Investigating the Androgen Metabolizing Enzymes HSD17B6 & DHRS7 in Prostate Cancer

Prostate cancer (adenocarcinoma of the prostate, PCa) is one of the most common cancers in the world. Several treatment options exist, including surgery and radiotherapy with curative intention, hormonal therapy, and chemotherapy. The etiology of PCa is heavily dependent on androgens activating the androgen receptor (AR) with most drugs and therapy approaches focusing on this aspect. Unfortunately, cancer progression to a state that is characterized by therapy resistance and metastases is common and this late stage of PCa is considered incurable. Therefore, there is a need to further understand the underlying biology of this malignancy in order to discover new therapeutic approaches.
The first project addressed the 17β-hydroxysteroid dehydrogenase (HSD17B) 6. This enzyme performs the oxidation of 3α-androstanediol (3α-Adiol) to 5α-dihydrotestosterone (5α-DHT), thereby providing the AR with its main ligand. This reaction is an essential step in the so-called “backdoor” pathway of androgen synthesis and is suspected to play a role in PCa. Other enzymes were reported to perform the same reaction and together with HSD17B6 they are referred to as 3α-hydroxysteroid dehydrogenases (3α-HSDs). It was the goal to compare the enzymes’ ability to oxidize
not only 3α-Adiol, but also 3β-Adiol as well as a potential reverse reaction by reducing 5α-DHT. For that purpose, a reporter gene assay (transactivation assay) was used in intact CV-1 cells. This revealed that several enzymes were able to cause an AR activation by oxidation of 3α-Adiol, but HSD17B6 was uniquely able to oxidize 3β-Adiol as well. Oxidation of 3α-androsterone (3α-ADT) leads to the formation of 5α-DHT as well (via the intermediate product 5α-androstanedione or 3α-Adiol). A preliminary experiment showed that HSD17B6 was again the only enzyme to use not only 3α-ADT as substrate, but also its isomer 3β-epiandrosterone (3β-ADT). Based on the finding that HSD17B6 has a wider range of substrates that can lead to AR activation, we were interested in finding potential inhibitors for HSD17B6. While colleagues of the Computational Pharmacy group of the University of Basel generated a homology model of HSD17B6 to perform a virtual screening, I generated different expression constructs that can be used to establish an enzymatic assay for testing any hits obtained from the screening. Discovery of an inhibitor can help to further understand the importance of androgen synthesis in PCa and ideally may even find clinical application. The main project investigated the role of the “orphan” enzyme dehydrogenase/reductase (DHRS) 7 in PCa. This enzyme has so far no assigned physiological substrate but it appears to be a tumor suppressor in the prostate. This is based on publications that showed decreased expression of DHRS7 in clinical PCa samples and that reduced expression in the PCa cell line LNCaP leads to increased
proliferation. In addition, it was shown that DHRS7 is capable of reducing, and therefore inactivating, the AR ligand 5α-DHT to 3α-Adiol. The goal of this project was to further understand the role of DHRS7 in PCa. Experiments indicated that the function of DHRS7 in PCa cells is surprisingly unrelated to AR signaling. In order to discover other potentially related molecular pathways, an siRNA-mediated knock-down of DHRS7 in LNCaP cells was performed and followed by proteomic analysis. We discovered an increased expression of the epidermal growth factor (EGF) receptor (EGFR), a well known drug target in other cancers with both antibodies and small molecule inhibitors used to treat patients. Our findings were confirmed by molecular biology techniques in the LNCaP cells and in two additional PCa cell lines. Treating the cells with EGF after knock-down of DHRS7 led to increased activation of the EGFR by phosphorylation. Trends of increased phosphorylation of downstream signaling proteins were observed as well. By using data obtained from clinical samples of PCa, we were able to show a negative correlation of DHRS7 and EGFR expression and finally, analysis of patient survival rates showed a positive correlation between high expression of DHRS7 and better survival rates while high expression of EGFR correlated negatively with survival rates. These results support the hypothesis that DHRS7 is a tumor suppressor in the prostate but future studies are required to further describe the relationship between DHRS7 and EGFR and to identify the substrate of DHRS7.