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

Jeter CR, Liu B, Liu X, Chen X, Liu C, Calhoun-Davis T, Repass J, Zaehres H, Shen JJ, Tang DG - Oncogene (2011)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Carcinogenesis, University of Texas MD Anderson Cancer Center, Science Park-Research Division, Smithville, USA. cjeter@mdanderson.org

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.

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NANOG overexpression promotes CSC characteristicsa-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.
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Figure 7: NANOG overexpression promotes CSC characteristicsa-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.

Mentions: To further evaluate the cellular and molecular consequences of NANOG overexpression in other contexts, we continued the in vitro and in vivo studies using our inducible Du145 and MCF7 cells. NANOGP8 induction in Du145 cells, which are AI PCa cells and lack AR expression, promoted tumor regeneration in both ectopic (i.e., s.c; Figure 7a) or orthotopic (i.e., dorsal prostate or DP; Figure 7b) sites. Curiously, pLVX-Nanog1 Du145 cells did not regenerate significantly larger tumors than pLVX control cells (Figure 7a-b) despite overall similar expression levels of NANOG mRNA (Supplemental Figure S6a, right) and protein (not shown) in pLVX-Nanog1 and pLVX-NanogP8 Du145 tumors. pLVX-NanogP8 Du145 cell-derived orthotopic tumors had higher levels of c-Myc protein in comparison to either pLVX-Nanog1 or pLVX-control cells (Figure 7c), perhaps partially accounting for the protumorigenic activity of NANOGP8.


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

Jeter CR, Liu B, Liu X, Chen X, Liu C, Calhoun-Davis T, Repass J, Zaehres H, Shen JJ, Tang DG - Oncogene (2011)

NANOG overexpression promotes CSC characteristicsa-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.
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Figure 7: NANOG overexpression promotes CSC characteristicsa-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.
Mentions: To further evaluate the cellular and molecular consequences of NANOG overexpression in other contexts, we continued the in vitro and in vivo studies using our inducible Du145 and MCF7 cells. NANOGP8 induction in Du145 cells, which are AI PCa cells and lack AR expression, promoted tumor regeneration in both ectopic (i.e., s.c; Figure 7a) or orthotopic (i.e., dorsal prostate or DP; Figure 7b) sites. Curiously, pLVX-Nanog1 Du145 cells did not regenerate significantly larger tumors than pLVX control cells (Figure 7a-b) despite overall similar expression levels of NANOG mRNA (Supplemental Figure S6a, right) and protein (not shown) in pLVX-Nanog1 and pLVX-NanogP8 Du145 tumors. pLVX-NanogP8 Du145 cell-derived orthotopic tumors had higher levels of c-Myc protein in comparison to either pLVX-Nanog1 or pLVX-control cells (Figure 7c), perhaps partially accounting for the protumorigenic activity of NANOGP8.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Carcinogenesis, University of Texas MD Anderson Cancer Center, Science Park-Research Division, Smithville, USA. cjeter@mdanderson.org

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.

Show MeSH
Related in: MedlinePlus