Paul Thelen

Prospects of estrogen receptor β activation in the treatment of castration-resistant prostate cancer

263 Background: ADT is the initial therapy for advanced prostate cancer, and androgen biosynthesis inhibitors (abiraterone-acetate, AA) reveal clinical survival benefit for CRPC. On CRPC progression, therapy sequencing towards androgen receptor (AR) antagonism is applied and AA withdrawal / re-challenge strategies (+AA-AA+AA) succeeded in several case reports. Here we established a VCaP-tumor mouse model for a +AA-AA+AA-sequence and external testosterone-boosts (+AA[-AA+T]+AA). Methods: 20 tumor bearing mice (from 10⁶ VCaP in Matrigel) received surgical ADT and then +AA-AA+AA, (AA intraperitoneal 70mg/kg, daily [Janssen-Cilag]), or +AA[-AA+T]+AA (+0.6 µl/kg Nebido [250mg testosterone-undecaonate/ml, Jenapharm]). Tumor growth was monitored with calipers. When tumors grew > 200 mm³, animals were castrated and treatment-sequences began when CRPC growth became evident (n = 15) or early one week after castration (n = 5). Expression analyses were performed with Experion, CSX-96 and ChemiDoc, chemicals, software (BioRad). Steroids were quantified by Dept. Clin. Chemistry. Results: 20 tumor-bearing mice received ADT. To interfere with ADT failure for 15 mice +AA-AA+AA (n = 7) or +AA[-AA+T]+AA (n = 8) commenced when tumors doubled within two weeks. Animals benefited a reduction of up to 210 mm³ receiving +AA-AA+AA, whereas a T-Boost in +AA[-AA+T]+AA revealed a loss of solid tumor mass as mean 42% (+/-21%) liquid effusion. No complete remission was observed, tumors > 1 cm³ were considered dead-of-disease (DOD). Another group (n = 5) was AA-treated one week after castration. There +AA-AA+AA and +AA[-AA+T]+AA resulted for one animal in DOD, however tumors of up to 990 mm³ underwent complete remissions (n = 4) and tumor-free untreated survival. AA-withdrawal is associated with progesterone decrease and DHEA increase. AR (+ AR-Vs) decreases to 95% expression immediately after -AA and to 39% (no AR-Vs) upon +T. Conclusions: Therapy sequences with AA-withdrawal/rechallenge may lead to tumor regression and complete tumor-free survival. Concomitant with tumor regression upon AA-withdrawal, AR overexpression is reduced, which may be boosted with testosterone supplementation.

Recent breakthrough therapies targeting androgen receptor signalling in castration resistant prostate cancer (CRPC) involve multifunctional androgen receptor (AR) blockade and exhaustive androgen deprivation. Nevertheless, limitations to an enduring effectiveness of new drugs are anticipated in resistance mechanisms occurring under such treatments. In this study we used CRPC cell models VCaP and LNCaP as well as AR-negative PC-3- and non-neoplastic epithelial BPH-1-cells treated with 5, 10 or 25 μmol/L abiraterone hydrolyzed from abiraterone acetate (AA). The origin of CYP17A1 up-regulation under AA treatment was investigated in CRPC cell models by qRT-PCR and western-blot procedures. AA treatments of AR positive CRPC cell models led to decreased expression of androgen regulated genes such as PSA. In these cells diminished expression of androgen regulated genes was accompanied by an up-regulation of CYP17A1 expression within short-term treatments. No such effects became evident in AR-negative PC-3 cells. AR directed siRNA (siAR) used in VCaP cells significantly reduced mRNA expression and AR protein abundance. Such interference with AR signalling in the absence of abiraterone acetate also caused a marked up-regulation of CYP17A1 expression. Down-regulation of androgen regulated genes occurs in spite of an elevated expression of CYP17A1, the very target enzyme for this drug. CYP17A1 up-regulation already takes place within such short treatments with AA and does not require adaptation events over several cell cycles. CYP17A1 is also up-regulated in the absence of AA when AR signalling is physically eliminated by siAR. These results reveal an immediate counter-regulation of CYP17A1 expression whenever AR-signalling is inhibited adequately but not a persisting adaptation yielding drug resistance.

154 Background: Androgen deprivation and direct androgen receptor (AR) antagonism are standard treatments for castration resistant prostate cancer (CRPC). The advent of new drugs such as abiraterone acetate (AA) and enzalutamide rendered highly efficient implementations of both anti-androgen strategies. However, reports of therapy resistance or cross resistance soon accompanied reports of successful clinical trials of these new drugs. Moreover, case reports of an AA-withdrawal phenomenon surfaced, an outcome normally associated with AR antagonism e.g. by bicalutamide with high AR-affinity and the potential to switch from antagonism to AR-activation. Methods: CRPC cell model VCaP and VCaP resistant to permanent treatment of 5 µmol/L AA (Janssen-Cilag, Germany) were analyzed under AA and for AA withdrawal. Expression of AR, AR-V7, androgen regulated genes, CYP17A1 and AKR1C3 was quantitated by real time RT-PCR. Further analyses were performed by western blots, prostate-specific antigen (PSA) ELISA kits and cell proliferation BrdU tests. Results: VCaP cells resistant to 5 µmol/L AA after 7 months revealed excessive AR and CYP17A1 expression and a marked decline of tumor cell proliferation in response to AA withdrawal or to a similar extent to added 1 nmol/L testosterone. Proliferation dropped to less than 50% when both procedures were combined. Elimination of tumor cells was accompanied by a massive release of PSA which was most pronounced in the combined treatment. Conclusions: Overexpression of AR turning into a liability as a therapeutic target for a testosterone surge has been reported by several research groups. AA withdrawal from AA resistant cells complies with these necessities by overexpression of AR and CYP17A1 formerly arrested by AA. In patients a PSA decline upon AA withdrawal may be explained by an excessive loss of PSA secreting tumor cells. Therefore, AA qualifies uniquely for effective intermittent androgen deprivation of CRPC.

BACKGROUND The primary therapeutic target for non-organ-confined prostate cancer is the androgen receptor (AR). Main strategies to ablate AR function are androgen depletion and direct receptor blockade by AR antagonists. However, incurable castration resistant prostate cancer (CRPC) develops resistance mechanisms to cope with trace amounts of androgen including AR overexpression and mutation in the AR ligand binding domain. METHODS The CRPC cell model VCaP derivative of a prostate cancer bone metastasis was used in vitro and in nude mice in vivo to examine the effects of immediate testosterone boost on CRPC cells. In addition, a testosterone tolerant cell model was established by incremental acclimatization of VCaP cells to 1 nM testosterone. The effects of androgen withdrawal and testosterone boosts on gene expression were assessed by quantitative real-time polymerase chain reaction, ELISA, and Western blots. Tumor cell proliferation was evaluated with a BrdU test. RESULTS Testosterone boosts on CRPC VCaP cells eliminate tumor cells to a higher extent than androgen withdrawal in androgen tolerant cells. The pronounced decrease of tumor cell proliferation was accompanied by a marked downregulation of AR expression regarding full-length AR and splice variant AR V7. CONCLUSIONS Acquiring castration resistance of prostate cancer cells by AR overexpression and amplification obviously sensitizes such cells to testosterone concentrations as low as physiological values. This introduces novel therapeutic means to treat CRPC with non-toxic measures and may find clinical implementation in intermittent androgen deprivation regimens. Prostate 73: 1699–1709, 2013.

130 Background: Recent breakthrough therapies targeting androgen receptor signalling in CRPC involve multifunctional androgen receptor (AR) blockade and exhaustive androgen deprivation. Nevertheless, limitations to an enduring effectiveness of new drugs are anticipated in resistance mechanisms occurring under such treatments. Here we specify the origin of CYP17A1 up-regulation under abiraterone acetate treatment in CRPC cell models. Methods: CRPC cell models VCaP and LNCaP as well as AR-negative PC-3- and non-neoplastic epithelial BPH-1-cells were treated with various concentrations of abiraterone hydrolyzed from abiraterone acetate (AA, Janssen-Cilag, Germany). Functional analyses by RNA interference were done with AR- and CYP17A1-siRNA. Expression of androgen regulated genes and CYP17A1, AKR1C3 was quantitated by real time PCR. Protein analyses were performed by western blots and PSA secretion was assessed by ELISA kits. Results: AA treatments (5 to 25 µM) of AR positive CRPC cell models led to decreased expression of androgen regulated genes such as PSA. In these cells diminished expression of androgen regulated genes was accompanied by an up-regulation of CYP17A1 expression (>2-fold at 25µM AA) within short treatments of 24 hours. No such effects became evident in AR-negative PC-3 cells. AR directed siRNA (siAR) used in VCaP cells significantly reduced mRNA expression and AR protein abundance. Such interference with AR signalling in the absence of AA also caused an (up to 8-fold) up-regulation of CYP17A1 expression. Conclusions: In AR-positive cells AA treatments cause a down-regulation of androgen regulated genes in spite of an elevated expression of CYP17A1, the very target enzyme for this drug. CYP17A1 up-regulation already occurs within 24 hour treatments of AA and does not require adaptation events over several cell cycles. CYP17A1 is also up-regulated in the absence of AA when AR signalling is physically eliminated by siAR. These results reveal an immediate counter-regulation of CYP17A1 expression whenever AR-signalling is inhibited adequately but not a persisting adaptation yielding drug resistance.

Testicular germ cell tumours (TGCTs) are the most common malignancy in young men aged 18–35 years. They are clinically and histologically subdivided into seminomas and non-seminomas. Cadherins are calcium-dependent transmembrane proteins of the group of adhesion proteins. They play a role in the stabilization of cell-cell contacts, the embryonic morphogenesis, in the maintenance of cell polarity and signal transduction. N-cadherin (CDH2), the neuronal cadherin, stimulates cell-cell contacts during migration and invasion of cells and is able to suppress tumour cell growth.

As the majority of prostate cancers (PC) express estrogen receptors, we evaluated the combination of radiation and estrogenic stimulation (estrogen and genistein) on the radiosensitivity of PC cells in vitro.

Oncological results of radiotherapy for locally advanced prostate cancer (PC) are significantly improved by simultaneous application of LHRH analoga (e.g. goserelin). As 85% of PC express LHRH receptors, we investigated the interaction of goserelin incubation with radiotherapy under androgen-deprived conditions in vitro.