Journal: Molecular & Cellular Proteomics : MCP

Article Title: A System-wide Approach to Monitor Responses to Synergistic BRAF and EGFR Inhibition in Colorectal Cancer Cells *

doi: 10.1074/mcp.RA117.000486

Figure Lengend Snippet: Feedback mechanisms aimed at restoring MAPK signaling activity. A, Log2-fold-changes of mRNA expression plotted against mRNA half-life of regulators of major signaling pathways at 2 h. Only short-lived negative regulators of MAPK signaling respond to growth factor stimulation or BRAF inhibition. In all conditions, DUSP1, DUSP8 and DUSP10 are up-regulated in response to serum stimulation at T = 0 h. DUSP4, DUSP6 and SPRY1 are downregulated only in BRAFi treated samples, in response to BRAF inhibition. B, Scaled mRNA expression levels of a cluster of RTKs. Of the 35 RTKs, 18 are up-regulated (INSRR, ROS1, STYK1, ERBB3, FGFR2, PTK7, EPHA4, TEK, EPHB2, ERBB2, LMTK3, MERTK, INSR, EPHA10, NTRK1, DDR1). C, mRNA expression of ERBB2. D, mRNA expression of ERBB3. E, mRNA expressions of ERBB2 and ERBB3 in SNU-C5, VACO432, and KM-20 BRAF(V600E) CRC cell lines. F, Phosphorylation expression of the negative regulator ERRFI1 is downregulated at residue S251 in BRAFi samples.

Article Snippet: Antibodies against HSP-90 (H-114), PTPN11 (SH-PTP2 C-18), ERK1 (C-16), ERK2 (C-14), and p-ERK1/2 (E4) were purchased from Santa Cruz. p-EGFR (Y1068, ab5644), p-SHP2 (Y542, ab62322) were purchased from Abcam. p-ERBB3 (Y1197, #4561), p-IGF1R (Y1135/1136, #3024), IGF1R (#3027), p90RSK (#8408), AKT 1/2 (#2920), p-AKT (S473, #4060) were purchased from Cell Signaling Technology.

Techniques: Activity Assay, Expressing, Inhibition

Journal: Molecular & Cellular Proteomics : MCP

Article Title: A System-wide Approach to Monitor Responses to Synergistic BRAF and EGFR Inhibition in Colorectal Cancer Cells *

doi: 10.1074/mcp.RA117.000486

Figure Lengend Snippet: Assessment of WiDr CRC cell growth by combination treatments of BRAFi and ERBB or metabolic inhibitors. A, Comparison of mono- and double therapy on WiDr CRC cells growth. All three graphs show inhibition of either EGFR, EGFR/ERBB2 or EGFR/ERBB2/ERBB3 is ineffective as a monotherapy. Moreover, concomitant inhibition of ERBB2 and ERBB3 does not provide further benefit to the synergistic effect of BRAF(V600E) and EGFR inhibitors. B, WiDr cell confluence is measured comparing double and triple treatments. The addition of etomoxir or DCA as third metabolic inhibitors after 96 h does not show additional benefit to the BRAFi+EGFRi treatment.

Article Snippet: Antibodies against HSP-90 (H-114), PTPN11 (SH-PTP2 C-18), ERK1 (C-16), ERK2 (C-14), and p-ERK1/2 (E4) were purchased from Santa Cruz. p-EGFR (Y1068, ab5644), p-SHP2 (Y542, ab62322) were purchased from Abcam. p-ERBB3 (Y1197, #4561), p-IGF1R (Y1135/1136, #3024), IGF1R (#3027), p90RSK (#8408), AKT 1/2 (#2920), p-AKT (S473, #4060) were purchased from Cell Signaling Technology.

Techniques: Inhibition

Journal: Journal of Thoracic Disease

Article Title: A subset of esophageal squamous cell carcinoma patient-derived xenografts respond to cetuximab, which is predicted by high EGFR expression and amplification

doi: 10.21037/jtd.2018.09.18

Figure Lengend Snippet: Correlation between protein level and tumor response

Article Snippet: For all of the samples, the relative EGFR protein expression level was determined by immunohistochemistry (IHC), anti-human antibodies including EGFR (CST, Beverly, MA, USA), P-EGFR (Abcam, Cambridge, MA, USA), HER3 (CST, Beverly, MA, USA), P-HER3 (CST, Beverly, MA, USA), MET (CST, Beverly, MA, USA), P-MET (CST, Beverly, MA, USA), Akt (CST, Beverly, MA, USA), P-Akt (CST, Beverly, MA, USA), ERK (CST, Beverly, MA, USA), P-ERK (CST, Beverly, MA, USA) were applied to stain the positive sections.

Techniques: Expressing

Journal: Scientific Reports

Article Title: Neuregulin 1 improves complex 2-mediated mitochondrial respiration in skeletal muscle of healthy and diabetic mice

doi: 10.1038/s41598-017-02029-z

Figure Lengend Snippet: NRG1 treatment increases ERBB4 activation in gastrocnemius muscle. C57BL/6JRJ (C57) control and db/db (Db) male mice were treated with vehicle (VHL; 0.9% NaCl solution; n = 8/each condition), or with NRG1 (50 μg . kg −1 ; n = 8/each condition), three days per week for eight weeks. Western blot analysis ( A ) cropped images) was used to quantify in gastrocnemius muscle samples the abundance of full length (115 kDa) ( B ) and cleaved (42 kDa) ( C ) NRG1 and the NRG1 cleavage index (the ratio between cleaved and full length NRG1) ( D ) as well as the abundance ( E ) and phosphorylation ratios ( F ) of ERBB2, ERBB3 and ERBB4. Results are the mean ± SEM (n = 8 per group) relative to the level in untreated healthy mice (C57-VHL, white bars). Diabetes (healthy vs db/db mice) and NRG1 (saline vs NRG1) effects were investigated with a 2 × 2 ANOVA. When a significant interaction was found, the Tuckey’s test was used for post-hoc multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, NS: not significant.

Article Snippet: The anti-porin (1/1000), -AKT (1/1000), -p-AKT Ser473 (1/1000), -p-AKT Thr308 (1/1000), -ERK (1/1000), -p-ERK, (1/1000), -AMPK (1/1000), -p-AMPK (1/1000), ACC (1/1000), -p-ACC (1/1000), -ACL (1/1000), -p-ACL (1/1000), -GLUT4 (1/1000), -ERBB3 (1/200) and -p-ERBB3 (1/200) antibodies were purchased from Cell Signaling (Beverly, MA, USA).

Techniques: Activation Assay, Western Blot

Journal: Scientific Reports

Article Title: Neuregulin 1 improves complex 2-mediated mitochondrial respiration in skeletal muscle of healthy and diabetic mice

doi: 10.1038/s41598-017-02029-z

Figure Lengend Snippet: Summary of the main effects of diabetes and NRG1 treatment in gastrocnemius muscle. The abundance of ERBB receptors is increased in diabetic db/db mice. This is associated with increased phosphorylation ratio of ERBB2 and decreased phosphorylation ratios of ERBB3 and ERBB4. Similarly, phosphorylation (activation) of the metabolic regulators AMPK, ACC and ACL is reduced in diabetic mice as well as Pparb and Tfam mRNA expression level. However, mitochondrial respiration in permeabilised fibres is similar in diabetic and control healthy mice. Chronic NRG1 treatment increases ERBB4 phosphorylation and tends to decrease ERBB3 phosphorylation. Among the regulators of the mitochondrial biogenesis pathway, only Pparb mRNA expression is slightly increased by NRG1. However, in NRG1-treated mice, complex 2-mediated mitochondrial respiration and complex 2 subunit abundance are increased. Effect of diabetes: red arrows. Effect of NRG1: green arrows. Figure was produced using Servier Medical Art image bank ( http://www.servier.com/Powerpoint-image-bank ), which is licensed under a Creative Commons Attribution 3.0 Unported License (http://creativecommons.org/licenses/by/3.0/).

Article Snippet: The anti-porin (1/1000), -AKT (1/1000), -p-AKT Ser473 (1/1000), -p-AKT Thr308 (1/1000), -ERK (1/1000), -p-ERK, (1/1000), -AMPK (1/1000), -p-AMPK (1/1000), ACC (1/1000), -p-ACC (1/1000), -ACL (1/1000), -p-ACL (1/1000), -GLUT4 (1/1000), -ERBB3 (1/200) and -p-ERBB3 (1/200) antibodies were purchased from Cell Signaling (Beverly, MA, USA).

Techniques: Activation Assay, Expressing, Produced

Journal: International Journal of Biological Sciences

Article Title: ESM1 facilitates the EGFR/HER3-triggered epithelial-to-mesenchymal transition and progression of gastric cancer via modulating interplay between Akt and angiopoietin-2 signaling

doi: 10.7150/ijbs.100276

Figure Lengend Snippet: Targeting the ESM1-epidermal growth factor receptor (EGFR) interaction by therapeutic peptides suppresses EGFR/human EGFR3 (HER3)-driven epithelial-mesenchymal transition (EMT) and cell mobility in gastric cancer (GC) cells. ( A ) Wild-type ESM1 (WT-ESM1) was introduced into AGS, Kato-III, and NCI-N87 cells, and then cells were subjected to a Western blot assay to evaluate the phosphorylation status of HER3. ( B, C ) Co-immunoprecipitation assays were conducted to assess the interaction between EGFR, ESM1, and HER3 in AGS/WT-ESM cells ( B ). Comparing the association of EGFR and HER3 in the AGS cells transfected with either WT-ESM1 or a control vector (Ctrl) ( C ). Subsequently, a Western blot analysis was performed to examine the formation of this complex. ( D ) Schematic diagram of two synthetic peptides including peptide 1 (1-27 aas) and peptide 2 (26-46 aas) of the ESM1 protein. ( E, F ) AGS cells were infected with a lentivirus carrying a control vector or WT-ESM1 following treatment of cells with synthetic peptides (1 µM) as indicated. The phosphorylation status of HER3, EGFR and Akt, and expressions of angiopoietin-2, Snail, and Slug were all detected by Western blotting ( E ). The migratory ability of cells was determined by a transwell migration assay ( F ). Differences are presented as the mean ± SD. *** p < 0.001, compared to the WT-ESM1-overexpressing only group.

Article Snippet: The following primary antibodies were used: endocan (LIA-1001; Lunginnov, Lille, France); phosphorylated (p)-EGFR (#3777), p-Akt (#9271), p-ERK (#4370), p-STAT3 (#9145), EGFR (#2239), Akt (#9272), STAT3 (#9139), ERK (#4695), E-cadherin (#3195), vimentin (#5741), Snail (#3879), Slug (#9585), p-HER3 (#2842), and HER3 (#12708) antibodies all obtained from Cell Signaling Technology (Danvers, MA, USA); angiopoietin-2 (bs-0677R-TR) and p-HER3 (bs-3491R) antibodies purchased from Bioss (Woburn, MA, USA); α-tubulin (#66031-1-Ig) antibodies respectively obtained from Proteintech (Rosemont, IL, USA).

Techniques: Western Blot, Phospho-proteomics, Immunoprecipitation, Transfection, Control, Plasmid Preparation, Infection, Transwell Migration Assay

Journal: International Journal of Biological Sciences

Article Title: ESM1 facilitates the EGFR/HER3-triggered epithelial-to-mesenchymal transition and progression of gastric cancer via modulating interplay between Akt and angiopoietin-2 signaling

doi: 10.7150/ijbs.100276

Figure Lengend Snippet: A working model shows the molecular mechanism underlying the ability of ESM1 to promote progression of gastric cancer (GC) cells. The oncogenic role of ESM1 was attributed to triggering the epithelial-to-mesenchymal transition (EMT) by activating epidermal growth factor receptor (EGFR)/human EGFR3 (HER3) and their downstream signal, Akt. Angiopoietin-2 was highly correlated with ESM1 and interplayed with Akt to promote EMT progression. Blocking the interaction of ESM1 and the EGFR by synthetic ESM1 peptides attenuated the EGFR/HER3 activation-driven EMT, cell motility, and proliferation. This schematic representation was created using BioRender software.

Article Snippet: The following primary antibodies were used: endocan (LIA-1001; Lunginnov, Lille, France); phosphorylated (p)-EGFR (#3777), p-Akt (#9271), p-ERK (#4370), p-STAT3 (#9145), EGFR (#2239), Akt (#9272), STAT3 (#9139), ERK (#4695), E-cadherin (#3195), vimentin (#5741), Snail (#3879), Slug (#9585), p-HER3 (#2842), and HER3 (#12708) antibodies all obtained from Cell Signaling Technology (Danvers, MA, USA); angiopoietin-2 (bs-0677R-TR) and p-HER3 (bs-3491R) antibodies purchased from Bioss (Woburn, MA, USA); α-tubulin (#66031-1-Ig) antibodies respectively obtained from Proteintech (Rosemont, IL, USA).

Techniques: Blocking Assay, Activation Assay, Software