Journal: Cell reports

Article Title: PRMT6 Regulates RAS/RAF Binding and MEK/ERK-Mediated Cancer Stemness Activities in Hepatocellular Carcinoma through CRAF Methylation.

doi: 10.1016/j.celrep.2018.09.053

Figure Lengend Snippet: Figure 1. PRMT6 Is Weakly Expressed and Negatively Correlated with Aggressive Cancer Features in HCC Patients (A and B) Scattered dot (A) and waterfall (B) plot analyses of PRMT6 mRNA levels in HCC and matched non-tumor liver specimens from 77 patient samples. Red and blue bars represent samples that show a relative PRMT6 fold change of R2 overexpression and underexpression, respectively (HCC/NT). (C) Scattered dot plot analysis of PRMT6 mRNA levels in normal (n = 50) and HCC (n = 371) tissues using information gathered from the TCGA HCC dataset. (D) PRMT6 immunostaining of tissue microarray comprising 83 paired non-tumor liver and HCC tissue samples. Shown are representative images of the immunostaining. Scale bar, 100 mm. (E) Graph indicates the percentage of cases displaying no and/or low or moderate and/or high staining intensity of PRMT6. (F) Proteomic expression of PRMT6 in HCC cell lines by western blot. Data are representative of two or more independent experiments. Bars and error represent mean ± SD of replicate measurements.

Article Snippet: Sections were subsequently incubated with anti-human/mouse PRMT6 (1:1000 for human, 1:100 for mouse, Abcam, ab47244), anti-human PRMT6 (1:300, Atlas Antibodies, HPA059424), anti-mouse SOX2 (1:100, R&D Systems, MAB2018) and anti-mouse p-ERK1/2 (1:100, Abcam, ab5011).

Techniques: Over Expression, Immunostaining, Microarray, Staining, Expressing, Western Blot

Journal: Cell reports

Article Title: PRMT6 Regulates RAS/RAF Binding and MEK/ERK-Mediated Cancer Stemness Activities in Hepatocellular Carcinoma through CRAF Methylation.

doi: 10.1016/j.celrep.2018.09.053

Figure Lengend Snippet: Figure 2. PRMT6 Negatively Regulates Cancer Properties in HCC (A) Representative images and quantification of number of cells that migrated and invaded in BEL7402 and Huh7 cells with or without PRMT6 expression modulated. **p < 0.01 and ***p < 0.001. Scale bar, 100 mm. (B) Percentage of annexin V-PI-positive cells in BEL7402 and Huh7 cells with or without PRMT6 expression modulated in the presence of 5-flurouracil, cisplatin, or sorafenib. (C) Xenograft tumors and tumor volume measurements of Huh7 cells with or without PRMT6 overexpressed. (D) Bioluminescence imaging and luciferase signal quantification of nude mice injected intrahepatically with luciferase-labeled BEL7402 cells with or without PRMT6 suppressed. (E) In vivo bioluminescence imaging of nude mice injected intrahepatically with luciferase-labeled MHCC97L cells with or without PRMT6 overexpressed. Ex vivo imaging of the lungs harvested. Representative H&E images of liver and lung tissues harvested. Scale bar, 100 mm. Bar chart summary of number of metastatic foci observed in lung. EV, empty vector control; NTC, non-target control; OE, overexpression. Data are representative of two or more independent experiments. Bars and error represent mean ± SD of replicate measurements.

Article Snippet: Sections were subsequently incubated with anti-human/mouse PRMT6 (1:1000 for human, 1:100 for mouse, Abcam, ab47244), anti-human PRMT6 (1:300, Atlas Antibodies, HPA059424), anti-mouse SOX2 (1:100, R&D Systems, MAB2018) and anti-mouse p-ERK1/2 (1:100, Abcam, ab5011).

Techniques: Expressing, Imaging, Luciferase, Injection, Labeling, In Vivo, Ex Vivo, Plasmid Preparation, Control, Over Expression

Journal: Cell reports

Article Title: PRMT6 Regulates RAS/RAF Binding and MEK/ERK-Mediated Cancer Stemness Activities in Hepatocellular Carcinoma through CRAF Methylation.

doi: 10.1016/j.celrep.2018.09.053

Figure Lengend Snippet: Figure 3. PRMT6 Negatively Regulates Cancer Stemness Properties in HCC (A) Oncosphere formation and serial passages of BEL7402 and Huh7 cells with or without PRMT6 expression modulated. ***p < 0.001. Scale bar, 100 mm. (B) Flow cytometry and western blot analyses for CD133 expression in Huh7 cells with or without PRMT6 overexpressed. (C) Western blot analysis for PRMT6, SOX2, and NANOG expression in BEL7402 and Huh7 cells with or without PRMT6 expression modulated. (D) Images of xenograft tumors (red arrows) formed in NOD/SCID mice injected subcutaneously with CD133+PRMT6low, CD133+PRMT6high, CD133PRMT6low, and CD133-PRMT6high cells isolated from Huh7 cells in primary passage (picture representative of 10,000 cells injected). Kaplan-Meier curves showing percentage of tumor-free survival of the annotated groups of primary and secondary recipient mice. n = 8. Engraftment rates, average tumor latency, and tumor- initiating frequency of CD133+ and CD133 subsets with or without PRMT6 overexpressed in Huh7 cells. EV, empty vector control; NTC, non-target control; OE, overexpression; TIC, tumor-initiating cells. Data are representative of two or more independent experiments. Bars and error represent mean ± SD of replicate measurements.

Article Snippet: Sections were subsequently incubated with anti-human/mouse PRMT6 (1:1000 for human, 1:100 for mouse, Abcam, ab47244), anti-human PRMT6 (1:300, Atlas Antibodies, HPA059424), anti-mouse SOX2 (1:100, R&D Systems, MAB2018) and anti-mouse p-ERK1/2 (1:100, Abcam, ab5011).

Techniques: Expressing, Flow Cytometry, Western Blot, Injection, Isolation, Plasmid Preparation, Control, Over Expression

Journal: Cell reports

Article Title: PRMT6 Regulates RAS/RAF Binding and MEK/ERK-Mediated Cancer Stemness Activities in Hepatocellular Carcinoma through CRAF Methylation.

doi: 10.1016/j.celrep.2018.09.053

Figure Lengend Snippet: Figure 4. The Catalytically Active Domain of PRMT6 Is Functionally Important in Contributing Augmented Aggressive Cancer Stemness Properties in HCC (A) Western blot analysis for ADMA expression in BEL7402 and Huh7 cells with or without PRMT6 expression modulated. (B) Schematic illustration of PRMT6 wild-type (WT) and PRMT6 catalytic inactive (mutant) constructs used in this study. (C–E) Representative images and quantification of number of cells that (C) migrated, (D) invaded, and (E) formed oncospheres in BEL7402 cells stably over- expressing EV control, WT, and mutant PRMT6. **p < 0.01 and ***p < 0.001. Scale bar, 100 mm. (F)PercentageofannexinV-PI-positivecells inBEL7402cellsstably overexpressingEVcontrol,WT,andmutantinthe presenceof5-flurouracil,cisplatin,orsorafenib. (G) Western blot analysis for PRMT6, SOX2, and NANOG expression in BEL7402 cells stably overexpressing EV control, WT, and mutant PRMT6. (H) In vivo bioluminescence imaging and luciferase signal quantification of nude mice injected intrahepatically with luciferase-labeled BEL7402 cells stably overexpressing EV control, WT, and mutant PRMT6. EV, empty vector control; mut, mutant; NTC, non-target control; OE, overexpression; WT, wild-type. Data are representative of two or more independent experiments. Bars and error represent mean ± SD of replicate measurements.

Article Snippet: Sections were subsequently incubated with anti-human/mouse PRMT6 (1:1000 for human, 1:100 for mouse, Abcam, ab47244), anti-human PRMT6 (1:300, Atlas Antibodies, HPA059424), anti-mouse SOX2 (1:100, R&D Systems, MAB2018) and anti-mouse p-ERK1/2 (1:100, Abcam, ab5011).

Techniques: Western Blot, Expressing, Mutagenesis, Construct, Stable Transfection, Control, In Vivo, Imaging, Luciferase, Injection, Labeling, Plasmid Preparation, Over Expression

Journal: Cell reports

Article Title: PRMT6 Regulates RAS/RAF Binding and MEK/ERK-Mediated Cancer Stemness Activities in Hepatocellular Carcinoma through CRAF Methylation.

doi: 10.1016/j.celrep.2018.09.053

Figure Lengend Snippet: Figure 5. PRMT6 Interacts Directly with CRAF and Methylates It on Arginine 100 Residue (A) Gene set enrichment analysis (GSEA) identified an enrichment of RAS signaling in PRMT6 silenced cells. (B) Co-immunoprecipitation analysis for validation of CRAF as a PRMT6 interacting protein partner in 293T cells expressing SFB-tagged PRMT6, 293T cells expressing SFB-tagged CRAF, BEL7402 cells expressing SFB-tagged PRMT6, and BEL7402 expressing endogenous PRMT6. (C) Left: western blot analysis for PRMT6-mediated incorporation of asymmetric arginine dimethylation in CRAF. Middle: western blot analysis of in vitro methylation assay of CRAF. Right: GAR (glycine-arginine-rich) sequence positive control in the in vitro methylation assay. (D) Schematic diagram illustrating of full-length (FL) and deletion mutations of CRAF used in this study. CRD, cysteine-rich domain; RBD, Ras binding domain. (E) Mapping of protein domain in CRAF methylated by PRMT6 through in vivo methylation assay. Red arrows indicate ADMA bands at the predicted size. White arrow indicates loss of ADMA band at the predicted size in D2 CRAF truncation mutant. Yellow arrows indicate successful FLAG pull-down. (F) Left: levels of ADMA in WT and site-directed mutants of CRAF. Right: in vitro methylation assay of CRAF R100K mutant versus WT. (G) Left: in vitro methylation assay with immunoprecipitated PRMT6 from 293T cells stably transfected with SFB-PRMT6. Assay was performed by adding no peptide, CAVFRLLHE peptide, or CAVFKLLHE mutant peptide. Right: fragmentation spectrum of the methylated peptide identified by liquid chromatography/ tandem mass spectrometry (LC-MS/MS). m/z, mass/charge ratio. ***p < 0.001. Endo, endogenous; Exo, exogenous; KD, knockdown; mut, mutant; NTC, non-target control; WT, wild-type. Data are representative of two or more independent experiments. Bars and error represent mean ± SD of replicate measurements.

Article Snippet: Sections were subsequently incubated with anti-human/mouse PRMT6 (1:1000 for human, 1:100 for mouse, Abcam, ab47244), anti-human PRMT6 (1:300, Atlas Antibodies, HPA059424), anti-mouse SOX2 (1:100, R&D Systems, MAB2018) and anti-mouse p-ERK1/2 (1:100, Abcam, ab5011).

Techniques: Residue, Immunoprecipitation, Biomarker Discovery, Expressing, Western Blot, In Vitro, Methylation, Sequencing, Positive Control, Binding Assay, In Vivo, Mutagenesis, Stable Transfection, Transfection, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Knockdown, Control

Journal: Cell reports

Article Title: PRMT6 Regulates RAS/RAF Binding and MEK/ERK-Mediated Cancer Stemness Activities in Hepatocellular Carcinoma through CRAF Methylation.

doi: 10.1016/j.celrep.2018.09.053

Figure Lengend Snippet: Figure 6. PRMT6 Methylation of CRAF at Arginine 100 Interferes with RAS/RAF Binding Domain and Inhibits MEK/ERK-Related Kinase Activity (A) CRAF kinase assay, western blot analysis for expression of phosphorylated and total MEK1/2, phosphorylated and total ERK1/2 and ERK kinase assay in BEL7402 and Huh7 cells with or without PRMT6 expression modulated. (B) Representative images and quantification of number of cells that migrated, invaded, and formed oncospheres in BEL7402 cells expressing NTC or shPRMT6 clone 956 that were treated with control or MEK inhibitor U0126. ***p < 0.001. Scale bar, 100 mm. (C) Percentage of annexin V-PI-positive cells in BEL7402 cells expressing NTC or shPRMT6 clone 956 that were treated with control or MEK inhibitor U0126, following sorafenib treatment. (D) Western blot analysis for expression of CRAF, phosphorylated and total ERK1/2, as well as ERK kinase assay in BEL7402 cells with EV control, CRAF WT, or CRAF R100K overexpressed. (E and F) Western blot analysis for the co-immunoprecipitation of CRAF and RAS in BEL7402 cells (E) with or without PRMT6 expression modulated and (F) with CRAF WT or R100K mutant. (G) In vitro methylation assay of CRAF WT and R100K mutant followed by RAS binding assay with immunoprecipitated MBP-RAS. EV, empty vector control; NTC, non-target control; OE, overexpression; WT, wild-type. Data are representative of two or more independent experiments. Bars and error represent mean ± SD of replicate measurements.

Article Snippet: Sections were subsequently incubated with anti-human/mouse PRMT6 (1:1000 for human, 1:100 for mouse, Abcam, ab47244), anti-human PRMT6 (1:300, Atlas Antibodies, HPA059424), anti-mouse SOX2 (1:100, R&D Systems, MAB2018) and anti-mouse p-ERK1/2 (1:100, Abcam, ab5011).

Techniques: Methylation, Binding Assay, Activity Assay, Kinase Assay, Western Blot, Expressing, Control, Immunoprecipitation, Mutagenesis, In Vitro, Plasmid Preparation, Over Expression

Journal: Cell reports

Article Title: PRMT6 Regulates RAS/RAF Binding and MEK/ERK-Mediated Cancer Stemness Activities in Hepatocellular Carcinoma through CRAF Methylation.

doi: 10.1016/j.celrep.2018.09.053

Figure Lengend Snippet: Figure 7. Loss of PRMT6 Expression Aggravates Liver Tumorigenesis in a DEN+CCL4 HCC-Induced PRMT6 Knockout Mouse Model, and PRMT6-Dependent CRAF/ERK Signaling Regulates HCC Stemness as Demonstrated in Patient-Derived Organoids (A) Representative pictures of livers harvested from WT and PRMT6 knockout (PRMT6/) mice that received DEN+CCl4 carcinogen induction. (B) Maximum size of HCC tumors and liver weight over body weight ratio. *p < 0.05. (C) Representative H&E and IHC images of PRMT6 and p-ERK1/2 expression of liver tissues harvested from WT or PRMT6/ mice treated with DEN+CCL4. Scale bar, 100 mm (inset, 20 mm). (D) Western blot analysis for expression of PRMT6, CD133, NANOG, SOX2, and phosphorylated and total ERK1/2 in non-tumor liver and HCC organoids with PRMT6 stably suppressed or overexpressed, respectively. (E) Percentage of viable cells in non-tumor liver organoids with PRMT6 suppressed, compared with controls, following 5-flurouracil, cisplatin, and sorafenib treatment. *p < 0.05, **p < 0.01, and ***p < 0.001. (F) Representative images and quantification of number of cells that migrated, invaded, and formed oncospheres in HCC organoids with PRMT6 overexpressed compared with controls. Scale bar, 100 mm. *p < 0.05, **p < 0.01, and ***p < 0.001. EV, empty vector control; NT, non-tumor liver; NTC, non-target control; OE, overexpression; T, tumor/HCC. Data are representative of two or more independent experiments. Bars and error represent mean ± SD of replicate measurements.

Article Snippet: Sections were subsequently incubated with anti-human/mouse PRMT6 (1:1000 for human, 1:100 for mouse, Abcam, ab47244), anti-human PRMT6 (1:300, Atlas Antibodies, HPA059424), anti-mouse SOX2 (1:100, R&D Systems, MAB2018) and anti-mouse p-ERK1/2 (1:100, Abcam, ab5011).

Techniques: Expressing, Knock-Out, Derivative Assay, Western Blot, Stable Transfection, Plasmid Preparation, Control, Over Expression