monoanionic phosphotyrosine mimics reported in the literature, was examined as a phosphotyrosine (pTyr) mimic in a non-peptidyl platform for PTP1B inhibition. The DFMS-bearing inhibitor was found to be an approximately 1000-fold poorer inhibitor than its phosphorus analogue. It was also found that the fluorines in the DFMS inhibitor contributed little to inhibitory potency. In addition, [sulfonamido(difluoromethyl)]-phenylalanine (F2Smp) was examined as a neutral pTyr mimic in commonly used hexapeptide and tripeptide platforms. F2Smp was found to be a poor pTyr mimic. These inhibition studies also revealed that the tripeptide platform is not suitable for assessing pTyr mimics for PTP1B inhibition. Taken together, the kinetic data on the inhibition of STS and PTP1B provide valuable information relevant for future design of inhibitors of these two therapeutic targets.

The genomes of the hemiascomycetes Saccharomyces cerevisiae and Ashbya gossypii have been completely sequenced, allowing a comparative analysis of these two genomes, which reveals that a small number of genes appear to have entered these genomes as a result of horizontal gene transfer from bacterial sources. One potential case of horizontal gene transfer in A. gossypii and 10 potential cases in S. cerevisiae were identified, of which two were investigated further. One gene, encoding the enzyme dihydroorotate dehydrogenase (DHOD), is potentially a case of horizontal gene transfer, as shown by sequencing of this gene from additional bacterial and fungal species to generate sufficient data to construct a well-supported phylogeny. The DHOD-encoding gene found in S. cerevisiae, URA1 (YKL216W), appears to have entered the Saccharomycetaceae after the divergence of the S. cerevisiae lineage from the Candida albicans lineage and possibly since the divergence from the A. gossypii lineage. This gene appears to have come from the Lactobacillales, and following its acquisition the endogenous eukaryotic DHOD gene was lost. It was also shown that the bacterially derived horizontally transferred DHOD is required for anaerobic synthesis of uracil in S. cerevisiae. The other gene discussed in detail is BDS1, an aryl- and alkyl-sulfatase gene of bacterial origin that we have shown allows utilization of sulfate from several organic sources. Among the eukaryotes, this gene is found in S. cerevisiae and Saccharomyces bayanus and appears to derive from the alpha-proteobacteria. / Dissertation

Cette étude a permis de démontrer les fonctions et les mécanismes de la 17bêtahydroxystéroïde déshydrogénase de type 1 (17β-HSD1) et de la stéroïde sulfatase (STS) au niveau du cancer du sein, y compris la cinétique moléculaire et cellulaire, la liaison du ligand étudiée par la titration de fluorescence, la régulation des stéroïdes et la régulation mutuelle entre les enzymes stéroïdiennes et les cellules cancéreuses du sein. 1), L’inhibition de la 17β-HSD1 par son substrat a été démontrée par la cinétique enzymatique au niveau cellulaire pour la première fois, soutenant ainsi la fonction biologique de l’inhibition produite par le substrat. 2), En tant qu’inhibiteur, la dihydrotestostérone (DHT) n’a pas affecté la concentration du substrat estrone (E1) à laquelle l’activité enzymatique a commencé à diminuer, mais certaines augmentations de vitesse ont été observées, suggérant une diminution significative de l’inhibition par le substrat. 3), Les résultats de la modulation de l’ARNm ont démontré que la transcription du gène codant la 17β-HSD7 diminuait en réponse à l’inhibition de la 17β-HSD1 ou au knockdown dans les cellules du cancer du sein par la modification estradiol (E2). 4), L’expression de la STS est stimulée par E2 de manière à générer une rétroaction positive, ce qui favorise la biosynthèse de E2 dans les cellules de cancer du sein. 5), L’inhibition conjointe de la STS et de la 17β-HSD7 pourrait bloquer leurs activités enzymatiques, diminuant ainsi la formation de E2, mais rétablissant la formation de DHT, réduisant de façon synergique la prolifération cellulaire et induisant l’arrêt du cycle cellulaire en G0 / G1. 6), Les 17β-HSD7 et STS synthétisent E2 et sont toutes deux régulées par E2. Ainsi, elles forment un groupe fonctionnel d’enzymes mutuellement positivement corrélées, l’inhibition de l’une peut réduire l’expression d’une autre, amplifiant ainsi potentiellement les traitements inhibiteurs. 7), Le recepteur estrogenique α ERα a été non seulement régulés à la baisse par E2, mais également réduits par la DHT grâce à l’activation des récepteurs aux androgènes (AR). En conclusion, la 17β-HSD1 et la 17β-HSD7 jouent des rôles essentiels dans la conversion et la régulation des hormones sexuelles, et l’inhibition conjointe de la STS et de la 17β-HSD7 constitue une nouvelle stratégie pour le traitement hormonal des cancers du sein sensibles aux estrogènes. / Human 17beta-hydroxysteroid dehydrogenase type 1 (17β-HSD1), 17betahydroxysteroid dehydrogenase type 7 (17β-HSD7) and steroid sulfatase (STS) play a crucial role in regulating estrogen synthesis for breast cancer (BC). However, mutual regulation of enzymes and the interaction of these steroids (estrogens, androgens and their precursor dehydroepiandrosterone (DHEA)) are not clear. This study demonstrated the functions and mechanisms including kinetics at molecular level and in cells, ligand binding using fluorescence titration, regulation of steroids and mutual regulation between steroid enzymes in BC cells: 1) Substrate inhibition of 17β-HSD1 was shown for the first time by enzyme kinetics at the cell level, supporting the biological function of substrate inhibition. 2) As an inhibitor, dihydrotestosterone (DHT) did not affect the estrone (E1) substrate concentration at which the enzyme activity started to decrease, but some increases in velocity were observed, suggesting a corresponding decrease in substrate inhibition 3) The mRNA modulation results demonstrated that 17β-HSD7 transcription decreased in response to 17β-HSD1 inhibition or knockdown in BC cells due to estradiol (E2) concentration decrease. 4) The expression of STS is stimulated by E2 in a positive-feedback manner which finally promotes E2 biosynthesis within BC cells. 5) The joint inhibition of STS and 17β-HSD7 could block the activities of these enzymes, thus decreasing E2 formation but restoring DHT formation, to synergistically reduce cell proliferation and induce G0/G1 cell cycle arrest. 6) 17β-HSD7 and STS can synthesize E2 and are all regulated by E2. Thus, they form a functional group of enzymes mutually positively correlated, inhibition of one can reduce the expression of the other, thereby potentially amplifying the inhibitory effects. 7) Estrogen Receptor α (ERα) is not only down-regulated by E2, but also reduced by DHT though androgen receptor (AR) activation. In conclusion, 17β-HSD1 and 17β-HSD7 play essential roles in sex-hormone conversion and regulation, and the joint inhibition of STS and 17β-HSD7 constitutes a novel strategy for hormonal treatment of estrogen-receptor positive BC

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