Nuclear receptor profiling in prostatospheroids and castration-resistant prostate cancer

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   Figure 1

   Prostate cancer cell line-derived prostatospheroids display enhanced expression of PCSC-associated markers. (A) Representative images of three selected prostate cancer cell lines (AR-positive: LNCaP and VCaP; AR-negative: DU145) grown under the conventional 2D-adherent culture conditions and their corresponding single-cell-derived prostatospheroids formed under the non-adherent 3D-culture conditions. (B) qRT-PCR analysis. Results showed that the expression levels of PCSC-associated markers were significantly upregulated in prostatospheroids. Fold changes of expression levels of PCSC-associated markers expressed in prostatospheroids were normalized to that in adherent 2D-cultured cells. *P < 0.05; **P < 0.01 vs corresponding 2D-cultured cells.
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   Figure 2

   Prostatospheroids display distinct expression profiles of NRs. (A, B and C) Histograms show the relative fold changes (RFC) of expressions of NRs in prostatospheroids derived from (A) LNCaP, (B) VCaP and (C) DU145 prostate cancer cell lines. NRs are grouped according to their ligand dependence as endocrine, adopted and orphan NRs. RFC of NR expressions were normalized to that in adherent 2D-cultured parental cells. The normalized NR expression levels are defined as upregulated if RFC values are ≥2.0, downregulated if RFC values ≤0.5, no significant change if RFC values within 0.5–2.0, and undetected if Ct value >34. Results showed that many members of NRs showed significant upregulation in prostatospheroids. (D) Left: data analysis summarized that a total of 21 NR members exhibited common upregulation in prostatospheroids derived from three prostate cancer cell lines. Middle: pie chart shows the number of endocrine, adopted and orphan NRs with common upregulation in prostatospheroids. Right: table shows the trivial and gene names of upregulated NRs in prostatospheroids.
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   Figure 3

   Castration-relapse VCaP-CRPC xenografts contain more population of prostate cancer stem-like cells. (A) VCaP-CRPC xenografts were established based on the castration-relapse growth of VCaP xenografts in castrated host SCID mice (n = 5). Left: Growth curve of VCaP tumor xenografts in host mice during 0–8 weeks after castration. Relapse of tumor growth occurred at 4-week post-castration. Right: qRT-PCR analysis. Results showed that the VCaP-CRPC xenografts expressed significantly higher mRNA levels of several PCSC-associated markers (CD44, CD24, OCT3/4, and CK5). (B) FACS analysis. Results showed that VCaP-CRPC tumor xenografts harvested at 4-day and 2-month post-castration contained increased population of CD44⁺/CD133⁺ cells as compared to tumors harvested before castration. (C) CD44 immunofluorescence. Results showed that VCaP-CRPC tumor xenografts harvested at 4-day and 2-month post-castration expressed higher CD44 immunosignals at tumor cell membranes as compared to tumor cells harvested at pre-castration. Bars = 20 μm. *P < 0.05; **P < 0.01 vs pre-castration.
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   Figure 4

   Castration-relapse VCaP-CRPC tumor xenografts display distinct expression profiles of NRs. (A and B) Histograms show the relative fold changes (RFC) of expression levels of NRs in VCaP-CRPC tumor xenografts harvested at 4-day and 2-month post-castration. Results revealed that over 20 NRs showing significant elevated expressions were identified in post-castrated VCaP-CRPC xenografts as compared to pre-castrated VCaP xenografts. (B) Left: data analysis showed that 14 NRs showing common upregulation were identified in VCaP-CRPC xenografts harvested at 4-day and 2-month post-castration. Right: a pie chart shows the NR numbers and a table shows the names of the identified upregulated NRs in the castration-relapse VCaP-CRPC tumor xenografts.
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   Figure 5

   Expression analyses of NRs showing common upregulation in both prostate cancer cell line-derived prostatospheroids and post-castrated VCaP-CRPC tumor xenografts. (A) Five orphan NRs (including RORβ, TLX, COUP-TFII, NURR1 and LRH-1) were identified to exhibit common upregulation in both prostate cancer cell line-derived prostatospheroids and castration-relapse VCaP-CRPC tumor xenografts. (B) qRT-PCR analysis validated the common upregulation of the identified orphan NRs in prostatospheroids and post-castrated VCaP-CRPC tumor xenografts. *P < 0.05; **P < 0.01 vs adherent parental cells or pre-castration. (C) Immunoblot analysis validated the elevated protein expressions of five identified orphan NRs (including RORβ, TLX, COUP-TFII, NURR1 and LRH-1) in LNCaP- and DU145-derived prostatospheroids, and VCaP-CRPC xenograft model. Their expressions became reduced in prostatospheroid-derived re-adherent cells cultured under 2D culture condition. Similar to LNCaP/DU145-derived prostatospheroids, increased protein expressions of five orphan NRs were shown in 2-month post-castration VCaP-CRPC tumors. Representative figures shown above the blots represent the β-actin normalized levels relative to either adherent cells grown under 2D culture condition or pre-castration VCaP xenograft tumors.
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   Figure 6

   Upregulation of five identified orphan NRs shown in abiraterone-treated VCaP-CRPC xenografts, FACS-sorted CD133⁺-primary prostate cancer cells and clinical prostate cancer samples. (A) Abiraterone treatment of castration-relapse VCaP-CRPC xenograft model. qRT-PCR results showed that the abiraterone-treated VCaP-CRPC tumors expressed higher levels of the five identified orphan NRs (RORβ, TLX, COUP-TFII, NURR1 and LRH-1) as compared to vehicle-treated tumors. *P < 0.05; **P < 0.01 vs vehicle treatment. (B) FACS-sorted CD133⁺-primary prostate cancer cells. qRT-PCR results showed that the FACS-sorted CD133⁺-primary prostate cancer cells showed higher levels of TLX, NURR1 and LRH-1 as compared to CD133⁻ cells. (C) Oncogenomic analysis of the five identified orphan NRs in prostate cancer clinical samples using the ONCOMINE datasets (http://www.oncomine.org). Results of expressions showed that higher log2 median-centered intensities of RORβ, TLX, COUP-TFII, NURR1 and LRH-1 were detected in clinical prostate cancer samples as compared to normal prostate gland tissues. Individual data points are presented as means (± s.e.m.) by boxplots of the log2 median-centered intensity where the greater intensity indicates greater gene expression. *P < 0.05; **P < 0.01 prostate carcinoma vs normal prostate.
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   Figure 7

   Stable prostate cancer infectants with overexpression of the five identified orphan NRs show increased expression of PCSC-associated markers and enhanced sphere formation capacity. (A) Immunoblot validation of DU145 infectants with stable overexpression of five respective orphan NRs (RORβ, TLX, COUP-TFII, NURR1 and LRH-1). (B) All five validated DU145 infectants showed increased expressions of multiple PCSC-associated markers as compared to their vector infectants. *P < 0.05 vs vector infectants. (C) Upper panels: representative images of 3D-cultured prostatospheroids formed by the orphan NR or vector infectants of DU145 and LNCaP cells. Lower panels: Quantitative analysis of prostatospheroids formed by the respective infectants. All five orphan NR infectants of DU145 and LNCaP cells showed significant enhanced sphere formation capacity than their respective vector infectants. *P < 0.05 vs vector infectants.

• © 2018 Society for Endocrinology