The atypical kinase right open reading frame kinase 1 suppresses glioma cell growth through mammalian target of rapamycin inhibition and apoptotic signaling activation

Introduction

Glioma represents the widespread form of primary malignant brain tumor in adults (1). Although therapy has advanced, there remains no substantial improvement in survival rates and quality of life among patients (2). For individuals with a diagnosis of glioblastoma (GBM), the median survival duration is 20.9 months when treated with a combination of conventional therapy and tumor-treating electric fields (3,4). Canonical protein kinases feature a highly conserved kinase catalytic domain (approximately 250–300 amino acids), consisting of two functional subdomains (N-terminal small and C-terminal large) and multiple strictly conserved key motifs, which act as the core for mediating phosphorylation reactions (5). Protein kinases modulate glioma progression through activating distinct substrate proteins, thereby orchestrating key oncogenic processes such as cell proliferation, invasion, and metabolic reprogramming (6). In recent years, the molecular pathology of glioma has made great progress. In the updated 5th edition of the World Health Organization (WHO) central nervous system (CNS) tumor classification, diffuse gliomas are subclassified into distinct entities according to isocitrate dehydrogenase 1 (IDH1) status and 1p/19q status (7). This paves the way for searching for specific molecular markers and targets of glioma.

Right open reading frame kinase 1 (RIOK1) belongs to RIO kinases family, which also includes RIOK2 and RIOK3 (8). These enzymes are often referred to as atypical kinases because they lack the characteristic domains typically found in conventional kinases (9). RIOK1 is an atypical serine/threonine kinase encoded by the RIOK1 gene in humans, which is localized to the short arm of chromosome 6, band 24 (6p24.3). Structurally, it comprises two key functional regions: an N-terminal region (approximately 1–480 amino acids) that harbors the catalytic domain of atypical serine protein kinases and a C-terminal region (approximately 480–568 amino acids) that serves as a structural platform for both the metal-binding loop and the catalytic loop (10). RIOKs are implicated in 18S ribosomal RNA (rRNA) processing and the assembly of small ribosomal subunit, thereby facilitating ribosome maturation in yeast and human (11-14). RIOK1 and RIOK2 can interact with diverse proteins, regulating cell cycle and cell proliferation (15,16). In yeast, silencing of RIOK1 causes arrest of the cell cycle in the S and mitotic phases (17). RIOK2 can be phosphorylated by polo-like kinase 1 (PLK1), regulating the mitosis progression (18).

Given the crucial involvement of RIOKs in ribosome biogenesis and cell cycle control, an increasing number of studies have focused on their involvement in the progression of malignant neoplasms. Silencing RIOK1 suppresses the proliferation and invasion ability of prostate, breast and lung cancer cells (19-21). RIOK1 or RIOK2 knockdown elicits apoptosis in GBM cells (12). Furthermore, RIOK1 facilitates the growth and metastasis behavior of colorectal cancer, gastric cancer and hepatocellular carcinoma (22,23). However, separate research also indicates that RIOK1 enhances the apoptotic process in colon cancer cells (24). Therefore, the role of RIOK1 in malignant tumors remains controversial.

Our previous studies revealed that RIOK2 and RIOK3 enhanced glioma cell proliferative, migratory, and invasive capacities (25,26). This study intended to examine the functional significance and molecular mechanism by which RIOK1 acts in glioma growth. Firstly, we examined the expression patterns of RIOK1 and its correlation with patient survival across different glioma subtypes using available datasets. In addition, we evaluated RIOK1 protein expression levels in both non-tumor specimens and glioma specimens through Western blot and immunohistochemical analyses. Then, stable cell lines with RIOK1 overexpression or downregulation were established, and the role of RIOK1 in regulating glioma cell proliferation was examined. Finally, we screened and validated the downstream signaling pathways that are regulated by RIOK1 in glioma cells using Western blot. We present this article in accordance with the MDAR and ARRIVE reporting checklists (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1599/rc).

Methods

Datasets and processing

Data on RNA expression levels and pathoclinical profiles of glioma patients were sourced from two databases: Gene Expression Omnibus (GEO) and Chinese Glioma Genome Atlas (CGGA). The patient survival was analyzed via Kaplan-Meier curves, with subsequent log-rank testing. Those with inadequate prognostic information were excluded from the analysis.

Patient samples

In the standard Western blot protocol, all human glioma tissues were rapidly frozen at −80 ℃ right after surgical resection. This was pivotal for immunohistochemical analysis, with samples undergoing rapid fixation in 10% buffered formalin and subsequent embedding in paraffin to prepare sections. The Affiliated Hospital of Xuzhou Medical University provided all glioma and non-tumor brain tissue samples, which were collected from patients undergoing decompressive surgery for cranial trauma. These samples had not undergone any preoperative treatments such as radiation, immunotherapy, or chemotherapy.

The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The collection and study of glioma samples strictly conformed to the ethical norms set by the Ethics Committee of The Affiliated Hospital of Xuzhou Medical University (No. XYFY2020-KL217-01). Informed consent was obtained from all individual participants.

Cell culture

Human glioma cell lines U251, U118, A172 and U87 were procured from the Shanghai Cell Bank of the Chinese Academy of Sciences. These cell lines were cultured in Dulbecco’s Modified Eagle Media (DMEM) medium containing 10% fetal bovine serum (Hyclone-GE Healthcare Life Sciences, Logan, UT, USA), whereas U87 cells were cultured in Minimum Essential Medium (MEM). All cells were cultured in a humidified incubator maintained at 37 ℃ with 5% carbon dioxide.

Antibodies

RIOK1 (1:500, sc-130253) and β-actin (1:1,500, sc-47778) primary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against signaling proteins were obtained from Cell Signaling Technology (Danvers, MA, USA) at a dilution of 1:1,000.

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA extraction was performed using TRIzol (Invitrogen, Waltham, MA, USA), followed by reverse transcription into complementary DNA (cDNA) with a commercial kit (Roche, Germany). qRT-PCR was performed following the same protocol and equipment as outlined before (26). The primer pairs were listed below: RIOK1, forward: 5’-GAAGACAGCCAAGACGAAAA-3’; reverse: 5’-TCCTCTGTCAACACCAGACA-3’; and β-actin, forward: 5’-CCAACCGCGAGAAGATGA-3’; reverse:5’-CCAGAGGCGTACAGGGATAG-3’. Relative RIOK1 expression level was quantified following our previous method (27).

Western blot analysis

We first extracted total protein and measured its concentration. Then, protein samples were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. Primary antibodies for RIOK1 and the signaling molecules were incubated with the PVDF membrane overnight at 4 ℃. On the following day, incubation with a secondary antibody was performed, and visualization was achieved via an enhanced chemiluminescence (ECL) method. We employed ImageJ software (National Institutes of Health, Bethesda, Maryland) for analyzing band intensity.

Immunohistochemistry

This procedure commenced with microwave-assisted antigen retrieval in a citric acid buffer (pH 6.0). Next, to block non-specific binding, the sections were incubated in tris-buffered saline (TBS) with 5% non-fat dry milk for 2 hours at room temperature. Subsequently, the tissue sections were treated with a 1:50 dilution of rabbit anti-human RIOK1 polyclonal antibody, with incubation at room temperature for 2 hours followed by an overnight period at 4 ℃. The ABC kit (Vector Laboratories, Burlingame, CA, USA) was used to detect antigen-antibody complexes according to the manufacturer’s instructions. After a 10-minute exposure to 3,3'-diaminobenzidine (DAB) substrate with 0.01% hydrogen peroxide, the sections were stained with methylene blue (Sigma).

Lentivirus construction and cell transfection

PCR amplification was used to obtain the full-length RIOK1 gene in HEK293T cells, which was then cloned into the pWPXLd-puro plasmid. The primers utilized in this study were—forward: 5'-CGCTTAATTAAATGGACTACCGGCGGCTT-3'; reverse: 5'-CGACGCGTCCTTTGCCTTTTTTCGTCTTGG-3'. The short hairpin RNA (shRNA) sequences targeting RIOK1 were designed and synthesized by Invitrogen. The primers used were—shRIOK1-1, forward: 5'-GATCCGCGCCAACGTCAATGATTTTTCAAGAGAAAATCATTGACGTTGGCGCTTTTTTG-3'; reverse: 5'-AATTCAAAAAAGCGCCAACGTCAATGATTTTCTCTTGAAAAATCATTGACGTTGGCGCG-3'; shRIOK1-2, forward: 5'-GATCCGAAATAGCATCTCAAAGGATTCAAGAGATCCTTTGAGATGCTATTTCTTTTTTG-3'; reverse: 5'-AATTCAAAAAAGAAATAGCATCTCAAAGGATCTCTTGAATCCTTTGAGATGCTATTTCG-3'; Scramble, forward: 5'-GATCCGCGTCGTCCAACATTATCATTTCAAGAGAATGATAATGTTGGACGACGTTTTTTG-3'; reverse: 5'-AATTCAAAAAACGTCGTCCAACATTATCATTCTCTTGAAATGATAATGTTGGACGACGCG-3', followed by cloning into the pLV-puro plasmid. Lentiviruses designed to overexpress RIOK1 and downregulate RIOK1 were utilized to infect glioma cells, as detailed in our previous research (26).

Cell growth assay

We evaluated cell viability with a Cell Counting Kit-8 (CCK8, Dojindo, Kumamoto, Japan) following the manufacturer’s recommended procedure. A 96-well plate was seeded with cells at 2×10⁴ cells per well, and then incubated for different time periods (24–72 h). Next, the plate was maintained at 37 ℃ for 2 hours (h) in an incubator upon addition of CCK8 reagent to the wells. We measured the optical density (OD) using a microplate spectrophotometer set to 450 nm.

5-ethynyl-2'-deoxyuridine (EdU) assay

A commercial EdU detection kit (Ribobio, Guangzhou, China) was used to assess cell proliferation. Cells were inoculated in a 24-well plate at 3×10⁴ cells/well. Following 72 hours of incubation, fixation was performed with 4% paraformaldehyde, followed by permeabilization with 0.5% Triton X-100. Staining of the cell nuclei was performed with 100 µL of Hoechst 33342 (5 µg/mL). A Leica microscope was utilized to capture fluorescent images, and EdU-positive cell proportion was determined by dividing the count of EdU-stained cells by the total Hoechst-stained cell count.

Cell hypoxia and living cell imaging

U251 cells were seeded in 6-well and 96-well plates after cell counting. Hypoxia preconditioning was achieved by placing cells in a tri-gas incubator (1% O₂, 5% CO₂, and 94% N₂). Cells were scanned using the EVOS FL Auto imaging system (Invitrogen), which allowed the scanning of 6-well plates in an automated manner. We captured images at different times following incubation under hypoxic conditions. Subsequently, the software of ImageJ was employed to quantify the gray values of fluorescence images and perform relative analysis.

Tumor xenografts in nude mice

Six-week-old male athymic BALB/c nude mice were purchased from the Experimental Animal Center of Xuzhou Medical University. To establish a subcutaneous xenograft model, U251 Scramble group cells (2×10⁶ cells in 100 µL DMEM medium) were subcutaneously injected into the left flank of each nude mouse, and U251 cells with downregulated RIOK1 (2×10⁶ cells in 100 µL DMEM medium) were subcutaneously injected into the right flank of each nude mouse. Starting from day 5, tumor size and nude mouse weight were measured every 3 days. Tumor size was calculated as volume (mm³) = (length × width²)/2. Two weeks later, the mice were sacrificed, the xenograft tumors were collected, and the tumor weight was measured. The animal experiments were performed under a project license (No. L20251010033) granted by the Ethics Committee of Xuzhou Medical University, in compliance with national guidelines for care and use of animals. A protocol was prepared before the study without registration.

Statistical analysis

The clinical data were presented in scatter plots with median. Two-group differences were evaluated using the Mann-Whitney U test, while comparisons across multiple groups were analyzed via the Kruskal-Wallis test (with the Mann-Whitney U test as a post-hoc measure). In vitro assays were performed in triplicate or more, with data reported as the mean ± standard error of the mean (SEM). Comparison of two groups was performed using t-tests. For analyzing variations across multiple groups, one-way ANOVA was employed, and Dunnett’s post hoc test was then used.

Results

RIOK1 is downregulated in IDH wild-type (IDHwt) gliomas

IDH1 serves as a crucial factor for pathological classification and prognostic evaluation of gliomas (28). We firstly examined the expression changes of RIOK1 between IDH mutation-type (IDHmt) and IDHwt in the GSE16011 and CGGA693 datasets. A marked decrease in messenger RNA (mRNA) levels of RIOK1 was noted in IDHwt gliomas relative to IDHmt gliomas across both datasets (Figure 1A,1B). According to the newest WHO classification criteria, gliomas are divided into different subtypes based on molecular pathology (29). In the GSE16011 dataset, RIOK1 mRNA expression was notably reduced in IDHwt GBM compared to low-grade glioma (LGG) with IDHmt and 1p/19q codeletion (Figure 1C). The IDHwt-LGG and -GBM also exhibited relatively low levels of RIOK1 mRNA in the CGGA693 dataset, but there was no statistically significant (Figure 1D). Survival analysis in the GSE16011 dataset revealed that patients with elevated RIOK1 mRNA levels exhibited more favorable prognoses in IDHwt glioma patients. However, with a larger sample size in the CGGA693 dataset, no significant statistical correlation was detected between RIOK1 expression and the overall survival of glioma patients (Figure 1E). These findings suggest that low expression of RIOK1 is indicative of IDHwt gliomas, but it may not provide significant value for glioma patients’ prognoses.

Figure 1 The clinical importance of RIOK1 expression in gliomas. (A,B) Changes of RIOK1 mRNA levels between IDHmt and IDHwt gliomas in the GSE16011 dataset (IDHmt, n=42; IDHwt, n=61) and in the CGGA693 dataset (IDHmt, n=185; IDHwt, n=142). (C,D) Differences in RIOK1 mRNA levels among distinct glioma subtypes in the GSE16011 dataset (IDHmt, 1p/19q-codel, LGG: n=22; IDHmt, 1p/19q-non-codel, LGG: n=11; IDHwt, LGG: n=23; IDHmt, GBM: n=9; IDHwt, GBM: n=38) and in the CGGA693 dataset (IDHmt, 1p/19q-codel, LGG: n=69; IDHmt, 1p/19q-non-codel, LGG: n=97; IDHwt, LGG: n=55; IDHmt, GBM: n=19; IDHwt, GBM: n=87). (E) The correlation between RIOK1 mRNA levels and overall survival of IDHwt and IDHmt glioma patients, respectively, in the GSE16011 and CGGA693 datasets. *, P<0.05; **, P<0.01. GBM, glioblastoma; HR, hazard ratio; IDHmt, isocitrate dehydrogenase mutation-type; IDHwt, isocitrate dehydrogenase wild-type; LGG, low-grade glioma; mRNA, messenger RNA; RIOK1, right open reading frame kinase 1.

RIOK1 is downregulated in glioma tissues

With the aim of exploring the RIOK1’s function in glioma, we initially assessed the differential expression of RIOK1 between non-tumor brain specimens and glioma specimens through Western blot and immunohistochemical analyses. Results from Western blot analyses indicated that the expression levels of RIOK1 protein were significantly lower in grade 3 gliomas compared to non-tumor tissues (Figure 2A,2B). The immunohistochemical staining demonstrated a notable reduction in the count of RIOK1-positive cells in grade 2 and 4 gliomas compared to non-tumor tissues (Figure 2C,2D). However, no significant differences were identified across different grades of glioma tissues. Collectively, these findings suggest that RIOK1 is significantly downregulated in glioma tissues.

Figure 2 Overview of RIOK1 expression in glioma samples. (A) A representative Western blot image of RIOK1 in non-tumor tissues (n=9), grade 2 (n=12), grade 3 (n=12), and grade 4 (n=9) glioma tissues. β-actin serves as the loading control. (B) The statistical diagram of the Western blot. (C) Representative immunohistochemistry images of RIOK1 in non-tumor tissues (n=11), grade 2 (n=10), grade 3 (n=12), and grade 4 (n=15) glioma tissues. (D) The statistical diagram of RIOK1-IR (immunoreactivity) cells. Scale bars: 100 and 50 µm; **, P<0.01; ***, P<0.001. RIOK1, right open reading frame kinase 1.

Establishment of lentivirus-mediated stable cell lines

We firstly measured RIOK1 mRNA levels through qRT-PCR in multiple glioma cell lines (U87, U118, A172, and U251). It was observed that RIOK1 was relatively highly expressed in U251 cells (Figure 3A). Subsequently, stable U251 cell lines were established via lentiviral infection to either overexpress or knock down RIOK1. Observation via fluorescence microscopy showed that 80% of the cells infected by the lentivirus overexpressing RIOK1 displayed green fluorescent protein (GFP) fluorescence (Figure 3B), indicating the infection was successful. Western blot assays revealed successful and abundant overexpression of the GFP-tagged RIOK1 protein in U251 cells (Figure 3C). Two RIOK1 shRNAs also showed an infection efficiency of more than 80% in U251 glioma cells, as indicated by GFP fluorescence (Figure 3D). qRT-PCR and Western blot data indicated that the mRNA and protein expression levels of RIOK1 were significantly diminished by two specific shRNAs designed to target RIOK1 (Figure 3E-3G). These results demonstrated that stable cell lines with lentivirus-mediated RIOK1 overexpression and downregulation were successfully constructed.

Figure 3 Establishment of stable U251 cell lines with lentivirus-mediated RIOK1 overexpression or downregulation. (A) mRNA expression levels of RIOK1 in different glioma cell lines. (B) BF and GFP fluorescence images showed the lentiviral transduction efficiency in both Vector and RIOK1 groups. (C) Western blot analysis verified that exogenous GFP-RIOK1 exhibited sufficient overexpression in U251 cells. (D) BF and GFP fluorescence images showed the lentiviral transduction efficiency in both Scramble and shRIOKs groups. (E) qRT-PCR demonstrated the downregulation efficiency of RIOK1 mRNA level by two shRNAs in U251 cells. (F) Western blot analysis revealed notable reductions in RIOK1 protein levels induced by the shRNAs. (G) The statistical diagram illustrated the downregulation of RIOK1 at protein level by the shRNAs. Scale bars: 200 µm; **, P<0.01; ***, P<0.001. BF, bright field; GFP, green fluorescent protein; mRNA, messenger RNA; qRT-PCR, quantitative real-time polymerase chain reaction; RIOK1, right open reading frame kinase 1; shRIOK1, short hairpin RNA targeting RIOK1; shRNA, short hairpin RNA.

RIOK1 exerts a suppressive effect on glioma cell proliferation

The function of RIOK1 in the growth and proliferation of glioma cells was examined using CCK-8 and EdU assays. Results from CCK8 assays indicated that RIOK1 upregulation diminished cell viability at 24, 48 and 72 h in U251 cells, whereas knockdown of RIOK1 increased cellular viability at 48 and 72 h (Figure 4A,4B). EdU assays (Figure 4C,4D) demonstrated that RIOK1-overexpressing cells exhibited a notable decrease in the ratio of EdU-positive cells (Figure 4E), whereas RIOK1 downregulation exerted the opposite effect (Figure 4F). These findings demonstrate that RIOK1 exerts a suppressive effect on glioma cell proliferation.

Figure 4 The effect of RIOK1 upregulation and downregulation on glioma cell growth and proliferative capacity. (A) CCK8 assays revealed the growth curves in the RIOK1-overexpressing and -downregulating U251 cell lines. (B-E) EdU assays demonstrated the impact of RIOK1 overexpression and downregulation on U251 cell proliferative capacity. Images of EdU-positive cells and nuclei-stained cells are depicted in (B,C). Quantitative results on the proportion of EdU-positive cells are presented in (D,E). Scale bar: 200 µm; *, P<0.05; **, P<0.01; ***, P<0.001. CCK8, Cell Counting Kit-8; EdU, 5-ethynyl-2'-deoxyuridine; OD, optical density; RIOK1, right open reading frame kinase 1.

Hypoxia results in low expression of RIOK1 in glioma cells

Solid tumors, gliomas among them, frequently exhibit hypoxia, or low oxygen levels (30). We next measured hypoxia-inducible factor 1α (HIF-1α) and RIOK1 protein expression levels in U251 cells at different time points following hypoxia. Western blot analysis revealed that hypoxia led to a marked elevation in the HIF-1α protein levels at 6 h, peaking at 12 h, which was gradually reduced with the hypoxia time in U251 cells, while the RIOK1 protein levels showed a significant decrease at 6 h and negatively correlated with the HIF-1α levels following hypoxia (Figure 5A,5B). These results indicate that hypoxia leads to decreased expression of RIOK1 in glioma cells.

Figure 5 Changes in RIOK1 protein expression under hypoxic conditions. (A,B) Western blot assay revealed changes in the protein levels of HIF-1α and RIOK1 in U251 cells over hypoxic time gradients. HIF-1α, hypoxia-inducible factor 1α; RIOK1, right open reading frame kinase 1.

RIOK1 overexpression inhibits glioma cell proliferation under hypoxic conditions

To reveal the function of RIOK1 under hypoxic conditions, living cell imaging was employed to observe how RIOK1 overexpression affects glioma cell proliferation after exposure to hypoxia (Figure 6A). As reflected by the GFP-positive cell intensity, hypoxia led to time-dependent induction of cell proliferation in the vector group, whereas overexpression of RIOK1 inhibited cell proliferation at 36 and 48 h post-hypoxia (Figure 6B). CCK8 assay demonstrated that, compared with the vector group, cell viability in the RIOK1 group exhibited a notable decline at 48 and 72 h following hypoxia (Figure 6C). These findings suggest that RIOK1 suppresses hypoxia-induced glioma cell proliferation.

Figure 6 The influence of increased RIOK1 expression on the proliferative capacity of glioma cells under hypoxic conditions. (A) U251 cells’ growth and proliferative activity were monitored by a living cell imaging system at various time points after hypoxia. (B) Quantification of the relative fluorescence intensity in images. (C) CCK8 assay showed the growth curves of U251 cells at a time gradient following hypoxia. Scale bar: 200 µm; *, P<0.05. BF, bright field; CCK8, Cell Counting Kit-8; GFP, green fluorescent protein; RIOK1, right open reading frame kinase 1.

RIOK1 regulates the mTOR and apoptosis signal pathways

To investigate the underlying mechanism by which RIOK1 suppresses glioma cell proliferation, Western blot analysis examined the changes of important signaling molecules in both RIOK1-overexpressing and RIOK1-downregulating cells (Figure 7A,7B). Upregulation of RIOK1 caused a notable reduction in phosphorylated mammalian target of rapamycin (p-mTOR), phosphorylated P70 ribosomal protein S6 kinase (p-P70S6K), and phosphorylated ribosomal protein S6 (p-S6) levels, whereas downregulation of RIOK1 elicited opposite effects (Figure 7C,7D). For the protein kinase B (Akt) signal pathway, RIOK1 overexpression caused a significant reduction in phosphorylated Akt at serine 308 (p-Akt308), while RIOK1 knockdown induced an increase in p-Akt473 and phosphorylated proline-rich Akt substrate of 40 kDa (p-PRAS40) levels rather than affecting p-Akt308 (Figure 7E,7F). Overexpression of RIOK1 stimulated apoptosis-related signaling pathway, as reflected by the increased levels of cleaved poly ADP-ribose polymerase (PARP) and phosphorylated P53 (p-P53) (Figure 8A,8B). Downregulation of RIOK1 was correlated with a significant decline in cleaved PARP levels (Figure 8A,8C). These observations indicate that RIOK1 may suppress glioma cell growth and proliferation through attenuating the mTOR pathway activity and triggering apoptotic signaling cascade.

Figure 7 The role of RIOK1 overexpression or downregulation in regulating mTOR and Akt signaling pathways. (A,B) Representative images of Western blot showed the changes in the protein activity of key molecules in the mTOR (A) and Akt (B) pathways following RIOK1 overexpression and knockdown. (C) The statistical diagram demonstrated a significant reduction in the p-mTOR, p-P70S6K, and p-S6 levels following RIOK1 overexpression. (D) The statistical diagram demonstrated a significant increase in the p-mTOR, p-P70S6K, and p-S6 levels following RIOK1 downregulation. (E) The statistical diagram indicated a notable reduction in the level of p-Akt308 following RIOK1 overexpression. (F) The statistical diagram indicated that RIOK1 downregulation led to significant elevations in p-Akt473 and p-PRAS40 levels, accompanied by a decrease in the p-Akt308 level. *, P<0.05; **, P<0.01. Akt, protein kinase B; p-Akt308, phosphorylated protein kinase B at serine 308; p-mTOR, phosphorylated mammalian target of rapamycin; p-P70S6K, phosphorylated P70 ribosomal protein S6 kinase; p-S6, phosphorylated ribosomal protein S6; RIOK1, right open reading frame kinase 1.

Figure 8 The effect of RIOK1 overexpression or knockdown on the apoptotic signaling molecules. (A) Representative Western blot images showed changes in apoptosis-related molecules upon overexpression or knockdown of RIOK1 in U251 cells. (B) The statistical diagram demonstrated a significant increase in the cleaved PARP and p-P53 levels following RIOK1 overexpression. (C) The statistical diagram demonstrated a significant reduction in the cleaved PARP protein level following RIOK1 downregulation. *, P<0.05; **, P<0.01. p-P53, phosphorylated P53; PARP, poly ADP-ribose polymerase; RIOK1, right open reading frame kinase 1.

Downregulation of RIOK1 promotes the growth of glioma xenografts

We further investigated the effect of RIOK1 downregulation on glioma tumorigenesis via in vivo experiments. Specifically, U251 Scramble cells were subcutaneously injected into the left flank of 6 nude mice, while RIOK1-downregulated U251 cells were injected into the right flank of the same mice (bilateral inoculation model). Starting from day 5 post-inoculation, tumor size and body weight of the nude mice were measured every 3 days. Two weeks after cell implantation, the xenograft tumors were dissected and collected for weight measurement (Figure 9A). The results showed that the xenograft tumors derived from shRIOK1 U251 cells exhibited significantly larger volumes on days 8, 11, and 14 compared with those from the scramble groups (Figure 9B). Consistent with the volume data, the final tumor weight measurement revealed that shRIOK1 xenografts were significantly heavier than the scramble counterparts (Figure 9C). Importantly, there was no significant difference in the body weight of tumor-bearing mice between the two experimental groups (Figure 9D). Collectively, these in vivo findings further demonstrate that RIOK1 downregulation promotes glioma tumorigenesis.

Figure 9 In vivo study on the effect of RIOK1 downregulation on glioma growth. (A) Tumor-bearing nude mice and tumors isolated from the scramble and shRIOK1 groups. (B) The statistical diagram of tumor volume on days 5, 8, 11, and 14. (C) The statistical diagram of tumor weight. (D) Changes in body weight of 6 nude mice. *, P<0.05; **, P<0.01. RIOK1, right open reading frame kinase 1.

Discussion

A previous study revealed that RIOK1 expression was elevated alongside increasing WHO grades of gliomas, and this was a prognostic factor for poor survival in glioma patients (31). However, in both the GSE16011 and CGGA693 datasets, we observed that RIOK1 expression was significantly higher in IDHmt gliomas compared to IDHwt gliomas. The results of the tissue specimen analysis indicated that glioma tissues exhibited a notably lower level of RIOK1 protein expression than non-tumor brain tissues. Additionally, RIOK1 expression showed no significant correlation with the glioma patients’ overall survival in the CGGA693 dataset, which had a larger sample size. The discrepancy may arise from the omission of molecular pathology characteristics of gliomas in previous studies. The 2021 WHO guidelines for CNS tumors categorize gliomas according to the mutational status of IDH and the codeletion status of 1p/19q (29). Diagnosis of glioma based solely on histological information, without considering the status of these molecular markers, may yield biased research outcomes.

RIOK1 belongs to the RIO kinase family, which also includes RIOK2 and RIOK3. The RIO kinases are pivotal to ribosome biogenesis and the progression of the cell cycle (32). Our previous study demonstrated that RIOK2 and RIOK3 exerted a facilitative effect on glioma cell proliferative activity and invasiveness (25,26), whereas the current study demonstrated that RIOK1 inhibits glioma cell growth. Indeed, the roles of RIO kinases in tumors are controversial. For example, RIOK3 promoted colon cancer cell growth under glucose starvation conditions (33). RIOK1 inhibited the survival of colon cancer cells (23). It was found that two molecules belonging to the same family exerted contrasting effects on glioma growth through complementary mechanisms (34). Therefore, we proposed that the diverse functions of RIO kinases in gliomas may result from competitive or complementary mechanisms. In this process, RIOK1 functioned as a monitoring molecule to prevent excessive activation of RIOK2 and RIOK3.

Akt/mTOR signaling pathway is commonly dysregulated in glioma; targeting Akt/mTOR signaling pathway is considered a potential treatment strategy for GBM (35). Current studies revealed that RIOK1 overexpression exerted a reducing effect on mTOR pathway activity, while RIOK1 downregulation had the opposite effect of increasing it. The findings from our previous study revealed that RIOK2 interacted with mTOR, thereby facilitating the progression of glioma (36). RIOK1 and RIOK2 exhibit a high degree of homology in their amino acid sequences (8). Therefore, stable overexpression of RIOK1 may lead to a reduction in the amount of RIOK2 binding to mTOR, thereby suppressing glioma cell growth. Taken together, RIOK1 may exert a suppressive effect on the growth of glioma cells via mTOR signaling pathway, but the exact mechanism awaits further elucidation.

The process of cell apoptosis is primarily regulated by caspases (37). Caspase-3, -8, and -10, the apoptotic proteases, play important roles in programmed cell death (38). PARP serves as a substrate for caspase-3, and elevated levels of cleaved PARP are indicative of caspase-3 activation, which in turn induces cell apoptosis (39). We found that RIOK1 overexpression induced an upregulation of cleaved PARP in U251 cells, while downregulation of RIOK1 showed the opposite result. Research findings have demonstrated that RIOK1 can trigger apoptosis in GBM U87 cells by activating procaspase-8 (24). RIOK3 interacts with caspase-10 and exerts a suppressive influence on the nuclear factor-kappa B (NF-κB) mediated apoptosis signaling pathway (40). Therefore, RIOK1 may inhibit glioma cell survival through the induction of caspase-mediated apoptosis. Further research is required to support this hypothesis.

Hypoxia is one of typical characteristics of glioma microenvironment (41). The presence of hypoxic conditions can facilitate the proliferation and dissemination of gliomas by inducing HIF-1α expression (42). Here, we observed that hypoxia induced downregulation of RIOK1 expression and upregulation of HIF-1α expression. The overexpression of RIOK1 effectively suppressed glioma cell proliferation under hypoxic conditions, as observed under a living cell microscope. In addition, hypoxia induced upregulation of RIOK3 expression, promoting the invasion and metastasis of breast cancer cells (43). These findings provide additional evidence for the tumor-suppressive function of RIOK1 in glioma cell growth.

Conclusions

We demonstrated that RIOK1 was downregulated in glioma tissues, particularly in IDHwt gliomas. RIOK1 suppressed glioma cell growth and proliferation under both normoxic and hypoxic conditions. The underlying mechanism may involve a reduction in mTOR signaling pathway activity and an increase in apoptotic signaling cascade activity. The in vivo experiments confirmed that RIOK1 downregulation promotes the growth of glioma xenografts. These findings indicate that RIOK1 acts as a biomarker and an effective target in IDHwt gliomas.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR and ARRIVE reporting checklists. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1599/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1599/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1599/prf

Funding: This work was supported by the Key Research & Development Plan of Xuzhou City (KC23211) and the Medical Science and Technology Innovation Project of Xuzhou Health Commission (XWKYSL20220288) and the Science & Technology Plan of Suzhou City (SYW2025170).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1599/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The collection and study of glioma samples strictly conformed to the ethical norms set by the Ethics Committee of The Affiliated Hospital of Xuzhou Medical University (No. XYFY2020-KL217-01). Informed consent was obtained from all individual participants. The animal experiments were performed under a project license (No. L20251010033) granted by the Ethics Committee of Xuzhou Medical University, in compliance with national guidelines for care and use of animals. A protocol was prepared before the study without registration.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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