Journal: Clinical Cancer Research

Article Title: Optimized EGFR Blockade Strategies in EGFR Addicted Gastroesophageal Adenocarcinomas

doi: 10.1158/1078-0432.ccr-20-0121

Figure Lengend Snippet: Figure 1. EGFR CNG. The graphs illustrate the percentage of tumors displaying EGFR CNG in four different cohorts. Real-time PCR analysis of IRCC gastric/gastroesophageal junction (GEJ) adenocarcinomas displaying EGFR gain (4–8 copies or >8 copies)or heterogeneity (significantly different EGFR CNG in diverse analyzed samples from the same tumor, with one tumor sample displaying >8 copies and one tumor sample having≤8 copies).CGP of FMI gastricand esophageal/gastroesophageal junction cases, FISH analysis of COG esophageal/gastroesophageal junction cases, and SISH analysis of INT gastric/gastroesophageal junction adenocarcinomas.

Article Snippet: Primary antibodies, anti-EGFR (1005: sc-03) and anti-Actin, were from Santa Cruz Biotechnology, and antibodies against phosphorylated EGFR (Tyr 845), ERK (Thr202/Tyr204), phosphorylated AKT (Ser473) (Clone D9E), total AKT, and ERK were from Cell Signaling Technology.

Techniques: Real-time Polymerase Chain Reaction

Journal: Clinical Cancer Research

Article Title: Optimized EGFR Blockade Strategies in EGFR Addicted Gastroesophageal Adenocarcinomas

doi: 10.1158/1078-0432.ccr-20-0121

Figure Lengend Snippet: Figure 2. Survival analysis of patients with EGFR CNG. A, The graphs show the cumulative survival (cum survival) of patients of the COG (left) and INT (right) cohorts related to EGFR CNG. B, The graphs show the OS (left) and the disease-free survival (right) of patients of the gastroesophageal TCGA dataset, related to EGFR CNG.

Article Snippet: Primary antibodies, anti-EGFR (1005: sc-03) and anti-Actin, were from Santa Cruz Biotechnology, and antibodies against phosphorylated EGFR (Tyr 845), ERK (Thr202/Tyr204), phosphorylated AKT (Ser473) (Clone D9E), total AKT, and ERK were from Cell Signaling Technology.

Techniques:

Journal: Clinical Cancer Research

Article Title: Optimized EGFR Blockade Strategies in EGFR Addicted Gastroesophageal Adenocarcinomas

doi: 10.1158/1078-0432.ccr-20-0121

Figure Lengend Snippet: Figure 3. Clinical history of patients treated with EGFR-targeted drugs. Summarized clinical course of INT patients with EGFR CNG. Red-lined boxes indicate periods of administration of the indicated therapeutic agents. Blue vertical lines indicate timing of tumor specimen acquisition from surgical procedures or biopsies, as well as dates of tumor assessment by CT scan. PD and SD according to RECIST 1.1. 5FU, 5-fluorouracil; CCDP, cisplatin, vinorelbine, ifosfamide, and epirubicin; EOX, epirubicin, oxaliplatin, and capecitabine; FOLFIRI, folinic acid, 5-fluorouracil, and irinotecan; OGD, esophago-gastro-duodenoscopy; TCF, docetaxel, carboplatin, and 5-fluorouracil; XELOX, capecitabine and oxaliplatin.

Article Snippet: Primary antibodies, anti-EGFR (1005: sc-03) and anti-Actin, were from Santa Cruz Biotechnology, and antibodies against phosphorylated EGFR (Tyr 845), ERK (Thr202/Tyr204), phosphorylated AKT (Ser473) (Clone D9E), total AKT, and ERK were from Cell Signaling Technology.

Techniques: Computed Tomography

Journal: Clinical Cancer Research

Article Title: Optimized EGFR Blockade Strategies in EGFR Addicted Gastroesophageal Adenocarcinomas

doi: 10.1158/1078-0432.ccr-20-0121

Figure Lengend Snippet: Figure 4. RTK/KRAS pathway activation in EGFR-amplified cases. A, The scatter plot shows a significant inverse correlation between the extent of RTKs coamplification (HER2, HER3, HER4, MET, FGFR1, FGFR2, FGFR3, FGFR4, IGF1R, PDGFR2, VEGFR1, VEGR2, and VEGFR3 ≥4 gene copies) and OS in 12 EGFR FISH–positive gastroesophageal adenocar- cinomas treated with gefitinib in the COG trial. Red dots indicate cases with high polysomy and blue dots represent cases with EGFR amplification. B, The graph shows the cooccurrence of EGFR amplification and genomic events affecting the RTK/KRAS pathway in EGFR-amplified gastro- esophageal adenocarcinoma tumors in the FMI dataset.

Article Snippet: Primary antibodies, anti-EGFR (1005: sc-03) and anti-Actin, were from Santa Cruz Biotechnology, and antibodies against phosphorylated EGFR (Tyr 845), ERK (Thr202/Tyr204), phosphorylated AKT (Ser473) (Clone D9E), total AKT, and ERK were from Cell Signaling Technology.

Techniques: Activation Assay

Journal: Clinical Cancer Research

Article Title: Optimized EGFR Blockade Strategies in EGFR Addicted Gastroesophageal Adenocarcinomas

doi: 10.1158/1078-0432.ccr-20-0121

Figure Lengend Snippet: Figure 5. Dual EGFR blockade is the most effective treatment in EGFR-amplified PDXs. Tumor growth curves in mice cohorts derived from GTR0060 (A), GTR0110 (C), and GTR0511 (D) patients treated with the EGFR inhibitors, cetuximab (CETUX), erlotinib (ERL), and lapatinib (LAP), alone or in combination, as indicated. The red lines indicate the day when treatment was started. The response in mice has been evaluated using RECIST 1.1–like criteria, that is, PD: ≥35% increase from baseline; PR: ≥ 50% reduction from baseline; and SD: intermediate variations from baseline. B, Spaghetti plot illustrating drug response in the xenotrial performed on the cohort of mice derived from GTR0060 PDX. Individual lines represent, for each mouse, the percentage variation in tumor burden, from start of treatment (day 0). Blue lines, cetuximab þ lapatinib–treated mice and red lines, cetuximab þ erlotinib–treated mice. Dashed line indicates treatment stop. E, Western blot analysis of the activation state of EGFR and its downstream targets (AKT, MAPK, and S6) in GTR0060 tumor–derived cells treated with the indicated drugs/drug combinations. Actin was used as loading control. Statistical significance is indicated (, P <0.01; , P < 0.001).

Article Snippet: Primary antibodies, anti-EGFR (1005: sc-03) and anti-Actin, were from Santa Cruz Biotechnology, and antibodies against phosphorylated EGFR (Tyr 845), ERK (Thr202/Tyr204), phosphorylated AKT (Ser473) (Clone D9E), total AKT, and ERK were from Cell Signaling Technology.

Techniques: Derivative Assay, Western Blot, Activation Assay, Control

Journal: Clinical Cancer Research

Article Title: Optimized EGFR Blockade Strategies in EGFR Addicted Gastroesophageal Adenocarcinomas

doi: 10.1158/1078-0432.ccr-20-0121

Figure Lengend Snippet: Figure 6. TSC2 inactivation is a mechanism of resistance to EGFR-targeted therapies. A, Tumor growth curves in the mice cohorts derived from GTR0078 and treated with the EGFR inhibitors, cetuximab (CETUX), erlotinib (ERL), and lapatinib (LAP), alone or in combination, as indicated. The red line indicates the day when treatment was started. B, The table shows the two TSC2 mutations identified in GTR0078 PDX. C, Tumor growth curves in the mice cohorts derived from GTR0078 and treated with erlotinib or the combination erlotinib þ everolimus (ERLþ EVEROL). The red line indicates the day when treatment was started. D, Western blot analysis of the activation state of EGFR and its downstream targets (AKT, MAPK, and S6) in GTR0078 tumor–derived cells treated with the indicated drugs/drug combinations. Actin was used as loading control. Statistical significance is indicated (, P < 0.001).

Article Snippet: Primary antibodies, anti-EGFR (1005: sc-03) and anti-Actin, were from Santa Cruz Biotechnology, and antibodies against phosphorylated EGFR (Tyr 845), ERK (Thr202/Tyr204), phosphorylated AKT (Ser473) (Clone D9E), total AKT, and ERK were from Cell Signaling Technology.

Techniques: Derivative Assay, Western Blot, Activation Assay, Control

Journal: Oncotarget

Article Title: CHIP is a novel tumor suppressor in pancreatic cancer and inhibits tumor growth through targeting EGFR

doi:

Figure Lengend Snippet: (A) The levels of proteins that were degraded by CHIP in other types of cells were determined by immunoblotting. (B) CHIP interacts with EGFR in an endogenous or exogenous way. (i)BxPC-3 cells were pretreated with MG132 (5 μM) for 6 h, and endogenous CHIP-EGFR interaction was examined by immunoprecipitation with CHIP or EGFR antibody. (ii)BxPC-3 cells were transfected with His-EGFR and Flag-CHIP, MG132 were used after 48 h of transfection, and two exogenous protein interactions were determined by His or Flag antibody. (C) CHIP promotes EGFR degradation in Panc-1 and BxPC-3 cells. The levels of EGFR were determined after cells were infected with scrambled controls, CHIP amiRNA or CHIP overexpression(CHIP OE ) lentiviruses. (D) CHIP enhances EGFR degradation in concentration and ubiquitination/proteasome dependent ways. BxPC-3 cells were co-transfected with HA-ubiquitin, His-EGFR and Flag-CHIP plasmids (0, 1 μg, 2 μg per well in a six-well plate). The levels of His-EGFR were determined by immunoblotting, using antibody against His-tag. Wells in another six-well plate were treated with geldanamycin (GA,1 μM) or MG132 (5 μM) for 6 h after 48 h co-transfection. The levels of His-EGFR and HA-ubiquitin were detected with anti-His or anti-HA antibody. (E) CHIP enhances EGFR degradation in a time-dependent manner. BxPC-3 cells were transfected with control,CHIP OE or CHIP amiRNA for 0, 4, 8, 12, or 24 h. The levels of EGFR were determined by immunoblotting.

Article Snippet: Antibodies and their sources were as follows: anti-CHIP antibody (Santa Cruz,California,USA); anti-EGFR antibody and anti-phosphorylated EGFR (Tyr845, Tyr1068, Tyr1173) antibody (Cell Signaling,Massachusettes,USA); anti-AKT antibody and anti-phosphorylated AKT (Ser473) antibody (Cell Signaling); anti-mTOR antibody and anti-phosphorylated mTOR (Ser2448) antibody (Cell Signaling); anti BAD antibody and anti-phosphorylated BAD (Ser136) antibody (Cell Signaling); anti-p21 antibody (Cell Signaling); anti-Src antibody and anti-phosphorylated Src (Tyr416) antibody (Cell Signaling); anti-FAK antibody and anti-phosphorylated FAK (Tyr 925) antibody (Cell Signaling); anti-paxillin antibody and anti-phosphorylated paxillin (Tyr118) antibody (Cell Signaling); anti-Erk1/2 antibody and anti-phosphorylated Erk1/2 (Thr202/Tyr204) antibody (Cell Signaling); anti-His antibody (Santa Cruz); anti-Flag antibody (Santa Cruz); anti ki67 antibody (Abcam,Cambridge,UK); and anti-Cleaved Caspase-3 antibody (Cell Signaling).

Techniques: Western Blot, Immunoprecipitation, Transfection, Infection, Over Expression, Concentration Assay, Cotransfection

Journal: Oncotarget

Article Title: CHIP is a novel tumor suppressor in pancreatic cancer and inhibits tumor growth through targeting EGFR

doi:

Figure Lengend Snippet: (A) (i)Two main functional domains of CHIP are illustrated schematically. (ii) The U-box domain of CHIP is required for degradation of EGFR. BxPC-3 cells were co-transfected with HA-ubiquitin, His-EGFR and Flag tagged CHIP-full length (CHIP FL ) or its Flag tagged domains (CHIP ΔU-box for CHIP protein without a U-box domain, CHIP ΔTPR for CHIP protein without a TPR domain) for 48 h. Then, the cells were treated with or without MG132 (5 μM). EGFR and ubiquitin were detected by immunoblotting with anti-His or anti-HA antibody. Anti-Flag antibody was used to test the expression of CHIP FL or its domains. (iii) CHIP FL is required to interact with EGFR. BxPC-3 cells were co-transfected with His-EGFR and Flag-CHIP FL , Flag-CHIP ΔU-box or Flag-CHIP ΔTPR for 48 h; then, the cells were treated with MG132 (5 μM). CHIP or its domains were combined by anti-Flag antibody (IP) and immunoprecipitated by A/G agarose beads. Immunoblotting (IB) using anti-His antibody was performed to determine the exogenous expression of EGFR. (B) The downstream pathways of EGFR are regulated by CHIP in Panc-1 and BxPC-3 cells. The lysate of stable CHIP knockdown cells or CHIP OE cells were used to determine the expression of AKT/mTOR, Src/FAK/paxillin,MAPK pathways by different antibodies. In the figure, p- represents phosphorylated, and t- represents total. (C) CHIP down-regulates phosphorylation of Tyr845 and Tyr 1068 of EGFR in Panc-1 and BxPC-3 cells. (D) CHIP is co-localized with EGFR in Bxpc-3 cells and attenuates the expression of EGFR. BxPC-3 cells were transfected with vector or Flag-CHIP plasmid combined with His-EGFR plasmid. After 48 h, the cells were treated with or without EGF (50 ng/mL for 30 min) and then were stained with anti-His or anti-Flag antibody. The white arrows indicate that EGFR was present or absent due to CHIP under- or over-expression in the cytoplasm or nucleus.

Article Snippet: Antibodies and their sources were as follows: anti-CHIP antibody (Santa Cruz,California,USA); anti-EGFR antibody and anti-phosphorylated EGFR (Tyr845, Tyr1068, Tyr1173) antibody (Cell Signaling,Massachusettes,USA); anti-AKT antibody and anti-phosphorylated AKT (Ser473) antibody (Cell Signaling); anti-mTOR antibody and anti-phosphorylated mTOR (Ser2448) antibody (Cell Signaling); anti BAD antibody and anti-phosphorylated BAD (Ser136) antibody (Cell Signaling); anti-p21 antibody (Cell Signaling); anti-Src antibody and anti-phosphorylated Src (Tyr416) antibody (Cell Signaling); anti-FAK antibody and anti-phosphorylated FAK (Tyr 925) antibody (Cell Signaling); anti-paxillin antibody and anti-phosphorylated paxillin (Tyr118) antibody (Cell Signaling); anti-Erk1/2 antibody and anti-phosphorylated Erk1/2 (Thr202/Tyr204) antibody (Cell Signaling); anti-His antibody (Santa Cruz); anti-Flag antibody (Santa Cruz); anti ki67 antibody (Abcam,Cambridge,UK); and anti-Cleaved Caspase-3 antibody (Cell Signaling).

Techniques: Functional Assay, Transfection, Western Blot, Expressing, Immunoprecipitation, Plasmid Preparation, Staining, Over Expression

Journal: Oncotarget

Article Title: CHIP is a novel tumor suppressor in pancreatic cancer and inhibits tumor growth through targeting EGFR

doi:

Figure Lengend Snippet: (A) CHIP suppresses cell growth rate. CHIP amiRNA, CHIP OE and their corresponding control Panc-1 or BxPC-3 cells were grown in 96-well plates for 1, 2, 3, 4, 5 and 6 days. Cell survival was detected by CCK-8 analysis (mean±standard deviation; **P<.01). (B) CHIP suppresses anchorage-independent growth in Panc-1 and BxPC-3 cells. Stable CHIP knockdown or CHIP OE cells were plated in a 6-well plate that contained soft agar. After incubation for 21 days, colonies were photographed and counted under the microscope (mean±standard deviation; *P<.05, **P<.01). (C)The stable CHIP knockdown or CHIP OE cells and their control BxPC-3 cells were subcutaneously injected into nude mice. Thirty-seven days after the injections, the mice were sacrificed, and tumor tissues were collected. The left panel shows tumor growth curves in nude mice; the middle and right panel shows the size and weight of the tumors after 37 days (mean±standard deviation; *P<.05, **P<.01). (D) CHIP OE decreases, but knockdown CHIP enhances the expression of EGFR and Ki67. Sections of tumors from injected nude mice were stained with CHIP, EGFR and Ki67 antibodies by immunohistochemistry (magnification ×100). The right panel shows the percentage of strongly Ki67 stained tumor cells (mean±standard deviation; *P<.05).

Article Snippet: Antibodies and their sources were as follows: anti-CHIP antibody (Santa Cruz,California,USA); anti-EGFR antibody and anti-phosphorylated EGFR (Tyr845, Tyr1068, Tyr1173) antibody (Cell Signaling,Massachusettes,USA); anti-AKT antibody and anti-phosphorylated AKT (Ser473) antibody (Cell Signaling); anti-mTOR antibody and anti-phosphorylated mTOR (Ser2448) antibody (Cell Signaling); anti BAD antibody and anti-phosphorylated BAD (Ser136) antibody (Cell Signaling); anti-p21 antibody (Cell Signaling); anti-Src antibody and anti-phosphorylated Src (Tyr416) antibody (Cell Signaling); anti-FAK antibody and anti-phosphorylated FAK (Tyr 925) antibody (Cell Signaling); anti-paxillin antibody and anti-phosphorylated paxillin (Tyr118) antibody (Cell Signaling); anti-Erk1/2 antibody and anti-phosphorylated Erk1/2 (Thr202/Tyr204) antibody (Cell Signaling); anti-His antibody (Santa Cruz); anti-Flag antibody (Santa Cruz); anti ki67 antibody (Abcam,Cambridge,UK); and anti-Cleaved Caspase-3 antibody (Cell Signaling).

Techniques: CCK-8 Assay, Standard Deviation, Incubation, Microscopy, Injection, Expressing, Staining, Immunohistochemistry

Journal: Cancer Research

Article Title: Comparison of Kinome Profiles of Barrett's Esophagus with Normal Squamous Esophagus and Normal Gastric Cardia

doi: 10.1158/0008-5472.can-06-1370

Figure Lengend Snippet: Figure 4. Immunoblot analysis of expression levels of several phosphorylation sites of the EGFR (A) in Barrett’s esophagus, normal squamous esophagus, and gastric cardia. A, high expression levels of phosphorylated EGFR (P-EGF Receptor) in squamous epithelium, lower expression in Barrett’s esophagus, and lowest in gastric cardia. h-Actin was used as a control. B, quantification of immunoblot results of phosphorylated EGFR Tyr845, Tyr992, and Tyr1068 and phosphorylated HER2 (P-HER) Tyr1245 in Barrett’s esophagus, squamous esophagus, and normal gastric cardia of 20 patients shows that these are significantly higher expressed in the normal squamous esophagus biopsies. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Phosphorylation levels of EGFR were also significantly higher in Barrett’s esophagus compared with gastric cardia. The phosphorylation levels were determined by the ratio of signal intensity of the protein to that of the h-actin. Columns, mean; bars, SE.

Article Snippet: Antibodies used were phosphorylated epidermal growth factor (EGF) receptor (EGFR) Tyr845 (1:1,000; Cell Signaling), phosphorylated EGFR Tyr992 (Cell Signaling), phosphorylated EGFR Tyr1068 (Cell Signaling), phosphorylated HER2 Tyr1248 (Cell Signaling), phosphorylated Src Tyr416 (Cell Signaling), phosphorylated extracellular signal-regulated kinase (ERK) Tyr204 (Santa Cruz Biotechnology, Santa Cruz, CA), protein kinase C (PKC)-h1 (Santa Cruz Biotechnology), phosphorylated phosphoinositide-dependent kinase (PDK) Ser241 (Cell Signaling), and h-actin I-19 (Santa Cruz Biotechnology).

Techniques: Western Blot, Expressing, Phospho-proteomics, Control

Journal: Cancer Research

Article Title: Comparison of Kinome Profiles of Barrett's Esophagus with Normal Squamous Esophagus and Normal Gastric Cardia

doi: 10.1158/0008-5472.can-06-1370

Figure Lengend Snippet: Figure 4. Immunoblot analysis of expression levels of several phosphorylation sites of the EGFR (A) in Barrett’s esophagus, normal squamous esophagus, and gastric cardia. A, high expression levels of phosphorylated EGFR (P-EGF Receptor) in squamous epithelium, lower expression in Barrett’s esophagus, and lowest in gastric cardia. h-Actin was used as a control. B, quantification of immunoblot results of phosphorylated EGFR Tyr845, Tyr992, and Tyr1068 and phosphorylated HER2 (P-HER) Tyr1245 in Barrett’s esophagus, squamous esophagus, and normal gastric cardia of 20 patients shows that these are significantly higher expressed in the normal squamous esophagus biopsies. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Phosphorylation levels of EGFR were also significantly higher in Barrett’s esophagus compared with gastric cardia. The phosphorylation levels were determined by the ratio of signal intensity of the protein to that of the h-actin. Columns, mean; bars, SE.

Article Snippet: Antibodies used were phosphorylated epidermal growth factor (EGF) receptor (EGFR) Tyr845 (1:1,000; Cell Signaling), phosphorylated EGFR Tyr992 (Cell Signaling), phosphorylated EGFR Tyr1068 (Cell Signaling), phosphorylated HER2 Tyr1248 (Cell Signaling), phosphorylated Src Tyr416 (Cell Signaling), phosphorylated extracellular signal-regulated kinase (ERK) Tyr204 (Santa Cruz Biotechnology, Santa Cruz, CA), protein kinase C (PKC)-h1 (Santa Cruz Biotechnology), phosphorylated phosphoinositide-dependent kinase (PDK) Ser241 (Cell Signaling), and h-actin I-19 (Santa Cruz Biotechnology).

Techniques: Western Blot, Expressing, Phospho-proteomics, Control