SHARPIN-mediated regulation of protein arginine methyltransferase 5 controls melanoma growth

Abstract

SHARPIN, an adaptor for the linear ubiquitin chain assembly complex (LUBAC), plays important roles in NF-κB signaling and inflammation. Here, we have demonstrated a LUBAC-independent role for SHARPIN in regulating melanoma growth. We observed that SHARPIN interacted with PRMT5, a type II protein arginine methyltransferase, and increased its multiprotein complex and methyltransferase activity. Activated PRMT5 controlled the expression of the transcription factors SOX10 and MITF by SHARPIN-dependent arginine dimethylation and inhibition of the transcriptional corepressor SKI. Activation of PRMT5 by SHARPIN counteracted PRMT5 inhibition by methylthioadenosine, a substrate of methylthioadenosine phosphorylase, which is codeleted with cyclin-dependent kinase inhibitor 2A (CDKN2A) in approximately 15% of human cancers. Collectively, we identified a LUBAC-independent role for SHARPIN in enhancing PRMT5 activity that contributes to melanomagenesis through the SKI/SOX10 regulatory axis.

Keywords: Cancer; Cell Biology; Melanoma; Oncology; Signal transduction.

Figure 1. SHARPIN is upregulated in melanoma.

(A) Upper panel: SHARPIN transcript levels in diverse cancer types (red dots, n = approximately 169–998, TCGA) and normal tissue (green dots, n = approximately 0–155) (2-tailed Mann-Whitney U test). Lower panel: HOIP, HOIL-1L, and SHARPIN transcript levels in normal tissue (n = 7), benign nevi (n = 18), and melanoma (n = 45) (GDS1375 data set). One-way ANOVA with Dunnett’s test. (B) Immunoblot (upper panel) and qPCR (lower panel) analysis of LUBAC components in melanocytes (NHEM) and the indicated melanoma cell lines. Tubulin was probed as a loading control. qPCR data are presented as relative mRNA levels compared with NHEM cells. Data are representative results from 2 experiments. **P < 0.005; ***P < 0.0005. NA, not applicable.

Figure 2. SHARPIN plays a role in melanoma growth.

(A) Colony-forming efficiency (CFE) assay of melanoma cell lines expressing scrambled (Scr) or HOIP-, HOIL-1L–, or SHARPIN-specific shRNA. Cells were seeded at 10³/well and incubated for 14 days. Upper panels show representative images on day 14, and lower graph shows quantification of CFE (ImageJ). KD efficiency of each gene was confirmed with immunoblotting (see Supplemental Figure 1D). (B) CFE assay of melanoma cell lines expressing scrambled or SHARPIN-specific shRNAs (nos. 1, 2, and 3). Cells were seeded at 2.5 × 10³/well and analyzed as in D. (C) Growth of WM115 cells (4 × 10⁶ cells) expressing scrambled or SHARPIN-specific shRNA after subcutaneous injection into female nude mice. Tumor volumes were measured at the indicated time points. Data are presented as mean ± SD. n = 8 mice/group. Statistical significance was calculated using 2-way ANOVA. (D) CFE assay of WM793 and WM35 cells treated as indicated in C except that SHARPIN was reexpressed (+ rescue) as indicated. Upper panel shows representative images of colonies on day 14 after seeding at 10³/well. Lower graph shows CFE quantification on day 14. All quantitation data are presented as mean ± SD (n = 3). (A, B, D) Statistical significance was calculated using 1-way ANOVA and Dunnett’s test. *P < 0.05; **P < 0.005; ***P < 0.0005 (2-tailed Student’s t test). (A and B) Data are representative of 3 experiments.

Figure 3. SHARPIN affects melanoma growth and survival through regulation of SOX10, PAX3, and MITF expression.

(A) Pigmentation (upper panels), immunoblot (lower panels), and qPCR (right panel) analysis of MITF and upstream/downstream regulatory proteins in WM115 or WM35 melanoma cells overexpressing SHARPIN or a control empty vector (EV). qPCR data are presented as relative mRNA levels compared with control cells. (B) Immunoblot (left panel) and qPCR (right panels) analysis of WM115 cells expressing scrambled or SHARPIN-specific siRNAs or shRNAs. Statistical significance was calculated using Student’s t test (unpaired, 2 tailed, siRNA) or 1-way ANOVA and Dunnett’s test (shRNA). (C) Immunoblot analysis of WM35, WM793 and WM115 cells expressing scrambled or SHARPIN-specific shRNAs. Arrow and NS indicate SHARPIN and nonspecific bands, respectively. (D) Immunoblot analysis and CFE assay of WM115 melanoma cells expressing scrambled or SHARPIN-specific shRNA and ectopically expressing control or SOX10 (rescue). Middle and right panels show representative images of colonies and quantification of CFE 14 days after cells were seeded at 10³/well. CFE (ratio to scrambled/control) was presented as mean ± SD, and statistical significance was calculated using 2-way ANOVA and Tukey’s test (n = 6 from 2 independent experiments). All qPCR data are presented as mean ± SD (n = 3).*P < 0.05; **P < 0.005; ***P < 0.0005 (2-tailed, Student’s t test). (A–D) Data are representative of 2 experiments.

Figure 4. SHARPIN facilitates PRMT5 complex formation and enhances PRMT5 methyltransferase activity.

(A) Immunoblot analysis of WM115 cell lysates immunoprecipitated with normal rabbit IgG, anti-PRMT5, or anti-SHARPIN antibodies and blotted for PRMT5 or SHARPIN. (B) Immunoblot analysis of gel filtration fractions from WM793 lysates (3 mg protein). (C) 2D Blue Native–PAGE/SDS-PAGE analysis of WM793 lysates (40 μg/lane). Asterisks indicate SHARPIN and PRMT5 in the complex. (D) Immunoblot analysis of V5-immunoprecipitates from HEK293T cells expressing empty vector or V5-tagged PRMT5 and coexpressing Myc-tagged SHARPIN full-length (FL), NZF-deleted (ΔNZF), UBL deleted (ΔUBL), or Flag-tagged CC–deleted (ΔCC) proteins. (E) Immunoblot analysis of Myc immunoprecipitates from HEK293T cells expressing empty vector or Myc-tagged full-length SHARPIN and coexpressing Flag-tagged full-length PRMT5, N terminus (N-term), middle region (middle), and C terminus (C-term). (F) qPCR analysis (left) of SHARPIN and the PRMT5 target genes ST7, RBL2, or NM23 in WM35 cells expressing scrambled or SHARPIN-specific shRNA. ChIP analysis (right) of arginine-methylated histone H4 at the enhancer/promoter of ST7 or NM23 genes. WM35 cells expressing scrambled or SHARPIN-specific shRNA were immunoprecipitated with an anti-H4R3me2s antibody, and coimmunoprecipitated ST7 or NM23 promoter sequences were quantified by qPCR. (G) Immunoblot analysis of WM793 or WM35 cells expressing scrambled or SHARPIN-specific shRNAs (nos. 1 and 3). (H) In vitro methylation of histone 4 by PRMT5/MEP50 after preincubation with buffer or purified full-length or ΔUBL SHARPIN proteins. (I) Blue Native–PAGE gel of lysates from WM793 cells expressing scrambled or SHARPIN-specific shRNAs (nos. 1 and 3). For immunoblot/immunoprecipitation analyses, input indicates 5% of lysate. qPCR data are presented as mean ± SD (n = 3). *P < 0.05; **P < 0.005; ***P < 0.0005, 2-tailed Student’s t test. (A–I) Data shown represents results of at least 2 independent experiments.

Figure 5. PRMT5 increases SOX10 and PAX3 expression by arginine methylation–dependent inhibition of SKI.

(A) Luciferase reporter assay of TGF-β signaling in WM793 cells expressing scrambled shRNA or HOIP-, HOIL-1L–, or SHARPIN-specific shRNAs. Cells transfected with TGF-β reporter (CAGAx9-luc) and pCMV-Cypridina-luc (internal control) reporter plasmids were treated with TGF-β (20 ng/ml) for 8 or 24 hours. (B) qPCR analysis of SOX10, PAX3, and MITF expression in WM115 cells expressing empty vector (control), SHARPIN, or PRMT5 expression plasmids and treated with TGF-β (20 ng/ml) for 0, 24, or 48 hours. (C) Immunoblot and qPCR analysis of WM115 cells transfected with scrambled (control) siRNA or SKI-specific SMARTpool siRNA. (D) ChIP analysis of WM115 cells expressing empty vector, SHARPIN, or PRMT5 and treated with vehicle (Veh) or TGF-β (20 ng/ml) for 18 hours. ChIP analysis was performed with anti-SKI antibody, and coimmunoprecipitated SOX10 or PAX3 promoter sequences were quantified by qPCR. (E) Immunoblot analysis of SKI and p53 in WM115 cell lysates immunoprecipitated with normal rabbit serum (NRS) or SYM10 antibody. (F) Analysis as in E except WM115 cells expressed scrambled or PRMT5-specific shRNA. (G) Immunoblot analysis of anti-Flag immunoprecipitates of A375 cells coexpressing WT, Arg8 mutated (R8K), or Arg658/660 mutated (RR/KK) SKI with either PRMT5 or SHARPIN. Asterisks and arrowhead indicate SHARPIN and tubulin, respectively. (H) Immunoblot analysis of anti-Myc immunoprecipitates of HEK293T cells expressing Myc-tagged SHARPIN and Flag-tagged WT, R8K mutant, or RR/KK mutant SKI proteins. (I) Immunoblot analysis of IgG or anti-SKI immunoprecipitates of SK-Mel-28 cells. (J) ChIP analysis as in D of WM115 cells expressing WT or R8K mutant SKI proteins. Lysates were immunoprecipitated with indicated antibodies. For immunoprecipitation analyses, input indicates 5% of lysates. Statistical significance was calculated using 2-way ANOVA (Dunnett’s test, A and B) or 2-tailed Student’s t test (C, D, J). Data are presented as mean ± SD (n = 3). *P < 0.05; **P < 0.005; ***P < 0.0005.

Figure 6. SHARPIN regulation of PRMT5 activity controls SOX10 expression and growth in MTAP-deleted human melanomas.

(A) Expression of MTAP and CDKN2A genes in melanoma patients (TCGA, n = 472). Green and red vertical lines indicate the cutoffs for classification as low-MTAP (n = 70) and high-MTAP (n = 50) melanomas, respectively. The green horizontal line indicates the expression level in normal tissue (TCGA, n = 1). (B) Pearson’s correlation coefficients for SHARPIN and SOX10 or SHARPIN and PAX3 expression in all (n = 472, blue), MTAP-low (n = 70, green), and MTAP-high (n = 50, red) melanoma samples. (C) Immunoblot analysis of the indicated proteins in melanoma cells with high, medium, or low sensitivity to growth inhibition by SHARPIN KD. (D) Immunoblot analysis (left upper) and colony-forming efficiency (lower) assay of WM35 cells expressing empty vector (control) or overexpressing SHARPIN. Cells were treated with vehicle (DMSO) or MTA (100 μM) for 72 hours. For CFE, cells were seeded at 10³/well and colonies were visualized and quantified (lower right) after 14 days in culture. PRMT5 activity (upper right) was assessed in anti-PRMT5–immunoprecipitated cell lysates. The quantitation data are presented as mean ± SD. n = 3 (lower right). n = 6 (upper right). Statistical significance was calculated using 2-way ANOVA (Tukey’s test). *P < 0.05; ***P < 0.0005. (C and D) Data shown represent at least 2 independent experiments.

Figure 7. MTAP expression determines sensitivity to SHAPIN-mediated growth inhibition of melanoma.

(A) Immunoblot analysis and CFE assay of WM35 cells stably expressing empty vector or MTAP expression plasmids plus scrambled or SHARPIN-specific shRNA. Cells were seeded at 10³/well and colonies were visualized and quantified after 14 days in culture. (B) Growth of WM35 cells (2.0 × 10⁶ cells) injected subcutaneously into female nude mice. Stable WM35 transfectants expressing empty vector or MTAP were subjected to KD with scrambled or SHARPIN-specific shRNA. Tumor volume (upper panel) and weight (lower panel) were measured at the indicated time. Immunoblot analysis (right panel) was performed using representative tumor lysates from each group. Data are presented as mean ± SD (at the end point of experiment). n = 8 mice/group. Two-way ANOVA test (tumor volume) or Welch’s test (tumor weight). (C) Cell growth/viability (ATPlite, left panel) of A375 cells expressing scrambled or SHARPIN shRNA, and then treated with indicated amount of MTA for 5 days. KD efficiency of SHARPIN was analyzed with qPCR (right panels). (D) As in C, qPCR analysis (left) and growth/viability assay (ATPlite, right) of A375 cells expressing shRNAs, scrambled, shSHARPIN, and/or shMTAP. (E) CFE assay of A375 cells expressing scrambled, SHARPIN, or MTAP (nos. 1 and 5) shRNAs. Cells (2.5 × 10³/well) were seeded and cultured for 14 days. Colonies were visualized and quantified (ImageJ). All quantitation data and qPCR data are presented as mean ± SD (n = 3). Statistical significance was calculated using 1-way (D, left) or 2-way (A and B, D, right, E) Tukey’s test. *P < 0.05; **P < 0.05; ***P < 0.0005. (A, C–E) Data represent results from 2 to 3 independent experiments.

Figure 8. SHARPIN expression in MTAP-deleted tumors correlates strongly with SOX10 expression and patient survival.

(A) Quantification of immunohistochemical staining of SHARPIN and SOX10 in a human melanoma tissue microarray (n = 438). Staining intensity was scored as 0, 1, 2, or 3 (see Supplemental Figure 6), and the data are expressed as the percentage of specimens with the indicated combinations of staining scores. (B) Pearson’s correlation coefficients for SHARPIN and SOX10 expression in all, low-MTAP, and high-MTAP melanomas. (C) Survival of patients from the TCGA data set (n = 281 completely sequenced tumors). Left: patients with tumors expressing normal (unaltered) or amplified/upregulated (altered) SOX10 levels (n = 281). Middle: patients with tumors expressing unaltered or altered SHARPIN levels and MTAP deletion (n = 75). Right: patients with tumors expressing unaltered or altered SHARPIN levels and normal MTAP levels (n = 196). (D) Survival of patients from the TMA data set (n = 50). Patients with tumors expressing low MTAP (IHC score = 0 or 1) and low (IHC score = 0 or 1) or high (IHC score = 2 or 3) levels of SOX10 (left, n = 50), SHARPIN (middle, n = 50), or both SHARPIN and SOX10 (right, n = 29). Statistical significance was calculated using Prism7.0 (log-rank test).