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. 2014 Nov;74(15):1530-43.
doi: 10.1002/pros.22870. Epub 2014 Aug 31.

The pluripotency factor Nanog is directly upregulated by the androgen receptor in prostate cancer cells

Steven Kregel  1 , Russell Z Szmulewitz, Donald J Vander Griend
Affiliations

The pluripotency factor Nanog is directly upregulated by the androgen receptor in prostate cancer cells

Steven Kregel et al. Prostate. 2014 Nov.

Abstract

Background: The Androgen Receptor (AR) is a nuclear hormone receptor that functions as a critical oncogene in all stages of prostate cancer progression, including progression to castration-resistance following androgen-deprivation therapy. Thus, identifying and targeting critical AR-regulated genes is one potential method to block castration-resistant cancer proliferation. Of particular importance are transcription factors that regulate stem cell pluripotency; many of these genes are emerging as critical oncogenes in numerous tumor cell types. Of these, Nanog has been previously shown to increase the self-renewal and stem-like properties of prostate cancer cells. Thus, we hypothesized that Nanog is a candidate AR target gene that may impart castration-resistance.

Methods: We modulated AR signaling in LNCaP prostate cancer cells and assayed for Nanog expression. Direct AR binding to the NANOG promoter was tested using AR Chromatin Immunoprecipation (ChIP) and analyses of publically available AR ChIP-sequencing data-sets. Nanog over-expressing cells were analyzed for cell growth and cytotoxicity in response to the AR antagonist enzalutamide and the microtubule stabilizing agent docetaxel.

Results: AR signaling upregulates Nanog mRNA and protein. AR binds directly to the NANOG promoter, and was not identified within 75 kb of the NANOGP8 pseudogene, suggesting the NANOG gene locus was preferentially activated. Nanog overexpression in LNCaP cells increases overall growth, but does not increase resistance to enzalutamide or docetaxel.

Conclusions: Nanog is a novel oncogenic AR target gene in prostate cancer cells, and stable expression of Nanog increases proliferation and growth of prostate cancer cells, but not resistance to enzalutamide or docetaxel.

Keywords: NANOGP8; androgen receptor; docetaxel; enzalutamide; nanog; prostate cancer; stem cell.

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Conflict of interest statement

Conflicts of Interest: None

Figures

Figure 1
Figure 1. Androgen Receptor (AR) activation leads to an increase in Nanog expression
A: Increased Nanog mRNA in LNCaP cells upon AR stimulation as measured by Q-RT-PCR. Levels at 0.25 hours and beyond represent a statistically significant increase in Nanog mRNA (* indicates p<0.05). A further statistically significant increase was observed at 3 hours compared to all other conditions (** indicates p<0.05). LNCaP cells were grown in phenol-red free RPMI media (Corning, Corning, NY) with 10% charcoal stripped serum (Atlanta Biologicals, Lawrenceville, GA), to decrease endogenous AR-ligands, as LNCaP cells have a mutation in the ligand binding domain (T877A) of the AR which allows for promiscuous ligand binding (23). Cells were pre-incubated in charcoal stripped serum at least 24 hours prior to any pharmacologic treatments. B: Increased expression of the established AR target gene PSA (KLK3) in LNCaP cells within three hours. C: Increased expression of Nanog protein (35–42kD) upon AR stimulation with physiologic levels of androgen (1 nM R1881) in androgen sensitive LNCaP prostate cancer cells. Protein lysates from cells treated at defined intervals (3–72 hours) after androgen treatment were subjected to western blotting. Nanog protein migrates multiple bands around 35–42kD via western blot using a variety of Nanog antibodies (27).
Figure 2
Figure 2. Androgen Receptor (AR)-mediated up-regulation of Nanog expression can be reversed by treatment with the Anti-Androgen Enzalutamide
A: LNCaP cells were grown in phenol-red free RPMI media (Corning, Corning, NY) with 10% charcoal stripped serum (Atlanta Biologicals, Lawrenceville, GA), to decrease endogenous AR-ligands, as LNCaP cells have a mutation in the ligand binding domain (T877A) of the AR which allows for promiscuous ligand binding (23). Cells were pre-incubated in charcoal stripped serum at least 24 hours prior to any pharmacologic treatments. To verify that the increase in Nanog mRNA and protein is specific to AR activation and can be reversed by an AR antagonist, cells were incubated 1 nm or 10 nm R1881, or vehicle for 24 hours, and then either 10 µM enzalutamide or vehicle was added to the culture medium for an additional 48 hours prior to lysis. An increase in Nanog mRNA with R1881 treatment was measured using qPCR (*p<0.05), which was brought back to control levels upon treatment with enzalutamide, in both LNCaP cells. 10nM R1881 was enough to overcome inhibition of Nanog mRNA upregulation with 10µM MDV3100 treatment in LNCaP cells. B: Western blots show an increase of Nanog protein with R1881 that can be decreased with addition of enzalutamide, with minimal changes in to AR protein levels, similar to mRNA. Nanog protein migrates multiple bands around 35–42kD via western blot using a variety of Nanog antibodies (27). β-Actin was used as a loading control.
Figure 3
Figure 3. Androgen Receptor Activation Increases both NANOG and NANOGP8 Transcripts in LNCaP Cells
A: Restriction fragment length polymorphism (RFLP) approach to identify multiple Nanog transcripts. Illustration of the regions of the three genes, NANOG, NANOGP7 and NANOGP8, flanking the AlwN1 digestion site, and the regions of NANOG and NANOGP8 flanking the Sma1 site in the 3’ UTRs of these transcripts. The AlwN1 digestion site exists only in the NANOGP8 transcript, and the Sma1 digestion site exists only in NANOG. B: RT-PCR products from regions around the unique restriction enzyme digestion site for AlwN1 in NANOGP8 as well as the same sized fragments (348 base pairs) of NANOG and NANOGP7, were obtained from in LNCaP cells after 3 hours of 1nM treatment of R1881 or vehicle as previously described, and WA01(H1) cells (as a positive control for NANOG). Cloned NANOG, NANOGP7 and NANOGP8 transcripts served as controls (negative, negative and positive, respectively). A purified Nanog overexpression plasmid (Genecopia) served as an additional control. All PCR products were digested with AlwN1, and only NANOGP8 is cut into two fragments from the initial 348bp into fragments sized 230 and 118 bp. Undigested controls are also shown. For the unique restriction enzyme cut site for Sma1 in the 3’ UTR of NANOG, products the same sized fragment (353 base pairs) of the NANOGP8 3’ UTR, and as with the AlwN1 digestion, fragments were cut out of NANOG sized 221 and 132 bp. GAPDH serves as a control for cDNA loading, utilizing the same primers used for Q-RT-PCR in Figures 1 and 2. Together, these treatments illustrate an increase in both NANOG and NANOGP8.
Figure 4
Figure 4. Androgen Receptor (AR) directly binds to the NANOG promoter
A: Schematic of AR binding sites identified at the promoter and cis-enhancer regions of NANOG (within 2kb of the Nanog transcriptional start site) in published ChIP-seq (Yu et al. 2010) and ChIP-chip (Wang et al. 2009) data sets. Primer sequences were designed to investigate AR binding to Androgen Response Elements (ARE) and verify published binding via ChIP-qPCR. B: AR Chromatin Immunoprecipitation (ChIP) documents direct binding of ligand-activated AR to the Nanog promoter region in response to AR stimulation by R1881. LNCaP cells were treated with vehicle control or 1 nM R1881, and enrichment of the Nanog promoter after AR-ChIP was verified by primer-directed qPCR. Normal Rabbit IgG and Histone H3 served as negative and positive controls, respectively. When compared to the IgG negative control, both the positive control Histone H3 and ligand-activated AR significantly enriched for the Nanog enhancer (p<0.05). Known AR recruitment to the PSA/KLK3 promoter served to validate the AR ChIP. Cells were grown in phenol-red free RPMI media (Corning, Corning, NY) with 10% charcoal stripped serum (Atlanta Biologicals, Lawrenceville, GA), to decrease endogenous AR-ligands, as LNCaP cells have a mutation in the ligand binding domain (T877A) of the AR which allows for promiscuous ligand binding (23). Cells were pre-incubated in charcoal stripped serum at least 24 hours prior to any pharmacologic treatments.
Figure 5
Figure 5. Nanog overexpression in LNCaP cells leads to an increase in growth, but cells remain sensitive to androgen, anti-androgen and chemotherapeutic drug treatments
A: Immunoblot validation of lentiviral overexpression of Nanog or GFP control. LNCaP cells were transduced with lentivirus for stabile overexpression of Nanog (LV-Nanog) or control GFP (LV-GFP), and treated with R1881 and enzalutamide, and Nanog and AR protein levels were analyzed. Cells were grown in 1 nm or 10 nm R1881, or vehicle for 24 hours, and then either 10 µM enzalutamide or vehicle was added to the culture medium for an additional 48 hours. β-Actin was used as a loading control. Cells were grown in phenol-red free RPMI media (Corning, Corning, NY) with 10% charcoal stripped serum (Atlanta Biologicals, Lawrenceville, GA), to decrease endogenous AR-ligands, as LNCaP cells have a mutation in the ligand binding domain (T877A) of the AR which allows for promiscuous ligand binding (23). Cells were pre-incubated in charcoal stripped serum at least 24 hours prior to any pharmacologic treatments in all experiments. Nanog protein migrates multiple bands around 35–42kD via western blot using a variety of Nanog antibodies (27). B: A schematic illustrates the infection, selection, pharmacologic treatment and analysis of growth of the transduced cells. C: LNCaP cells transduced with lentivirus for stabile overexpression of Nanog or control GFP were assayed for cell density, growth and cytotoxicity using a Sulforhodamine B (SRB) assay (calculated by change in absorbance) over 7 day treatment with 10 µM enzalutamide, 1nM R1881 or vehicle control (* indicates p<0.05, when compared to GFP control, † indicates p<0.05, when compared to vehicle). D: LNCaP GFP or Nanog overexpressing cells were assayed for sensitivity to Docetaxel included cytotoxicity using SRB assay over 7 day treatment with 1nM, 5nM or 10nM Docetaxel or vehicle control (* indicates p<0.05, when compared to GFP control, † indicates p<0.05, when compared to vehicle).
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