Defective p53 antiangiogenic signaling in glioblastoma

Abstract

Previous findings suggest an angiogenesis-regulating function of the p53 tumor suppressor protein in various malignancies. With several antiangiogenic agents entering the clinic, we assessed the value of the TP53 status in predicting angiogenesis in glioblastoma in vivo and examined underlying angiogenic-signaling pathways in vitro. We identified 26 TP53 wild-type and 9 TP53 mutated treatment-naïve, primary, isocitrate dehydrogenase 1 (IDH1) wild-type glioblastoma specimens by sequence analysis and quantified vascularization. P53 responsiveness of the angiogenesis-related target genes, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), thrombospondin 1 (TSP-1), brain-specific angiogenesis inhibitor 1 (BAI1), and collagen prolyl-4-hydroxylase alpha 2 (P4HA2), was evaluated by (i) overexpression of wild-type p53 in homozygously TP53-deleted LN-308 cells; (ii) shRNA-mediated p53 knockdown in the TP53 wild-type LNT-229 cells; and (iii) chemical induction of wild-type p53 expression in LNT-229 cells by camptothecin. Irrespective of the TP53 status, vascularization did not differ significantly between the two groups of glioblastoma specimens. Of all target genes, only P4HA2 mRNA was upregulated through wild-type p53. As opposed to several nonglial tumors, in glioblastoma cells, p53-mediated transcriptional induction of P4HA2 mRNA neither resulted in increased levels of P4HA2 protein or antiangiogenic endostatin nor did it influence endothelial cell sprouting, viability, or transmigration in vitro. Moreover, p53-uncoupled stable overexpression of P4HA2 in LN-308 cells did not affect endothelial cell viability. These data challenge the view of p53 as an angiogenesis-regulator in glioblastoma in that relevant signaling pathways are silenced, potentially contributing to the angiogenic switch during malignant progression.

Fig. 1.

The extent of vascularization in primary glioblastomas is independent of the TP53 mutational status. (A) Photomicrographs of CD31-stained primary glioblastoma specimens carrying either the TP53 wild-type (TP53wt; 26 of 35, 74%) or TP53-mutated (TP53mut; 9 of 35, 26%) gene. Micrographs were subjected to automated image analyses. The left panel shows a representative TP53wt tumor (Specimen 3), the right panel represents a TP53mut tumor (Specimen 22). Magnification 100-fold. (B and C) Vessel quantification in TP53wt and TP53mut primary glioblastoma specimens depicted as total area of vascular structures as percentage of total analyzed tumor region (B, P = .31), and total number of vascular structures (C, P = .4). Data are mean ± standard deviation.

Fig. 2.

p53 activates transcription of P4HA2 in glioblastoma cells. (A) Expression levels of p53 (upper left panel), p21^CIP1/WAF1 (upper right panel), and mRNAs of several proangiogenic (VEGF and bFGF) and antiangiogenic (TSP-1, BAI1, P4HA2) factors plus P4HA1 (lower panel) measured by quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR) in homozygously TP53-deleted (p53^−/−) LN-308 glioblastoma cells following transfection with a p53-encoding expression vector (p53) or the empty vector as a control (Ctrl.). (B) qRT-PCR-measured expression levels of p53 (upper panel), p21^CIP1/WAF1 (upper middle panel), P4HA2 (lower middle panel), and P4HA1 (lower panel) mRNAs in the p53^−/− H1299 lung carcinoma cell line transfected as LN-308 cells in (A), in p53-silenced (p53sh) or control (Ctrl.) LNT-229 glioblastoma cells (TP53 wild-type, p53wt), and in LNT-229 cells treated with increasing concentrations (1, 10, and 100 nM) of camptothecin or dimethyl sulfoxide as a vehicle control (0 nM). GAPDH-normalized expression of each transcript in (A) and (B) is expressed relative to control conditions set as 1. The gap in the x-axis of each panel indicates that the absolute baseline (control) expression levels of each transcript differ between H1299 and LNT-229 cells and were set to 1 for each cell line individually. Data are mean ± standard deviation. *P < .05. Experiments were repeated three times with similar results. (C) Methylation analysis of the P4HA2 promoter region. Upper panel: schematic diagram of the P4HA2 promoter region from position −467 to −442, showing the positions of the putative partially overlapping p53-binding half sites at positions −442 to −450, −449 to −457, and −458 to −467, as indicated with brackets, and the genomic and deduced bisulfite DNA sequences in case of methylation (a) or not (b) including the critical CpG site at position −458/−457 (marked by red rectangle) relative to the transcription start site. Middle panel: methylation-specific polymerase chain reaction (MSP) products at 271 bp using bisulfite-treated genomic DNA derived from LN-308 and LNT-229 glioblastoma cells as a template and primer pairs specific for annealing to methylated (M) and unmethylated (U) bisulfite DNA. CpGenome Universal Methylated (Meth) or Unmethlated (Unmeth) DNA (Millipore, Temecula, California) served as positive or negative controls for both primer pairs. A control reaction without any template DNA (H₂O) was included as an additional negative control. PCR products were separated on a 3% agarose gel. First lane left, 100 bp DNA marker. Lower panel: methylation-specific sequence of bisulfite-treated DNA of the P4HA2 promoter region (position −467 to −442) in LN-308 and LNT-229 cells. The methylation status of the critical CpG site at position −458/−457 is marked by a red square corresponding to the deduced bisulfite DNA sequence depicted in the upper panel. TSS, transcription start site.

Fig. 3.

p53-dependent antiangiogenic signaling via P4HA2 is defective in glioblastoma cells. (A) Immunoblot analyses demonstrating expression levels of p53, p21^CIP1/WAF1, P4HA2, and P4HA1 proteins in human glioblastoma cell lines. Left panel: lysates of homozygously TP53-deleted (p53^−/−) LN-308 cells transiently transfected with an expression vector containing either p53-encoding (p53) or no insert cDNA (Ctrl.). Middle panel: lysates of TP53 wild-type (p53wt) LNT-229 cells stably transfected with a p53-silencing plasmid (p53sh) or an empty control plasmid (Ctrl.), respectively. Right panel: lysates of LNT-229 cells treated with increasing concentrations (1, 10, and 100 nM) of camptothecin or with dimethyl sulfoxide as a vehicle control (0 nM). Actin, GAPDH, and α-tubulin served as loading controls. (B) ELISA-based quantification of endostatin concentrations in conditioned supernatants of LN-308 and LNT-229 cells transfected as in (A). (C and E) In vitro angiogenesis assays testing conditioned supernatants derived from either p53-overexpressing LN-308 cells (LN-308/p53), p53-silenced LNT-229 cells (LNT-229/p53sh), or control cells as in (A) for HUVEC sprouting (C), HUVEC and hCMEC/D3 viability (D), or HUVEC transmigration (E). Treatment with 10 µg/mL recombinant human endostatin (rhEndo) served as a positive control in each assay. Responses of HUVEC and hCMEC/D3 to supernatants from p53-overexpressing and p53-silenced glioblastoma cells or recombinant endostatin is expressed relative to supernatants derived from respective control cells or the vehicle for endostatin set as 100%. Data are mean ± standard deviation. *P < .05. Experiments were repeated three times with similar results. Insets depict representative endothelial cell responses towards the respective treatment as indicated. Scale bars, 100 µm.

Fig. 4.

p53-uncoupled P4HA2-mediated antiangiogenic signaling through endostatin is functional in carcinoma but not in glioblastoma cells. (A) Immunoblot analysis confirming overexpression of P4HA2 protein in both p53^−/− H1299 lung carcinoma (left panel) and p53^−/− LN-308 glioblastoma cells (right panel) following stable transfection with an expression vector encoding P4HA2 (P4HA2) and an empty vector (Ctrl.), respectively. α-Tubulin or GAPDH served as loading controls. (B) ELISA-based quantification of endostatin concentrations in conditioned supernatants of either H1299 or LN-308 cells transfected as in (A). (C) Viability of HUVEC exposed to conditioned supernatants from either H1299 or LN-308 cells transfected as in (A). HUVEC viability at exposure to supernatants derived from P4HA2-overexpressing tumor cells is expressed relative to supernatants from empty vector controls set as 100%. Data are mean ± standard deviation. *P < .05. Experiments were repeated three times with similar results.