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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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GFP mRNA is not expressed in Oct4GFP and NanogGFP transgenic MT/ret, MMTV-PyMT or MMTV-Neu tumors.Relative GFP mRNA expression levels of (A) MT/ret, (B) MMTV-PyMT and (C) MMTV-Neu compound tumors with indicated genotypes and Oct4GFP+ and NanogGFP+ testes were analysed by qPCR. For each tumor model, GFP mRNA expression levels of compound tumors and Oct4GFP+ and NanogGFP+ testes were compared to one control tumor which was set to 1. Circles represent individual samples, the bar indicates the mean value of all samples. The number of analysed samples is as follows: testes: Oct4GFP+/− n = 7, NanogGFP+ n = 5. (A) MT/ret: control n = 4, Oct4GFP+/− n = 6, Oct4GFP+/+ n = 6, NanogGFP+ n = 6 (B) MMTV-PyMT: control n = 5, Oct4GFP+/− n = 6, Oct4GFP+/+ n = 6, NanogGFP+ n = 6 (C) MMTV-Neu: control n = 5, Oct4GFP+/− n = 5, Oct4GFP+/+ n = 5, NanogGFP+ n = 9.
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pone-0057465-g004: GFP mRNA is not expressed in Oct4GFP and NanogGFP transgenic MT/ret, MMTV-PyMT or MMTV-Neu tumors.Relative GFP mRNA expression levels of (A) MT/ret, (B) MMTV-PyMT and (C) MMTV-Neu compound tumors with indicated genotypes and Oct4GFP+ and NanogGFP+ testes were analysed by qPCR. For each tumor model, GFP mRNA expression levels of compound tumors and Oct4GFP+ and NanogGFP+ testes were compared to one control tumor which was set to 1. Circles represent individual samples, the bar indicates the mean value of all samples. The number of analysed samples is as follows: testes: Oct4GFP+/− n = 7, NanogGFP+ n = 5. (A) MT/ret: control n = 4, Oct4GFP+/− n = 6, Oct4GFP+/+ n = 6, NanogGFP+ n = 6 (B) MMTV-PyMT: control n = 5, Oct4GFP+/− n = 6, Oct4GFP+/+ n = 6, NanogGFP+ n = 6 (C) MMTV-Neu: control n = 5, Oct4GFP+/− n = 5, Oct4GFP+/+ n = 5, NanogGFP+ n = 9.

Mentions: To further examine whether or not GFP-positive cells were present in the GFP-knockin tumors, we analysed GFP mRNA expression in the tumors using qPCR. In comparison to GFP expression in testes from Oct4GFP and NanogGFP mice, expression levels in the Oct4GFP+ and NanogGFP+ tumors was 100–10 000 times lower depending on the analysed tumor model (Figure 4). Furthermore, no significant increase in GFP expression compared to GFP-negative control tumors could be detected in either the Oct4GFP+ and NanogGFP+ MT/ret and MMTV-Neu tumors. Indeed, in the case of the melanomas a significant decrease in GFP levels was observed in the Oct4GFP+ and NanogGFP+ tumors compared to the negative controls (Figure 4A), indicating that the expression of GFP transcription was below the threshold of detectability. For the MMTV-PyMT tumors, a three-fold significant increase in GFP expression was observed in the Oct4GFP+ and NanogGFP+ tumors compared to GFP negative tumors (Figure 4B), albeit from a very low basal level close to the noise threshold. Together these data suggest again that very few if any GFP-expressing cells were present in the Oct4GFP+ and NanogGFP+ tumors.


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)

GFP mRNA is not expressed in Oct4GFP and NanogGFP transgenic MT/ret, MMTV-PyMT or MMTV-Neu tumors.Relative GFP mRNA expression levels of (A) MT/ret, (B) MMTV-PyMT and (C) MMTV-Neu compound tumors with indicated genotypes and Oct4GFP+ and NanogGFP+ testes were analysed by qPCR. For each tumor model, GFP mRNA expression levels of compound tumors and Oct4GFP+ and NanogGFP+ testes were compared to one control tumor which was set to 1. Circles represent individual samples, the bar indicates the mean value of all samples. The number of analysed samples is as follows: testes: Oct4GFP+/− n = 7, NanogGFP+ n = 5. (A) MT/ret: control n = 4, Oct4GFP+/− n = 6, Oct4GFP+/+ n = 6, NanogGFP+ n = 6 (B) MMTV-PyMT: control n = 5, Oct4GFP+/− n = 6, Oct4GFP+/+ n = 6, NanogGFP+ n = 6 (C) MMTV-Neu: control n = 5, Oct4GFP+/− n = 5, Oct4GFP+/+ n = 5, NanogGFP+ n = 9.
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pone-0057465-g004: GFP mRNA is not expressed in Oct4GFP and NanogGFP transgenic MT/ret, MMTV-PyMT or MMTV-Neu tumors.Relative GFP mRNA expression levels of (A) MT/ret, (B) MMTV-PyMT and (C) MMTV-Neu compound tumors with indicated genotypes and Oct4GFP+ and NanogGFP+ testes were analysed by qPCR. For each tumor model, GFP mRNA expression levels of compound tumors and Oct4GFP+ and NanogGFP+ testes were compared to one control tumor which was set to 1. Circles represent individual samples, the bar indicates the mean value of all samples. The number of analysed samples is as follows: testes: Oct4GFP+/− n = 7, NanogGFP+ n = 5. (A) MT/ret: control n = 4, Oct4GFP+/− n = 6, Oct4GFP+/+ n = 6, NanogGFP+ n = 6 (B) MMTV-PyMT: control n = 5, Oct4GFP+/− n = 6, Oct4GFP+/+ n = 6, NanogGFP+ n = 6 (C) MMTV-Neu: control n = 5, Oct4GFP+/− n = 5, Oct4GFP+/+ n = 5, NanogGFP+ n = 9.
Mentions: To further examine whether or not GFP-positive cells were present in the GFP-knockin tumors, we analysed GFP mRNA expression in the tumors using qPCR. In comparison to GFP expression in testes from Oct4GFP and NanogGFP mice, expression levels in the Oct4GFP+ and NanogGFP+ tumors was 100–10 000 times lower depending on the analysed tumor model (Figure 4). Furthermore, no significant increase in GFP expression compared to GFP-negative control tumors could be detected in either the Oct4GFP+ and NanogGFP+ MT/ret and MMTV-Neu tumors. Indeed, in the case of the melanomas a significant decrease in GFP levels was observed in the Oct4GFP+ and NanogGFP+ tumors compared to the negative controls (Figure 4A), indicating that the expression of GFP transcription was below the threshold of detectability. For the MMTV-PyMT tumors, a three-fold significant increase in GFP expression was observed in the Oct4GFP+ and NanogGFP+ tumors compared to GFP negative tumors (Figure 4B), albeit from a very low basal level close to the noise threshold. Together these data suggest again that very few if any GFP-expressing cells were present in the Oct4GFP+ and NanogGFP+ tumors.

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