Systematic Evaluation for the Influences of the SOX17/Notch Receptor Family Members on Reversing Enzalutamide Resistance in Castration-Resistant Prostate Cancer Cells

Abstract

The treatment of castration-resistant prostate cancer (CRPC) remains challenging due to the failure of androgen deprivation therapy (ADT); hence the search for other molecular therapeutic targets besides androgen receptor signaling is ongoing. This study systematically investigated the expression of SOX17 and Notch receptors in CRPC tissues and cells in vitro, showing that consistent clinical CRPC, SOX17/Notch1, and Notch4 were responsible for enzalutamide resistance in CRPC cells. The γ secretase inhibitors, BMS-708163, GSI-IX, PF-3084014, and RO4929097 abrogated the enzalutamide resistance by inhibiting Notch1 or/and Notch4 in vitro, with GSI-IX and RO4929097 being more effective than BMS-708163 and PF-3084014 in reliving bone metastasis in vivo. In conclusion, the Notch1 and Notch4 inhibitors GSI-IX and RO4929097 are promising therapeutic agents for the treatment of CRPC.

Keywords: Notch receptor family members; SOX17; castration-resistant prostate cancer; enzalutamide; γ secretase inhibitors.

Figure 1

The expression of SOX17 and Notch receptors in samples of benign prostatic hyperplasia (BPH), prostate cancer (PCa) and castration-resistant prostate cancer (CRPC). (A–E) The expression levels of SOX17, Notch1 and Notch2, Notch3, Notch4 were detected by using immunohistochemistry (×200). (F) The positive expression of SOX17 in cancer thrombus of CRPC (red arrows) (×200). (G–K) Average staining scores for SOX17, Notch1, Notch2, Notch3, Notch4 BPH, PPC and CRPC samples. Staining scoring, according to staining intensity, was defined as 0, no staining; 1, weak staining; 2, light staining; 3, moderate staining; and 4, strong staining. Staining scores of ≤1 were defined as negative expression, while staining scores of ≥2 were defined as positive expression. (L–O) The correlation curve analysis for SOX17 staining scores versus Notch1 and Notch2, Notch3, Notch4 staining scores in CRPC tissues. Values of P < 0.05 were considered to be statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001, ns, no significance.

Figure 2

Kaplan-Meier survival analysis for the progression-free survival (PFS) of 33 patients with CRPC. (A) 21 patients with SOX17-positive, 12 patients with SOX17-negative, (B) 23 patients with Notch1-positive, 10 patients with Notch1-negative, (C) 21 patients with Notch2-positive, 12 patients with Notch2-negative, (D) 25 patients with Notch3-positive, eight patients with Notch3-negative, (E) 23 patients with Notch4-positive, 10 patients with Notch4-negative. P < 0.05 was considered statistically significant.

Figure 3

Expression of SOX17 and Notch receptors was detected in Enza-R cells and knocking down them inhibited the viability of Enza-R cells. (A) Identification for CRPC cells, both LNCaP and Enza-R cells were treated with increasing concentrations of enzalutamide for 24 h and IC50 was detected by cell counting kit-8 (CCK-8) assay. (B–D) Expression of SOX17, Notch receptors in both mRNA and protein level was detected by RT-PCR, Western blot and Immunofluorescence assay (magnification, ×200). (E–I) After incubated for 96 h, the viability of Enza-R cells, treated with shSOX17, shNotch1, shNotch2, shNotch3, and shNotch4 respectively, was evaluated by CCK-8 assay. (J) Enza-R cells, infected with LV-NC or LV-shSOX17, were treated with increasing concentrations of enzalutamide for 24 h, and the half maximal inhibitory concentration (IC50) was determined by CCK-8 assay. Enza-R: enzalutamide-resistant LNCaP cells. *P < 0.05, **P < 0.01, ***P < 0.001, ns, no significance.

Figure 4

Knocking down SOX17 reducing the expression of EMT related proteins, AR, Notch receptor members. (A) Expression of E-cadherin, N-cadherin, Vimentin and Zeb-1 in Enza-R cells, infected with LV-NC or LV-shSOX17, was examined by Western blot assay. GAPDH served as a loading control. (B–D) The expression of AR in Enza-R or/and DU145 cells, infected with LV-NC or LV-shSOX17, was evaluated by using RT-PCR, Western blot and Immunofluorescence (magnification, ×200) assay. GAPDH served as a loading control. (E–G) Expression of Notch1, Notch2, Notch3, Notch4 were investigated by using RT-PCR, Western blot and Immunofluorescence (magnification, ×200) assay in Enza-R cells treated with LV‐NC or LV‐shSOX17, *P < 0.05, **P < 0.01, ns, no significance; AR, androgen receptor; Enza-R, enzalutamide-resistant LNCaP cells.

Figure 5

Knocking down Notch receptors inhibited the expression of AR and reversed the enzalutamide resistance in Enza-R cells. (A–C) Expression of AR in Enza-R cells infected with LV-shNotch1, LV-shNotch2, LV-shNotch3, LV-shNotch4 were respectively determined by using RT-PCR, Western blot and Immunofluorescence (magnification, ×200) assay. *P < 0.05, **P < 0.01, ns, no significance. (B–G) The half maximal inhibitory concentration (IC50) was determined by CCK-8 assay after the cells were treated with increasing concentrations of enzalutamide for 24 h. Enza-R cells were infected with LV-NC or LV-shNotch1, LV-shNotch2, LV-shNotch3, LV-shNotch4 respectively. AR, androgen receptor; Enza-R, enzalutamide-resistant LNCaP cells.

Figure 6

γ-secretase inhibitors reversed the resistance by decreasing activities of Notch receptors. (A–C) The half maximal inhibitory concentration (IC50) was determined by CCK-8 assay after the Enza-R cells were treated with enzalutamide and γ-secretase inhibitors, LY3039478, BMS-708163, LY-450139, GSI-IX, PF-3084014, RO4929097, LY01027 respectively for 24 h. (D–J) Activities of each Notch receptor member were determined in Enza-R cells after treated them with various γ-secretase inhibitors respectively. The cleaved Notch 1–4 means the intracellular domain part of Notch receptor members spliced by γ-secretase, which realizes the function of notch receptor members. GAPDH served as a loading control.

Figure 7

The combination of γ-secretase inhibitors and enzalutamide suppressed growth and bone metastasis of Enza-R cells in vivo. The nude mice with subcutaneous or bone xenograft were respectively treated 10 mg/kg enzalutamide and 50 mg/kg various γ-secretase inhibitors. (A, B) Tumor growth curve (C) Weight of tumors (D) Images of the recovered tumors (E) X-ray for bone metastasis (F) H&E staining for bone metastasis (upper×100, lower ×200). (G) The quantitative results of bone metastasis based on bone metastasis. *P < 0.05, **P < 0.01, ***P < 0.001.