The β-Secretase 1 Enzyme as a Novel Therapeutic Target for Prostate Cancer

Abstract

Recent studies have demonstrated the association of APP and Aβ with cancer, suggesting that BACE1 may play an important role in carcinogenesis. In the present study, we assessed BACE1's usefulness as a therapeutic target in prostate cancer (PCa). BACE1 expression was observed in human PCa tissue samples, patient-derived xenografts (PDX), human PCa xenograft tissue in nude mice, and transgenic adenocarcinoma of the mouse prostate (TRAMP) tissues by immunohistochemistry (IHC) analysis. Additionally, the downstream product of BACE1 activity, i.e., Aβ1-42 expression, was also observed in these PCa tissues by IHC as well as by PET imaging in TRAMP mice. Furthermore, BACE1 gene expression and activity was confirmed in several established PCa cell lines (LNCaP, C4-2B-enzalutamide sensitive [S], C4-2B-enzalutamide resistant [R], 22Rv1-S, 22Rv1-R, PC3, DU145, and TRAMP-C1) by real-time PCR and fluorometric assay, respectively. Treatment with a pharmacological inhibitor of BACE1 (MK-8931) strongly reduced the proliferation of PCa cells in in vitro and in vivo models, analyzed by multiple assays (MTT, clonogenic, and trypan blue exclusion assays and IHC). Cell cycle analyses revealed an increase in the sub-G1 population and a significant modulation in other cell cycle stages (G1/S/G2/M) following MK-8931 treatment. Most importantly, in vivo administration of MK-8931 intraperitoneal (30 mg/kg) strongly inhibited TRAMP-C1 allograft growth in immunocompetent C57BL/6 mice (approximately 81% decrease, p = 0.019). Furthermore, analysis of tumor tissue using the prostate cancer-specific pathway array revealed the alteration of several genes involved in PCa growth and progression including Forkhead O1 (FOXO1). All together, these findings suggest BACE1 as a novel therapeutic target in advanced PCa.

Keywords: BACE1; MK-8931; amyloid β peptide; prostate cancer.

Figure 1

BACE1 and Aβ1-42 expression in PCa. (A) Representative IHC images showing the expression of BACE1 observed in normal seminal vesicles (NSVs) (n = 4), normal prostate tissue (NP), (n = 17) and localized PCa (n = 28) presented on a tissue array. The scale bar (100 µm) is shown on the images. Histogram representing IHC score for BACE1 expression calculated from the IHC images as mentioned in the methods. Analysis was performed using unpaired t-test with Welch’s correction and results are plotted as mean ± SEM, ** p < 0.001. (B) Representative immunofluorescence images showing the BACE1 expression (green) in TRAMP primary tumors (n = 3); DAPI was used as nuclear counterstain (blue). The scale bar (10 µm) is shown on the images. (C) Representative images of BACE1 expression in TRAMP primary tumors (n = 5), TRAMP abdominal cavity metastasis tumors (n = 3), 22Rv1 xenografts (n = 3), PC3-PTXR xenografts (n = 3), PDX118 xenografts (n = 3), and PDX174 (n = 3) xenografts. (D) Representative Aβ1-42 IHC images for TRAMP primary tumors (n = 5); TRAMP abdominal cavity metastasis tumors (n = 3); and 22Rv1 (n = 3), PC3-PTXR (n = 3), PDX118 (n = 3), and PDX174 (n = 3) xenografts. The scale bar (100 µm) is shown on the images. (E) Representative microPET/CT image of TRAMP PCa tumors 0–30 min after [¹¹C]PiB injection in TRAMP mice (n = 2).

Figure 2

The gene expression and activity of BACE1 in multiple PCa cell lines. (A) The gene expression of BACE1 in various PCa cells was analyzed by RT-PCR; results are plotted as fold change compared to BACE1 expression in non-neoplastic PWR-1E cells. Each bar represents mean ± SEM (n = 3). (B) BACE1 activity of PCa cells. Each bar represents mean ± SEM (n = 3).

Figure 3

BACE1 inhibitor treatment reduces viability in multiple PCa cell lines. (A–H) The mentioned PCa cells (n = 6 replicates per group, repeated at least once) were treated with vehicle control (DMSO) or MK-8931 (25–200 μM) for 24–72 h, and cell viability was analyzed in MTT assay. Analysis was performed using one-way ANOVA, and results are plotted as mean ± SEM. * p < 0.05, ** p < 0.001, *** p < 0.001, and **** p < 0.0001.

Figure 4

BACE1 inhibitor treatment reduces clonogenicity in multiple PCa cell lines. (A–H) The mentioned PCa cells (n = 3 per group, performed twice) were treated with vehicle control (DMSO) or MK-8931 (25–100 μM) for the defined duration, and the number of colonies (≥50 cells) was counted at the end. Analysis was performed by one-way ANOVA and presented as mean ± SEM. * p < 0.05, *** p < 0.001, and **** p < 0.0001.

Figure 5

The effect of BACE1 inhibition on cell proliferation and death in enzalutamide-sensitive and -resistant 22Rv1 PCa cells. (A) 22Rv1-S and (B) 22Rv1-R cells were treated with vehicle control (DMSO) or MK-8931 (25–100 μM) for 24–72 h. At the end of each time point, total live cells and % dead cells were counted (n = 3/group). Analysis was performed using one-way ANOVA and results are plotted as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Figure 6

The effect of BACE1 inhibition on cell cycle distribution in enzalutamide-sensitive and -resistant 22Rv1 PCa cells. (A) 22Rv1-S and (B) 22Rv1-R cells were treated with vehicle control (DMSO) or MK-8931 (25–100 μM) for 72 h. At the end, cells (n = 3 replicates/group) were collected and analyzed for cell cycle distribution as described in the methods. Experiment was performed in triplicate (repeated once). Analysis was performed using one-way ANOVA, and results are plotted as mean ± SEM. * p < 0.05, ** p < 0.01 and *** p < 0.001.

Figure 7

BACE1 inhibitor treatment reduces the in vivo growth of TRAMP-C1 allografts in syngeneic murine model. (A) Schematic representation of the experimental design. TRAMP-C1 cells were engrafted bilaterally into the flanks of wild-type C57BL6 mice. After one week of engraftment, mice were treated with either vehicle control or MK-8931 (30 mg/kg, IP) for 5 weeks. (B,C) Animal body weight and tumor volume was measured over the course of the study. Data are presented as mean ± SEM. Analysis was performed using multiple t-test analysis. Significant comparisons are outlined in dashed boxes for control versus MK-8931, p < 0.05. (D,E) Allografts were excised; volume (mm³) and weight (mg) were measured ex vivo. Data are presented as mean ± SEM. Analysis was performed using Student’s t-test. ** p = 0.019. (F) IHC analysis for PCNA expression in the tumor tissue of vehicle- and MK-8931 treated mice. Data are presented as mean ± SEM. Analysis was performed using Student’s t-test. (G) Pathway analysis of genes related to mouse prostate cancer pathway. Data are presented as volcano plot; significantly altered genes are highlighted as purple solid circles.