Toward the identification of a function of the “orphan” enzyme DHRS7 Inauguraldissertation zur Erlangung der Würde eines Doktors der Philosophie vorgelegt der Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel von Selene Araya, aus Lugano, Tessin Basel, 2018 Originaldokument gespeichert auf dem Dokumentenserver der Universität Basel edoc.unibas.ch Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät auf Antrag von Prof. Dr. Alex Odermatt (Fakultätsverantwortlicher) und Prof. Dr. Michael Arand (Korreferent) Basel, den 26.6.2018 ________________________ Dekan Prof. Dr. Martin Spiess I. List of Abbreviations 3α/βAdiol 3α/β-Androstanediol (5α-Androstane-3α/β,17β-diol) 3α/βHSD 3α/β-hydroxysteroid dehydrogenase 17β-HSD 17β-Hydroxysteroid Dehydrogenase 17αOHProg 17α-Hydroxyprogesterone 20α/βOHProg 20α/β-Hydroxyprogesterone 17α,20α/βdiOHProg 20α/βdihydroxyprogesterone ADT Androgen deprivation therapy ANOVA Analysis of variance AR Androgen Receptor AKR Aldo-Keto Reductase ATCC American Type Culture Collection CAM Cell Adhesion Molecule CYP Cytochrome P450 CBR1 Carbonyl reductase 1 CRPC Castration resistant prostate cancer Ct-value Cycle threshold-value DHRS7 (B/C) Dehydrogenase/Reductase Short Chain Dehydrogenase Family Member 7 (B/C) DHEA Dehydroepiandrosterone DHP Dehydroprogesterone DHT 5α-Dihydrotestosterone DMEM Dulbecco's Modified Eagle's Medium DMSO Dimethyl Sulfoxide DTT Dithiothreitol E1 Estrone E2 Estradiol ECM Extracellular Membrane EDTA Ethylenediaminetetraacetic acid EMT Epithelial-mesenchymal transition ER Endoplasmic Reticulum ERα/β Estrogen Receptor α/β FBS Fetal Bovine Serum 3 FDR False discovery rate FGF Fibroblast growth factor HEPES 4-(2-Hydroxyethyl)-1-Piperazineethanesulfonic Acid HMDB Human Metabolome Database HPLC High Performance Liquid Chromatography HSD Hydroxysteroid Dehydrogenase IC50 Half-Maximal Inhibitory Concentration LNCaP Lymph node carcinoma of the prostate mRNA Messenger Ribonucleic Acid n.d. Not Detected NADPH Nicotinamide Adenine Dinucleotide Phosphate 6 NR Nuclear Receptor MAPK Mitogen-activated protein kinase MEM Minimum Essential Medium MW Molecular Weight PCa Prostate cancer PCR Polymerase Chain Reaction PPAR Peroxisome Proliferator-Activated Receptor PVDF Polyvinylidene fluoride RIPA Radioimmunoprecipitation assay RT Room Temperature SD Standard Deviation SDS Sodium dodecyl sulfate SDR Short-Chain Dehydrogenase/Reductase T Testosterone TBS-T Tris-buffered saline with 0.1% (v/v) Tween 20 TEMED Tetramethylethylenediamine TLC Thin-Layer Chromatography Tris Tris(hydroxymethyl)aminomethane (UHP)LC-MS/MS (Ultra High Performance) Liquid Chromatography/tandem Mass Spectrometry 4 Table of Contents I. List of Abbreviations .............................................................................................................. 3 1 Summary ............................................................................................................................... 6 2 Introduction........................................................................................................................... 7 2.1 Prostate Cancer ...................................................................................................................... 7 2.2 Associations of Short Chain Dehydrogenases (SDRs) and Aldo Keto Reductases (AKRs) ............ with Cancer .......................................................................................................................... 11 2.3 Carbonyl Reductases of the SDR and AKR Families ................................................................ 17 2.4 The “Orphan” Enzyme DHRS7 ............................................................................................... 21 2.5 Deorphanization ................................................................................................................... 25 3 Aims of the Thesis ................................................................................................................ 27 4 Chapter 1: Toward the Identification of Substrates of DHRS7 ............................................... 29 4.1 Published article: DHRS7 (SDR34C1) - a New Player in the Regulation of Androgen .................. Receptor Function by Inactivation of 5α-Dihydrotestosterone? ............................................ 34 4.2 Further Characterization of DHRS7 Activity ........................................................................... 43 5 Chapter 2: Functional and Phenotypical Characterization Following DHRS7 Depletion ......... 67 5.1 Assessing the Phenotype of Breast and Adrenal Cancer Cell Lines under siRNA ........................ mediated DHRS7 Silencing .................................................................................................... 69 5.2 Assessing the Phenotype of LNCaP Prostate Cancer Cells under siRNA ..................................... mediated DHRS7 Silencing by Untargeted Proteomics .......................................................... 81 6 General Discussion ............................................................................................................. 115 7 Acknowledgements ........................................................................................................... 118 8 Literature ........................................................................................................................... 119 9 Supplementary Data .......................................................................................................... 134 5 1 Summary The short chain dehydrogenase DHRS7 has been previously described as a possible tumor suppressor, regulated during prostate cancer progression, with the potential of being a marker of prostate cancer. However, the function of DHRS7 and substrates with good affinity to be of potential physiological meaning remains unknown leaving it still classified as an “orphan” enzyme. These observations furthered the need to identify physiologically relevant substrates and understand the mechanisms affected by DHRS7 in endogenously expressing cell lines. In this thesis, in vitro assays were performed to help to characterize the activity of DHRS7. They showed DHRS7 has 3α and 20β reductase activities on the carbonyl of steroidal substrates, and interestingly revealed conversion of the main ligand of the androgen receptor dihydrotestosterone (DHT) toward the inactive 5α-androstane3α,17β-diol (3α-Adiol). This activity was further characterized through androgen receptor (AR) transactivation activity in an overexpressing system and biochemically through kinetic enzyme turnover assays. Moreover, this activity allowed to develop a novel screening lysate assay for substrates and inhibitors identification. However, no other promising physiologically relevant substrates were revealed. In the second part, the phenotypic changes upon DHRS7 silencing were investigated in endogenous cell models by functional cancer assays, mass spectrometry and untargeted proteomics supported by cell cycle analysis, immunofluorescence, real time qPCR and western blot. These results disproved the modulation of the endogenous AR in the prostate cancer cell line LNCaP under DHRS7 depletion but supported the hypothesis of DHRS7 having a tumor suppressor role with protein changes observed for cell cycle, adhesion and migration relevant to the phenotype. Interestingly, protein changes involved in mechanisms relevant for tumor biogenesis were observed. In conclusion, the results presented in this thesis extend the knowledge about DHRS7 in vitro activity, provide the characterization of an in vitro tool to test hypothesized substrates and inhibitors and suggest further investigation toward androgen receptor independent mechanisms. 6 2 Introduction 2.1 Prostate Cancer Prostate cancer is the second most common cancer in men and is the leading cause of cancer related death among men globally (1.1 million cases, 307’000 deaths per year) [1]. Even though the prognosis is improving (the 5 years survival rate in Europe is currently about 93%) [2], some patients develop an aggressive form of the disease, despite primary treatment, leading to disease progression and death. The etiology of prostate cancer is complex but known to be associated with the non-modifiable risk factors age, ethnicity, and a family history of the disease [3, 4], and with environmental risk factors such as insulin- like growth factor-I (IGF-I) [5]. Inherent to this complexity seems to be the diversity of involved genetic and environmental factors. The prostate is an exocrine gland in the male reproductive system located underneath the urinary bladder, in front of the rectum, and that surrounds the urethra. Usually, the cellular origin of prostate cancer is attributed to the epithelial cells of the peripheral zone (PZ) [6] (Figure 1A). The prostate zones consist of the prostatic epithelial acini, which are glandular structures arranged in a fibromuscular stromal network composed of columnar luminal and basal layer cells (Figure 1B). The basal layer is populated by stem cells, transit amplifying cells and committed basal cells. The prostatic epithelial acini are responsible for prostate secretions that drain into the urethra together with the spermatozoa and secretions from the seminal vesicles [7]. Prostatic acini, which progress into cancerous acini, are identified by specific micro environmental and molecular changes and with luminal hyperproliferation. The luminal epithelial cells, which in the healthy acini represent up to 60% of the