The pluripotency factor nanog promotes breast cancer tumorigenesis and metastasis

Abstract

Nanog is a transcription factor required for maintaining the pluripotency of embryonic stem cells, and is not expressed in most normal adult tissues. However, recent studies have indicated that Nanog is overexpressed in many types of human cancers, including breast cancer. To elucidate the physiological roles of Nanog in tumorigenesis, we developed an inducible Nanog transgenic mouse model, in which the expression of Nanog in adult tissues can be induced via LoxP/Cre-mediated deletion. Our findings indicate that overexpression of Nanog in the mammary gland is not sufficient to induce mammary tumor. However, when coexpressed with Wnt-1 in the mouse mammary gland, it promotes mammary tumorigenesis and metastasis. In this context, Nanog promotes the migration and invasion of breast cancer cells. Microarray analysis has shown that the ectopic expression of Nanog deregulates the expression of numerous genes associated with tumorigenesis and metastasis, such as the PDGFRα gene. Our findings demonstrate the involvement of Nanog in breast cancer metastasis, and provide the basis for the reported correlation between Nanog expression and poor prognosis of human breast cancer patients. As Nanog is not expressed in most adult tissues, these findings identify Nanog as a potential therapeutic target in the treatment of Nanog-expressing metastatic breast cancer.

Figure 1

Ectopic expression of Nanog in mammary epithelial cells of TN mice. (a) Scheme of the Nanog transgenic construct. The Nanog expression cassette contains the constitutive active cytomegalovirus (CMV) early enhancer element and chicken beta-actin promoter (CAG P), a LoxP flanked Neo- Stop cassette, the Nanog full-length cDNA (Nanog) and SV40 poly A. LoxP/Cre-mediated deletion of transcription stop element permits Nanog expression. P1, P2, P3 are the primers used for genotyping. (b) Nanog is ectopically expressed in mammary glands of TN mice, as determined by Western blotting. (c) Confocal immunofluorescence images show that Nanog is ectopically expressed in the mammary epithelial cells of two-month-old TN mice.

Figure 2

Ectopic expression of Nanog does not induce mammary tumor but accelerates mammary tumorigenesis. (a) Survival curves of TN, TNanog, and MMTV-Cre mice. (b) Survival curves of TNW, MMTV-Cre/mWnt-1, and mWnt-1 mice. “N” indicates the number of mice monitored. TNW mice died of mammary tumor earlier than MMTV-Cre/mWnt-1 and mWnt-1 mice (p = 0.0013 and 0.0016, respectively). P values were calculated using log-rank test analysis. (c) Whole mount showing hyperplasia in a Nanog- and mWnt-1-expressing mouse mammary gland. (d) H&E-stained mammary gland sections show alveolar expansion in a typical TNW mammary gland compared to a typical MMTV-Cre control mammary gland (magnification ×100). Quantification of epithelial versus fat and stromal content taken from H&E-stained mammary gland sections from MMTV-Cre/mWnt-1 and TNW mice glands (Left). The bars represent the mean ± SD. “*” indicates P < 0.05. The P value was obtained from an unpaired, 2-tailed t test with Welch’s correction.

Figure 3

Ectopic expression of Nanog promotes lung metastasis of mammary tumor in mWnt-1 female mice. (a) Percentage of mice with lung metastasis. Two-tailed P-values were obtained from Fisher’s exact test. Asterisks (***) indicate p < 0.0001; “NS” indicates no significant difference. P values were calculated using Fisher’s exact test. (b) Representative lung tissue with mammary tumor metastasis in TNW mice. (c) Representative H&E-stained sections of lung tissue with metastatic mammary adenocarcinoma of a TNW mouse. MT: metastatic tumor. (d) Q-PCR analysis showing that Nanog is expressed in noncancerous breast tissue, primary mammary tumor and metastatic mammary tumor of TNW mice.

Figure 4

Over-expression of Nanog promotes migration and invasion of mammary tumor cells. (a) Western blot detecting Nanog expression in mammary tumor cells from TNanog/mWnt-1 mice following transduction with a lentivirus expressing GFP-Cre recombinase or control GFP. (b) Primary TNanog/mWnt-1 mammary tumor cells expressing GFP or GFP-Cre were induced to migrate toward serum. The migrated cells were counted and compared. (c) Primary TNanog/mWnt-1 mammary tumor cells expressing GFP or GFP-Cre were induced to invade through the matrigel toward serum. Invasive cells were counted and compared. The bars represent the mean ± SD. “*” indicates P < 0.05. P value was obtained from unpaired, 2-tailed t test with Welch’s correction.

Figure 5

Overexpression of human Nanog (hNanog) enhanced colony formation and migration of MCF7 breast cancer cells. (a) Western blot detecting hNanog-HA expression in MCF7 cells. (b) Soft agar assay showing that overexpression of hNanog enhances the colony formation ability of MCF7. (c and d) Overexpression of Nanog promotes MCF7 cell migration and invasion. (e) Images of tumors formed by MCF-7 cells with the overexpression of Nanog versus mock-infected MCF7 cells, orthotopically transplanted in the second mammary gland of SCID mice. (f) Weights of tumors formed by MCF-7 cells with the overexpression of Nanog versus mock-infected MCF7 cells. (g) Typical H&E-stained sections of primary mammary tumors formed by MCF-7 with the overexpression of Nanog versus mock-infected MCF7 cells. Arrows indicate areas of stromal invasion. The bars represent the mean ± SD “*” indicates P < 0.05. “***” indicates P < 0.0001 P value were obtained from unpaired, 2-tailed t test with Welch’s correction.

Figure 6

Identification of genes differentially expressed between mammary tumors with the overexpression of Nanog and control tumors with no Nanog expression. (a) Heatmap diagram showing relative differences in expression levels for the 60 most differentially expressed genes. In this diagram, the difference in the expression level of a particular gene compared to the average level across all samples is indicated by color (increased expression, yellow; decreased expression, blue) and similarity in gene expression patterns is indicated by clustering. (b) Q-PCR confirmed Pdgfrα gene expression in high-Nanog-expressing mammary tumors and the control MMTV-Cre/mWnt-1 mammary tumors. (c) Q-PCR analysis detects Pdgfrα gene expression in the human breast cancer cell line MCF7 with Nanog overexpression or mock-transduction. (d) Schematic diagram showing genes and canonical pathways differentially affected by Nanog overexpression in breast tumors.