PAX6 suppression of glioma angiogenesis and the expression of vascular endothelial growth factor A

Abstract

We reported that PAX6 suppresses glioblastoma cell growth in vivo and anchorage-independent growth without significant alteration of cell proliferation in vitro, suggesting that PAX6 may alter the tumor microenvironment. Because we found that PAX6 downregulates expression of the gene encoding vascular endothelial growth factor A (VEGFA) in glioma cells, we used a subcutaneous xenograft model to verify PAX6 suppression of VEGFA-induced angiogenesis based on CD31-immunostaining of endothelial cells. The results showed a significant reduction of VEGFA at the transcription level in PAX6-transfected cells in xenografts and PAX6 has a suppressive effect on the microvascular amplification typically seen in glioblastoma. We showed that PAX6 suppression of VEGFA expression requires its DNA binding-domain. The C-terminal truncation mutant of PAX6, however, did not show the dominant negative function in regulating VEGFA expression that it showed previously in regulating MMP2 expression. In the glioma cell line U251HF, we further determined that blocking the PI3K/Akt signaling pathway with either adenoviral-mediated PTEN expression or LY294002 enhanced PAX6-mediated suppression of VEGFA in an additive manner; thus, PAX6-mediated suppression of VEGFA is not via the canonical pathway through HIF1A. These two VEGFA-regulatory pathways can also be similarly modulated in another malignant glioma cell line, U87, but not in LN229 where the basal VEGFA level is low and PTEN is wild-type. PAX6 suppression of VEGFA appears to be blocked in LN229. In conclusion, our data showed that PAX6 can initiate in glioma cells a new signaling pathway independent of PI3K/Akt-HIF1A signaling to suppress VEGFA expression.

Fig. 1

PAX6 overexpression in glioma cell line U251HF suppresses cell tumorigenicity, tumor angiogenesis and VEGFA expression. a Comparison of the weights of U251HF (n = 8) and its PAX6 stable transfectant PAX6-2.2 xenografts (n = 24) 24 days after subcutaneously implantation in nude mice. Fold change is 3.85; P < 0.0001 by two-sample t-test. b Immunofluroscent analysis of the xenografts from a for expression of PAX6 and CD31, with counterstaining of nuclei by DAPI. See [21] for image analysis procedure and definitions of microvascular parameters. c Comparison of blood vessel density (BVD) normalized to peripheral region of U251HF tumor. d Comparison of total hemoglobin (THb) concentration and tissue oxygen saturation (StO₂) in the two groups of s.c. xenografts (four tumors each group, two mice per group) by modulated imaging, all were subjected to CD31-IHC for BVD data above. Two-sample t-test P values were 0.043 for THb, and 0.70 for StO₂, after Tukey-adjusted for comparisons among groups. e VEGFA expression in the s.c. xenografts from a by real-time AqRT-PCR. Fold change is 2.52 as shown; P = 0.025 by two-sample t-test

Fig. 2

Decrease of PAX6 is related to increase of VEGFA expression during tumor growth. a–b Comparison of PAX6 and VEGFA mRNA levels in U251HF and PAX6-2.2 cells grown in vitro or in vivo under nude mice skin at various time. The gene expression was quantified by real-time qRT-PCR, normalized to GAPDH, and compared with U251HF arbitrarily set to unity. c Comparison of tumor volumes that were measured 6 days after s.c. implantation of U251HF and PAX6-2.2. d Co-immunostaining PAX6_2.2 xenografts 24 days after implantation showing single pictures of PAX6 staining and VEGFA staining and merge picture of PAX6 and VEGFA, with cell nucleus counterstained by DAPI

Fig. 3

PAX6 regulation of VEGFA expression. a Comparison of secreted VEGFA quantified by VEGF-165 ELISA from condition medium of cells expressing transfected cDNA constructs of wild-type or two mutant forms of PAX6 (dmt and 344) to the parental glioma cell U251HF, which was arbitrarily set to unity. Bonferroni adjusted P-value with fold of increase (+) and decrease (−) were shown with averages for the clones use for comparison. On top depicts the PAX6 mutants in functional domains, with PAI and RED and HD represent three DNA-binding domain of PAX6, with two point mutations in the paired domain R26G and I87R reported to cause loose of in vitro DNA binding ability [7]. b Luciferase assays of VEGF promoter activities in U251HF and three of U251HF PAX6 transfected clones, with comparison of −52/+144 VEGF promoter activities between parental and PAX6-transfected cells. Mean and SD were from 4 to 8 repeats of transfection and luciferase assay, which were carried out in 2–4 independent experiments

Fig. 4

PAX6 does not alter PI3K/Akt signaling and HIF1A level in glioma cells under normoxic condition. By Western blot assay, phosphorylation status of Akt and the downstream target GSK-3β (a) and HIF1A level (b) were shown unchanged in U251HF cells 48 h after treatment of cells with of Ad-PAX6. Analysis of cells with Ad-PTEN infection was positive control for antibodies, and detections of Actin as control equal protein loading while PAX6 and PTEN for positive infections. c AqRT-PCR revealed the level of VHL expression relative to ENO1 in U251HF cells with or without adenoviral infections. The mean and SD are based on data of two independent experiments, with viral dose of 10 viral partial per cell, cultured for 48 h under serum-free condition after 1 h of infection. P-values are shown for comparisons to mock infection, and are computed using Dunnett’s ANOVA post-hoc procedure

Fig. 5

PAX6 suppression of VEGFA expression was enhanced by co-expression of PTEN or blocking Akt-signaling pathway in U251HF cells under normoxic condition. Comparison of VEGFA expression in U251HF cells after treatment Ad-PAX6 and/or Ad-PTEN, and/or PI3K inhibitor LY294002 at mRNA level (a) and at secreted protein level (b). Bonferroni adjusted P value were shown

Fig. 6

Regulation of VEGFA expression in U87 and LN229 by PAX6 or blocking of PI3K/Akt signaling. a VEGF-165 ELISA data on the secreted VEGFA protein in condition medium of cells infected with Ad-PAX6 alone or together with Ad-PTEN simultaneously or separate with 1 day apart, under normoxic and hypoxic conditions. Mean and SD were from 1 to 2 independent experiments, with P-values shown for comparison to mock infection. b Comparison of VEGFA expression in LN229 cells after treatment Ad-PAX6 and/or Ad-PTEN, and/or PI3K inhibitor LY294002 at mRNA level. c comparison of PAX6 and VEGFA expressions in U251HF, U87 and LN229. X marks zero expression value for PAX6 in LN229; PTEN status (mt, mutant or wt, wild type) in these cell lines is based on information in [23]