anti her3 antibody  (Cell Signaling Technology Inc)

Journal: Clinical Cancer Research

Article Title: PI3Kα Inhibitor and Degrader Inavolisib Can Co-opt FGFR2 to Enhance Responses in Patients with PIK3CA -Mutated Solid Tumors and in Preclinical Models

doi: 10.1158/1078-0432.CCR-25-1459

Figure Lengend Snippet: Inavolisib sensitivity depends on high FGFR2 expression. A, Legend related to panels in B–I . B, Cell lines were treated with 0.03 μmol/L of the FGFR2i or 2-μmol/L lapatinib for 1 hour followed by immunoblotting with the antibodies indicated at left. Representative results from experiments ( n = 2). C, Cell lines were treated with inavolisib or the FGFR2i at various concentrations for 1 hour. Cell lysates were immunoprecipitated with an antibody against p85β, followed by immunoblotting with the antibodies indicated at left. Representative results from experiments ( n = 2). IB, immunoblotting. D, Following RAS-GTP pulldown, cell lines were treated with the FGFR2i or lapatinib for different durations and immunoblotted with the antibodies indicated at left. Representative results from experiments ( n = 2). E, Cell lysates from cells treated with inavolisib alone or in combination with the FGFR2i or lapatinib for 4 hours were immunoprecipitated with RAS antibody and blotted with p110α antibody. F, Mechanistic model of the effects of FGFR2 and HER2 inhibition on HER3 and RAS activity. FGFR2-high–expressing cell lines induced PI3K signaling through both HER3 and WT RAS activity (top) compared with HER2-induced PI3K signaling through HER3 but not RAS activity (bottom). G, FGFR2-high–expressing cell lines, MFM223 and SUM52PE, were treated with inavolisib, FGFR2i, or lapatinib for 1 hour. Membrane fractions were analyzed by reciprocal co-IP with one another using HER3 or FGFR2 antibody and Western blotting with FGFR2, HER3, RAS, and p85β antibody. Representative results from experiments ( n = 2). H, SUM52PE, MFM223, and MFE280 cells were treated with inavolisib single-agent or in combination with the FGFR2i or lapatinib for 6 hours. Ubiquitinated proteins were pulled down from the membrane fraction with TUBE1 reagent and blotted with p110α antibody. Representative results from experiments ( n = 2). I, Western blots of the inhibitor response in PI3K signaling (pHER3 and pAKT) in PIK3CA mutant MFM223 and PIK3CA WT SUM52PE; cell lines were treated with 0.5-μmol/L inavolisib or 1-μmol/L alpelisib for different durations. Representative results from experiments ( n = 2). J, Ratio of inavolisib and alpelisib GR 50 values in FGFR2-high ( n = 12) vs. FGFR2-low ( n = 9) expressing cell lines harboring PIK3CA mutations, as assessed in a 5-day viability assay. Data are represented as median (center line) ± IQR (25th to 75th percentile, box) and ± full range (minimum to maximum, whiskers). P value was calculated using Wilcoxon rank-sum test. Representative results from experiments ( n = 2). WB, Western blotting.

Article Snippet: Antibodies to p110α (cat. No. 4249, RRID: AB_2165248), pAKT Ser473 (cat. No. 4060, RRID: AB_2315049), pS6 S235/236 (cat. No. 2211, RRID: AB_331679), HER3 (cat. No. 12708, RRID: AB_2721919), HER2 (cat. No. 2242, RRID: AB_331015), pHER2 Y1221/Y1222 (cat. No. 2243, RRID: AB_490899), pHER3 Y128 (cat. No. 4791, RRID: AB_2099709), pHER3 Y1328 (cat. No. 14525, RRID: AB_2798501), pPLCγ Y783 (cat. No. 14008, RRID: AB_2728690), pERK T202/T204 (cat. No. 9101, RRID: AB_331646), p4EBP T37/46 (cat. No. 9459, RRID: AB_330985), FGFR1 (cat. No. 9740, RRID: AB_11178519), FGFR2 (cat. No. 11835, RRID: AB_2797742), FGFR3 (cat. No. 4574, RRID: AB_2246903), FGFR4 (cat. No. 8562, RRID: AB_10891199), pFGFR Y653/654 (cat. No. 3476, RRID: AB_331369), and pFRS2A Y196 (cat. No. 3864, RRID: AB_2106222) were obtained from Cell Signaling Technology.

Techniques: Expressing, Western Blot, Immunoprecipitation, Inhibition, Activity Assay, Membrane, Co-Immunoprecipitation Assay, Mutagenesis, Viability Assay

Journal: Oncogene

Article Title: Hotspot mutations in HER2 interfaces destabilize structure, causing breast cancer treatment failure

doi: 10.1038/s41388-025-03653-0

Figure Lengend Snippet: A Representative immunofluorescence photomicrographs of MCF-7 cells overexpressing wild-type HER2 (H2-WT) or DDMs, showing HER2:HER3 PLA puncta (red) after heregulin-1β stimulation. Scale bar: 10 μm. B Graph showing quantified number of puncta as in (A). Each dot represents a field with at least 5 cells. C Schematic depicting the establishment method of H2-WT or DDM expressing cell lines using HER2-high AU-565 and HER2-low ZR-75-1 cells. D Co-immunoprecipitation result using HER2 mAb in AU-565 cells. BO: bead only control, meaning lysate without HER2 mAb. Side panel showing immunoblot analysis for phospho- and pan-HER2 and HER3 in AU-565 cells. SS-Control: serum starved BC cells, EV: empty pc-DNA3.1 plasmid vector. E Reverse co-immunoprecipitation (co-IP) using HER3 mAb in ZR-75-1 cells showing physical interaction of H2-WT and DDMs with HER3 in presence of heregulin-1β ligand; side panel shows immunoblot analysis for phospho- and total HER2 and HER3 levels in ZR-75-1 cells. F Immunofluorescence photomicrographs showing HER2:HER3 PLA puncta (red) in ZR-75-1 cells with stable overexpression of H2-WT or DDMs after heregulin-1β stimulation; cross-stained for F-actin (green) and DAPI (blue); scale bar: 20 μm. G Graph showing average numbers of puncta counted as in ( F ). H PLA assessment results in ZR-75-1 cells for HER2:pHER2 homodimer in the absence of a ligand. I Graph showing average numbers of puncta counted as in ( H ). Each dot represents a field with at least 5 cells. Error bars represent ± standard deviation; * P < 0.05; ** P < 0.01; *** P < 0.001, **** P < 0.0001; ns non-significant.

Article Snippet: Similarly, HER2 primary mAb (# MA5-13675), goat anti-rabbit HRP secondary antibody (# 31460), goat anti-mouse DyLight 633 secondary antibody (# 35512), and goat anti-rabbit DyLight 633 secondary antibody (# 35562) were obtained from Thermo Fisher Scientific (Waltham, MA, USA); HER3 mAb (# 12708S), phospho-HER2 mAb (# 2247), phospho-HER3 mAb (# 4791), phospho-ERK mAb (# 4370) pan-ERK mAb (# 4695), phospho-AKT mAb (# 9271), pan-AKT mAb (# 4691), cleaved PARP (# 5625), and α-tubulin mAb (# 2125) from Cell Signaling Technology (Danvers, USA), and anti-mouse HRP secondary antibody (# ab6728) were from Abcam (Cambridge, UK).

Techniques: Immunofluorescence, Expressing, Immunoprecipitation, Control, Western Blot, Plasmid Preparation, Co-Immunoprecipitation Assay, Over Expression, Staining, Standard Deviation

Journal: Oncogene

Article Title: Hotspot mutations in HER2 interfaces destabilize structure, causing breast cancer treatment failure

doi: 10.1038/s41388-025-03653-0

Figure Lengend Snippet: A Western blot showing phospho- and panAKT, phospho- and panERK level in ZR-75-1 cells with overexpression of H2-WT and the DDMs as marked. HER2 and α-tubulin control blots were shown as well. Heatmap showing ERK activation measured using p-ERK bioluminescence resonance energy transfer (BRET) sensor ( B ) and AKT activation measured using p-AKT BRET sensor ( C ) after heregulin-1β stimulation in ZR-75-1 cells overexpressing wild-type HER2 (H2-WT) and DDMs. Each cell represents the mean of 3 replicates. D , E Representative array and heatmap showing the fold-change in phosphorylated proteins in H2-WT and its DDMs expressing cells. Respective arrays involved candidate proteins of MAPK and AKT signaling cascade. F Heatmap showing the corresponding transcript level measured for MAPK and AKT signaling molecules in H2-WT and its DDMs harboring patients with breast cancer; each column represents a patient from METABRIC dataset. G Co-immunoprecipitation result using HER2 mAb showing physical interaction specificity of HER2 DDMs with HER3 after HER3-TRIM-ing in ZR-75-1 clonal cells as marked. Loading amount was one-fifth of the amount used for immunoprecipitation; BO: bead only. Bottom immunoblot panels showing levels of phospho- and total AKT and ERK in H2-WT and its DDMs expressing cells after ligand stimulation and HER3-TRIM-ing; Control-SS: serum starved ZR-75-1 cells, EV: pcDNA-3.1 empty vector.

Article Snippet: Similarly, HER2 primary mAb (# MA5-13675), goat anti-rabbit HRP secondary antibody (# 31460), goat anti-mouse DyLight 633 secondary antibody (# 35512), and goat anti-rabbit DyLight 633 secondary antibody (# 35562) were obtained from Thermo Fisher Scientific (Waltham, MA, USA); HER3 mAb (# 12708S), phospho-HER2 mAb (# 2247), phospho-HER3 mAb (# 4791), phospho-ERK mAb (# 4370) pan-ERK mAb (# 4695), phospho-AKT mAb (# 9271), pan-AKT mAb (# 4691), cleaved PARP (# 5625), and α-tubulin mAb (# 2125) from Cell Signaling Technology (Danvers, USA), and anti-mouse HRP secondary antibody (# ab6728) were from Abcam (Cambridge, UK).

Techniques: Western Blot, Over Expression, Control, Activation Assay, Bioluminescence Resonance Energy Transfer, Expressing, Immunoprecipitation, Plasmid Preparation

Journal: Oncogene

Article Title: Hotspot mutations in HER2 interfaces destabilize structure, causing breast cancer treatment failure

doi: 10.1038/s41388-025-03653-0

Figure Lengend Snippet: A Immunofluorescence images comparing HER2:HER3 PLA puncta (red) after trastuzumab treatment (300 μg/mL) for 24 h in ZR-75-1 cells stably expressing H2-WT, G309A, S310Y, S310F or P523S mutation. Scale bar: 20 μm B Graph showing the number of puncta counted from images as in (A); “+/-” represent trastuzumab treatment. Error bars represent ± standard deviation; ns non-significant. Each dot represents a field with at least 5 cells. C Western blots showing levels of phospho- and pan-HER2, HER3, AKT, and ERK, along with α-tubulin loading control in ZR-75-1 cells after neratinib treatment. D Immunoblots showing levels of phospho- and pan-HER2, HER3, AKT, and ERK, along with α-tubulin loading control in AU-565 cells expressing H2-WT, S305C, S310Y, S310F and P523S, after treatment of different doses of neratinib for 12 and 24 h. Pan-blots (total) are used for quantification of the respective phospho-protein in both ( C , D ).

Article Snippet: Similarly, HER2 primary mAb (# MA5-13675), goat anti-rabbit HRP secondary antibody (# 31460), goat anti-mouse DyLight 633 secondary antibody (# 35512), and goat anti-rabbit DyLight 633 secondary antibody (# 35562) were obtained from Thermo Fisher Scientific (Waltham, MA, USA); HER3 mAb (# 12708S), phospho-HER2 mAb (# 2247), phospho-HER3 mAb (# 4791), phospho-ERK mAb (# 4370) pan-ERK mAb (# 4695), phospho-AKT mAb (# 9271), pan-AKT mAb (# 4691), cleaved PARP (# 5625), and α-tubulin mAb (# 2125) from Cell Signaling Technology (Danvers, USA), and anti-mouse HRP secondary antibody (# ab6728) were from Abcam (Cambridge, UK).

Techniques: Immunofluorescence, Stable Transfection, Expressing, Mutagenesis, Standard Deviation, Western Blot, Control

Journal: Oncogene

Article Title: Hotspot mutations in HER2 interfaces destabilize structure, causing breast cancer treatment failure

doi: 10.1038/s41388-025-03653-0

Figure Lengend Snippet: A Representative bioluminescence images of H2-WT tumor-bearing mice in untreated and treated arms. B Graph representing quantitative photonic signal (average radiance) from tumor site of the mice over time. C Representative bioluminescence images of S310F tumor-bearing mice in untreated and treated arms. D Graph representing quantitative photonic signal (average radiance) from tumor site of the mice over time. E Representative bioluminescence images of P523S tumor-bearing mice in untreated and treated arms. F Graph representing quantitative photonic signal (average radiance) from tumor site of the mice over time. Error bars represent ± standard deviation for five mice/group. G Western blots showing levels of p-HER2, pan-HER2, p-HER3, and pan-HER3 measured from harvested H2-WT, S310F, and P523S tumor samples treated or untreated with trastuzumab. H Graph showing measured average radiance values from metastatic nodules in the harvested critical organs like lungs, liver, bone, and brain at the end point of the above three mice cohorts.

Article Snippet: Similarly, HER2 primary mAb (# MA5-13675), goat anti-rabbit HRP secondary antibody (# 31460), goat anti-mouse DyLight 633 secondary antibody (# 35512), and goat anti-rabbit DyLight 633 secondary antibody (# 35562) were obtained from Thermo Fisher Scientific (Waltham, MA, USA); HER3 mAb (# 12708S), phospho-HER2 mAb (# 2247), phospho-HER3 mAb (# 4791), phospho-ERK mAb (# 4370) pan-ERK mAb (# 4695), phospho-AKT mAb (# 9271), pan-AKT mAb (# 4691), cleaved PARP (# 5625), and α-tubulin mAb (# 2125) from Cell Signaling Technology (Danvers, USA), and anti-mouse HRP secondary antibody (# ab6728) were from Abcam (Cambridge, UK).

Techniques: Standard Deviation, Western Blot

Journal: Oncogene

Article Title: Hotspot mutations in HER2 interfaces destabilize structure, causing breast cancer treatment failure

doi: 10.1038/s41388-025-03653-0

Figure Lengend Snippet: a In HER2-positive breast cancer with overexpression of the wild-type HER2 receptor, HER2:HER2 homo-dimer forms leading to constitutive activation of downstream ERK signaling. The monoclonal antibody drug, trastuzumab, binds to the extracellular domain of HER2 can prevent the dimerization and therefore downstream signaling activation. b In presence of hotspot mutations in the dimerization domain II and IV (i.e., G309A, S310F, S310Y, P523S), structural change in the dimer face of HER2 potentiate HER2:HER3 heterodimerization and activate both PI3K–AKT signaling axis, thereby turn trastuzumab treatment ineffective. In the altered scenario, as the small molecule tucatinib inhibits kinase activity of HER2:HER3 heterodimer, this drug shows growth inhibitory effect.

Article Snippet: Similarly, HER2 primary mAb (# MA5-13675), goat anti-rabbit HRP secondary antibody (# 31460), goat anti-mouse DyLight 633 secondary antibody (# 35512), and goat anti-rabbit DyLight 633 secondary antibody (# 35562) were obtained from Thermo Fisher Scientific (Waltham, MA, USA); HER3 mAb (# 12708S), phospho-HER2 mAb (# 2247), phospho-HER3 mAb (# 4791), phospho-ERK mAb (# 4370) pan-ERK mAb (# 4695), phospho-AKT mAb (# 9271), pan-AKT mAb (# 4691), cleaved PARP (# 5625), and α-tubulin mAb (# 2125) from Cell Signaling Technology (Danvers, USA), and anti-mouse HRP secondary antibody (# ab6728) were from Abcam (Cambridge, UK).

Techniques: Over Expression, Activation Assay, Activity Assay