The Ig superfamily protein PTGFRN coordinates survival signaling in glioblastoma multiforme

Abstract

Glioblastoma multiforme (GBM) is the most malignant primary brain tumor with a median survival of approximately 14 months. Despite aggressive treatment of surgical resection, chemotherapy and radiation therapy, only 3-5% of GBM patients survive more than 3 years. Contributing to this poor therapeutic response, it is believed that GBM contains both intrinsic and acquired mechanisms of resistance, including resistance to radiation therapy. In order to define novel mediators of radiation resistance, we conducted a functional knockdown screen, and identified the immunoglobulin superfamily protein, PTGFRN. In GBM, PTGFRN is found to be overexpressed and to correlate with poor survival. Reducing PTGFRN expression radiosensitizes GBM cells and potently decreases the rate of cell proliferation and tumor growth. Further, PTGFRN inhibition results in significant reduction of PI3K p110β and phosphorylated AKT, due to instability of p110β. Additionally, PTGFRN inhibition decreases nuclear p110β leading to decreased DNA damage sensing and DNA damage repair. Therefore overexpression of PTGFRN in glioblastoma promotes AKT-driven survival signaling and tumor growth, as well as increased DNA repair signaling. These findings suggest PTGFRN is a potential signaling hub for aggressiveness in GBM.

Keywords: AKT; Glioblastoma multiforme; PI3K p110β; PTGFRN; Radiation.

Figure 1:

Relevance of PTGFRN to GBM A, Oncomine data of PTGFRN expression in brain tumors versus normal brain tissue in four studies. B, PrognoScan survival curves of GBM patients whose tumors express high versus low levels of PTGFRN. C, From TCGA LGG+GBM database, Kaplan Maier survival plot for low and high grade gliomas dichotomized by PTGFRN expression. D, The expression of PTGFRN in low and high grade gliomas from the TCGA database. Pairwise statistical comparisons of each subtype of low grade glioma to GBM are indicated (student’s t-tests).

Figure 2:

PTGFRN is required for cell proliferation and tumor growth. A, C, Cell proliferation assay in A172MG and U87MG GBM knockdown cells. B, D, qRT-PCR for samples in A and C. E, Subcutaneous U87 shRNA tumor growth curve. F, Kaplan-Meier curve for mice in E. G, Bioluminescence of intracranial tumor growth of shRNA neurospheres. H, Kaplan-Meier curve of mice in G. I, PTGFRN expression from U87 cells before injection and from U87 subcutaneous tumors. J, PTGFRN expression from GBM0913 cells before injection and from GBM0913 intracranial tumors. ***, p<0.001; ****, p<0.0001 by two-way ANOVA (A, C, E, G) or log rank (F, H).

Figure 3:

PTGFRN reduction sensitizes GBM cells to IR. Clonogenic assays of A172 (A), U87MG (B), GBM0821 (C) and GBM0913 neurospheres (D) cells after indicated doses of IR. qRT-PCR of expression normalized to GAPDH can be found in supplemental figure 3. E, qRTPCR of GBM0913 Tet-on-shRNA cells with or without doxycycline in vitro. F, qRT-PCR of PTGFRN expression in animals injected with GBM0913 Tet-On-shRNA cells treated with doxycycline. G, Kaplan-Meier survival curve of GBM0913 Tet-onshRNA intracranial xenograft.*, p<0.05; **, p<0.01; ***, p<0.001 by student’s t tests (A, B, C, D) or log rank (E).

Figure 4:

PTGFRN depletion decreases p110β levels. A, B, C, Western blot evaluating basal P-AKT signaling in different GBM shPTGFRN cells compared to shGFP control. D, E, Western blot assessing p110β protein levels in GBM0821 and GBM0913 neurosphere cell lines. F, qRT-PCR for p85α and p110β in 3 different GBM shPTGFRN cell lines. Expression normalized to GAPDH. G, H, Immunoblot analyses of GBM0913 shRNA cells treated with cycloheximide alone (G) or in combination with proteasome inhibitor, MG132 (H). I, Proximity ligation assay of GBM0913 shPTGFRN or shGFP cells; negative control contained no primary antibody; positive control comprised two different antibodies targeting PTGFRN; * p<0.05 by student’s t test (above). Immunoblot of cells used for the PLA assay (below). J, Cellular fractionation assay in GBM0913 shRNA neurosphere cells using a-tubulin, Histone H3 and Na/K-ATPase as cellular fractionation internal and loading controls.

Figure 5:

PTGFRN promotes DNA damage sensing. A, Western blot of GBM0913 shRNA cells that received either no treatment (NT) or radiation (10 Gy) and collected after 30 minutes. B, Quantification of γH2AX foci following IR (2 Gy) in U87 shRNA cells. C, Immunofluorescence of γH2AX (red) foci. D, qRTPCR of targeted genes in HR assay. Expression normalized to b-actin. E, DR-GFP HR reporter assay in siPTGFRN, siGAPDH (negative control), siBRCA1 (positive control), or siRISC (control) U87 cells.