Journal: bioRxiv

Article Title: Oxytocin receptor and HER2 interactions in breast cancer

doi: 10.1101/2025.03.09.642136

Figure Lengend Snippet: A-D . Relapse-free survival (RFS) probability curves of HER2/basal/luminal A/luminal B subtype breast cancer patients with high or low OT/OTR expression. The auto-selected best cut-off button was chosen so that patients were stratified into high- and low-level groups according to the best cut-off, which was the OT/OTR expression value that had the best-performing threshold in survival analysis. The ‘Number at risk’ (shown below the plot to the corresponding time point) indicates the number of patients that are not censored or dead at the given time point (0, 50, 100, 150, 200, and 250 months). HR, hazard ratio; OT, gene ID, 207576_x_at; OTR, gene ID, 206825_at. E . Upper quartile survival summary of patients with low or high OT and OTR levels in different breast cancer subtypes. # , the upper quartile survival was measured as months and rounded off from data obtained from KM-Plotter data analysis. F . Effects of OT treatment on in vitro cell viability of breast cancer cell lines using the MTT assay. Cells were incubated with the indicated doses of OT for 72 h, and cell viability was assessed using the MTT assay after the treatment. Statistical analysis used One-way ANOVA analysis followed by Tukey’s multiple comparisons test. G . Effects of OTR expression modulation on SK-BR-3 cell growth. Cells were seeded in a 96-well plate and transfected with the pCMV6-OTR plasmid for OTR overexpression or siRNA SMARTpool for OTR knockdown (siOTR). After 24 h, the transfected cells were allowed to continue in culture for 48 h before adding MTT. Statistical analysis was using the Student’s t-test. **, p < 0.01 vs control (vector group was the control for pCMV-OTR group, and scRNA (scrambled non-targeting RNA) group was the control for siOTR group). *, p < 0.05; **, p < 0.01; ***, p < 0.001 vs control; n ≥ 3.

Article Snippet: The membrane was probed with the following primary antibodies: mouse-antibody for DYKDDDDK tag (FLAG) (MA191878, Invitrogen, Rockford, IL, USA), rabbit anti-HER2 (2165; Cell Signaling Technology, Beverly, MA, USA), rabbit anti-p-HER2 Tyr 1248 (2274; Cell Signaling Technology), mouse anti-p44/42 MAPK (Erk1/2) (4696; Cell Signaling Technology), rabbit anti-Phospho-p44/42 MAPK (Erk1/2) (Thr 202 /Tyr 204 ) (4370; Cell Signaling Technology), rabbit-anti-Akt (4691; Cell Signaling Technology), rabbit anti-Phospho-Akt (4060; Cell Signaling Technology), mouse anti-tubulin (M30109S; Abmart), and mouse anti-GAPDH (sc-47724; Santa Cruz).

Techniques: Expressing, In Vitro, MTT Assay, Incubation, Transfection, Plasmid Preparation, Over Expression, Knockdown, Control

Journal: bioRxiv

Article Title: Oxytocin receptor and HER2 interactions in breast cancer

doi: 10.1101/2025.03.09.642136

Figure Lengend Snippet: A . Correlation between OTR and HER2 mRNA levels in 57 breast cancer cell lines from the Cancer Cell Line Encyclopedia as analysed by Pearson correlation analysis. Each dot indicates one cell line, with the SK-BR-3 cell line labelled grey. B & C . HER2 expression on mRNA and protein levels in OTR-overexpressing SK-BR-3 cells. Cells were transfected with the pCMV6 plasmid vector or the pCMV6-OTR plasmid for the control group or OTR overexpression, respectively. HER2 mRNA and protein levels were analysed by qPCR and western blot assay 24 h after transfection (GAPDH was used as an internal control for gene expression, and tubulin was used as an internal control for protein expression, n = 3). D - F . Effects of Pertuzumab on SK-BR-3 cell proliferation with or without OT. The Student’s t-test was used for statistical analysis *, p < 0.05; **, p < 0.01; ****, p < 0.0001.

Article Snippet: The membrane was probed with the following primary antibodies: mouse-antibody for DYKDDDDK tag (FLAG) (MA191878, Invitrogen, Rockford, IL, USA), rabbit anti-HER2 (2165; Cell Signaling Technology, Beverly, MA, USA), rabbit anti-p-HER2 Tyr 1248 (2274; Cell Signaling Technology), mouse anti-p44/42 MAPK (Erk1/2) (4696; Cell Signaling Technology), rabbit anti-Phospho-p44/42 MAPK (Erk1/2) (Thr 202 /Tyr 204 ) (4370; Cell Signaling Technology), rabbit-anti-Akt (4691; Cell Signaling Technology), rabbit anti-Phospho-Akt (4060; Cell Signaling Technology), mouse anti-tubulin (M30109S; Abmart), and mouse anti-GAPDH (sc-47724; Santa Cruz).

Techniques: Expressing, Transfection, Plasmid Preparation, Control, Over Expression, Western Blot, Gene Expression

Journal: bioRxiv

Article Title: Oxytocin receptor and HER2 interactions in breast cancer

doi: 10.1101/2025.03.09.642136

Figure Lengend Snippet: A . Time course of HER2 phosphorylation in response to OT (1 μM) treatment detected by western blot. The bar graph represents the quantification of the phosphorylation indicated by the pHER2:HER2 ratio. One-way ANOVA analysis followed by Tukey’s multiple comparisons test was used for statistical analysis. *, p < 0.05 vs. 0 min group without OT treatment; n ≥ 3. B . Confocal microscope images of immunofluorescence for HER2 (green) with DAPI nucleus staining (blue) in SK-BR-3 cells treated with OT (1 μM) for 5 min and 24 h. HER2 was visualised with a primary rabbit antibody that specifically binds to the HER2 protein, followed by an Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody using a Fluoro 2 – Zeiss Apotome 2.0 confocal microscope. The magnification for the top panel images is 630× (scale bar 20 μm), and the bottom panel images represent enlargements of the boxed areas from the top panel. Control, cells without OT treatment. C . Co-immunoprecipitation (Co-IP) demonstrated interaction of OTR with HER2. Total protein extracts from the cell lysis (input) were immunoprecipitated with anti-FLAG antibody (targeting the OTR-FLAG) and the mouse IgG (negative control) and resolved on SDS-PAGE gel. Protein interaction was immunodetected using HER2 and FLAG antibodies. Heterodimer of HER2 and OTR-FLAG was observed (output).

Article Snippet: The membrane was probed with the following primary antibodies: mouse-antibody for DYKDDDDK tag (FLAG) (MA191878, Invitrogen, Rockford, IL, USA), rabbit anti-HER2 (2165; Cell Signaling Technology, Beverly, MA, USA), rabbit anti-p-HER2 Tyr 1248 (2274; Cell Signaling Technology), mouse anti-p44/42 MAPK (Erk1/2) (4696; Cell Signaling Technology), rabbit anti-Phospho-p44/42 MAPK (Erk1/2) (Thr 202 /Tyr 204 ) (4370; Cell Signaling Technology), rabbit-anti-Akt (4691; Cell Signaling Technology), rabbit anti-Phospho-Akt (4060; Cell Signaling Technology), mouse anti-tubulin (M30109S; Abmart), and mouse anti-GAPDH (sc-47724; Santa Cruz).

Techniques: Western Blot, Microscopy, Immunofluorescence, Staining, Control, Immunoprecipitation, Co-Immunoprecipitation Assay, Lysis, Negative Control, SDS Page

Journal: bioRxiv

Article Title: Oxytocin receptor and HER2 interactions in breast cancer

doi: 10.1101/2025.03.09.642136

Figure Lengend Snippet: A . Proposed model of HER2-OTR internalisation. OTR activation induces ERK1/2 and Akt phosphorylation. HER2-mediated signalling is either initiated by homodimerization when HER2 is expressed at high levels or by ligand-induced heterodimerization with other HER family receptors (HER1/HER3/HER4). Receptor dimerization causes transphosphorylation of tyrosine residues within the intracellular domains of the HER family receptors. Phosphorylation of HER2 reveals its binding sites for adaptor or effector proteins such as Ras/Raf and PI3K, which further activate ERK and Akt pathways, promoting cell survival and proliferation. B & C . Representative western blot images of total and phosphorylated ERK1/2 and Akt induced by OT treatment (1 μM). Column figures represent the quantification of the phosphorylation indicated by pERK1/2:ERK1/2 or pAkt:Akt ratio compared with 0 min group (n = 3 independent experiments). One-way ANOVA analysis followed by Tukey’s multiple comparisons test was used for statistical analysis. *, p < 0.05, **, p < 0.001 vs 0 min group (without OT treatment).

Article Snippet: The membrane was probed with the following primary antibodies: mouse-antibody for DYKDDDDK tag (FLAG) (MA191878, Invitrogen, Rockford, IL, USA), rabbit anti-HER2 (2165; Cell Signaling Technology, Beverly, MA, USA), rabbit anti-p-HER2 Tyr 1248 (2274; Cell Signaling Technology), mouse anti-p44/42 MAPK (Erk1/2) (4696; Cell Signaling Technology), rabbit anti-Phospho-p44/42 MAPK (Erk1/2) (Thr 202 /Tyr 204 ) (4370; Cell Signaling Technology), rabbit-anti-Akt (4691; Cell Signaling Technology), rabbit anti-Phospho-Akt (4060; Cell Signaling Technology), mouse anti-tubulin (M30109S; Abmart), and mouse anti-GAPDH (sc-47724; Santa Cruz).

Techniques: Activation Assay, Binding Assay, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: IQGAP1 Protein Binds Human Epidermal Growth Factor Receptor 2 (HER2) and Modulates Trastuzumab Resistance

doi: 10.1074/jbc.M111.220939

Figure Lengend Snippet: Knockdown of IQGAP1 reduces HER2 expression, phosphorylation, and signaling and enhances the inhibitory effects of trastuzumab. A, SkBR3 cells were transiently transfected with siRNA against renilla luciferase (siRen) or siRNAs against IQGAP1 (siIQ12 and siIQ14). 36 h after siRNA transfection, cells were serum-starved in the presence of vehicle (−) or 21 μg/ml trastuzumab (+) for 48 h. Equal amounts of protein were resolved by SDS-PAGE, transferred to PVDF membranes, and probed with anti-IQGAP1, anti-pHER2, anti-HER2, anti-pAKT, anti-AKT, anti-pERK, anti-ERK, and anti-β-tubulin antibodies. The data are representative of five independent experiments. B, the amounts of IQGAP1 and HER2 were quantified by densitometry and corrected for the amount of β-tubulin in the corresponding lysate. The amounts of pHER2 and pAKT were quantified by densitometry and corrected for the amounts of total HER2 and total AKT, respectively, in the corresponding lysate. Samples that were treated with vehicle are depicted by black bars, and those treated with trastuzumab are depicted by white bars. The data, expressed relative to the amount of each protein in control cells transfected with siRen and treated with vehicle represent the mean ± S.E. (n = 5). *, significantly different from control cells transfected with siRen and treated with vehicle (p < 0.05); θ, significantly different from cells treated with vehicle (p < 0.05). C, SkBR3 cells were transiently transfected with siRNA against renilla luciferase and vector (siRen and Vector), siRNA against IQGAP1 and vector (siIQ12 and Vector), or siRNA against IQGAP1 and siRNA-resistant IQGAP1 (siIQ12 and IQGAP1). 36 h after transfection, cells were serum-starved in the presence of vehicle (−) or 21 μg/ml trastuzumab (+) for 48 h. Equal amounts of protein were resolved by SDS-PAGE, transferred to PVDF membranes, and probed with anti-IQGAP1, anti-pHER2, anti-HER2, anti-pAKT, anti-AKT, anti-pERK, anti-ERK, and anti-β-tubulin antibodies. The data are representative of three independent experiments. D, the amounts of IQGAP1 and HER2 were quantified by densitometry and corrected for the amount of β-tubulin in the corresponding lysate. The amounts of pHER2 and pAKT were quantified by densitometry and corrected for the amounts of total HER2 and total AKT, respectively, in the corresponding lysate. Samples that were treated with vehicle are depicted by black bars, and those treated with trastuzumab are depicted by white bars. The data, expressed relative to the amount of each protein in control cells transfected with siRen and vector and treated with vehicle, represent the mean ± S.E. (n = 3). *, significantly different from control cells transfected with siRen and vector and treated with vehicle (p < 0.05); θ, significantly different from cells treated with vehicle (p < 0.05); #, significantly different from cells transfected with siIQ12 and vector (p < 0.05).

Article Snippet: Anti-pHER2 (Tyr 1221 /Tyr 1222 ), anti-HER2, anti-pAKT (Ser 473 ), anti-AKT, anti-pERK (Thr 202 /Tyr 204 ), anti-ERK, anti-p27, and anti-β-tubulin antibodies and a glutathione S -transferase (GST) fusion construct encoding the entire HER2 intracellular domain (designated GST-HER2; residues 676–1255 (numbering corresponds to the immature sequence)) were obtained from Cell Signaling Technology (Danvers, MA).

Techniques: Expressing, Transfection, Luciferase, SDS Page, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: IQGAP1 Protein Binds Human Epidermal Growth Factor Receptor 2 (HER2) and Modulates Trastuzumab Resistance

doi: 10.1074/jbc.M111.220939

Figure Lengend Snippet: Knockdown of IQGAP1 reduces HER2 expression, phosphorylation, and signaling and restores trastuzumab sensitivity to trastuzumab-resistant SkBR3 cells. A, trastuzumab-resistant SkBR3 cells were transiently transfected with siRNA against renilla luciferase (siRen) or siRNAs against IQGAP1 (siIQ12 and siIQ14). 36 h after siRNA transfection, cells were serum-starved in the presence of vehicle (−) or 21 μg/ml trastuzumab (+) for 48 h. Equal amounts of protein were resolved by SDS-PAGE, transferred to PVDF membranes, and probed with anti-IQGAP1, anti-pHER2, anti-HER2, anti-pAKT, anti-AKT, anti-pERK, anti-ERK, anti-p27, and anti-β-tubulin antibodies. The data are representative of five independent experiments. B, the amounts of IQGAP1, HER2, and p27 were quantified by densitometry and corrected for the amount of β-tubulin in the corresponding lysate. The amounts of pHER2 and pAKT were quantified by densitometry and corrected for the amounts of total HER2 and total AKT, respectively, in the corresponding lysate. Samples that were treated with vehicle are depicted by black bars, and those treated with trastuzumab are depicted by white bars. The data, expressed relative to the amount of each protein in control cells transfected with siRen and treated with vehicle, represent the mean ± S.E. (n = 5). *, significantly different from control cells transfected with siRen and treated with vehicle (p < 0.05); θ, significantly different from cells treated with vehicle (p < 0.05).

Article Snippet: Anti-pHER2 (Tyr 1221 /Tyr 1222 ), anti-HER2, anti-pAKT (Ser 473 ), anti-AKT, anti-pERK (Thr 202 /Tyr 204 ), anti-ERK, anti-p27, and anti-β-tubulin antibodies and a glutathione S -transferase (GST) fusion construct encoding the entire HER2 intracellular domain (designated GST-HER2; residues 676–1255 (numbering corresponds to the immature sequence)) were obtained from Cell Signaling Technology (Danvers, MA).

Techniques: Expressing, Transfection, Luciferase, SDS Page