Journal: Cancer research

Article Title: Regulation of ERBB2 Receptor by t-DARPP Mediates Trastuzumab Resistance in Human Esophageal Adenocarcinoma

doi: 10.1158/0008-5472.CAN-12-1119

Figure Lengend Snippet: A) Significant mRNA overexpression of t-DARPP and ERBB2 in adenocarcinomas of the esophagus and stomach (141 tumors and 51 normal tissue samples) (p<0.001). B) Spearman’s correlation coefficient and correlation test where the cutoff gene expression is ≥log(5,2)=2.32, show that t-DARPP and ERBB2 overexpression levels are significantly correlated in tumors (r=0.58, p=0.003). C) The multivariate regression model analysis indicates that tumor stage has a significant effect on t-DARPP mRNA gene expression levels (p=0.02). D) Left panel, cell viability of OE19 and OE33 cells in response to trastuzumab treatment was evaluated by Trypan blue staining. OE19 cells were two-fold more sensitive to trastuzumab than OE33 cells (p<0.001). Right panel, Western blot analysis demonstrates higher protein expression of ERBB2 in OE19 cells than OE33 cells. In contrast, t-DARPP expression was undetectable in OE19 cells but highly expressed in OE33 cells.

Article Snippet: DARPP-32 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and P-ERBB2(Y1248) antibody was obtained from Abcam (San Francisco, CA).

Techniques: Over Expression, Expressing, Staining, Western Blot

Journal: Cancer research

Article Title: Regulation of ERBB2 Receptor by t-DARPP Mediates Trastuzumab Resistance in Human Esophageal Adenocarcinoma

doi: 10.1158/0008-5472.CAN-12-1119

Figure Lengend Snippet: A) ERBB2 protein stability in OE19 cells stably expressing t-DARPP or pcDNA3 empty vector was evaluated by Western blot analysis after treatment with 80 μg/ml CHX to block new protein synthesis for the indicated times. The protein degradation data indicate that t-DARPP expression extended the protein half-life of ERBB2 from 30.8 h to 42.2 h relative to control (lower panel). B) ERBB2 protein stability in parental and trastuzumab resistant OE19 cells was assessed by Western blot analysis after treatment with CHX (80 μg/ml) for the indicated times. The protein degradation data show that endogenous t-DARPP expression in resistant cells was associated with increased ERBB2 protein half-life (60.8 h) relative to parental cells (31.3 h) (lower panel). C) Western blot analysis of p-ERBB2 (Y1248), ERBB2, p-AKT (S473), AKT, and t-DARPP proteins in OE19 cells infected with control (10 MOI) or t-DARPP (10 MOI) adenoviruses after treatment with vehicle or trastuzumab (20 μg/ml) for 24 h. The data indicate that transient expression of t-DARPP increased p-ERBB2(Y1248) and p-AKT(S473) basal protein levels, and blocked trastuzumab-dependent dephosphorylation of ERBB2 and AKT proteins. D) Western blot analysis of p-ERBB2 (Y1248), ERBB2, p-AKT (S473), AKT, and t-DARPP proteins in OE19 cells stably expressing t-DARPP or pcDNA3 vector after treatment with vehicle or trastuzumab (20 μg/ml) for 24 h. The results show that stable expression of t-DARPP increased basal levels of p-ERBB2(Y1248) and p-AKT(S473), and inhibited trastuzumab-dependent dephosphorylation of ERBB2 and AKT proteins. E) Western blot analysis of p-ERBB2(Y1248), ERBB2, p-AKT(S473), AKT, and t-DARPP proteins in parental or trastuzumab resistant OE19 cells following treatment with vehicle or trastuzumab (20 μg/ml) for 24 h. The results indicate that endogenous t-DARPP expression was associated with increased basal levels of p-ERBB2(Y1248) and p-AKT(S473), and suppression of trastuzumab-dependent dephosphorylation of ERBB2 and AKT proteins.

Article Snippet: DARPP-32 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and P-ERBB2(Y1248) antibody was obtained from Abcam (San Francisco, CA).

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Western Blot, Blocking Assay, Infection, De-Phosphorylation Assay

Journal: Cancer research

Article Title: Regulation of ERBB2 Receptor by t-DARPP Mediates Trastuzumab Resistance in Human Esophageal Adenocarcinoma

doi: 10.1158/0008-5472.CAN-12-1119

Figure Lengend Snippet: A) Western blot analysis of p-ERBB2(Y1248), ERBB2, p-AKT(S473), AKT, and t-DARPP proteins in OE33 cells transfected with control siRNA or t-DARPP siRNA and treated with vehicle or trastuzumab (20 μg/ml) for 48 h. The data indicate that knockdown of endogenous t-DARPP increased trastuzumab-dependent dephosphorylation of ERBB2 and AKT proteins. B) Cell viability of OE33 cells transfected with control siRNA or t-DARPP siRNA in response to treatment with vehicle or trastuzumab (20 μg/ml) for 48 h, was evaluated by CellTiter-Glo Luminescent CellViability Assay. The results revealed that knockdown of endogenous t-DARPP with treatment induced a significant decrease in cell survival (p<0.01).

Article Snippet: DARPP-32 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and P-ERBB2(Y1248) antibody was obtained from Abcam (San Francisco, CA).

Techniques: Western Blot, Transfection, De-Phosphorylation Assay

Journal: Cancer research

Article Title: Regulation of ERBB2 Receptor by t-DARPP Mediates Trastuzumab Resistance in Human Esophageal Adenocarcinoma

doi: 10.1158/0008-5472.CAN-12-1119

Figure Lengend Snippet: A) Western blot analysis of co-immunoprecipitated exogenous t-DARPP and endogenous ERBB2 proteins with M2-flag or trastuzumab antibodies in OE19 cells infected with t-DARPP-flag adenovirus (10 MOI). The data demonstrate protein association of ERBB2 with t-DARPP. B) Western blot analysis of immunoprecipitated endogenous ERBB2 protein with trastuzumab antibody in OE19 cells infected with control (10 MOI) or t-DARPP (10 MOI) adenoviruses. Pulled-down ERBB2 band intensity was depicted as a ratio relative to input ERBB2 protein. The results show that exogenous t-DARPP expression blocked binding of trastuzumab to ERBB2 receptor relative to control. C) Western blot analysis of immunoprecipitated endogenous ERBB2 protein with trastuzumab antibody in parental or trastuzumab resistant OE19 cells. The band intensity of immunoprecipitated ERBB2 protein was shown as a ratio relative to input ERBB2. The data indicate that endogenous t-DARPP expression in trastuzumab-resistant cells was associated with a significant decrease in trastuzumab/ERBB2 protein interaction relative to control.

Article Snippet: DARPP-32 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and P-ERBB2(Y1248) antibody was obtained from Abcam (San Francisco, CA).

Techniques: Western Blot, Immunoprecipitation, Infection, Expressing, Binding Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Inhibition of HER2 Increases Jagged1-dependent Breast Cancer Stem Cells: Role for Membrane Jagged1

doi: 10.1158/1078-0432.CCR-17-1952

Figure Lengend Snippet: A, HCC1954 B, MCF-7-HER2 or C, MCF-7 cells were treated with 2 μM Lapatinib or vehicle (DMSO) for 4 days. Cells were harvested and 100,000 cells/well were plated into 6 well ultra-low attachment plates containing mammosphere forming medium-supplemented with 5 μM MRK-003 GSI or vehicle (DMSO). After 7 days of plating, the mammospheres were collected and counted in order to determine the mammosphere forming efficiency (MFE). Scale bars = 100 μm. Bar graphs show mean %MFE ± S.D. from at least three independent experiments. Statistical significance was calculated using ANOVA with a Tukey post-hoc test for multiple comparisons. *, P < 0.05; **, P < 0.01; ***, P < 0.001 and ****, P <0.0001. Lysates from each treated cell lines were harvested for total protein and subjected to Western blotting to detect phosphorylated forms of HER2 and EGFR. Actin was detected as a loading control.

Article Snippet: HER2 siRNA (rGrCrCrArArCrArArArGrArArArUrCrUrUrArGrArCrGrAAG) was purchased from Origene, Rockville, MD (Cat. SR301443).

Techniques: Western Blot

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Inhibition of HER2 Increases Jagged1-dependent Breast Cancer Stem Cells: Role for Membrane Jagged1

doi: 10.1158/1078-0432.CCR-17-1952

Figure Lengend Snippet: HER2+ HCC1954 (A), MCF-7-HER2 (B), MDA-MB-453 (C), and HER2 wild type expressing MCF-7 (D) cells were treated for four days with 2 μM Lapatinib or vehicle (DMSO). Cells were harvested and stained for Jagged1 followed by flow cytometry. E. HCC1954 cells were stained for Jagged1 and then sorted by flow cytometry on the basis of Jagged1 surface expression. The Jagged1 -/low or high population was sorted from both vehicle and lapatinib treated cells. The schematic shows the gating for the cells that were sorted. 35,000 HCC1954 cells were sorted into a well of a 24 well ultra-low attachment plate containing mammosphere forming medium. After 7 days, the mammospheres were harvested and %MFE was determined. Scale bars = 100 μm.

Article Snippet: HER2 siRNA (rGrCrCrArArCrArArArGrArArArUrCrUrUrArGrArCrGrAAG) was purchased from Origene, Rockville, MD (Cat. SR301443).

Techniques: Expressing, Staining, Flow Cytometry

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Inhibition of HER2 Increases Jagged1-dependent Breast Cancer Stem Cells: Role for Membrane Jagged1

doi: 10.1158/1078-0432.CCR-17-1952

Figure Lengend Snippet: HCC1954 (A) or MCF-7-HER2 (B) cells were treated with DMSO (Vehicle) or lapatinib for 4 days followed by flow cytometry to detect cell surface expression of Jagged1. Cells were sorted for low versus high Jagged1 expressing cells and plated onto low attachment plates-containing mammosphere forming medium-supplemented with DMSO (Control) or 5μM MRK-003 GSI. %MFE was assessed at day 7. Scale bar = 100μm. *Denotes statistical significance of P < 0.05; **, P < 0.01; ***, P < 0.001 and ****, P <0.0001. One way ANOVA was performed followed by a Tukey post-hoc test.

Article Snippet: HER2 siRNA (rGrCrCrArArCrArArArGrArArArUrCrUrUrArGrArCrGrAAG) was purchased from Origene, Rockville, MD (Cat. SR301443).

Techniques: Flow Cytometry, Expressing

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Inhibition of HER2 Increases Jagged1-dependent Breast Cancer Stem Cells: Role for Membrane Jagged1

doi: 10.1158/1078-0432.CCR-17-1952

Figure Lengend Snippet: A, Tissue microarray (TMA) was performed on the Nottingham cohort of 145 primary, invasive stage II-III HER2+ breast cancer tissues. Jagged1 staining was performed by immunohistochemistry and Jagged1 was assessed to be localized near the perinucleus, cytoplasm or cell membrane. Jagged1 staining was scored 0.0 for low/negative staining and 1.0 for high/positive staining. A Kaplan-Meier curve was plotted for overall survival and statistics was performed using the log-rank (Mantel-Cox) test. The panels are representative of negative staining, strong membrane + cytoplasmic, cytoplasmic, or perinuclear + cytoplasmic staining. The upper panels are representative images at low magnification with enlarged regions (lower panels) expanded from boxed areas. B. Working model of the effects of anti-HER2 therapy on bulk breast cancer cells to enrich for Jagged1-high expressing CSCs, possibly responsible for resistance and tumor recurrence. A combination approach of anti-HER2 and anti-Jagged1 could potentially prevent this enrichment and drug resistance.

Article Snippet: HER2 siRNA (rGrCrCrArArCrArArArGrArArArUrCrUrUrArGrArCrGrAAG) was purchased from Origene, Rockville, MD (Cat. SR301443).

Techniques: Microarray, Staining, Immunohistochemistry, Negative Staining, Expressing

Journal: Ecotoxicology and environmental safety

Article Title: HER2 overexpression triggers the IL-8 to promote arsenic-induced EMT and stem cell-like phenotypes in human bladder epithelial cells.

doi: 10.1016/j.ecoenv.2020.111693

Figure Lengend Snippet: Fig. 4. HER2 is essential for IL-8- mediated EMT and CD44 expression levels. (A) T-SV-HUC-1 cells were treated with lapatinib (1 μM) or recombinant human HER2 (100 ng/L) for 24 h. The mRNA expression levels and contents of IL-8 were shown. (B) T-SV-HUC-1 cells were treated with saracatinib (1 μM), U0126, SB203580, SP600125, GSK3β inhibitor LiCl (10 mM), LY294002 or DMSO for 24 h. The mRNA expression levels and contents of IL-8 were shown. (C) T-SV-HUC-1 cells were treated with recombinant human HER2 for 30 min, then exposed to U0126, SB203580, SP600125, LiCl or LY294002 for 24 h, respectively. The mRNA expression levels and contents of IL-8 were shown. (D and E) T-SV-HUC-1 cells were treated with lapatinib or recombinant human HER2 for 24 h. The protein levels of E- cadherin, vimentin, Snail, Slug, Twist and CD44 were shown. (F and G) The protein levels of E-cadherin, vimentin, Snail, Slug, Twist and CD44 in T-SV-HUC-1 cells were treated with lapatinib and/or recombinant human IL-8. (H) The protein levels of phosphorylated and total- ERK, AKT and STAT3 in T-SV-HUC-1 cells were treated with lapatinib and/or recombinant human IL-8. (I) The cellular migration abilities were analyzed by wound healing assays; Scale bar, 200 µm. Data are expressed as mean ±SD, n=3. *P < 0.05, **P < 0.01. T-SV-HUC-1: Chronic As -treated SV-HUC-1 cells; lap: lapatinib; HER2: recombinant human HER2; IL-8: re combinant human IL-8.

Article Snippet: The ERK inhibitor U0126 (Cat. #S1102), p38 inhibitor SB203580 (Cat. #S1076), JNK inhibitor SP600125 (Cat. #S1460), AKT inhibitor LY294002 (Cat. #S1105), STAT3 inhibitor Stattic (Cat. #S7024), Src inhibitor Saracatinib (Cat. #S1006), HER2 inhibitor Lapatinib (Cat. #S2111) and IL-8 receptor inhibitor Reparixin (Cat. #S8640) were purchased from Selleckchem.com (TX, USA).

Techniques: Expressing, Recombinant, Migration

Journal: Ecotoxicology and environmental safety

Article Title: HER2 overexpression triggers the IL-8 to promote arsenic-induced EMT and stem cell-like phenotypes in human bladder epithelial cells.

doi: 10.1016/j.ecoenv.2020.111693

Figure Lengend Snippet: Fig. 6. Genistein inhibits arsenic -induced SV-HUC-1 cell migration, EMT and CD44 expression levels by inhibiting HER2 phosphorylation. (A and B) T-SV-HUC-1 cells were treated with genistein (10, 20, 40 μM) or DMSO for 24 h. The protein levels of p-HER2, p-ERK, ERK, p-AKT, AKT, p-STAT3, STAT3 were detected by western blots. (C–E) T- SV-HUC-1 cells were treated with recombinant human HER2 for 30 min before co-exposure to genistein (20 μM) for 24 h, the protein levels of E-cadherin, vimentin, Snail, Slug, CD44, Cyclin D1 and PCNA were shown. (F) The cellular migration abilities were analyzed by wound healing assays; Scale bar, 200 µm. (G) Transwell assays were performed to detect cell migratory capacities; Scale bar, 100 µm. Data are expressed as the mean ±SD, n=3. *P < 0.05, **P < 0.01. T-SV-HUC-1: Chronic As -treated SV-HUC-1 cells.

Article Snippet: The ERK inhibitor U0126 (Cat. #S1102), p38 inhibitor SB203580 (Cat. #S1076), JNK inhibitor SP600125 (Cat. #S1460), AKT inhibitor LY294002 (Cat. #S1105), STAT3 inhibitor Stattic (Cat. #S7024), Src inhibitor Saracatinib (Cat. #S1006), HER2 inhibitor Lapatinib (Cat. #S2111) and IL-8 receptor inhibitor Reparixin (Cat. #S8640) were purchased from Selleckchem.com (TX, USA).

Techniques: Migration, Expressing, Phospho-proteomics, Western Blot, Recombinant

Journal: Ecotoxicology and environmental safety

Article Title: HER2 overexpression triggers the IL-8 to promote arsenic-induced EMT and stem cell-like phenotypes in human bladder epithelial cells.

doi: 10.1016/j.ecoenv.2020.111693

Figure Lengend Snippet: Fig. 7. Combination of genistein and lapatinib inhibits the arsenic -induced EMT phenotype and CD44 expression levels. T- SV-HUC-1 cells were treated with lapatinib (1 μM) and/or genistein (20 μM) for 24 h. (A–C) The protein levels of E-cadherin, vimentin, Snail, Slug, CD44, Cyclin D1 and PCNA were shown. (D) The cellular migration abilities were analyzed by wound healing assays; Scale bar, 200 µm. (E) Transwell assays were performed to detect cell migratory capacities; Scale bar, 100 µm. Data are expressed as the mean ±SD, n=3. *P < 0.05, **P < 0.01. T-SV-HUC-1: Chronic As -treated SV-HUC-1 cells.

Article Snippet: The ERK inhibitor U0126 (Cat. #S1102), p38 inhibitor SB203580 (Cat. #S1076), JNK inhibitor SP600125 (Cat. #S1460), AKT inhibitor LY294002 (Cat. #S1105), STAT3 inhibitor Stattic (Cat. #S7024), Src inhibitor Saracatinib (Cat. #S1006), HER2 inhibitor Lapatinib (Cat. #S2111) and IL-8 receptor inhibitor Reparixin (Cat. #S8640) were purchased from Selleckchem.com (TX, USA).

Techniques: Expressing, Migration

Journal: PLoS ONE

Article Title: Photo-Activated Psoralen Binds the ErbB2 Catalytic Kinase Domain, Blocking ErbB2 Signaling and Triggering Tumor Cell Apoptosis

doi: 10.1371/journal.pone.0088983

Figure Lengend Snippet: (A) 8MOP interacts with three peptide regions within the ErbB2 catalytic kinase domain. Qualitative peptide identifications within the ErbB2 catalytic kinase domain following LC-MS/MS analysis of a streptavidin pull-down of biotinylated-8MOP bait (see ). The transmembrane domain is indicated (red diamond) and the five C-terminus tyrosine autophosphorylation sites are indicated (p). (B) Non-reducing Western blot analysis of the interaction of 8MOP with ErbB2. BT474 cells were treated with 800dye-8MOP (Promega) or with vehicle (0.01% DMSO) alone served as control for 48 hr and then exposed to UV irradiation (2J) prior to Western blot analysis. The image on the left shows the Western blot for ErbB2 (red). The image on the right shows the same membrane directly scanned for the presence of 800dye-8MOP (green), which overlays the ErbB2 signal. The results are representative of three independent experiments.

Article Snippet: ErbB2+ (BT474; SKBR3) and ErbB2 negative (MCF-7; T47D) human breast cancer cell lines, and the human foreskin fibroblast (HFF) cell line were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Liquid Chromatography with Mass Spectroscopy, Western Blot, Control, Irradiation, Membrane

Journal: PLoS ONE

Article Title: Photo-Activated Psoralen Binds the ErbB2 Catalytic Kinase Domain, Blocking ErbB2 Signaling and Triggering Tumor Cell Apoptosis

doi: 10.1371/journal.pone.0088983

Figure Lengend Snippet: The growth and viability of BT474 and SKBR3 cells (top bar graphs) after being subjected to the indicated treatment conditions. The combination of PUVA plus neratinib: P <0.0005 (BT474 and SKBR3 cells). Results represent the mean +/− standard error of triplicate samples, and are representative of three independent experiments. (B) Western blot analysis showing steady-state ErbB2, ErbB3, and phospho-Akt (S473) protein levels in BT474 and SKBR3 cells treated according to the indicated treatment conditions. Vehicle alone (0.01% DMSO) served as a control. Steady-state actin protein levels served as a control for equal loading of protein. The results are representative of three independent experiments.

Article Snippet: ErbB2+ (BT474; SKBR3) and ErbB2 negative (MCF-7; T47D) human breast cancer cell lines, and the human foreskin fibroblast (HFF) cell line were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Western Blot, Control

Journal: PLoS ONE

Article Title: Photo-Activated Psoralen Binds the ErbB2 Catalytic Kinase Domain, Blocking ErbB2 Signaling and Triggering Tumor Cell Apoptosis

doi: 10.1371/journal.pone.0088983

Figure Lengend Snippet: Top bar graph shows the results of the growth assays performed in T47D and stably transfected T47D cell line. T47D cells expressing p85 ErbB2 were pretreated with 5 µM lapatinib or 5 µM 8MOP for 4 hr followed by irradiation in a UV Stratalinker 1800 (Statagene). Cells transfected with empty vector (T47D/Vector), and those treated with vehicle alone (0.01% DMSO) served as controls. The effects of the treatments on cell growth and viability are shown in the bar graph. P<0.0071 (8MOP + UVA irradiation). Results represent the mean +/− standard error of triplicate samples, and are representative of three independent experiments. Steady-state phospho-p85 ErbB2 protein levels (dotted arrow) and phospho-p185 ErbB2 (solid arrow) are shown by Western blot. Actin steady-state protein levels served as a control to ensure for equal loading of protein. Results are representative of three independent experiments.

Article Snippet: ErbB2+ (BT474; SKBR3) and ErbB2 negative (MCF-7; T47D) human breast cancer cell lines, and the human foreskin fibroblast (HFF) cell line were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Stable Transfection, Transfection, Expressing, Irradiation, Plasmid Preparation, Western Blot, Control