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.
Highlights
The idea that cancer is driven by a subpopulation of tumor cells with stem cell properties was proposed around 150 years ago [1]
To this end we used ‘‘knockin’’ transgenic mice that express GFP under the control of the endogenous Oct4 or Nanog promoters [39,40]. These mice were crossed with MT/ret mice that develop melanocytic tumors in response to transgenic expression of the Ret oncogene [41], with MMTV-PyMT transgenic mice that develop polyomavirus middle T oncogene-driven multifocal mammary adenocarcinomas [42], and with MMTV-Neu mice that develop polyclonal mammary adenocarcinomas due to expression of the activated Neu oncogene in the mammary epithelium [43]
These mice were crossed with MT/ ret [41], MMTV-PyMT [42] and MMTV-Neu [43] transgenic mice to generate compound mice that spontaneously developed melanomas or mammary tumors, respectively, and that expressed GFP under the control of the endogenous Nanog or Oct4 promoter
Summary
The idea that cancer is driven by a subpopulation of tumor cells with stem cell properties was proposed around 150 years ago [1]. Cancer stem cells are a subset of tumor cells that are able to self-renew, give rise to heterogeneous progeny, and initiate the growth of new tumors [3]. Stem cell properties such as indefinite growth, growth under non-adherent conditions, drug resistance and asymmetrical division have been attributed to CSCs [4]. These characteristics suggest that targeting of CSCs should be an effective anti-cancer strategy [2,5]. New ways of reliably identifying CSCs to facilitate their isolation and characterisation are required
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