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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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Oct4GFP and NanogGFP are expressed in mouse testes.(A) Cells from testes of GFP−/− control transgenic mice, Oct4GFP+/−, Oct4GFP+/+ and NanogGFP+ animals were isolated and analysed for GFP positivity by FACS. Representative flow cytometry analyses in which sideward scatter is plotted against GFP fluorescence are shown for each genotype. The percentage of GFP+ cells compared to GFP negative control testes is indicated in the gate. (B) GFP mRNA expression in control (n = 3), Oct4GFP+/− (n = 6), Oct4GFP+/+ (n = 2) and NanogGFP+ (n = 5) testes was analysed by qPCR. Circles represent individual samples, the bar indicates the mean value of all samples. (C) GFP+ cells can be detected in Oct4GFP+ and NanogGFP+ testes but not in control testis. Representative pictures of immunofluorescence staining with anti-GFP antibody are shown. Magnification 200×. The framed region is enlarged in the insert.
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pone-0057465-g002: Oct4GFP and NanogGFP are expressed in mouse testes.(A) Cells from testes of GFP−/− control transgenic mice, Oct4GFP+/−, Oct4GFP+/+ and NanogGFP+ animals were isolated and analysed for GFP positivity by FACS. Representative flow cytometry analyses in which sideward scatter is plotted against GFP fluorescence are shown for each genotype. The percentage of GFP+ cells compared to GFP negative control testes is indicated in the gate. (B) GFP mRNA expression in control (n = 3), Oct4GFP+/− (n = 6), Oct4GFP+/+ (n = 2) and NanogGFP+ (n = 5) testes was analysed by qPCR. Circles represent individual samples, the bar indicates the mean value of all samples. (C) GFP+ cells can be detected in Oct4GFP+ and NanogGFP+ testes but not in control testis. Representative pictures of immunofluorescence staining with anti-GFP antibody are shown. Magnification 200×. The framed region is enlarged in the insert.

Mentions: First we verified that the GFP reporter is expressed appropriately in these mice. As expected, GFP expression could be readily observed in the inner cell mass of transgenic blastocysts (Figure 1). Furthermore, we examined GFP expression in the testis as Oct4 and Nanog are expressed in spermatogonia [45], [46]. Using FACS analysis, GFP-positive cells were found to be present in the testes from both Oct4GFP and NanogGFP compound mice (Figure 2A). Furthermore, GFP mRNA expression could be readily detected in testis by qPCR analysis (Figure 2B), and single GFP positive cells were observed in testis sections using immunofluorescence staining, consistent with the expression of Oct4 and Nanog in early type-A spermatogonia (Figure 2C).


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)

Oct4GFP and NanogGFP are expressed in mouse testes.(A) Cells from testes of GFP−/− control transgenic mice, Oct4GFP+/−, Oct4GFP+/+ and NanogGFP+ animals were isolated and analysed for GFP positivity by FACS. Representative flow cytometry analyses in which sideward scatter is plotted against GFP fluorescence are shown for each genotype. The percentage of GFP+ cells compared to GFP negative control testes is indicated in the gate. (B) GFP mRNA expression in control (n = 3), Oct4GFP+/− (n = 6), Oct4GFP+/+ (n = 2) and NanogGFP+ (n = 5) testes was analysed by qPCR. Circles represent individual samples, the bar indicates the mean value of all samples. (C) GFP+ cells can be detected in Oct4GFP+ and NanogGFP+ testes but not in control testis. Representative pictures of immunofluorescence staining with anti-GFP antibody are shown. Magnification 200×. The framed region is enlarged in the insert.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585372&req=5

pone-0057465-g002: Oct4GFP and NanogGFP are expressed in mouse testes.(A) Cells from testes of GFP−/− control transgenic mice, Oct4GFP+/−, Oct4GFP+/+ and NanogGFP+ animals were isolated and analysed for GFP positivity by FACS. Representative flow cytometry analyses in which sideward scatter is plotted against GFP fluorescence are shown for each genotype. The percentage of GFP+ cells compared to GFP negative control testes is indicated in the gate. (B) GFP mRNA expression in control (n = 3), Oct4GFP+/− (n = 6), Oct4GFP+/+ (n = 2) and NanogGFP+ (n = 5) testes was analysed by qPCR. Circles represent individual samples, the bar indicates the mean value of all samples. (C) GFP+ cells can be detected in Oct4GFP+ and NanogGFP+ testes but not in control testis. Representative pictures of immunofluorescence staining with anti-GFP antibody are shown. Magnification 200×. The framed region is enlarged in the insert.
Mentions: First we verified that the GFP reporter is expressed appropriately in these mice. As expected, GFP expression could be readily observed in the inner cell mass of transgenic blastocysts (Figure 1). Furthermore, we examined GFP expression in the testis as Oct4 and Nanog are expressed in spermatogonia [45], [46]. Using FACS analysis, GFP-positive cells were found to be present in the testes from both Oct4GFP and NanogGFP compound mice (Figure 2A). Furthermore, GFP mRNA expression could be readily detected in testis by qPCR analysis (Figure 2B), and single GFP positive cells were observed in testis sections using immunofluorescence staining, consistent with the expression of Oct4 and Nanog in early type-A spermatogonia (Figure 2C).

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