Combinations of genetic mutations in the adult neural stem cell compartment determine brain tumour phenotypes.

It has been suggested that intrinsic brain tumours originate from a neural stem/progenitor cell population in the subventricular zone of the post-natal brain. However, the influence of the initial genetic mutation on the phenotype as well as the contribution of mature astrocytes to the formation of brain tumours is still not understood. We deleted Rb/p53, Rb/p53/PTEN or PTEN/p53 in adult subventricular stem cells; in ectopically neurografted stem cells; in mature parenchymal astrocytes and in transplanted astrocytes. We found that only stem cells, but not astrocytes, gave rise to brain tumours, independent of their location. This suggests a cell autonomous mechanism that enables stem cells to generate brain tumours, whereas mature astrocytes do not form brain tumours in adults. Recombination of PTEN/p53 gave rise to gliomas whereas deletion of Rb/p53 or Rb/p53/PTEN generated primitive neuroectodermal tumours (PNET), indicating an important role of an initial Rb loss in driving the PNET phenotype. Our study underlines an important role of stem cells and the relevance of initial genetic mutations in the pathogenesis and phenotype of brain tumours.

Bone morphogenetic protein 7 in the development and treatment of bone metastases from breast cancer.

Bone morphogenetic protein 7 (BMP7) counteracts the physiological epithelial-to-mesenchymal transition (EMT), a process that is indicative of epithelial plasticity. Because EMT is involved in cancer, we investigated whether BMP7 plays a role in breast cancer growth and metastasis. In this study, we show that decreased BMP7 expression in primary breast cancer is significantly associated with the formation of clinically overt bone metastases in patients with > or = 10 years of follow-up. In line with these clinical observations, BMP7 expression is inversely related to tumorigenicity and invasive behavior of human breast cancer cell lines. Moreover, BMP7 decreased the expression of vimentin, a mesenchymal marker associated with invasiveness and poor prognosis, in human MDA-MB-231 (MDA-231)-B/Luc(+) breast cancer cells under basal and transforming growth factor-beta (TGF-beta)-stimulated conditions. In addition, exogenous addition of BMP7 to TGF-beta-stimulated MDA-231 cells inhibited Smad-mediated TGF-beta signaling. Furthermore, in a well-established bone metastasis model using whole-body bioluminescent reporter imaging, stable overexpression of BMP7 in MDA-231 cells inhibited de novo formation and progression of osteolytic bone metastases and, hence, their metastatic capability. In line with these observations, daily i.v. administration of BMP7 (100 mug/kg/d) significantly inhibited orthotopic and intrabone growth of MDA-231-B/Luc(+) cells in nude mice. Our data suggest that decreased BMP7 expression during carcinogenesis in the human breast contributes to the acquisition of a bone metastatic phenotype. Because exogenous BMP7 can still counteract the breast cancer growth at the primary site and in bone, BMP7 may represent a novel therapeutic molecule for repression of local and bone metastatic growth of breast cancer.

Advances in optical imaging and novel model systems for cancer metastasis research.

Research into the genetic and physiological interactions of tumours with their host environment requires in vivo assays to address molecular expression patterns and function. In recent years much of this work has been performed using bioluminescent and fluorescent imaging techniques that allow real-time and non-invasive imaging of gene expression and (tumour) tissue development. Luminescence imaging has until now been more or less the only tool that allows the imaging of intra-osseous breast cancer cells and indeed this technique has been pioneered in our laboratory. Here we summarise some recent innovations and developments using cancer cells and some of the first imaging models of multimodal dual luminescence and luminescence combined with fluorescence of intra-osseous tumours. We further engineered our models to incorporate a specific insertion site in the genome and will discuss some of the possible applications. These include the insertion of signalling pathway-specific reporters and studying the fate of multiple injected populations in a single mouse. We conclude that recent improvements in luminescence- and fluorescence-detection platforms now clearly allow multimodal imaging which will greatly enhance our ability to assess gene function and for the first time to visualise multiple gene- and cellular interactions in real time and in vivo.

Generation of brain tumours in mice by Cre-mediated recombination of neural progenitors in situ with the tamoxifen metabolite endoxifen.

Targeted cell- or region-specific gene recombination is widely used in the functional analysis of genes implicated in development and disease. In the brain, targeted gene recombination has become a mainstream approach to study neurodegeneration or tumorigenesis. The use of the Cre-loxP system to study tumorigenesis in the adult central nervous system (CNS) can be limited, when the promoter (such as GFAP) is also transiently expressed during development, which can result in the recombination of progenies of different lineages. Engineering of transgenic mice expressing Cre recombinase fused to a mutant of the human oestrogen receptor (ER) allows the circumvention of transient developmental Cre expression by inducing recombination in the adult organism. The recombination of loxP sequences occurs only in the presence of tamoxifen. Systemic administration of tamoxifen can, however, exhibit toxicity and might also recombine unwanted cell populations if the promoter driving Cre expression is active at the time of tamoxifen administration. Here, we report that a single site-specific injection of an active derivative of tamoxifen successfully activates Cre recombinase and selectively recombines tumour suppressor genes in neural progenitor cells of the subventricular zone in mice, and we demonstrate its application in a model for the generation of intrinsic brain tumours.

Comparative expression analysis reveals lineage relationships between human and murine gliomas and a dominance of glial signatures during tumor propagation in vitro.

Brain tumors are thought to originate from stem/progenitor cell populations that acquire specific genetic mutations. Although current preclinical models have relevance to human pathogenesis, most do not recapitulate the histogenesis of the human disease. Recently, a large series of human gliomas and medulloblastomas were analyzed for genetic signatures of prognosis and therapeutic response. Using a mouse model system that generates three distinct types of intrinsic brain tumors, we correlated RNA and protein expression levels with human brain tumors. A combination of genetic mutations and cellular environment during tumor propagation defined the incidence and phenotype of intrinsic murine tumors. Importantly, in vitro passage of cancer stem cells uniformly promoted a glial expression profile in culture and in brain tumors. Gene expression profiling revealed that experimental gliomas corresponded to distinct subclasses of human glioblastoma, whereas experimental supratentorial primitive neuroectodermal tumors (sPNET) correspond to atypical teratoid/rhabdoid tumor (AT/RT), a rare childhood tumor.

Cytokine-mediated inhibition of apoptosis in non-transformed T cells and neutrophils can be dissociated from protein kinase B activation.

In the absence of survival-inducing cytokines activated T cells and neutrophils enter apoptosis spontaneously. Phosphatidylinositol 3-kinase (PI3 K) activation and signaling through PKB/AKT have been widely linked to the inhibition of apoptosis by cytokines. Here we have investigated the role of PKB in the inhibition of spontaneous apoptosis of activated human CD4+ T cells and neutrophils. We used a range of cytokines known to induce survival and/or activation of PKB. We found activation of PKB in T cells treated with IL-2 and insulin, and neutrophils cultured with N-formyl-Met-Leu-Phe (fMLP), insulin or granulocyte-macrophage colony-stimulating factor. Insulin did not inhibit apoptosis in neutrophils or T cells and fMLP did not delay neutrophil apoptosis. Intriguingly, IFN-beta induced PI3 K-dependent survival in both cell types, but did not activate PKB. IL-2 mediated rescue of T cells from apoptosis but no induction of proliferation occurred in thepresence of LY294002, an inhibitor of PI3 K, which also blocked subsequent PKB activation. The main role of PI3 K in IL-2-mediated signaling may therefore be in the regulation of proliferation. These findings suggest that activation of PKB and inhibition of apoptosis can be dissociated in cytokine-mediated rescue of non-transformed CD4+ T cells and neutrophils.