Small molecule BKM1972 inhibits human prostate cancer growth and overcomes docetaxel resistance in intraosseous models

Abstract

Bone metastasis is a major cause of prostate cancer (PCa) mortality. Although docetaxel chemotherapy initially extends patients' survival, in most cases PCa becomes chemoresistant and eventually progresses without a cure. In this study, we developed a novel small-molecule compound BKM1972, which exhibited potent in vitro cytotoxicity in PCa and other cancer cells regardless of their differences in chemo-responsiveness. Mechanistic studies demonstrated that BKM1972 effectively inhibited the expression of anti-apoptotic protein survivin and membrane-bound efflux pump ATP binding cassette B 1 (ABCB1, p-glycoprotein), presumably via signal transducer and activator of transcription 3 (Stat3). BKM1972 was well tolerated in mice and as a monotherapy, significantly inhibited the intraosseous growth of chemosensitive and chemoresistant PCa cells. These results indicate that BKM1972 is a promising small-molecule lead to treat PCa bone metastasis and overcome docetaxel resistance.

Keywords: Bone metastasis; Chemoresistance; Preclinical model; Prostate cancer; Small-molecule therapy.

Figure 1.. BKM1972 suppresses survivin expression and inhibits the in vitro viability of human cancer cell lines.

(A) Chemical structures of BKM570 and its analog BKM1972. Note both compounds have a 3-moiety “A-B-C” core structure. (B) PCR analysis of the effect of several BKM570 analogs on survivin mRNA expression in C4–2 cells (5 μM, 24 h). (C) A summary of the in vitro cytotoxicity of BKM1972 in the NCI-60 panel (48 h). GI₅₀ is the concentration that causes 50% growth inhibition, relative to the vehicle control. (D) MTS assay of the in vitro cytotoxicity of BKM1972 in established PCa cell lines (72 h). (E) MTS assay of the in vitro cytotoxicity of BKM1972 in lineage-derived KB cells (72 h).

Figure 2.. BKM1972 inhibits Stat3 signaling and suppresses survivin transcription in PCa cells.

(A) Flow cytometry assay of Annexin V expression in C4–2 cells treated with varying concentrations of BKM1972 (72 h). *: p < 0.001. (B) Left: protein expression of p-PKC-ɛ(S729), p-Stat3, Stat3 and survivin in C4–2 and ARCaP_M cells treated with 5 μM BKM1972 for the indicated times. β-actin was used as the loading control; Right: Expression of p-Stat3 and Stat3 in the cytosolic and nuclear lysates from C4–2 cells treated with BKM1972 (5 μM). TATAbinding protein (TBP) was used as the loading control for nuclear proteins. (C) PCR analysis of survivin mRNA levels in C4–2 cells treated with BKM1972 (5 μM) at the indicated times. *, **: p < 0.05. (D) Upper: Human survivin reporter pSurvivin-Luc1430 contains two putative Stat3-binding cis-elements (blue bars), which are absent in the deletion construct pSurvivin-Luc230. The transcriptional start site was as +1; Bottom: Relative luciferase activity of pSurvivin-Luc1430 and pSurvivin-Luc230 in C4–2 cells treated with BKM1972 at the indicated concentrations (48 h). *: p < 0.01.

Figure 3.. LG1980 inhibits the expression of survivin and ABCB1 and induces apoptosis in chemoresistant PCa cells.

(A) Flow cytometry assay of Annexin V expression in C4–2B-TaxR cells treated with varying concentrations of BKM1972 (72 h). *, **: p < 0.01. (B) Left: protein expression of p-PKC-ε(Ser729), p-Stat3(Ser727), survivin and ABCB1 in C4–2B-TaxR and parental C4–2B cells. Α-tubulin and β-actin were used as the loading controls; Right: Western blot analyses of nuclear expression of p-Stat3(S727) in C4–2B and C4–2B-TaxR cells. H3 histone was used as the loading control for nuclear proteins. (C) Western blot analyses of C4–2B-TaxR cells treated with BKM1972 (5 μM) for the indicated times. (D) Western blot analyses of the cytosolic and nuclear proteins in C4–2B-TaxR cells treated with BKM1972 (5 μM) for the indicated times. (E) PCR analysis of mRNA expression of survivin and ABCB1 in C4–2B-TaxR cells treated with BKM1972 (5 μM) for the indicated times. *, **: p < 0.05. (F) Left: The sketch map of putative Stat3 binding motif (blue bar) within human ABCB1 promoter. The transcriptional start site was as +1; Right: Relative luciferase activity of ABCB1 reporters (pMDR1–221 and pMDR1–1202) in C4–2B-TaxR cells treated with varying concentrations of BKM1972 (48 h). *, **: p < 0.01. (G) Fluorescence microscopy images of cellular uptake of Oregon Green 488-paclitaxel in C4–2B-TaxR cells. Cells were first treated with varying concentrations of BKM1972 for 72 h prior to paclitaxel incubation for the indicated times. (H) Flow cytometry assay of Oregon Green 488-paclitaxel in C4–2B-TaxR cells pre-treated with BKM1972 (5 μM) for 72 h prior to paclitaxel incubation for the indicated times. *, **: p < 0.05.

Figure 4.

Average body weights of athymic nude mice treated with BKM1972 (10 mg/kg or 20 mg/kg) or vehicle control, via intraperitoneal injection, three times per week, for 3 weeks (n = 5 per group).

Figure 5.. BKM1972 inhibits the intratibial growth of C4–2 tumors in athymic nude mice.

(A) Left: Serum PSA levels of C4–2 tumor-bearing mice treated with vehicle control (n = 5), 5 mg/kg BKM1972 (n = 5) or 10 mg/kg BKM1972 (n = 6) for 8 weeks. Two-way ANOVA was used for all pairwise comparisons; Right: Average body weights of C4–2 tumor-bearing mice treated with vehicle or BKM1972 (5 mg/kg or 10 mg/kg). (B) X-ray radiography of tumor-bearing bones from different treatment groups. Red arrows indicate tumor-induced osteolytic lesions. (C) H&E and TRAP staining of C4–2 bone tumors from different treatment groups. Scale bar is 50 μM for H&E and 100 μM for TRAP respectively.

Figure 6.. BKM1972 inhibits the intratibial growth of C4–2B-TaxR tumors in athymic nude mice.

(A) Left: Serum PSA levels of C4–2B-TaxR tumor-bearing mice treated with vehicle control (n = 5), docetaxel (2.5 mg/kg, n = 7) or BKM1972 (15 mg/kg, n = 7) for 11 weeks. Two-way ANOVA was used for all pairwise comparisons; Right: Average body weights of C4–2B-TaxR tumor-bearing mice treated with vehicle, docetaxel or BKM1972. (B) X-ray radiography of tumor-bearing bones from different treatment groups. Red arrows: osteolytic lesions. (C) H&E and TRAP staining of C4–2B-TaxR bone tumors from different treatment groups. Scale bar is 50 μM for H&E and 100 μM for TRAP respectively.