Journal: Molecular Cancer Therapeutics

Article Title: Thio-2 Inhibits Key Signaling Pathways Required for the Development and Progression of Castration-resistant Prostate Cancer

doi: 10.1158/1535-7163.MCT-23-0354

Figure Lengend Snippet: BAG-1 is highly expressed and associates with signaling pathways critical for the development and progression of CRPC. A, Two independent CRPC patient transcriptome cohorts were used in this study. The SU2C/PCF patient cohort included RNA-seq on 159 CRPC biopsies and the ICR/RMH patient cohort included RNA-seq on 95 CRPC biopsies. SU2C/PCF ( B ) and ICR/RMH ( C ) CRPC transcriptome analyses for BAG-1 mRNA expression compared with the 20,000 highest expressed genes divided into very high (upper 25% expressed genes), medium high (50%−75% expressed genes), medium low (25%−50% expressed genes), and very low (lower 25% expressed genes). GESA shows BAG-1 mRNA levels association with hallmark pathways in SU2C/PCF ( D ) and ICR/RMH ( E ) cohorts. NESs and FDRs are shown. Hallmark pathways significantly (FDR ≤ 0.05) enriched and de-enriched with BAG-1 mRNA expression are shown.

Article Snippet: Pan-mouse-BAG-1 (panmoBAG-1, mouse, goat polyclonal, AF815, R&D Systems), mouse/human AR-FL [AR-FL, mouse/human, rabbit monoclonal, EPR1535 , Abcam] and pan-BAG-1 (panBAG-1, human, rabbit monoclonal, RM356, RevMAb) were all validated and optimized for IHC in this study.

Techniques: RNA Sequencing Assay, Expressing

Journal: Molecular Cancer Therapeutics

Article Title: Thio-2 Inhibits Key Signaling Pathways Required for the Development and Progression of Castration-resistant Prostate Cancer

doi: 10.1158/1535-7163.MCT-23-0354

Figure Lengend Snippet: Druggability assessment of the BAG domain of BAG-1. A, Visualization of the BAG domain cavity of interest identified by canSAR using the 44 3D HSC70-BAG domain structures available mapped onto the representative structure (PDB ID 3FZLB). The cavity of interest as volume surface (in yellow) is shown on the BAG domain (violet) of BAG-1. B, Key geometric and physicochemical parameters for the cavity of interest within the BAG domain (blue), a druggable protein–protein interaction (BCL-2; orange) and the druggable kinase ATP site (green) are shown as violin plots. P values were calculated using the Kruskal–Wallis (K-W) test. C, Monte Carlo simulations identified 449 models with 4,489 cavities for the original 44 3D HSC70-BAG domain structures.

Article Snippet: Pan-mouse-BAG-1 (panmoBAG-1, mouse, goat polyclonal, AF815, R&D Systems), mouse/human AR-FL [AR-FL, mouse/human, rabbit monoclonal, EPR1535 , Abcam] and pan-BAG-1 (panBAG-1, human, rabbit monoclonal, RM356, RevMAb) were all validated and optimized for IHC in this study.

Techniques:

Journal: Molecular Cancer Therapeutics

Article Title: Thio-2 Inhibits Key Signaling Pathways Required for the Development and Progression of Castration-resistant Prostate Cancer

doi: 10.1158/1535-7163.MCT-23-0354

Figure Lengend Snippet: BAG-1 KO male mice are fertile and viable with reduced prostate weight. A, BAG-1 KO mouse strain Bag1tm1a(EUCOMM)Hmgu was developed by the EUCOMM program by insertion of an artificial exon containing the coding sequence of beta-Geo, a fusion protein of beta-Galactosidase (LacZ) and neomycin (neo) followed by a stop codon and polyadenylation (pA) sequence between exon 2 and exon 3 flanked by Flp recognition sites disrupts the expression of the WT gene, replacing the endogenous BAG-1 expression by a fusion protein of the BAG-1 N-terminal sequence encoded in exon 1 and 2 and beta-Geo. Mouse prostates from BAG-1 KO and BAG-1 WT male mice were analyzed for BAG-1 mRNA (qRT-PCR) levels. BAG-1 exon 1 to 2 (Ex 1–2), exon 1 to 3 (Ex 1–3), and exon 5 to 7 (Ex 5–7) mRNA was quantified for BAG-1 KO (red bars; n = 3) and BAG-1 WT (gray bars; n = 3) mice. mRNA expression was calculated relative to mouse GAPDH and normalized to BAG-1 WT. Mean levels from three prostates are shown. P values were calculated for BAG-1 KO compared with BAG-1 WT mice using unpaired Student t test. P values ≤ 0.05 are shown. B, Prostates from BAG-1 KO mouse strain Bag1tm1a(EUCOMM)Hmgu and BAG-1 WT male mice were analyzed for mouse BAG-1 protein (IHC) levels. Representative micrographs of BAG-1 detection in mouse prostates by panmoBAG-1 antibody IHC are shown. Scale bar, 200 μm. C, Mouse prostates from BAG-1 KO (red bars; n = 5) and BAG-1 WT (gray bars; n = 6) male mice at age 12 weeks were taken and samples prepared for RNA-seq. Median BAG isoforms and CHMP5 mRNA levels (fragments per kilobase of transcript per million reads; FPKM) with interquartile range, and smallest and largest value, is shown. P values were calculated for BAG-1 KO compared with BAG-1 WT mice using unpaired Student t test. P values ≤ 0.05 are shown. D, Overview of GSEA using MsigDB (v7.0) functional pathways: H, Hallmark; C2, Curated Gene Sets (including KEGG, Biocarta, Reactome). BAG-1 KO and BAG-1 WT RNA-seq analysis was compared, change in gene expression was ranked by log 2 fold change, then tested for enrichment against functional gene sets (H, C2), using GSEA. The distribution of resulting FDR values (log 10 ) is shown in red, and the threshold for significance (FDR 0.05) is shown by dotted grey line. None of the pathways tested reached the significance threshold. E, Kaplan–Meier curves of overall survival (OS) of BAG-1 KO (red line; n = 9) and BAG-1 WT (gray line; n = 8) male mice from birth. Median OS, HR with 95% confidence intervals and P values for univariate Cox survival model are shown. F, The body weight of male BAG-1 KO (red bars) and BAG-1 WT (gray bars) male mice at 12 weeks and 12 months was determined. Median body weight with interquartile range, and smallest and largest value, is shown. P values were calculated for BAG-1 KO compared with BAG-1 WT mice using unpaired Student t test. P values ≤ 0.05 are shown. G, The weight of the genitourinary tract, kidney, testis, prostate, and serum levels of testosterone, from male BAG-1 KO (red bar) and BAG-1 WT (gray bar) male mice at age 3 months and older was determined. Median weight or serum testosterone levels with interquartile range, and smallest and largest value, is shown. P values were calculated for BAG-1 KO compared with BAG-1 WT mice using unpaired Student t test. P values ≤ 0.05 are shown.

Article Snippet: Pan-mouse-BAG-1 (panmoBAG-1, mouse, goat polyclonal, AF815, R&D Systems), mouse/human AR-FL [AR-FL, mouse/human, rabbit monoclonal, EPR1535 , Abcam] and pan-BAG-1 (panBAG-1, human, rabbit monoclonal, RM356, RevMAb) were all validated and optimized for IHC in this study.

Techniques: Sequencing, Expressing, Quantitative RT-PCR, RNA Sequencing Assay, Functional Assay

Journal: Molecular Cancer Therapeutics

Article Title: Thio-2 Inhibits Key Signaling Pathways Required for the Development and Progression of Castration-resistant Prostate Cancer

doi: 10.1158/1535-7163.MCT-23-0354

Figure Lengend Snippet: BAG-1 isoform knockdown induces a limited phenotype in the LNCaP cell line prostate cancer model. A, LNCaP shRNA clones were collected and prepared for RNA-seq. Mean BAG-1 mRNA levels (FPKM) in control (gray bars; n = 3) and BAG-1 (red bars; n = 3) shRNA clones is shown. P values were calculated for BAG-1 shRNA (shBAG-1) clones compared with control shRNA (shCnt) clones using unpaired Student t test. P values ≤ 0.05 are shown. Analysis of RNA-seq with GESA shows BAG-1 knockdown associates with hallmark pathways. NESs and FDRs are shown. Hallmark pathways significantly (FDR ≤ 0.05) enriched and de-enriched with BAG-1 knockdown are shown. B, LNCaP shRNA clones were grown in starved media (10% charcoal-stripped serum) for 72 hours prior to treatment with vehicle (gray bars; Ethanol 0.1%) or 10 nmol/L DHT (red bars) for 16 hours and BAG-1, PSA, TMPRSS2 and FKBP5 mRNA expression was determined. Mean mRNA expression (normalized to average of GAPDH/B2M/HRPT1/RPLP0 and shCnt/vehicle; defined as 1) with SD from three individual experiments is shown. P values were calculated for the impact of BAG-1 knockdown on DHT stimulation using unpaired Student t test. P values ≤ 0.05 are shown. C, NSG male mice were inoculated with LNCaP shRNA control (shCnt; gray line; n = 10) or BAG-1 (shBAG-1; red line; n = 10) clones and growth was measured between days 16 and 27. Growth of all tumors (left) and mean growth (right) is shown. P values were calculated comparing shCnt and shBAG-1 at day 27 using unpaired Student t test.

Article Snippet: Pan-mouse-BAG-1 (panmoBAG-1, mouse, goat polyclonal, AF815, R&D Systems), mouse/human AR-FL [AR-FL, mouse/human, rabbit monoclonal, EPR1535 , Abcam] and pan-BAG-1 (panBAG-1, human, rabbit monoclonal, RM356, RevMAb) were all validated and optimized for IHC in this study.

Techniques: shRNA, Clone Assay, RNA Sequencing Assay, Expressing

Journal: Molecular Cancer Therapeutics

Article Title: Thio-2 Inhibits Key Signaling Pathways Required for the Development and Progression of Castration-resistant Prostate Cancer

doi: 10.1158/1535-7163.MCT-23-0354

Figure Lengend Snippet: Thio-2 inhibits the growth of CRPC patient-derived models with associated suppression of AR target genes. A, CP50, CP89, and CP142 PDX-Os were treated with vehicle (Veh, DMSO 0.1%), various concentrations (5, 10, 25, and 50 μmol/L) of Thio-2 or various concentrations (1 and 10 μmol/L) of enzalutamide (E), and growth determined after 5 days by CellTiter-Glo 3D Cell Viability Assay. Mean fold change in growth (compared with day 0) with SD from a single experiment with six replicates is shown. P values were calculated for each condition compared with vehicle at 5 days using unpaired Student t test. P values ≤ 0.05 are shown. B, CP50 PDX-O were treated with vehicle (DMSO 0.1%) or various concentrations (25 and 50 μmol/L) of Thio-2 for 17 hours. The effect of each condition on AR-FL, BAG-1, and GAPDH protein expression was determined. Single Western blot analysis with triplicates is shown. C, Representative micrographs of AR-FL, AR-V7, and pan BAG-1 (panBAG1) detection by IHC of formalin-fixed paraffin-embedded CP50 PDX-O treated with vehicle (DMSO 0.1%) or various concentrations (25 and 50 μmol/L) of Thio-2 for 17 hours are shown. Scale bar: 50 μm. D, CP50 PDX-O were treated with vehicle (DMSO 0.1%) or various concentrations (25 and 50 μmol/L) of Thio-2 for 17 hours. The effect of each condition on PSA, TMPRSS2 and FKBP5 mRNA expression was determined. Mean mRNA expression (normalized to average of GAPDH/B2M/HRPT1/RPLP0 and vehicle treatment; defined as 1) with SD from a single experiment with six replicates is shown. P values were calculated for each condition compared with vehicle using unpaired Student t test. P values ≤ 0.05 are shown. E, CP50 PDXs were treated with 15 mg/kg Thio-2 ( n = 7) or vehicle ( n = 6) once daily intraperitoneally for 14 days. Mean growth (normalized to day 0; defined as 1) with SD was determined on day 14. P values were calculated comparing 15 mg/kg Thio-2 IP OD treatment arm with vehicle control using unpaired Student t test. P values ≤ 0.05 are shown. F, The doubling time (2-fold growth) for CP50 PDXs were used as a surrogate endpoint for overall survival (OS). Median OS, HR with 95% confidence intervals and P values for univariate cox survival model are shown. G, The effect of 15 mg/kg Thio-2 OD IP compared with vehicle on serum PSA was determined at 5 days. Mean serum PSA with SD was determined for each mouse. P values were calculated for vehicle compared with 15 mg/kg Thio-2 once daily intraperitoneally using unpaired Student t test. P values ≤ 0.05 are shown.

Article Snippet: Pan-mouse-BAG-1 (panmoBAG-1, mouse, goat polyclonal, AF815, R&D Systems), mouse/human AR-FL [AR-FL, mouse/human, rabbit monoclonal, EPR1535 , Abcam] and pan-BAG-1 (panBAG-1, human, rabbit monoclonal, RM356, RevMAb) were all validated and optimized for IHC in this study.

Techniques: Derivative Assay, Viability Assay, Expressing, Western Blot, Formalin-fixed Paraffin-Embedded

Journal: Molecular Cancer Therapeutics

Article Title: Thio-2 Inhibits Key Signaling Pathways Required for the Development and Progression of Castration-resistant Prostate Cancer

doi: 10.1158/1535-7163.MCT-23-0354

Figure Lengend Snippet: Thio-2 downregulates androgen receptor signaling and inhibits growth of prostate cancer cell lines through a BAG-1–independent mechanism. LNCaP ( A ), 22Rv1 ( D ), and LNCaP95 ( G ) prostate cancer cells were transfected with 50 nmol/L of either control (siCnt; clear bars) or BAG-1 (siBAG-1; red bars) siRNA for 72 hours prior to treatment with vehicle (DMSO 0.1%) or various concentrations (5, 10, 25, and 50 μmol/L) of Thio-2 and growth was determined after 6 days by CellTiter-Glo Luminescent Cell Viability Assay. Mean fold change in growth (compared with day 0) with SD from a single experiment with six replicates is shown. P values were calculated for each condition compared with vehicle in siCnt and siBAG-1 cells, and between vehicle-treated siCnt and siBAG-1 cells (dotted lines), using unpaired Student t test. P values ≤ 0.05 are shown. LNCaP ( B ) 22Rv1 ( E ), and LNCaP95 ( H ) prostate cancer cells were transfected with 50 nmol/L of either siCnt or siBAG-1 siRNA for 55 hours prior to treatment with vehicle (DMSO 0.1%) or various concentrations (5 and 50 μmol/L) of Thio-2 for 17 hours (total 72 hours) and AR-FL, AR-V7, PSA, BAG-1, and GAPDH protein expression was determined. Single Western blot analysis is shown. LNCaP ( C ), 22Rv1 ( F ), and LNCaP95 ( I ) prostate cancer cells were transfected with 50 nmol/L of either siCnt or siBAG-1 siRNA for 55 hours prior to treatment with vehicle (DMSO 0.1%) or various concentrations (5 and 50 μmol/L) of Thio-2 for 17 hours (total 72 hours) and PSA, TMPRSS2, and FKBP5 mRNA expression was determined. Mean mRNA expression (normalized to average of GAPDH/B2M/HRPT1/RPLP0 and siCnt/vehicle; defined as 1) with SD from a single experiment with six replicates is shown. P values were calculated for each condition compared with vehicle in siCnt and siBAG-1 cells, and between vehicle-treated siCnt and siBAG-1 cells (dotted lines), using unpaired Student t test. P values ≤ 0.05 are shown.

Article Snippet: Pan-mouse-BAG-1 (panmoBAG-1, mouse, goat polyclonal, AF815, R&D Systems), mouse/human AR-FL [AR-FL, mouse/human, rabbit monoclonal, EPR1535 , Abcam] and pan-BAG-1 (panBAG-1, human, rabbit monoclonal, RM356, RevMAb) were all validated and optimized for IHC in this study.

Techniques: Transfection, Cell Viability Assay, Expressing, Western Blot

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Proteostasis network response to environmental chronic stress: linking survival to protein aggregation in a human neuroblastoma cellular model

doi: 10.1007/s00018-025-05884-6

Figure Lengend Snippet: Analysis of the BAG1/BAG3 triage system. a Western blot analysis of the expression of BAG1 in the RIPA-soluble protein fraction after 24 h ( n = 3) and b 72 h ( n = 4). c Analysis of the expression of soluble BAG3 after 24 h ( n = 4) and d 72 h ( n = 4). e. Analysis of BAG3 in the insoluble fraction after 72 h of treatment ( n = 3). Data were normalized against Vinculin as loading control. For the insoluble fraction, the same amount of proteins quantified in the respective soluble fractions were considered. Data are expressed as fold increase ± S.E.M and compared to normalized control, shown as dashed line. Statistical significance was determined after one-way ANOVA with Dunnet’s multiple comparison test of each treatment against control. *** p < 0.001; ** p < 0.01; * p < 0.05

Article Snippet: Primary antibodies utilized were: PARP-1, Cell Signaling Technology, 9542; BiP/Grp78, Abcam, ab32618; p-eIF2⍺, Cell Signaling Technology, 9721; eIF2⍺, Cell Signaling Technology, 9722; Ubiquitin, antibodies.com, A85455; BAG3, Invitrogen, MA5-32706; BAG1, Proteintech, 19064–1-AP; LC3, RBC Lifescience, PD014; p62/SQSTM1, Cell Signaling Technology, 8025; pTDP-43, Proteintech, 80007–1-RR −1-AP; TDP-43, Proteintech, 12892–1-AP; β-Actin, Sigma-Aldrich, A5316; Vinculin, Sigma-Aldrich, V9131.

Techniques: Western Blot, Expressing, Control, Comparison