Glioma is formed by active Akt1 alone and promoted by active Rac1 in transgenic zebrafish

Abstract

Background: Ongoing characterization of glioma has revealed that Akt signaling plays a crucial role in gliomagenesis. In mouse models, however, Akt alone was not sufficient to induce glioma.

Methods: We established transgenic zebrafish that overexpressed dominant-active (DA) human Akt1 or Rac1(G12V) (DARac1) at ptf1a domain and investigated transgenic phenotypes and mechanisms leading to gliomagenesis.

Results: Transgene expressions were spatiotemporally restricted without any developmental abnormality of embryos and persisted at cerebellum and medulla in adult zebrafish. DAAkt1 alone induced glioma (with visible bumps at the head), with incidences of 36.6% and 49% at 6 and 9 months, respectively. Histologically, gliomas showed various histologic grades, increased proliferation, and frequent invasion into the fourth ventricle. Preferential location of small tumors at periventricular area and coexpression of Her4 suggested that tumors originated from Ptf1a- and Her4-positive progenitor cells at ventricular zone. Gliomagenesis was principally mediated by activation of survival pathway through upregulation of survivin genes. Although DARac1 alone was incapable of gliomagenesis, when coexpressed with DAAkt1, gliomagenesis was accelerated, showing higher tumor incidences (62.0% and 73.3% at 6 and 9 months, respectively), advanced histologic grade, invasiveness, and shortened survival. DARac1 upregulated survivin2, cyclin D1, β-catenin, and snail1a but downregulated E-cadherin, indicating that DARac1 promotes gliomagenesis by enhancing proliferation, survival, and epithelial-to-mesenchymal transition. On pharmacologic tests, only Akt1/2 inhibitor effectively suppressed gliomagenesis, inhibited cellular proliferation, and induced apoptosis in established gliomas.

Conclusions: The zebrafish model reinforces the pivotal role of Akt signaling in gliomagenesis and suggests Rac1 as an important protein involved in progression.

Fig. 1.

Targeted expression of transgenes in embryos. (A) Transgenesis strategy. Gal4-UAS system allows targeted expression of transgenes. (B) Inverted fluorescence images (upper column, dorsal views; lower column, lateral views). Transgenic GFP expression is spatiotemporally restricted to the Ptf1a domain. Exocrine pancreatic expression of DARac1 disturbs the posterior expansion of the exocrine pancreas. The morphology of the hindbrain is not altered by either DAAkt1 or DARac1 expression. (C) Whole-mount ISH at 2 dpf (dorsal views) and 4 dpf (lateral views). (D) Confocal images showing membrane localization of GFP fused with DARac1. Abbreviations: H, hindbrain; R, retina; P, exocrine pancreas. Bars, 20 μm.

Fig. 2.

Transgene expression in juvenile and adult zebrafish. (A) Merged bright and fluorescence images showing GFP expression at the cerebellum and medulla. Depigmented phenotypes were obtained by successive crossing with the mitfa^w2;roy^a9 line. Although 1-month-old zebrafish showed sustained GFP expression at the cerebellum and medulla, transgene expression was more localized at the cerebellum at 3 months. The top is anterior. Inlets are lateral views. (B) ISH for Ptf1a and transgenes. Transgene expression is stronger at the intermediate layer and the ventricular zone. Inlets are enlarged views of the ventricular area. (C) Coronal images of 1-month-old Ptf1a^Gal4/UAS^GFP zebrafish of (A). (b) GFP expression is robust along the midline and at the dorsal lining of the fourth ventricle. (D) Immunofluorescence for S100 at the similar plane level of (Cb). S100-positive cells are concentrated at the ventral lining of the fourth ventricle and the intermediate layer and scattered in the granular layer. Strong Ptf1a expression (GFP) persists in cells at intermediate layer, ventricular zone, and along the midline. Images reveal cerebellar cells positive for either GFP or S100 (red arrowheads) alone or for both (white arrowheads). (E–G) Coexpression analyses. During embryonic development, Ptf1a expression is noted in cells at the dorsum of cerebellum and inferior olive nucleus (IO). (E) GFAP^GFP/Ptf1a^Gal4/UAS^mCherry zebrafish. Most cells expressing Ptf1a (RFP) also express GFAP (GFP). (F) Olig2^dRFP/Ptf1a^Gal4/UAS^GFP zebrafish. Cerebellar cells do not coexpress Olig2 and Ptf1a both in embryo and adult. (G) Her4^dRFP/Ptf1a^Gal4/UAS^GFP zebrafish. During embryonic development, ptf1a-positive cells (GFP) are at just underneath Her4-expressing (RFP) cells and rarely coexpress Her4. In adults, Ptf1a-positive cerebellar cells at ventricular and supraventricular areas occasionally coexpress both Ptf1a and Her4. Bars, 50 μm.

Fig. 3.

DAAkt1-induced gliomas. (A) Gross and histologic findings. (a,b) Glioma-bearing 3-month-old Ptf1a^Gal4/UAS^GFP-UAS^DAAkt1 zebrafish with bent body and visible bumps at the head, showing strong GFP expression on merged bright and fluorescence images (inlets). (c) Hematoxylin and eosin stains of cerebellum in control zebrafish. (d–i) Hematoxylin and eosin stains of gliomas. (d and e) Small tumors (boundaries by red arrowheads) at periventricular area showing invasion into 4th ventricle. (f) A large glioma replacing almost the whole cerebellum. (g) A large glioma showing invasion into the midbrain. (h) A high-grade glioma showing increased vascularity (black arrowhead). (i) A high-grade glioma showing necrosis (red arrowhead). (j) IHC for GFAP showing robust expression. (k) IHC for Akt1 on a small tumor invading 4th ventricle showing expression at tumor. (l) ISH for DAAkt1 showing stronger expression at the hypercellular area. (B) Increased proliferation in DAAkt1-induced gliomas. IHC indicates highly frequent positivity to PCNA, which is even higher in high-grade tumor. Tumors occasionally show positive staining to PHH3, a mitotic marker. BrdU labeling also reveals frequently positive cells. (C) Proliferation analyses in the pre-neoplastic cerebellum of 2-month-old zebrafish. Reactivity at the intestinal cells is used as an internal positive control (Inlets). Cells at the ventricular lining and the intermediate layer frequently reveal immunoreactivity to proliferative markers. The non–tumor-bearing cerebellum of DAAkt1-expressing zebrafish is not associated with increased proliferation. Average numbers of positive cells counted at the periventricular are 4.5 ± 2.2 and 4.7 ± 2.6 for PHH3 and 18.7 ± 6.8 and 19.9 ± 6.9 for BrdU in Ptf1a^Gal4/UAS^GFP and Ptf1a^Gal4/UAS^GFP-UAS^DAAkt1 zebrafish, respectively, which are not significantly different. Bars, 50 μm. (D) Activation of Akt downstream components in nonneoplastic DAAkt1-expressing cerebellum. IHC analyses reveal slightly increased expression of phospho-mTOR, -RS6K, and -4EBP1 in cells at the ventricular and periventricular zone.

Fig. 4.

DARac1 accelerates DAAkt1-induced glioma. (A) Two-month-old zebrafish showing bumps at the head. Inlets are merged bright field and fluorescence images. (B) Histologic findings of gliomas in Ptf1a^Gal4/UAS^GFP-UAS^DAAkt1/UAS^GFP-DARac1 zebrafish. (a-g) Hematoxylin and eosin staining. Tumors frequently reveal heterogeneous grade of glioma showing a hypercellular nest within the tumor (red arrowheads) and increased vascularity (black arrowheads). (g and h) IHC and ISH for transgenes in a high-grade glioma. Inlets of (g) and (h) are IHC for Akt1 and ISH for DARac1, respectively. (i and j) ISH for DARac1. Cells at the invasion front show stronger expression of DARac1. Arrows indicate the direction of invasion. (k and l) Proliferation analyses reveal increased positivity to PCNA and PHH3, especially at the hypercellular areas, which accompanies a more robust expression of DARac1 on ISH (inlet). (C) Histologic grade of gliomas. Coexpression of DARac1 increased not only the tumor incidence but also the histologic grades of gliomas (D) Tumor incidence and mortality. 112 zebrafish from each Ptf1a^Gal4/UAS^GFP-UAS^DAAkt1 and Ptf1a^Gal4/UAS^GFP-UAS^DAAkt1/UAS^GFP-DARac1 line were followed. Tumor incidence was estimated by body bending, which preceded the appearance of an obvious bump in the head. The incidence rates of glioma in Ptf1a^Gal4/UAS^GFP-UAS^DAAkt1 and Ptf1a^Gal4/UAS^GFP-UAS^DAAkt1/UAS^GFP-DARac1 zebrafish are 14.3% and 44.4% at 3 months, 36.6% and 62.0% at 6 months, and 49.1% and 73.2% at 9 months, respectively. The mortality rates are also increased by the coexpression of DARac1: 0.9% vs. 22.2% at 3 months, 15.2% vs. 50.9% at 6 months, and 21.4% vs. 60.2% at 9 months. The tumor-free survival rates are much lower in the Ptf1a^Gal4/UAS^GFP-UAS^DAAkt1/UAS^GFP-DARac1 line. Bars, 50 μm.

Fig. 5.

Differential genes involved in gliomagenesis. (A) Real-time RT-PCR using dissected glioma and control cerebellum shows changes in transcripts. Survivin1 and p21 are upregulated in pre-neoplastic cerebellum by DAAkt1 expression. DAAkt1-induced glioma shows upregulation of cyclin D1, p21, Survivin1, 2, and snail1a. Coexpression of DARac1 reveals further upregulation of survivin2 and snail1a, and downregulation of E-cadherin. *P< 0.05. Mann-Whitney U test was used for statistical differences. Electrophoretic (B) and Western blot (C) images confirm quantitative RT-PCR findings. C, control; A, Ptf1a^Gal4/UAS^GFP-UAS^DAAkt1 without tumor; A(T), Ptf1a^Gal4/UAS^GFP-UAS^DAAkt1 with tumor; Ptf1a^Gal4/UAS^GFP-UAS^DAAkt1/UAS^GFP-DARac1 with tumor. (D) Expression analyses of differential genes by IHC (c, g, i, k, l, n, p, and q) or ISH (a, b, d, e, f, h, j, m, o, and r). Dotted red lines indicate tumor boundaries. Gliomas coexpressing DAAkt1 and DARac1 show stronger expression of survivin2, snail1a, β-catenin, and cyclin D1, especially at the hypercellular area (red arrowheads). Expression of pan-cadherin and E-cadherin is downregulated in glioma coexpressing DARac1, especially at the higher-grade area (* in p). Bars, 50 μm.

Fig. 6.

Treatment with Akt pathway inhibitors in groups of ptf1a^Gal4/UAS^GFP-UAS^DAAkt1 larvae. (A and B) Prolonged treatment from 2 weeks to 2 months. (A) Tumor incidence is significantly lowered by Akt1/2 inhibitor but not by miltefosine or rapamycin (χ² test). (B) Expression of Akt1 downstream component. Untreated glioma cells express phospho-mTOR, -4EBP1, and RS6K, suggesting activation of Akt1 signaling. Although rapamycin treatment decreased phospho-mTOR expression, miltefosine and Akt1/2 inhibitor decreased phospho-RS6K expression. (C, D) Treatment in 3-month-old zebrafish with glioma. (C) Counted PCNA-positive cells from 10 high-power fields are significantly lowered by treatment with rapamycin or Akt1/2 inhibitor (ANOVA test). (D) IHC analyses indicated increased cells positive for active caspases in gliomas treated with Akt1/2 inhibitor and decreased cells positive for PCNA in gliomas treated with Akt1/2 inhibitor or rapamycin. Bars, 50 μm.