The Oncogenic Protein Kinase/ATPase RIOK1 Is Up-Regulated via the c-myc/E2F Transcription Factor Axis in Prostate Cancer

Abstract

The atypical protein kinase/ATPase RIO kinase (RIOK)-1 is involved in pre-40S ribosomal subunit production, cell-cycle progression, and protein arginine N-methyltransferase 5 methylosome substrate recruitment. RIOK1 overexpression is a characteristic of several malignancies and is correlated with cancer stage, therapy resistance, poor patient survival, and other prognostic factors. However, its role in prostate cancer (PCa) is unknown. In this study, the expression, regulation, and therapeutic potential of RIOK1 in PCa were examined. RIOK1 mRNA and protein expression were elevated in PCa tissue samples and correlated with proliferative and protein homeostasis-related pathways. RIOK1 was identified as a downstream target gene of the c-myc/E2F transcription factors. Proliferation of PCa cells was significantly reduced with RIOK1 knockdown and overexpression of the dominant-negative RIOK1-D324A mutant. Biochemical inhibition of RIOK1 with toyocamycin led to strong antiproliferative effects in androgen receptor-negative and -positive PCa cell lines with EC₅₀ values of 3.5 to 8.8 nmol/L. Rapid decreases in RIOK1 protein expression and total rRNA content, and a shift in the 28S/18S rRNA ratio, were found with toyocamycin treatment. Apoptosis was induced with toyocamycin treatment at a level similar to that with the chemotherapeutic drug docetaxel used in clinical practice. In summary, the current study indicates that RIOK1 is a part of the MYC oncogene network, and as such, could be considered for future treatment of patients with PCa.

Figure 1

RIOK1 is overexpressed in PCa at the mRNA level. A and B: Re-analysis of the single-cell RNA-seq GSE193337 (https://www.ncbi.nlm.nih.gov/geo; accession number GSE193337) containing four PCa and adjacent benign tissue samples showing the clustering/cell type marker gene expression (A) and log₂ RIOK1 mRNA expression as shown in a two-dimensional uniform manifold approximation and projection (UMAP) plot (B). C: Bar graph, after pseudobulk summarization, of RIOK1 mRNA expression (linear scale) across different cell types. D: RIOK1 bulk mRNA expression and meta-analysis–based differential gene expression statistics in PCa and benign prostate tissue of four independent public transcriptome data sets from The Cancer Genome Atlas (TCGA) (https://portal.gdc.cancer.gov/projects/TCGA-PRAD, last accessed May 30, 2023; accession TCGA-PRAD), and Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo; accession numbers GSE62872, GSE21034, and GSE35988). Data are expressed as means (C) or as medians (interquartile range) [minimum, maximum] and outliers (D). ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. AMACR, α-methylacyl–coenzyme A racemase; Hill., Hillock; KLK, kallikrein; KRT, keratin; SCGB1A1, secretoglobin family 1A member 1; scRNA, small conditional RNA; SMC, structural maintenance of chromosomes protein.

Figure 2

RIOK1 is strongly overexpressed in PCa at the protein level. A: IHC RIOK1 staining of PCa and adjacent benign prostate tissue from two patients. Boxed regions in upper panels correspond to lower panels at higher magnification. B: Quantification of IHC analysis RIOK1 staining (immunoreactivity score) in epithelial cells in the tissue microarray. C: Heatmap showing the expression (z-score–normalized pseudobulk) of four post-translational RIOK1 regulators across different cell types in the re-analyzed single-cell RNA-seq data set (https://www.ncbi.nlm.nih.gov/geo; accession number GSE193337). D: Log₂ mRNA expression of the post-translational RIOK1 regulator lysine-specific histone demethylase (KDM)-1A. Data are expressed as medians (interquartile ranges) [minimum, maximum]. ∗∗∗∗P < 0.0001. Scale bars: 100 μm (original magnification, ×20; A, upper panel); 20 μm (original magnification, ×63; A, lower panel). CSNK2, casein kinase II; FBXO, F-box only protein; SETD7, histone-lysine N-methyltransferase SETD7; SMC, structural maintenance of chromosomes protein; UMAP, uniform manifold approximation and projection.

Figure 3

RIOK1 is downstream target of the c-myc/E2F transcription factor axis. A: Correlation of RIOK1 mRNA expression (z-score) with c-myc mRNA expression and MYC/E2F target gene set activity (GSVA software version 1.44.4). B: Histone marks and transcription factor chromatin immunoprecipitation sequencing peaks in the genomic region surrounding RIOK1 from public data sets available via the University of California–Santa Cruz genome browser and ENCODE project (https://genome.ucsc.edu/ENCODE, last accessed May 30, 2023). C: Detection of RIOK1 protein expression by Western blot analysis upon siRNA-mediated knockdown of c-myc in PC3 cells at 72 hours after transfection. Data are expressed as means (95% CI). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗∗P < 0.0001. ChIP, chromatin immunoprecipitation; CTCF, transcriptional repressor CTCF; GAPDH, glyceraldehyde phosphate dehydrogenase; MAX, protein max; TFs, transcription factors.

Figure 4

RIOK1 is an essential gene in PCa. A:RIOK1 gene dependency in 1086 cell lines of a public clustered regularly interspaced short palindromic repeats (CRISPR)-knockout (KO) data set. B: Correlation of RIOK1 gene dependency against all other genes summarized as co-dependency network of the top three pathways. C and D: RIOK1 protein expression (C) and population growth curves (D) upon CRISPR interference–based RIOK1 knockdown in PC3 cells. E: Exemplary Western blot showing RIOK1 and cleaved PARP protein expression upon overexpression (oe) of wild type and dominant-negative (D324A mutant) RIOK1 in PC3 cells. F: Proliferation of PC3 cells upon overexpression of wild type and dominant-negative (D324A mutant) RIOK1 after 72 hours after transfection. Data are expressed as means (95% CI). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗∗P < 0.0001. GAPDH, glyceraldehyde phosphate dehydrogenase; GFP, green fluorescent protein; sg, single-guide; WT, wild-type.

Figure 5

Toyocamycin is a potent inhibitor of PCa cell growth. A: Dose-response curves after treatment of various PCa cell lines with the RIOK1 inhibitor toyocamycin after 72 hours. B: Bar plots showing the fold-changes at selected toyocamycin concentrations at 72 hours after treatment. Data are expressed as means (95% CI). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001.

Figure 6

Toyocamycin reduces rRNA levels and induces apoptosis in PCa cell lines. A–D: RIOK1 protein expression (A), total rRNA content per cell (B), rRNA profile (C), and quantification (D) of the 28S/18S rRNA ratio at 24 hours after treatment with 100 nmol/L toyocamycin (Toyo.) or docetaxel (Doc.). E: Percentage sub-G₁ cells after 72 hours of 100 nmol/L toyocamycin or docetaxel treatment. Data are expressed as means (95% CI). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde phosphate dehydrogenase.