Dysregulation of spliceosome gene expression in advanced prostate cancer by RNA-binding protein PSF

Abstract

Developing therapeutic approaches are necessary for treating hormone-refractory prostate cancer. Activation of androgen receptor (AR) and its variants' expression along with the downstream signals are mostly important for disease progression. However, the mechanism for marked increases of AR signals and its expression is still unclear. Here, we revealed that various spliceosome genes are aberrantly induced by RNA-binding protein PSF, leading to enhancement of the splicing activities for AR expression. Our high-speed sequence analyses identified global PSF-binding transcripts. PSF was shown to stabilize and activate key long noncoding RNAs and AR-regulated gene expressions in prostate cancer cells. Interestingly, mRNAs of spliceosome-related genes are putative primary targets of PSF. Their gene expressions are up-regulated by PSF in hormone-refractory prostate cancer. Moreover, PSF coordinated these spliceosome proteins to form a complex to promote AR splicing and expression. Thus, targeting PSF and its related pathways implicates the therapeutic possibility for hormone-refractory prostate cancer.

Keywords: NONO; PSF; RNA-binding protein; androgen receptor; prostate cancer.

Fig. 1.

Clinical significance of PSF expression in prostate cancer progression and CRPC tumor growth. (A) PSF is up-regulated in a subset of prostate cancer samples. Immunohistochemistry of PSF in prostate cancer and benign prostate tissues (n = 102) was performed. A negative control using normal rabbit IgG as a primary antibody in the case with PSF positive staining is shown. (B) Higher expression of PSF (n = 51) is a prognostic factor for prostate cancer patients. Kaplan–Meier analysis using the log-rank test was performed. (C) PSF expression levels in metastatic prostate cancer tissues were analyzed using public database (GSE35988, GSE3325, GSE21034). Meta, metastatic cancer; Localized, stage < T1; Advanced, stage > T2. (D) Nude mice were inoculated with 22Rv1 cells. After tumor development, we performed castration and divided into two groups randomly. Tumor growth of xenografted 22Rv1 cells in nude mice treated with siControl or siPSF is shown (n = 8). Representative views of tumors in nude mice are shown. (E) Western blot analysis was performed to evaluate PSF and its downstream signals in tumors. Values represent the mean ± SD. *P < 0.05, **P < 0.01.

Fig. 2.

Global analysis of PSF-binding RNAs revealed roles of PSF in the spliceosome and lncRNA maturation. (A) Schematic diagram of global analysis of PSF-binding RNAs. (B) Identification of RefSeq and NONCODE transcripts bound with PSF. (C) Global mapping of PSF-binding transcripts in CRPC cells. RIP-seq was performed in LNCaP, LTAD, and 22Rv1 cells. The number of significant PSF-binding genes annotated in RefSeq are shown. (D) Representative mapping of PSF-binding RNAs in the CTBP1-AS locus. We present the results of RIP-seq, CLIP-seq, and ChIP-seq (AR, AcH3) in LNCaP cells. Arrows indicate the direction of CTBP1 and CTBP1-AS. (E) Genomic location of the identified peaks in the presence and absence of androgen. TTS, terminal transcription site; 5′UTR, 5′ untranslated region; 3′UTR, 3′ untranslated region. (F) The difference of PSF-binding genes identified by CLIP in the absence and presence of DHT. (G) PSF binds to androgen-induced genes significantly in the presence of DHT. The number of PSF-binding genes was counted, and χ² test was performed. (H) Overlap of identified genes by CLIP-seq with RIP-seq. (I) Regulation of PSF-binding genes in prostate cancer cells. LNCaP cells were treated with siControl or siPSF#1. After 48 h incubation, cells were treated with vehicle or DHT for 24 h. PSF-binding genes identified by RIP-seq were selected, and changes in their expression levels by PSF knockdown are summarized. (J) PSF regulates androgen-dependent gene activation. Heatmap results of RNA-seq in androgen-regulated genes are shown. (K) Regulation of lncRNAs by PSF binding. PSF-binding lncRNAs identified by CLIP-seq and RIP-seq are selected. (L) The ratio of miRNAs and lncRNAs in PSF-binding lncRNAs identified by CLIP. (M) Expressions of androgen-induced miRNAs (miR-125b2, miR-99a, and miR-21) were repressed by knockdown of PSF. LNCaP cells were treated with siControl, siPSF#1, or siPSF#2 for 48 h. We then measured miRNA expression levels by qRT-PCR in cells after treatment of vehicle or DHT for 24 h (n =3). (N) mRNA stability of SchLAP1 was decreased by knockdown of PSF. LNCaP cells are treated with siControl, siPSF #1, or siPSF #2 for 48 h. To inhibit transcription, actinomycin-D (1 nM) was added. After incubation for indicated times, mRNA expression levels were measured by qRT-PCR (n = 3). Values represent the mean ± SD. *P < 0.05, **P < 0.01.

Fig. 3.

PSF bindings to the target transcripts are enhanced in CRPC. (A) Pathway analysis of PSF-binding genes in 22Rv1 cells is shown. (B) Representative mapping of PSF-binding RNAs in AR locus. (C) Expression levels of PSF, NONO, AR, and AR-V7 in prostate cancer cells. Lysates from LNCaP, LTAD, VCaP, and 22Rv1 cells were used for immunoblots with indicated antibodies. (D) Regulation of AR and AR-V7 expression by PSF and NONO in CRPC model cells. (Left) The 22Rv1 cells were treated with siPSF or siNONO for 72 h. AR and AR-V7 mRNA levels were measured by qRT-PCR (n = 3). (Right) Lysates from 22Rv1 cells transfected with siPSF or siNONO are used for immunoblots to detect AR and AR-V7 protein levels. (E) mRNA stability of AR and AR-V7 was decreased by knockdown of PSF. The 22Rv1 cells are treated with siControl, siPSF #1, or siPSF #2 for 48 h. To inhibit transcription, actinomycin-D (1 nM) was added. After incubation for indicated times, mRNA expression levels were measured by qRT-PCR (n = 3). (F) Immunohistochemistry of AR-V7 in prostate cancer tissues (n = 102) was performed. (G) AR-V7 expression (high, n = 18; low, n = 86) is a strong prognostic factor for prostate cancer patients. Cases with labeling index (LI) > 10% were determined to be AR-V7 high expression. (H) Positive correlation of PSF expression with AR and AR-V7 levels in prostate cancer tissues. LI in each group is shown. (I) NONO as well as PSF was up-regulated in metastatic prostate cancer tissues. Heatmap results using microarray database (GSE35988) is shown. (J) Immunohistochemistry of NONO in prostate cancer tissues (n = 102). (K) NONO expression (high, n = 51; low, n = 51) is a prognostic factor of poor outcome of prostate cancer patients. Values represent the mean ± SD. *P < 0.05, **P < 0.01.

Fig. 4.

Spliceosome genes targeted by PSF are up-regulated in CRPC and promote AR splicing by forming a complex with PSF. (A) Expression levels of spliceosome genes (42 genes) targeted by PSF. Microarray expression data were downloaded from GEO database (GSE35988). Meta, metastatic cancer. (B) Overexpression of PSF and NONO enhances the expression level of spliceosome genes. We measured mRNA expression levels of spliceosome genes by qRT-PCR in LNCaP cells stably expressing PSF or NONO and control cells (n =3). For analyzing AR and AR-V7 expression levels, we transfected transiently control vector or NONO to LNCaP cells overexpressing PSF. (C) Expression level of spliceosome genes in AR-V7 positive prostate cancer cells. In LNCaP, VCaP, and 22Rv1 cells, mRNA levels of spliceosome genes targeted by PSF were measured by qRT-PCR (n = 3). (D) Negative regulation of spliceosome genes by knockdown of PSF in CRPC cells. We treated 22Rv1 cells with siPSF #1, #2, or siControl for 72 h. mRNA level of each spliceosome gene was measured by qRT-PCR (n = 3). (E) Regulation of AR and AR-V7 mRNA expression by splicing factors regulated by PSF in CRPC model cells. The 22Rv1 cells were treated with siRNA targeting spliceosome genes for 72 h. AR and AR-V7 mRNA levels were measured by qRT-PCR (n = 3). (F) Forming complex of PSF with splicing factors for modulating AR expression. Lysates of 22Rv1 transfected with siControl or siPSF for 72 h are used for immunoprecipitation. (G) Immunofluorescence analysis of splicing factors and PSF in 22Rv1 cells. (H) RIP analysis of splicing factors in 22Rv1 cells (n = 3). (I) Spliceosome genes regulate CRPC cell growth. Growth of 22Rv1 prostate cancer cells after transfection of siControl or siRNA targeting splicing factors (n = 4). (J) Schematic model of PSF via DNA binding and RNA binding ability. Values represent the mean ± SD. *P < 0.05, **P < 0.01. MB, myoglobin; Vec, vector.