Impact of short‐term Dutasteride treatment on prostate‐specific membrane antigen expression in a mouse xenograft model

Abstract Background Dutasteride has been shown to increase expression of the prostate‐specific membrane antigen (PSMA) in prostate cancer cells in previous in vitro studies. This 5‐alpha‐reductase inhibitor is commonly used for the treatment of symptomatic benign prostatic enlargement. The modulation of PSMA expression might affect PSMA‐based prostate cancer imaging and therapy. Aim The purpose of this work was to further analyze concentration‐dependent effects of Dutasteride on PSMA expression in a mouse xenograft model. Methods and results Four groups of mice bearing LNCaP xenografts were treated for 14 days with daily intraperitoneal injections of either vehicle control or different concentrations of Dutasteride (0.1, 1, 10 mg/kg). Total expression of PSMA, androgen receptor (AR), and caspase‐3 protein was analyzed using immunoblotting (WES). In addition, PSMA, cleaved caspase‐3 and Ki‐67 expression was assessed and quantified by immunohistochemistry. Tumor size was measured by caliper on day 7 and 14, tumor weight was assessed following tissue harvesting. The mean PSMA protein expression in mice increased significantly after treatment with 1 mg/kg (10‐fold) or 10 mg/kg (sixfold) of Dutasteride compared to vehicle control. The mean fluorescence intensity significantly increased by daily injections of 0.1 mg/kg Dutasteride (1.6‐fold) as well as 1 and 10 mg/kg Dutasteride (twofold). While the reduction in tumor volume following treatment with high concentrations of 10 mg/kg Dutasteride was nonsignificant, no changes in AR, caspase‐3, cleaved caspase‐3, and Ki‐67 expression were observed. Conclusion Short‐term Dutasteride treatments with concentrations of 1 and 10 mg/kg significantly increase the total PSMA protein expression in a mouse LNCaP xenograft model. PSMA fluorescence intensity increases significantly even using lower daily concentrations of 0.1 mg/kg Dutasteride. Further investigations are needed to elucidate the impact of Dutasteride treatment on PSMA expression in patients.


| INTRODUCTION
Prostate-specific membrane antigen (PSMA) 1 is a type II transmembrane glycoprotein which is highly expressed in 85%-95% of all prostate cancer lesions. 2,3 Small molecules targeting PSMA are increasingly used for imaging and also therapy of advanced prostate cancer. 4 However, the performance of PSMA targeting applications is directly correlated to the PSMA expression of the respective tumor tissue. 5 Upregulation of cellular PSMA expression following androgen deprivation therapy (ADT) has initially been described in 1996 in primary tumor tissue samples. 6 In vitro and in vivo studies confirmed an increased PSMA expression also following treatment with novel antiandrogens. [7][8][9] In addition, retrospective and primary prospective studies suggested an altered PSMA uptake in patients following ADT or a direct androgen receptor (AR) blockade. [10][11][12] Since the exact regulatory cascade involved is still unknown, indepth knowledge on function and regulation of PSMA may be used to increase the performance of PSMA-based prostate cancer imaging and therapy.
In our previous research, we demonstrated that upregulation of PSMA expression can not only be achieved by the androgen receptor blocker Enzalutamide but also by high concentrations of Dutasteride in vitro. 13 In addition, the effect of Dutasteride on PSMA expression was observed to be time-dependent in LNCaP cells. 14 Dutasteride, a 5-alpha-reductase inhibitor with a welltolerable risk profile is usually prescribed for the treatment of bladder outlet obstructions due to benign prostatic hyperplasia. 15 Considering the low risk of severe side effects, Dutasteride is an interesting candidate to potentially induce PSMA expression prior to PSMA targeting applications.
In this study, we aimed to analyze concentration-dependent effects of Dutasteride on PSMA expression in an LNCaP-bearing mouse xenograft model. In addition, the impact on tumor growth and weight, AR expression, and apoptosis were analyzed.

| Sample processing and immuno-/histological assessment
All harvested tissue samples were divided into two pieces: one part was snap frozen for protein analysis, the other part was fixed in 10%

| Protein simple immunoblotting (WES)
Tumor samples were crushed in liquid nitrogen and resuspended in modified lysis buffer supplemented with a protease inhibitor cocktail (Sigma-Aldrich, Buchs, Switzerland). Afterward, samples were centrifuged for 20 minutes at 13 000 rpm and the supernatant was collected for protein determination. Total protein was measured using a BCA protein assay kit (Thermo Scientific, Lausanne, Switzerland). F I G U R E 3 PSMA and AR expression. Total PSMA protein expression (A) and total AR protein expression (B) were measured on harvested tumor tissue from mice treated with daily intraperitoneal injections of vehicle, 0.1 mg/kg Dutasteride, 1.0 mg/kg Dutasteride, and 10.0 mg/kg Dutasteride for 14 days. PSMA and AR expression is presented as percentage compared to vehicle control. Data are shown as mean ± SE of the mean (SEM) of three to six independent experiments. Statistical analysis was performed using Kruskal-Wallis test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Protein simple immunoblotting (WES) from one representative experiment (C). PSMA fluorescence intensity was measured and quantified using image J software. The results are presented as percentage of PSMA expression compared to vehicle control. Data are shown as mean ± SE of the mean (SEM) of five to seven measurements. Statistical analysis was performed using ANOVA test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (D). Visualization of PSMA expression using immunohistochemistry (E). Representative confocal images of PSMA surface staining. Tumor tissue sections were stained with primary anti-PSMA antibody and detected using FITC (green) conjugated secondary antibody and DAPI (blue, 4 0 ,6-diamidino-2-phenylindole) The samples were mixed and heated at 95 C for 5 minutes to denaturate the proteins and obtain a better protein separation. Then, individual glass microcapillaries were loaded with stacking and separation matrices followed by protein sample loading. During capillary electrophoresis, the proteins were separated by size and then immobilized to the capillary wall. The separated proteins were then   and was significantly higher compared to vehicle control (201 mg ± 22 mg, P < 0.05). Groups treated with 0.1 mg/kg (264 mg ± 55 mg) or 10 mg/kg (199 mg ± 36 mg) showed no significant difference in mean tumor weight compared to vehicle control ( Figure 2B). Proper tumor formation was confirmed by H&E stainings performed on representative samples from all experimental conditions ( Figure 2C).
In addition, immunohistological PSMA staining and quantification of PSMA fluorescence intensity were performed ( Figure 3C,D,E).
Treatment with different concentrations of Dutasteride did not significantly alter apoptosis compared to vehicle control. In mice treated with 0.1 mg/kg Dutasteride, the mean normalized apoptosis was 1.1 ± 0.2 compared to 1.1 ± 0.1 in mice treated with vehicle control.
In the groups treated with 1 and 10 mg/kg Dutasteride, a mean apoptosis of 1.4 ± 0.1 and 1.3 ± 0.1 was observed, respectively.

| DISCUSSION
In this in vivo study, we demonstrate for the first time that daily intraperitoneal injections of 1 and 10 mg/kg of Dutasteride in an LNCaPbearing mouse xenograft model significantly increase total PSMA protein expression. PSMA fluorescence intensity was significantly increased using even lower concentrations of 0.1 mg/kg Dutasteride daily. In addition, we show that Dutasteride does not significantly alter AR, caspase-3, cleaved caspase-3, and Ki-67 expression in vivo.
Applications targeting PSMA especially for imaging, but also for therapy of prostate cancer, is increasingly used. However, the exact biologic function and regulation of PSMA is not yet fully understood. 16 It has been shown that glycosylation is necessary for the enzymatic activity of PSMA, and that after internalization, it follows intracellular pathways to the endosomal compartment. [17][18][19] Furthermore, in vitro data demonstrated that PSMA cross-linking activates AKT, mTOR, and MAPK pathway. 20  Apart from preclinical data, a feasibility study including five patients suggested a possible PSMA enhancing effect using ADT for a median of 9 days (range 6-11 days). 22 Rosar et al demonstrated an increase of PSMA expression of 45-55% in 10 patients planned for radioligand therapy scanned 2-3 weeks following treatment start with Enzalutamide. 11 However, due to small patient cohorts, definitive conclusions cannot be drawn. Others reported inconclusive prospective data in 9 men scanned 3-4 weeks following start of treatment with ADT. 12 Further, retrospective data from patients scanned 3 months following start of treatment with ADT could not demonstrate any significant PSMA alterations, indirectly indicating that a possible PSMA flare up phenomenon might reach its maximum after 2-4 weeks. 10,23 While the influence of molecules altering the AR axis on PSMA expression is increasingly established, we were able to demonstrate that also Dutasteride significantly increases PSMA expression in vitro. 13 Dutasteride is a 5-alpha-reductase inhibitor with a wellknown risk profile used for the treatment of lower urinary tract symptoms due to benign prostatic enlargement. 15

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.