Protein arginine methyltransferase 5 is a key regulator of the MYCN oncoprotein in neuroblastoma cells

Abstract

Approximately half of poor prognosis neuroblastomas (NBs) are characterized by pathognomonic MYCN gene amplification and MYCN over-expression. Here we present data showing that short-interfering RNA mediated depletion of the protein arginine methyltransferase 5 (PRMT5) in cell-lines representative of NBs with MYCN gene amplification leads to greatly impaired growth and apoptosis. Growth suppression is not apparent in the MYCN-negative SH-SY5Y NB cell-line, or in two immortalized human fibroblast cell-lines. Immunoblotting of NB cell-lines shows that high PRMT5 expression is strongly associated with MYCN-amplification (P < 0.004, Mann–Whitney U-test) and immunohistochemical analysis of primary NBs reveals that whilst PRMT5 protein is ubiquitously expressed in the cytoplasm of most cells, MYCN-amplified tumours exhibit pronounced nuclear PRMT5 staining. PRMT5 knockdown in MYCN-overexpressing cells, including the SHEP-21N cell-line with inducible MYCN expression leads to a dramatic decrease in MYCN protein and MYCN-associated cell-death in SHEP-21N cells. Quantitative gene expression analysis and cycloheximide chase experiments suggest that PRMT5 regulates MYCN at a post-transcriptional level. Reciprocal co-immunoprecipitation experiments demonstrated that endogenous PRMT5 and MYCN interact in both SK-N-BE(2)C and NGP cell lines. By using liquid chromatography – tandem mass spectrometry (LC-MS/MS) analysis of immunoprecipitated MYCN protein, we identified several potential sites of arginine dimethylation on the MYCN protein. Together our studies implicate PRMT5 in a novel mode of MYCN post-translational regulation and suggest PRMT5 plays a major role in NB tumorigenesis. Small-molecule inhibitors of PRMT5 may therefore represent a novel therapeutic strategy for neuroblastoma and other cancers driven by the MYCN oncogene.

Anguilla japonica lectin 1 delivery through adenovirus vector induces apoptotic cancer cell death through interaction with PRMT5

“…Through cooperation with cyclin D1, PRMT5 inhibits p53‐dependent tumor suppression, thereby facilitating lymphomagenesis . In addition, PRMT5 has been extensively studied as a therapeutic target in various cancers, including colorectal cancer , neuroblastoma and B‐cell lymphoma . Transcription factor E2F‐1 has been determined to be a direct substrate for PRMT1 and PRMT5, and E2F‐1 methylated by PRMT1 prevents its methylation by PRMT5 and induces E2F‐1‐dependent cell apoptosis, whereas E2F‐1 methylated by PRMT5 antagonizes its methylation by PRMT1 and favors cell proliferation , suggesting a key role of differed arginine methylation in determining E2F‐1 biological activities.…”

“…Through cooperation with cyclin D1, PRMT5 inhibits p53-dependent tumor suppression, thereby facilitating lymphomagenesis [53]. In addition, PRMT5 has been extensively studied as a therapeutic target in various cancers, including colorectal cancer [54], neuroblastoma [55] and B-cell lymphoma [56].…”

A Wnt-BMP4 signalling axis induces MSX and NOTCH proteins and promotes growth suppression and differentiation in neuroblastoma

“…Cell proliferation was monitored real time by using Incucyte Live Cell Imaging system using cell confluence as surrogate for growth, according to manufacturer's instructions. Propidium-iodide labelling and fluorescence activated cell sorting (FACS) analysis to detect cell cycle phases was performed as previously described (32).…”

“…Cells were lysed in Radioimmunoprecipitation assay (RIPA) buffer and protein concentration was determined by using Micro BCA TM protein assay kit (Thermo Fisher). Western blot was performed as described previously (32). The antibodies used are listed in Supplementary Table 1.…”

Small Molecule Inhibitors of Protein Arginine Methyltransferases

“…PRMT-related pathologies include virus-related diseases [37–39], inflammatory response [40, 41], cardiovascular disease [42, 43], renal disease [44, 45], diabetes [46], pulmonary disorders [47] and the most actively studied area, carcinogenesis [2, 3, 27, 48–53] (Table 1). Arginine methylation levels might be used as diagnostic biomarkers.…”