mda mb 453 ar v7 geo database gse244283 spatial genomics geo database gse245202 mda mb 453 xenograft geo database gse244283  (ATCC)


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    ATCC mda mb 453 ar v7 geo database gse244283 spatial genomics geo database gse245202 mda mb 453 xenograft geo database gse244283
    A. Structure of AR and AR-SV (biorender.com). B. Kaplan Meier relapse free survival plot of patients with high (red) and low (black) AR (211621_at) expression (kmplot.com). C. Patient demographics. D-G. AR and AR-SV gene expression in specimens from TNBC patients. RNA was extracted (n=52) from TNBC patient specimens and real-time PCR was performed with AR-NTD -binding probe (D), AR-NTD - and LBD-binding taqman probes (F), and <t>AR-V7</t> probe (G). IHC was performed with AR NTD -binding antibody (E). Percent of patients positive for AR (>10% cells positive for AR) is shown at the bottom of the graph. H. Flow chart of breast cancer subtypes and TNBC subtypes, including AR-SV-positive TNBC. IHC- immunohistochemistry; NTD- N-terminus domain; CTD- C-terminus domain; AA- African American; CA-Caucasian American; DBD- DNA binding domain; Hin- Hinge; LBD- Ligand Binding Domain; U- Unique cryptic exon; AF-1- Activation Function-1 Domain; AR-FL- androgen receptor full length; AR-SV- androgen receptor splice variant; RV1– 22RV1 prostate cancer cells; LN- LNCaP prostate cancer cells. Values are expressed as mean -/+ SEM. *-p<0.05; **-p<0.01; ***-p<0.001; ****-p<0.0001 (t-test).
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    1) Product Images from "Identification of a Targetable JAK-STAT Enriched Androgen Receptor (AR) and AR Splice Variant Positive Triple Negative Breast Cancer Subtype"

    Article Title: Identification of a Targetable JAK-STAT Enriched Androgen Receptor (AR) and AR Splice Variant Positive Triple Negative Breast Cancer Subtype

    Journal: Cell reports

    doi: 10.1016/j.celrep.2023.113461

    A. Structure of AR and AR-SV (biorender.com). B. Kaplan Meier relapse free survival plot of patients with high (red) and low (black) AR (211621_at) expression (kmplot.com). C. Patient demographics. D-G. AR and AR-SV gene expression in specimens from TNBC patients. RNA was extracted (n=52) from TNBC patient specimens and real-time PCR was performed with AR-NTD -binding probe (D), AR-NTD - and LBD-binding taqman probes (F), and AR-V7 probe (G). IHC was performed with AR NTD -binding antibody (E). Percent of patients positive for AR (>10% cells positive for AR) is shown at the bottom of the graph. H. Flow chart of breast cancer subtypes and TNBC subtypes, including AR-SV-positive TNBC. IHC- immunohistochemistry; NTD- N-terminus domain; CTD- C-terminus domain; AA- African American; CA-Caucasian American; DBD- DNA binding domain; Hin- Hinge; LBD- Ligand Binding Domain; U- Unique cryptic exon; AF-1- Activation Function-1 Domain; AR-FL- androgen receptor full length; AR-SV- androgen receptor splice variant; RV1– 22RV1 prostate cancer cells; LN- LNCaP prostate cancer cells. Values are expressed as mean -/+ SEM. *-p<0.05; **-p<0.01; ***-p<0.001; ****-p<0.0001 (t-test).
    Figure Legend Snippet: A. Structure of AR and AR-SV (biorender.com). B. Kaplan Meier relapse free survival plot of patients with high (red) and low (black) AR (211621_at) expression (kmplot.com). C. Patient demographics. D-G. AR and AR-SV gene expression in specimens from TNBC patients. RNA was extracted (n=52) from TNBC patient specimens and real-time PCR was performed with AR-NTD -binding probe (D), AR-NTD - and LBD-binding taqman probes (F), and AR-V7 probe (G). IHC was performed with AR NTD -binding antibody (E). Percent of patients positive for AR (>10% cells positive for AR) is shown at the bottom of the graph. H. Flow chart of breast cancer subtypes and TNBC subtypes, including AR-SV-positive TNBC. IHC- immunohistochemistry; NTD- N-terminus domain; CTD- C-terminus domain; AA- African American; CA-Caucasian American; DBD- DNA binding domain; Hin- Hinge; LBD- Ligand Binding Domain; U- Unique cryptic exon; AF-1- Activation Function-1 Domain; AR-FL- androgen receptor full length; AR-SV- androgen receptor splice variant; RV1– 22RV1 prostate cancer cells; LN- LNCaP prostate cancer cells. Values are expressed as mean -/+ SEM. *-p<0.05; **-p<0.01; ***-p<0.001; ****-p<0.0001 (t-test).

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Binding Assay, Immunohistochemistry, Ligand Binding Assay, Activation Assay, Variant Assay

    TNBC cell lines were stably transfected with AR-V7 using lentivirus. A. BrdU assay of control and AR-V7 lentivirus transfected TNBC cell lines MDA-MB-453 (453), MDA-MB-231 (231), BT549, and MFM223. Cells were plated in growth medium and BrDU assay was performed after 72 hours (n=4/group). B. Scratch assay in MDA-MB-453 and MDA-MB-453-V7. Cells were plated in growth medium and imaged at the start and after 72 hours, and the gap closure measured by imaging software (n=4/group). C. Ki67 staining of TNBC patient specimens (23 (AR-positive (AR+)), and 11 (AR and AR-SV -positive (AR+/AR-SV+)). D and E. RNA sequencing was performed with 453 and 453-V7 cell lines and AR+ and AR+/AR-SV+ patient specimens. Gene set from the Molecular Signatures Database is reported in 453 cells compared to 453-V7 cells and AR+ patient specimens compared to AR+/AR-SV+ patient specimens. Values are expressed as mean -/+ SEM. Experiments in panels A-C were reproduced at least three times and representative experiment is shown. * p<0.05, ** p<0.01, ****p<0.00001 (t-test).
    Figure Legend Snippet: TNBC cell lines were stably transfected with AR-V7 using lentivirus. A. BrdU assay of control and AR-V7 lentivirus transfected TNBC cell lines MDA-MB-453 (453), MDA-MB-231 (231), BT549, and MFM223. Cells were plated in growth medium and BrDU assay was performed after 72 hours (n=4/group). B. Scratch assay in MDA-MB-453 and MDA-MB-453-V7. Cells were plated in growth medium and imaged at the start and after 72 hours, and the gap closure measured by imaging software (n=4/group). C. Ki67 staining of TNBC patient specimens (23 (AR-positive (AR+)), and 11 (AR and AR-SV -positive (AR+/AR-SV+)). D and E. RNA sequencing was performed with 453 and 453-V7 cell lines and AR+ and AR+/AR-SV+ patient specimens. Gene set from the Molecular Signatures Database is reported in 453 cells compared to 453-V7 cells and AR+ patient specimens compared to AR+/AR-SV+ patient specimens. Values are expressed as mean -/+ SEM. Experiments in panels A-C were reproduced at least three times and representative experiment is shown. * p<0.05, ** p<0.01, ****p<0.00001 (t-test).

    Techniques Used: Stable Transfection, Transfection, BrdU Staining, Wound Healing Assay, Imaging, Software, Staining, RNA Sequencing Assay

    A. Structure of UT-105. B. UT-105 inhibits wildtype and mutant AR transactivation. AR or AR F876L (50 ng), 0.25 μg GRE-LUC, and 10 ng CMV-renilla-LUC were transfected into COS7 cells. Cells were treated 24 hours after transfection with a dose response of UT-105 or enzalutamide in the presence of 0.1 nM R1881, and luciferase assay was performed 24 hours after treatment. Firefly luciferase values were normalized to renilla luciferase. Numbers provided in bracket are IC50 values. C. UT-105 degrades AR. LNCaP PCa cells were maintained in charcoal-stripped FBS-containing medium for 2 days before treating with UT-105 in the presence of 0.1 nM R1881. Cells were harvested 24 hours after treatment, and Western blot for AR and GAPDH was performed. D. Top. UT-105 binds to AR-NTD. Recombinant purified AR NTD was incubated with DMSO or 10 μM UT-105 overnight at 4°C. SYPRO orange dye was added to the mixture and a PCR was performed with increasing temperature. SYPRO orange signal was monitored. Bottom. UT-105 stabilized purified AF-1 and AR-V7 recombinant protein. Recombinant purified AF-1 or AR-V7 protein (5 ng) were incubated at room temperature for 4 hours with DMSO or 100 μM of UT-105 or UT-34. Proteins were fractionated on an SDS-PAGE and Western blot was performed with AR antibody (AR-441). E. Illustration depicting the binding regions of UT-105 and enzalutamide. F. UT-105 irreversibly inhibits AR. Transactivation assay was performed with AR with a dose response of R1881 in the presence of 3 and 10 μM UT-105 as indicated in panel B. G. UT-105 inhibits AR-target genes. MDA-MB-453 cells in charcoal stripped serum-containing medium were treated for 16–20 hours. RNA was extracted and expression of FKBP5, TMPRSS2, and STEAP4 was quantified by real-time PCR and normalized to GAPDH (n=4/group). * p<0.05 (one-way ANOVA) H. RNA-seq. MDA-MB-453 cells maintained in charcoal-stripped serum-containing medium for 2 days were treated with vehicle or 3 μM UT-105 in the presence of 0.1 nM R1881 for 20 hours (n=3/group). Cells were harvested, RNA extracted, and sequenced. Heatmap of global gene expression changes, top GSEA pathways enriched, and heatmap and bar graphs of AR signaling pathway are represented. I. UT-105 inhibits proliferation of TNBC cells. MDA-MB-453 cells plated in charcoal stripped serum-containing medium and treated for seven days with medium change and retreatment after day 3 (n=3/group). Sulforhodamine B (SRB) colorimetric assay was performed to measure cell viability. J. UT-105 inhibits clonogenicity. TNBC cells stably expressing AR-V7 were plated in 6-well plates and treated with 10 μM of the indicated compounds for two weeks. Colonies were imaged and the number of colonies formed was counted using an imaging software (n=4/group). Panels B-D and F, G, I-J are representatives of at least three independent replicates. * p<0.05, ** p<0.01 (one-way ANOVA). Enza- enzalutamide; V7- AR-V7 splice variant. Values are expressed as mean -/+ SEM.
    Figure Legend Snippet: A. Structure of UT-105. B. UT-105 inhibits wildtype and mutant AR transactivation. AR or AR F876L (50 ng), 0.25 μg GRE-LUC, and 10 ng CMV-renilla-LUC were transfected into COS7 cells. Cells were treated 24 hours after transfection with a dose response of UT-105 or enzalutamide in the presence of 0.1 nM R1881, and luciferase assay was performed 24 hours after treatment. Firefly luciferase values were normalized to renilla luciferase. Numbers provided in bracket are IC50 values. C. UT-105 degrades AR. LNCaP PCa cells were maintained in charcoal-stripped FBS-containing medium for 2 days before treating with UT-105 in the presence of 0.1 nM R1881. Cells were harvested 24 hours after treatment, and Western blot for AR and GAPDH was performed. D. Top. UT-105 binds to AR-NTD. Recombinant purified AR NTD was incubated with DMSO or 10 μM UT-105 overnight at 4°C. SYPRO orange dye was added to the mixture and a PCR was performed with increasing temperature. SYPRO orange signal was monitored. Bottom. UT-105 stabilized purified AF-1 and AR-V7 recombinant protein. Recombinant purified AF-1 or AR-V7 protein (5 ng) were incubated at room temperature for 4 hours with DMSO or 100 μM of UT-105 or UT-34. Proteins were fractionated on an SDS-PAGE and Western blot was performed with AR antibody (AR-441). E. Illustration depicting the binding regions of UT-105 and enzalutamide. F. UT-105 irreversibly inhibits AR. Transactivation assay was performed with AR with a dose response of R1881 in the presence of 3 and 10 μM UT-105 as indicated in panel B. G. UT-105 inhibits AR-target genes. MDA-MB-453 cells in charcoal stripped serum-containing medium were treated for 16–20 hours. RNA was extracted and expression of FKBP5, TMPRSS2, and STEAP4 was quantified by real-time PCR and normalized to GAPDH (n=4/group). * p<0.05 (one-way ANOVA) H. RNA-seq. MDA-MB-453 cells maintained in charcoal-stripped serum-containing medium for 2 days were treated with vehicle or 3 μM UT-105 in the presence of 0.1 nM R1881 for 20 hours (n=3/group). Cells were harvested, RNA extracted, and sequenced. Heatmap of global gene expression changes, top GSEA pathways enriched, and heatmap and bar graphs of AR signaling pathway are represented. I. UT-105 inhibits proliferation of TNBC cells. MDA-MB-453 cells plated in charcoal stripped serum-containing medium and treated for seven days with medium change and retreatment after day 3 (n=3/group). Sulforhodamine B (SRB) colorimetric assay was performed to measure cell viability. J. UT-105 inhibits clonogenicity. TNBC cells stably expressing AR-V7 were plated in 6-well plates and treated with 10 μM of the indicated compounds for two weeks. Colonies were imaged and the number of colonies formed was counted using an imaging software (n=4/group). Panels B-D and F, G, I-J are representatives of at least three independent replicates. * p<0.05, ** p<0.01 (one-way ANOVA). Enza- enzalutamide; V7- AR-V7 splice variant. Values are expressed as mean -/+ SEM.

    Techniques Used: Mutagenesis, Transfection, Luciferase, Western Blot, Recombinant, Purification, Incubation, SDS Page, Binding Assay, Transactivation Assay, Expressing, Real-time Polymerase Chain Reaction, RNA Sequencing Assay, Colorimetric Assay, Stable Transfection, Imaging, Software, Variant Assay

    (A-F) RNA was extracted from MDA-MB-453 tumors (shown in Fig. 4B) and sequenced (n = 3–5/group). A. Differentially expressed genes (DEGs) are visualized as a heatmap. The number of significant DEGs in each treatment compared to the vehicle control is shown below. B. GSEA analysis was performed on UT-105-treated tumors. Pathways with an FDR<0.25 are shown with corresponding normalized enrichment scores (NES). C. Enrichment plots are shown for interferon ɤ (top) and interferon α (bottom) Hallmark response pathways. D. Fold change in gene expression from RNA sequencing with MDA-MB-453 tumors for STAT signaling pathway genes IRF1, IRF7, and IRF9 is shown. E. A heatmap of chemokine normalized means from the RNA-seq data performed in MDA-MB-453 xenograft tumors. F. Western immunoblot of phospho-STAT1, phospho-STAT3, and phospho-AKT in representative samples from vehicle and UT-105-treated MDA-MB-453 xenograft tumors (from Fig 4B). G. Effect of UT-105 and ruxolitinib on STAT1 phosphorylation in MDA-MB-453 cells. MDA-MB-453 cells were maintained in csFBS-containing medium for 48 hours. Cells were treated with 3 μM UT-105 or ruxolitinib for 24 hours before the induction of STAT1 phosphorylation with interferon α (1000 units). Cells were harvested 30 minutes after induction, protein was extracted, and Western blot for pSTAT1 and GAPDH was performed. Representative blot is shown. The bands were quantified and fold change from vehicle is provided under the blots. H. Effect of UT-105 and ruxolitinib on cell proliferation. Cells were plated in growth medium and treated with vehicle, UT-105, or JAK-STAT inhibitor ruxolitinib for seven days with medium change and retreatment after day 3 (n=4/group; representative of three replicates; one-way ANOVA). SRB assay was performed. I. AR and AR-SV -positive TNBC specimens are enriched for JAK-STAT pathway. RNA was extracted from TNBC specimens (n=41) shown in Fig. 1C and sequenced. The top pathways enriched in AR and AR-SV compared to AR negative specimens obtained from GSEA (FDR <0.25) are shown as a bar graph. J. Enrichment plots are shown. Rux- ruxolitinib; enza- enzalutamide. Mean -/+ SEM: * p<0.05, ** p<0.01, *** p<0.001, **** p<0.00001.
    Figure Legend Snippet: (A-F) RNA was extracted from MDA-MB-453 tumors (shown in Fig. 4B) and sequenced (n = 3–5/group). A. Differentially expressed genes (DEGs) are visualized as a heatmap. The number of significant DEGs in each treatment compared to the vehicle control is shown below. B. GSEA analysis was performed on UT-105-treated tumors. Pathways with an FDR<0.25 are shown with corresponding normalized enrichment scores (NES). C. Enrichment plots are shown for interferon ɤ (top) and interferon α (bottom) Hallmark response pathways. D. Fold change in gene expression from RNA sequencing with MDA-MB-453 tumors for STAT signaling pathway genes IRF1, IRF7, and IRF9 is shown. E. A heatmap of chemokine normalized means from the RNA-seq data performed in MDA-MB-453 xenograft tumors. F. Western immunoblot of phospho-STAT1, phospho-STAT3, and phospho-AKT in representative samples from vehicle and UT-105-treated MDA-MB-453 xenograft tumors (from Fig 4B). G. Effect of UT-105 and ruxolitinib on STAT1 phosphorylation in MDA-MB-453 cells. MDA-MB-453 cells were maintained in csFBS-containing medium for 48 hours. Cells were treated with 3 μM UT-105 or ruxolitinib for 24 hours before the induction of STAT1 phosphorylation with interferon α (1000 units). Cells were harvested 30 minutes after induction, protein was extracted, and Western blot for pSTAT1 and GAPDH was performed. Representative blot is shown. The bands were quantified and fold change from vehicle is provided under the blots. H. Effect of UT-105 and ruxolitinib on cell proliferation. Cells were plated in growth medium and treated with vehicle, UT-105, or JAK-STAT inhibitor ruxolitinib for seven days with medium change and retreatment after day 3 (n=4/group; representative of three replicates; one-way ANOVA). SRB assay was performed. I. AR and AR-SV -positive TNBC specimens are enriched for JAK-STAT pathway. RNA was extracted from TNBC specimens (n=41) shown in Fig. 1C and sequenced. The top pathways enriched in AR and AR-SV compared to AR negative specimens obtained from GSEA (FDR <0.25) are shown as a bar graph. J. Enrichment plots are shown. Rux- ruxolitinib; enza- enzalutamide. Mean -/+ SEM: * p<0.05, ** p<0.01, *** p<0.001, **** p<0.00001.

    Techniques Used: Expressing, RNA Sequencing Assay, Western Blot, Sulforhodamine B Assay

    A. Schematic representation of CDX and PDX experiments (biorender.com). B. Left. MDA-MB-453 orthotopic xenograft was conducted by implanting 5 million cells into the mammary fat pad of female NSG mice. Once the tumors reach 100–300 mm3, the animals (n=8–10/group) were randomized and treated orally with UT-105 (60 mg/kg/day) or vehicle control (DMSO + PEG-300) for 28 days. Tumor volume was measured by digital caliper twice weekly and the percent change in tumor volume is represented in the graph. Right. MDA-MB-453 tumor-bearing female NSG mice (n=8–10/group) were treated with enzalutamide (60 mg/kg/day), bicalutamide (60 mg/kg/day), or vehicle control for 28 days. C. Change in body weight of MDA-MB-453 tumor-bearing mice. D-E. UT-1355 TNBC PDX characterization. Protein was extracted from UT-1355 PDX tumor fragments and Western blot with AR NTD-binding antibody or AR-V7 antibody, and GAPDH antibody was performed. LNCaP and 22RV1 prostate cancer cells were used as control for AR and AR-SV, respectively. Representative blots shown. F. UT-105 completely inhibits UT-1355 PDX tumor growth. UT-1355 PDX tumor fragments (1 mm3) were orthotopically implanted into the mammary fat pad in female NSG mice (n=8–10/group), and a xenograft experiment was performed as indicated above for MDA-MB-453. G-H. Change in body weight of UT-1355-bearing mice and tumor weight. Mean -/+ SEM is shown with One way ANOVA conducted in Graph Pad Prism: * p<0.05, ** p<0.01, *** p<0.001.
    Figure Legend Snippet: A. Schematic representation of CDX and PDX experiments (biorender.com). B. Left. MDA-MB-453 orthotopic xenograft was conducted by implanting 5 million cells into the mammary fat pad of female NSG mice. Once the tumors reach 100–300 mm3, the animals (n=8–10/group) were randomized and treated orally with UT-105 (60 mg/kg/day) or vehicle control (DMSO + PEG-300) for 28 days. Tumor volume was measured by digital caliper twice weekly and the percent change in tumor volume is represented in the graph. Right. MDA-MB-453 tumor-bearing female NSG mice (n=8–10/group) were treated with enzalutamide (60 mg/kg/day), bicalutamide (60 mg/kg/day), or vehicle control for 28 days. C. Change in body weight of MDA-MB-453 tumor-bearing mice. D-E. UT-1355 TNBC PDX characterization. Protein was extracted from UT-1355 PDX tumor fragments and Western blot with AR NTD-binding antibody or AR-V7 antibody, and GAPDH antibody was performed. LNCaP and 22RV1 prostate cancer cells were used as control for AR and AR-SV, respectively. Representative blots shown. F. UT-105 completely inhibits UT-1355 PDX tumor growth. UT-1355 PDX tumor fragments (1 mm3) were orthotopically implanted into the mammary fat pad in female NSG mice (n=8–10/group), and a xenograft experiment was performed as indicated above for MDA-MB-453. G-H. Change in body weight of UT-1355-bearing mice and tumor weight. Mean -/+ SEM is shown with One way ANOVA conducted in Graph Pad Prism: * p<0.05, ** p<0.01, *** p<0.001.

    Techniques Used: Western Blot, Binding Assay

    A. STAT1 increases R1881-induced AR transactivation. AR transactivation was performed in HEK-293 cells with 0.1 μg vector or STAT1. Numbers provided in bracket are R1881’s EC50 values. B. STAT1 and AR interact. AR and STAT1 (5 μg) were transfected into HEK-293 cells. Cells were treated 48 hours after transfection with 2000 IU interferon α and 10 nM R1881. Cells were harvested 4 hours after treatment, immunoprecipitation was performed with IgG or AR antibody, and Western blot was performed for STAT1. C. AR and STAT1 interact in MDA-MB-453 cells. MDA-MB-453 cells were maintained in charcoal-stripped serum-containing medium for two days and treated with interferon α and R1881 for 4 hours. Cells were fixed and immunostained for AR (red) and STAT1 (green), and confocal microscopy was performed. Scale is 2 μm. D. STAT1 is recruited to ARE (on FKBP5 regulatory region). MDA-MB-453 cells were treated as indicated. The cells were crosslinked, sheared, and ChIP PCR was performed with the STAT1 and IgG antibodies. PCR was performed using the primers for the indicated regions. E. AR is recruited to STAT1 responsive gene regulatory region. Data from AR ChIP-seq performed in PCa cells (n=2) were loaded in IGV browser and CXCL8 (IL8) and FKBP5 regulatory regions were scanned to determine the binding of AR. F. UT-105 inhibits STAT1 recruitment to STAT1RE and ARE. MDA-MB-453 cells maintained in charcoal-stripped serum-containing medium were treated with interferon α (1000 units), R1881 (1 nM), combination in the presence and absence of UT-105 (10 μM) for 4 hours. ChIP assay was performed with STAT1 antibody and real-time PCR was performed with primers specific for hIL8 STATRE and FKBP5 ARE. Representative experiment is shown in the figure. G. Ruxolitinib inhibits AR-target gene expression. MDA-MB-453 cells maintained in charcoal stripped serum-containing medium were treated with R1881 (0.1 nM) alone or in combination with ruxolitinib (3 μM) for 24 hours. RNA was extracted, and real-time PCR for FKBP5 was performed (n=4/group; one-way ANOVA). H. UT-105 inhibits STAT1-dependent coactivation of AR. AR transactivation was performed in HEK-293 cells in the presence or absence of 0.1 μg STAT1. I. Effect of AR and JAK inhibitors on the proliferation of patient tumors explants growth. Illustration of gelatin sponge culture (Biorender.com). Tumor tissue from two patients (1473 and 1474) were placed on pre-soaked gelatin sponges and treated as indicated for 48 hours. The tissues were fixed and stained with Ki67. The bar graphs show the percentage of cells that stained positively for Ki67. J. Model. A schematic of the proposed mechanism of action of SARDs in TNBC tumors (biorender.com). The model summarizes the choice of drug for inhibiting the growth of LAR TNBC tumors. This would depend on the specific mechanisms involved in AR activation and tumor progression. Enzalutamide may work if AR activity is the primary driver, but its efficacy could be limited by androgen surges. STAT inhibitors like ruxolitinib may not be effective if AR activation is independent of STAT1. Degraders like UT-105 seem promising as they degrade AR directly and could prevent AR or AR splice variant activation, offering a more comprehensive approach to inhibit tumor growth. However, the actual effectiveness of these drugs would need to be studied in clinical settings and may vary from patient to patient. Panels A-H representative of three independent experiments is shown.
    Figure Legend Snippet: A. STAT1 increases R1881-induced AR transactivation. AR transactivation was performed in HEK-293 cells with 0.1 μg vector or STAT1. Numbers provided in bracket are R1881’s EC50 values. B. STAT1 and AR interact. AR and STAT1 (5 μg) were transfected into HEK-293 cells. Cells were treated 48 hours after transfection with 2000 IU interferon α and 10 nM R1881. Cells were harvested 4 hours after treatment, immunoprecipitation was performed with IgG or AR antibody, and Western blot was performed for STAT1. C. AR and STAT1 interact in MDA-MB-453 cells. MDA-MB-453 cells were maintained in charcoal-stripped serum-containing medium for two days and treated with interferon α and R1881 for 4 hours. Cells were fixed and immunostained for AR (red) and STAT1 (green), and confocal microscopy was performed. Scale is 2 μm. D. STAT1 is recruited to ARE (on FKBP5 regulatory region). MDA-MB-453 cells were treated as indicated. The cells were crosslinked, sheared, and ChIP PCR was performed with the STAT1 and IgG antibodies. PCR was performed using the primers for the indicated regions. E. AR is recruited to STAT1 responsive gene regulatory region. Data from AR ChIP-seq performed in PCa cells (n=2) were loaded in IGV browser and CXCL8 (IL8) and FKBP5 regulatory regions were scanned to determine the binding of AR. F. UT-105 inhibits STAT1 recruitment to STAT1RE and ARE. MDA-MB-453 cells maintained in charcoal-stripped serum-containing medium were treated with interferon α (1000 units), R1881 (1 nM), combination in the presence and absence of UT-105 (10 μM) for 4 hours. ChIP assay was performed with STAT1 antibody and real-time PCR was performed with primers specific for hIL8 STATRE and FKBP5 ARE. Representative experiment is shown in the figure. G. Ruxolitinib inhibits AR-target gene expression. MDA-MB-453 cells maintained in charcoal stripped serum-containing medium were treated with R1881 (0.1 nM) alone or in combination with ruxolitinib (3 μM) for 24 hours. RNA was extracted, and real-time PCR for FKBP5 was performed (n=4/group; one-way ANOVA). H. UT-105 inhibits STAT1-dependent coactivation of AR. AR transactivation was performed in HEK-293 cells in the presence or absence of 0.1 μg STAT1. I. Effect of AR and JAK inhibitors on the proliferation of patient tumors explants growth. Illustration of gelatin sponge culture (Biorender.com). Tumor tissue from two patients (1473 and 1474) were placed on pre-soaked gelatin sponges and treated as indicated for 48 hours. The tissues were fixed and stained with Ki67. The bar graphs show the percentage of cells that stained positively for Ki67. J. Model. A schematic of the proposed mechanism of action of SARDs in TNBC tumors (biorender.com). The model summarizes the choice of drug for inhibiting the growth of LAR TNBC tumors. This would depend on the specific mechanisms involved in AR activation and tumor progression. Enzalutamide may work if AR activity is the primary driver, but its efficacy could be limited by androgen surges. STAT inhibitors like ruxolitinib may not be effective if AR activation is independent of STAT1. Degraders like UT-105 seem promising as they degrade AR directly and could prevent AR or AR splice variant activation, offering a more comprehensive approach to inhibit tumor growth. However, the actual effectiveness of these drugs would need to be studied in clinical settings and may vary from patient to patient. Panels A-H representative of three independent experiments is shown.

    Techniques Used: Plasmid Preparation, Transfection, Immunoprecipitation, Western Blot, Confocal Microscopy, ChIP-sequencing, Binding Assay, Real-time Polymerase Chain Reaction, Expressing, Staining, Activation Assay, Activity Assay, Variant Assay

    Key resources table
    Figure Legend Snippet: Key resources table

    Techniques Used: Virus, Plasmid Preparation, Recombinant, RNA Sequencing Assay, Synthesized, Expressing, Construct, Software

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    ATCC mda mb 453 ar v7 geo database gse244283 spatial genomics geo database gse245202 mda mb 453 xenograft geo database gse244283
    A. Structure of AR and AR-SV (biorender.com). B. Kaplan Meier relapse free survival plot of patients with high (red) and low (black) AR (211621_at) expression (kmplot.com). C. Patient demographics. D-G. AR and AR-SV gene expression in specimens from TNBC patients. RNA was extracted (n=52) from TNBC patient specimens and real-time PCR was performed with AR-NTD -binding probe (D), AR-NTD - and LBD-binding taqman probes (F), and <t>AR-V7</t> probe (G). IHC was performed with AR NTD -binding antibody (E). Percent of patients positive for AR (>10% cells positive for AR) is shown at the bottom of the graph. H. Flow chart of breast cancer subtypes and TNBC subtypes, including AR-SV-positive TNBC. IHC- immunohistochemistry; NTD- N-terminus domain; CTD- C-terminus domain; AA- African American; CA-Caucasian American; DBD- DNA binding domain; Hin- Hinge; LBD- Ligand Binding Domain; U- Unique cryptic exon; AF-1- Activation Function-1 Domain; AR-FL- androgen receptor full length; AR-SV- androgen receptor splice variant; RV1– 22RV1 prostate cancer cells; LN- LNCaP prostate cancer cells. Values are expressed as mean -/+ SEM. *-p<0.05; **-p<0.01; ***-p<0.001; ****-p<0.0001 (t-test).
    Mda Mb 453 Ar V7 Geo Database Gse244283 Spatial Genomics Geo Database Gse245202 Mda Mb 453 Xenograft Geo Database Gse244283, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    A. Structure of AR and AR-SV (biorender.com). B. Kaplan Meier relapse free survival plot of patients with high (red) and low (black) AR (211621_at) expression (kmplot.com). C. Patient demographics. D-G. AR and AR-SV gene expression in specimens from TNBC patients. RNA was extracted (n=52) from TNBC patient specimens and real-time PCR was performed with AR-NTD -binding probe (D), AR-NTD - and LBD-binding taqman probes (F), and AR-V7 probe (G). IHC was performed with AR NTD -binding antibody (E). Percent of patients positive for AR (>10% cells positive for AR) is shown at the bottom of the graph. H. Flow chart of breast cancer subtypes and TNBC subtypes, including AR-SV-positive TNBC. IHC- immunohistochemistry; NTD- N-terminus domain; CTD- C-terminus domain; AA- African American; CA-Caucasian American; DBD- DNA binding domain; Hin- Hinge; LBD- Ligand Binding Domain; U- Unique cryptic exon; AF-1- Activation Function-1 Domain; AR-FL- androgen receptor full length; AR-SV- androgen receptor splice variant; RV1– 22RV1 prostate cancer cells; LN- LNCaP prostate cancer cells. Values are expressed as mean -/+ SEM. *-p<0.05; **-p<0.01; ***-p<0.001; ****-p<0.0001 (t-test).

    Journal: Cell reports

    Article Title: Identification of a Targetable JAK-STAT Enriched Androgen Receptor (AR) and AR Splice Variant Positive Triple Negative Breast Cancer Subtype

    doi: 10.1016/j.celrep.2023.113461

    Figure Lengend Snippet: A. Structure of AR and AR-SV (biorender.com). B. Kaplan Meier relapse free survival plot of patients with high (red) and low (black) AR (211621_at) expression (kmplot.com). C. Patient demographics. D-G. AR and AR-SV gene expression in specimens from TNBC patients. RNA was extracted (n=52) from TNBC patient specimens and real-time PCR was performed with AR-NTD -binding probe (D), AR-NTD - and LBD-binding taqman probes (F), and AR-V7 probe (G). IHC was performed with AR NTD -binding antibody (E). Percent of patients positive for AR (>10% cells positive for AR) is shown at the bottom of the graph. H. Flow chart of breast cancer subtypes and TNBC subtypes, including AR-SV-positive TNBC. IHC- immunohistochemistry; NTD- N-terminus domain; CTD- C-terminus domain; AA- African American; CA-Caucasian American; DBD- DNA binding domain; Hin- Hinge; LBD- Ligand Binding Domain; U- Unique cryptic exon; AF-1- Activation Function-1 Domain; AR-FL- androgen receptor full length; AR-SV- androgen receptor splice variant; RV1– 22RV1 prostate cancer cells; LN- LNCaP prostate cancer cells. Values are expressed as mean -/+ SEM. *-p<0.05; **-p<0.01; ***-p<0.001; ****-p<0.0001 (t-test).

    Article Snippet: Biological samples 52 TNBC Patient specimens IRB 14–03113-XP UTHSC N/A PDX UT-1355 IRB 14–03113-XP UTHSC N/A Chemicals, peptides, and recombinant proteins Enzalutamide Medkoo 201821 Bicalutamide AK Scientific 90357–06-5 Ruxlotinib AmBeed A272323 Interferon Gift from Dr. Pfeffer (which was a generous gift from Amgen) 84 N/A BrDU Cell signaling 6813s R1881 Sigma R0908–10MG Sypro Orange Thermofisher S6650 Matrigel fisherscientific 8774552 Gelatin dental sponge (Vetspon Dental Cubes) fisherscientific NC0654350 Critical commercial assays Kinome Scan DiscoverX Eurofins N/A GPCR Scan DiscoverX Eurofins N/A Deposited data RNA Seq for MDA-MB-453 and MDA-MB-453-AR-V7 GEO database GSE244283 Spatial genomics GEO database GSE245202 MDA-MB-453 xenograft GEO database GSE244283 41 patient specimens GEO database GSE244283 MDA-MB-453 cell line RNA sequencing GEO database GSE245554 Experimental models: Cell lines LNCaP ATCC CRL-1740 22RV1 ATCC CRL-2505 MDA-MB-453 ATCC HTB-131 MDA-MB-231 ATCC CRM-HTB-26 BT549 ATCC HTB-122 PC3 ATCC CRL-1435 MFM223 Sigma Aldrich SKU 98050130–1VL Experimental models: Organisms/strains NSG mice JAX labs 005557 Sprague Dawley rats Charles River N/A Oligonucleotides TaqMan primers and probe Androgen Receptor N-terminus Life Technologies Hs00907242_m1 TaqMan primers and probe Androgen Receptor C-terminus Life Technologies Hs00171172_m1 TaqMan primers and probe FKBP5 Life Technologies Hs00188025_m1 TaqMan primers and probe TMPRSS2 Life Technologies Hs00237175_m1 TaqMan primers and probe STEAP4 Life Technologies Hs01026584_m1 TaqMan primers and probe IRF-1 Life Technologies Hs00971965_m1 TaqMan primers and probe IRF-7 Life Technologies Hs01014809_g1 TaqMan primers and probe IRF-9 Life Technologies Hs00196051_m1 TaqMan primers and probe GAPDH Life Technologies Hs00266705_g1 Primers and probe AR-V7 Custom synthesized Recombinant DNA STAT-1 plasmid Gift from Dr. Pfeffer N/A Androgen receptor Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A GRE-LUC Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A Glucocorticoid receptor Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A Mineralocorticoid receptor Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A STAT-1RE LUC James E. Darnell, Rockefeller University N/A AR-LBD bacterial expression vector Constructed in our lab. N/A AF-1 bacterial expression vector Constructed in our lab. N/A AR-V7 bacterial expression vector Constructed in our lab. N/A Software and algorithms Other Open in a separate window Key resources table.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Binding Assay, Immunohistochemistry, Ligand Binding Assay, Activation Assay, Variant Assay

    TNBC cell lines were stably transfected with AR-V7 using lentivirus. A. BrdU assay of control and AR-V7 lentivirus transfected TNBC cell lines MDA-MB-453 (453), MDA-MB-231 (231), BT549, and MFM223. Cells were plated in growth medium and BrDU assay was performed after 72 hours (n=4/group). B. Scratch assay in MDA-MB-453 and MDA-MB-453-V7. Cells were plated in growth medium and imaged at the start and after 72 hours, and the gap closure measured by imaging software (n=4/group). C. Ki67 staining of TNBC patient specimens (23 (AR-positive (AR+)), and 11 (AR and AR-SV -positive (AR+/AR-SV+)). D and E. RNA sequencing was performed with 453 and 453-V7 cell lines and AR+ and AR+/AR-SV+ patient specimens. Gene set from the Molecular Signatures Database is reported in 453 cells compared to 453-V7 cells and AR+ patient specimens compared to AR+/AR-SV+ patient specimens. Values are expressed as mean -/+ SEM. Experiments in panels A-C were reproduced at least three times and representative experiment is shown. * p<0.05, ** p<0.01, ****p<0.00001 (t-test).

    Journal: Cell reports

    Article Title: Identification of a Targetable JAK-STAT Enriched Androgen Receptor (AR) and AR Splice Variant Positive Triple Negative Breast Cancer Subtype

    doi: 10.1016/j.celrep.2023.113461

    Figure Lengend Snippet: TNBC cell lines were stably transfected with AR-V7 using lentivirus. A. BrdU assay of control and AR-V7 lentivirus transfected TNBC cell lines MDA-MB-453 (453), MDA-MB-231 (231), BT549, and MFM223. Cells were plated in growth medium and BrDU assay was performed after 72 hours (n=4/group). B. Scratch assay in MDA-MB-453 and MDA-MB-453-V7. Cells were plated in growth medium and imaged at the start and after 72 hours, and the gap closure measured by imaging software (n=4/group). C. Ki67 staining of TNBC patient specimens (23 (AR-positive (AR+)), and 11 (AR and AR-SV -positive (AR+/AR-SV+)). D and E. RNA sequencing was performed with 453 and 453-V7 cell lines and AR+ and AR+/AR-SV+ patient specimens. Gene set from the Molecular Signatures Database is reported in 453 cells compared to 453-V7 cells and AR+ patient specimens compared to AR+/AR-SV+ patient specimens. Values are expressed as mean -/+ SEM. Experiments in panels A-C were reproduced at least three times and representative experiment is shown. * p<0.05, ** p<0.01, ****p<0.00001 (t-test).

    Article Snippet: Biological samples 52 TNBC Patient specimens IRB 14–03113-XP UTHSC N/A PDX UT-1355 IRB 14–03113-XP UTHSC N/A Chemicals, peptides, and recombinant proteins Enzalutamide Medkoo 201821 Bicalutamide AK Scientific 90357–06-5 Ruxlotinib AmBeed A272323 Interferon Gift from Dr. Pfeffer (which was a generous gift from Amgen) 84 N/A BrDU Cell signaling 6813s R1881 Sigma R0908–10MG Sypro Orange Thermofisher S6650 Matrigel fisherscientific 8774552 Gelatin dental sponge (Vetspon Dental Cubes) fisherscientific NC0654350 Critical commercial assays Kinome Scan DiscoverX Eurofins N/A GPCR Scan DiscoverX Eurofins N/A Deposited data RNA Seq for MDA-MB-453 and MDA-MB-453-AR-V7 GEO database GSE244283 Spatial genomics GEO database GSE245202 MDA-MB-453 xenograft GEO database GSE244283 41 patient specimens GEO database GSE244283 MDA-MB-453 cell line RNA sequencing GEO database GSE245554 Experimental models: Cell lines LNCaP ATCC CRL-1740 22RV1 ATCC CRL-2505 MDA-MB-453 ATCC HTB-131 MDA-MB-231 ATCC CRM-HTB-26 BT549 ATCC HTB-122 PC3 ATCC CRL-1435 MFM223 Sigma Aldrich SKU 98050130–1VL Experimental models: Organisms/strains NSG mice JAX labs 005557 Sprague Dawley rats Charles River N/A Oligonucleotides TaqMan primers and probe Androgen Receptor N-terminus Life Technologies Hs00907242_m1 TaqMan primers and probe Androgen Receptor C-terminus Life Technologies Hs00171172_m1 TaqMan primers and probe FKBP5 Life Technologies Hs00188025_m1 TaqMan primers and probe TMPRSS2 Life Technologies Hs00237175_m1 TaqMan primers and probe STEAP4 Life Technologies Hs01026584_m1 TaqMan primers and probe IRF-1 Life Technologies Hs00971965_m1 TaqMan primers and probe IRF-7 Life Technologies Hs01014809_g1 TaqMan primers and probe IRF-9 Life Technologies Hs00196051_m1 TaqMan primers and probe GAPDH Life Technologies Hs00266705_g1 Primers and probe AR-V7 Custom synthesized Recombinant DNA STAT-1 plasmid Gift from Dr. Pfeffer N/A Androgen receptor Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A GRE-LUC Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A Glucocorticoid receptor Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A Mineralocorticoid receptor Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A STAT-1RE LUC James E. Darnell, Rockefeller University N/A AR-LBD bacterial expression vector Constructed in our lab. N/A AF-1 bacterial expression vector Constructed in our lab. N/A AR-V7 bacterial expression vector Constructed in our lab. N/A Software and algorithms Other Open in a separate window Key resources table.

    Techniques: Stable Transfection, Transfection, BrdU Staining, Wound Healing Assay, Imaging, Software, Staining, RNA Sequencing Assay

    A. Structure of UT-105. B. UT-105 inhibits wildtype and mutant AR transactivation. AR or AR F876L (50 ng), 0.25 μg GRE-LUC, and 10 ng CMV-renilla-LUC were transfected into COS7 cells. Cells were treated 24 hours after transfection with a dose response of UT-105 or enzalutamide in the presence of 0.1 nM R1881, and luciferase assay was performed 24 hours after treatment. Firefly luciferase values were normalized to renilla luciferase. Numbers provided in bracket are IC50 values. C. UT-105 degrades AR. LNCaP PCa cells were maintained in charcoal-stripped FBS-containing medium for 2 days before treating with UT-105 in the presence of 0.1 nM R1881. Cells were harvested 24 hours after treatment, and Western blot for AR and GAPDH was performed. D. Top. UT-105 binds to AR-NTD. Recombinant purified AR NTD was incubated with DMSO or 10 μM UT-105 overnight at 4°C. SYPRO orange dye was added to the mixture and a PCR was performed with increasing temperature. SYPRO orange signal was monitored. Bottom. UT-105 stabilized purified AF-1 and AR-V7 recombinant protein. Recombinant purified AF-1 or AR-V7 protein (5 ng) were incubated at room temperature for 4 hours with DMSO or 100 μM of UT-105 or UT-34. Proteins were fractionated on an SDS-PAGE and Western blot was performed with AR antibody (AR-441). E. Illustration depicting the binding regions of UT-105 and enzalutamide. F. UT-105 irreversibly inhibits AR. Transactivation assay was performed with AR with a dose response of R1881 in the presence of 3 and 10 μM UT-105 as indicated in panel B. G. UT-105 inhibits AR-target genes. MDA-MB-453 cells in charcoal stripped serum-containing medium were treated for 16–20 hours. RNA was extracted and expression of FKBP5, TMPRSS2, and STEAP4 was quantified by real-time PCR and normalized to GAPDH (n=4/group). * p<0.05 (one-way ANOVA) H. RNA-seq. MDA-MB-453 cells maintained in charcoal-stripped serum-containing medium for 2 days were treated with vehicle or 3 μM UT-105 in the presence of 0.1 nM R1881 for 20 hours (n=3/group). Cells were harvested, RNA extracted, and sequenced. Heatmap of global gene expression changes, top GSEA pathways enriched, and heatmap and bar graphs of AR signaling pathway are represented. I. UT-105 inhibits proliferation of TNBC cells. MDA-MB-453 cells plated in charcoal stripped serum-containing medium and treated for seven days with medium change and retreatment after day 3 (n=3/group). Sulforhodamine B (SRB) colorimetric assay was performed to measure cell viability. J. UT-105 inhibits clonogenicity. TNBC cells stably expressing AR-V7 were plated in 6-well plates and treated with 10 μM of the indicated compounds for two weeks. Colonies were imaged and the number of colonies formed was counted using an imaging software (n=4/group). Panels B-D and F, G, I-J are representatives of at least three independent replicates. * p<0.05, ** p<0.01 (one-way ANOVA). Enza- enzalutamide; V7- AR-V7 splice variant. Values are expressed as mean -/+ SEM.

    Journal: Cell reports

    Article Title: Identification of a Targetable JAK-STAT Enriched Androgen Receptor (AR) and AR Splice Variant Positive Triple Negative Breast Cancer Subtype

    doi: 10.1016/j.celrep.2023.113461

    Figure Lengend Snippet: A. Structure of UT-105. B. UT-105 inhibits wildtype and mutant AR transactivation. AR or AR F876L (50 ng), 0.25 μg GRE-LUC, and 10 ng CMV-renilla-LUC were transfected into COS7 cells. Cells were treated 24 hours after transfection with a dose response of UT-105 or enzalutamide in the presence of 0.1 nM R1881, and luciferase assay was performed 24 hours after treatment. Firefly luciferase values were normalized to renilla luciferase. Numbers provided in bracket are IC50 values. C. UT-105 degrades AR. LNCaP PCa cells were maintained in charcoal-stripped FBS-containing medium for 2 days before treating with UT-105 in the presence of 0.1 nM R1881. Cells were harvested 24 hours after treatment, and Western blot for AR and GAPDH was performed. D. Top. UT-105 binds to AR-NTD. Recombinant purified AR NTD was incubated with DMSO or 10 μM UT-105 overnight at 4°C. SYPRO orange dye was added to the mixture and a PCR was performed with increasing temperature. SYPRO orange signal was monitored. Bottom. UT-105 stabilized purified AF-1 and AR-V7 recombinant protein. Recombinant purified AF-1 or AR-V7 protein (5 ng) were incubated at room temperature for 4 hours with DMSO or 100 μM of UT-105 or UT-34. Proteins were fractionated on an SDS-PAGE and Western blot was performed with AR antibody (AR-441). E. Illustration depicting the binding regions of UT-105 and enzalutamide. F. UT-105 irreversibly inhibits AR. Transactivation assay was performed with AR with a dose response of R1881 in the presence of 3 and 10 μM UT-105 as indicated in panel B. G. UT-105 inhibits AR-target genes. MDA-MB-453 cells in charcoal stripped serum-containing medium were treated for 16–20 hours. RNA was extracted and expression of FKBP5, TMPRSS2, and STEAP4 was quantified by real-time PCR and normalized to GAPDH (n=4/group). * p<0.05 (one-way ANOVA) H. RNA-seq. MDA-MB-453 cells maintained in charcoal-stripped serum-containing medium for 2 days were treated with vehicle or 3 μM UT-105 in the presence of 0.1 nM R1881 for 20 hours (n=3/group). Cells were harvested, RNA extracted, and sequenced. Heatmap of global gene expression changes, top GSEA pathways enriched, and heatmap and bar graphs of AR signaling pathway are represented. I. UT-105 inhibits proliferation of TNBC cells. MDA-MB-453 cells plated in charcoal stripped serum-containing medium and treated for seven days with medium change and retreatment after day 3 (n=3/group). Sulforhodamine B (SRB) colorimetric assay was performed to measure cell viability. J. UT-105 inhibits clonogenicity. TNBC cells stably expressing AR-V7 were plated in 6-well plates and treated with 10 μM of the indicated compounds for two weeks. Colonies were imaged and the number of colonies formed was counted using an imaging software (n=4/group). Panels B-D and F, G, I-J are representatives of at least three independent replicates. * p<0.05, ** p<0.01 (one-way ANOVA). Enza- enzalutamide; V7- AR-V7 splice variant. Values are expressed as mean -/+ SEM.

    Article Snippet: Biological samples 52 TNBC Patient specimens IRB 14–03113-XP UTHSC N/A PDX UT-1355 IRB 14–03113-XP UTHSC N/A Chemicals, peptides, and recombinant proteins Enzalutamide Medkoo 201821 Bicalutamide AK Scientific 90357–06-5 Ruxlotinib AmBeed A272323 Interferon Gift from Dr. Pfeffer (which was a generous gift from Amgen) 84 N/A BrDU Cell signaling 6813s R1881 Sigma R0908–10MG Sypro Orange Thermofisher S6650 Matrigel fisherscientific 8774552 Gelatin dental sponge (Vetspon Dental Cubes) fisherscientific NC0654350 Critical commercial assays Kinome Scan DiscoverX Eurofins N/A GPCR Scan DiscoverX Eurofins N/A Deposited data RNA Seq for MDA-MB-453 and MDA-MB-453-AR-V7 GEO database GSE244283 Spatial genomics GEO database GSE245202 MDA-MB-453 xenograft GEO database GSE244283 41 patient specimens GEO database GSE244283 MDA-MB-453 cell line RNA sequencing GEO database GSE245554 Experimental models: Cell lines LNCaP ATCC CRL-1740 22RV1 ATCC CRL-2505 MDA-MB-453 ATCC HTB-131 MDA-MB-231 ATCC CRM-HTB-26 BT549 ATCC HTB-122 PC3 ATCC CRL-1435 MFM223 Sigma Aldrich SKU 98050130–1VL Experimental models: Organisms/strains NSG mice JAX labs 005557 Sprague Dawley rats Charles River N/A Oligonucleotides TaqMan primers and probe Androgen Receptor N-terminus Life Technologies Hs00907242_m1 TaqMan primers and probe Androgen Receptor C-terminus Life Technologies Hs00171172_m1 TaqMan primers and probe FKBP5 Life Technologies Hs00188025_m1 TaqMan primers and probe TMPRSS2 Life Technologies Hs00237175_m1 TaqMan primers and probe STEAP4 Life Technologies Hs01026584_m1 TaqMan primers and probe IRF-1 Life Technologies Hs00971965_m1 TaqMan primers and probe IRF-7 Life Technologies Hs01014809_g1 TaqMan primers and probe IRF-9 Life Technologies Hs00196051_m1 TaqMan primers and probe GAPDH Life Technologies Hs00266705_g1 Primers and probe AR-V7 Custom synthesized Recombinant DNA STAT-1 plasmid Gift from Dr. Pfeffer N/A Androgen receptor Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A GRE-LUC Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A Glucocorticoid receptor Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A Mineralocorticoid receptor Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A STAT-1RE LUC James E. Darnell, Rockefeller University N/A AR-LBD bacterial expression vector Constructed in our lab. N/A AF-1 bacterial expression vector Constructed in our lab. N/A AR-V7 bacterial expression vector Constructed in our lab. N/A Software and algorithms Other Open in a separate window Key resources table.

    Techniques: Mutagenesis, Transfection, Luciferase, Western Blot, Recombinant, Purification, Incubation, SDS Page, Binding Assay, Transactivation Assay, Expressing, Real-time Polymerase Chain Reaction, RNA Sequencing Assay, Colorimetric Assay, Stable Transfection, Imaging, Software, Variant Assay

    (A-F) RNA was extracted from MDA-MB-453 tumors (shown in Fig. 4B) and sequenced (n = 3–5/group). A. Differentially expressed genes (DEGs) are visualized as a heatmap. The number of significant DEGs in each treatment compared to the vehicle control is shown below. B. GSEA analysis was performed on UT-105-treated tumors. Pathways with an FDR<0.25 are shown with corresponding normalized enrichment scores (NES). C. Enrichment plots are shown for interferon ɤ (top) and interferon α (bottom) Hallmark response pathways. D. Fold change in gene expression from RNA sequencing with MDA-MB-453 tumors for STAT signaling pathway genes IRF1, IRF7, and IRF9 is shown. E. A heatmap of chemokine normalized means from the RNA-seq data performed in MDA-MB-453 xenograft tumors. F. Western immunoblot of phospho-STAT1, phospho-STAT3, and phospho-AKT in representative samples from vehicle and UT-105-treated MDA-MB-453 xenograft tumors (from Fig 4B). G. Effect of UT-105 and ruxolitinib on STAT1 phosphorylation in MDA-MB-453 cells. MDA-MB-453 cells were maintained in csFBS-containing medium for 48 hours. Cells were treated with 3 μM UT-105 or ruxolitinib for 24 hours before the induction of STAT1 phosphorylation with interferon α (1000 units). Cells were harvested 30 minutes after induction, protein was extracted, and Western blot for pSTAT1 and GAPDH was performed. Representative blot is shown. The bands were quantified and fold change from vehicle is provided under the blots. H. Effect of UT-105 and ruxolitinib on cell proliferation. Cells were plated in growth medium and treated with vehicle, UT-105, or JAK-STAT inhibitor ruxolitinib for seven days with medium change and retreatment after day 3 (n=4/group; representative of three replicates; one-way ANOVA). SRB assay was performed. I. AR and AR-SV -positive TNBC specimens are enriched for JAK-STAT pathway. RNA was extracted from TNBC specimens (n=41) shown in Fig. 1C and sequenced. The top pathways enriched in AR and AR-SV compared to AR negative specimens obtained from GSEA (FDR <0.25) are shown as a bar graph. J. Enrichment plots are shown. Rux- ruxolitinib; enza- enzalutamide. Mean -/+ SEM: * p<0.05, ** p<0.01, *** p<0.001, **** p<0.00001.

    Journal: Cell reports

    Article Title: Identification of a Targetable JAK-STAT Enriched Androgen Receptor (AR) and AR Splice Variant Positive Triple Negative Breast Cancer Subtype

    doi: 10.1016/j.celrep.2023.113461

    Figure Lengend Snippet: (A-F) RNA was extracted from MDA-MB-453 tumors (shown in Fig. 4B) and sequenced (n = 3–5/group). A. Differentially expressed genes (DEGs) are visualized as a heatmap. The number of significant DEGs in each treatment compared to the vehicle control is shown below. B. GSEA analysis was performed on UT-105-treated tumors. Pathways with an FDR<0.25 are shown with corresponding normalized enrichment scores (NES). C. Enrichment plots are shown for interferon ɤ (top) and interferon α (bottom) Hallmark response pathways. D. Fold change in gene expression from RNA sequencing with MDA-MB-453 tumors for STAT signaling pathway genes IRF1, IRF7, and IRF9 is shown. E. A heatmap of chemokine normalized means from the RNA-seq data performed in MDA-MB-453 xenograft tumors. F. Western immunoblot of phospho-STAT1, phospho-STAT3, and phospho-AKT in representative samples from vehicle and UT-105-treated MDA-MB-453 xenograft tumors (from Fig 4B). G. Effect of UT-105 and ruxolitinib on STAT1 phosphorylation in MDA-MB-453 cells. MDA-MB-453 cells were maintained in csFBS-containing medium for 48 hours. Cells were treated with 3 μM UT-105 or ruxolitinib for 24 hours before the induction of STAT1 phosphorylation with interferon α (1000 units). Cells were harvested 30 minutes after induction, protein was extracted, and Western blot for pSTAT1 and GAPDH was performed. Representative blot is shown. The bands were quantified and fold change from vehicle is provided under the blots. H. Effect of UT-105 and ruxolitinib on cell proliferation. Cells were plated in growth medium and treated with vehicle, UT-105, or JAK-STAT inhibitor ruxolitinib for seven days with medium change and retreatment after day 3 (n=4/group; representative of three replicates; one-way ANOVA). SRB assay was performed. I. AR and AR-SV -positive TNBC specimens are enriched for JAK-STAT pathway. RNA was extracted from TNBC specimens (n=41) shown in Fig. 1C and sequenced. The top pathways enriched in AR and AR-SV compared to AR negative specimens obtained from GSEA (FDR <0.25) are shown as a bar graph. J. Enrichment plots are shown. Rux- ruxolitinib; enza- enzalutamide. Mean -/+ SEM: * p<0.05, ** p<0.01, *** p<0.001, **** p<0.00001.

    Article Snippet: Biological samples 52 TNBC Patient specimens IRB 14–03113-XP UTHSC N/A PDX UT-1355 IRB 14–03113-XP UTHSC N/A Chemicals, peptides, and recombinant proteins Enzalutamide Medkoo 201821 Bicalutamide AK Scientific 90357–06-5 Ruxlotinib AmBeed A272323 Interferon Gift from Dr. Pfeffer (which was a generous gift from Amgen) 84 N/A BrDU Cell signaling 6813s R1881 Sigma R0908–10MG Sypro Orange Thermofisher S6650 Matrigel fisherscientific 8774552 Gelatin dental sponge (Vetspon Dental Cubes) fisherscientific NC0654350 Critical commercial assays Kinome Scan DiscoverX Eurofins N/A GPCR Scan DiscoverX Eurofins N/A Deposited data RNA Seq for MDA-MB-453 and MDA-MB-453-AR-V7 GEO database GSE244283 Spatial genomics GEO database GSE245202 MDA-MB-453 xenograft GEO database GSE244283 41 patient specimens GEO database GSE244283 MDA-MB-453 cell line RNA sequencing GEO database GSE245554 Experimental models: Cell lines LNCaP ATCC CRL-1740 22RV1 ATCC CRL-2505 MDA-MB-453 ATCC HTB-131 MDA-MB-231 ATCC CRM-HTB-26 BT549 ATCC HTB-122 PC3 ATCC CRL-1435 MFM223 Sigma Aldrich SKU 98050130–1VL Experimental models: Organisms/strains NSG mice JAX labs 005557 Sprague Dawley rats Charles River N/A Oligonucleotides TaqMan primers and probe Androgen Receptor N-terminus Life Technologies Hs00907242_m1 TaqMan primers and probe Androgen Receptor C-terminus Life Technologies Hs00171172_m1 TaqMan primers and probe FKBP5 Life Technologies Hs00188025_m1 TaqMan primers and probe TMPRSS2 Life Technologies Hs00237175_m1 TaqMan primers and probe STEAP4 Life Technologies Hs01026584_m1 TaqMan primers and probe IRF-1 Life Technologies Hs00971965_m1 TaqMan primers and probe IRF-7 Life Technologies Hs01014809_g1 TaqMan primers and probe IRF-9 Life Technologies Hs00196051_m1 TaqMan primers and probe GAPDH Life Technologies Hs00266705_g1 Primers and probe AR-V7 Custom synthesized Recombinant DNA STAT-1 plasmid Gift from Dr. Pfeffer N/A Androgen receptor Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A GRE-LUC Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A Glucocorticoid receptor Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A Mineralocorticoid receptor Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A STAT-1RE LUC James E. Darnell, Rockefeller University N/A AR-LBD bacterial expression vector Constructed in our lab. N/A AF-1 bacterial expression vector Constructed in our lab. N/A AR-V7 bacterial expression vector Constructed in our lab. N/A Software and algorithms Other Open in a separate window Key resources table.

    Techniques: Expressing, RNA Sequencing Assay, Western Blot, Sulforhodamine B Assay

    A. Schematic representation of CDX and PDX experiments (biorender.com). B. Left. MDA-MB-453 orthotopic xenograft was conducted by implanting 5 million cells into the mammary fat pad of female NSG mice. Once the tumors reach 100–300 mm3, the animals (n=8–10/group) were randomized and treated orally with UT-105 (60 mg/kg/day) or vehicle control (DMSO + PEG-300) for 28 days. Tumor volume was measured by digital caliper twice weekly and the percent change in tumor volume is represented in the graph. Right. MDA-MB-453 tumor-bearing female NSG mice (n=8–10/group) were treated with enzalutamide (60 mg/kg/day), bicalutamide (60 mg/kg/day), or vehicle control for 28 days. C. Change in body weight of MDA-MB-453 tumor-bearing mice. D-E. UT-1355 TNBC PDX characterization. Protein was extracted from UT-1355 PDX tumor fragments and Western blot with AR NTD-binding antibody or AR-V7 antibody, and GAPDH antibody was performed. LNCaP and 22RV1 prostate cancer cells were used as control for AR and AR-SV, respectively. Representative blots shown. F. UT-105 completely inhibits UT-1355 PDX tumor growth. UT-1355 PDX tumor fragments (1 mm3) were orthotopically implanted into the mammary fat pad in female NSG mice (n=8–10/group), and a xenograft experiment was performed as indicated above for MDA-MB-453. G-H. Change in body weight of UT-1355-bearing mice and tumor weight. Mean -/+ SEM is shown with One way ANOVA conducted in Graph Pad Prism: * p<0.05, ** p<0.01, *** p<0.001.

    Journal: Cell reports

    Article Title: Identification of a Targetable JAK-STAT Enriched Androgen Receptor (AR) and AR Splice Variant Positive Triple Negative Breast Cancer Subtype

    doi: 10.1016/j.celrep.2023.113461

    Figure Lengend Snippet: A. Schematic representation of CDX and PDX experiments (biorender.com). B. Left. MDA-MB-453 orthotopic xenograft was conducted by implanting 5 million cells into the mammary fat pad of female NSG mice. Once the tumors reach 100–300 mm3, the animals (n=8–10/group) were randomized and treated orally with UT-105 (60 mg/kg/day) or vehicle control (DMSO + PEG-300) for 28 days. Tumor volume was measured by digital caliper twice weekly and the percent change in tumor volume is represented in the graph. Right. MDA-MB-453 tumor-bearing female NSG mice (n=8–10/group) were treated with enzalutamide (60 mg/kg/day), bicalutamide (60 mg/kg/day), or vehicle control for 28 days. C. Change in body weight of MDA-MB-453 tumor-bearing mice. D-E. UT-1355 TNBC PDX characterization. Protein was extracted from UT-1355 PDX tumor fragments and Western blot with AR NTD-binding antibody or AR-V7 antibody, and GAPDH antibody was performed. LNCaP and 22RV1 prostate cancer cells were used as control for AR and AR-SV, respectively. Representative blots shown. F. UT-105 completely inhibits UT-1355 PDX tumor growth. UT-1355 PDX tumor fragments (1 mm3) were orthotopically implanted into the mammary fat pad in female NSG mice (n=8–10/group), and a xenograft experiment was performed as indicated above for MDA-MB-453. G-H. Change in body weight of UT-1355-bearing mice and tumor weight. Mean -/+ SEM is shown with One way ANOVA conducted in Graph Pad Prism: * p<0.05, ** p<0.01, *** p<0.001.

    Article Snippet: Biological samples 52 TNBC Patient specimens IRB 14–03113-XP UTHSC N/A PDX UT-1355 IRB 14–03113-XP UTHSC N/A Chemicals, peptides, and recombinant proteins Enzalutamide Medkoo 201821 Bicalutamide AK Scientific 90357–06-5 Ruxlotinib AmBeed A272323 Interferon Gift from Dr. Pfeffer (which was a generous gift from Amgen) 84 N/A BrDU Cell signaling 6813s R1881 Sigma R0908–10MG Sypro Orange Thermofisher S6650 Matrigel fisherscientific 8774552 Gelatin dental sponge (Vetspon Dental Cubes) fisherscientific NC0654350 Critical commercial assays Kinome Scan DiscoverX Eurofins N/A GPCR Scan DiscoverX Eurofins N/A Deposited data RNA Seq for MDA-MB-453 and MDA-MB-453-AR-V7 GEO database GSE244283 Spatial genomics GEO database GSE245202 MDA-MB-453 xenograft GEO database GSE244283 41 patient specimens GEO database GSE244283 MDA-MB-453 cell line RNA sequencing GEO database GSE245554 Experimental models: Cell lines LNCaP ATCC CRL-1740 22RV1 ATCC CRL-2505 MDA-MB-453 ATCC HTB-131 MDA-MB-231 ATCC CRM-HTB-26 BT549 ATCC HTB-122 PC3 ATCC CRL-1435 MFM223 Sigma Aldrich SKU 98050130–1VL Experimental models: Organisms/strains NSG mice JAX labs 005557 Sprague Dawley rats Charles River N/A Oligonucleotides TaqMan primers and probe Androgen Receptor N-terminus Life Technologies Hs00907242_m1 TaqMan primers and probe Androgen Receptor C-terminus Life Technologies Hs00171172_m1 TaqMan primers and probe FKBP5 Life Technologies Hs00188025_m1 TaqMan primers and probe TMPRSS2 Life Technologies Hs00237175_m1 TaqMan primers and probe STEAP4 Life Technologies Hs01026584_m1 TaqMan primers and probe IRF-1 Life Technologies Hs00971965_m1 TaqMan primers and probe IRF-7 Life Technologies Hs01014809_g1 TaqMan primers and probe IRF-9 Life Technologies Hs00196051_m1 TaqMan primers and probe GAPDH Life Technologies Hs00266705_g1 Primers and probe AR-V7 Custom synthesized Recombinant DNA STAT-1 plasmid Gift from Dr. Pfeffer N/A Androgen receptor Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A GRE-LUC Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A Glucocorticoid receptor Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A Mineralocorticoid receptor Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A STAT-1RE LUC James E. Darnell, Rockefeller University N/A AR-LBD bacterial expression vector Constructed in our lab. N/A AF-1 bacterial expression vector Constructed in our lab. N/A AR-V7 bacterial expression vector Constructed in our lab. N/A Software and algorithms Other Open in a separate window Key resources table.

    Techniques: Western Blot, Binding Assay

    A. STAT1 increases R1881-induced AR transactivation. AR transactivation was performed in HEK-293 cells with 0.1 μg vector or STAT1. Numbers provided in bracket are R1881’s EC50 values. B. STAT1 and AR interact. AR and STAT1 (5 μg) were transfected into HEK-293 cells. Cells were treated 48 hours after transfection with 2000 IU interferon α and 10 nM R1881. Cells were harvested 4 hours after treatment, immunoprecipitation was performed with IgG or AR antibody, and Western blot was performed for STAT1. C. AR and STAT1 interact in MDA-MB-453 cells. MDA-MB-453 cells were maintained in charcoal-stripped serum-containing medium for two days and treated with interferon α and R1881 for 4 hours. Cells were fixed and immunostained for AR (red) and STAT1 (green), and confocal microscopy was performed. Scale is 2 μm. D. STAT1 is recruited to ARE (on FKBP5 regulatory region). MDA-MB-453 cells were treated as indicated. The cells were crosslinked, sheared, and ChIP PCR was performed with the STAT1 and IgG antibodies. PCR was performed using the primers for the indicated regions. E. AR is recruited to STAT1 responsive gene regulatory region. Data from AR ChIP-seq performed in PCa cells (n=2) were loaded in IGV browser and CXCL8 (IL8) and FKBP5 regulatory regions were scanned to determine the binding of AR. F. UT-105 inhibits STAT1 recruitment to STAT1RE and ARE. MDA-MB-453 cells maintained in charcoal-stripped serum-containing medium were treated with interferon α (1000 units), R1881 (1 nM), combination in the presence and absence of UT-105 (10 μM) for 4 hours. ChIP assay was performed with STAT1 antibody and real-time PCR was performed with primers specific for hIL8 STATRE and FKBP5 ARE. Representative experiment is shown in the figure. G. Ruxolitinib inhibits AR-target gene expression. MDA-MB-453 cells maintained in charcoal stripped serum-containing medium were treated with R1881 (0.1 nM) alone or in combination with ruxolitinib (3 μM) for 24 hours. RNA was extracted, and real-time PCR for FKBP5 was performed (n=4/group; one-way ANOVA). H. UT-105 inhibits STAT1-dependent coactivation of AR. AR transactivation was performed in HEK-293 cells in the presence or absence of 0.1 μg STAT1. I. Effect of AR and JAK inhibitors on the proliferation of patient tumors explants growth. Illustration of gelatin sponge culture (Biorender.com). Tumor tissue from two patients (1473 and 1474) were placed on pre-soaked gelatin sponges and treated as indicated for 48 hours. The tissues were fixed and stained with Ki67. The bar graphs show the percentage of cells that stained positively for Ki67. J. Model. A schematic of the proposed mechanism of action of SARDs in TNBC tumors (biorender.com). The model summarizes the choice of drug for inhibiting the growth of LAR TNBC tumors. This would depend on the specific mechanisms involved in AR activation and tumor progression. Enzalutamide may work if AR activity is the primary driver, but its efficacy could be limited by androgen surges. STAT inhibitors like ruxolitinib may not be effective if AR activation is independent of STAT1. Degraders like UT-105 seem promising as they degrade AR directly and could prevent AR or AR splice variant activation, offering a more comprehensive approach to inhibit tumor growth. However, the actual effectiveness of these drugs would need to be studied in clinical settings and may vary from patient to patient. Panels A-H representative of three independent experiments is shown.

    Journal: Cell reports

    Article Title: Identification of a Targetable JAK-STAT Enriched Androgen Receptor (AR) and AR Splice Variant Positive Triple Negative Breast Cancer Subtype

    doi: 10.1016/j.celrep.2023.113461

    Figure Lengend Snippet: A. STAT1 increases R1881-induced AR transactivation. AR transactivation was performed in HEK-293 cells with 0.1 μg vector or STAT1. Numbers provided in bracket are R1881’s EC50 values. B. STAT1 and AR interact. AR and STAT1 (5 μg) were transfected into HEK-293 cells. Cells were treated 48 hours after transfection with 2000 IU interferon α and 10 nM R1881. Cells were harvested 4 hours after treatment, immunoprecipitation was performed with IgG or AR antibody, and Western blot was performed for STAT1. C. AR and STAT1 interact in MDA-MB-453 cells. MDA-MB-453 cells were maintained in charcoal-stripped serum-containing medium for two days and treated with interferon α and R1881 for 4 hours. Cells were fixed and immunostained for AR (red) and STAT1 (green), and confocal microscopy was performed. Scale is 2 μm. D. STAT1 is recruited to ARE (on FKBP5 regulatory region). MDA-MB-453 cells were treated as indicated. The cells were crosslinked, sheared, and ChIP PCR was performed with the STAT1 and IgG antibodies. PCR was performed using the primers for the indicated regions. E. AR is recruited to STAT1 responsive gene regulatory region. Data from AR ChIP-seq performed in PCa cells (n=2) were loaded in IGV browser and CXCL8 (IL8) and FKBP5 regulatory regions were scanned to determine the binding of AR. F. UT-105 inhibits STAT1 recruitment to STAT1RE and ARE. MDA-MB-453 cells maintained in charcoal-stripped serum-containing medium were treated with interferon α (1000 units), R1881 (1 nM), combination in the presence and absence of UT-105 (10 μM) for 4 hours. ChIP assay was performed with STAT1 antibody and real-time PCR was performed with primers specific for hIL8 STATRE and FKBP5 ARE. Representative experiment is shown in the figure. G. Ruxolitinib inhibits AR-target gene expression. MDA-MB-453 cells maintained in charcoal stripped serum-containing medium were treated with R1881 (0.1 nM) alone or in combination with ruxolitinib (3 μM) for 24 hours. RNA was extracted, and real-time PCR for FKBP5 was performed (n=4/group; one-way ANOVA). H. UT-105 inhibits STAT1-dependent coactivation of AR. AR transactivation was performed in HEK-293 cells in the presence or absence of 0.1 μg STAT1. I. Effect of AR and JAK inhibitors on the proliferation of patient tumors explants growth. Illustration of gelatin sponge culture (Biorender.com). Tumor tissue from two patients (1473 and 1474) were placed on pre-soaked gelatin sponges and treated as indicated for 48 hours. The tissues were fixed and stained with Ki67. The bar graphs show the percentage of cells that stained positively for Ki67. J. Model. A schematic of the proposed mechanism of action of SARDs in TNBC tumors (biorender.com). The model summarizes the choice of drug for inhibiting the growth of LAR TNBC tumors. This would depend on the specific mechanisms involved in AR activation and tumor progression. Enzalutamide may work if AR activity is the primary driver, but its efficacy could be limited by androgen surges. STAT inhibitors like ruxolitinib may not be effective if AR activation is independent of STAT1. Degraders like UT-105 seem promising as they degrade AR directly and could prevent AR or AR splice variant activation, offering a more comprehensive approach to inhibit tumor growth. However, the actual effectiveness of these drugs would need to be studied in clinical settings and may vary from patient to patient. Panels A-H representative of three independent experiments is shown.

    Article Snippet: Biological samples 52 TNBC Patient specimens IRB 14–03113-XP UTHSC N/A PDX UT-1355 IRB 14–03113-XP UTHSC N/A Chemicals, peptides, and recombinant proteins Enzalutamide Medkoo 201821 Bicalutamide AK Scientific 90357–06-5 Ruxlotinib AmBeed A272323 Interferon Gift from Dr. Pfeffer (which was a generous gift from Amgen) 84 N/A BrDU Cell signaling 6813s R1881 Sigma R0908–10MG Sypro Orange Thermofisher S6650 Matrigel fisherscientific 8774552 Gelatin dental sponge (Vetspon Dental Cubes) fisherscientific NC0654350 Critical commercial assays Kinome Scan DiscoverX Eurofins N/A GPCR Scan DiscoverX Eurofins N/A Deposited data RNA Seq for MDA-MB-453 and MDA-MB-453-AR-V7 GEO database GSE244283 Spatial genomics GEO database GSE245202 MDA-MB-453 xenograft GEO database GSE244283 41 patient specimens GEO database GSE244283 MDA-MB-453 cell line RNA sequencing GEO database GSE245554 Experimental models: Cell lines LNCaP ATCC CRL-1740 22RV1 ATCC CRL-2505 MDA-MB-453 ATCC HTB-131 MDA-MB-231 ATCC CRM-HTB-26 BT549 ATCC HTB-122 PC3 ATCC CRL-1435 MFM223 Sigma Aldrich SKU 98050130–1VL Experimental models: Organisms/strains NSG mice JAX labs 005557 Sprague Dawley rats Charles River N/A Oligonucleotides TaqMan primers and probe Androgen Receptor N-terminus Life Technologies Hs00907242_m1 TaqMan primers and probe Androgen Receptor C-terminus Life Technologies Hs00171172_m1 TaqMan primers and probe FKBP5 Life Technologies Hs00188025_m1 TaqMan primers and probe TMPRSS2 Life Technologies Hs00237175_m1 TaqMan primers and probe STEAP4 Life Technologies Hs01026584_m1 TaqMan primers and probe IRF-1 Life Technologies Hs00971965_m1 TaqMan primers and probe IRF-7 Life Technologies Hs01014809_g1 TaqMan primers and probe IRF-9 Life Technologies Hs00196051_m1 TaqMan primers and probe GAPDH Life Technologies Hs00266705_g1 Primers and probe AR-V7 Custom synthesized Recombinant DNA STAT-1 plasmid Gift from Dr. Pfeffer N/A Androgen receptor Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A GRE-LUC Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A Glucocorticoid receptor Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A Mineralocorticoid receptor Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A STAT-1RE LUC James E. Darnell, Rockefeller University N/A AR-LBD bacterial expression vector Constructed in our lab. N/A AF-1 bacterial expression vector Constructed in our lab. N/A AR-V7 bacterial expression vector Constructed in our lab. N/A Software and algorithms Other Open in a separate window Key resources table.

    Techniques: Plasmid Preparation, Transfection, Immunoprecipitation, Western Blot, Confocal Microscopy, ChIP-sequencing, Binding Assay, Real-time Polymerase Chain Reaction, Expressing, Staining, Activation Assay, Activity Assay, Variant Assay

    Key resources table

    Journal: Cell reports

    Article Title: Identification of a Targetable JAK-STAT Enriched Androgen Receptor (AR) and AR Splice Variant Positive Triple Negative Breast Cancer Subtype

    doi: 10.1016/j.celrep.2023.113461

    Figure Lengend Snippet: Key resources table

    Article Snippet: Biological samples 52 TNBC Patient specimens IRB 14–03113-XP UTHSC N/A PDX UT-1355 IRB 14–03113-XP UTHSC N/A Chemicals, peptides, and recombinant proteins Enzalutamide Medkoo 201821 Bicalutamide AK Scientific 90357–06-5 Ruxlotinib AmBeed A272323 Interferon Gift from Dr. Pfeffer (which was a generous gift from Amgen) 84 N/A BrDU Cell signaling 6813s R1881 Sigma R0908–10MG Sypro Orange Thermofisher S6650 Matrigel fisherscientific 8774552 Gelatin dental sponge (Vetspon Dental Cubes) fisherscientific NC0654350 Critical commercial assays Kinome Scan DiscoverX Eurofins N/A GPCR Scan DiscoverX Eurofins N/A Deposited data RNA Seq for MDA-MB-453 and MDA-MB-453-AR-V7 GEO database GSE244283 Spatial genomics GEO database GSE245202 MDA-MB-453 xenograft GEO database GSE244283 41 patient specimens GEO database GSE244283 MDA-MB-453 cell line RNA sequencing GEO database GSE245554 Experimental models: Cell lines LNCaP ATCC CRL-1740 22RV1 ATCC CRL-2505 MDA-MB-453 ATCC HTB-131 MDA-MB-231 ATCC CRM-HTB-26 BT549 ATCC HTB-122 PC3 ATCC CRL-1435 MFM223 Sigma Aldrich SKU 98050130–1VL Experimental models: Organisms/strains NSG mice JAX labs 005557 Sprague Dawley rats Charles River N/A Oligonucleotides TaqMan primers and probe Androgen Receptor N-terminus Life Technologies Hs00907242_m1 TaqMan primers and probe Androgen Receptor C-terminus Life Technologies Hs00171172_m1 TaqMan primers and probe FKBP5 Life Technologies Hs00188025_m1 TaqMan primers and probe TMPRSS2 Life Technologies Hs00237175_m1 TaqMan primers and probe STEAP4 Life Technologies Hs01026584_m1 TaqMan primers and probe IRF-1 Life Technologies Hs00971965_m1 TaqMan primers and probe IRF-7 Life Technologies Hs01014809_g1 TaqMan primers and probe IRF-9 Life Technologies Hs00196051_m1 TaqMan primers and probe GAPDH Life Technologies Hs00266705_g1 Primers and probe AR-V7 Custom synthesized Recombinant DNA STAT-1 plasmid Gift from Dr. Pfeffer N/A Androgen receptor Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A GRE-LUC Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A Glucocorticoid receptor Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A Mineralocorticoid receptor Gift from Dr. Nancy Weigel, Baylor College of Medicine N/A STAT-1RE LUC James E. Darnell, Rockefeller University N/A AR-LBD bacterial expression vector Constructed in our lab. N/A AF-1 bacterial expression vector Constructed in our lab. N/A AR-V7 bacterial expression vector Constructed in our lab. N/A Software and algorithms Other Open in a separate window Key resources table.

    Techniques: Virus, Plasmid Preparation, Recombinant, RNA Sequencing Assay, Synthesized, Expressing, Construct, Software