Journal: Cancer Discovery

Article Title: PTEN Loss Promotes PI3Kβ Phosphorylation and EPHA2/SRC/p-PI3Kβ Y962 Complex Assembly to Drive Tumorigenesis

doi: 10.1158/2159-8290.CD-25-1126

Figure Lengend Snippet: A SRC–EPHA2–PI3Kβ tripartite complex drives oncogenic signaling in PTEN-null tumors. A, A schematic of the experiment for the targeted compound library screening to identify PI3Kβ phosphorylation inhibitors. Endogenous PI3Kβ-depleted BT549 or PC3 cells replaced with PI3Kβ-WT or PI3Kβ-Y962F mutants were treated with compounds from the library at 0.1 μmol/L. Cell viability was assessed by CCK-8 assays at 48 hours. B and C, Results of the kinase inhibitor screening in BT549 ( B ) and PC3 ( C ) cells. SRC inhibitors dasatinib and KX2-391 were among the top three significant inhibitors in both cells. D and E, Dasatinib effectively inhibited p-SRC, p-PI3Kβ Y962 , p-ERK/c-Myc, and pAKT in PTEN-null BT549 and PC3 cells. F, KX2-391 effectively inhibited p-SRC, p-PI3Kβ Y962 , p-ERK/c-Myc, and pAKT in PTEN-null BT549 cells. G, Dasatinib and KX2-391 dramatically reduced the growth of PPB cells with addback of PI3Kβ-WT but was not as effective on PPB cells with addback of PI3Kβ-Y956F. H, Knockdown of SRC or EPHA2 decreased phosphorylation of PI3Kβ-Y962 and c-MYC in BT549 cells. I, SRC coordinated with EPHA2 to phosphorylate PI3Kβ. SRC-Flag and EPHA2 plasmids were overexpressed in PTEN-WT and PTEN-KO HEK293T cells, and Flag was immunoprecipitated, followed by Western blot analysis. J, SRC and EPHA2 phosphorylated PI3Kβ-Y962 peptides in vitro . GST-SRC or GST-EPHA2 was incubated with synthetic Y962 containing PI3Kβ peptides in phosphorylation buffer, and the resulting peptides were analyzed by MS spectrum. K, In vitro phosphorylation assays showed that SRC and EPHA2 directly phosphorylated PI3Kβ-Y962, and the phosphorylation activity was blunted on PI3Kβ mutant at Y962. Particularly, SRC could strongly phosphorylate PI3Kβ-Y962. An in vitro kinase assay was performed by mixing GST-SRC or GST-EPHA2 with Flag-PI3Kβ-WT or Flag-PI3Kβ-Y962F proteins in the presence of ATP. Anti–phospho-PI3Kβ-Y962 antibody was used to detect the phosphorylated PI3Kβ-Y962. L, Knockdown of SRC (shSRC) dramatically decreased signaling of the p-PI3Kβ Y962 –pERK/c-Myc axis in BT549 cells; this effect can be slightly rescued by exogenous expression of EPHA2. M, Knockdown of EPHA2 (shEPHA2) decreased signaling of the p-PI3Kβ Y962 –pERK/c-Myc axis in BT549 cells; exogenous expression of SRC was unable to rescue the effect by sh EPHA2 in BT549 cells. N, SRC expression elevated p-ERK and c-Myc levels in BT549 cells, which were abolished by EPHA2 KO. O, A mechanism model to show that SRC collaborates with EPHA2 to phosphorylate PI3Kβ-Y962, whereas PTEN loss abolished PI3Kβ dephosphorylation, leading to PI3Kβ hyperphosphorylation and subsequent SRC–EPHA2–p-PI3Kβ Y962 complex formation to upregulate p-ERK/c-Myc signaling, accompanying with enhanced accessibility of PI3Kβ to phosphorylate PIP2 on the cell membrane to upregulate pAKT. Values were presented as the mean ± SEM. P values were determined by unpaired two-tailed t test (B, C). **** P < 0.0001.

Article Snippet: For peptide reactions, a 30 μL reaction system containing kinase reaction buffer added with 100 μmol/L ATP, 1 mmol/L DTT 250 ng of purified GST–SRC or GST-EPHA2 (MCE), and 0.05 mg/mL synthetic nonphosphorylated peptide were prepared.

Techniques: Drug discovery, Phospho-proteomics, CCK-8 Assay, Knockdown, Immunoprecipitation, Western Blot, In Vitro, Incubation, Activity Assay, Mutagenesis, Kinase Assay, Expressing, De-Phosphorylation Assay, Membrane, Two Tailed Test

Journal: The Journal of International Medical Research

Article Title: Identification of therapeutic targets and prognostic biomarkers of the ephrin receptor subfamily in pancreatic adenocarcinoma

doi: 10.1177/03000605231218559

Figure Lengend Snippet: EphA2 mRNA expression profiles of various tumor samples and paired normal tissues. The height of the bar represents the median expression level (in transcripts per million (TPM)) in each tumor or normal tissue type.

Article Snippet: A mouse anti-EphA2 antibody (MAB3035, 1:50, R&D Systems, Boston, MA, USA) was used.

Techniques: Expressing

Journal: The Journal of International Medical Research

Article Title: Identification of therapeutic targets and prognostic biomarkers of the ephrin receptor subfamily in pancreatic adenocarcinoma

doi: 10.1177/03000605231218559

Figure Lengend Snippet: EphA2 expression patterns in pancreatic adenocarcinoma (PAAD) patients. (a) EphA2 transcriptional levels in PAAD and normal pancreatic tissues and (b) EphA2 mRNA levels in PAAD primary tumors and normal tissues.

Article Snippet: A mouse anti-EphA2 antibody (MAB3035, 1:50, R&D Systems, Boston, MA, USA) was used.

Techniques: Expressing

Journal: The Journal of International Medical Research

Article Title: Identification of therapeutic targets and prognostic biomarkers of the ephrin receptor subfamily in pancreatic adenocarcinoma

doi: 10.1177/03000605231218559

Figure Lengend Snippet: Associations between pancreatic adenocarcinoma (PAAD) clinical pathological stage and EphA2 expression.

Article Snippet: A mouse anti-EphA2 antibody (MAB3035, 1:50, R&D Systems, Boston, MA, USA) was used.

Techniques: Expressing

Journal: The Journal of International Medical Research

Article Title: Identification of therapeutic targets and prognostic biomarkers of the ephrin receptor subfamily in pancreatic adenocarcinoma

doi: 10.1177/03000605231218559

Figure Lengend Snippet: The prognostic value of EphA2 expression in pancreatic adenocarcinoma (PAAD) patients. Kaplan–Meier plots are shown for (a) overall survival and (b) disease-free survival.

Article Snippet: A mouse anti-EphA2 antibody (MAB3035, 1:50, R&D Systems, Boston, MA, USA) was used.

Techniques: Expressing

Journal: The Journal of International Medical Research

Article Title: Identification of therapeutic targets and prognostic biomarkers of the ephrin receptor subfamily in pancreatic adenocarcinoma

doi: 10.1177/03000605231218559

Figure Lengend Snippet: EphA2 promoter DNA methylation patterns in pancreatic adenocarcinoma (PAAD) primary tumors and normal tissues.

Article Snippet: A mouse anti-EphA2 antibody (MAB3035, 1:50, R&D Systems, Boston, MA, USA) was used.

Techniques: DNA Methylation Assay

Journal: The Journal of International Medical Research

Article Title: Identification of therapeutic targets and prognostic biomarkers of the ephrin receptor subfamily in pancreatic adenocarcinoma

doi: 10.1177/03000605231218559

Figure Lengend Snippet: Correlations of immune cell infiltration and EphA2 expression in pancreatic adenocarcinoma (PAAD) patients.

Article Snippet: A mouse anti-EphA2 antibody (MAB3035, 1:50, R&D Systems, Boston, MA, USA) was used.

Techniques: Expressing

Journal: The Journal of International Medical Research

Article Title: Identification of therapeutic targets and prognostic biomarkers of the ephrin receptor subfamily in pancreatic adenocarcinoma

doi: 10.1177/03000605231218559

Figure Lengend Snippet: Multivariate Cox proportional risk model of pancreatic adenocarcinoma (PAAD).

Article Snippet: A mouse anti-EphA2 antibody (MAB3035, 1:50, R&D Systems, Boston, MA, USA) was used.

Techniques: Expressing

Journal: The Journal of International Medical Research

Article Title: Identification of therapeutic targets and prognostic biomarkers of the ephrin receptor subfamily in pancreatic adenocarcinoma

doi: 10.1177/03000605231218559

Figure Lengend Snippet: Protein-protein interaction (PPI) analysis of EphA2 in pancreatic adenocarcinoma (PAAD) patients. (a) PPI network of EphA2. (b) Potential functions of the proteins of interest and (c) Molecular complex detection components identified in the gene lists.

Article Snippet: A mouse anti-EphA2 antibody (MAB3035, 1:50, R&D Systems, Boston, MA, USA) was used.

Techniques:

Journal: The Journal of International Medical Research

Article Title: Identification of therapeutic targets and prognostic biomarkers of the ephrin receptor subfamily in pancreatic adenocarcinoma

doi: 10.1177/03000605231218559

Figure Lengend Snippet: Functional enrichment analysis of EphA2 in pancreatic adenocarcinoma (PAAD) patients.

Article Snippet: A mouse anti-EphA2 antibody (MAB3035, 1:50, R&D Systems, Boston, MA, USA) was used.

Techniques: Functional Assay

Journal: The Journal of International Medical Research

Article Title: Identification of therapeutic targets and prognostic biomarkers of the ephrin receptor subfamily in pancreatic adenocarcinoma

doi: 10.1177/03000605231218559

Figure Lengend Snippet: Analysis of EphA2 protein expression using immunohistochemistry (IHC) in tissue samples from pancreatic adenocarcinoma (PAAD) patients. Representative images are shown at 100x magnification. (a) High EphA2 protein expression in pancreatic cancer tissue and (b) Low EphA2 protein expression in paracancerous tissue.

Article Snippet: A mouse anti-EphA2 antibody (MAB3035, 1:50, R&D Systems, Boston, MA, USA) was used.

Techniques: Expressing, Immunohistochemistry

Journal: PLoS ONE

Article Title: Lithocholic Acid Is an Eph-ephrin Ligand Interfering with Eph-kinase Activation

doi: 10.1371/journal.pone.0018128

Figure Lengend Snippet: 96 well ELISA high binding plates were incubated O/N with EphA2-Fc and the following day washed and blocked with PBS +0.5% BSA for 1 hour at 37°C. Compounds were added in the wells at proper concentrations 1 hour before the addition of biotinylated ephrinA1-Fc. After 4 hours wells were washed and incubated with a streptavidin-HRP solution for 20 minutes at room temperature. Wells were washed again and incubated with tetra-methylbenzidine. The reaction was stopped with 3N HCl and the absorbance was measured at 450 nm. A, lithocholic acid dose-dependently displaced binding of ephrin-A1-Fc ectodomain from immobilized EphA2-Fc ectodomain. B, binding of ephrin-A1-Fc ectodomain to immobilized EphA2-Fc ectodomain in presence of different concentration of lithocholic acid. C, The dissociation constants (Kd) from the previous plot were used to calculate Log (Dose-ratio - 1) and to graph the Schild plot. pKi of lithocholic acid was estimated by the intersection of the interpolated line with the X-axis. The slope of the interpolated line can be related to the nature of the binding. A slope between 0.8 and 1.2 is related to a competitive binding whereas higher numbers are related to non-specific interactions. D, EphA2-ephrinA1 binding in presence of 200 µM LCA with or without washing three times with PBS.

Article Snippet: EphA2-, EphB4- and EGFR-phosphorylation were measured in cell lysates using DuoSet®IC Sandwich ELISA (RnD Systems, #DYC4056, #DYC4057 and #DYC1095, respectively) following manufacturer's protocol.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Incubation, Concentration Assay

Journal: Oncoimmunology

Article Title: Utilization of universal-targeting mSA2 CAR-T cells for the treatment of glioblastoma

doi: 10.1080/2162402X.2025.2518631

Figure Lengend Snippet: GB cell line screening for target selection and generation of OE and KO cell line models. (a) Expression of glioma-associated membrane antigens in primary and conventional GB cell lines by RT-qPCR. N = 2 technical replicates (two independent qPCR reactions) per gene per cell line. (b) Protein expression levels of EPHA2, CD276, IL13Ra2 and CD70 (blue histograms) in primary GB cell lines, measured by flow cytometry. (c) Evaluation of EPHA2, CD276, IL13Ra2 and CD70 protein levels (blue histograms) on the surface of generated tumor cell models by flow cytometry. For (b and c), isotype control antibodies (red histograms) were used, and data were gated on live single cells. For (b and c), indicative histograms from N = 3 biological replicates per marker per cell line and N = 3 independent experimental repeats. Results from independent experiments are shown; no data pooling was performed.

Article Snippet: Cells were then incubated with 10 μg/mL biotinylated anti-human CD70 antibody (#MA5–17726, Invitrogen), or a 1:11 dilution of biotinylated anti-human CD276 antibody (#130–095–514, Miltenyi Biotec), or a 1:11 dilution of biotinylated anti-human CD213a2 antibody (a-IL13Ra2; #130–104–503, Miltenyi Biotec), or 7.6 μg/mL biotinylated anti-human EPHA2 antibody (#BAF3035, R&D Systems) or 10 μg/mL of a biotinylated non-targeting control antibody (#13–4714–85, Thermo Fisher Scientific) at 4 °C for 30 min. All antibodies used in this study are listed in Supplementary Table S3.

Techniques: Selection, Expressing, Membrane, Quantitative RT-PCR, Flow Cytometry, Generated, Control, Marker

Journal: Oncoimmunology

Article Title: Utilization of universal-targeting mSA2 CAR-T cells for the treatment of glioblastoma

doi: 10.1080/2162402X.2025.2518631

Figure Lengend Snippet: In vitro evaluation of the mSA2 CAR-T cell specificity and killing potency. (a) In vitro experimental pipeline. (b) mSA2 CAR-T cell activation after co-culture with GB models, determined by flow cytometry. N = 3 biological replicates (three independent co-cultures) per group. Data gated on live single CD3 + cells (NT) or live single CD3 + /EGFP + cells (SFG, mSA2_h28z, mSA2_hBBz). An isotype control antibody was used for gating. An unpaired two-tailed student’s t-test was used to evaluate statistical significance. N = 2 independent T-Cell donors. Representative results from N = 3 independent experimental repeats. (c) Confocal IF images of P3/CD70 (CD70 + /CD276 + /EPHA2 + ) cells co-cultured with mSA2_hBBz cells. For a-EPHA2: t 0 = 0 min, t 1 = 140 min, t 2 = 280 min, t 3 = 420 min, t 4 = 560 min. For a-CD276: t 0 = 0 min, t 1 = 220 min, t 2 = 440 min, t 3 = 660 min, t 4 = 880 min. For a-CD70: t 0 = 0 min, t 1 = 350 min, t 2 = 700 min, t 3 = 1050 min, t 4 = 1400 min. N = 2 biological replicates (two separate co-cultures) per group. N = 1 T-Cell donor. (d) Quantification of tumor cell signal from (c). A Welch’s ANOVA test with a post-hoc Dunnett T3 test for multiple comparisons was performed to assess statistical significance at the t = 560 min mark. Data presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; n.s., not significant. Results from independent experiments are shown; no data pooling was performed.

Article Snippet: Cells were then incubated with 10 μg/mL biotinylated anti-human CD70 antibody (#MA5–17726, Invitrogen), or a 1:11 dilution of biotinylated anti-human CD276 antibody (#130–095–514, Miltenyi Biotec), or a 1:11 dilution of biotinylated anti-human CD213a2 antibody (a-IL13Ra2; #130–104–503, Miltenyi Biotec), or 7.6 μg/mL biotinylated anti-human EPHA2 antibody (#BAF3035, R&D Systems) or 10 μg/mL of a biotinylated non-targeting control antibody (#13–4714–85, Thermo Fisher Scientific) at 4 °C for 30 min. All antibodies used in this study are listed in Supplementary Table S3.

Techniques: In Vitro, Activation Assay, Co-Culture Assay, Flow Cytometry, Control, Two Tailed Test, Cell Culture

Journal: Oncoimmunology

Article Title: Utilization of universal-targeting mSA2 CAR-T cells for the treatment of glioblastoma

doi: 10.1080/2162402X.2025.2518631

Figure Lengend Snippet: Investigation of the mSA2 CAR-T cell capacity to address tumor heterogeneity in vitro . (a) Co-culture pipeline. (b) Apoptotic tumor cell fraction after co-culture with mSA2 CAR-T cells, measured by flow cytometry. Data gated on live single EGFP − cells. N = 1 T-Cell donor. N = 3 biological replicates (three independent co-cultures) per group. A Welch’s ANOVA test with a post-hoc Dunnett T3 test for multiple comparisons was used for statistical significance. (c) Analysis of the Annexin-V-incorporating fraction from (b) by flow cytometry. Data gated on live single EGFP − /Annexin-V high tumor cells. (d) Quantification of Annexin-V incorporation from (c). (e) Confocal live cell if images of P3/CD276_KO : P3/EPHA2_KO cells (left panel) and P3/CD70 : P3/IL13Ra2 cells (right panel) after 48 h co-culture with mSA2_h28z cells, after incubation with combinations of biotinylated antibodies, or a biotinylated isotype control antibody. Indicative images from N = 2 biological replicates (two independent co-cultures) and N = 2 independent T-Cell donors per group. (f) Quantification of tumor cell signal over time from the co-culture in (e). For (d and f), an unpaired two-tailed student’s t-test was used to evaluate statistical significance. For (f), statistical significance was assessed at the t = 750 min mark. Data presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; n.s., not significant. Results from independent experiments are shown; no data pooling was performed.

Article Snippet: Cells were then incubated with 10 μg/mL biotinylated anti-human CD70 antibody (#MA5–17726, Invitrogen), or a 1:11 dilution of biotinylated anti-human CD276 antibody (#130–095–514, Miltenyi Biotec), or a 1:11 dilution of biotinylated anti-human CD213a2 antibody (a-IL13Ra2; #130–104–503, Miltenyi Biotec), or 7.6 μg/mL biotinylated anti-human EPHA2 antibody (#BAF3035, R&D Systems) or 10 μg/mL of a biotinylated non-targeting control antibody (#13–4714–85, Thermo Fisher Scientific) at 4 °C for 30 min. All antibodies used in this study are listed in Supplementary Table S3.

Techniques: In Vitro, Co-Culture Assay, Flow Cytometry, Incubation, Control, Two Tailed Test