biotin conjugated her3  (OriGene)

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: For HER3 and FGFR2 IP, lysates were incubated overnight with a biotin-conjugated HER3 or FGFR2 antibody, respectively (Invitrogen, cat. No. MA5-13037, RRID: AB_10983790; and OriGene, cat. No. TA502917AM), which was followed by the addition of 50 μL of streptavidin agarose beads to each sample and an additional 2 hours of incubation.

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

Journal: The Journal of biological chemistry

Article Title: Androgen receptor transcriptional activity is required for heregulin-1β-mediated nuclear localization of the HER3/ErbB3 receptor tyrosine kinase.

doi: 10.1016/j.jbc.2023.104973

Figure Lengend Snippet: Figure 1. Expression of ErbB3 in the nucleus is exclusively seen in malignant, but not benign, prostate epithelia and correlates with AR expression but not that of AR target genes. A, prostatectomy samples from tumor and nontumor sections of the prostates of 78 patients were laid out in a tissue microarray (TMA) and sections from the TMA stained with anti-ErbB3 (C-terminal, kinase domain) antibody. ErbB3 stained exclusively the epithelial cells with no staining in the stroma. Bar represents the length of an epithelial cell at 20×, 40×, and 100× magnification. Upper panel: sections containing no tumor tissue (benign) showed ErbB3 expression exclusively in the cytoplasm. Lower panel: sections containing tumor tissue (malignant) expressed higher cyto- plasmic ErbB3 but in addition, also showed nuclear localization of ErbB3. B, boxplot demonstrating median levels of ErbB3 in the cytoplasmic and nuclear compartments of tumor and nontumor sections. C, upper panel: negative correlation between cytoplasmic ErbB3 in the cancer cells and preprostatectomy PSA (p = 0.049, Spearman’s rho = −0.25). Lower panel: no correlation between nuclear ErbB3 in the cancer cells and preprostatectomy PSA. D, positive correlation between cytoplasmic ErbB3 in the cancer cells and nuclear AR expression (Spearman’s rho = 0.32; p = 0.013). AR, androgen receptor; PSA, prostate-specific antigen.

Article Snippet: Immunohistochemistry was conducted using an ErbB3 antibody directed toward the C-terminal portion of the receptor (sc-7390, Santa Cruz Technology).

Techniques: Expressing, Microarray, Staining

Journal: The Journal of biological chemistry

Article Title: Androgen receptor transcriptional activity is required for heregulin-1β-mediated nuclear localization of the HER3/ErbB3 receptor tyrosine kinase.

doi: 10.1016/j.jbc.2023.104973

Figure Lengend Snippet: Figure 2. HRG treatment, but not DHT, in LNCaP cells cultured in FBS, but not CSS, causes ErbB3 nuclear translocation. LNCaP cells were cultured in complete media (containing growth factors, steroid hormones, FBS, etc) or media lacking those constituents (CSS) for 72 h with or without ligands specific to activating the androgen receptor (AR) or ErbB3. Dihydrotestosterone (DHT, 1 nM, dissolved in 200-proof ethanol, EtOH) and heregulin-1β (HRG, dissolved in sterile PBS) were used to activate AR and ErbB3, respectively. Cells were separated into cytoplasmic and nuclear fractions and probed with antibodies to AR, ErbB3, and its downstream targets phosphorylated AKT and phosphorylated ERK. Twenty micrograms of protein lysates were loaded in each lane. Lamin and GAPDH were used as protein loading controls for crude nuclear and cytoplasmic fractions, respectively. B, quantitation of ErbB3 bands in the nucleus (N-ErbB3) and the cytoplasm (C-ErbB3). C, quantitation of AR band in the nucleus (N-AR) and the cytoplasm (C-AR). D, high-magnification microscopy showing the cytoplasmic localization of ErbB3 and an increase in ErbB3 protein when treated with HRG for 72 h. Cells were grown on coverslips and fixed for 10 min at room temperature with 100% ice-cold methanol before being incubated with ErbB3-specific antibodies approved for immunofluorescence. The scale bars represent 4 μm. E, LNCaP cells display increased AR transcriptional activity on a human PSA promoter when cultured with 1 nM DHT despite decreased viability, and this effect persists regardless of the presence of androgens. HRG treatment decreases AR transcriptional activity in the presence and absence of androgens. The PSA promoter sequence was housed in a pGL4 backbone (Promega). Promoter activity was recorded for individual samples using relative luminescent units. The results are from experiments performed in triplicate. Error bars represent SD from the mean. Table below shows p- values with respect to FBS alone. CSS, charcoal-stripped serum; FBS, fetal bovine serum; PSA, prostate-specific antigen.

Article Snippet: Immunohistochemistry was conducted using an ErbB3 antibody directed toward the C-terminal portion of the receptor (sc-7390, Santa Cruz Technology).

Techniques: Cell Culture, Translocation Assay, Sterility, Quantitation Assay, Microscopy, Incubation, Activity Assay, Sequencing

Journal: The Journal of biological chemistry

Article Title: Androgen receptor transcriptional activity is required for heregulin-1β-mediated nuclear localization of the HER3/ErbB3 receptor tyrosine kinase.

doi: 10.1016/j.jbc.2023.104973

Figure Lengend Snippet: Figure 3. HRG treatment in cells cultured in FBS, but not in CSS, causes an increase in AR transcriptional activity. A, C4-2 cells, a subline derived from LNCaP cells, also display predominantly cytoplasmic ErbB3 whose levels increase with HRG but not DHT. AR protein remains nuclear in its localization. C4- 2 cells were grown on coverslips for 72 h and treated with ligands as previously described (legend, Fig. 2). The scale bars represent 4 μm. B, in CRPC C4- 2 cells derived from LNCaP, AR transcriptional activation on a human PSA promoter appears to increase when cytoplasmic ErbB3 staining becomes fainter. HRG is unable to transcriptionally activate the AR in the absence of androgens relative to the presence of androgens. All experiments were performed in triplicate. Error bars represent SD from the mean. Table below shows statistical analysis (Student t test, 2-tailed, equal variance). AR, androgen receptor; CSS, charcoal-stripped serum; DHT, dihydrotestosterone; FBS, fetal bovine serum; HRG, heregulin-1β; PSA, prostate-specific antigen.

Article Snippet: Immunohistochemistry was conducted using an ErbB3 antibody directed toward the C-terminal portion of the receptor (sc-7390, Santa Cruz Technology).

Techniques: Cell Culture, Activity Assay, Derivative Assay, Activation Assay, Staining

Journal: The Journal of biological chemistry

Article Title: Androgen receptor transcriptional activity is required for heregulin-1β-mediated nuclear localization of the HER3/ErbB3 receptor tyrosine kinase.

doi: 10.1016/j.jbc.2023.104973

Figure Lengend Snippet: Figure 4. HRG treatment, but not AR activity, affects ErbB3 localization in CRPC 22Rv1 cells, while in parental HSPC CWR22 tumors, castration suppresses ErbB3 nuclear localization. A, 22Rv1 cells were cultured and treated with ligands, fractionated, and probed as previously described. Castration-resistant cells express very little nuclear ErbB3, whose levels are increased by HRG but not DHT and are unaffected by the presence of androgens. B, 22Rv1 cells were treated with increasing concentrations of HRG for 15 min before being collected, lysed, and analyzed by immunoblot as previously described. C, 4- to 5-week-old nu/nu mice were implanted (subcutaneous) with CWR22 tumor cells (AD) (n = 24) or their relapsed and androgen-resistant subline 22Rv1 cells (CRPC) (n = 24). CWR22 cells are an androgen-dependent, serially transplantable xenograft derived from a primary human prostate cancer. CWR22 tumors regress markedly after androgen withdrawal (or castration) but recur (in some animals) in 2 to 7 months. The 22Rv1 cell line was derived from this relapsed tumor, no longer responds to androgen withdrawal, and is used to model CRPC. This model behaved differently from the LNCaP xenografts and castration decreased levels of ErbB3 protein in CWR22 xenografts but appeared to have little effect in the CRPC CWR22-Rv1 xenografts. CWR22-Rv1 xenografts also appeared to display faint membrane ErbB3 staining, unlike CWR22 xenografts where ErbB3 was largely cytoplasmic. D and E, when quantified in CWR22 (D) and 22Rv1 tumor(E), these differences were found to be significant only in the former (p < 0.05). AR, androgen receptor; C, cytoplasmic; CRPC, castration-resistant prostate cancer; DHT, dihydrotestosterone; HRG, heregulin-1β; HSPC, hormone-sensitive prostate cancer; N, nuclear.

Article Snippet: Immunohistochemistry was conducted using an ErbB3 antibody directed toward the C-terminal portion of the receptor (sc-7390, Santa Cruz Technology).

Techniques: Activity Assay, Cell Culture, Western Blot, Derivative Assay, Membrane, Staining

Journal: The Journal of biological chemistry

Article Title: Androgen receptor transcriptional activity is required for heregulin-1β-mediated nuclear localization of the HER3/ErbB3 receptor tyrosine kinase.

doi: 10.1016/j.jbc.2023.104973

Figure Lengend Snippet: Figure 5. AR introduction in AR-null castration-resistant prostate cells restores regulation of ErbB3 localization and downstream signaling. A, AR- null PC3 cells (“PC3”) and PC3 cells stably transfected with full-length WT AR (“PC3-wt-AR”) were cultured, treated, and fractionated as previously described. ErbB3 levels and localization are unaltered in PC3 cells but increase in response to HRG in PC3-wt-AR cells. B, quantitation of ErbB3 bands in PC-3 cells in the nucleus (N-ErbB3) and the cytoplasm (C-ErbB3) C, quantitation of ErbB3 bands in PC-3-wt-AR in the nucleus (N-ErbB3) and the cytoplasm (C-ErbB3). D, quantitation of AR bands in PC-3-wt-AR cells in the nucleus (N-ErbB3) and the cytoplasm (C-ErbB3) using Image (E) PC3-wt-AR cells possess greater transcriptional activity than PC3 cells, and this transcriptional activity remains elevated in the absence of androgens in PC3-wt-AR cells but not in PC3 cells. Luciferase assays were performed as described. All experiments were performed in triplicate. Error bars represent SD from the mean. Table shows p-values (Student t test, 2-tailed, equal variance). F, C-terminal ErbB3 staining (CS-12708) appears diffused throughout the cytoplasm in AR-null PC3 cells. G, C- terminal ErbB3 appears to be localized in the cell membrane in PC3-wt-AR cells. High-magnification microscopy was performed as previously described. AR, androgen receptor; HRG, heregulin-1β.

Article Snippet: Immunohistochemistry was conducted using an ErbB3 antibody directed toward the C-terminal portion of the receptor (sc-7390, Santa Cruz Technology).

Techniques: Stable Transfection, Transfection, Cell Culture, Quantitation Assay, Activity Assay, Luciferase, Staining, Membrane, Microscopy

Journal: The Journal of biological chemistry

Article Title: Androgen receptor transcriptional activity is required for heregulin-1β-mediated nuclear localization of the HER3/ErbB3 receptor tyrosine kinase.

doi: 10.1016/j.jbc.2023.104973

Figure Lengend Snippet: Figure 6. Divergent effect of AR transcriptional activity and AR expression on heregulin-induced ErbB3 nuclear localization. A, LNCaP cells were transfected with AR- or ErbB3-specific siRNA (Dharmacon) for 48 h and treated with ligands for 24 h prior to collection. Enzalutamide (Enz) is an FDA- approved AR inhibitor and was used to dissolve in 100% dimethyl sulfoxide. AR knockdown but not ErbB3 knockdown decreased PSA protein. AR knockdown increased cytoplasmic ErbB3, and ErbB3 knockdown increased cytoplasmic AR. B, quantitation of ErbB3 bands and (C) AR bands from (A) in the nucleus (N-ErbB3) and the cytoplasm (C-ErbB3). D, AR silencing decreases AR transcriptional activity on a human PSA promoter and this cannot be rescued by DHT or HRG. Luciferase assays were performed as described in detail in the legend for Figure 2. Table alongside shows relevant p-values. AR, androgen receptor; DHT, dihydrotestosterone; HRG, heregulin-1β; PSA, prostate-specific antigen.

Article Snippet: Immunohistochemistry was conducted using an ErbB3 antibody directed toward the C-terminal portion of the receptor (sc-7390, Santa Cruz Technology).

Techniques: Activity Assay, Expressing, Transfection, Knockdown, Quantitation Assay, Luciferase

Journal: The Journal of biological chemistry

Article Title: Androgen receptor transcriptional activity is required for heregulin-1β-mediated nuclear localization of the HER3/ErbB3 receptor tyrosine kinase.

doi: 10.1016/j.jbc.2023.104973

Figure Lengend Snippet: Figure 7. Differential effects of AR transcriptional activity and AR expression on heregulin-induced ErbB3 and mutant ErbB3 nuclear localization. A, schematic of full-length ErbB3 showing mutations created on known phosphorylation sites (black). B, LNCaP cells were transfected with full-length, WT AR (“AR”), ErbB3 (“B3”), or mutated ErbB3 (“mB3” or “mErbB3”) plasmids housed in a pcDNA3 (“EV”) construct. Cells were transfected for 48 h and treated with ligands (1 nM DHT or 50 ng/μl HRG) for 24 h prior to collection. Lysates were separated into cytoplasmic and nuclear fractions as previously described and probed with the appropriate antibodies. C, quantitation of ErbB3 bands and (D) AR bands from (B) in the nucleus (N-ErbB3) and the cytoplasm (C-ErbB3). E, the transcriptional activity of the AR was assayed using a PSA luciferase construct in cells that had been transfected with ErbB3 or mB3 plasmids and treated with ligands as previously detailed. HRG was unable to rescue the decreased AR transcriptional activity that resulted from ErbB3 transfection. AR tran- scriptional activity was modestly increased by the transfection of mutated ErbB3 as compared to WT ErbB3. Readings represent the average of experiments carried out in triplicate, and error bars depict SD. Cell viability is (F) increased in LNCaP cells by transfection of full-length, WT ErbB3 but (G) decreased by mutated ErbB3 in 22Rv1 cells. All experiments were performed in triplicate. Error bars indicate SD. * indicates p < 0.05. AR, androgen receptor; DHT, dihydrotestosterone; ECD, extracellular domain; EV, empty vector; HRG, heregulin-1β; TK, tyrosine kinase domain; TM, transmembrane domain; PSA, prostate-specific antigen.

Article Snippet: Immunohistochemistry was conducted using an ErbB3 antibody directed toward the C-terminal portion of the receptor (sc-7390, Santa Cruz Technology).

Techniques: Activity Assay, Expressing, Mutagenesis, Phospho-proteomics, Transfection, Construct, Quantitation Assay, Luciferase, Plasmid Preparation

Journal: The Journal of biological chemistry

Article Title: Androgen receptor transcriptional activity is required for heregulin-1β-mediated nuclear localization of the HER3/ErbB3 receptor tyrosine kinase.

doi: 10.1016/j.jbc.2023.104973

Figure Lengend Snippet: Figure 8. Schematic of proposed model of AR-ErbB3 signaling in hormone-sensitive prostate cancer versus castration-resistant prostate cancer. A, loss of AR transcriptional activity activates a negative feedback loop that results in an increase in AR expression. B, increased AR expression promotes the levels of ErbB3, likely by suppressing its degradation via regulation of its posttranslational modification as shown by us previously (22). We now see that the AR regulates ErbB3 localization through both androgen-dependent and androgen-independent pathways (AR LBD is present only in full-length AR but not in AR variants). C, HRG binding enables ErbB3 nuclear localization in both HSPC and CRPC cells. However, (D) in HSPC, androgen-dependent AR tran- scriptional activity is required for ErbB3 nuclear localization despite HRG stimulation, but in CRPC cells, this effect is androgen-independent. E, AR also regulates ErbB3 export from the nucleus to the cytoplasm in a ligand-independent manner. F, ErbB3 then signals to the PI3K/Akt pathway, regulating cell viability. G, on the other hand, HRG binding to ErbB3, which results in ErbB3 nuclear localization, is shown to result in enhanced AR transcriptional activity, indicating that nuclear ErbB3 may act as an AR coregulator. AR, androgen receptor; CRPC, castration-resistant prostate cancer; HRG, heregulin-1β; HSPC, hormone-sensitive prostate cancer; LBD, ligand-binding domain.

Article Snippet: Immunohistochemistry was conducted using an ErbB3 antibody directed toward the C-terminal portion of the receptor (sc-7390, Santa Cruz Technology).

Techniques: Activity Assay, Expressing, Binding Assay, Ligand Binding Assay