Journal: Signal Transduction and Targeted Therapy

Article Title: Erianin suppresses constitutive activation of MAPK signaling pathway by inhibition of CRAF and MEK1/2

doi: 10.1038/s41392-023-01329-3

Figure Lengend Snippet: Erianin inhibits MAPK signaling pathway through suppressing CRAF and MEK1/2 but not BRAF kinase activity. a , b The inhibitory effect of erianin on the activity of MEK1 and MEK2 kinase. Active GST-MEK1 full length or GST-MEK2 full length (60 ng) and various doses of erianin were incubated with inactive GST-ERK1 or tag free ERK2 (400 ng) as substrate at 30 °C for 30 min. The phosphorylation of ERK1/2 (Thr202/Tyr204) was detected by western blotting. c The inhibitory effect of erianin on the activity of CRAF kinase. Active CRAF (306-end) (50 ng) and various doses of erianin were incubated with inactive GST-MEK1 (600 ng) as substrate at 30 °C for 30 min. d – f Quantifications of integrated density in ( a – c ) were performed. Data were shown as means ± S.D. of three independent experiments. The asterisks (* p < 0.05, ** p < 0.01, *** p < 0.001) indicate a significant difference in the expression of phosphorylation of ERK1 or ERK2 vs total ERK1 or ERK2 in control and erianin-treated group. g The luminescent ADP detection assay was developed to detect the luminescence signal of ATP-to-ADP using the same concentration kinases and substrates described in above kinase assay. Three independent repeats were conducted in this experiment. h Immunoprecipitation (IP)/WB of endogenous CRAF from lysates of SK-MEL-2 (NRAS mut) and A375 (BRAF V600E) cells treated with DMSO or erianin at 12.5, 25, 50 nM for 24 h. Total lysates were immunoblotted for BRAF, CRAF, and MEK1. i IP/WB of endogenous MEK1 from lysates of SK-MEL-2 and A375 cells treated with DMSO or erianin at 12.5, 25, 50 nM for 24 h. Total lysates were immunoblotted for CRAF and MEK1. j , k Western blotting of phospho-CRAF, phospho-MEK1/2 and phospho-ERK1/2 by erianin, vemurafenib, cobimetinib or LY3009120 at indicated concentration for 24 h in NRAS mutant SK-MEL-2 and BRAF V600E mutant A375 cell lines. l Western blotting of MAPK signa l ing pathway by erianin, vemurafenib, cobimetinib, or LY3009120 at indicated concentrations for 24 h in KRAS mutant HCT116 cell line

Article Snippet: Active BRAF (#B08-11BG), active BRAF V600E (#B08-12G), active RAF1 (EE) (#R01-13G), corresponding RAF substrate inactive MEK1 (#M02-14BG), as well as active MEK1 (#M02-10G), active MEK2 (#M03-10G), inactive ERK1 protein (#M29-14G), and inactive ERK2 protein (#M28-14U) were obtained from Signal Chem.

Techniques: Activity Assay, Incubation, Phospho-proteomics, Western Blot, Expressing, Control, Detection Assay, Concentration Assay, Kinase Assay, Immunoprecipitation, Mutagenesis

Journal: Signal Transduction and Targeted Therapy

Article Title: Erianin suppresses constitutive activation of MAPK signaling pathway by inhibition of CRAF and MEK1/2

doi: 10.1038/s41392-023-01329-3

Figure Lengend Snippet: Erianin inhibits proliferation in BRAF V600E or RAS mutant cell lines. a Chemical structure of erianin. b Cytotoxicity of erianin in normal NHEM and NHDF cell lines using MTT assay. c The left panel shows representative dose–response curves by MTT assay. The SK-MEL-2 (NRAS mut), HCT116 (KRAS mut), A375 (BRAF V600E), and SK-MEL-28 (BRAF V600E) cell lines were exposed to erianin for 72 h. The concentrations are transformed to Log10 values; the Y -axis shows the corresponding relative cell viability. The right panel shows IC50 values of erianin, three BRAF inhibitors (vemurafenib, dabrafenib and encorafenib) and three MEK inhibitors (cobimetinib, trametinib, and binimetinib) calculated in GraphPad Prism 7.0. d The effect of erianin on growth of SK-MEL-2, A375, SK-MEL-28, and HCT 116 cells was estimated by MTT assay at 24, 48, or 72 h. Data were shown as means ± S.D. e The effect of erianin on anchorage-independent growth in above cells was evaluated. Data were shown as means ± S.D. Scale bars: 400 μm. The colonies numbers were calculated in Image-Pro Plus software. * p < 0.05; ** p < 0.01; *** p < 0.001. f Synergism effect of erianin and vemurafenib in SK-MEL-2, A375, SK-MEL-28, and HCT 116. The synergism and antagonism (CI value) were determined and analyzed using CompuSyn 1.0. CI value > 1.1 indicates antagonism, 1.1 ≥ CI value > 0.9 shows addictive effect and CI value ≤ 0.9 indicates synergism

Article Snippet: Active BRAF (#B08-11BG), active BRAF V600E (#B08-12G), active RAF1 (EE) (#R01-13G), corresponding RAF substrate inactive MEK1 (#M02-14BG), as well as active MEK1 (#M02-10G), active MEK2 (#M03-10G), inactive ERK1 protein (#M29-14G), and inactive ERK2 protein (#M28-14U) were obtained from Signal Chem.

Techniques: Mutagenesis, MTT Assay, Transformation Assay, Software

Journal: Signal Transduction and Targeted Therapy

Article Title: Erianin suppresses constitutive activation of MAPK signaling pathway by inhibition of CRAF and MEK1/2

doi: 10.1038/s41392-023-01329-3

Figure Lengend Snippet: Erianin suppresses either BRAF V600E or RAS mutant cell growth in CDX model. a NOD-SCID mice were injected subcutaneously with SK-MEL-2 (NRAS mut, 5 × 10 6 cell/mouse), A375 (BRAF V600E, 1 × 10 7 cell/mouse), SK-MEL-28 (BRAF V600E, 5 × 10 6 cell/mouse) and HCT116 (KRAS mut, 1 × 10 7 cell/mouse) cells mixed with Matrigel (1:1); erianin (50 mg/kg), vemurafenib (50 mg/kg) or the combine was given through oral gavage and the size of the tumors was monitored twice per week. Tumor volume (mm 3 ) = (length × width × height) × 0.52. SK-MEL-2: n = 10; A375 and SK-MEL-28: n = 8; HCT116: n = 9. b The photographs show tumors from CDX mice treated with vehicle, erianin, vemurafenib, or the combination. c The weight of the tumors was quantified and expressed as the treatment groups compared with the vehicle-treated group. Data were presented as mean ± S.D. One-way ANOVA test. * p < 0.05; ** p < 0.01. d Western blotting shows the expression of phospho-MEK1/2 and phospho-ERK1/2 by erianin in SK-MEL-2, A375, and SK-MEL-28 CDX tumor tissues. The tissue lysates were prepared from CDX tumor tissues in each treatment group. Three samples were randomly prepared for each group and every blot shows one sample. e The quantization (IOD values) of IHC staining in the treatment groups compared with the vehicle-treated group. Each point represents the IOD values of four quantified data from one mouse. Scale bars: 50 μm. One-way ANOVA test. *** p < 0.001. f Kaplan–Meier curve depicting tumors less than 1000 mm 3 in the treatment groups compared with the vehicle-treated group

Article Snippet: Active BRAF (#B08-11BG), active BRAF V600E (#B08-12G), active RAF1 (EE) (#R01-13G), corresponding RAF substrate inactive MEK1 (#M02-14BG), as well as active MEK1 (#M02-10G), active MEK2 (#M03-10G), inactive ERK1 protein (#M29-14G), and inactive ERK2 protein (#M28-14U) were obtained from Signal Chem.

Techniques: Mutagenesis, Injection, Western Blot, Expressing, Immunohistochemistry

Journal: Signal Transduction and Targeted Therapy

Article Title: Erianin suppresses constitutive activation of MAPK signaling pathway by inhibition of CRAF and MEK1/2

doi: 10.1038/s41392-023-01329-3

Figure Lengend Snippet: Erianin exerts antitumor efficacy in melanoma and colorectal cancer in vivo. a Tumor pharmacodynamic assay was performed in tumor-bearing NPG mice (tumor has been passaged from melanoma patient to mice for three generations). The photographs show tumors from melanoma PDX mice treated with vehicle or drugs. b The effect of erianin on the volume of PDX tumors over time (within 78 days) was plotted. Vehicle, erianin (50 mg/kg, once a day), vemurafenib (50 mg/kg, once a day), erianin and vemurafenib combination therapy, cobimetinib (5 mg/kg, twice a week) or vemurafenib and cobimetinib combination therapy (once a day and twice a week, respectively) were administered by oral gavage, n = 8 in each group. Tumor volume was measured once a week. One-way ANOVA test. * p < 0.05; ** p < 0.01. c Tumor weight was measured after treatment on the last day of the study. d The expression of phospho-MEK1/2 and phospho-ERK1/2 were examined by immunofluorescence analysis. Scale bars: 20 μm. One-way ANOVA test. *** p < 0.001. e Antitumor efficacy of erianin with or without immunity using B16F10 cell xenograft in C57BL-6J mouse. f , g Trend of tumor volume over time and tumor weight was measured after treatment on the last day of the study. One-way ANOVA test. * p < 0.05; ** p < 0.01. h The model depicts that erianin suppresses constitutive activation of MAPK signaling pathway in either BRAF V600E or RAS mutant cancers (Created with BioRender.com). Through inhibition of CRAF and MEK1/2 kinases, erianin suppresses phospho-MEK1/2 and phospho-ERK1/2 without paradoxical activation in vitro and in vivo

Article Snippet: Active BRAF (#B08-11BG), active BRAF V600E (#B08-12G), active RAF1 (EE) (#R01-13G), corresponding RAF substrate inactive MEK1 (#M02-14BG), as well as active MEK1 (#M02-10G), active MEK2 (#M03-10G), inactive ERK1 protein (#M29-14G), and inactive ERK2 protein (#M28-14U) were obtained from Signal Chem.

Techniques: In Vivo, Expressing, Immunofluorescence, Activation Assay, Mutagenesis, Inhibition, In Vitro

Journal: Cancers

Article Title: Nuclear Localization of BRAF V600E Is Associated with HMOX-1 Upregulation and Aggressive Behavior of Melanoma Cells.

doi: 10.3390/cancers14020311

Figure Lengend Snippet: Figure 2. Transcriptomic analysis on public datasets highlighted the role of HMOX-1 in aggressiveness and BRAF inhibitor resistance in melanoma. (A,B) Kaplan–Meier curves representing the disease- free survival (DFS) of patients with a high expression of HMOX-1 compared to samples with low expression of HMOX-1 in a TCGA melanoma cohort (406 samples with survival information) (A) and in BRAF mutant samples (165 samples) (B) HMOX-1 expression was stratified as “high” and “low” with an optimized cut-off. (C) Boxplot representing the distribution of HMOX-1 expression in sensitive and resistant mice after A375 cell inoculation and BRAF inhibitor treatment (GSE74729, 4 sensitive and 5 resistant samples). (D) Boxplot representing the distribution of HMOX-1 expression in sensitive and resistant patients in a merge of 3 melanoma patient datasets (43 BRAF inhibitor sensitive (before treatment) and 48 resistant and relapse of disease (after treatment)). FC, fold change; DFS, disease-free survival.

Article Snippet: MEF (BRAF KO) and MV3 (BRAF wild-type) melanoma cells expressing nuclear localization signal (NLS)-BRAFV600E and BRAFV600E were established by infecting the cells with lentiviral particles expressing human BRAFV600E with NLS sequence pLV[Exp]Neo-CMV>DsRed_Express2: ORF_2373bp/ Myc(hBRAFV600E/3xNLS) or without NLS pLV[Exp]-NeoCMV>DsRed_ Express2:ORF_2301bp/Myc(hBRAFV600E) tagged with DsRed fluorescent protein according to the standard protocols (Cyagen, Santa Clara, CA, USA).

Techniques: Expressing, Mutagenesis

Journal: Cancers

Article Title: Nuclear Localization of BRAF V600E Is Associated with HMOX-1 Upregulation and Aggressive Behavior of Melanoma Cells.

doi: 10.3390/cancers14020311

Figure Lengend Snippet: Figure 3. Nuclear BRAFV600E and HMOX-1 expression in xenograft mouse and human melanoma tissue cores. (A) The plasmid (BRAFV600E) or (3XNLS-BRAFV600E), containing BRAFV600E with nu- clear localization sequences (NLS), was transfected into the melanoma cell line MV3 (BRAFwt/wt) and selected with antibiotic G418. (B) MV3 cells transduced with BRAFV600E, NLS-BRAFV600E, and control cells were injected in mice (n = 5 for each group), and representative tumor images were taken. Xenograft mouse tissues were stained with an anti-HOMX1 antibody. Mag. 400×. Scale bars = 50 µm (C) Human malignant melanoma tissue array: immunoreactivity to BRAFV600E and HMOX1 in the human melanoma cores was detected and counterstained for the nuclear and cy- toplasm location. Mag. 400×. Scale bars = 50 µm. (Figure S3) provides the whole IHC analysis. (D) phosphoERK and phosphoAKT were assessed in MEF stably transfected with BRAFV600E or NLS-BRAFV600E after treatment with 5 µM PLX-4032 at different time points. A representative gel is shown. Two additional independent experiments provided similar results.

Article Snippet: MEF (BRAF KO) and MV3 (BRAF wild-type) melanoma cells expressing nuclear localization signal (NLS)-BRAFV600E and BRAFV600E were established by infecting the cells with lentiviral particles expressing human BRAFV600E with NLS sequence pLV[Exp]Neo-CMV>DsRed_Express2: ORF_2373bp/ Myc(hBRAFV600E/3xNLS) or without NLS pLV[Exp]-NeoCMV>DsRed_ Express2:ORF_2301bp/Myc(hBRAFV600E) tagged with DsRed fluorescent protein according to the standard protocols (Cyagen, Santa Clara, CA, USA).

Techniques: Expressing, Plasmid Preparation, Transfection, Transduction, Control, Injection, Staining, Stable Transfection

Journal: Cancers

Article Title: Nuclear Localization of BRAF V600E Is Associated with HMOX-1 Upregulation and Aggressive Behavior of Melanoma Cells.

doi: 10.3390/cancers14020311

Figure Lengend Snippet: Figure 6. Pathway analysis revealed a key role of HMOX-1 in BRAF inhibitor therapy resistance in the xenograft model and in melanoma patients. (A) Dotplot representing the enrichment score obtained for the common pathways identified in the xenograft model and melanoma patients. (B) Heatmap representing the correlation matrix between the 36 genes involved in the 3 selected pathways (ferroptosis, fluid shear stress and atherosclerosis, and hepatocellular carcinoma) common in the xenograft model and melanoma patients. Values are from the patient dataset. (C) Network representation of the 36 genes (green nodes) involved in the 3 selected pathways (light yellow nodes), with their Spearman correlation as edges. Dark red ellipse, genes belonging to “fluid shear stress and atherosclerosis”; orange, genes belonging to “ferroptosis”; blue ellipse, genes belonging to “hepatocellular carcinoma”; thicker edges, correlation related to HMOX1; blue edges, negative correlation; red edges, positive correlation; edge transparency proportional to correlation value; blue node border, negative correlation with HMOX1; red node border, positive correlation with HMOX1. Correlation values are from the patient dataset.

Article Snippet: MEF (BRAF KO) and MV3 (BRAF wild-type) melanoma cells expressing nuclear localization signal (NLS)-BRAFV600E and BRAFV600E were established by infecting the cells with lentiviral particles expressing human BRAFV600E with NLS sequence pLV[Exp]Neo-CMV>DsRed_Express2: ORF_2373bp/ Myc(hBRAFV600E/3xNLS) or without NLS pLV[Exp]-NeoCMV>DsRed_ Express2:ORF_2301bp/Myc(hBRAFV600E) tagged with DsRed fluorescent protein according to the standard protocols (Cyagen, Santa Clara, CA, USA).

Techniques: Shear