BackgroundIn analogy to normal stem cell differentiation, the current cancer stem cell (CSC) model presumes a hierarchical organization and an irreversible differentiation in tumor tissue. Accordingly, CSCs should comprise only a small subset of the tumor cells, which feeds tumor growth. However, some recent findings raised doubts on the general applicability of the CSC model and asked for its refinement.Methodology/Principal FindingsIn this study we analyzed the CSC properties of mammary carcinoma cells derived from transgenic (WAP-T) mice. We established a highly tumorigenic WAP-T cell line (G-2 cells) that displays stem-like traits. G-2 cells, as well as their clonal derivates, are closely related to primary tumors regarding histology and gene expression profiles, and reflect heterogeneity regarding their differentiation states. G-2 cultures comprise cell populations in distinct differentiation states identified by co-expression of cytoskeletal proteins (cytokeratins and vimentin), a combination of cell surface markers and a set of transcription factors. Cellular subsets sorted according to expression of CD24a, CD49f, CD61, Epcam, Sca1, and Thy1 cell surface proteins, or metabolic markers (e.g. ALDH activity) are competent to reconstitute the initial cellular composition. Repopulation efficiency greatly varies between individual subsets and is influenced by interactions with the respective complementary G-2 cellular subset. The balance between differentiation states is regulated in part by the transcription factor Sox10, as depletion of Sox10 led to up-regulation of Twist2 and increased the proportion of Thy1-expressing cells representing cells in a self-renewable, reversible, quasi-mesenchymal differentiation state.Conclusions/SignificanceG-2 cells constitute a self-reproducing cancer cell system, maintained by bi- and unidirectional conversion of complementary cellular subsets. Our work contributes to the current controversial discussion on the existence and nature of CSC and provides a basis for the incorporation of alternative hypotheses into the CSC model.

Simian virus 40 (SV40) is a powerful tool to study cellular transformation in vitro, as well as tumor development and progression in vivo. Various cellular kinases, among them members of the CK1 family, play an important role in modulating the transforming activity of SV40, including the transforming activity of T-Ag, the major transforming protein of SV40, itself. Here we characterized the effects of mutant CK1δ variants with impaired kinase activity on SV40-induced cell transformation in vitro, and on SV40-induced mammary carcinogenesis in vivo in a transgenic/bi-transgenic mouse model. CK1δ mutants exhibited a reduced kinase activity compared to wtCK1δ in in vitro kinase assays. Molecular modeling studies suggested that mutation N172D, located within the substrate binding region, is mainly responsible for impaired mutCK1δ activity. When stably over-expressed in maximal transformed SV-52 cells, CK1δ mutants induced reversion to a minimal transformed phenotype by dominant-negative interference with endogenous wtCK1δ. To characterize the effects of CK1δ on SV40-induced mammary carcinogenesis, we generated transgenic mice expressing mutant CK1δ under the control of the whey acidic protein (WAP) gene promoter, and crossed them with SV40 transgenic WAP-T-antigen (WAP-T) mice. Both WAP-T mice as well as WAP-mutCK1δ/WAP-T bi-transgenic mice developed breast cancer. However, tumor incidence was lower and life span was significantly longer in WAP-mutCK1δ/WAP-T bi-transgenic animals. The reduced CK1δ activity did not affect early lesion formation during tumorigenesis, suggesting that impaired CK1δ activity reduces the probability for outgrowth of in situ carcinomas to invasive carcinomas. The different tumorigenic potential of SV40 in WAP-T and WAP-mutCK1δ/WAP-T tumors was also reflected by a significantly different expression of various genes known to be involved in tumor progression, specifically of those involved in wnt-signaling and DNA repair. Our data show that inactivating mutations in CK1δ impair SV40-induced cellular transformation in vitro and mouse mammary carcinogenesis in vivo.

The tumor suppressor p53 was discovered in 1979 as a cellular protein complexed to the simian virus 40 (SV40) large tumor antigen (LT) (49, 52). Based on the seeming analogy to the complex of the polyoma virus middle T-antigen with the cellular Src protein (12, 13), p53 initially was considered a cellular oncoprotein recruited by LT. This assumption was further supported by experiments demonstrating that p53 was able to immortalize certain primary cells and to cooperate with activated Ras in cellular transformation (27,65,72). However, these initial experiments had been performed with mutant p53 genes, and in 1989 the true nature of p53 as a tumor suppressor was established (31, 42). The discovery of p53 as a tumor suppressor not only spurred research on the role of p53 in tumorigenesis but also led to a renaissance of DNA tumor virus research, as the transforming proteins of polyoma-, papilloma-, and adenoviruses all target the tumor suppressor proteins p53 and pRb (16,22,39,50,53,64,92). Consistent with the functional inactivation of p53 in human and animal tumors, a wealth of evidence demonstrated that the interaction of transforming proteins with p53 also inactivated p53 function.Cellular transformation by SV40 differs from cellular transformation by most other DNA tumor viruses insofar as SV40 LT, the major transforming protein of SV40, is able to perform both major steps of cellular transformation in vitro, immortalization and phenotypic transformation, by itself, while other DNA tumor viruses require the cooperation of at least two viral transforming proteins (11,23,44,48). For example, immortalization of primary cells by the E1A protein of adenovirus or the E7 protein of human papilloma viruses (18,69) requires the functional elimination of the proapoptotic, senescence-inducing functions of p53 by other viral proteins that inactivate p53 (e.g., its sequestration, degradation by the E1B 55K and E4orf6 proteins of adenovirus, or its degradation by the E6 protein of human papillomaviruses) (36,68,77,85,97). Thus, while it is undisputed that SV40 LT eliminates p53 tumor suppressor functions during cellular immortalization, the role of the LT-p53 complex in phenotypic transformation seems to be more complex (33,61). In this respect, our laboratory already in 1987 provided evidence that metabolic stabilization of p53 is not simply a consequence of its physical interaction with LT (19,20) but is an active cellular process that correlates with cellular transformation (21, 87-89, 94, 96). The finding raised the question of why the p53 protein, when destined to be functionally eliminated, should be stabilized during cellular transformation. Also, animal experiments revealed that SV40 is more efficient in promoting tumor growth when wt p53 is present (41). The suggestion that p53 in complex with SV40 LT might support SV40 phenotypic transformation has been received with due skepticism. However, recently it has been reported that human papillomavirus type 16 E6 protein mediated degradation of p53 in SV40-transformed...