Endothelial nitric oxide synthase (eNOS)-NO signaling axis functions to promote the growth of prostate cancer stem-like cells

Abstract

Background: Accumulating evidence supports that prostate cancer stem-like cells (PCSCs) play significant roles in therapy resistance and metastasis of prostate cancer. Many studies also show that nitric oxide (NO) synthesized by NO synthases can function to promote tumor progression. However, the exact roles of NOSs and NO signaling in the growth regulation of PCSCs and castration-resistant prostate cancer (CRPC) are still not fully understood.

Methods: The regulatory functions of NOS-NO signaling were evaluated in prostate cancer cells, especially in PCSCs enriched by 3D spheroid culture and CD133/CD44 cell sorting. The molecular mechanisms of NOS-NO signaling in PCSCs growth regulation and tumor metastasis were investigated in PCSCs and mice orthotopic prostate tumor model.

Results: Endothelial NOS (eNOS) exhibited a significant upregulation in high-grade prostate cancer and metastatic CRPC. Xenograft models of CRPC exhibited notable increased eNOS expression and higher intracellular NO levels. PCSCs isolated from various models displayed significant enhanced eNOS-NO signaling. Functional analyses demonstrated that increased eNOS expression could promote in vivo tumorigenicity and metastatic potential of prostate cancer cells. Characterization of eNOS-NO involved downstream pathway which confirmed that enhanced eNOS signaling could promote the growth of PCSCs and antiandrogen-resistant prostate cancer cells via an activated downstream NO-sGC-cGMP-PKG effector signaling pathway. Interestingly, eNOS expression could be co-targeted by nuclear receptor ERRα and transcription factor ERG in prostate cancer cells and PCSCs.

Conclusions: Enhanced eNOS-NO signaling could function to promote the growth of PCSCs and also the development of metastatic CRPC. Besides eNOS-NO as potential targets, targeting its upstream regulators (ERRα and ERG) of eNOS-NO signaling could also be the therapeutic strategy for the management of advanced prostate cancer, particularly the aggressive cancer carrying with the TMPRSS2:ERG fusion gene.

Keywords: Castration resistance; ERG; ERRα; NO; Prostate cancer; Prostate cancer stem-like cells; eNOS.

Fig. 1

Increased eNOS expression in high-grade prostate cancer and metastatic CRPC. a Comparison of mRNA expressions of three NOS isoforms showed that eNOS exhibited a consistent elevated expression pattern in metastatic CRPC tissues as compared to that in benign hyperplastic prostates and localized prostate cancer tissues, as revealed by two microarray expression datasets (GSE32269, GSE35988). b eNOS displayed statistical higher mRNA expression in high Gleason score prostate cancers, as revealed by a microarray expression dataset GSE21032. c Kaplan–Meier survival curves of cohort from TCGA using the GEPIA2 analysis tool revealed that prostate cancer patients with high eNOS expression level (top 20%) would have a significant shorter disease-free overall survival than patients with low eNOS expression level (bottom 20%). *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 2

CRPC models contain more PCSC populations with enhanced eNOS expression and higher intracellular NO levels. a, b Androgen-sensitive and castration relapse VCaP and CWR22 xenografts. q-PCR analysis showed that eNOS exhibited a progressive increase in mRNA levels in xenograft tumors at 4 days (Post-Cas) and 2 months post-castration (Relapse) as compared to tumors before host castration. Results also revealed that the castration relapse CWR22-CRPC xenograft tumors expressed significant higher levels of multiple PCSC-associated biomarkers. c, d Microscopic detection of intracellular NO by NO fluorescent probe DAF-FM in bicalutamide-resistant LNCaP-BC32 cells-derived and FACS-sorted CD133⁻/CD44⁻ and CD133⁺/CD44⁺ cell populations, grown under either adherent 2D culture or non-adherent 3D culture (spheroids) condition and upon treatments with NOS inhibitor (L-NAME, 100 μM) or substrate (L-Arginine, 0.5 mM). c Representative micrographs show the intracellular NO-activated DAF-FM signals. Bars: 50 μm. d Semiquantitative analysis of NO levels (DAF-FM fluorescence signals). Results showed that 3D culture spheroids (derived from CD133⁺/CD44⁺ cells) displayed higher intense basal NO signals as compared to adherent 2D culture CD133⁻/CD44⁻ cells without treatment with L-NAME or L-Arginine. Their NO levels as detected in both 3D culture spheroids and adherent 2D culture cells were significantly reduced or abolished upon treatment with L-NAME but significantly intensified upon L-Arginine treatment. e FACS CD133-CD44 sorting of LNCaP-BC and LNCaP cells showed that the LNCaP-BC32 cells contained more CD133⁺-CD44⁺ subpopulation than their parental LNCaP cells. The CD133⁺-CD44⁺ subpopulation was significantly reduced/lessened upon treatment with L-NAME (100 μM). Results repeated at least three times are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 3

PCSCs exhibit significant activation of eNOS-NO signaling. a–e Characterization of 3D culture spheroids and SORE6⁺ cells on their PCSC phenotype. a Representative micrographs of prostate spheroids. Bars: 2D cultures, 100 μm; spheroids, 200 μm. b RT-qPCR analysis of PCSC-associated markers. The spheroids expressed higher levels of multiple PCSC-associated transcription factors (SOX2, OCT4, KLF4, NANOG) and membrane antigens (CD44, CD133). c FACS CD133-CD44 sorting of LNCaP cells. Results showed that the spheroids contained more subpopulations of CD133⁺/CD44⁺ cells. d Fluorescent detection of SORE6-GFP signals in spheroids. Left: The spheroids expressed intense GFP signals. Upon re-adherent differentiation culture for 48 h, 2D cultures still contained small population of SORE6-GFP⁺ cells. Bars: 200 μm. Right: FACS-sorted SORE6⁺- and SORE6⁻-LNCaP cells were re-plated at 2D culture condition for 48 h, and GFP signals were detected in SORE6⁺ LNCaP cells but absent in SORE⁻-LNCaP cells. Bars: 100 μm. e In vivo tumorigenicity assay by low-cell-number inoculations (1 × 10⁴ cells per site) of spheroids (inoculation site: right flank, red arrows) versus 2D cultures (left flank). Duration for xenograft tumor growth: DU145 cells for 7 weeks, VCaP cells for 12 weeks. Results showed that almost all spheroids could form xenograft tumors but not 2D cultures. f RT-qPCR and immunoblot analyses of eNOS expression. Results showed that the spheroids exhibited significant higher mRNA and protein levels. The immunoblots (IB) were cropped around the bands at 133 kDa and 42 kDa molecular weight markers from different membrane blots. g Microscopic detection of intracellular NO using DAF-FM in spheroids versus 2D cultures. Results showed that all spheroids showed more intense NO signal. Bars: 50 μm. h, i RT-qPCR analysis of FACS-sorted SORE6⁺ cells showed that SORE6⁺ cells expressed significant higher eNOS levels as compared to SORE6⁻ cells. CD133⁺ cells sorted from primary-cultured prostate cancer tissues showed higher eNOS levels as compared to CD133⁻ cells. Results repeated at least three times are expressed as mean ± SD, *P < 0.05, **P < 0.01

Fig. 4

eNOS can promote EMT of PCSCs and in vivo prostate tumor growth and metastasis. a, b RT-qPCR analysis of stemness-associated genes in adherent 2D culture cells and 3D spheroids with eNOS transgene overexpression. Results showed that eNOS overexpression induced significant upregulation of stemness genes in adherent 2D culture DU145 cells and also 3D culture LNCaP spheroids. c RT-qPCR analysis of four EMT-associated markers [mesenchymal markers: CHD2 (N-cadherin), EMT-inducing factors: transcription factor ZEB1 and CLDN1 (claudin-1); epithelial marker: CDH1 (E-cadherin], in LNCaP-eNOS cells. Results showed that eNOS overexpression could induce significant upregulation of CDH2, ZEB1 and CLDN1 but downregulation of CDH1 in 3D culture spheroids formed by LNCaP-eNOS cells as compared to spheroids formed by the LNCaP vectors cells. d, e Wound healing assay. d Representative images of PC-3 M-vector/-eNOS/-sheNOS-transduced cells taken at 0 h and 41 h time point. Bar: 200 μm. e Semiquantitative analysis of wound closure determined by measurement of width of wounds. Results showed that PC-3 M-eNOS cells showed significant higher migration capacity, whereas PC-3 M-sh-eNOS cells showed reduced capacity, as compared to PC-3 M-vector cells. f Luciferase-based bioluminescence in vivo imaging. Representative bioluminescence pictures of mice at 5 weeks post-inoculation of PC-3 M-vector/eNOS/sh-eNOS cells. Intense bioluminescence tumor growth signals were detected in the prostates of mice which had received orthotopic inoculation of PC-3 M-eNOS cells. Moderate and very weak bioluminescence signals were detected in mice bearing inoculations of PC-3 M-vector or PC-3 M-sh-eNOS cells, respectively. g Photograph shows the dissected prostate tumors formed by the inoculated PC-3 M-vector/eNOS/sh-eNOS cells in mice. Significant larger tumors were formed by PC-3 M-eNOS cells. h Table summarizes the tumor weights and detected metastasis to para-aortic lymph nodes in host mice bearing xenografts of PC-3 M-vector/eNOS/sh-eNOS cells. Results repeated at least three times are expressed as mean ± SD, *P < 0.05, **P < 0.01

Fig. 5

Activation of eNOS-NO signaling and its downstream effectors in PCSCs and antiandrogen-resistant prostate cancer cells. a Cell viability assay. DU145-sh-eNOS cells grew at the same proliferation rate as DU145-sh-scramble cells under adherent 2D culture condition, suggesting that knockdown of eNOS induced no significant impact on proliferation of DU145 cells. b–d 3D culture spheroid formation assay performed on DU145 and LNCaP cells. b Knockdown of eNOS by sh-eNOS could significantly suppress the spheroid formation capacity of DU145 and LNCaP cells. c Schematic diagram illustrates the actions of different used selective activator and inhibitors of regulators of the eNOS-NO-sGC-cGMP-PKG signaling pathway. d Treatment with eNOS inhibitors, L-NAME and L-NIO (100 μM, respectively), could significantly suppress the spheroid formation capacity of DU145 cells. e Treatment with an NOS3 enhancer AVE3085 (5 μM) could significantly enhance the spheroid formation capacity of DU145 cells. However, treatments with selective sGC inhibitor ODQ (20 μM) and PKG inhibitor KT5823 (10 μM) could significantly suppress the spheroid formation capacity of DU145 cells. f Measurement of cGMP levels in DU145-eNOS and LNCaP-eNOS-transduced cells by direct immunoassay. Results showed that both DU145-eNOS and LNCaP-eNOS cells contained significant higher cGMP levels than their corresponding parental cells, grown as either adherent 2D culture cells or 3D culture spheroids, and with such increased cGMP levels being suppressed by L-NIO treatment (100 μM). g Cell viability assay performed in LNCaP-BC32 cells. Analysis showed that treatments with inhibitors targeting to eNOS (L-NAME, 100 μM; L-NIO, 200 μM) and sGC (ODQ, 20 μM) could induce more growth inhibition on LNCaP-BC32 cells than LNCaP cells. Results repeated at least three times are expressed as mean ± SD, *P < 0.05, **P < 0.01

Fig. 6

ERRα and ERG can act to co-activate the eNOS-NO signaling in prostate cancer cells. a RT-qPCR analysis of ERRα and eNOS expression in prostate 3D culture spheroids versus adherent 2D culture cells. Left: spheroids (Sp) grown from DU145 or LNCaP cells exhibited higher ERRα expression levels as compared to their corresponding 2D cultures. Right: spheroids exhibited significant higher eNOS expression as compared to their parental cells. Overexpression of ERRα in 2D-cutured cells could significantly upregulate eNOS expression (blue bars). b Immunoblot analysis of eNOS, ERG and ERRα in DU145 and LNCaP cells. The blots were cropped around the bands at 133, 55, 50 and 42 kDa molecular weight markers from different membrane blots. Results showed that transient overexpression of either ERG or ERRα could significantly enhance protein expression of eNOS. c Measurement of cGMP levels in 3D culture spheroids derived from DU145-ERG-transduced cells. Analysis showed that DU145-ERG spheroids contained higher cGMP concentration levels than DU145-vector spheroids, and with such increased cGMP levels being eliminated by L-NIO treatment. d Immunoblot analysis of eNOS, ERG and ERRα in VCaP cells upon treatments with ERRα inverse agonist XCT790 (5 μM), ERG inhibitory peptide EIP1 (50 μM) or its negative control muEIP1 (50 μM). The blots were cropped around the bands at 133, 55, 50 and 42 kDa molecular weight markers from different membrane blots. Results showed that inhibition of ERRα by XCT790 and also ERG by EIP1 could moderately reduce the eNOS levels in VCaP cells. e 3D culture spheroid formation assay performed on ERRα-overexpressed prostate cancer cells. Results showed that overexpression of ERRα could significantly promote the spheroid formation capacities of both DU145 and LNCaP cells. However, their spheroid formation capacities could be weakened by treatments with inhibitors of NOS (L-NIO, 100 μM) or sGC (ODQ, 20 μM). Results repeated at least three times are expressed as mean ± SD, *P < 0.05, **P < 0.01. f Schematic diagram illustrates the contribution of regulatory loop between ERRα and ERG in the activation of eNOS-NO-sGC-cGMP-PKG signaling pathway in the regulation of PCSCs and CRPC