Development of Bag-1L as a therapeutic target in androgen receptor-dependent prostate cancer
- Dana-Farber Cancer Institute and Harvard Medical School, United States
- Dana-Farber Cancer Institute, United States
- Karlsruhe Institute of Technology, Germany
- Institute of Cancer Research and The Royal Marsden NHS Foundation Trust, United Kingdom
- The Barcelona Institute of Science and Technology, Spain
- PRP-ENV/SERIS/LECO, France
- Institute of Cancer Research, United Kingdom
- Innsbruck Medical University, Austria
- University of Illinois at Urbana-Champaign, United States
- The Institute of Cancer Research, United Kingdom
- Passeig Lluís Companys, Spain
Figures
Figure 1 with 3 supplements
Loss of Bag-1L inhibits PCa cell growth and reduces the AR cistrome and transcriptome.
(A) Schematic of the TALEN approach to knockout Bag-1L expression. The TALEN target sequences are highlighted in grey and the start codon (CTG) of Bag-1L is highlighted in yellow. Note, the start …
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Figure 1—source data 1
Direct AR-target genes in TALEN control compared with Bag-1L KO cell lines.
- https://doi.org/10.7554/eLife.27159.007
Figure 1—figure supplement 1
Signal profiles and heatmaps of AR ChIP-seq data in Bag-1L control, KO and rescue cell lines in the presence of vehicle.
Signals are shown for sites with an AR peak in one or more cell lines. Cells were treated with vehicle (ETOH) for 4 hr prior to ChIP-seq.
Figure 1—figure supplement 2
ChIP qPCR validation of three AR enhancer sites in Bag-1L control and KO cells.
Directed AR qPCR for indicated gene targets in hormone-depleted control (black) and Bag-1L KO (grey) LNCaP cells treated with 10 nM DHT for indicated times. Results are presented as the mean of …
Figure 1—figure supplement 3
Top five HALLMARK terms (GSEA) associated with direct AR-target genes lost in Bag-1L KO compared to control cells.
Direct AR-target genes were defined as genes with DHT-induced differential expression (p≤0.05, fold change ≥1.5) that harbor a DHT-responsive AR binding site within 50 kb of the TSS of said gene.
Figure 2 with 6 supplements
The Bag-1L:AR interaction is mediated by K231/232/279 in the BAG domain of Bag-1L.
(A, B) Mammalian one-hybrid assay in HeLa cells transfected with indicated AR domains linked to Gal4 DBD, subjected to increasing concentration of Bag-1L. The results are the mean of three …
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Figure 2—source data 1
List of Bag-1L interactors altered by the BAG domain mutation (≥2 standard deviations of Bag-1L/IgG control RIME).
- https://doi.org/10.7554/eLife.27159.015
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Figure 2—source data 2
Associated functions (FuncAssociate) of Bag-1L interactors altered by the BAG domain mutation.
- https://doi.org/10.7554/eLife.27159.016
Figure 2—figure supplement 1
Schematic of the SPOT synthesis technology.
Mutant Bag-1L sequences (alanine substitutions shown in red) spanning the entire BAG domain of Bag-1L (residues 219–345) were bound to a membrane, incubated with GST-AR τau-5 and subsequently probed …
Figure 2—figure supplement 2
Overlap between AR cistromes in wild-type and CMut Bag-1L-expressing LNCaP cells.
Venn diagram of AR cistromes in wild-type (WT; red) and K231A/K232A/K279A mutant Bag-1L (CMut; blue) LNCaP cells, treated with 10 nM DHT for 4 hr. The union of binding sites is indicated.
Figure 2—figure supplement 3
Top ten GO-terms (GSEA) associated with direct AR-target genes lost in the Bag-1L CMut- compared to the wild-type Bag-1L-expressing cells.
Direct AR-target genes were defined as genes with DHT-induced differential expression (p≤0.05, fold change ≥1.5) that harbor a DHT-responsive AR binding site within 50 kb of the TSS of said gene. …
Figure 2—figure supplement 4
Conserved BAG domain mutations that inhibit the AR AF-1 transactivation.
Top, amino acid sequence of the BAG domain of Bag-1L (residues 219–345) with evolutionarily conserved residues highlighted in red and CMut (K231A/K232A/K279A) shown in blue. Residues involved in …
Figure 2—figure supplement 5
CBCACONH data of wild-type and CMut Bag-1L.
Superposition of CBCACONH of Bag-1L WT (black) and the K231/232/279A mutant (Bag-1L CMut; red). 1H 13C planes were extracted from the 3D spectrum at the indicated 15N values.
Figure 2—figure supplement 6
15N-HSQC spectra of wild-type and CMut Bag-1L.
Superposition of 15N-HSQC spectra of the BAG domain of Bag-1L WT (black) and the K231/232/279A mutant (Bag-1L CMut; red).
Figure 3
The BAG domain mutations decrease AR binding and overlap a druggable pocket.
(A) Top left: Normalized peak intensities of AR-AF-1 (residues 142–448) in the presence of GST (grey), or the GST-fused wild-type (blue) or K231/232/279A mutant BAG domain (red) of Bag-1L. A …
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Figure 3—source data 1
Distinct 3D-snapshots of the BAG domain of Bag1/Bag-1L.
- https://doi.org/10.7554/eLife.27159.018
Figure 4
The thioflavin Thio-2 inhibits AR function and AR-dependent PCa growth.
(A) Co-immunoprecipitation of endogenous Bag-1L and AR in LNCaP cells, treated with or without 5 μM Thio-2 for 16 hr. A Bag-1L-specific antibody was employed for the IP and an antibody against AR …
Figure 5 with 5 supplements
Bag-1L enhances hormone-dependent and -independent PCa cell growth.
LNCaP Bag-1L KO (Bag-1L KO (A)) or parental LNCaP cells (LNCaP (B)) were transfected with an empty retroviral construct (+Vector; black), or expression vectors for wild-type Bag-1L (+WT; red) or the …
Figure 5—figure supplement 1
Western blot of Bag-1L KO rescue cell lines.
LNCaP Bag-1L KO cells were stably transfected with an empty retroviral vector (Vec) or expression vectors for FLAG-HA-tagged wild-type Bag-1L (WT) or the Bag-1L K231/232/279A mutant (CMut). …
Figure 5—figure supplement 2
Western blot of Bag-1L over-expression cell lines.
LNCaP cells were stably transfected with an empty retroviral vector (Vec) or expression vectors for FLAG-HA-tagged wild-type Bag-1L (WT) or the Bag-1L K231/232/279A mutant (CMut). Expression levels …
Figure 5—figure supplement 3
Western blot of wild-type Bag-1L over-expression CRPC lines.
LNCaP-abl (Abl) or LNCaP95 cells (LN95) were stably transfected with an empty retroviral vector (Vec) or expression vectors for FLAG-HA-tagged wild-type Bag-1L (WT). Expression levels of Bag-1L were …
Figure 5—figure supplement 4
Western blot comparison of endogenous Bag-1L levels in PCa and CRPC lines.
Endogenous Bag-1L levels, as determined by an antibody against Bag-1, in hormone-dependent LNCaP (LN) cells and in the CRPC lines LNCaP-abl (Abl) and LNCaP95 (LN95). AR levels and equal protein …
Figure 5—figure supplement 5
Western blot of BAG domain mutant Bag-1L over-expression CRPC lines.
LNCaP-abl (Abl) or LNCaP95 cells (LN95) were stably transfected with an empty retroviral vector (Vec) or expression vectors for FLAG-HA-tagged Bag-1L K231/232/279A mutant (CMut). Expression levels …
Figure 6 with 5 supplements
Nuclear Bag-1 levels increased from hormone naïve to CRPC status in PCa patients.
(A) Representative immunohistochemistry images of AR and Bag-1 detection in HSPC and CRPC. Scale bars: 20 μm. (B, C) Expression (H-score) of nuclear and cytoplasmic Bag-1 (B) and AR (C) in 43 …
Figure 6—figure supplement 1
IHC control in response to Bag-1 knock-down.
IHC of HeLa cells transfected with either control siRNA or Bag-1 siRNA and probed with anti-Bag-1 antibody. Scale bars represent 20 μm.
Figure 6—figure supplement 2
Cell line validation of Bag-1 antibody for IHC.
IHC of Bag-1 in indicated cell lines depleted with GST-Bag-1L or GST alone, as a control. Negative (no antibody added) and positive controls (no antibody depletion) are shown. SK-MEL-2 is a cell …
Figure 6—figure supplement 3
Validation of Bag-1 antibody for IHC using patient samples.
IHC of Bag-1 in patient samples depleted with GST-Bag-1L or GST alone, as a control. Scale bars represent 20 μm.
Figure 6—figure supplement 4
Correlation between nuclear Bag-1 and AR expression in HSPC and CRPC patient samples.
Spearman’s rank correlation test is shown.
Figure 6—figure supplement 5
Kaplan-Meier curve of overall survival on abiraterone treatment.
Data is shown for nuclear Bag-1 positive (red; n = 29) and negative (grey; n = 9) patients (at HSPC). Hazard ratio (HR) with 95% confidence intervals (95% CI) and p-value for univariate cox survival …
Figure 7
The Bag-1L is a promising target for the inhibition of the AR NTD.
The function of Bag-1L in PCa is to promote the correct folding of the AR, augment its affinity for chromatin and regulate its transcriptional activity. Loss of Bag-1L, or mutation or inhibition of …
Tables
Table 1
Clinical characteristics of patients at HSPC biopsy and initiation of abiraterone treatment.
HSPC: hormone sensitive biopsy, CRPC: castration-resistant prostate cancer, ECOG PS: Eastern Cooperative Oncology Group performance status, IQR: interquartile range, SD: standard deviation, NA: not …
| Overall 38 pts | Bag-1 negative 9 pts | Bag-1 positive 29pts | p-value | ||
|---|---|---|---|---|---|
| At diagnostic (archival) HSPC biopsy | Biopsy Gleason score, n (%) | ||||
| ≤6 | 2 (5) | 0 (0) | 2 (7) | 0.40† | |
| 7 | 7 (18) | 4 (44) | 3 (10) | ||
| 8–10 | 27 (71) | 5 (56) | 22 (76) | ||
| NA | 2 (5) | 0 (0) | 2 (7) | ||
| Metastatic at diagnosis, n (%) | |||||
| No | 17 (45) | 2 (22) | 15 (52) | 0.25§ | |
| Yes | 14 (37) | 4 (44) | 10 (34) | ||
| NA | 7 (18) | 3 (33) | 4 (14) | ||
| Primary therapy, n (%) | |||||
| Prostatectomy | 4 (11) | 0 (0) | 4 (14) | 0.65§ | |
| Radiotherapy | 13 (34) | 3 (33) | 10 (34) | ||
| Systemic therapy | 21 (55) | 6 (67) | 15 (52) | ||
| PSA at diagnosis, μg/L | |||||
| Median | 46.0 | 61.0 | 29.0 | 0.30* | |
| IQR | 13.1–105.9 | 28.8–150.0 | 10.0–96.6 | ||
| At initiation of abiraterone treatment | Age, yr | ||||
| Median | 69.2 | 69.5 | 69.0 | 0.59* | |
| IQR | 65.5–73.3 | 61.6–74.1 | 66.1–73.3 | ||
| Sites of metastasis, n (%) | |||||
| Node only | 4 (11) | 0 (0) | 4 (14) | 0.57§ | |
| Bone only | 28 (74) | 7 (78) | 21 (72) | ||
| Visceral (with/without bone) | 6 (16) | 2 (22) | 4 (14) | ||
| ECOG PS, n (%) | |||||
| 0 | 12 (32) | 6 (67) | 6 (21) | 0.05§ | |
| 1 | 24 (63) | 3 (33) | 21 (72) | ||
| 2 | 2 (5) | 0 (0) | 2 (7) | ||
| PSA, μg/L | |||||
| Median | 185.5 | 222.0 | 147.0 | 0.77* | |
| IQR | 83.8–445.8 | 51.2–781.5 | 88.0–363.0 | ||
| Hemoglobin, g/L | |||||
| Mean | 118.1 | 124.4 | 116.2 | 0.18‡ | |
| SD | 16.0 | 16.3 | 15.7 | ||
| Alkaline phosphatase, U/L | |||||
| Median | 131.0 | 133.0 | 129.0 | 0.85* | |
| IQR | 69.0–230.5 | 64.5–250.5 | 70.0–231.0 | ||
| Lactate dehydrogenase, U/L | |||||
| Median | 178.0 | 161.0 | 192.0 | 0.08* | |
| IQR | 155.5–247.0 | 149.5–190.0 | 160.0–287.0 | ||
| NA | 2 | 0 | 2 | ||
| Albumin, g/L | |||||
| Mean | 35.8 | 37.8 | 35.2 | 0.17‡ | |
| SD | 4.8 | 2.4 | 5.2 | ||
| Previous treatments for CRPC, n (%) | |||||
| Docetaxel | 27 (71) | 5 (55) | 22 (76) | 0.40§ | |
| Enzalutamide | 2 (5) | 0 (0) | 2 (7) | 1.00§ | |
| Cabazitaxel | 5 (13) | 0 (0) | 5 (17) | 0.31§ | |
| Subsequent treatments for CRPC, n (%) | |||||
| Docetaxel | 8 (21) | 4 (44) | 4 (14) | 0.07§ | |
| Enzalutamide | 7 (18) | 1 (11) | 6 (21) | 1.00§ | |
| Cabazitaxel | 15 (39) | 5 (55) | 10 (34) | 0.44§ | |
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*Mann-Whitney test
†Chi-square test for trend
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‡Unpaired t test
§Fisher’s exact test
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Table 1—source data 1
Association of nuclear Bag-1 or AR expression with clinical benefits from abiraterone therapy.
HSPC: hormone sensitive prostate cancer, CRPC: castration-resistant prostate cancer, PSA: prostate specific antigen, HR: hazard ratio, 95% CI: 95% confidence intervals. a Univariate cox survival model.
- https://doi.org/10.7554/eLife.27159.033
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Table 1—source data 2
Clinical characteristics of patients at time of castration-resistant prostate cancer biopsy.
CRPC: castration-resistant prostate cancer, ECOG PS: Eastern Cooperative Oncology Group performance status, IQR: interquartile range, SD: standard deviation, PSA: prostate specific antigen, n: number, pts: patients. at-test from linear regression model of Nuclear Bag-1 H-score at the time of CRPC biopsy bWald test from linear regression model of Nuclear Bag-1 H-score at the time of CRPC biopsy
- https://doi.org/10.7554/eLife.27159.034
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.27159.036
