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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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Only very rare GFP+ cells can be detected in compound MMTV-PyMT or MMTV-Neu tumors.Sections of Oct4GFP+ or NanogGFP+ MT/ret, MMTV-PyMT and MMTV-Neu tumors were stained with an anti-GFP antibody and analysed for GFP positive cells. (A) Representative pictures of GFP immunofluorescence stainings showing the GFP negativity of virtually all sections from the compound tumors. (B) Pictures of the only GFP+ cells detected in single sections from Oct4GFP+ and NanogGFP+ MMTV-PyMT and MMTV-Neu tumors. The framed region is enlarged in the insert. (C) Table summarizing the immunofluorescence analysis, showing the number of analysed tumors per tumor model, the total number of analysed sections, and the total number of detected GFP+ cells per tumor model.
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pone-0057465-g005: Only very rare GFP+ cells can be detected in compound MMTV-PyMT or MMTV-Neu tumors.Sections of Oct4GFP+ or NanogGFP+ MT/ret, MMTV-PyMT and MMTV-Neu tumors were stained with an anti-GFP antibody and analysed for GFP positive cells. (A) Representative pictures of GFP immunofluorescence stainings showing the GFP negativity of virtually all sections from the compound tumors. (B) Pictures of the only GFP+ cells detected in single sections from Oct4GFP+ and NanogGFP+ MMTV-PyMT and MMTV-Neu tumors. The framed region is enlarged in the insert. (C) Table summarizing the immunofluorescence analysis, showing the number of analysed tumors per tumor model, the total number of analysed sections, and the total number of detected GFP+ cells per tumor model.

Mentions: Finally we examined GFP expression in tumor sections. No direct GFP signal could be observed in sections from Oct4GFP+ and NanogGFP+ tumors (data not shown). We therefore stained the sections with anti-GFP antibody in an attempt to increase the sensitivity of detection. Tumors from between 3 to 6 animals were analysed per animal model, with at least 45 sections per tumor type analysed in total. In MT/ret primary tumors no NanogGFP or Oct4GFP positive cells could be detected (Figure 5A and C). The vast majority of analysed sections of MMTV-PyMT and MMTV-Neu Oct4GFP+ and NanogGFP+ tumors were also negative. However, a single Oct4GFP-positive cell and two NanogGFP-positive cells were detected in single sections from MMTV-PyMT tumors (Figure 5B and C). A single NanogGFP positive cell was also detected in a section from one MMTV-Neu tumor. These data again suggest that Oct4 and Nanog were expressed at virtually non-detectable levels in the MT/ret melanoma and MMTV-PyMT and MMTV-Neu mammary tumor cells.


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)

Only very rare GFP+ cells can be detected in compound MMTV-PyMT or MMTV-Neu tumors.Sections of Oct4GFP+ or NanogGFP+ MT/ret, MMTV-PyMT and MMTV-Neu tumors were stained with an anti-GFP antibody and analysed for GFP positive cells. (A) Representative pictures of GFP immunofluorescence stainings showing the GFP negativity of virtually all sections from the compound tumors. (B) Pictures of the only GFP+ cells detected in single sections from Oct4GFP+ and NanogGFP+ MMTV-PyMT and MMTV-Neu tumors. The framed region is enlarged in the insert. (C) Table summarizing the immunofluorescence analysis, showing the number of analysed tumors per tumor model, the total number of analysed sections, and the total number of detected GFP+ cells per tumor model.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0057465-g005: Only very rare GFP+ cells can be detected in compound MMTV-PyMT or MMTV-Neu tumors.Sections of Oct4GFP+ or NanogGFP+ MT/ret, MMTV-PyMT and MMTV-Neu tumors were stained with an anti-GFP antibody and analysed for GFP positive cells. (A) Representative pictures of GFP immunofluorescence stainings showing the GFP negativity of virtually all sections from the compound tumors. (B) Pictures of the only GFP+ cells detected in single sections from Oct4GFP+ and NanogGFP+ MMTV-PyMT and MMTV-Neu tumors. The framed region is enlarged in the insert. (C) Table summarizing the immunofluorescence analysis, showing the number of analysed tumors per tumor model, the total number of analysed sections, and the total number of detected GFP+ cells per tumor model.
Mentions: Finally we examined GFP expression in tumor sections. No direct GFP signal could be observed in sections from Oct4GFP+ and NanogGFP+ tumors (data not shown). We therefore stained the sections with anti-GFP antibody in an attempt to increase the sensitivity of detection. Tumors from between 3 to 6 animals were analysed per animal model, with at least 45 sections per tumor type analysed in total. In MT/ret primary tumors no NanogGFP or Oct4GFP positive cells could be detected (Figure 5A and C). The vast majority of analysed sections of MMTV-PyMT and MMTV-Neu Oct4GFP+ and NanogGFP+ tumors were also negative. However, a single Oct4GFP-positive cell and two NanogGFP-positive cells were detected in single sections from MMTV-PyMT tumors (Figure 5B and C). A single NanogGFP positive cell was also detected in a section from one MMTV-Neu tumor. These data again suggest that Oct4 and Nanog were expressed at virtually non-detectable levels in the MT/ret melanoma and MMTV-PyMT and MMTV-Neu mammary tumor cells.

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