Enzalutamide as an androgen receptor inhibitor prevents urothelial tumorigenesis

Abstract

Emerging preclinical evidence suggests the critical role of androgen-mediated androgen receptor (AR) signals in the development of bladder cancer. However, little is known about the efficacy of enzalutamide, an AR signaling inhibitor, in androgen-induced urothelial tumorigenesis. We therefore aimed to assess the effects of enzalutamide on neoplastic transformation of urothelial cells. An immortalized normal urothelial cell line SVHUC stably expressing wild-type AR (SVHUC-AR) was exposed to a chemical carcinogen 3-methylcholanthrene (MCA) to induce neoplastic transformation, and subsequently cultured for 6 weeks in the presence of anti-androgens, including enzalutamide, hydroxyflutamide, and bicalutamide. Tumorigenesis was then monitored, using plate and soft agar colony formation assays as well as mouse xenograft models. In SVHUC-AR cells exposed to MCA, each anti-androgen inhibited AR-mediated transcriptional activity, but only enzalutamide prevented AR nuclear translocation. In vitro transformation showed that treatment with each anti-androgen during the process of neoplastic transformation reduced the efficiency of colony formation in vitro. Compared with mock treatment, culture with enzalutamide (P = 0.028), hydroxyflutamide (P = 0.033), or bicalutamide (P = 0.038) also resulted in prevention/retardation of tumor formation in male NOD-SCID mice. In addition, anti-androgens up-regulated the expression of several molecules that play a protective role in bladder tumorigenesis, including p53, p21, and PTEN, and down-regulated that of several oncogenic genes, such as c-myc, cyclin D1, and cyclin E, in MCA-exposed SVHUC-AR cells. Thus, enzalutamide, flutamide, and bicalutamide were found to similarly prevent neoplastic transformation of urothelial cells. These findings offer a potential chemopreventive approach for urothelial tumors using AR antagonists.

Keywords: Bicalutamide; enzalutamide; flutamide; neoplastic transformation; urothelial cancer.

Figure 1

Effects of enzalutamide on AR in MCA-exposed urothelial cells. A. Cell extracts from SVHUC-AR without MCA exposure or SVHUC-control/SVHUC-AR exposed to MCA and subsequently treated with ethanol (mock), hydroxyflutamide (HF; 5 μM), bicalutamide (BC; 5 μM), or enzalutamide (EZ; 5 μM) for 48 hours were analyzed on western blotting, using an antibody to AR (110 kDa). GAPDH (37 kDa) served as an internal control. The means of densitometry values for AR standardized by GAPDH that are relative to the value of mock treatment (6th lane; set as 100%) from three independent experiments are included below the lanes. B. Luciferase reporter gene assay was performed in SVHUC-control or SVHUC-AR exposed to MCA, transfected with MMTV-Luc and pRL-TK, and subsequently treated with ethanol (mock), hydroxyflutamide (HF; 5 μM), bicalutamide (BC; 5 μM), or enzalutamide (EZ; 5 μM) for 24 hours. Luciferase activity is presented relative to that of mock treatment in SVHUC-AR cells. Each value represents the mean + standard deviation from three independent experiments. C. Immunofluorescence of AR in SVHUC-AR cells exposed to MCA and subsequently treated with ethanol (mock), hydroxyflutamide (HF; 5 μM), bicalutamide (BC; 5 μM), or enzalutamide (EZ; 5 μM) for 24 hours. We merged the images between AR and DAPI that was used to visualize nuclei, and the proportion of cells with AR expression only in their nuclei was quantitated. Each value represents the mean + standard deviation of 6 determinants. *P < 0.05 (vs. mock treatment in SVHUC-AR).

Figure 2

Effects of enzalutamide on anchorage-dependent growth of MCA-exposed urothelial cells. SVHUC-control or SVHUC-AR exposed to MCA and subsequently cultured for 6 weeks in the presence of ethanol (mock), hydroxyflutamide (HF; 5 μM), bicalutamide (BC; 5 μM), or enzalutamide (EZ; 5 μM) was seeded for plate colony formation assay (cultured for additional 2 weeks). The number and area of the colonies were counted and are presented relative to those in mock-treated SVHUC-AR cells. Each value represents the mean + standard deviation from three independent experiments. *P < 0.05 (vs. mock treatment in SVHUC-AR).

Figure 3

Effects of enzalutamide on anchorage-independent growth of MCA-exposed urothelial cells. SVHUC-control or SVHUC-AR exposed to MCA and subsequently cultured for 6 weeks in the presence of ethanol (mock), hydroxyflutamide (HF; 5 μM), bicalutamide (BC; 5 μM), or enzalutamide (EZ; 5 μM) was seeded for soft agar colony formation assay (cultured for additional 3 weeks). The number of the colonies was counted and is presented relative to that in mock-treated SVHUC-AR cells. Each value represents the mean + standard deviation from three independent experiments. *P < 0.05 (vs. mock treatment in SVHUC-AR).

Figure 4

Effects of enzalutamide on urothelial tumor formation in mouse xenograft models. SVHUC-control or SVHUC-AR exposed to MCA and subsequently cultured for 6 weeks in the presence of ethanol (mock), hydroxyflutamide (HF; 5 μM), bicalutamide (BC; 5 μM), or enzalutamide (EZ; 5 μM) was suspended, mixed with Matrigel (1 × 10⁶ cells/100 μL), and subcutaneously implanted into the flank of 6-week-old male NOD-SCID mice (n = 8 in each group). The endpoint for this study was tumor formation (exceeding 10 mm³ in its estimated volume [by the following formula: (short diameter)² × (longest diameter) × 0.5] or 5 mm in greatest dimension). Comparisons were made by log-rank test.

Figure 5

Effects of enzalutamide on the expression of tumor suppressors and oncogenes in MCA-exposed urothelial cells. SVHUC-control or SVHUC-AR exposed to MCA and subsequently cultured for 6 weeks in the presence of ethanol (mock), hydroxyflutamide (HF; 5 μM), bicalutamide (BC; 5 μM), or enzalutamide (EZ; 5 μM) was subjected to a quantitative RT-PCR for p53, p27, and PTEN (A) as well as c-myc, cyclin D1, and cyclin E (B). Expression of each gene was normalized to that of GAPDH. Transcription amount is presented relative to that in mock-treated SVHUC-AR cells. Each value represents the mean + standard deviation from three independent experiments. *P < 0.05 (vs. mock treatment in SVHUC-AR).