ADAR2 Protein Is Associated with Overall Survival in GBM Patients and Its Decrease Triggers the Anchorage-Independent Cell Growth Signature

Abstract

Background: Epitranscriptomic mechanisms, such as A-to-I RNA editing mediated by ADAR deaminases, contribute to cancer heterogeneity and patients' stratification. ADAR enzymes can change the sequence, structure, and expression of several RNAs, affecting cancer cell behavior. In glioblastoma, an overall decrease in ADAR2 RNA level/activity has been reported. However, no data on ADAR2 protein levels in GBM patient tissues are available; and most data are based on ADARs overexpression experiments.

Methods: We performed IHC analysis on GBM tissues and correlated ADAR2 levels and patients' overall survival. We silenced ADAR2 in GBM cells, studied cell behavior, and performed a gene expression/editing analysis.

Results: GBM tissues do not all show a low/no ADAR2 level, as expected by previous studies. Although, different amounts of ADAR2 protein were observed in different patients, with a low level correlating with a poor patient outcome. Indeed, reducing the endogenous ADAR2 protein in GBM cells promotes cell proliferation and migration and changes the cell's program to an anchorage-independent growth mode. In addition, deep-seq data and bioinformatics analysis indicated multiple RNAs are differently expressed/edited upon siADAR2.

Conclusion: ADAR2 protein is an important deaminase in GBM and its amount correlates with patient prognosis.

Keywords: ADAM12; ADAR2; PTPX3; RNA editing; anchorage-independent growth; cancer.

Figure 1

ADAR2 correlates with patients’ overall survival. (a) IHC analysis (H&E and the corresponding ADAR2 antibody staining) of two glioblastoma tissues (examples of high and low ADAR2 levels). Subpanels A and B show a representative GBM case with high ADAR2 expression; in subpanels C and D, a GBM case with low ADAR2 expression is shown; scale bar: 200 µm. (b) Kaplan–Meier curve comparing the survival of GBM patients (n = 39) stratified by ADAR2 levels. The red and blue lines represent low and high ADAR2 expression, respectively, following the scores indicated in Material and Methods (p = 0.0005; HR 3.954; 95% CI from 1834 to 8525).

Figure 2

ADAR2 expression level and activity across GBM cell lines. (a) ADAR2 expression (qRT-PCR) in normal brain (fetal and adult), astrocytes, and glioblastoma cell lines (T98G, U138-MG, U87-MG, U118-MG, LN-18, and A172). Ct values were normalized to GAPDH mRNA levels. Mean  ±  standard deviation (n  =  3), values are representative as means ± SD, * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001. Of note, ADAR2 activity is more robust in the whole brain (either adult or fetal) because of neuronal cells, where ADAR2 is highly active. (b) Western blotting analysis showing the ADAR2 level in glioblastoma cell lines. No control was added as the ADAR2 level was too high. (c) The recoding editing index (REI) in normal astrocytes (×10), T98-G, U87-MG, U118-MG, LN-18, and A172 glioblastoma cells lines is shown. REI values were calculated as the weighted average of editing levels over all known recording sites from the highly accurate list.

Figure 3

ADAR2 loss promotes proliferation, migration, and anchorage-independent growth of cancer cells. ADAR2 knockdown (qRT-PCR at 96 h, on the left) and cell proliferation (MTS, on the right) are shown in A172 (a) and U87-MG (b) cell lines. Ct values were normalized to GAPDH mRNA levels. Mean ±  standard deviation (n  =  3), t-test p  <  0.01 **. (c) ADAR2 mRNA in A172 untreated cells (black), scrambles (SCR-A, -B in light and dark grey), and different levels of ADAR2 knockdown (siAD2-A,-B in light and dark blue; siAD2-C,-D in light and dark red) are shown in A172 cells. Mean ± standard error of mean (s.e.m.) p  <  0.01 **. Ct values were normalized to β-actin levels. The expression levels were calculated as a relative-fold increase compared with the untreated cells arbitrarily set to 1. Below, 5 × 10⁴ of A172 un-transfected (untreated), A172 scramble (SCR-A and SCR-B), and silenced ADAR2 cells (siAD2-A, -B, -C, -D) were seeded and proliferation was monitored over 4 days. Mean ± s.e.m (n = 3), t-test, p < 0.05 *, p < 0.01 **. (d) Colony formation ability of A172 scramble (scr) and siADAR2 (siAD2) cells 10 days post-seeding (left panel, a photograph of three representative plates per cell line) quantification is shown on the right, mean  ±  standard deviation (n  =  4), t-test, p  <  0.01 **. (e) siADAR2 and control cells were seeded (2 × 10⁵ cells/well) to monitor the formation of anchorage-independent colonies after 15 days. Mean  ±  standard deviation (n  =  3), t-test p  <  0.01 **. (f) Representative photographs of scramble (scr) and siADAR2 (siAD2) A172 cells at 0, 12, and 22 hours after scratching the surface of a monolayer of cells. The wound-healing assay was performed within an interval time in which the cells do not divide.

Figure 4

Gene expression profile and RNA editing pattern changes upon ADAR2 silencing. (a) Volcano plots showing the effect of ADAR2 silencing on the A172 and U87-MG cell lines transcriptome in terms of significance levels and fold changes in gene expression as calculated by DESeq2. The cut-off for log2FC is >|2| and the cut-off for the p.adj value is 1 × 10⁻⁶. (b) Gene enrichment analysis showing the pathways in which common genes are significantly upregulated in A172 siADAR2 and U87MG siADAR2, * p < 0.05. (c) qRT-PCR showing the expression levels of PTX3 and ADAM12 transcripts in control and siADAR2 A172 and U87-MG cells. Mean ± standard deviation t-test, ** p ≤ 0.01, *** ≤ 0.001. (d) Scatter plots reporting the editing levels (%) for sites significantly (t-test, p-value < 0.05) modulated between scramble (scr) versus silenced ADAR2 (siA2) cell lines. The under-edited sites in siADAR2 are shown in light red, while the upregulated sites are shown in cyan.