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PRMT1 Is a Novel Regulator of Epithelial-Mesenchymal-Transition in Non-small Cell Lung Cancer.

Avasarala S, Van Scoyk M, Karuppusamy Rathinam MK, Zerayesus S, Zhao X, Zhang W, Pergande MR, Borgia JA, DeGregori J, Port JD, Winn RA, Bikkavilli RK - J. Biol. Chem. (2015)

Bottom Line: Taken together, we show that PRMT1 is a novel regulator of EMT and arginine 34 (Arg-34) methylation of Twist1 as a unique "methyl arginine mark" for active E-cadherin repression.Therefore, targeting PRMT1-mediated Twist1 methylation might represent a novel strategy for developing new anti-invasive/anti-metastatic drugs.Moreover, methylated Twist1 (Arg-34), as such, could also emerge as a potential important biomarker for lung cancer.

View Article: PubMed Central - PubMed

Affiliation: From the Division of Pulmonary, Critical Care, Sleep and Allergy, and.

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PRMT-mediated Twist1 methylation is required for E-cadherin repression.A, A549 cells were transiently transfected with either Myc-tagged wild-type or R34K mutant of Twist1. The lysates were later probed for the expression of E-cadherin and N-cadherin via immunoblotting with specific antibodies. B, A549 cells transfected with empty vector, Myc-tagged wild-type or R34K mutant of Twist1 were fixed, permeabilized, and co-immunostained with anti-myc antibodies and anti-E-cadherin antibodies, and the expressions of E-cadherin and Twist1 (Myc) were visualized by indirect immunofluorescence and confocal microscopy. Scale bar 5 μm. C, MCF7 cells were transiently transfected with either Myc-tagged wild-type or R34K mutant of Twist1. The lysates were later probed for the expression of E-cadherin and N-cadherin via immunoblotting with specific antibodies. D, MCF7 cells transfected with empty vector, Myc-tagged wild-type or R34K mutant of Twist1 were fixed, permeabilized and co-immunostained with anti-myc antibodies and anti-E-cadherin antibodies, and the expressions of E-cadherin and Twist1 (Myc) were visualized by indirect immunofluorescence and confocal microscopy. Scale bar 5 μm. E, migration of A549 cells transfected with empty vector, Myc-tagged wild-type or R34K mutant of Twist1 were determined using trans-well assays as described under “Experimental Procedures.” Top panel represents the number of cells migrated, whereas representative images are displayed in the lower panel. ##, p < 0.01; **, p < 0.01; versus control. F, A549 cells were co-transfected with Twist1 siRNA and either wild-type or R34K mutant of Twist1. The lysates were later probed for the expression of E-cadherin, Myc-Twist1 with specific antibodies. G, A549 cells were co-transfected with Myc-tagged Twist1 and either control siRNA or PRMT1 siRNA. Total lysates were later probed for the expression of E-cadherin, N-cadherin, PRMT1, and Twist1 (Myc). H, A549 cells transiently transfected with either Myc-tagged Twist1 or R34K mutant were treated with cycloheximide (100 μg/ml) for indicated periods of time, followed by immunoblotting and densitometric scanning. Upper panel represents the normalized Twist1 (Myc)/actin levels, whereas representative images are displayed in the lower panel.
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Figure 6: PRMT-mediated Twist1 methylation is required for E-cadherin repression.A, A549 cells were transiently transfected with either Myc-tagged wild-type or R34K mutant of Twist1. The lysates were later probed for the expression of E-cadherin and N-cadherin via immunoblotting with specific antibodies. B, A549 cells transfected with empty vector, Myc-tagged wild-type or R34K mutant of Twist1 were fixed, permeabilized, and co-immunostained with anti-myc antibodies and anti-E-cadherin antibodies, and the expressions of E-cadherin and Twist1 (Myc) were visualized by indirect immunofluorescence and confocal microscopy. Scale bar 5 μm. C, MCF7 cells were transiently transfected with either Myc-tagged wild-type or R34K mutant of Twist1. The lysates were later probed for the expression of E-cadherin and N-cadherin via immunoblotting with specific antibodies. D, MCF7 cells transfected with empty vector, Myc-tagged wild-type or R34K mutant of Twist1 were fixed, permeabilized and co-immunostained with anti-myc antibodies and anti-E-cadherin antibodies, and the expressions of E-cadherin and Twist1 (Myc) were visualized by indirect immunofluorescence and confocal microscopy. Scale bar 5 μm. E, migration of A549 cells transfected with empty vector, Myc-tagged wild-type or R34K mutant of Twist1 were determined using trans-well assays as described under “Experimental Procedures.” Top panel represents the number of cells migrated, whereas representative images are displayed in the lower panel. ##, p < 0.01; **, p < 0.01; versus control. F, A549 cells were co-transfected with Twist1 siRNA and either wild-type or R34K mutant of Twist1. The lysates were later probed for the expression of E-cadherin, Myc-Twist1 with specific antibodies. G, A549 cells were co-transfected with Myc-tagged Twist1 and either control siRNA or PRMT1 siRNA. Total lysates were later probed for the expression of E-cadherin, N-cadherin, PRMT1, and Twist1 (Myc). H, A549 cells transiently transfected with either Myc-tagged Twist1 or R34K mutant were treated with cycloheximide (100 μg/ml) for indicated periods of time, followed by immunoblotting and densitometric scanning. Upper panel represents the normalized Twist1 (Myc)/actin levels, whereas representative images are displayed in the lower panel.

Mentions: Since Twist1 is methylated by PRMT1 specifically at arginine 34 (Arg-34, Fig. 5), we sought to interrogate the functional significance of R34 methylation of Twist1 on E-cadherin repression. For these experiments, we generated myc-epitope tagged versions of wild-type and R34K mutant of Twist1 for mammalian expression (Fig. 6). Expression of wild-type Twist1, as expected, repressed E-cadherin expression (Fig. 6A). Expression of R34K mutant, in contrast, failed to impact E-cadherin expression (Fig. 6A), suggesting that the Arg-34 methylation of Twist1 is required for E-cadherin repression. In addition, we also observed a corresponding increase in N-cadherin, a mesenchymal cell marker, by the wild-type Twist1 but not by the R34K mutant of Twist1 (Fig. 6A). Similar effects of R34K mutant of Twist1 on E-cadherin repression were also observed in H2122 cells (data not shown). We also confirmed the effects of Twist1 expression on E-cadherin repression via indirect immunofluorescence (Fig. 6B). A549 cells expressing wild-type Twist1 (Fig. 6B, Myc panel) displayed reduced E-cadherin staining, while A549 cells expressing R34K mutant displayed positive staining for E-cadherin (Fig. 6B). To ensure that the Twist1 arginine methylation-mediated E-cadherin repression was not restricted to the epithelium of the lung, we also tested the effects of wild-type and methylation-deficient mutants of Twist1 on E-cadherin repression in another epithelial cell-derived cancer cell line, MCF7 (a human breast cancer cell line). Consistent with the effects of the Twist1 mutants on E-cadherin repression in NSCLC cells, expression of the R34K mutant also failed to repress E-cadherin in MCF7 cells, as determined by Western blot (Fig. 6C), and indirect immunofluorescence (Fig. 6D).


PRMT1 Is a Novel Regulator of Epithelial-Mesenchymal-Transition in Non-small Cell Lung Cancer.

Avasarala S, Van Scoyk M, Karuppusamy Rathinam MK, Zerayesus S, Zhao X, Zhang W, Pergande MR, Borgia JA, DeGregori J, Port JD, Winn RA, Bikkavilli RK - J. Biol. Chem. (2015)

PRMT-mediated Twist1 methylation is required for E-cadherin repression.A, A549 cells were transiently transfected with either Myc-tagged wild-type or R34K mutant of Twist1. The lysates were later probed for the expression of E-cadherin and N-cadherin via immunoblotting with specific antibodies. B, A549 cells transfected with empty vector, Myc-tagged wild-type or R34K mutant of Twist1 were fixed, permeabilized, and co-immunostained with anti-myc antibodies and anti-E-cadherin antibodies, and the expressions of E-cadherin and Twist1 (Myc) were visualized by indirect immunofluorescence and confocal microscopy. Scale bar 5 μm. C, MCF7 cells were transiently transfected with either Myc-tagged wild-type or R34K mutant of Twist1. The lysates were later probed for the expression of E-cadherin and N-cadherin via immunoblotting with specific antibodies. D, MCF7 cells transfected with empty vector, Myc-tagged wild-type or R34K mutant of Twist1 were fixed, permeabilized and co-immunostained with anti-myc antibodies and anti-E-cadherin antibodies, and the expressions of E-cadherin and Twist1 (Myc) were visualized by indirect immunofluorescence and confocal microscopy. Scale bar 5 μm. E, migration of A549 cells transfected with empty vector, Myc-tagged wild-type or R34K mutant of Twist1 were determined using trans-well assays as described under “Experimental Procedures.” Top panel represents the number of cells migrated, whereas representative images are displayed in the lower panel. ##, p < 0.01; **, p < 0.01; versus control. F, A549 cells were co-transfected with Twist1 siRNA and either wild-type or R34K mutant of Twist1. The lysates were later probed for the expression of E-cadherin, Myc-Twist1 with specific antibodies. G, A549 cells were co-transfected with Myc-tagged Twist1 and either control siRNA or PRMT1 siRNA. Total lysates were later probed for the expression of E-cadherin, N-cadherin, PRMT1, and Twist1 (Myc). H, A549 cells transiently transfected with either Myc-tagged Twist1 or R34K mutant were treated with cycloheximide (100 μg/ml) for indicated periods of time, followed by immunoblotting and densitometric scanning. Upper panel represents the normalized Twist1 (Myc)/actin levels, whereas representative images are displayed in the lower panel.
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Figure 6: PRMT-mediated Twist1 methylation is required for E-cadherin repression.A, A549 cells were transiently transfected with either Myc-tagged wild-type or R34K mutant of Twist1. The lysates were later probed for the expression of E-cadherin and N-cadherin via immunoblotting with specific antibodies. B, A549 cells transfected with empty vector, Myc-tagged wild-type or R34K mutant of Twist1 were fixed, permeabilized, and co-immunostained with anti-myc antibodies and anti-E-cadherin antibodies, and the expressions of E-cadherin and Twist1 (Myc) were visualized by indirect immunofluorescence and confocal microscopy. Scale bar 5 μm. C, MCF7 cells were transiently transfected with either Myc-tagged wild-type or R34K mutant of Twist1. The lysates were later probed for the expression of E-cadherin and N-cadherin via immunoblotting with specific antibodies. D, MCF7 cells transfected with empty vector, Myc-tagged wild-type or R34K mutant of Twist1 were fixed, permeabilized and co-immunostained with anti-myc antibodies and anti-E-cadherin antibodies, and the expressions of E-cadherin and Twist1 (Myc) were visualized by indirect immunofluorescence and confocal microscopy. Scale bar 5 μm. E, migration of A549 cells transfected with empty vector, Myc-tagged wild-type or R34K mutant of Twist1 were determined using trans-well assays as described under “Experimental Procedures.” Top panel represents the number of cells migrated, whereas representative images are displayed in the lower panel. ##, p < 0.01; **, p < 0.01; versus control. F, A549 cells were co-transfected with Twist1 siRNA and either wild-type or R34K mutant of Twist1. The lysates were later probed for the expression of E-cadherin, Myc-Twist1 with specific antibodies. G, A549 cells were co-transfected with Myc-tagged Twist1 and either control siRNA or PRMT1 siRNA. Total lysates were later probed for the expression of E-cadherin, N-cadherin, PRMT1, and Twist1 (Myc). H, A549 cells transiently transfected with either Myc-tagged Twist1 or R34K mutant were treated with cycloheximide (100 μg/ml) for indicated periods of time, followed by immunoblotting and densitometric scanning. Upper panel represents the normalized Twist1 (Myc)/actin levels, whereas representative images are displayed in the lower panel.
Mentions: Since Twist1 is methylated by PRMT1 specifically at arginine 34 (Arg-34, Fig. 5), we sought to interrogate the functional significance of R34 methylation of Twist1 on E-cadherin repression. For these experiments, we generated myc-epitope tagged versions of wild-type and R34K mutant of Twist1 for mammalian expression (Fig. 6). Expression of wild-type Twist1, as expected, repressed E-cadherin expression (Fig. 6A). Expression of R34K mutant, in contrast, failed to impact E-cadherin expression (Fig. 6A), suggesting that the Arg-34 methylation of Twist1 is required for E-cadherin repression. In addition, we also observed a corresponding increase in N-cadherin, a mesenchymal cell marker, by the wild-type Twist1 but not by the R34K mutant of Twist1 (Fig. 6A). Similar effects of R34K mutant of Twist1 on E-cadherin repression were also observed in H2122 cells (data not shown). We also confirmed the effects of Twist1 expression on E-cadherin repression via indirect immunofluorescence (Fig. 6B). A549 cells expressing wild-type Twist1 (Fig. 6B, Myc panel) displayed reduced E-cadherin staining, while A549 cells expressing R34K mutant displayed positive staining for E-cadherin (Fig. 6B). To ensure that the Twist1 arginine methylation-mediated E-cadherin repression was not restricted to the epithelium of the lung, we also tested the effects of wild-type and methylation-deficient mutants of Twist1 on E-cadherin repression in another epithelial cell-derived cancer cell line, MCF7 (a human breast cancer cell line). Consistent with the effects of the Twist1 mutants on E-cadherin repression in NSCLC cells, expression of the R34K mutant also failed to repress E-cadherin in MCF7 cells, as determined by Western blot (Fig. 6C), and indirect immunofluorescence (Fig. 6D).

Bottom Line: Taken together, we show that PRMT1 is a novel regulator of EMT and arginine 34 (Arg-34) methylation of Twist1 as a unique "methyl arginine mark" for active E-cadherin repression.Therefore, targeting PRMT1-mediated Twist1 methylation might represent a novel strategy for developing new anti-invasive/anti-metastatic drugs.Moreover, methylated Twist1 (Arg-34), as such, could also emerge as a potential important biomarker for lung cancer.

View Article: PubMed Central - PubMed

Affiliation: From the Division of Pulmonary, Critical Care, Sleep and Allergy, and.

Show MeSH
Related in: MedlinePlus