Trapping the mouse genome to hunt human alterations.

G lioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors. Genetic alterations and their consequences in these malignant astrocytomas have been studied extensively and include (i) overexpression of growth factors and their corresponding receptors (fibroblast growth factor, epidermal growth factor, and platelet-derived growth factors), (ii) abnormalities of transduction signaling pathways (activation of PI3 kinase/AKT, RAS/MAP kinase, and protein kinase C), or (iii) disruption of cell cycle arrest (loss of p16INK4A and p14ARF, mutations in p53 protein, and PTEN) (1). Whether these modifications are causative or participate in tumor progression is a pivotal question that can best be answered by modeling glioma formation in mice. In this issue of PNAS, Kamnasaran et al. (2) combine genetically engineered murine (GEM) models of gliomas with a retroviral gene-trapping approach to identify new molecular alterations in human gliomas. There has been substantial progress made recently in developing GEM models that recapitulate the genesis and progression of human malignancies. In such an effort, several publications from Guha (e.g., ref. 3) have described an astrocytoma mouse model using embryonic stem (ES) cell transgenesis to overexpress oncogenic Ras under the control of a GFAP promoter. These mice express various levels of oncogenic Ras where the highest producers develop astrocytomas with similarities to glioblastomas. Consequently, one strain of the model relies on the development of low-grade astrocytomas (LGA) that progress to anaplastic astrocytomas [high-grade astrocytomas (HGA)]. An alternative approach to inducing tumor formation in mice consists of somaticcell gene transfer using tissue-specific, replication-competent avian leukosis virus-based retroviral vectors. In such a model, the combined transfer of genes encoding activated Ras and Akt to nestin-expressing CNS progenitors leads to the formation of gliomas (4). However, contrary to the mouse model of Guha, neither Ras nor Akt alone is sufficient to generate glioblastomas. The requirement for combined Ras and Akt signaling implies that these pathways may coordinately regulate some critical process that leads to glioma formation. In this line of view, in the GFAP-Ras model, astrocytoma cell lines established from transgenic newborn mice (B8-P0) showed moderate expression of the RAS transgene and were nontumorigenic, as opposed to those coming from 3-month-old mice (B8-3mth) that carry multiples genetic abnormalities. These findings directed the authors to hypothesize that Ras is not sufficient to transform a given astrocyte but provides a lower threshold for transformation. The laboratory of Guha used the unbiased approach of gene trapping to discover novel molecular alterations in a murine Ras cancer model. The study led to the identification of GATA6 as a new tumor suppressor for gliomas. More importantly, it permitted the discovery of novel genetic alterations in corresponding human cancers. Basically, a retroviral gene trap cassette was used to infect B8-P0 astrocytes, triggering the transformation of a small subset of astrocytes that grew in anchorageindependent assays in soft agarose. Analyses of the gene-trapped astrocytes identified GATA6 in the majority of infected clones. Interestingly, in the more malignant astrocytomas, i.e., coming from B8-3mth, GATA6 expression was totally absent. GATA6 has been identified as a member of the GATA family of zinc-finger transcription factors. In previous reports, Koutsourakis et al. (5) had shown that GATA6 is involved in embryonic development. Moreover, GATA6 knockout mice arrest during the early gastrula stage and show defects in endoderm differentiation. All data converge to an essential role for GATA6 in lineage determination during development and maintenance of cell differentiation in adult tissues. At the brain level, GATA6 is normally expressed in the adult mouse and human brain, where nuclear expression was detected in neurons, astrocytes, and endothelial cells (6). The present study of Kamnasaran et al. (2) brings a role for GATA6 acting as a tumor suppressor in gliomas: The trapped clones expressing a lower level of GATA6 showed increased proliferation and were able to grow intracranially as invasive malignant astrocytomas after injection into mice. A novel function arose after comparison of the level of GATA6 with tumor grade: GATA6 is abundantly present in LGA, whereas it is absent in HGA. This suggests an important role for the loss of GATA6 expression in tumor progression. One of the most promising findings in the study of Kamnasaran et al. comes from the use of the gene-trapping approach in GEM models that led to the identification of GATA6 alterations in the corresponding human GBM. As observed in the Ras astrocytomas model, GATA6 expression was absent in a panel of human GBM cell lines and human GBM operative specimens. Of particular interest, the authors elucidated one mechanism responsible for the loss of GATA6 expression in mouse and human gliomas. In the B8-3mth astrocytes, they identified a mutation in the DNA binding domain of the transcription factor. Similarly, in human GBM, mutations were detected within the DNA binding domain and the C terminus of the protein and were associated with LOH. In transactivation assay, mutations in human GATA6 resulted in decreased activity. This emphasizes the relevance of loss of GATA6 expression and function in human GBM and the further selection of the population during tumor progression. In the literature, great discrepancy arises from the level of GATA6 expression in different cancer types. Similar to glioblastomas, in ovarian cancer (7) and adrenocortical (8) tumors, GATA6 is absent or mislocalized, whereas normal tissues express abundant levels. A close-up at the cancer profiling database ONCOMINE (www.oncomine.org) revealed a highly significant down-regulation of GATA6 in lung and ovarian tumors compared with the normal tissues (Fig. 1A). On the other hand, GATA6 was found to be overexpressed in human colon cancer (9) and ubiquitously expressed in esophageal cancer (10). Overall, these data suggest that the regulation of GATA6 is tumor-type-specific. Looking further at the biological function of GATA6, the induction of differentiation during embryonic development