Fig 2: CREB inhibitor 666-15 blocks prostate cancer cell and CRPC tumor growth(A) Indicated prostate cancer cell lines were treated with dose responses of 666-15 for 7 days and IC50 values were quantified. Data are represented as mean ± SEM.(B) Cell growth curve of LNCaP and C4-2 cells treated with 666-15.(C) Cell growth curves of Cas9 (control), CREB1 KO, ATF1 SKO, and CREB1/ATF1 DKO C4-2 cells treated with 666-15.(D) BrdU cell-cycle analysis of 666-15-treated C4-2 cells after 12 h.(E and F) Tumor growth (data are represented as mean ± SEM. One-way ANOVA; **p < 0.01) (E) and KM survival curves (F) of C4-2 xenograft mice (castrated) treated with vehicle or 666-15.(G) IHC staining of BrdU for tumor samples from (E) and (F). Left, representative staining (100×); right, quantification. Data are represented as mean ± SEM. Student’s t test; **p < 0.01, compared with vehicle treatment. Scale bar, 100 μM.(H and I) Tumor growth curves of PDX models MDA-PCa-274-2 (H) and MDA-PCa-180-30 (I) treated with vehicle, 666-15, or enzalutamide as indicated. Data are represented as mean +SEM. One-way ANOVA; *p < 0.05, ns = not significant.

Fig 3: CAMKIα is the predominant kinase mediating AR-CAMKK2-induced phosphorylation and activation of CREB in prostate cancer cells(A) Illustration of the downstream kinases of CAMKK2.(B) LNCaP cells were transfected with mock (M) or siRNAs targeting scramble control (NC) or PRKAA1 (encoding the major AMPKα catalytic subunit isoform in prostate cancer cells), and immunoblot analysis was performed on transfected cells that were treated ± 10 nM R1881 for 72 h.(C and D) LNCaP (C) and VCaP (D) cells were transfected with siRNAs targeting mock (M), scramble control (NC), or CAMKIα and immunoblot analysis of transfected cells that were treated ±10 nM R1881 for 72 h.(E) C4-2 (left) and 22Rv1 (right) cells were transfected with siRNAs targeting mock (M), scramble control (NC), or CAMKIα and subjected to immunoblot analysis after 72 h. C4-2 and 22Rv1 experiments were done in the absence of androgens.(F) CRE-luciferase activity of LNCaP cells treated with control (NC) or siCAMK1α and vehicle or R1881 (10 nM) for 72 h. Data are represented as mean ± SEM. One-way ANOVA; **p < 0.01, compared with NC + R1881. *p < 0.05, compared with NC + R1881.

Fig 4: ATF1 and CREB1 exhibit functional redundancy in CRPC(A) Alignment of human CREB1 and ATF1 proteins. Percentages denote the amino acid homology between CREB1 and ATF1 in KID and bZIP domains.(B) Cell growth curve of CRISPR-modified C4-2 cells with ATF1 KO compared with parental C4-2 and C4-2 Cas9 cells. Data are represented as mean ± SEM. One-way ANOVA; **p < 0.01.(C) Heatmaps of differentially expressed genes in CREB1 KO (left) and ATF1 KO (right) cells, compared with Cas9 control (LFC ≥ 1 and padj < 0.01). The color scale indicates the Z score (blue, decreased expression, red, increased expression).(D) Immunoblot analysis of CRISPR-mediated C4-2 cells with ATF1 KO.(E) The number of downregulated genes in CREB1 single knockout (SKO), ATF1 SKO, and CREB1/ATF1 double KO (DKO) cells. The result shows the overlap gene list from two sgRNAs.(F–I) CRISPR-mediated CREB1 SKO and CREB1/ATF1 DKO C4-2 cells were subcutaneously injected into castrated NSG mice (n = 10/group).(F) Tumor growth. Data are represented as mean ± SEM. One-way ANOVA; **p < 0.01. ##p < 0.01, compared to CREB1 KO-1.(G) KM plot of tumor incidence. Log rank test, **p < 0.01, compared with Cas9 control. ##p < 0.01, compared with CREB1 KO-1.(H) KM plot of survival. Three of the DKO mice survived more than 8 months. Log rank test, **p < 0.01, compared with Cas9 control. ##p < 0.01, compared with CREB1 KO-1.(I) IHC staining of p-CREB and BrdU for tumor samples (100×). Data are represented as mean ± SEM. One-way ANOVA; **p < 0.01, *p < 0.05, compared with Cas9 control. Scale bar, 100 μM. Experiments were done in the absence of androgens.

Fig 5: CREB phosphorylation and activity correlate with CAMKK2 and disease progression in diverse preclinical prostate cancer cell and animal models(A) GSEA analysis of cAMP pathway and CREB motif signatures along the stat score rank of transcripts expressed in CAMKK2 KO compared with Cas9 control (WT) C4-2 cells.(B) Immunoblot and densitometry (left) and densitometry Pearson correlation (right) analysis of AR, CAMMK2, and p-CREB (Ser133) levels in 11 prostate cancer PDX models.(C) Immunoblot analysis of LNCaP and LNCaP-derived CRPC cells (C4-2 and C4-2B).(D) Immunohistochemical (IHC) staining of p-CREB (representative in dissected prostate lobes: left, 100×; quantification: right) in 40-week-old Pb-Cre4+/+;Ptenf/f (n = 3) and Pb-CreTg/+;Ptenf/f (n = 3) mice. Scale bar, 100 μM.(E) IHC staining of p-CREB (representative prostate staining: left, 100×; quantification: right) in 30-week-old WT (n = 5) and TRAMP (n = 5) mice. Scale bar, 100 μM.