Integrative analysis of cell adhesion molecules in glioblastoma identified prostaglandin F2 receptor inhibitor (PTGFRN) as an essential gene

Abstract

Background: Glioblastoma (GBM) is the most common primary malignant brain tumor in adults exhibiting infiltration into surrounding tissues, recurrence, and resistance to therapy. GBM infiltration is accomplished by many deregulated factors such as cell adhesion molecules (CAMs), which are membrane proteins that participate in cell-cell and cell-ECM interactions to regulate survival, proliferation, migration, and stemness.

Methods: A comprehensive bioinformatics analysis of CAMs (n = 518) in multiple available datasets revealed genetic and epigenetic alterations among CAMs in GBM. Univariate Cox regression analysis using TCGA dataset identified 127 CAMs to be significantly correlated with survival. The poor prognostic indicator PTGFRN was chosen to study its role in glioma. Silencing of PTGFRN in glioma cell lines was achieved by the stable expression of short hairpin RNA (shRNA) against the PTGFRN gene. PTGFRN was silenced and performed cell growth, migration, invasion, cell cycle, and apoptosis assays. Neurosphere and limiting dilution assays were also performed after silencing of PTGFRN in GSCs.

Results: Among the differentially regulated CAMs (n = 181, 34.9%), major proportion of them were found to be regulated by miRNAs (n = 95, 49.7%) followed by DNA methylation (n = 32, 16.7%), and gene copy number variation (n = 12, 6.2%). We found that PTGFRN to be upregulated in GBM tumor samples and cell lines with a significant poor prognostic correlation with patient survival. Silencing PTGFRN diminished cell growth, colony formation, anchorage-independent growth, migration, and invasion and led to cell cycle arrest and induction of apoptosis. At the mechanistic level, silencing of PTGFRN reduced pro-proliferative and promigratory signaling pathways such as ERK, AKT, and mTOR. PTGFRN upregulation was found to be due to the loss of its promoter methylation and downregulation of miR-137 in GBM. PTGFRN was also found to be higher in glioma stem-like cells (GSCs) than the matched differentiated glioma cells (DGCs) and is required for GSC growth and survival. Silencing of PTGFRN in GSCs reduced transcript levels of reprogramming factors (Olig2, Pou3f2, Sall2, and Sox2).

Conclusion: In this study, we provide a comprehensive overview of the differential regulation of CAMs and the probable causes for their deregulation in GBM. We also establish an oncogenic role of PTGFRN and its regulation by miR-137 in GBM, thus signifying it as a potential therapeutic target.

Keywords: CAM; GSC; Glioblastoma; Growth; Migration; PTGFRN; miR-137.

Fig. 1

Transcriptional aberrations identified in CAMs in GBM predict the pro-tumorigenic potential of PTGFRN in GBM. A Volcano graph depicting upregulated (red), downregulated (green), and unregulated (black) CAMs in GBM samples (n = 572) as compared to control samples (n = 10). The horizontal line separates the CAMs having a significant difference in expression (p-value≤0.05). Vertical lines show the cut-off value ≤− 0.58 or ≥0.58 log₂ ratio for classifying differentially regulated CAMs. B Kaplan-Meier curve shows the overall survival difference between PTGFRN-high and low transcript groups of GBM. Scatter plots show the transcript level of PTGFRN in GBM in - C TCGA Agilent, TCGA RNA-Seq, and REMBRANDT, D TCGA GBM subtypes: classical, mesenchymal, neural, and proneural, E mut-IDH1 and wt-IDH1, G-CIMP+ and G-CIMP–, and MGMT methylated and MGMT unmethylated groups. F Immunohistochemical (IHC) analysis for PTGFRN in GBM tissues, percent tumor cell positivity is indicated, G quantification shows percent tumor cell positivity in a given number of tissues samples. H Bar graph shows transcript level of PTGFRN in GBM cell lines and immortalized human astrocytes (SVG and IHA), and control brain samples as measured by RT-qPCR. I Immunoblot shows protein level of PTGFRN in GBM cell lines and immortalized human astrocytes and β-Actin served as a loading control (required portion of the blot is shown after cropping from the whole blot for both the proteins). The normalized protein levels are shown in the bar diagram. The significance was tested using the Wilcoxon-Mann-Whitney test and the symbols are indicated as follows: (ns) not significant; (*) p ≤ 0.05; (**) p ≤ 0.01 and (***) p ≤ 0.001

Fig. 2

Knockdown of PTGFRN diminishes cell growth, migration, and invasion in GBM. In U373 cells PTGFRN silenced with either shNT or shPTGFRN, A immunoblot shows protein levels of PTGFRN and β-Actin served as a loading control (required portion of the blot is shown after cropping from the whole blot for both the proteins), B line graph shows the relative cell viability. Representative images show (C) colony number, (D) soft agar colony number, (E) migration, and (F) invasion after silencing PTGFRN in U373, and quantification is shown as bar graphs. (G) Histograms represent the DNA content (stained with PI) in control and PTGFRN silenced cells in U373 and the bar graph represents the quantification of percentage of cells in different phases of cell cycle. (H) Flow cytometry dot plots represent the Annexin-V positive cells in control and PTGFRN silenced cells in U373 and quantification showed as a bar graph, for quantification UR and LR regions of the plot were considered. (I) Immunoblots show the protein levels of PTGFRN, p-ERK, ERK, p-AKT, AKT, p-p70S6, p70S6, p-4EBP1, and 4EBP1 after silencing PTGFRN in U373 and T98G. β-Actin is used as a loading control in western blotting (the required portion of the blot is shown after cropping from the whole blot for all the proteins). The quantification for each blot is given below the blot. The significance was tested using the Student’s t-test and the symbols are indicated as follows: (ns) not significant; (*) p ≤ 0.05; (**) p ≤ 0.01 and (***) p ≤ 0.001

Fig. 3

Regulation of PTGFRN by promoter methylation and miR-137. A Scatter plots depicting the beta values for CpGs cg22448232 in control and GBM samples in TCGA 450 K, GSE60274, and GSE79122 datasets. B The correlation graph shows the correlation between CpG methylation of cg22448232 and the expression of PTGFRN in TCGA GBM samples. C Scatter plot depicting the transcript levels of miR-137 in control and GBM samples in TCGA dataset. D Dot plot represents the correlation between expression of PTGFRN and miR-137 in TCGA GBM samples. E The bar graph shows the transcript levels of miR-137 in IHA and GBM cell lines. F The correlation graph shows the correlation between protein levels of PTGFRN (normalized values from Fig. 1I) and the miR-137 levels (log₂ ratio values from Fig. 3E) in GBM cell lines. G Schematic shows miR-137 targeting sites on the 3’UTR of PTGFRN and base pairing between miR-137 and targeted sequence in the 3’UTR of PTGFRN. (H) Immunoblot shows PTGFRN protein levels in vector and miR-137 overexpression in U373 and β-Actin was used as a loading control (required portion of the blot is shown after cropping from the whole blot for both the proteins). I The bar graph is depicting the normalized luciferase activity of pmiR-GLO-3’UTR of PTGFRN in pcDNA3.2/V5-Vector and pcDNA3.2/V5-miR-137 overexpression conditions. The significance was performed using the Wilcoxon-Mann-Whitney test or Student's t-test and the symbols are indicated as follows: (ns) not significant; (*) p ≤ 0.05; (**) p ≤ 0.01 and (***) p ≤ 0.001

Fig. 4

PTGFRN is upregulated in GSCs and required for its growth. A Heatmaps representing the differentially expressed CAMs in GSCs as compared to NSCs (left) and in GSCs as compared to DGCs (right). GSE46016 (GSC vs NSC, gene microarray) and GSE54791 (GSC vs DGC, RNA-Seq) datasets were used for the analysis. Red and green color indicates the upregulated and downregulated CAMs, respectively. B Scatter plots represent the transcript levels of PTGFRN in different datasets GSE119834, GSE31262, and GSE54791. C Immunoblot shows the protein levels of PTGFRN in GSCs and corresponding DGCs in MGG8, MGG6, MGG4, and 1035 and β-Actin was used as a loading control (required portion of the blot is shown after cropping from the whole blot for both the proteins). GSCs cultured as neurospheres indicated as Sph (Spheroid culture) and DGCs cultured as monolayer and indicated as Diff (Differentiated cells). D The bar graph shows the transcript levels of PTGFRN in shNT and shPTGFRN in MGG6 and MGG8. E Representative images of neurospheres and their quantification in MGG6 and MGG8 after silencing PTGFRN. F Line graphs show the limiting dilution analysis in MGG6 and MGG8 after silencing PTGFRN. G Bar diagram shows the transcript levels of Olig2, Pou3f2, Sall2, and Sox2 in shNT and shPTGFRN (represented as shPT) in MGG6 and MGG8. The significance was performed using the Wilcoxon-Mann-Whitney test or Student’s t-test and the symbols are indicated as follows: (ns) not significant; (*) p ≤ 0.05; (**) p ≤ 0.01 and (***) p ≤ 0.001