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Autochthonous mouse melanoma and mammary tumors do not express the pluripotency genes Oct4 and Nanog.

Schreiber C, Kuch V, Umansky V, Sleeman JP - PLoS ONE (2013)

Bottom Line: The homeodomain transcription factors Oct4 and Nanog maintain pluripotency and self-renewal in embryonic stem cells.However, we could find no evidence for expression of the GFP reporter above background levels in tumors using FACS, qPCR and immunohistochemistry.Furthermore, cultivation of Oct4GFP and NanogGFP MMTV-PyMT tumor cells either adherently or as spheroids had no effect on the expression of the GFP reporter.

View Article: PubMed Central - PubMed

Affiliation: Centre for Biomedicine and Medical Technology Mannheim, Medical Faculty Mannheim, University Heidelberg, Mannheim, Germany. caroline.schreiber@medma.uni-heidelberg.de

ABSTRACT
The homeodomain transcription factors Oct4 and Nanog maintain pluripotency and self-renewal in embryonic stem cells. In somatic cells, inappropriate expression of these genes has been associated with loss of differentiation, malignant transformation, and the acquisition of cancer stem cell-like properties. As cancer stem cells have been suggested to underlie the growth and malignancy of tumors, Oct4 and Nanog may represent therapeutic targets. Their expression could also act as a marker of the cancer stem cell population, permitting its isolation and characterisation. Nevertheless, the existence of multiple pseudogenes and isoforms of these genes has complicated the interpretation of the data that supports a role for Oct4 and Nanog in the cancer context. Here we addressed this issue using knockin mice in which IRES elements are used to allow GFP expression under the control of the endogenous Oct4 or Nanog promoters, while maintaining correct expression of the Oct4 or Nanog gene. These mice were crossed with MT/ret mice that develop melanomas, and with MMTV-PyMT mice and MMTV-Neu mice that develop mammary adenocarcinomas. We analysed the tumors that developed in these compound mice for GFP expression. In this way we could assess transcription of Oct4 and Nanog in autochthonous cancers without the complication of factors such as pseudogene expression, alternative splicing and antibody specificity. Both the Oct4 and Nanog knockin tumor-bearing mice expressed GFP in blastocysts and testes as expected. However, we could find no evidence for expression of the GFP reporter above background levels in tumors using FACS, qPCR and immunohistochemistry. Furthermore, cultivation of Oct4GFP and NanogGFP MMTV-PyMT tumor cells either adherently or as spheroids had no effect on the expression of the GFP reporter. Together these data suggest that Oct4 and Nanog are not expressed in tumor cells that arise in the autochthonous cancer models studied here.

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Spheroid cell culture does not induce Oct4GFP or NanogGFP expression in MMTV-PyMT or MMTV-Neu tumor cells.(A) No GFP+ cells can be detected in spheroids of Oct4GFP+ or NanogGFP+ MMTV-PyMT tumor cells. Oct4GFP+ or NanogGFP+ MMTV-PyMT tumor cells were freshly isolated from tumors, grown as spheres for one passage when pictures were taken. Representative bright field (left side) and fluorescent (right side) pictures are shown. Scale bar = 200 µm. (B-D) Freshly isolated Oct4GFP+ or NanogGFP+ MMTV-PyMT and MMTV-Neu tumor cells were either grown adherently (passage 1–4) or as spheres (passage 0) and analysed for relative expression of (B) GFP, (C) Oct4 and (D) Nanog mRNA by qPCR. mRNA expression of NanogGFP+ testis and compound MMTV-PyMT and MMTV-Neu tumor cells was compared to mRNA expression of control MMTV-PyMT tumor cells, which was set to 1. The analysis was performed in triplicate. The mean ± SEM is shown.
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pone-0057465-g006: Spheroid cell culture does not induce Oct4GFP or NanogGFP expression in MMTV-PyMT or MMTV-Neu tumor cells.(A) No GFP+ cells can be detected in spheroids of Oct4GFP+ or NanogGFP+ MMTV-PyMT tumor cells. Oct4GFP+ or NanogGFP+ MMTV-PyMT tumor cells were freshly isolated from tumors, grown as spheres for one passage when pictures were taken. Representative bright field (left side) and fluorescent (right side) pictures are shown. Scale bar = 200 µm. (B-D) Freshly isolated Oct4GFP+ or NanogGFP+ MMTV-PyMT and MMTV-Neu tumor cells were either grown adherently (passage 1–4) or as spheres (passage 0) and analysed for relative expression of (B) GFP, (C) Oct4 and (D) Nanog mRNA by qPCR. mRNA expression of NanogGFP+ testis and compound MMTV-PyMT and MMTV-Neu tumor cells was compared to mRNA expression of control MMTV-PyMT tumor cells, which was set to 1. The analysis was performed in triplicate. The mean ± SEM is shown.

Mentions: Non-adherent culture of tumor cells as spheres has been reported to enrich for CSCs and to increase the expression of genes such as Oct4 and Nanog [20], [47], [48]. We therefore freshly isolated cells from Oct4GFP+ and NanogGFP+ MMTV-PyMT or MMTV-Neu tumors and plated them either adherently or as suspension spheres. Bright field and fluorescent pictures of the spheres were taken, then GFP expression was determined by qPCR analysis. No GFP+ cells were detected using fluorescent microscopy in spheres derived from Oct4GFP+ and NanogGFP+ MMTV-PyMT or MMTV-Neu tumors (Figure 6A and data not shown). Furthermore, no increase in expression of GFP, Oct4 or Nanog was observed in qPCR analysis of either adherent or spheroid-cultured Oct4GFP+ and NanogGFP+ cells (Figure 6B–D). Together the data suggest that cultivation of tumor cells from the autochthonous cancer models studied here, even under conditions that might be expected to increase CSC numbers, does not result in enhanced expression of Oct4 or Nanog.


Autochthonous mouse melanoma and mammary tumors do not express the pluripotency genes Oct4 and Nanog.

Schreiber C, Kuch V, Umansky V, Sleeman JP - PLoS ONE (2013)

Spheroid cell culture does not induce Oct4GFP or NanogGFP expression in MMTV-PyMT or MMTV-Neu tumor cells.(A) No GFP+ cells can be detected in spheroids of Oct4GFP+ or NanogGFP+ MMTV-PyMT tumor cells. Oct4GFP+ or NanogGFP+ MMTV-PyMT tumor cells were freshly isolated from tumors, grown as spheres for one passage when pictures were taken. Representative bright field (left side) and fluorescent (right side) pictures are shown. Scale bar = 200 µm. (B-D) Freshly isolated Oct4GFP+ or NanogGFP+ MMTV-PyMT and MMTV-Neu tumor cells were either grown adherently (passage 1–4) or as spheres (passage 0) and analysed for relative expression of (B) GFP, (C) Oct4 and (D) Nanog mRNA by qPCR. mRNA expression of NanogGFP+ testis and compound MMTV-PyMT and MMTV-Neu tumor cells was compared to mRNA expression of control MMTV-PyMT tumor cells, which was set to 1. The analysis was performed in triplicate. The mean ± SEM is shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585372&req=5

pone-0057465-g006: Spheroid cell culture does not induce Oct4GFP or NanogGFP expression in MMTV-PyMT or MMTV-Neu tumor cells.(A) No GFP+ cells can be detected in spheroids of Oct4GFP+ or NanogGFP+ MMTV-PyMT tumor cells. Oct4GFP+ or NanogGFP+ MMTV-PyMT tumor cells were freshly isolated from tumors, grown as spheres for one passage when pictures were taken. Representative bright field (left side) and fluorescent (right side) pictures are shown. Scale bar = 200 µm. (B-D) Freshly isolated Oct4GFP+ or NanogGFP+ MMTV-PyMT and MMTV-Neu tumor cells were either grown adherently (passage 1–4) or as spheres (passage 0) and analysed for relative expression of (B) GFP, (C) Oct4 and (D) Nanog mRNA by qPCR. mRNA expression of NanogGFP+ testis and compound MMTV-PyMT and MMTV-Neu tumor cells was compared to mRNA expression of control MMTV-PyMT tumor cells, which was set to 1. The analysis was performed in triplicate. The mean ± SEM is shown.
Mentions: Non-adherent culture of tumor cells as spheres has been reported to enrich for CSCs and to increase the expression of genes such as Oct4 and Nanog [20], [47], [48]. We therefore freshly isolated cells from Oct4GFP+ and NanogGFP+ MMTV-PyMT or MMTV-Neu tumors and plated them either adherently or as suspension spheres. Bright field and fluorescent pictures of the spheres were taken, then GFP expression was determined by qPCR analysis. No GFP+ cells were detected using fluorescent microscopy in spheres derived from Oct4GFP+ and NanogGFP+ MMTV-PyMT or MMTV-Neu tumors (Figure 6A and data not shown). Furthermore, no increase in expression of GFP, Oct4 or Nanog was observed in qPCR analysis of either adherent or spheroid-cultured Oct4GFP+ and NanogGFP+ cells (Figure 6B–D). Together the data suggest that cultivation of tumor cells from the autochthonous cancer models studied here, even under conditions that might be expected to increase CSC numbers, does not result in enhanced expression of Oct4 or Nanog.

Bottom Line: The homeodomain transcription factors Oct4 and Nanog maintain pluripotency and self-renewal in embryonic stem cells.However, we could find no evidence for expression of the GFP reporter above background levels in tumors using FACS, qPCR and immunohistochemistry.Furthermore, cultivation of Oct4GFP and NanogGFP MMTV-PyMT tumor cells either adherently or as spheroids had no effect on the expression of the GFP reporter.

View Article: PubMed Central - PubMed

Affiliation: Centre for Biomedicine and Medical Technology Mannheim, Medical Faculty Mannheim, University Heidelberg, Mannheim, Germany. caroline.schreiber@medma.uni-heidelberg.de

ABSTRACT
The homeodomain transcription factors Oct4 and Nanog maintain pluripotency and self-renewal in embryonic stem cells. In somatic cells, inappropriate expression of these genes has been associated with loss of differentiation, malignant transformation, and the acquisition of cancer stem cell-like properties. As cancer stem cells have been suggested to underlie the growth and malignancy of tumors, Oct4 and Nanog may represent therapeutic targets. Their expression could also act as a marker of the cancer stem cell population, permitting its isolation and characterisation. Nevertheless, the existence of multiple pseudogenes and isoforms of these genes has complicated the interpretation of the data that supports a role for Oct4 and Nanog in the cancer context. Here we addressed this issue using knockin mice in which IRES elements are used to allow GFP expression under the control of the endogenous Oct4 or Nanog promoters, while maintaining correct expression of the Oct4 or Nanog gene. These mice were crossed with MT/ret mice that develop melanomas, and with MMTV-PyMT mice and MMTV-Neu mice that develop mammary adenocarcinomas. We analysed the tumors that developed in these compound mice for GFP expression. In this way we could assess transcription of Oct4 and Nanog in autochthonous cancers without the complication of factors such as pseudogene expression, alternative splicing and antibody specificity. Both the Oct4 and Nanog knockin tumor-bearing mice expressed GFP in blastocysts and testes as expected. However, we could find no evidence for expression of the GFP reporter above background levels in tumors using FACS, qPCR and immunohistochemistry. Furthermore, cultivation of Oct4GFP and NanogGFP MMTV-PyMT tumor cells either adherently or as spheroids had no effect on the expression of the GFP reporter. Together these data suggest that Oct4 and Nanog are not expressed in tumor cells that arise in the autochthonous cancer models studied here.

Show MeSH
Related in: MedlinePlus