
==== Front
Neoplasia
Neoplasia
Neoplasia (New York, N.Y.)
1522-8002
1476-5586
Neoplasia Press

S1476-5586(24)00087-3
10.1016/j.neo.2024.101045
101045
Original Research
Regulation of prostate-specific membrane antigen (PSMA) expression in prostate cancer cells after treatment with dutasteride and lovastatin
Kuzmanov Aleksandar
Salemi Souzan
Eberli Daniel
Kranzbühler Benedikt benedikt.kranzbuehler@usz.ch
⁎
Department of Urology, University Hospital Zürich, University of Zurich, Laboratory for Urologic Oncology and Stem Cell Therapy, Zurich, Switzerland
⁎ Corresponding author at: Department of Urology, University Hospital Zürich. Frauenklinikstrasse 10, 8091 Zürich, Switzerland. benedikt.kranzbuehler@usz.ch
05 9 2024
11 2024
05 9 2024
57 1010451 7 2024
31 7 2024
26 8 2024
© 2024 Published by Elsevier Inc.
2024

https://creativecommons.org/licenses/by-nc/4.0/ This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-nc/4.0/).
PSMA expression gradually increases from benign prostatic hyperplasia to adenocarcinoma of the prostate and is therefore used for the development of improved diagnostic (PSMA)‐based prostate cancer imaging tools. Pharmacological induction of PSMA is therefore eminent to further improve the detection rate of PSMA-based imaging. Our previous studies have demonstrated that lovastatin (Lova) and dutasteride (Duta) are able to induce PSMA expression. However, the mechanisms by which PSMA is regulated in prostate cancer remain poorly understood. Androgen receptor (AR) and homeobox B13 (HOXB13) are the best known regulators of PSMA, hence in the present study we aimed to explore the PSMA regulation by HOXB13 and AR signaling in LNCaP and VCaP cells following treatments with Lova and Duta. Furthermore, our previous research revealed a growth arrest in prostate cancer cells after Lova, but not after Duta treatment. To understand this discrepancy, we explored the influence of Lova and Duta on well known tumor growth promoters, such as AR, the mTOR/Akt signaling pathways and Cyclin D1. Our results showed that treatment with Lova leads to a significant inhibition of the investigated tumor promoters and results in growth regression of LNCaP and VCaP cells. In contrast, Duta does not show these effects. Furthermore, we confirm the cooperative effect of HOXB13 and AR in regulating PSMA in LNCaP cells, and extend the investigations to an additional prostate cancer cell line (VCaP).

Keywords

Lovastatin (LOVA)
Dutasteride (DUTA)
Androgen receptor (AR)
HOXB13
mTOR/ Akt signaling pathways
Cyclin D1
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pmcIntroduction

Prostate cancer remains one of the leading causes of cancer-related death among men in developed countries and one of the most frequently diagnosed cancers in men worldwide [1,2]. Patients with localized prostate cancer generally have a good prognosis. However, overall survival is significantly reduced in patients presenting with metastatic prostate cancer at diagnosis [3]. Surgical or chemical castration targeting the androgen receptor (AR) signaling axis is still the mainstay of treatment in advanced prostate cancer. However, resistance mechanisms limit the efficacy of new treatment compounds [4].

Prostate-specific membrane antigen (PSMA) represents a promising novel target for imaging and therapy of prostate cancer [5]. Interestingly, the PSMA regulation itself remains poorly understood. Besides an indirect regulation via AR, Bakht et al. identified HOXB13 as a direct positive regulator of PSMA in LNCaP cells [6]. In addition, we previously showed a significant PSMA upregulation after lovastatin and dutasteride treatment [7].

Lovastatin (Lova) is a cholesterol lowering compound that inhibits 3-hydroxy 3-methylglutaryl coenzyme A (HMG-CoA) reductase, which in turn is the rate-limiting enzyme of the mevalonate pathway, [8]. Furthermore, Lova inhibits the growth of RWPE-1 and 22Rv1 cells in vitro [[9], [10], [11]]. Our own research confirmed the inhibitory effect of Lova on the AR expression and on cell growth of LNCaP, C4-2 and VCaP prostate cancer cells [7].

Dutasteride (Duta) is a well-established 5α-reductase inhibitor, which is regularly used for the treatment of benign prostatic enlargement [12]. Several studies investigated the effect of Duta in localized prostate cancer [[13], [14], [15]]. In our previous study, we did not observe any effect of Duta on the growth of LNCaP, C4-2 and VCaP prostate cancer cells [7].

In the present study, we aim to explore differences in PSMA regulation via HOXB13 and AR signaling in LNCaP and VCaP cells following treatment with Lova and Duta. In addition, knowing that activation of mTOR and Akt signaling pathways are frequent events in prostate cancer and facilitate tumor formation and disease progression [[16], [17], [18], [19]], we aim to investigate whether these signaling pathways are targeted by Duta or Lova.

Materials and methods

Cell culture

Prostate cancer cells (LNCaP and VCaP) were purchased from American Type Culture Collection (ATCC, Manassas, USA). PNT1A epithelial cells were a generous gift from Pirkko Härkönen (Institute of Biomedicine, University of Turku, Finnland). As PSMA/AR are not expressed in PNT1A cells, these cells were used as negative control when PSMA status was explored. LNCaP and PNT1A cells were cultured in RPMI Medium 1640 (1x) [+] L-Glutamine (Gibco) supplemented with 10 % FBS and 1 % penicillin/streptomycin (P/S). VCaP cells, derived from a vertebral metastatic prostate cancer lesion [8], were grown in DMEM (BioConcept) High Glucose (4,5g/l) with stable Glutamine and Sodiumpyruvate, supplemented with 10 % FBS and 1 % penicillin/streptomycin (P/S). All cells were incubated at 37°C with 5 % CO2. Cells were used for up to 10 passages. Medium was changed twice a week.

Treatment with pharmacological compounds

LNCaP cells were treated for up to 7 days with different concentrations of dutasteride (0.25 μM, 0.5 μM and 1 μM) or lovastatin (0.5 μM, 1 μM, 2 μM, 5 μM and 10 μM) (both purchased from Selleckchem, Luzern, Switzerland) according to the experimental conditions listed above. VCaP cells were treated for 14 days (due to slow growth rate) with the same combination of compounds. The vehicle used was 0.1 % dimethyl sulfoxide (DMSO). Medium containing compounds was generally changed on day 3 after initial treatment. All experiments were performed in triplicate.

Protein simple WES immunoblotting

After 7 and 14 days of culturing respectively, LNCaP and VCaP cells were washed with cold PBS supplemented with a protease inhibitor cocktail (Sigma-Aldrich) and lysed with modified lysis buffer. Total protein was measured with the BCA Protein Assay Kit (ThermoFisher SCIENTIFIC, Lausanne, Switzerland). Protein at a concentration of 1 mg/ml was used for the WES sample preparation using the 12-230 kDa cartridge kit. Proteins were separated in WES with a capillary cartridge according to the manufacturer's protocols (Protein Simple WES, Germany). Primary antibodies used were mouse anti-PSMA/FOLH1 (R&D Systems, used 4:100), rabbit anti-AR (Cell Signaling, Cat. N: D6F11, used 1:100), rabbit anti-HOXB13 (Cell Signaling, Cat. N: 90944, used 1:50), rabbit anti-Cyclin D1 (Cell Signaling, Cat. N: 2922, used 1:50), rabbit anti-4EBP1 (Cell Signaling, Cat. N: 9644, used 1:50), rabbit anti-eIF4E (Cell Signaling, Cat. N: 9742, used 1:50), rabbit anti-peIF4G (Cell Signaling, Cat. N: 2441, used 1:50), mouse anti-pAkt (Cell Signaling, Cat. N: 4051, used 1:50), rabbit anti-Akt (Cell Signaling, Cat. N: 4691, used 1:50), rabbit anti-p4EBP1 (236B4) (Cell Signaling, Cat. N: 2855, used 1:100). Mouse anti-GAPDH (Novus Biologicals Europe, Cat. N: NB300-221, used 1:100) served as internal control.

Statistical analysis

Data analysis was performed using GraphPad Prism (GraphPad Software, Inc., La Jolla, CA, version 7). Mann-Whitney nonparametric t-test was performed to determine statistical significance. P-values < 0.05 were considered significant. All data presented is expressed as means with corresponding standard error of the mean (± SEM).

Results

Effect of lovastatin and dutasteride on PSMA, AR and HOXB13 protein expression in prostate cancer cells

Initial experiments using protein simple immunoblotting were performed to assess the effect of Lova and Duta on PSMA, AR and HOXB13 protein expression in LNCaP and VCaP cells.

Lovastatin - Impact on PSMA, AR and HOXB13 protein expression

PSMA expression was significantly increased following a 7-day treatment of LNCaP cells with Lova at concentrations of 1 μM (160 % ± 30 %, p < 0.01), 2 μM (220 % ± 50 %, p < 0.001), 5 μM (238 % ± 61 %, p < 0.001) and 10 μM (259 % ± 70 %, p < 0.001) compared to control (Fig. 1A). A 14-day of treatment of VCaP cells led to a similar PSMA induction (Fig. 1B).Fig. 1 Whole PSMA, AR and HOXB13 protein expression analyzed by protein simple immunoblotting. (A) LNCaP cells were treated for 7 days with different concentrations of Lova (0.5, 1, 2, 5 and 10 μM). (B) VCaP cells were treated for 14 days with the same Lova concentrations. PSMA, AR and HOXB13 expression is presented as the percentage of protein expression compared to vehicle control. Data is shown as mean with standard error of the mean (± SEM) of three independent experiments. *P < .05, **P < .01, ***P < .001.

Fig 1

AR expression was significantly inhibited in LNCaP cells after a 7-day treatment with Lova at concentrations of 1 μM (86 % ± 4 %, p < 0.05), 2 μM (74 % ± 4 %, p < 0.05), 5 μM (51 % ± 4, p < 0.01) and 10 μM (25 % ± 2 %, p < 0.001) (Fig. 1A). A 14-day treatment of VCaP cells led to a significant reduction of AR expression only in the cells treated with Lova concentrations of 10 μM (80 % ± 3 %, p < 0.05) (Fig. 1B).

Furthermore, a significant increase in total HOXB13 protein expression resulted from a treatment of LNCaP cells with Lova at concentrations of 0.5 μM (258 % ± 54 %, p < 0.001) and 1 μM (359 % ± 42 %, p < 0.001) (Fig. 1A). In VCaP cells, a significant upregulation of HOXB13 was detected when cells were treated with Lova at concentrations of 1 μM (201 % ± 20 %, p < 0.01), 2 μM (260 % ± 25 %, p < 0.01), 5 μM (280 % ± 30 %, p < 0.01) and 10 μM (340 % ± 42 %, p < 0.001) (Fig. 1B).

Dutasteride - Impact on PSMA, AR and HOXB13 protein expression

Total PSMA expression was significantly increased after a 7-day treatment of LNCaP cells with Duta at concentrations of 0.25 μM (168 % ± 24 %, p < 0.05), 0.5 μM (215 % ± 42 %, p < 0.01) and 1 μM (339 % ± 68 %, p < 0.001) compared to vehicle control (Fig. 2A). In VCaP cells, an induced total PSMA expression was observed after a 14-day treatment with Duta at concentrations of 0.5 μM (134 % ± 26 %, p < 0.01) and 1 μM (255 % ± 60 %, p < 0.001) compared to vehicle control (Fig. 2B).Fig. 2 Whole PSMA, AR and HOXB13 protein expression analyzed by protein simple immunoblotting. (A) LNCaP cells were treated for 7 days with different concentrations of Duta (0.25, 0.5 and 1 μM). (B) VCaP cells were treated for 14 days with the same Duta concentrations. Data is shown as mean with standard error of the mean (± SEM) of three independent experiments. *P < .05, **P < .01, ***P < .001.

Fig 2

In contrast to PSMA, AR expression showed no significant change in both LNCaP and VCaP cells following treatment with different concentrations of Duta (Fig. 2, Fig. 2).

In addition, the total HOXB13 expression was significantly increased after a 7-day treatment of LNCaP cells with Duta at concentrations of 0.25 μM (306 % ± 43 %, p < 0.001), 0.5 μM (309 % ± 37 %, p < 0.001) and 1 μM (295 % ± 35 %, p < 0.001) (Fig. 2A). Similarly, in VCaP cells the HOXB13 expression was significantly induced after a 14-day of treatment with Duta at concentrations of 0.5 μM (301 % ± 37 %, p < 0.001) and 1 μM (310 % ± 40 %, p < 0.001) compared to vehicle control (Fig. 2B).

Effect of lovastatin and dutasteride on mTOR/Akt signaling pathways

Protein simple immunoblotting was conducted to analyze the impact of Lova and Duta on the mTOR/Akt signaling pathways in LNCaP and VCaP cells.

Lovastatin - Impact on protein expression of mTOR signaling members

The total p4EBP1 protein expression was significantly decreased after a 7-day treatment of LNCaP cells with Lova at concentrations of 1 μM (55 % ± 12 %, p < 0.05), 2 μM (30 % ± 10 %, p < 0.01), 5 μM (28 % ± 9 %, p < 0.01) and 10 μM (17 % ± 7 %, p < 0.001) compared to vehicle control (Fig. 3A). Similar result were observed for peIF4G when LNCaP cells were treated with Lova at concentrations of 1 μM (55 % ± 14 %, p < 0.05), 2 μM (59 % ± 17 %, p < 0.05), 5 μM (45 % ± 12 %, p < 0.01) and 10 μM (47 % ± 11 %, p < 0.01) (Fig. 3A). Contrary to p4EBP1 and peIF4G, eIF4E was significantly upregulated when cells were treated with Lova at concentrations of 0.5 μM (210 % ± 28 %, p < 0.001), 1 μM (211 % ± 29 %, p < 0.001), 2 μM (213 % ± 30 %, p < 0.001), 5 μM (220 % ± 35 %, p < 0.001) and 10 μM (222 % ± 33 %, p < 0.001) compared to control (Fig. 3A). 4EBP1 protein expression was not affected in LNCaP cells treated with Lova at all tested concentrations (Fig. 3A).Fig. 3 Whole AR, p4EBP1, 4EBP1, eIF4E, peEF4G, pAkt and Akt protein expression analyzed by protein simple immunoblotting. (A) LNCaP cells were treated for 7 days with different concentrations of Lova (0.5, 1, 2, 5 and 10 μM). (B) VCaP cells were treated for 14 days with the same Lova concentrations. Data is shown as mean with standard error of the mean (± SEM) of three independent experiments. *P < .05, **P < .01, ***P < .001.

Fig 3

In VCaP cells, significant inhibitions in total p4EBP1 (32 % ± 7 %, p < 0.001) and peIF4G (25 % ± 8 %, p < 0.001) protein expression were detected after 14-day applications of Lova at 10 μM compared to vehicle control (Fig. 3B). In contrast, eIF4E was significantly upregulated after a treatment of cells with Lova at concentrations of h 0.5 μM (220 % ± 30 %, p < 0.001), 1 μM (200 % ± 28 %, p < 0.001), 2 μM (219 % ± 29 %, p < 0.001), 5 μM (222 % ± 15 %, p < 0.001) and10 μM (215 % ± 16 %, p < 0.001) compared to control (Fig. 3B). As already observed in LNCaP cells, treatments of VCaP cells with Lova at all tested concentrations did not affect 4EBP1 protein expression (Fig. 3B).

Lovastatin - Impact on protein expression of Akt signaling members

pAkt protein expression was significantly decreased following a 7-day treatment of LNCaP cells with Lova at concentrations of 1 μM (65 % ± 14 %, p < 0.05), 2 μM (64 % ± 15 %, p < 0.05), 5 μM (45 % ± 10 %, p < 0.01) and 10 μM (40 % ± 8 %, p < 0.01) compared to control (Fig. 3A). Total Akt was also inhibited when the cells were treated with Lova at concentrations of 1 μM (40 % ± 15 %, p < 0.01), 2 μM (20 % ± 8 %, p < 0.001), 5 μM (19 % ± 7 %, p < 0.001) and 10 μM Lova (21 % ± 8 %, p < 0.001) (Fig. 3A).

In VCaP cells, the 14-day application of Lova at concentrations of 5 μM (45 % ± 7 %, p < 0.01) and 10 μM (35 % ± 8 %, p < 0.001) significantly inhibited pAkt protein expression (Fig. 3B). We observed a similar outcome for total Akt in these cells after treatment with Lova at 10 μM (52 % ± 8 %, p < 0.01) (Fig. 3B).

Dutasteride – Impact on protein expression of mTOR signaling members

Both, the 7-day treatment of LNCaP (Fig. 4A) and the 14-day treatment of VCaP cells (Fig. 4B) with Duta at concentrations of 0.25, 0.5 and 1 μM had no effect on p4EBP1, peIF4G, 4EBP1, eIF4E protein expression.Fig. 4 Whole AR, p4EBP1, 4EBP1, eIF4E, peEF4G, pAkt and Akt protein expression analyzed by protein simple immunoblotting. (A) LNCaP cells were treated for 7 days with different concentrations Duta (0.25, 0.5 and 1 μM). (B) VCaP cells were treated for 14 days with the same Duta concentrations. Data is shown as mean with standard error of the mean (± SEM) of three independent experiments. *P < .05, **P < .01, ***P < .001.

Fig 4

Dutasteride - Impact on protein expression of Akt signaling members

The treatments of Duta at concentrations of 0.25, 0.5 and 1 μM had no effect on pAkt and total Akt protein expression in both LNCaP (Fig. 4A) and VCaP cells (Fig. 4B).

Effect of lovastatin and dutasteride treatment on Cyclin D1 protein expression in prostate cancer cells

As Cyclin D1 is known to be positively regulated by the mTOR/Akt signaling pathways, we explored its protein expression in LNCaP and VCaP cells after treatment with different concentrations of Lova or Duta.

Lovastatin - Impact on Cyclin D1 protein expression

In LNCaP cells, the total Cyclin D1 protein expression was significantly decreased after a 7-day treatment with Lova at concentrations of 1 μM (70 % ± 12 %, p < 0.05), 2 μM (45 % ± 9 %, p < 0.01), 5 μM (19 % ± 7 %, p < 0.001) and 10 μM (12 % ± 5 %, p < 0.001) compared to vehicle control (Fig. 5A). In VCaP cells, comparable effects were observed only after a 14-day treatment with Lova at 10 μM (35 % ± 5 %, p < 0.001) (Fig. 5B).Fig. 5 Whole Cyclin D1 protein expression analyzed by protein simple immunoblotting. (A) LNCaP cells were treated for 7 days with different concentrations of Lova (0.5, 1, 2, 5 and 10 μM). (B) VCaP cells were treated for 14 days with the same Lova concentrations. Data is shown as mean with standard error of the mean (± SEM) of three independent experiments. *P < .05, **P < .01, ***P < .001.

Fig 5

Dutasteride - Impact on Cyclin D1 protein expression

No effect on Cyclin D1 protein expression was observed after the treatment of both LNCaP (Fig. 6A) and VCaP cells (Fig. 6B) with Duta at all concentrations (0.25, 0.5 and 1 μM) for both 7- and 14-day treatments.Fig. 6 Whole Cyclin D1 protein expression analyzed by protein simple immunoblotting. (A) LNCaP cells were treated for 7 days with different concentrations of Duta (0.25, 0.5 and 1 μM). (B) VCaP cells were treated for 14 days with the same Duta concentrations. Data is shown as mean with standard error of the mean (± SEM) of three independent experiments. *P < .05, **P < .01, ***P < .001.

Fig 6

Discussion

In the present study, we explored the differences in PSMA regulation mediated via HOXB13 and AR signaling in LNCaP and VCaP cells following treatment with Lova and Duta. In addition, we demonstrated for the first time that Lova promotes cell growth arrest via the AR - mTOR/Akt - Cyclin D1 axis.

AR-mediated PSMA regulation has been extensively described [20,21]. Already in 1996, a first study reported PSMA induction following androgen deprivation therapy (ADT) in primary prostate cancer tissue samples [21]. Thereafter, Meller et al. showed an increased PSMA expression following short-term treatment of prostate cancer cells with abiraterone as a second generation ADT [22]. A different study demonstrated a time-dependent AR inhibition using enzalutamide and PSMA induction in vitro [23]. However, ADT is associated with significant side effects in patients and high costs for the health care system. Thus, these compounds are not suited to pharmacologically induce PSMA expression prior to imaging, especially in patients with primary prostate cancer or early biochemical recurrence. Therefore, we focused on exploring alternative, well-established compounds with a low toxicity profile and desired properties on PSMA stimulation.

Lovastatin (Lova) is such an alternative compound with a low toxicity profile that is commonly used to reduce blood lipid concentrations in patients with hypercholesterolemia [10,24]. We focused our investigations on clinically achievable serum concentrations below 10 μM [10].

Our results showed a significant and concentration-dependent AR inhibition following a 7-day treatment of LNCaP cells with Lova concentrations of 1, 2, 5 and 10 μM. Similar results were described by Yang et al. [10]. They reported a significant inhibition of AR expression following treatment of LNCaP cells with ≥ 2 μM Lova for 72 hours.

In VCaP cells, only the highest concentrations of Lova (10 μM) decreased the AR expression. However, PSMA was also induced when cells were treated with the lower concentrations of Lova (1, 2 and 5 μM). These data suggest that there are other critical regulators of PSMA besides AR. In 2023, Bakht et al. have shown that HOXB13 directly interacts with the PSMA gene body and its upstream enhancer in the human carcinoma cell line 22Rv1, thereby promoting PSMA expression [6]. Our findings are in line with Bakht, M. K. et al. [6], by showing that Lova treatments (1, 2, 5 and 10μM) of VCaP cells lead to increased protein levels of both PSMA and HOXB13. Moreover, by revealing that both AR and HOXB13 influence PSMA regulation, our data confirm previously published findings showing the cooperative effect of AR and HOXB13 in driving downstream AR-regulated programs [[25], [26], [27]].

Dutasteride (Duta) is another available compound with an acceptable toxicity profile that is widely used for the treatment of lower urinary tract symptoms caused by benign prostatic enlargement [12]. Given that the highest serum concentrations achieved in men by daily oral doses of 5 mg Duta reach 1 µM [13], we focused on Duta concentrations of up to 1 µM in our current study. Contrary to Lova, our results showed that 7-day treatments of LNCaP and 14-day treatments of VCaP cells with Duta concentrations of 0.25 μM, 0.5 μM and 1 μM do not affect AR expression. Supporting results were observed by Chhipa et al., reporting that mutations in the ligand-binding domain of the AR do not significantly influence the inhibitory effect of Duta on the AR [28]. Despite the lack of influence on AR, our present study demonstrated an induction of both PSMA and HOXB13 protein expression in LNCaP and VCaP cells. This finding further corroborates the conclusion of Bakht et al. suggesting that HOXB13 is a positive regulator of PSMA expression.

Several in vitro studies demonstrated that Lova has a significant inhibitory effect on cell viability in different solid cancers such as breast, liver, cervical, lung, and colon cancer [24]. We were recently able to show the same effect of Lova on growth in a variety of prostate cancer cells such as LNCaP, C4-2 and VCaP cells [7]. Duta, however, did not affect prostate cancer cell growth at all [7]. The mechanisms by which Lova is able to influence prostate cancer growth, while Duta does not, remain at present unknown. To explore this question, we investigated the AR and its downstream targets. Since prostate cancer cell growth is AR-dependent [10,29], we considered that the inhibition of AR expression post Lova treatment is responsible for the reduction of prostate cancer cell growth we observed. Furthermore, we contemplate that the lack of AR expression changes post Duta treatment is accountable for the absence of cell viability variations. In addition, AR is described in prostate cancer as a positive regulator of the mTOR signaling pathway, which is a well-known critical factor for prostate cancer formation and disease progression [[16], [17], [18], [19]]. Our present results confirmed the inhibitory effect of Lova on AR in LNCaP and VCaP cells, expanding to show that this AR reduction is accompanied by mTOR inhibition. Members of the mTOR signaling cascade such as p4EBP1 and peIF4G were significantly downregulated in both LNCaP and VCaP cells post Lova treatment. Both p4EBP1 and peIF4G are known to promote tumor growth [30]. The phosphorylated form of 4EBP1 (p4EBP1) is described to lead to loss of its repressor functions over another mTOR member, namely eIF4E [30]. eIF4E is implicated in promoting malignant transformation, cell proliferation and growth [30]. As p4EBP1 is down regulated by Lova treatment and 4EBP1 is expressed in abundance, we consider that the repressor functions of 4EBP1 over eIF4E are present in both LNCaP and VCaP cells, thus promoting cell growth arrest.

Contrary to Lova, our study demonstrated that Duta does not affect AR protein expression in LNCaP and VCaP cells. Furthermore, the lack of effect also on the mTOR signaling pathway explains the lack of growth alteration in prostate cancer cells post Duta treatment.

Additionally, two studies demonstrated that the inhibition of mTOR by rapamycin produces an increase in the protein level or activity of AR [31,32]. Our results revealed that Lova inhibits mTOR, however, this does not induce AR expression as described previously. This discrepancy might be attributed to the fact that compared to rapamycin, Lova might potentially influence additional AR-regulating pathways (other than mTOR).

The Akt signaling pathway has previously been described to regulate AR expression in prostate cancer. Akt inhibition decreases AR expression, suggesting that Akt activity is required for regulation of AR protein levels [33]. In addition, AR inhibition has been shown to activate Akt signaling by reducing the expression of the Akt phosphatase PHLPP [34]. AR blockade reduces FKBP5 levels, subsequently impairing PHLPP function and leading to upregulation of pAkt [35]. Our study has revealed that the Lova treatment of LNCaP and VCaP cells promotes inhibition of both Akt/pAkt and AR, supporting the idea that Akt is a positive regulator of AR protein expression.

Cyclin D1 is a member of the cyclin protein family, which is well known to promote growth in variety of cancer types including prostate cancer [[36], [37], [38]]. Our study revealed that Lova treatment leads to Cyclin D1 inhibition accompanied by mTOR and Akt repression in LNCaP and VCaP cells. Interestingly, many previous reports describe Cyclin D1 to be under the regulation of both mTOR and Akt signaling pathways. mTOR and Akt activation lead to increased synthesis of Cyclin D1, which promotes the transition from the G1 to S phase of the cell cycle by binding to CDK4/6 and Rb protein phosphorylation, finally resulting in cancer progression [[39], [40], [41]]. Thus, our data suggest that the cell growth impairment that we observed when treating prostate cancer cells with Lova results from the inhibition of AR, mTOR and Akt signaling pathways, consequently promoting Cyclin D1 repression and cell growth arrest. The fact that Duta does not have these effects and consequently does not affect prostate cancer cell growth, confirms the importance of the Lova - mTOR/Akt - Cyclin D1 axis for prostate cancer cell growth.

In summary, we were able to confirm the cooperative effect of HOXB13 and AR in regulating PSMA in LNCaP cells [6], and show that these mechanisms of PSMA regulation also apply to an additional prostate cancer cell line (VCaP). Furthermore, we are the first to demonstrate that Lova treatment (but not Duta treatment) of prostate cancer cells leads to inhibition of the AR - mTOR/Akt – Cyclin D1 axis and promotes cell growth arrest.

Funding

Max and Hedwig Niedermaier-Stiftung and Lotte and Adolf HOTZ-SPRENGER STIFTUNG.

CRediT authorship contribution statement

Aleksandar Kuzmanov: Writing – original draft, Investigation. Souzan Salemi: Writing – review & editing. Daniel Eberli: Data curation. Benedikt Kranzbühler: Writing – review & editing, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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