Small-molecule blocks malignant astrocyte proliferation and induces neuronal gene expression - PubMed (original) (raw)

Small-molecule blocks malignant astrocyte proliferation and induces neuronal gene expression

Ling Zhang et al. Differentiation. 2011 Apr.

Abstract

In the central nervous system (CNS), neural stem cells (NSCs) differentiate into neurons, astrocytes, and oligodendrocytes--these cell lineages are considered unidirectional and irreversible under normal conditions. The introduction of a defined set of transcription factors has been shown to directly convert terminally differentiated cells into pluripotent stem cells, reinforcing the notion that preserving cellular identity is an active process. Indeed, recent studies highlight that tumor suppressor genes (TSGs) such as Ink4a/Arf and p53, control the barrier to efficient reprogramming, leaving open the question whether the same TSGs function to maintain the differentiated state. During malignancy or following brain injury, mature astrocytes have been reported to re-express neuronal genes and re-gain neurogenic potential to a certain degree, yet few studies have addressed the underlying mechanisms due to a limited number of cellular models or tools to probe this process. Here, we use a synthetic small-molecule (isoxazole) to demonstrate that highly malignant EGFRvIII-expressing Ink4a/Arf(-/-); Pten(-/-) astrocytes downregulated their astrocyte character, re-entered the cell cycle, and upregulated neuronal gene expression. As a collateral discovery, isoxazole small-molecules blocked tumor cell proliferation in vitro, a phenotype likely coupled to activation of neuronal gene expression. Similarly, histone deacetylase inhibitors induced neuronal gene expression and morphologic changes associated with the neuronal phenotype, suggesting the involvement of epigenetic-mediated gene activation. Our study assesses the contribution of specific genetic pathways underlying the de-differentiation potential of astrocytes and uncovers a novel pharmacological tool to explore astrocyte plasticity, which may bring insight to reprogramming and anti-tumor strategies.

Published by Elsevier B.V.

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Figures

Figure 1

1 activates neuronal genes in Ink4a/Arf-/-; PTEN-/-; EGFRvIII astrocytes (SS05 cells). (A) Chemical structure of lead isoxazole (1). (B) 1 induces a concentration-dependent increase in both GluR2- and _NR1_-luciferase (luc) reporters. (C-D) 1 promotes neuronal gene expression (Tuj 1) in SS05 cells compared to vehicle control (DMSO) in 4-day cultures. (E-F) 1 blocks proliferation of SS05 cells, compared to vehicle control (DMSO) in 4-day cultures. Scale bar: 25 μm. (G) Quantification of Ki67+ and Tuj1+ cells after 4 days of vehicle or 1 treatment is shown. (**p < 0.001, _t_-test, data in graphs represent ± s.d. from three independent experiments).

Figure 2

Comparison of 1's effects in primary mouse astrocytes carrying glioma-relevant mutations. (A-E) Representative images of vehicle or 1-treated astrocytes with indicated Ink4a/Arf; Pten; EGFRvIII genotypes or wild-type (wt) astrocytes and immunostaining for Ki67 in 4-day cultures. (F-J) Representative images of vehicle or 1-treated astrocytes with indicated Ink4a/Arf; Pten; EGFRvIII genotypes or wild-type (wt) astrocytes and immunostaining for Tuj1 or GFAP in 4-day cultures. Scale bar: 25 μm. (K-N) Quantification of Ki67+ or Tuj1+ primary astrocytes after 4 days of vehicle or 1 treatment is shown. (**p < 0.001, _t_-test, data in graphs represent ± s.d. from three independent experiments).

Figure 3

Compound 1 induces Tuj1 at the expense of GFAP expression in primary _Ink4a/Arf/-_astrocytes. (A) Comparison of vehicle (passage 2) or 1 (passage 2 and 3) treatment in Ink4a/Arff/f and Ink4a/Arf-/- astrocytes. Note co-expression of Tuj1 and GFAP in a subset of 1-treated cells (arrows). Scale bar: 25 μm. (B-C) Quantification of Tuj1+ or GFAP+ in passage 2 or 3 astrocytes after 4 days of vehicle or 1 treatment is shown. (*p<0.05, **p < 0.001, _t_-test, data in graphs represent ± s.d. from three independent experiments).

Figure 4

1 prevents proliferation, but not survival, of Ink4a/Arf-/-; PTEN-/-; EGFRvIII astrocytes (SS05 cells). (A) Total number of cells/plate treated with different concentrations of 1 and FBS after 4 days. (B) Increasing concentrations (>40 μM) of 1 induces death of SS05 cells over time. (C) Protein blotting time-course analysis of cell cycle regulatory genes in SS05 cells treated with 1 compared to vehicle control. (D) Cdk4 and Tuj1 protein levels in SS05 cells treated with vehicle or 1 for 4 days, then cultured for 3 additional days after washout of drug, in the presence of 1% FBS. Gapdh served as a normalization control. (Data in graphs represent ± s.d. from three independent experiments).

Figure 5

Ink4a/Arf-/-; PTEN-/-; EGFRvIII astrocytes (SS05 cells) display an irreversible block in proliferation with 1 treatment. (A) Quantification of the total number of cells/field after vehicle or 1 pre-treatment is shown. (B-D) Shown are representative images of SS05 cells treated with 1 for 4 days, pre-treated with vehicle for 4 days and post-treated with 10% FBS for 4 days, or pre-treated with 1 for 4 days and post-treated with 10% FBS for 4 days. Note in (D): 1 pre-treated cultures and post-treated with FBS contain a mixture of Ki67+ cells with small round nuclei (arrowhead) and Ki67- cells with large flat nuclei (arrow). Scale bar: 10 μm. (E) Quantification of the percentage of Ki67+ cells is shown. (F) Quantification of the percentage of clones expressing Tuj1 is shown. (*p < 0.05 or **p < 0.001, _t_-test, data in graphs represent ± s.d. from three independent experiments).

Figure 6

Stem cell and lineage-specific gene expression in primary Ink4a/Arf-/-; PTENf/f, Ink4a/Arf-/-; PTENf/f; EGFRvIII and Ink4a/Arf-/-; PTEN-/-; EGFRvIII astrocytes after 1 treatment. (A-E) Q-PCR analysis of stem cell, pro-neuronal, and pro-astrocytic gene expression in glioma stem cells treated with vehicle or 1 for 24 hours. Gapdh served as a normalization control. (F) Histone deacetylase inhibitors TSA or VPA induce Tuj1 in SS05 cells. Scale bar: 25 μm. (G) Histone H3 and H4 acetylation levels in SS05 cells after 4 days of treatment with various compounds. (Data in graphs represent ± s.e.m. from three independent experiments).

Figure 7

Model showing isoxazole SCM-mediated growth arrest and neuronal differentiation in malignant astrocytes. Upon loss of Ink4a/Arf tumor suppressor genes, wild-type astrocytes undergo de-differentiation into glioma “stem” cells. The inclusion of SCMs (e.g., 1 or 2) can be used to block proliferation and induce terminal neuronal differentiation, alone or in conjunction with other small-molecules/drugs that induce cell death, will likely facilitate successful GBM differentiation-based therapy. The subsequent activation of potent oncogenes in Ink4a/Arf-/- astrocytes (e.g., EGFRvIII) may promote the emergence of 1-resistant cells that may require additional loss of tumor suppressor genes (e.g., Pten) to sensitize 1-resistant cells and enhance the growth inhibitory/pro-differentiation effects of 1.

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Small-molecule blocks malignant astrocyte proliferation and induces neuronal gene expression - PubMed (original) (raw)