The central role of Sphingosine kinase 1 in the development of neuroendocrine prostate cancer (NEPC): A new targeted therapy of NEPC

Abstract

Background: Neuroendocrine prostate cancer (NEPC) is often diagnosed as a sub-type from the castration-resistant prostate cancer (CRPC) recurred from the second generation of anti-androgen treatment and is a rapidly progressive fatal disease. The molecular mechanisms underlying the trans-differentiation from CRPC to NEPC are not fully characterized, which hampers the development of effective targeted therapy.

Methods: Bioinformatic analyses were conducted to determine the clinical correlation of sphingosine kinase 1 (SphK1) in CRPC progression. To investigate the transcriptional regulation SphK1 and neuroendocrine (NE) transcription factor genes, both chromosome immunoprecipitation and luciferase reporter gene assays were performed. To demonstrate the role of SphK1 in NEPC development, neurosphere assay was carried out along with several biomarkers determined by quantitative PCR and western blot. Furthermore, in vivo NEPC xenograft models and patient-derived xenograft (PDX) model were employed to determine the effect of SphK1 inhibitors and target validation.

Results: Significant prevalence of SphK1 in NEPC development is observed from clinical datasets. SphK1 is transcriptionally repressed by androgen receptor-RE1-silencing transcription factor (REST) complex. Furthermore, sphingosine 1-phosphate produced by SphK1 can modulate REST protein turnover via MAPK signaling pathway. Also, decreased REST protein levels enhance the expression of NE markers in CRPC, enabling the transition to NEPC. Finally, specific SphK1 inhibitors can effectively inhibit the growth of NEPC tumors and block the REST protein degradation in PDX.

Conclusions: SphK1 plays a central role in NEPC development, which offers a new target for this lethal cancer using clinically approved SphK1 inhibitors.

Keywords: Sphingosine kinase 1; neuroendocrine prostate cancer; targeted therapy; therapy and castration resistant prostate cancer.

FIGURE 1

The association of Sphk1 expression with NEPC progression. (A) The frequency of SphK1 gene alterations (green: mutation; blue: deletion; red: amplification) in PCa patients (cBioPortal database). (B) The positive correlation of mRNA expression between SphK1 and NE‐related genes (BRN2, FOXA2, SOX2, CgA, Syp) in PCa patients (Betastasis Database). (C) The positive correlation of SphK1 and Syp expression using IHC on PCa TMA (n = 44). (D) Increased NETFs (BRN2 and FOXA2) and NE marker (Syp) protein expression in patient‐derived explants treated with 100 μM S1P for 24 h. (E) The induction of NETFs and NE markers in LNCaP cells expressing CA‐SphK1. (F) The stimulatory effect of CA‐SphK1 on the growth of LNCaP under androgen deprived condition (20 μM Enzalutamide). Left top panel: Scheme of co‐culture of VC cells (mCherry⁺) and CA‐SphK1 cells (GFP⁺) at ratio 100:1 incubated with 20 μM Enzalutamide for 3 weeks. Left bottom panel: Cell growth of two different LNCaP sub‐lines treated with Enzalutamide. Right panel: Images of two different LNCaP sub‐lines treated with Enzalutamide. *p < .05; **p < .01

FIGURE 2

The suppressive effect of AR on Sphk1 gene expression. (A) An inverse correlation of mRNA expression between SphK1 and AR‐regulated genes (AR, ABCC4, APPBP2, TMPRSS2 and TDD52) from TCGA database. (B) Decreased SphK1 gene expression in LNCaP cells treated with vehicle or DHT for 24 h (GSE62454, GSE436). (C) Time course effect of R1881 on the expression of SphK1 and TMPRSS2 genes in LNCaP cells (GSE14097). (D) The opposite effect of 10 nM DHT or 20 μM Enzalutamide on SphK1 mRNA expression in LNCaP cells. (E) The impact of 10 nM DHT or 20 μM Enzalutamide on the binding of AR for predict androgen recognition element (ARE, underline) in SphK1 promoter at P1 (A, B) and P2 (C, D) region. (F) The impact of 10 nM DHT or 20 μM Enzalutamide on the interaction between AR and REST proteins. (G) The impact of 10 nM DHT or 20 μM Enzalutamide on the binding of AR‐REST complex to predict ARE in SphK1 promoter. *p < .05; **p < .01

FIGURE 3

The role of SphK1 in the onset of NEPC. (A, B, C) Top panel: Decreased SphK1 protein expression in several SphK1 knockout (sgSphK1) PCa cells. Middle panel: Decreased mRNA expression of BRN2, EZH2, FOXA2, CHA and SYP gene in sgSphK1 cells. Bottom panel: Decreased protein expression of BRN2, EZH2, FOXA2 and SOX2 protein level in in sgSphK1 cells. (D, E) The effect of SphK1 on neurosphere formation of IIG5 or 22RV1 cells

FIGURE 4

The S1P‐elicited signaling pathway leading to the onset of NEPC. (A, B) Left panel: The specificity of PI3K inhibitor (LY294002) or MEK inhibitor (PD98059) on inhibiting the activation of respective kinase (Akt S473 phosphorylation or Erk phosphorylation). Right panel: The effect of LY294002 or PD98059 on S1P‐induced NETF protein expression. (C) Accumulation of REST protein in SphK1 knockout‐ PC3, 22RV1 and IIG5 cells. (D) The effect of REST mutant (S861/864A) on NETF protein expression in PC3, 22RV1 and IIG5 cells. (E) The antagonistic effect of REST mutant (S861/864A) on S1P‐elicited NETF expression in PC3 cells. *p < .05; **p < .01

FIGURE 5

The suppressive role of REST in S1P‐induced NETF genes transcription. (A) Annotation of REST binding sites in each NETF gene promoter. (B) ChIP‐qPCR analyses of REST occupancy at promoter region of NETF genes in PC3 and IIG5 cells. (C, D) The effect of REST mutant (S861/864A) on S1P‐induced SOX2, BRN2, EZH2 and FOXA2 promoter activity in SphK1 knockout—PC3 and IIG5 cells. *p < .05; **p < .01; ***p < .001; ****p < .0001

FIGURE 6

The potency of SphK1 inhibitors on NEPC therapy. (A, B, C) Left and Middle panel: The in vivo potency of SphK1 inhibitors in Enzalutamide‐resistant NEPC tumor models. Right panel: Target validation in tumor specimens harvested from the end of each treatment. (D) The inhibitory effect of SphK1 inhibitors on NETF protein expression in PDX models. **p < .01; ***p < .001

FIGURE 7

The role of SphK1 in PCa progression. (A) The scheme of the reciprocal regulator network among SphK1, AR and REST in NEPC development. (B) Clinical correlation of SphK1 and REST with overall survival of PCa patients