Genetic validation of the protein arginine methyltransferase PRMT5 as a candidate therapeutic target in glioblastoma

Abstract

Glioblastoma is the most common and aggressive histologic subtype of brain cancer with poor outcomes and limited treatment options. Here, we report the selective overexpression of the protein arginine methyltransferase PRMT5 as a novel candidate theranostic target in this disease. PRMT5 silences the transcription of regulatory genes by catalyzing symmetric dimethylation of arginine residues on histone tails. PRMT5 overexpression in patient-derived primary tumors and cell lines correlated with cell line growth rate and inversely with overall patient survival. Genetic attenuation of PRMT5 led to cell-cycle arrest, apoptosis, and loss of cell migratory activity. Cell death was p53-independent but caspase-dependent and enhanced with temozolomide, a chemotherapeutic agent used as a present standard of care. Global gene profiling and chromatin immunoprecipitation identified the tumor suppressor ST7 as a key gene silenced by PRMT5. Diminished ST7 expression was associated with reduced patient survival. PRMT5 attenuation limited PRMT5 recruitment to the ST7 promoter, led to restored expression of ST7 and cell growth inhibition. Finally, PRMT5 attenuation enhanced glioblastoma cell survival in a mouse xenograft model of aggressive glioblastoma. Together, our findings defined PRMT5 as a candidate prognostic factor and therapeutic target in glioblastoma, offering a preclinical justification for targeting PRMT5-driven oncogenic pathways in this deadly disease.

Figure 1. PRMT5 over-expression correlates with human GBM cell proliferation, and inversely correlates with patient survival

(A) PRMT5 expression correlates with cell growth (r=0.81 with p-value<0.0001). (B) H&E staining of GBM. (D-G) PRMT5 is selectively expressed in high grade (grade III and IV) astrocytomas (F,G), while normal brain (C) and low or intermediate grade astrocytomas (D,E) do not. (H) Kaplan Meier plots by glioma grade. (I) PRMT5 expression index by glioma grade. (J) Kaplan Meier plots of overall survival of GBM patients as a function of PRMT5 protein expression index. (K) Time to death and PRMT5 level in patients who died with GBM. PRMT5 level is continuously associated with time to death (Spearman’s rho = −0.57, p=0.0001).

Figure 2. siRNA-mediated silencing of PRMT5 expression

(A) Human GBM cell lines were treated as indicated and expression of PRMT5 detected by real-time PCR and Western blot. (B) si-PRMT5 reduced expression of PRMT5 and its epigenetic mark S2Me-H4R3 as determined by immunofluresence microscopy.

Figure 3. Consequences of PRMT5 silencing in GBM in vitro

(A) Proliferation was evaluated by MTS assay. (B) Change of cell morphology with PRMT5 knockdown. (C) PRMT5 silencing leads to decreased migratory activity of GBM cells measured by transwell assay. (D) Cell cycle analysis by flow cytometry.

Figure 4. PRMT5 Knockdown promotes cell death of human GBM cells

(A) U251 cells were treated as indicated and cell death assessed flow cytometry. (B) A172, U1242 and U251 were treated as indicated resulting in decreased cell viability of siPRMT5 compared to scrRNA (p=0.002). (C) Western blot for BCL2 and BAX. (D) Caspase inhibitors blocked siPRMT5-induced cell death. (E) Cell death of three human GBM cell lines pretreated with caspase inhibitors. (F) Cells treated with Temozolomide, replated, treated as indicated and cell death determined by flow cytometry.

Figure 5. si-PRMT5 induced cell death is P53-independent

(A) P53-wild type cell line, A172, treated as indicated and cell death assessed in triplicate by flow cytometry. (B) A172 cells treated as in (A) and P53 detected by Western blot. (C) The 2024 cell line treated as indicated and irradiated with a X-ray ionizing radiation (8Gy) to introduce DNA damage signal. Cell death was assessed by flow cytometry. (D) Western blot for PRMT5, P53 and β-ACTIN.

Figure 6. ST7 is a PRMT5 target gene

(A) Kaplan-Meier plot for GBM cases with differential ST7 gene expression (Low ST7 n=169; High ST7 n=12). (B) ChIP assays using IgG control or anti-PRMT5 antibody performed on chromatin from U251 or U1242 treated as indicated. Fold-enrichment with each antibody was calculated relative to the IgG control (in triplicate). The data were normalized by the input DNA (Delta Ct=Ct(IP)-Ct(input)) and one-way ANOVA was used to analyze the normalized data for each cell line. (C) Expression of ST7 is up-regulated by si-PRMT5 determined by by real-time PCR and (D) Western blot.

Figure 7. PRMT5 knockdown improves survival in vivo

(A) Representative brain MRI images taken on day 28 of mice in control and siPRMT5 treated groups. (B) Kaplan Meier plot of animals in control and siPRMT5 treated groups (n=8/grp).