Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Sep 8;30(36):3833-45.
doi: 10.1038/onc.2011.114. Epub 2011 Apr 18.

NANOG promotes cancer stem cell characteristics and prostate cancer resistance to androgen deprivation

C R Jeter  1 , B Liu, X Liu, X Chen, C Liu, T Calhoun-Davis, J Repass, H Zaehres, J J Shen, D G Tang
Affiliations

NANOG promotes cancer stem cell characteristics and prostate cancer resistance to androgen deprivation

C R Jeter et al. Oncogene. .

Abstract

Cancer cell molecular mimicry of stem cells (SC) imbues neoplastic cells with enhanced proliferative and renewal capacities. In support, numerous mediators of SC self-renewal have been evinced to show oncogenic potential. We have recently reported that short-hairpin RNA-mediated knockdown of the embryonic stem cell (ESC) self-renewal gene NANOG significantly reduced the clonogenic and tumorigenic capabilities of various cancer cells. In this study, we sought to test the potential pro-tumorigenic functions of NANOG, particularly, in prostate cancer (PCa). Using qRT-PCR, we first confirmed that PCa cells expressed NANOG mRNA primarily from the NANOGP8 locus on chromosome 15q14. We then constructed a lentiviral promoter reporter in which the -3.8-kb NANOGP8 genomic fragment was used to drive the expression of green fluorescence protein (GFP). We observed that NANOGP8-GFP(+) PCa cells showed cancer stem cell (CSC) characteristics such as enhanced clonal growth and tumor regenerative capacity. To further investigate the functions and mechanisms of NANOG in tumorigenesis, we established tetracycline-inducible NANOG-overexpressing cancer cell lines, including both PCa (Du145 and LNCaP) and breast (MCF-7) cancer cells. NANOG induction promoted drug resistance in MCF-7 cells, tumor regeneration in Du145 cells and, most importantly, castration-resistant tumor development in LNCaP cells. These pro-tumorigenic effects of NANOG were associated with key molecular changes, including an upregulation of molecules such as CXCR4, IGFBP5, CD133 and ALDH1. The present gain-of-function studies, coupled with our recent loss-of-function work, establish the integral role for NANOG in neoplastic processes and shed light on its mechanisms of action.

PubMed Disclaimer

Conflict of interest statement

Conflict of Interest: Authors declare no conflict of interest.

Figures

Figure 1
Figure 1. NANOG genomic loci and NANOG mRNA expression in PCa cells
a) Schematic of NANOG1 and NANOGP8 gene structures. Chr, chromosome; E, exon; UTR, untranslated region. The 22-bp region unique to NANOG1 (vertical bar) was used to design primers/probes for NANOG1 and NANOGP8 specific PCR. b) Quantitative PCR (qPCR) detection of NANOGP8 mRNA expression in various cancer cells, normalized to GAPDH. The normalized NANOGP8 mRNA levels in LNCaP cells were set at 1 and NANOGP8 mRNA levels in breast (MCF7), colon (Colo320) and PCa cell lines (LNCaP, Du145 and PC3) and xenografts (LAPC-4 and LAPC-9) were presented relative to LNCaP. c) NANOGP8 mRNA levels in a cohort of primary patient tumors (HPCa) as determined by qPCR. HPCa56P1 and HPCa101P0 are two early-passage (P) xenografts established in our lab.
Figure 2
Figure 2. NANOGP8-expressing PCa cells possess CSC properties
a) Schematic of RRL-GFP lentiviral reporter constructs. GFP, green fluorescence protein; PGK, phosphoglycerate kinase; NP8, NANOGP8 promoter. b) FACS analysis of the percentage of GFP+ cells following transduction with the promoter reporter lentiviruses. 50K cells/well in 12-well dishes plated 1-day earlier were transduced with the indicated volumes of lentivirus. Cultured cells were maintained in fresh media and passaged until FACS analysis ∼ 5 d post-transduction. c) Immunostaining of NP8-GFP transduced LNCaP cells for NANOG (red) demonstrates co-localization of GFP and NANOG expression. Original magnifications, x200. (d-f) FACS purified viable (7AAD-) NP8-GFP+ PCa cells transduced (7-10 d prior) with the NP8-GFP lentivirus exhibit enhanced cloning efficiency (C.E.). d) NP8-Du145 cells (200 cells/well in 6-well culture plate) scored 14 d post-plating: holo, holoclones; mero, meroclones; para, paraclones. *P <0.001. e) NP8-PC3 cells (200 cells/well in 6-well culture plate) scored 14 d post-plating. f) LNCaP cells assayed in parallel at clonal density in androgen-deprived conditions (CDSS + 20 μM bicalutamide) versus standard growth media (2K cells/well and 200 cells/well, respectively) scored 14 d post-plating. NP8-GFP+ LNCaP cells displayed increased C.E. only in androgen-deprivation conditions. g-h) NP8-GFP+ PCa cells are more tumorigenic than NP8-GFP- cells. FACS-purified GFP+/- cells were injected s.c in Matrigel in NOD/SCID-γ recipients. In g), 5K each of NP8-GFP+/- Du145 cells were injected and tumors harvested at ∼2 months. In h), 1K each of purified NP8+/- PC3 cells were injected s.c in the 1° and 2° tumor transplantation experiments (harvested at d 56 and day 49, respectively). Shown are the pooled data.
Figure 3
Figure 3. Establishment and characterization of a dox-inducible NANOG overexpression system
a) Schematic of pLVX-TetON (Clontech) binary system to express inducible NANOG1 or NANOGP8: CMV, cytomegalovirus promoter; Dox, doxycycline; TRE, tetracycline responsive element. b-c) Western blot analysis of protein extracts (80 μg whole cell lysate) demonstrates dox-inducible NANOG overexpression. N.S, non-specific band. b) Western analysis of NANOG in extracts derived from control pLVX MCF-7, Du145 and LNCaP cells or respective pLVX-NANOG1/P8 clones, either untreated or induced with doxycycline (500 ng/ml; 48 h). c) Doxycycline titration in MCF-7 pLVX cells as indicated. Note that in both Western analyses, the endogenous NANOG protein was barely detectable due to NANOG-expressing cells being very rare (see below). d-e) NANOG IF staining using a Rb polyclonal anti-NANOG antibody (H-155; Santa Cruz) revealed nuclear NANOG expression in the majority of clonally-derived cells following dox induction in pLVX-NANOG1/P8 Du145 (d) or LNCaP (e) cells.
Figure 4
Figure 4. Overexpressed NANOG1 and NANOGP8 proteins in cancer cells bind the expected molecular targets
a) ChIP assays in N-TERA human embryonal carcinoma cells. Chromatin prepared from N-TERA cells was used in immunoprecipitations with a Rb polyclonal anti-NANOG Ab or, as control, Rb IgG. DNA co-precipitated in ChIP was then used in PCR analysis of the promoter region of the indicated molecules. The results were expressed as NANOG binding to the targets relative to RbIgG (plotted as 1). b) pLVX-NANOG1/NANOGP8 LNCaP cells (clone D2) were either untreated or induced with dox (500 ng/ml) for 72 h. Chromatin was prepared and ChIP assays were performed similarly to those in N-TERA cells. c) Examples of ChIP assays with GAPDH ChIP as negative control.
Figure 5
Figure 5. NANOG overexpression promotes AI phenotypes in LNCaP cells
a-d) Enhanced androgen-independent (AI) growth of NANOG-overexpressing LNCaP cells relative to the empty vector (pLVX) control. a) pLVX- NANOGP8 expression promotes LNCaP sphere formation. 2.5K cells (Expt. #1) or 1K (Expt. #2) cells were plated in a 24-well dish in Methocult supplemented with B27 and N2 and 500 ng/ml doxycycline. Spheres (> 100 μM) were scored ∼2 weeks later. Shown below are two representative microphotographs of spheres formed in methycellulose. b-d) NANOG1 and NANOGP8 overexpression promotes the outgrowth of AI LNCaP cells. The three types of LNCaP cells (2K cells/well, plated in a 6-well TC dish) were cultured in CDSS-containing media for up to 30 d, feeding (+ 500 ng/ml doxycycline) every 3-5 d. Bright-field microscopy was used to capture AI foci and floating spheres (b). Shown in c) is quantification of AI-foci at two different time points and in d) is the total number of live cells/well at d30. e) NANOG-overexpressing LNCaP cells (1 × 106/injection) implanted s.c in NOD/SCID-γ males generated larger tumors than the pLVX-control cells (all harvested at d35). f-g) NANOG1/P8 overexpression promotes castration-resistant LNCaP tumor regeneration. The three types of pLVX LNCaP cells were implanted (10K/injection) s.c in either intact or fully castrated (i.e., surgical castration plus bicalutamide treatment) NOD/SCID-γ mice and tumors were harvested 41 (intact) and 61 (castrated) days after implantation. Shown are average tumor weight (f) and tumor images (g). h) NANOGP8-expressing LNCaP cells derived from AI tumors exhibit enhanced migratory properties as assessed by time-lapse video microscopy-assisted ‘scratch’ assays. Shown are representative time-lapse images for pLVX and pLVX-NANOGP8 LNCaP cells (left panels) and quantification of cell migration in all three groups (right).
Figure 6
Figure 6. Molecular changes associated with NANOG-promoted AI phenotypes in LNCaP cells
a) H&E staining and IHC analysis of the molecules indicated in three LNCaP AI tumors (tumor tag numbers indicated in parentheses). b-c) Western blotting analysis of c-Myc (b) and AR (c) in AI LNCaP tumors. N1, pLVX-NANOG1 LNCaP AI tumor; NP8, pLVX-NANOGP8 LNCaP AI tumor. N.S, non-specific band (served as loading control). d) Western blotting analysis of Nanog (top; N.S, non-specific band) and AR in clone B2 LNCaP cells treated by the indicated concentrations of dox. e-f) Heatmap presentation of gene expression changes of 11 molecules in LNCaP cells cultured in either androgen-deprived conditions (e) or regular serum cultures (f). The mRNA levels for these and other molecules (see Supplemental Table S1 and S3 for details) were determined by qRT-PCR and the heatmaps generated using the Matrix2.png software. The color-coded bars (scale below) indicate the mRNA level in the NANOG overexpressing cells cultured under the indicated conditions relative to pLVX control cells cultured under the same conditions.
Figure 7
Figure 7. NANOG overexpression promotes CSC characteristics
a-b) NANOGP8-overexpressing Du145 cells generate larger tumors than the pLVX-control cells. a) Subcutaneous (s.c) injection of the three types of pLVX Du145 cells (500K) in Matrigel into NOD/SCID hosts, harvested at d 79. b) Orthotopic transplantation of pLVX Du145 cells (1 million) in Matrigel into the dorsal prostate (D.P) of NOD/SCID-γ mice, harvested at d 40. c) Western blot analysis of c-Myc protein levels in the orthotopic Du145 tumors (above). The ERK and E-cadherin levels were unchanged and shown as loading controls. d) NANOG-overexpressing pLVX-MCF7 cells were resistant to the chemotherapeutic drugs doxorubicine (200 nM) and paclitaxel (25 nM). 35K cells plated per well (n = 3) in 6-well dish were induced with 500 ng/ml dox for 48 h followed by drug treatment (96 h). Cells were trypsinized and counted and the number of cells was presented as a percentage relative to the control. *P<0.01; #P<0.001 (Student's t-test). e) Heatmap presentation of gene expression changes of 14 molecules in the pLVX-Nanog1 (N1) or pLVX-NanogP8 (NP8) MCF7 cells induced with dox (500 ng/ml) for 3, 5, and 14 d. The mRNA levels for these and other molecules (see Supplemental Table S1 and S3 for details) were determined by qRT-PCR and the heatmaps generated using the Matrix2.png software. f) IF staining of CD133 protein in pLVX MCF7 cells as indicated. Images were captured on a confocal microscope. g) NANOG1/P8 induction increases the ALDEFLUOR-positive cells. The three types of pLVX-MCF7 cells were treated with dox (500 ng/ml, 10 d) and then used in Aldefluor assays. The mean percentages of ALDEFLUOR-positive cells were indicated.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci USA. 2003;100:3983–3988. - PMC - PubMed
    1. Ben-Porath I, Thomson MW, Carey VJ, Ge R, Bell GW, Regev A, et al. An embryonic stem cell-like gene expression signature in poorly differentiated aggressive human tumors. Nat Genet. 2008;40:499–507. - PMC - PubMed
    1. Boiko AD, Razorenova OV, van de Rijn M, Swetter SM, Johnson DL, Ly DP, et al. Human melanoma-initiating cells express neural crest nerve growth factor receptor CD271. Nature. 2010;466:133–7. - PMC - PubMed
    1. Booth HA, Holland PW. Eleven daughters of NANOG. Genomics. 2004;84:229–38. - PubMed
    1. Bourguignon LY, Peyrollier K, Xia W, Gilad E. Hyaluronan-CD44 iN-TERAction activates stem cell marker Nanog, Stat-3-mediated MDR1 gene expression, and ankyrin-regulated multidrug efflux in breast and ovarian tumor cells. J Biol Chem. 2008;283:17635–51. - PMC - PubMed

Publication types

MeSH terms