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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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Molecular changes associated with NANOG-promoted AI phenotypes in LNCaP cellsa) 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.
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Figure 6: Molecular changes associated with NANOG-promoted AI phenotypes in LNCaP cellsa) 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.

Mentions: Given that NANOG-overexpressing LNCaP cells both in vitro and in vivo exhibited enhanced hormone refractory growth, we wondered what molecular mechanisms could account for this resistance to androgen-deprivation? IHC analysis of AI tumors developed in castrated hosts (Figure 5g) revealed significantly more Ki-67+ cells in both pLVX-Nanog8 and pLVX-Nanog1 tumors (Figure 6a), suggesting that NANOG overexpression promotes cell proliferation in vivo. Consistent with this conclusion, the pLVX-Nanog1 AI tumors, and, in particular, the pLVX-NanogP8 AI tumors, showed higher levels of c-Myc compared to the control tumors (Figure 6b). In contrast, we did not observe obvious differences in Ki-67+ cells in the LNCaP tumors developed in intact male mice (Supplemental Figure S6c). Previously, we provided preliminary evidence that NANOG and androgen receptor (AR) seemed to be reciprocally expressed in HPCa cells in patient tumors (Jeter et al., 2009). IHC (Figure 6a) and Western blotting analysis (Figure 6c) detected reduced AR levels in pLVX-Nanog1/NanogP8 LNCaP AI tumors. In fact, even in short-term in vitro assays, both clone B2 (Figure 6d) and clone D2 (Supplemental Figure S5c) LNCaP cells, upon NANOG1/NANOGP8 induction, demonstrated subtle but reproducible decreases in AR. Consistent with reduced AR levels, the pLVX-Nanog1/NanogP8 LNCaP AI tumors also showed reduced levels of PSA (Figure 6a). Pilot experiments with a PSA promoter-driven GFP reporter also revealed that the pLVX-Nanog1/NanogP8 LNCaP cell cultures had ∼20% and 18%, respectively, less GFP+ cells than pLVX control LNCaP cells (data not shown). In principle, increased cell proliferation (i.e., increased Ki-67+ cells associated with c-Myc upregulation) and inhibition of differentiation (i.e., reduced AR and PSA) could help explain NANOG1/NANOGP8-promoted LNCaP AI tumor regeneration.


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)

Molecular changes associated with NANOG-promoted AI phenotypes in LNCaP cellsa) 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.
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Figure 6: Molecular changes associated with NANOG-promoted AI phenotypes in LNCaP cellsa) 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.
Mentions: Given that NANOG-overexpressing LNCaP cells both in vitro and in vivo exhibited enhanced hormone refractory growth, we wondered what molecular mechanisms could account for this resistance to androgen-deprivation? IHC analysis of AI tumors developed in castrated hosts (Figure 5g) revealed significantly more Ki-67+ cells in both pLVX-Nanog8 and pLVX-Nanog1 tumors (Figure 6a), suggesting that NANOG overexpression promotes cell proliferation in vivo. Consistent with this conclusion, the pLVX-Nanog1 AI tumors, and, in particular, the pLVX-NanogP8 AI tumors, showed higher levels of c-Myc compared to the control tumors (Figure 6b). In contrast, we did not observe obvious differences in Ki-67+ cells in the LNCaP tumors developed in intact male mice (Supplemental Figure S6c). Previously, we provided preliminary evidence that NANOG and androgen receptor (AR) seemed to be reciprocally expressed in HPCa cells in patient tumors (Jeter et al., 2009). IHC (Figure 6a) and Western blotting analysis (Figure 6c) detected reduced AR levels in pLVX-Nanog1/NanogP8 LNCaP AI tumors. In fact, even in short-term in vitro assays, both clone B2 (Figure 6d) and clone D2 (Supplemental Figure S5c) LNCaP cells, upon NANOG1/NANOGP8 induction, demonstrated subtle but reproducible decreases in AR. Consistent with reduced AR levels, the pLVX-Nanog1/NanogP8 LNCaP AI tumors also showed reduced levels of PSA (Figure 6a). Pilot experiments with a PSA promoter-driven GFP reporter also revealed that the pLVX-Nanog1/NanogP8 LNCaP cell cultures had ∼20% and 18%, respectively, less GFP+ cells than pLVX control LNCaP cells (data not shown). In principle, increased cell proliferation (i.e., increased Ki-67+ cells associated with c-Myc upregulation) and inhibition of differentiation (i.e., reduced AR and PSA) could help explain NANOG1/NANOGP8-promoted LNCaP AI tumor regeneration.

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