Small-molecule blocks malignant astrocyte proliferation and induces neuronal gene expression
Introduction
It is well established that mature astrocytes lack neurogenic potential, especially during late postnatal stages and in adult brain (Costa et al., 2010). Elegant work using genetic fate-mapping strategies confirmed that mature cortical astrocytes are largely quiescent and non-neurogenic, but retain the ability to proliferate and upregulate GFAP and other classical markers of reactive glia (Buffo et al., 2008, Buffo et al., 2005). Despite a few studies that examine the ability of glia to give rise to neuronal cells, this appears to only occur by forced expression of neuronal transcription factors in vitro, while the potential for this is drastically reduced in vivo, likely due to the non-neurogenic microenvironment (Blum et al., 2010, Heins et al., 2002, Berninger et al., 2007). Moreover, the finding that brain tumors often contain a mixture of neuronal and glial cell types has raised the notion that these tumors either contain multipotent or restricted stem/progenitors, or, arise from de-differentiated mature cell types, such as astrocytes (Stiles and Rowitch, 2008). Clearly, there is an urgent need to understand the cellular and molecular mechanisms underlying the proliferation and de-differentiation potential of mature astrocytes. Understanding this process, and especially, developing new strategies or tools to explore the extent of astrocyte plasticity may be relevant for designing neuroregenerative approaches and treating brain tumors.
Recent work from multiple labs indicates that reprogramming to pluripotent stem cells is markedly enhanced with the loss of tumor suppressor genes Ink4a or p53 (Li et al., 2009, Utikal et al., 2009). These data reinforce the connection between maintaining the differentiated state and initiating tumorigenesis. The Ink4a/Arf locus encodes two key tumor suppressor proteins (p16Ink4a and p19Arf) that, respectively, engage two critical anti-proliferative pathways, the retinoblastoma (Rb) and p53 pathways, both important for G1 checkpoint control (Sharpless, 2005, Serrano et al., 1993). Ink4a (as well as its other Ink4 orthologs, Ink4b, Ink4c, and Ink4d) bind and inhibit the D-type cyclin-dependent kinases Cdk4 and Cdk6 that, in turn, relieve the cell-cycle inhibitory activity of Rb. On the other hand, Arf binds to and inactivates the Mdm2 protein, which is an E3 ubiquitin ligase that destabilizes p53. Both expression of p16Ink4a and p19ARF are critical for effective tumor suppression—including GBM, which frequently harbors homozygous deletions of the Ink4a/Arf locus (Hall and Peters, 1996, Kamb et al., 1994, Ueki et al., 1996). Indeed, our previous studies indicate that Ink4a/Arf−/− astrocytes can undergo de-differentiation to a stem-like glioma cell and re-express progenitor markers such as Nestin and A2B5, retaining a capacity to become differentiated glial and neuronal progeny (Bachoo et al., 2002).
Several key questions are raised by these studies: (1) are there specific tumor suppressor genes and/or oncogenes that govern the differentiation potential of malignant astrocytes, and (2) what is the extent of phenotypic plasticity of malignant astrocytes and is it reversible? In this report, we use a synthetic small-molecule 3,5-disubstituted isoxazole (compound 1), identified in a previous high-throughput chemical compound screen for inducers of differentiation of P19 embryonal carcinoma cells (Sadek et al., 2008, Schneider et al., 2008), to interrogate the molecular pathways that control the lineage plasticity of malignant astrocytes. We demonstrate that Ink4a/Arf, Pten, and EGFRvIII pathways interact to maintain the differentiated state of astrocytes, and that in this context isoxazole acts as a stem cell modulator (SCM) to trigger neuronal gene expression and block tumor cell proliferation. Our findings provide novel insights into Ink4a/Arf-mediated de-differentiation of malignant astrocytes and identify a potential starting point for future glioma therapeutic drug design. Most importantly, we demonstrate the use of a novel pharmacological tool to explore the phenotypic plasticity of astrocytes, which is relevant in the context of cellular de-differentiation, reprogramming, and malignancy.
Section snippets
Astrocyte cell culture
SS05 cells or primary astrocytes were isolated from cerebral cortices of 5-day-old wild-type, Ink4a/Arff/f, Ptenf/f; Ink4a/Arf−/−; Ptenf/f, or p53−/−; Ptenf/f pups according to previous methods (Bachoo et al., 2002). The floxed Ink4a/Arf or Pten allele was deleted using an adenovirus expressing Cre. Infection of astrocytes with lentiviruses expressing constitutively active EGFR (EGFRvIII) has previously been described (Bachoo et al., 2002). Cells were cultured in 10% FBS in DMEM:F12 media
Isoxazole SCMs block cell proliferation and induce neuronal gene expression in malignant astrocytes
To explore the lineage potential of malignant astrocyte cultures, we used a continuous line of cultured astrocytes (SS05) derived from genetically engineered mice with homozygous deletion of both Ink4a/Arf and Pten tumor suppressor genes. SS05 cells initially expressed the astrocyte marker (GFAP), but downregulated their astrocyte phenotype and increased proliferation during in vitro cell culture with 10% FBS (data not shown). SS05 cells also harbor constitutively active epidermal growth factor
Discussion
One of the most surprising and exciting discoveries in neural developmental biology this past decade is the finding that NSCs possess characteristics of glial identity (Goldman, 2003, Mori et al., 2005). Radial glia give rise to neurons in the developing telecenphalon across vertebrate species and radial-like stem cells in the postnatal hippocampus and lateral ventricle subependymal zone differentiate into neurons throughout life (Kriegstein and Alvarez-Buylla, 2009). Why do some glial cells
Acknowledgements
We thank Yan Jiang, Yan Wu, and HK Jeong for technical assistance and Jose Cabrera for artwork. We also thank Gerard Evan, Sarah Comerford, Chun-li Zhang, and Ondine Cleaver for helpful comments on the manuscript. This work was funded by grants from the Cancer Prevention & Research Institute of Texas (RP100674, J.H.), the Welch Foundation (I-1660, J.H.), and National Institutes of Health/National Institute on Aging (AG032383, J.H.).
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Equal-author contribution.