Journal: Molecular Cancer Therapeutics

Article Title: Glucocorticoid Receptor (GR) Activation Is Associated with Increased cAMP/PKA Signaling in Castration-Resistant Prostate Cancer

doi: 10.1158/1535-7163.MCT-22-0479

Figure Lengend Snippet: GR-mediated cAMP signaling pathway-related genes ( n = 224) with co-treatment of SGRMs. A, Heat map of IPA “cAMP signaling pathway gene expression” in LAPC4 cells treated with Dex and ± GR antagonism (as in ). B, LAPC4 cells were cultured in media containing R1881 and enzalutamide for 7 days, and then treated for 6 hours with either Veh, Dex, Dex+C297, or Dex+C335 and qRT-PCR was performed for a cAMP pathway gene previously associated with prostate cancer biology , PKIB . PKIB steady-state mRNA expression (left: *, P < 0.05; **, P < 0.005; Welch t test). Western blot was performed for PKIB protein expression (right). Densitometric analysis of PKIB vs. β-tubulin is shown below each lane (Western blot is representative of three independent experiments which are quantified in Supplementary Fig. S3A). C, Left, LAPC4 cells were cultured as above, and then treated with ± Dex (8 hours). Cells were probed with either α-PKIB (top) or α-PKA-c antibody (third row) and counterstained with DAPI (600X magnification, scale bar = 25 μm). Right, Bar graph of nuclear PKIB expression per FOV (top) and the ratio of the average PKA-c nuclear to cytoplasmic intensity per FOV following Veh or Dex treatment (bottom). FOV per condition were measured for average intensity: a.u., arbitrary unit; ****, P < 0.0001 (Welch t test).

Article Snippet: Probes used were PKIB (Hs00261162_m1) and ACTB (Hs99999903_m1) as control.

Techniques: Gene Expression, Cell Culture, Quantitative RT-PCR, Expressing, Western Blot

Journal: Molecular Cancer Therapeutics

Article Title: Glucocorticoid Receptor (GR) Activation Is Associated with Increased cAMP/PKA Signaling in Castration-Resistant Prostate Cancer

doi: 10.1158/1535-7163.MCT-22-0479

Figure Lengend Snippet: A downstream cAMP-activated transcription factor, CREB, is phosphorylated at Ser133 following GR activation. A, Western blot of subcellular distribution of pCREBSer133, total CREB, PKA-c, and PKIB in LAPC4 cells cultured for 3 days in R1881 and enzalutamide (RE) media and then treated with Veh or Dex (8 hours). Densitometric analyses are listed below for each lane. is representative of three independent experiments. B, LAPC4 cells were cultured as above in RE media for 7 days, and then treated with either Veh, Dex, Dex+C297, Dex+C335 (8 hours) and a Western blot was performed. Densitometric analysis of phospho-CREB versus total CREB intensity is listed below each lane. The western blot shown is representative of three independent experiments. C, LAPC4 cells were cultured in RE media for 7 days, and then treated with either Veh or Dex (8 hours). Forskolin (FSK, 10 μmol/L) was used as a positive control for PKA-mediated CREB phosphorylation. Left, Cells were probed with α-pCREBSer133 antibody and stained with DAPI (600X magnification, scale bar = 25 μm). Right: Bar graph shows average CREB phosphorylation by mean immunofluorescent intensity per 8 to 13 FOVs; ***, P < 0.001 (Welch t test). D, Dot bar graphs (8–13 FOV per condition) show mean immunofluorescent intensity of whole cell or nuclear CREB and pCREB in LAPC4 cells.

Article Snippet: Probes used were PKIB (Hs00261162_m1) and ACTB (Hs99999903_m1) as control.

Techniques: Activation Assay, Western Blot, Cell Culture, Positive Control, Phospho-proteomics, Staining

Journal: Molecular Cancer Therapeutics

Article Title: Glucocorticoid Receptor (GR) Activation Is Associated with Increased cAMP/PKA Signaling in Castration-Resistant Prostate Cancer

doi: 10.1158/1535-7163.MCT-22-0479

Figure Lengend Snippet: Addition of SGRM to enzalutamide therapy delays tumor growth and inhibits PKIB expression in castration-resistant LAPC4 tumor xenografts. A, Schema of experiment shows that athymic nude mice underwent orchiectomy and immediate testosterone pellet implantation to allow initial LAPC4 xenograft growth. Following the establishment of primary LAPC4 xenograft tumors (in the presence of testosterone), removal of the testosterone pellet left castrate levels of androgen (“a”). Mice with resultant castration-resistant LAPC4 tumors (∼500 mm 3 ) began treatment with (“b”) enzalutamide (30 mg/kg chow), ± C297 (C297, 20 mg/kg/day), or C335 (C335, 20 mg/kg/day). Mice were sacrificed at tumor doubling (∼1000 mm 3 ) which was considered CRPC progression (“c”). Below, Kaplan–Meier progression-free survival curves show time to CRPC doubling/progression. Log-rank P values are shown. B, LAPC4 representative anti-GR and anti-Ki67 immunohistochemistry with a magnified inset. C, Percentage of GR+ (left: *, P < 0.05; Welch t test) and Ki67+ cells (right: **, P < 0.005; Welch t test) shown for at least 3 tumors in each treatment group. D, Dot plot with mean and S.E.M of fold-change of PKIB mRNA measured by qRT-PCR from LAPC4 xenograft tumors normalized to reference gene ( ACTB ). N = 7–8 tumors per treatment.

Article Snippet: Probes used were PKIB (Hs00261162_m1) and ACTB (Hs99999903_m1) as control.

Techniques: Expressing, Immunohistochemistry, Quantitative RT-PCR

Journal: Molecular Cancer Therapeutics

Article Title: Glucocorticoid Receptor (GR) Activation Is Associated with Increased cAMP/PKA Signaling in Castration-Resistant Prostate Cancer

doi: 10.1158/1535-7163.MCT-22-0479

Figure Lengend Snippet: GR-mediated cAMP signaling pathway-related genes ( n = 224) with co-treatment of SGRMs. A, Heat map of IPA “cAMP signaling pathway gene expression” in LAPC4 cells treated with Dex and ± GR antagonism (as in ). B, LAPC4 cells were cultured in media containing R1881 and enzalutamide for 7 days, and then treated for 6 hours with either Veh, Dex, Dex+C297, or Dex+C335 and qRT-PCR was performed for a cAMP pathway gene previously associated with prostate cancer biology , PKIB . PKIB steady-state mRNA expression (left: *, P < 0.05; **, P < 0.005; Welch t test). Western blot was performed for PKIB protein expression (right). Densitometric analysis of PKIB vs. β-tubulin is shown below each lane (Western blot is representative of three independent experiments which are quantified in Supplementary Fig. S3A). C, Left, LAPC4 cells were cultured as above, and then treated with ± Dex (8 hours). Cells were probed with either α-PKIB (top) or α-PKA-c antibody (third row) and counterstained with DAPI (600X magnification, scale bar = 25 μm). Right, Bar graph of nuclear PKIB expression per FOV (top) and the ratio of the average PKA-c nuclear to cytoplasmic intensity per FOV following Veh or Dex treatment (bottom). FOV per condition were measured for average intensity: a.u., arbitrary unit; ****, P < 0.0001 (Welch t test).

Article Snippet: Antibodies and concentrations were: anti-pan-phospho-PKA substrate [100G7E, 1:1,000, Cell Signaling Technology (CST)], anti-phospho-cAMP response element-binding protein (CREB) Ser133 (87G3, 1:1,000, CST), anti-CREB (86B10, 1:1,000, CST), anti-α-tubulin (DM1A, 1:5,000, CST), anti-beta actin (8H10D10, 1:5,000, CST), anti-PKA catalytic subunit (anti–PKA-c; 1:1,000, Santa Cruz Biotechnology sc-28315), anti-PKIB (1:1,000, Abcam ab233521), anti-Vinculin (13901, 1:1,000, CST), anti-Lamin B1 (13435, 1:1,000, CST).

Techniques: Expressing, Cell Culture, Quantitative RT-PCR, Western Blot

Journal: Molecular Cancer Therapeutics

Article Title: Glucocorticoid Receptor (GR) Activation Is Associated with Increased cAMP/PKA Signaling in Castration-Resistant Prostate Cancer

doi: 10.1158/1535-7163.MCT-22-0479

Figure Lengend Snippet: A downstream cAMP-activated transcription factor, CREB, is phosphorylated at Ser133 following GR activation. A, Western blot of subcellular distribution of pCREBSer133, total CREB, PKA-c, and PKIB in LAPC4 cells cultured for 3 days in R1881 and enzalutamide (RE) media and then treated with Veh or Dex (8 hours). Densitometric analyses are listed below for each lane. is representative of three independent experiments. B, LAPC4 cells were cultured as above in RE media for 7 days, and then treated with either Veh, Dex, Dex+C297, Dex+C335 (8 hours) and a Western blot was performed. Densitometric analysis of phospho-CREB versus total CREB intensity is listed below each lane. The western blot shown is representative of three independent experiments. C, LAPC4 cells were cultured in RE media for 7 days, and then treated with either Veh or Dex (8 hours). Forskolin (FSK, 10 μmol/L) was used as a positive control for PKA-mediated CREB phosphorylation. Left, Cells were probed with α-pCREBSer133 antibody and stained with DAPI (600X magnification, scale bar = 25 μm). Right: Bar graph shows average CREB phosphorylation by mean immunofluorescent intensity per 8 to 13 FOVs; ***, P < 0.001 (Welch t test). D, Dot bar graphs (8–13 FOV per condition) show mean immunofluorescent intensity of whole cell or nuclear CREB and pCREB in LAPC4 cells.

Article Snippet: Antibodies and concentrations were: anti-pan-phospho-PKA substrate [100G7E, 1:1,000, Cell Signaling Technology (CST)], anti-phospho-cAMP response element-binding protein (CREB) Ser133 (87G3, 1:1,000, CST), anti-CREB (86B10, 1:1,000, CST), anti-α-tubulin (DM1A, 1:5,000, CST), anti-beta actin (8H10D10, 1:5,000, CST), anti-PKA catalytic subunit (anti–PKA-c; 1:1,000, Santa Cruz Biotechnology sc-28315), anti-PKIB (1:1,000, Abcam ab233521), anti-Vinculin (13901, 1:1,000, CST), anti-Lamin B1 (13435, 1:1,000, CST).

Techniques: Activation Assay, Western Blot, Cell Culture, Positive Control, Staining

Journal: Molecular Cancer Therapeutics

Article Title: Glucocorticoid Receptor (GR) Activation Is Associated with Increased cAMP/PKA Signaling in Castration-Resistant Prostate Cancer

doi: 10.1158/1535-7163.MCT-22-0479

Figure Lengend Snippet: GR-mediated cAMP signaling pathway-related genes ( n = 224) with co-treatment of SGRMs. A, Heat map of IPA “cAMP signaling pathway gene expression” in LAPC4 cells treated with Dex and ± GR antagonism (as in ). B, LAPC4 cells were cultured in media containing R1881 and enzalutamide for 7 days, and then treated for 6 hours with either Veh, Dex, Dex+C297, or Dex+C335 and qRT-PCR was performed for a cAMP pathway gene previously associated with prostate cancer biology , PKIB . PKIB steady-state mRNA expression (left: *, P < 0.05; **, P < 0.005; Welch t test). Western blot was performed for PKIB protein expression (right). Densitometric analysis of PKIB vs. β-tubulin is shown below each lane (Western blot is representative of three independent experiments which are quantified in Supplementary Fig. S3A). C, Left, LAPC4 cells were cultured as above, and then treated with ± Dex (8 hours). Cells were probed with either α-PKIB (top) or α-PKA-c antibody (third row) and counterstained with DAPI (600X magnification, scale bar = 25 μm). Right, Bar graph of nuclear PKIB expression per FOV (top) and the ratio of the average PKA-c nuclear to cytoplasmic intensity per FOV following Veh or Dex treatment (bottom). FOV per condition were measured for average intensity: a.u., arbitrary unit; ****, P < 0.0001 (Welch t test).

Article Snippet: The following antibodies and reagents were used: anti-PKA-c (Santa Cruz Biotechnology, sc-28315), anti-PKIB (ThermoFisher Scientific, PA538783), anti-CREB (CST, 9104), anti-phospho-CREB S133 (CST, 9198), goat anti-rabbit and anti-mouse secondary antibodies (ThermoFisher Scientific, A32732, A32723), mounting medium with DAPI (ThermoFisher Scientific, P36941), and normal goat serum (CST, 5425S).

Techniques: Expressing, Cell Culture, Quantitative RT-PCR, Western Blot

Journal: Molecular Cancer Therapeutics

Article Title: Glucocorticoid Receptor (GR) Activation Is Associated with Increased cAMP/PKA Signaling in Castration-Resistant Prostate Cancer

doi: 10.1158/1535-7163.MCT-22-0479

Figure Lengend Snippet: A downstream cAMP-activated transcription factor, CREB, is phosphorylated at Ser133 following GR activation. A, Western blot of subcellular distribution of pCREBSer133, total CREB, PKA-c, and PKIB in LAPC4 cells cultured for 3 days in R1881 and enzalutamide (RE) media and then treated with Veh or Dex (8 hours). Densitometric analyses are listed below for each lane. is representative of three independent experiments. B, LAPC4 cells were cultured as above in RE media for 7 days, and then treated with either Veh, Dex, Dex+C297, Dex+C335 (8 hours) and a Western blot was performed. Densitometric analysis of phospho-CREB versus total CREB intensity is listed below each lane. The western blot shown is representative of three independent experiments. C, LAPC4 cells were cultured in RE media for 7 days, and then treated with either Veh or Dex (8 hours). Forskolin (FSK, 10 μmol/L) was used as a positive control for PKA-mediated CREB phosphorylation. Left, Cells were probed with α-pCREBSer133 antibody and stained with DAPI (600X magnification, scale bar = 25 μm). Right: Bar graph shows average CREB phosphorylation by mean immunofluorescent intensity per 8 to 13 FOVs; ***, P < 0.001 (Welch t test). D, Dot bar graphs (8–13 FOV per condition) show mean immunofluorescent intensity of whole cell or nuclear CREB and pCREB in LAPC4 cells.

Article Snippet: The following antibodies and reagents were used: anti-PKA-c (Santa Cruz Biotechnology, sc-28315), anti-PKIB (ThermoFisher Scientific, PA538783), anti-CREB (CST, 9104), anti-phospho-CREB S133 (CST, 9198), goat anti-rabbit and anti-mouse secondary antibodies (ThermoFisher Scientific, A32732, A32723), mounting medium with DAPI (ThermoFisher Scientific, P36941), and normal goat serum (CST, 5425S).

Techniques: Activation Assay, Western Blot, Cell Culture, Positive Control, Staining

Journal: Molecular Cancer Therapeutics

Article Title: Glucocorticoid Receptor (GR) Activation Is Associated with Increased cAMP/PKA Signaling in Castration-Resistant Prostate Cancer

doi: 10.1158/1535-7163.MCT-22-0479

Figure Lengend Snippet: Addition of SGRM to enzalutamide therapy delays tumor growth and inhibits PKIB expression in castration-resistant LAPC4 tumor xenografts. A, Schema of experiment shows that athymic nude mice underwent orchiectomy and immediate testosterone pellet implantation to allow initial LAPC4 xenograft growth. Following the establishment of primary LAPC4 xenograft tumors (in the presence of testosterone), removal of the testosterone pellet left castrate levels of androgen (“a”). Mice with resultant castration-resistant LAPC4 tumors (∼500 mm 3 ) began treatment with (“b”) enzalutamide (30 mg/kg chow), ± C297 (C297, 20 mg/kg/day), or C335 (C335, 20 mg/kg/day). Mice were sacrificed at tumor doubling (∼1000 mm 3 ) which was considered CRPC progression (“c”). Below, Kaplan–Meier progression-free survival curves show time to CRPC doubling/progression. Log-rank P values are shown. B, LAPC4 representative anti-GR and anti-Ki67 immunohistochemistry with a magnified inset. C, Percentage of GR+ (left: *, P < 0.05; Welch t test) and Ki67+ cells (right: **, P < 0.005; Welch t test) shown for at least 3 tumors in each treatment group. D, Dot plot with mean and S.E.M of fold-change of PKIB mRNA measured by qRT-PCR from LAPC4 xenograft tumors normalized to reference gene ( ACTB ). N = 7–8 tumors per treatment.

Article Snippet: The following antibodies and reagents were used: anti-PKA-c (Santa Cruz Biotechnology, sc-28315), anti-PKIB (ThermoFisher Scientific, PA538783), anti-CREB (CST, 9104), anti-phospho-CREB S133 (CST, 9198), goat anti-rabbit and anti-mouse secondary antibodies (ThermoFisher Scientific, A32732, A32723), mounting medium with DAPI (ThermoFisher Scientific, P36941), and normal goat serum (CST, 5425S).

Techniques: Expressing, Immunohistochemistry, Quantitative RT-PCR