Journal: Oncogene

Article Title: An ErbB-3 antibody, MP-RM-1, inhibits tumor growth by blocking ligand-dependent and independent activation of ErbB-3/Akt signaling.

doi: 10.1038/onc.2011.322

Figure Lengend Snippet: Figure 1 NRG-1b-dependent ErbB-3/Akt activation is inhibited by MP-RM-1. (a) pSuper 4Mut and shErbB-3 infected MDA-MB- 435 and IR-8 cells were grown in 0.2% FBS RPMI 1640 for 24 h and then stimulated with 10 ng/ml of NRG-1b for 5 min before lysis. Cell lysates where then probed with the indicated antibodies. (b) MDA-MB-435 cells were grown in 0.2% FBS RPMI 1640 for 24 h and then incubated with 10 mg/ml of MP-RM-1 for the indicated time before NRG-1b stimulation. Cell lysates were immunoblotted for phosphorylated ErbB-3 and Akt expression levels. The same filter was reprobed with anti-ErbB-3 and anti-Akt antibodies for a loading control. (c) MDA-MB-435 cells were grown in 0.2% FBS RPMI 1640 for 24 h and then incubated with the indicated amount of MP- RM-1 for 2 h before NRG-1b stimulation. Cell lysates were immunoblotted for phosphorylated ErbB-3 and Akt expression levels. The same filter was reprobed with anti-ErbB-3 and anti-Akt antibodies for a loading control. Anti-p-ErbB-3 and anti-p-Akt immunoreactivity was quantified and plotted after normalization for total ErbB-3 and Akt immunoreactiviy (right panels). (d) IR-8 human melanoma cancer cells were grown in 0.2% FBS RPMI 1640 for 24 h and then incubated with the 10 mg/ml of MP-RM-1 for 2 h before NRG-1b stimulation. Cell lysates were immunoblotted for phosphorylated ErbB-3 and Akt expression levels. The same filter was reprobed with anti-ErbB-3 and anti-Akt antibodies for a loading control.

Article Snippet: Antibodies were as follows: phosphorylated ErbB-3 (Tyr1289), phosphorylated Akt (Ser473), Akt, PLCg1, phosphorylated MET (Tyr1234 1235) from Cell Signaling (Boston, MA, USA); C-17 ErbB-3 and MET from Santa Cruz Biotechnology (Santa Cruz, CA, USA); Actin from Sigma-Aldrich (St Louis, MO, USA); anti-ErbB-2 antibody 300G9 was obtained as described in Digiesi et al. (1992) rhodamine[125I]-labeled phalloidin from Sigma; Draq5 from Alexis; Yo-Pro dye from Invitrogen (Carlsbad, CA, USA).

Techniques: Activation Assay, Infection, Lysis, Incubation, Expressing, Control

Journal: Oncogene

Article Title: An ErbB-3 antibody, MP-RM-1, inhibits tumor growth by blocking ligand-dependent and independent activation of ErbB-3/Akt signaling.

doi: 10.1038/onc.2011.322

Figure Lengend Snippet: Figure 5 MP-RM-1inhibits ligand-indipendent activation of ErbB-3. (a) pSuper 4Mut and shErbB-3 infected MKN-45 cancer cells were grown in serum-free DMEM for 24 h and then lysed for immunoblot analysis. (b) MKN-45 cells were grown in serum-free DMEM for 24 h and then exposed to MP-RM-1 for the indicated time. Lysates were analyzed by immunoblot for phosphorylated ErbB-3 and phosphorylated Akt. The same filter was reprobed with the indicated antibodies for a loading control. (c) MKN-45 cells were grown in serum free DMEM for 24 h and then incubated for 2 h with trastuzumab (10 mg/ml), MP-RM-1 (10 mg/ml) or increasing amount of MET inhibitor SU11274. Lysates were analyzed by immunoblot with the indicated antibodies. (d) MKN-45 cells were grown in serum-free DMEM for 24 h and then incubated with 10 mg/ml of MP-RM-1 for 15 and 120 min. Lysates were analysed by immunoblot with the indicated antibodies (lower panel) or immunoprecipitated with an anti-phosphorylated MET antibody and then probed with an anti-ErbB-3 antibody (upper panel). (e) MKN-45 cells were grown in serum free DMEM for 24 h and then incubated with 1 mg/ml of SU1174 alone or together with 10 mg/ml of MP-RM-1. After 2 h, NRG-1b (10 ng/ml) was added, as indicated.

Article Snippet: Antibodies were as follows: phosphorylated ErbB-3 (Tyr1289), phosphorylated Akt (Ser473), Akt, PLCg1, phosphorylated MET (Tyr1234 1235) from Cell Signaling (Boston, MA, USA); C-17 ErbB-3 and MET from Santa Cruz Biotechnology (Santa Cruz, CA, USA); Actin from Sigma-Aldrich (St Louis, MO, USA); anti-ErbB-2 antibody 300G9 was obtained as described in Digiesi et al. (1992) rhodamine[125I]-labeled phalloidin from Sigma; Draq5 from Alexis; Yo-Pro dye from Invitrogen (Carlsbad, CA, USA).

Techniques: Activation Assay, Infection, Western Blot, Control, Incubation, Immunoprecipitation

Journal: Biochemical Journal

Article Title: Autoregulation of the MET receptor tyrosine kinase by its intracellular juxtamembrane domain

doi: 10.1042/BCJ20253378

Figure Lengend Snippet: ( A ) Linear domain schematic of the MET intracellular fragment with key regions and amino acid numbers annotated. Key regulatory phospho-sites are indicated for reference: S985, which is phosphorylated by PKC, and Y1003, which recruits c-Cbl when phosphorylated. TMD stands for the transmembrane domain. ( B ) Conservation of the MET JM sequence was assessed using the Aminode algorithm. Values on Y-axis represent the relative substitution score for each amino acid number, with higher numbers indicating less sequence conservation. Yellow bars mark evolutionary conserved regions (ECRs) within the JM. ( C ) Disorder prediction for the MET JM using the IUPred2A algorithm. Dotted line indicates the order-disorder threshold; higher values reflect greater disorder. N-terminal region of the kinase domain fold is included for comparison. ( D ) AlphaFold3 model of the MET JM domain, colored by pLDDT (predicted local distance difference test) confidence scores. The location of the predicted N-terminal JM helix, the helix αJM, and the boundary residues of the N- and C-terminal JM residues are annotated. ( E ) JM domain sequence alignment of selected MET orthologs is shown with subdomain regions annotated as in ( A ). Known phosphorylation sites are marked in red or orange. Dotted box marks the predicted N-terminal JM helix. Relative secondary structure predictions (marked red for α helix and green for β sheet) and the corresponding confidence scores are shown below the sequence alignment.

Article Snippet: Phosphorylation at MET Y1003 (#3135, CST) was also measured to confirm complete dephosphorylation of the recombinant MET protein (Supp ).

Techniques: Sequencing, Comparison, Phospho-proteomics

Journal: Clinical Cancer Research

Article Title: Nicotine Reduces Survival via Augmentation of Paracrine HGF–MET Signaling in the Pancreatic Cancer Microenvironment

doi: 10.1158/1078-0432.ccr-15-1256

Figure Lengend Snippet: Figure 1. A, continued smoking reduces overall survival in patients with resected pancreatic cancer. Patients with pancreatic adenocarcinoma were followed postoperatively after pancreatic resection and a multivariate Cox proportional hazards model was used to evaluate the effects of continued tobacco abuse on overall survival. Continued smoking correlated with reduced survival in pancreatic cancer (HR 1.93; P ¼ 0.040). P < 0.05 was considered statistically significant. B, TAS induces gemcitabine chemoresistance and Id1 expression in pancreatic cancer cells. TAS cells were cultured from human pancreatic tumors using the outgrowth method. Pancreatic cancer cells were cocultured with or without TAS in the presence or absence of gemcitabine at concentrations corresponding to LD50 values for each pancreatic cancer cell line. C, TAS coculture induces Id1 expression in pancreatic cancer cells. b-Actin was used as a loading control. D–F, TAS cells secrete HGF while pancreatic cancer cells express c-Met and Id1. D, qRT- pancreatic cancer R (left) and ELISA (right) were performed for HGF expression and secretion in TAS and pancreatic cancer cells. Data, mean SD of three independent experiments (, P < 0.01 TAS vs. pancreatic cancer cell lines). E, protein lysates from TAS and pancreatic cancer cell lines indicated were harvested and probed for Id1 (right) and c-Met (left). b-Actin was used as a loading control. F, immunofluorescent stains viewed at 20 magnification were performed on cells in culture of the indicated cell type. c-Met is depicted here in red, while phalloidin staining appears in green. Phalloidin stains are displayed to indicate the characteristic stress fiber organization of TAS cells. Goat anti-mouse AF647-stained controls are displayed in the far right column. Scale bar, 10 mm.

Article Snippet: Phospho-Met was determined using the PathScan Phospho-Met (panTyr) Sandwich ELISA (Cell Signaling Technology) according to the manufacturer's instructions. siRNA knockdown/pharmacologic inhibition of c-Met expression/activity Pancreatic cancer cells were transfected with control (100 pmol/L), c-Met (100pmol/L), or c-Src (100pmol/L) siRNA (Santa Cruz Biotechnology) using Oligofectamine reagent (Invitrogen).

Techniques: Expressing, Cell Culture, Control, Enzyme-linked Immunosorbent Assay, Staining

Journal: Clinical Cancer Research

Article Title: Nicotine Reduces Survival via Augmentation of Paracrine HGF–MET Signaling in the Pancreatic Cancer Microenvironment

doi: 10.1158/1078-0432.ccr-15-1256

Figure Lengend Snippet: Figure 2. Nicotine augments expression of HGF and c-Met in TAS and pancreatic cancer cells, respectively. A, TAS were stimulated with nicotine and RNA, supernatants or protein lysates were harvested for ELISA (left) and qRT-PCR (right) for analysis of HGF expression. B, pancreatic cancer cells were stimulated with nicotine and Western blot analysis of c-Met induction was performed. b-Actin was used as a loading control. C, c-Met activity was evaluated in pancreatic cancer cells alone and direct TAS/pancreatic cancer cell coculture at 48 hours with and without nicotine stimulation (1 mmol/L) and crizotinib treatment (200 nmol/L). Phospho-Met staining (top, green) was superimposed upon phase-contrast images with DAPI stains shown separately (bottom, blue). D, qRT-PCR was also utilized to evaluate the expression of nicotinic AchR subunits within the TAS in the absence of nicotine exposure. Data, mean SD of three independent experiments.

Article Snippet: Phospho-Met was determined using the PathScan Phospho-Met (panTyr) Sandwich ELISA (Cell Signaling Technology) according to the manufacturer's instructions. siRNA knockdown/pharmacologic inhibition of c-Met expression/activity Pancreatic cancer cells were transfected with control (100 pmol/L), c-Met (100pmol/L), or c-Src (100pmol/L) siRNA (Santa Cruz Biotechnology) using Oligofectamine reagent (Invitrogen).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Western Blot, Control, Activity Assay, Staining

Journal: Clinical Cancer Research

Article Title: Nicotine Reduces Survival via Augmentation of Paracrine HGF–MET Signaling in the Pancreatic Cancer Microenvironment

doi: 10.1158/1078-0432.ccr-15-1256

Figure Lengend Snippet: Figure 4. Nicotine augments tumor growth and metastasis in a patient-derived pancreatic adenocarcinoma xenograft model. A, primary human pancreatic cancer specimens and normal pancreas controls were subjected to IHC analysis of a5 and a7 nicotinic AchR subunit staining. Images are viewed at 10 magnification. B, animals with palpable patient-derived tumors were administered physiologic doses of nicotine or control. Tumor dimensions were measured with calipers 3 times per week, and volumes were calculated over 45 days. Data, mean SEM (, P < 0.05 vs. control). C–E, xenograft lysates from nicotine-treated and control animals were subjected to qRT-PCR for HGF expression (C), ELISA (D), and Western blot analysis (E) for c-Met activation. F, two representative lungs from nicotine-treated and control animals were subjected to H&E analysis, viewed at 20 magnification.

Article Snippet: Phospho-Met was determined using the PathScan Phospho-Met (panTyr) Sandwich ELISA (Cell Signaling Technology) according to the manufacturer's instructions. siRNA knockdown/pharmacologic inhibition of c-Met expression/activity Pancreatic cancer cells were transfected with control (100 pmol/L), c-Met (100pmol/L), or c-Src (100pmol/L) siRNA (Santa Cruz Biotechnology) using Oligofectamine reagent (Invitrogen).

Techniques: Derivative Assay, Staining, Control, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Activation Assay

Journal: Clinical Cancer Research

Article Title: Nicotine Reduces Survival via Augmentation of Paracrine HGF–MET Signaling in the Pancreatic Cancer Microenvironment

doi: 10.1158/1078-0432.ccr-15-1256

Figure Lengend Snippet: Figure 5. Activated c-Met in pancreatic cancer specimens correlates with reduced postoperative survival. A, intratumoral c-Met activation was measured using a phospho-Met ELISA on tumor lysates, and representative samples were verified using IHC (insets). Kaplan– Meier survival curves were generated through stratification of expression or not of phospho-Met within the primary tumor of 26 patients with pancreatic adenocarcinoma for whom clinical follow-up was obtained. Significance was determined using log-rank analysis. B, a summary model is depicted by which nicotine stimulates HGF secretion in TAS while directly inducing c-Met expression in pancreatic cancer cells, thereby augmenting c-Met activation in a paracrine manner.

Article Snippet: Phospho-Met was determined using the PathScan Phospho-Met (panTyr) Sandwich ELISA (Cell Signaling Technology) according to the manufacturer's instructions. siRNA knockdown/pharmacologic inhibition of c-Met expression/activity Pancreatic cancer cells were transfected with control (100 pmol/L), c-Met (100pmol/L), or c-Src (100pmol/L) siRNA (Santa Cruz Biotechnology) using Oligofectamine reagent (Invitrogen).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Generated, Expressing

Journal: International Journal of Oncology

Article Title: Use of IHC and newly designed matriptase inhibitors to elucidate the role of matriptase in pancreatic ductal adenocarcinoma

doi: 10.3892/ijo_00000346

Figure Lengend Snippet: Figure 5. Effects of various matriptase inhibitors on the HGFR/cMet tyrosine phosphorylation in the human pancreatic adenocarcinoma cell line AsPC-1. The human pancreatic cell line AsPC-1 was stimulated by administration of 100 ng/ml recombinant pro-HGF/SF in the absence or presence of 0.1 μM of various matriptase-selective inhibitors. After 1 h, HGFR/cMet phosphorylation was determined with a HGFR/cMet phosphorylation ELISA. Samples without pro-HGF/SF administration measured the basal level of HGFR/cMet phosphorylation and served as negative control. Basal levels were substracted from all measurements. Samples with pro-HGF/SF stimulation without inhibitor were the positive control and defined as 100% HGFR/cMet phosphorylation. The graph shows mean values of three independent experiments performed as triplicates with standard deviations. Significant results (p<0.05) are marked with an asterix.

Article Snippet: Phosphorylation of human HGFR/cMet was determined using the human phospho-HGFR/cMet ELISA (R&D Systems Inc., USA) according to the manufacturer's protocol.

Techniques: Phospho-proteomics, Recombinant, Enzyme-linked Immunosorbent Assay, Negative Control, Positive Control