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

Park JH, Szemes M, Vieira GC, Melegh Z, Malik S, Heesom KJ, Von Wallwitz-Freitas L, Greenhough A, Brown KW, Zheng YG, Catchpoole D, Deery MJ, Malik K - Mol Oncol (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Cancer Epigenetics Laboratory University of Bristol, Bristol BS8 1TD, UK.

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PRMT5 and MYCN protein expression correlations in neuroblastoma. (A) Immunoblotting of PRMT5 in NB cell-lines without MYCN amplification (MYCN-un) and cells with amplification (MYCN-A). Vinculin is used as a loading control. (B) A box plot showing PRMT5 levels normalised to vinculin in cell-lines demonstrates a significant over-expression of PRMT5 in MYCN-A lines (P < 0.004, Mann–Whitney U test). (C) Immunohistochemical staining of NB sections for PRMT5 protein: the top row shows a normal ganglion (g) with cytoplasmic PRMT5 staining arrowed, and Schwannian stroma (ss), followed by a ganglioneuroma with differentiating neuroblasts (d) with cytoplasmic PRMT5 staining arrowed, and finally a differentiating NB, again with cytoplasmic PRMT5. The second row shows differentiating neuroblastomas without MYCN amplification showing predominantly cytoplasmic PRMT5 expression. Neuroblasts (n) and Homer Wright rosettes (hw) are indicated. The third row shows poorly differentiated neuroblastomas with MYCN amplification displaying intense nuclear PRMT5 staining. For PRMT5 immunohistochemistry controls, we used skeletal muscle which is negative for PRMT5 and normal prostate where PRMT5 expression has been reported to be strong in the nucleus of the epithelial cells (Gu et al., 2012) (Figure S3).
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fig3: PRMT5 and MYCN protein expression correlations in neuroblastoma. (A) Immunoblotting of PRMT5 in NB cell-lines without MYCN amplification (MYCN-un) and cells with amplification (MYCN-A). Vinculin is used as a loading control. (B) A box plot showing PRMT5 levels normalised to vinculin in cell-lines demonstrates a significant over-expression of PRMT5 in MYCN-A lines (P < 0.004, Mann–Whitney U test). (C) Immunohistochemical staining of NB sections for PRMT5 protein: the top row shows a normal ganglion (g) with cytoplasmic PRMT5 staining arrowed, and Schwannian stroma (ss), followed by a ganglioneuroma with differentiating neuroblasts (d) with cytoplasmic PRMT5 staining arrowed, and finally a differentiating NB, again with cytoplasmic PRMT5. The second row shows differentiating neuroblastomas without MYCN amplification showing predominantly cytoplasmic PRMT5 expression. Neuroblasts (n) and Homer Wright rosettes (hw) are indicated. The third row shows poorly differentiated neuroblastomas with MYCN amplification displaying intense nuclear PRMT5 staining. For PRMT5 immunohistochemistry controls, we used skeletal muscle which is negative for PRMT5 and normal prostate where PRMT5 expression has been reported to be strong in the nucleus of the epithelial cells (Gu et al., 2012) (Figure S3).

Mentions: Both SK-N-BE(2)C and NGP cells are known to have MYCN amplification and high MYCN protein expression, whereas SH-SY5Y cells do not, suggesting a relation between PRMT5 and MYCN expression. Examination of PRMT5 expression in NB cell-lines confirmed that MYCN over-expression was associated with higher PRMT5 protein levels (P = 0.004, Mann–Whitney U test) (Figure 3A–B). At the RNA level, in silico analysis shows that high PRMT5 transcripts also correlate with MYCN amplification status (P = 2.0e−08) and with poor overall prognosis (P = 2.2e−08) (Figure S2A). Notably, however, elevated PRMT5 mRNA levels were also associated with poor outcome in MYCN-unamplified tumours (P = 1.7e−06) (Figure S2B), alluding to a possible MYCN-independent oncogenic role for PRMT5 in NB. For example, PRMT5 has been shown to regulate cell-cycle progression (Scoumanne et al., 2009) and modify other proteins involved in NB tumorigenesis, such as E2F-1 and p53 (Jansson et al., 2008; Cho et al., 2012).


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

Park JH, Szemes M, Vieira GC, Melegh Z, Malik S, Heesom KJ, Von Wallwitz-Freitas L, Greenhough A, Brown KW, Zheng YG, Catchpoole D, Deery MJ, Malik K - Mol Oncol (2014)

PRMT5 and MYCN protein expression correlations in neuroblastoma. (A) Immunoblotting of PRMT5 in NB cell-lines without MYCN amplification (MYCN-un) and cells with amplification (MYCN-A). Vinculin is used as a loading control. (B) A box plot showing PRMT5 levels normalised to vinculin in cell-lines demonstrates a significant over-expression of PRMT5 in MYCN-A lines (P < 0.004, Mann–Whitney U test). (C) Immunohistochemical staining of NB sections for PRMT5 protein: the top row shows a normal ganglion (g) with cytoplasmic PRMT5 staining arrowed, and Schwannian stroma (ss), followed by a ganglioneuroma with differentiating neuroblasts (d) with cytoplasmic PRMT5 staining arrowed, and finally a differentiating NB, again with cytoplasmic PRMT5. The second row shows differentiating neuroblastomas without MYCN amplification showing predominantly cytoplasmic PRMT5 expression. Neuroblasts (n) and Homer Wright rosettes (hw) are indicated. The third row shows poorly differentiated neuroblastomas with MYCN amplification displaying intense nuclear PRMT5 staining. For PRMT5 immunohistochemistry controls, we used skeletal muscle which is negative for PRMT5 and normal prostate where PRMT5 expression has been reported to be strong in the nucleus of the epithelial cells (Gu et al., 2012) (Figure S3).
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fig3: PRMT5 and MYCN protein expression correlations in neuroblastoma. (A) Immunoblotting of PRMT5 in NB cell-lines without MYCN amplification (MYCN-un) and cells with amplification (MYCN-A). Vinculin is used as a loading control. (B) A box plot showing PRMT5 levels normalised to vinculin in cell-lines demonstrates a significant over-expression of PRMT5 in MYCN-A lines (P < 0.004, Mann–Whitney U test). (C) Immunohistochemical staining of NB sections for PRMT5 protein: the top row shows a normal ganglion (g) with cytoplasmic PRMT5 staining arrowed, and Schwannian stroma (ss), followed by a ganglioneuroma with differentiating neuroblasts (d) with cytoplasmic PRMT5 staining arrowed, and finally a differentiating NB, again with cytoplasmic PRMT5. The second row shows differentiating neuroblastomas without MYCN amplification showing predominantly cytoplasmic PRMT5 expression. Neuroblasts (n) and Homer Wright rosettes (hw) are indicated. The third row shows poorly differentiated neuroblastomas with MYCN amplification displaying intense nuclear PRMT5 staining. For PRMT5 immunohistochemistry controls, we used skeletal muscle which is negative for PRMT5 and normal prostate where PRMT5 expression has been reported to be strong in the nucleus of the epithelial cells (Gu et al., 2012) (Figure S3).
Mentions: Both SK-N-BE(2)C and NGP cells are known to have MYCN amplification and high MYCN protein expression, whereas SH-SY5Y cells do not, suggesting a relation between PRMT5 and MYCN expression. Examination of PRMT5 expression in NB cell-lines confirmed that MYCN over-expression was associated with higher PRMT5 protein levels (P = 0.004, Mann–Whitney U test) (Figure 3A–B). At the RNA level, in silico analysis shows that high PRMT5 transcripts also correlate with MYCN amplification status (P = 2.0e−08) and with poor overall prognosis (P = 2.2e−08) (Figure S2A). Notably, however, elevated PRMT5 mRNA levels were also associated with poor outcome in MYCN-unamplified tumours (P = 1.7e−06) (Figure S2B), alluding to a possible MYCN-independent oncogenic role for PRMT5 in NB. For example, PRMT5 has been shown to regulate cell-cycle progression (Scoumanne et al., 2009) and modify other proteins involved in NB tumorigenesis, such as E2F-1 and p53 (Jansson et al., 2008; Cho et al., 2012).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Cancer Epigenetics Laboratory University of Bristol, Bristol BS8 1TD, UK.

Show MeSH
Related in: MedlinePlus