Journal: Nature communications

Article Title: Peptidomimetic targeting of critical androgen receptor-coregulator interactions in prostate cancer.

doi: 10.1038/ncomms2912

Figure Lengend Snippet: Figure 1 | Structural design and synthesis of the LXXLL peptidomimetic D2. (a) D2 was structurally designed to disrupt the interaction between AR and NR box proteins containing the LXXLL motif such as PELP1. (b) Chemical structure of the bis-benzamide-based peptidomimetic D2 (right) and its inactive relative D1 (left), and (c) the lowest energy conformation of D2. (d) Overlay of the energy-minimized D2 (orange) over the a-helical LXXLL motif (white) (e) Docking studies of D2 on the AF2 domain of AR using AutoDock: Two isobutyl groups of D2 corresponding to the leucines at the i and i þ 4 positions of the LXXLL motif fit into the hydrophobic pocket in the AF2 domain in a manner similar to the known interactions between the LXXLL motifs and the AR. Crystal structure (PDB code 1T63) of the LXXLL motif bound on the AF2 domain (left). D2 docked in the same binding pocket (middle). Superimposition of the docked D2 (green) over the crystal structure of the LXXLL motif (purple) (right). (f) D2 directly binds to AR. The total cell lysate from C4-2 cells (200 mg), cultured in T-media with 10% FBS, was incubated with 1.5 nmoles of biotinylated D2 (Biotin-D2) alone or in the presence of tenfold excess unbiotinylated D2 (15 nmoles) or unbiotinylated D1 (15 nmoles). Input is shown in the right hand panel. (g) Effect of DHTon D2 binding to AR: LNCaP cells were serum starved overnight in pfRPMI þ 3% CFS in the presence or absence of 10 nM DHT and the cell lysate collected. Immunoprecipitation experiments were performed using Biotin-D2. Input lanes are also shown.

Article Snippet: The AR and b-actin antibodies were purchased from Sigma (St Louis, MO); PELP1 antibody from Bethyl Laboratories (Montgomery, TX); AR-C19, GAPDH, Histone H3, tERK, tSrc, tAkt, ERb and PSA antibodies from Santa Cruz Biotechnology (Santa Cruz, CA). pAkt, pSrc (T262/Y204) and pSrc (Y416) from Cell Signaling Technology (Danvers, MA).

Techniques: Binding Assay, Cell Culture, Incubation, Immunoprecipitation

Journal: Nature communications

Article Title: Peptidomimetic targeting of critical androgen receptor-coregulator interactions in prostate cancer.

doi: 10.1038/ncomms2912

Figure Lengend Snippet: Figure 2 | D2 blocks AR protein–protein interactions with PELP1. (a) LNCaP cells were pretreated with DMSO, D1 (100 nM) control or increasing concentrations of D2 (1, 10, 50, 100 and 200 nM) for 2 h and subsequently cultured in the presence or absence of 10 nM DHT for 24 h. Immunoprecipitation (IP) was performed by incubating cell lysates with specific antibodies for AR (top panel) or PELP1 (middle panel) and rabbit IgG DynaBeads for 60 min each at 4 C. Immunocomplexes were resolved by SDS–polyacrylamide gel electrophoresis and blots hybridized with AR, PELP1, hsp27 and ERb antibodies. Input is shown in the lower panel. Transient overexpression of (b) AR or (c) PELP1 by transfecting LNCaP cells with indicated amounts (in mg per well in a six-well plate) of corresponding expression plasmid rescued D2 (100 nM) -induced suppression of AR–PELP1 complex formation.

Article Snippet: The AR and b-actin antibodies were purchased from Sigma (St Louis, MO); PELP1 antibody from Bethyl Laboratories (Montgomery, TX); AR-C19, GAPDH, Histone H3, tERK, tSrc, tAkt, ERb and PSA antibodies from Santa Cruz Biotechnology (Santa Cruz, CA). pAkt, pSrc (T262/Y204) and pSrc (Y416) from Cell Signaling Technology (Danvers, MA).

Techniques: Protein-Protein interactions, Control, Cell Culture, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Over Expression, Expressing, Plasmid Preparation

Journal: Nature communications

Article Title: Peptidomimetic targeting of critical androgen receptor-coregulator interactions in prostate cancer.

doi: 10.1038/ncomms2912

Figure Lengend Snippet: Figure 3 | D2 blocks AR transcriptional activity. LAPC4 cells were cotransfected with 0.5 mg of a minimal ARE-luciferase reporter construct containing three copies of ARE upstream of -luciferase reporter gene and indicated amounts (mg per well in a six-well plate) of plasmids encoding (a) AR (ph5HBAR) or (b) PELP1 (pPELP1-GFP). Cells were then pretreated with DMSO, D1 (100 nM) or D2 (100 nM) for 2 h followed by culture in the presence or absence of 10 nM DHT. Luciferase activity measured after 48 h shows that overexpression of AR or PELP1 can rescue D2 suppression of DHT-induced AR transcriptional activity. Data are normalized to untreated cells and presented as relative luciferase units. Error bars represent the mean±s.d. *t-test, Po0.05 peptidomimetic treatment versus DHT alone. AR (B110 kDa) and PELP1 (B170 kDa) protein expression was visualized by immunoblot analysis of an independent set of cells treated in the same way. (c) Dose response of D1 and D2 on ARE-luciferase activity: LNCaP cells were transfected with 0.5 mg of ARE-luciferase reporter construct and were treated with indicated concentrations (nM) of D1 or D2 before treatment with 10 nM DHT. Luciferase activity was measured after 48 h and normalized to total protein concentration. (d) Left: gene expression profiling was performed in C4-2 cells treated with vehicle, DHT (10 nM) or DHT (10 nM) þ D2 (100 nM). Heat map display indicates that D2 blocked a majority of DHT-induced transcription. Right: expression levels of specific androgen-regulated genes and genes involved in cell proliferation in C4-2 cells treated with DHT (10 nM) or D2 (100 nM) þ DHT (10 nM) relative to untreated cells, as determined by gene expression profiling. The colour scale calibrates the fold differences in gene expression from baseline. (e) Validation of PSA and TMPRSS2 gene expression by QPCR analysis in an independent set of RNA. Actin mRNA level was used to demonstrate specificity of AR-regulated genes. Data is expressed as fold expression over untreated cells. Error bars represent the mean±s.d. of biological triplicates. *t-test, Po0.05 D2 versus DMSO. (f) Western blotting for PSA, AR and PELP1 in LAPC4 cells pretreated with DMSO, D1 (100 nM) or D2 (100 nM) for 2 h before the addition of 10 nM DHT. Actin was used as a loading control.

Article Snippet: The AR and b-actin antibodies were purchased from Sigma (St Louis, MO); PELP1 antibody from Bethyl Laboratories (Montgomery, TX); AR-C19, GAPDH, Histone H3, tERK, tSrc, tAkt, ERb and PSA antibodies from Santa Cruz Biotechnology (Santa Cruz, CA). pAkt, pSrc (T262/Y204) and pSrc (Y416) from Cell Signaling Technology (Danvers, MA).

Techniques: Activity Assay, Luciferase, Construct, Over Expression, Expressing, Western Blot, Transfection, Protein Concentration, Gene Expression, Biomarker Discovery, Control

Journal: Nature communications

Article Title: Peptidomimetic targeting of critical androgen receptor-coregulator interactions in prostate cancer.

doi: 10.1038/ncomms2912

Figure Lengend Snippet: Figure 6 | D2 inhibits prostate cancer cell growth in vitro and in vivo. (a) A panel of prostate cancer cells pretreated with DMSO, 100 nM D1 or D2 for 2 h before 10 nM DHT treatment for 72 h were assessed by MTTproliferation assay. Data were normalized to the DMSO treatment for each cell line. (b) Effect of pretreatment with 100 nM of D1 and D2 on DHT-induced proliferation in LNCaP cells by CyQuant assay. (c) Dose–response curve of D2 on the proliferation of LAPC4 cells. (d) Rescue of DHT-induced proliferation by overexpression of PELP1 by transfecting LNCaP cells with increasing amounts of pPELP1-GFP following suppression with 100 nM D2. (e) Subcutaneous C4-2 tumours were allowed to establish in SCID mice until palpable and detected on BLI. The basal rate of growth of these tumours was established for 1 week, followed daily intratumoural (i.t.) injections of DMSO, 10 mM D1 or D2 dissolved in 100 ml of 5% dextrose for 5 weeks. Effects on tumor growth were serially followed by BLI (shown in e after 5 weeks of daily i.t. injections) and graphically represented as fold change±s.d. over bioluminescence measured at the start of treatment (f). (g) AR was immunoprecipitated from mouse tumors by incubating extracts with an AR antibody conjugated to rabbit IgG DynaBeads for 60 min at 4 C. Immunocomplexes were resolved by SDS–polyacrylamide gel electrophoresis and blots hybridized for AR, PELP1 and actin. Input is shown in the lower panels. Data in a–d and f are representative of three independent experiments and is shown as mean±s.d., *Po0.05.

Article Snippet: The AR and b-actin antibodies were purchased from Sigma (St Louis, MO); PELP1 antibody from Bethyl Laboratories (Montgomery, TX); AR-C19, GAPDH, Histone H3, tERK, tSrc, tAkt, ERb and PSA antibodies from Santa Cruz Biotechnology (Santa Cruz, CA). pAkt, pSrc (T262/Y204) and pSrc (Y416) from Cell Signaling Technology (Danvers, MA).

Techniques: In Vitro, In Vivo, CyQUANT Assay, Over Expression, Immunoprecipitation, Polyacrylamide Gel Electrophoresis

Journal: Nature communications

Article Title: Peptidomimetic targeting of critical androgen receptor-coregulator interactions in prostate cancer.

doi: 10.1038/ncomms2912

Figure Lengend Snippet: Figure 7 | D2 inhibits AR–PELP1 interaction in prostate cancer cell explants. (a) Following radical prostatectomy, the extirpated prostate is bivalved and a core of tissue corresponding to the area of the tumour is removed by the pathologist. The tissue is then transferred to the laboratory and dissected into 1 mm3 fragments. The fragments are then cultured on gelatin sponge submerged in media supplemented with 10% fetal calf serum. Following incubation with media for a defined time period (2 days), the explants are then processed for further evaluation. (b) Immunoprecipitation (IP) analyses of these prostate explants demonstrate the PPIs between AR and PELP1 in the presence of D1, DMSO but not D2 (100 nM). (c) Representative AR immunohistochemistry (IHC) staining in prostate cancer explants following incubation for 2 days in either complete media in the presence of 100 nM DMSO, D1, D2 or 10 mM of MDV3100 reveals that D2 blocks nuclear AR expression. (d) Quantitation of AR immunostaining in prostate explants is shown for DMSO, D1, D2 or MDV3100 as a control (Po0.05). Data is shown as mean±s.d., n ¼ 7, *Po0.05.

Article Snippet: The AR and b-actin antibodies were purchased from Sigma (St Louis, MO); PELP1 antibody from Bethyl Laboratories (Montgomery, TX); AR-C19, GAPDH, Histone H3, tERK, tSrc, tAkt, ERb and PSA antibodies from Santa Cruz Biotechnology (Santa Cruz, CA). pAkt, pSrc (T262/Y204) and pSrc (Y416) from Cell Signaling Technology (Danvers, MA).

Techniques: Cell Culture, Incubation, Immunoprecipitation, Immunohistochemistry, Expressing, Quantitation Assay, Immunostaining, Control

Journal: Oncogene

Article Title: Molecular Basis of a Novel Oncogenic Mutation in GNAO1

doi: 10.1038/onc.2010.645

Figure Lengend Snippet: Rat Gαo was cloned into pcDNA3.1(+) (Eco RI/Not I), and R243H and Q205L mutants were generated using a Quikchange mutagenesis kit. NIH3T3 cells were transfected with the indicated plasmids using Genejuice as previously described (Ghosh et al., 2010; Ghosh et al., 2008) and maintained in DMEM media supplemented with 10% FBS. Approximately 32 h after transfection the cells were switched to DMEM media supplemented with 2% FBS and cultured overnight. Cells were lysed in sample buffer, boiled and proteins separated by SDS-PAGE. Samples were analyzed by immunobloting (IB) for Y705 phospho-STAT3 (pSTAT3, rabbit pAb, Cell Signaling), total STAT3 (tSTAT3 mouse mAb, Santa Cruz Biotechnology), Y416 phospho-Src (pSrc, rabbit pAb, Cell Signaling), total Src (tSrc, mouse mAb, Santa Cruz Biotechnology), phospho-ERK1/2 (pERK1/2, rabbit pAb, Cell Signaling), S473 phospho-Akt (pAkt, rabbit pAb, Cell Signaling) and Gαo (rabbit pAb, Santa Cruz Biotechnology). When the Gαo R243H (lane 3) and Gαo Q205L (lane 4) mutants were expressed the levels of pSTAT3 and pSrc increased approximately 3 times compared to vector transfected controls (lane 1), whereas wt Gαo (lane 2) had no effect. There was no effect on Akt and ERK1/2 phosphorylation when the different Gαo constructs were expressed.

Article Snippet: Samples were analyzed by immunobloting (IB) for Y705 phospho-STAT3 (pSTAT3, rabbit pAb, Cell Signaling), total STAT3 (tSTAT3 mouse mAb, Santa Cruz Biotechnology), Y416 phospho-Src (pSrc, rabbit pAb, Cell Signaling), total Src (tSrc, mouse mAb, Santa Cruz Biotechnology), phospho-ERK1/2 (pERK1/2, rabbit pAb, Cell Signaling), S473 phospho-Akt (pAkt, rabbit pAb, Cell Signaling) and Gαo (rabbit pAb, Santa Cruz Biotechnology).

Techniques: Clone Assay, Generated, Mutagenesis, Transfection, Cell Culture, SDS Page, Western Blot, Plasmid Preparation, Construct