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. 2019 May 7;47(8):4211-4225.
doi: 10.1093/nar/gkz108.

LncRNA PCAT1 Activates AKT and NF-κB Signaling in Castration-Resistant Prostate Cancer by Regulating the PHLPP/FKBP51/IKKα Complex

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Free PMC article

LncRNA PCAT1 Activates AKT and NF-κB Signaling in Castration-Resistant Prostate Cancer by Regulating the PHLPP/FKBP51/IKKα Complex

Zhiqun Shang et al. Nucleic Acids Res. .
Free PMC article

Abstract

In PTEN-deficient prostate cancers, AKT signaling may be activated upon suppression of androgen receptor signaling. Activation of AKT as well as NF-κB signaling involves a key regulatory protein complex containing PHLPP, FKBP51 and IKKα. Here, we report a critical role of lncRNA PCAT1 in regulating the PHLPP/FKBP51/IKKα complex and progression of castration-resistant prostate cancer (CRPC). Using database queries, bioinformatic analyses, as well as RIP and RNA pull-down assays, we discovered and validated that the lncRNA-PCAT1 perturbs the PHLPP/FKBP51/IKKα complex and activates AKT and NF-κB signaling. Expression of lncRNA-PCAT1 is positively linked to CRPC progression. PCAT1 binds directly to FKBP51, displacing PHLPP from the PHLPP/FKBP51/IKKα complex, leading to activation of AKT and NF-κB signaling. Targeting PCAT1 restores PHLPP binding to FKBP1 leading to suppression of AKT signaling. Preclinical study in a mouse model of CRPC suggests therapeutic potential by targeting lncRNA PCAT1 to suppress CRPC progression. Together, the newly identified PCAT1/FKBP51/IKKα complex provides mechanistic insight in the interplay between AKT, NF-κB and AR signaling in CRPC, and the preclinical studies suggest that a novel role for PCAT1 as a therapeutic target.

Figures

Figure 1.
Figure 1.
LncRNA-PCAT1 expression is correlated with castration-resistant prostate cancer. (A) Kaplan–Meier curve of the recurrence-free survival rates in prostate cancer patients with and without genetic amplification of PCAT1 (P = 0.021). Those without PCAT1 amplification included samples with deep deletion (n = 3) as well as those without alteration of PCAT1. The Cancer Genome Atlas data were retrieved from cBioPortal. (B) Kaplan–Meier curve of the overall survival rates in prostate cancer patients with and without genetic amplification of PCAT1 (P < 0.001). Those without PCAT amplification included samples with deep deletion (n = 3) as well as those without alteration of PCAT1. The Cancer Genome Atlas data were retrieved from cBioPortal. (C) Median expression of PCAT1 from two independent PCa patients’ RNA-seq data sets based on the value of FPKM. TCGA, The Cancer Genome Atlas; ADPC, androgen dependent prostate cancer; CRPC, castration-resistant prostate cancer. In dot plots, the center line is the median, with each dot depicting the FPKM value of each patient. The P value was determined by two-tailed t-tests. (D) RISH detection of PCAT1 expression in ADPC versus CRPC. Left panel: representative images; right panel: statistical analysis of five ADPC patient specimens and five CRPC patient specimens. (E) RT-PCR analysis of PCAT1 in fresh surgical specimens from patients with CRPC (n = 6) and ADPC (n = 8). GAPDH was used as a loading control. (F) Flowchart showing establishment of the androgen-independent LNCaP-AI cell line. The androgen-independent LNCaP-AI cell line was generated by long-term culture of androgen-dependent LNCaP cells in RPMI-1640 medium containing charcoal-stripped serum. LNCaP-AD, Androgen-dependent LNCaP cell line; LNCaP-AI, Androgen-independent LNCaP cell line. P, passage. (G) Scatter plots of lncRNAs significantly upregulated (blue) or downregulated (orange) in LNCaP-AI compared to LNCaP cells. X and Y axes are normalized signal values (log2 scaled) for each gene, with PCAT1 labeled by a red plot. (H) qRT-PCR detection of PCAT1 expression in LNCaP and LNCaP-AI cell lines, normalized by the level of GAPDH. A P-value of <0.05 was considered significant. *represents P < 0.05, **represents P < 0.01 and ***represents P < 0.001.
Figure 2.
Figure 2.
PCAT1 regulates AKT and NF-κB signaling pathways. (A) Heat map of key genes regulated positively by AKT or NF-κB signal pathways. These genes have decreased expression in PCAT1 depleted LNCaP-AI cells (P < 0.05). RNA-Seq data are displayed with log2 scaled FPKM (fragments per kilobase of transcript per million mapped reads) values for each gene in each sample. The gene names and other details for each specific gene were shown in Supplementary Table S4. (B) RT-PCR detection of PCAT1 and IB detection of indicated proteins in LNCaP-AI cells transfected with PCAT1 siRNAs. GAPDH was used as a loading control. (C) RT-PCR detection of PCAT1 and IB detection of AKT and NF-κB signaling molecules in C4-2 cells transfected with PCAT1 shRNAs. GAPDH was used as a loading control. (D) RT-PCR detection of PCAT1 in LNCaP-AI cell line with PCAT1 overexpression and IB detection of AKT and NF-κB signaling molecules. GAPDH was used as a loading control. (E) RT-PCR detection of PCAT1 in C4-2 cell line with PCAT1 overexpression and western blotting analysis of indicated proteins expression in PCAT1-overexpressed C4-2 cells. GAPDH was used as a loading control.
Figure 3.
Figure 3.
Bioinformatic identification and biochemical characterization of PCAT1-associated proteins. (A) Commonly upregulated (fold change > 2.0-fold, P < 0.01) lncRNAs (n = 245) in our Arraystar Human LncRNA Microarray V3.0 data (details in Supplementary Table S5) and possible lncRNAs (n = 92) interacted with FKBP51 protein predicted by the catRAPID omics module. (B) CatRAPID signature module prediction of the RNA-binding propensity for FKBP51 protein followed by prediction of RNA-binding regions. Overall interaction scores above 50% indicate propensity to bind. (C) CatRAPID fragments module prediction of the interaction profile and matrix between FKBP51 protein and PCAT1. (D) IB detection of proteins retrieved by in vitro-transcribed desthiobiotinylated PCAT1 from LNCaP-AI cell lysates. (E) RIP detection of the interaction between FKBP51 and PCAT1 by FKBP51 antibody in LNCaP-AI cells. The level of PCAT1 was determined by qRT-PCR and normalized by the input levels. A P-value of <0.05 was considered significant. *represents P < 0.05, **represents P < 0.01 and ***represents P < 0.001. (F) RT-PCR detection of PCAT1 in LNCaP-AI cell line after PCAT1 knockdown and IB detection of FKBP51, IKKα and PHLPP proteins expression after transfection with lentiviruses carrying PCAT1 shRNA in LNCaP-AI cells.
Figure 4.
Figure 4.
PCAT1/FKBP51 interaction perturbs FKPB51/IKKα/PHLPP protein complex in CRPC. (A) IP and IB detection of FKBP51/IKKα and FKBP51/PHLPP interactions in LNCaP cells, LNCaP-AD P30 cells and LNCaP-AI cells. LNCaP-AD, Androgen-dependent LNCaP cell line; LNCaP-AI, Androgen-independent LNCaP cell line. (B) IP and IB detection of FKBP51/IKKα and FKBP51/PHLPP interactions in LNCaP-AI cells transfected with indicated siRNAs. (C) Western blotting analysis of indicated proteins in LNCaP cells with PHLPP knockdown via PHLPP siRNAs. (D) Lack of PCAT1/FKBP51 interaction involving the truncation mutants. GST-tag (GST), GST-tagged full-length FKBP51 (GST-FKBP51-WT) and GST-tagged FKBP51 truncated mutant (GST-FKBP51-MUT) (Δ251–390AA) were transfected into PCAT1-MUT overexpressed LNCaP-AI cells (LNCaP-AI PCAT1-MUT). RIP assays were performed with the GST-tag antibody, and the levels of PCAT1 and PCAT1-MUT were determined by RT-PCR. PCAT1-MUT, PCAT1 truncated mutant (Δ1001–1400bp). (E) Overexpression of PCAT1, but not the mutant PCAT1, rescues the effect of PCAT1 silencing. PCAT1-truncated mutant (PCAT1-MUT) (Δ1001–1400bp) and wild-type PCAT1 were transfected into LNCaP-AI shPCAT1 stable cell line, and expression of PCAT1 and PCAT1-MUT were detected by RT-PCR (upper panel). FKBP51/IKKα and FKBP51/PHLPP interactions were determined in these cells by IP and IB assays. (F) A model for PCAT1-mediated activation of AKT and NF-κB signal pathways by perturbing FKPB51/IKKα/PHLPP protein complex in the progression from ADPC to CRPC after androgen deprivation therapy.
Figure 5.
Figure 5.
PCAT1 functions through the PCAT1/FKBP51/IKKα complex to promote CRPC progression. (A) MTT assays in LNCaP-AI and C4-2 cells infected with lentiviruses carrying shPCAT1. Left panel: qRT-PCR detection of PCAT1 expression after transfection with shPCAT1; right panel: cell growth assessed daily for 3 days using an MTT assay. Data were obtained from three independent experiments with samples in triplicate. One-way analysis of variance (ANOVA) and paired Student’s t-test was carried out using SPSS 22 statistical software. A P-value of <0.05 was considered significant. *represents P < 0.05, **represents P < 0.01 and ***represents P < 0.001. (B) MTT assays in LNCaP-AI and C4-2 cells infected with lentiviruses overexpressing PCAT1. Left panel: qRT-PCR detection of PCAT1 expression after overexpression of PCAT1; right panel: cell growth was assessed daily for 3 days using an MTT assay. A P-value of <0.05 was considered significant. *represents P < 0.05, **represents P < 0.01 and ***represents P < 0.001. (C) Immunofluorescence assays in PCAT1-deficient LNCaP-AI cells. For each group, representative images were randomly chosen under fluorescent microscopy with 200-fold magnification. (D) Hoechst33258-PI Staining assays in PCAT1-deficient LNCaP-AI cells. For each group, representative images were randomly chosen under fluorescent microscopy with 200-fold magnification. (E) Immunofluorescence assays in PCAT1-deficient C4-2 cells. For each group, representative images were randomly chosen under fluorescent microscopy with 200-fold magnification. (F) Hoechst33258-PI Staining assays in PCAT1-deficient C4-2 cells. For each group, representative images were randomly chosen under fluorescent microscopy with 200-fold magnification. (G) Statistical analysis of Figure 5C–F. A P-value of <0.05 was considered significant. *represents P < 0.05, **represents P < 0.01 and ***represents P < 0.001. (H) IB detection of FKBP51 and MTT assay detection of cells growth after FKBP51 knocked down in PCAT1 overexpressed LNCaP-AI cells. A P-value of <0.05 was considered significant. *represents P < 0.05, **represents P < 0.01 and ***represents P < 0.001.
Figure 6.
Figure 6.
Preclinical studies targeting the lncRNA PCAT1 in a CRPC animal model. (A and B) Suppression of CRPC tumor growth in animals treated with PCAT1 shRNA (n = 4) versus scrambled shRNA (n = 4) for 6 days. Tumor sizes were measured for 6 days (A), at the time of tumor removal (B). A P-value of <0.05 was considered significant. *represents P < 0.05, **represents P < 0.01 and ***represents P < 0.001. (C) RISH detection of PCAT1 expression in the two indicated groups of mouse tumor specimens. For each group, six different fields were randomly chosen and counted under microscopy with 400-fold magnification. Representative images and statistical analysis are shown. Evaluation not blinded. A P-value of <0.05 was considered significant. *represents P < 0.05, **represents P < 0.01 and ***represents P < 0.001. (D) IHC staining of Ki67, p-AKT (Ser473), p-NF-κB p65 (Ser536) and indicated proteins in the two indicated groups of mouse tumor specimens. For each group, six different fields were randomly chosen and counted under microscopy with 400-fold magnification. Representative images and statistical analysis are shown. Positive rate (%) = number of positive cells/ number of total cells × 100%. Evaluation not blinded. A P-value of <0.05 was considered significant. *represents P < 0.05, **represents P < 0.01 and ***represents P < 0.001.

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