Uridine-cytidine kinase 2 promotes glycolysis and reprograms glioblastoma stem cell characteristics under hypoxic conditions through the PI3K/Akt/HIF-1α pathway

ABSTRACT

This study aimed to explore key regulatory molecules involved in metabolic alterations clarify the heterogeneity of glioblastoma and develop novel therapeutic strategies. The microarray dataset GSE45117 was retrieved from the Gene Expression Omnibus database to analyze differentially expressed genes (DEGs) glioma stem cell (GSC) populations were enriched via microsphere suspension culture and ALDH+ cell sorting in vitro with the expression of the uridine-cytidine kinase 2 (UCK2) gene compared between stemness and non-stemness populations the UCK2 gene was stably knocked down or overexpressed in GSCs to assess cell invasion migration glucose uptake lactate production and ATP levels. Database analysis revealed high UCK2 expression in cancer stem cells (CSCs) manipulating UCK2 levels affected stemness factors and cell behaviors including proliferation migration invasion and tumor growth UCK2 was more abundant in hypoxic central tumor regions promoting increased glucose uptake and energy production knocking down UCK2 reduced glycolysis and stem cell properties under hypoxia mechanistically UCK2 stabilizes PI3K protein through deubiquitination thereby activating the Akt/HIF-1α pathway. UCK2 plays a pivotal role as a metabolic regulator in glucose metabolism by stabilizing PI3K protein expression via deubiquitination which in turn activates the Akt/HIF-1α signaling pathway.

1. Introduction

Glioma represents the most common primary tumors within the central nervous system, and glioblastoma (WHO grade IV) is the most malignant, highly aggressive, and prone to recurrence [1]. Despite the advancements in glioma treatment in recent years, encompassing the maximum extent of surgical resection, postoperative adjuvant radiotherapy and chemotherapy, the prognosis for glioma patients remains dismal, with the median survival time still falling short of 15 months and showing no significant extension [2]. The key reason lies in the fact that almost all patients with glioblastoma experience relapse after surgery. The recurrence of glioma is primarily ascribed to the highly invasive growth of the tumors, making complete resection unachievable through surgery. The efficacy of chemotherapy is not notable because the blood – brain barrier impedes the adequate transportation of anticancer drugs and the drug resistance of tumors [3]. Radiotherapy is the most important postoperative adjuvant treatment. Nevertheless, due to the radiation resistance of certain tumors cells and the potential damage to the surrounding normal brain tissue upon increasing the therapeutic dose. This study offers experimental support and a theoretical foundation for further exploring the characteristics of glioma, delving into the potential mechanism of malignant biological behavior, elaborating the mechanism of glioma resistance to treatment, identifying effective prognostic marker molecules for glioma, and developing specific targeted therapeutic approaches.

Recent studies have identified a distinct subpopulation of cells within glioma tissues that express related markers of normal embryonic stem cells and have the ability to undergo multidirectional differentiation and self-renewal. This subset of cells is defined as cancer stem cells (CSCs) [4]. Glioma stem cells (GSCs), a type of solid tumors stem cell, were recently isolated and identified by the American Academy of Cancer Research. In recent years, a large number of experiments have shown that GSCs are a special cell subset closely related to the occurrence and progression of glioblastoma and are the key cell population leading to glioblastoma drug resistance, radiotherapy resistance and recurrence after treatment [5]. GSCs were first identified in 2003, and their surface molecular markers such as CD133, CD15, A2B5, nestin and ALDH1 [6]. The ability of suspension culture to form spheres in vitro and the high tumorigenicity of xenograft tumors in nude mice can reflect the characteristics of CSCs. Previous studies have shown that GSCs are regulated by complex components of the tumor microenvironment, including heterogeneous cells, cytokines and metabolites [7]. As these components directly affect the properties of CSCs, they also open new research directions for exploring effective therapeutic targets for CSCs.

The reprogramming of energy metabolism is a hallmark that distinguishes tumor cells from their normal counterparts. An increasing number of studies have shown that abnormally rapid metabolism, such as aerobic glycolysis, is associated with tumor growth and tumor chemotherapy resistance [8]. CSCs exhibit a high degree of plasticity in metabolic mechanisms, which can rely on glycolysis or oxidative phosphorylation. As an important glucose metabolic process, glycolysis can promote the stemness maintenance of tumor cells in special microenvironments, such as hypoxia and nutrient deficiency [9]. Studies have shown that CSCs exhibit more glycolytic phenotypes than differentiated cells. Similarly, glycolysis-related processes, such as glucose uptake, glycolysis rate-limiting enzyme expression, lactate production and ATP production, are significantly increased in cancer cells and associated with decreased mitochondrial oxidative metabolism [10]. Conversely, inhibition of glycolysis inhibited the stemness maintenance of CSCs. For example, 2-DG, an inhibitor of glycolysis, can significantly reduce the proportion of glioma cells in individual populations and reduce tumorigenesis in vivo [11]. Lactate dehydrogenase (LDHA) knockdown in a K-ras-driven NSCLC mouse model led to impaired tumor sphere formation and tumor proliferation [12]. These studies suggest that glycolysis plays a crucial role in the maintenance of cancer stemness, but the underlying mechanisms remain unclear and require further investigation.

Taken together, the overarching objective of glioma research and therapeutics is to uncover highly sensitive prognostic and predictive biomarkers and explore key signaling pathways for identifying markers that could enable patient stratification based on the risk of progression and predicted treatment response. Consequently, in this study, the expression levels of key genes in GSCs were predicted to identify new targets for determining diagnostic indicators of glioma, developing drugs to treat glioma and providing new ideas and a basis for treating glioma.

2. Materials and methods

2.1. Cell culture

The human glioma cell lines U87 and GBM-1 and normal human astrocytes (NHAs) were obtained from the American Type Culture Collection. Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS, Thermo Fisher, USA) and 1% (10 mg/mL) penicillin/streptomycin dual antibody was used for cell culture. The cells were maintained at 37°C in a humidified atmosphere with 5% CO₂. When the cells reached a confluence of approximately 80–90%, they were carefully washed with phosphate – buffered saline (PBS) twice. Then, 0.25% trypsin – EDTA solution was added to the culture flask, and the flask was incubated at 37°C for 3–5 minutes. The digestion process was monitored under a microscope until the cells started to detach from the flask surface. After that, an equal volume of complete medium was added to inactivate the trypsin. The cell suspension was gently pipetted to ensure single – cell dispersion and then seeded into six – well plates at a density of 1 × 10⁵ cells per well. The cells were allowed to adhere and grow for 24–48 hours until they reached a density of 90%–95% under the same culture conditions before being subjected to subsequent treatments and collections for cell – behavior detection. U87 and GBM-1 cells were selected to establish uridine-cytidine kinase 2 (UCK2) knockdown and overexpression cell models. For the PI3K/Akt/HIF-1α pathway experimental part, 10 μM pathway inhibitor LY2 (DA, MedChem Express, USA) and 20 μM pathway activator 1,3-Dicaffeoylquinic acid (DA, MedChem Express, USA) were introduced to intervene the cells, respectively.

2.2. Bioinformatics analysis

The Gene Expression Omnibus public database (https://www.ncbi.nlm.nih.gov/gds) was used to retrieve CSC differences in gene expression profiles and related clinical data from the following retrieval platforms: GPL6244 and GSE45117. The GSE45117 dataset was generated with the GPL6244 platform, included four GSC samples and four glioma parent cell samples. Before data analysis, the dataset underwent background correction using the “normalizeBetweenArrays” function in the Limma R package. Then, linear modeling was performed with the “lmFit” function, and moderated t – statistics were calculated using the “eBayes” function. The standard for identifying differentially expressed genes (DEGs) was set as |log fold change| ≥ 0.5 and adjusted p < 0.05. These parameters were chosen based on previous studies in the field and were optimized to ensure the reliability of the differentially expressed gene identification. The resulting DEGs were then further analyzed for subsequent research.

2.3. Genetic overexpression and knockdown

The sequence of the UCK2 (gene ID: 7371) mRNA was obtained from the NCBI database and combined with the coding DNA sequence region of the target gene for shRNA design and synthesis of three UCK2 shRNAs for subsequent experiments. The UCK2 full sequence was ligated into the pcDNA3.1 plasmid (Ke Lei Biological Technology Co., Ltd., Shanghai, China). U87 cells were transfected with sh-UCK2#1 or sh-UCK2#2 by using Lipofectamine 3000 transfection reagent, and pcDNA-UCK2 was transfected into GBM-1 cells in the same manner. After transfection, the cells were cultured in a medium containing 800 μg/mL G418 for 2–3 weeks to select stable UCK2 - overexpressing or knockdown cell lines. During this selection process, the medium was changed every 3–4 days. The surviving colonies were then expanded, and the expression levels of UCK2 in these cells were verified by Western blotting and RT – qPCR to ensure the successful establishment of the stable cell lines. Subsequently, after all the cells were incubated at 37°C with 5% CO₂ for the indicated time, they were collected for subsequent experiments.

2.4. Colony-formation assay

Cells from each group with good growth status were seeded into 12-well plates at 1 × 10³ cells/well with three replicate wells for each group and cultured in a 5% CO₂, 37°C incubator with fluid changes every 3 or 4 days. Colony formation was illustrated by the appearance of macroscopically visible cell colonies after approximately 2 weeks of culture. Subsequently, after the cells were collected and fixed using 4% paraformaldehyde for 30 min, they were stained with 0.1% crystal violet for 15 min. PBS was used to wash nonspecific material, such as dyes, to dryness and, finally, to acquire images and perform clone counting.

2.5. Transwell assay

Matrigel was smeared on the bottom of the Transwell upper chamber and placed in a 37°C incubator for 30 min to ensure that the Matrigel was evenly distributed. Cells from each treatment group were suspended in 200 μL of serum-free DMEM at 5 × 10⁴/well and subsequently transferred to the pretreated upper chamber; the lower chamber was filled with 600 μL of DMEM (20% FBS). After the cells incubated for 48 h, 4% paraformaldehyde was used to fix them for 30 min at room temperature, followed by 0.1% crystal violet staining for 15 min. Subsequently, the cells on the upper chamber surface were removed and observed under a light microscope (10×), after which the cells were counted. Cell migration ability assay was completed without using Matrigel to pretreat the chambers, and the remaining steps were the same as above.

2.6. Glycolytic assay

Cell glucose uptake, lactate production and ATP levels were measured using a glucose assay kit (mlbio, Shanghai, China), a lactate detection kit (BioVision, Mountain View, CA, USA), and CellTiter-Glo luminescent cell viability assay (mlbio, Shanghai, China), respectively, in accordance with the manufacturer’s instructions. For glucose uptake measurement, cells were seeded in 96 - well plates at a density of 2 × 10⁵ cells per well and incubated overnight. The next day, the medium was replaced with fresh medium containing a known concentration of glucose. After a specific incubation time (usually 1–2 hours), the cells were washed with PBS, and the glucose uptake was determined according to the kit protocol. For lactate production assay, the cell culture supernatant was collected at the end of the experiment, and the lactate concentration was measured following the kit instructions. For ATP level measurement, cells were lysed directly in the 96 - well plates using the provided lysis buffer in the CellTiter – Glo kit, and the luminescence signal was measured using a microplate reader, which was proportional to the ATP content in the cells.

2.7. RT–qPCR

Total RNA was extracted from the PC cells by the TRIzol method. Briefly, cells were lysed directly in the culture wells with 1 mL of TRIzol reagent. After thorough mixing, 200 μL of chloroform was added, and the mixture was centrifuged at 12,000 rpm for 15 minutes at 4°C. The upper aqueous phase containing RNA was carefully transferred to a new tube, and 500 μL of isopropanol was added to precipitate the RNA. After centrifugation at 12,000 rpm for 10 minutes at 4°C, the RNA pellet was washed with 75% ethanol, air – dried, and dissolved in 30 μL of RNase – free water. The concentration of the extracted RNA was determined via an ultraviolet spectrophotometer, and the OD260/OD280 values were required to be between 1.8 and 2.1 to ensure the purity of the RNA. Total RNA (0.5 μg) was reverse – transcribed to cDNA using a reverse transcription kit following the manufacturer’s protocol. The reaction conditions included incubation at 37°C for 15 minutes for reverse transcription and 85°C for 5 seconds for enzyme inactivation. The expression levels of UCK2 and GAPDH were determined using an ABI7500 PCR instrument with GAPDH serving as an internal control for UCK2. The primers for UCK2 and GAPDH were designed based on their gene sequences. The PCR reaction was carried out in a total volume of 20 μL containing 10 μL of SYBR Green Master Mix, 0.5 μM of each primer, and 2 μL of cDNA template. The cycling conditions were: initial denaturation at 95°C for 30 seconds, followed by 40 cycles of denaturation at 95°C for 5 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds. The results were expressed using the 2^−ΔΔCT method.

2.8. Western blotting

Total protein was extracted with RIPA lysis buffer (Solarbio, Beijing, China) containing protease and phosphatase inhibitors. Cells were washed twice with ice – cold PBS and then lysed directly in the culture dishes with 100–200 μL of RIPA lysis buffer on ice for 30 minutes. The cell lysate was scraped off the dish and transferred to a microcentrifuge tube. After centrifugation at 12,000 rpm for 15 minutes at 4°C, the supernatant containing the total protein was collected. The protein concentration was determined via BCA assay following the manufacturer’s instructions. Briefly, a series of protein standards with known concentrations (0, 25, 50, 100, 200, 400, 800, 1600 μg/mL) were prepared, and the samples and standards were mixed with the BCA working reagent in a 96 - well plate. After incubation at 37°C for 30 minutes, the absorbance was measured at 562 nm using a microplate reader. A standard curve was generated based on the absorbance values of the protein standards, and the protein concentration of the samples was calculated accordingly. Five micrograms of protein was added to each loading well of a 10% SDS–polyacrylamide gel. After electrophoresis at 80 V for the first 30 minutes and then 120 V for 1–1.5 hours, the proteins were transferred to a PVDF membrane by the wet – transfer method at a constant voltage of 100 V for 1.5 hours. The membrane was then blocked with 5% skim milk powder in Tris – buffered saline with Tween 20 (TBST) for 2 h at room temperature to prevent non – specific binding. Subsequently, primary antibodies were introduced at an appropriate dilution (UCK2 antibody at 1:1000, C – myc antibody at 1:1000, OCT4 antibody at 1:1000, LIN28 antibody at 1:1000, β - actin antibody at 1:5000) and incubated at 4°C overnight. The next day, the membrane was washed three times with TBST for 10 minutes each time, and then horseradish peroxidase – labeled secondary antibody IgG was added at a dilution of 1:5000 and incubated at room temperature for 1 h. After washing the membrane three times again with TBST, ECL imaging was performed using an ECL detection kit. The chemiluminescent signal was detected using a gel imaging system, and grayscale analysis was performed with ImageJ software. The relative expression level of the target protein was calculated as the ratio of the gray value of the target protein band to that of the β - actin protein band.

2.9. Tumour-bearing mouse experiments

Twenty nude rats aged 5 or 6 weeks old and weighing 18–22 g were provided by Taixing People’s Hospital, and the animal experiments were conducted in compliance with the international requirements of experimental animal ethics. The nude mice were kept in a sterile environment, the room temperature was controlled at 23 °C–25°C, the humidity was maintained at 50%–60%, the light duration was 12 h, autoclaved water and sterile feed were provided freely and the experiments were carried out after 7 days of acclimatization. The nude mice were randomly divided into two groups (n = 10): the sh-NC group and the sh-UCK2 group. U87 cells transfected with the UCK2 shRNA#2 sequence were injected subcutaneously into the flanks of the nude mice at a cell count of 2 × 10⁶; these cells comprised the sh-UCK2 group. Sh-NC was injected subcutaneously, and equal numbers of cells were transfected with sh-NC. The mice were housed for 42 days, and tumor volume was monitored every 7 days beginning on day 7. After 42 days, the mice were sacrificed by cervical dislocation, and the tumors were surgically excised, photographed, weighed and subjected to paraffin-embedded sectioning. Animal experiments were approved by the Animal Ethics Committee of the Taixing People’s Hospital (IACUC approval protocol number: [YX2023-LL-0017], approval date: 2023–07–18). All methods were carried out in accordance with relevant guidelines and regulations.

2.10. Isolation of primary cells

The removed organs were washed three times with sterilized PBS, after which the tumors were carefully cut into pieces with ophthalmic scissors until they became small pieces of approximately 1 mm³. The tissues were subsequently washed with PBS until the tissue pieces turned white. The mixture was transferred to a sterile centrifuge tube and allowed to stand for several minutes to allow the tissue block to settle naturally to the bottom of the tube, after which the supernatant was discarded. A sterile centrifuge tube containing tissue blocks was treated with hyaluronic acid-collagenase at 37°C for 2 h, followed by centrifugation at 1000 rpm for 5 min. Then, 2 mL of trypsin was added for digestion at 37°C for 3 min, followed by subculturing into cell culture dishes for 12 h at 37°C under 5% CO₂. The cell state background in the cell culture dish was observed for impurities. The culture medium was then discarded, and the cells were added to the cell culture dish along the wall with sterilized PBS. The culture dish was gently shaken until the bottom of the dish was covered with PBS. Next, the PBS was discarded, and the cells were washed two times with PBS. Then, 2 mL of trypsin was added, and the mixture was digested at 37°C for 1 min. The trypsin was discarded, and the plates were washed three times with PBS as described above to thoroughly remove stromal cells. After washing, an appropriate amount of complete medium was added to the cell culture dish, and the adherent tumor cells were further cultured at 37°C in a 5% CO₂ incubator for 24 h.

2.11. Co-immunoprecipitation (co-IP) assay

Firstly, primary cells isolated from the central region of mouse transplantation tumors were lysed with RIPA buffer (Beyotime, Shanghai, China), and supernatants were collected. To each centrifuge tube, 400 µl of PBS (provided by Thermo Fisher, USA) was added, followed by the introduction of magnetic beads (courtesy of Takara Biotechnology, Dalian, China). Next, they were vigorously mixed using a vortex for 4 min to ensure a uniform blend. Post-magnetic separation, the supernatant was removed, and the magnetic beads were resuspended with an additional 400 µl of PBS for subsequent use. In each tube, 500 µl of PBS was combined with either PI3K or UCK2 antibodies at a final concentration of 1 μg/mL. The mixture underwent magnetic separation, followed by gentle pipetting of the magnetic beads with 400 µl of PBS to wash away any unbound antibodies. This washing step was repeated two times, with the supernatant being discarded after each round. Subsequently, 500 µl of 0.5% BSA blocking solution was introduced, and the mixture was incubated on a rotating shaker at room temperature for 1.5 h to allow for thorough blocking. At the conclusion of this process, the protein samples were carefully collected and preserved at − 20°C.

2.12. Statistical analysis

GraphPad statistical software (version 7.0) was used to analyze data. The measurement data are expressed as the mean ± SEM. T test was used for comparisons between groups, one-way analysis of variance (ANOVA) was used for comparisons amongst multiple groups, repeated measures ANOVA was used for comparisons at different timepoints and the SNK-Q test was used for pairwise comparisons. A difference of p < 0.05 was considered to indicate statistical significance.

3. Results

3.1. UCK2 is preferentially expressed in GSCs

In the GSE157506 dataset analysis, a significant number of DEGs were identified in GSCs, with 3404 upregulated and 3560 downregulated DEGs (Figure 1(A)). For validation, we focused on the top 10 upregulated DEGs with the most significant differences: HES5, OPRD1, EPB41, NKAIN1, FOXN2, UCK2, MAGI2, MAST1, ATP1A2, and ILDR2.

Figure 1.

UCK2 is preferentially expressed in GSCs.

A: Volcano plot of DEGs in GSE157506 dataset, which included three sample sets of GSCs and three sample sets of neural stem cells. The abscissal axis indicates the log2 (fold change) value. B: RT-qPCR analysis of the mRNA levels of HES5, OPRD1, EPB41, NKAIN1, FOXN2, UCK2, MAGI2, MAST1, ATP1A2 and ILDR2 in GSCs and neural stem cells. C: UCK2 levels in GSC samples from GSE45117 dataset (biological replicates, n = 3, t-test). D: RT-qPCR analysis of the mRNA levels of UCK2 in ALDH-negative and -positive U87 cells (biological replicates, n = 3, t-test). E: Western blotting analysis of the protein levels of UCK2 in NHAs, U87 cells and GBM-1 cells (biological replicates, n = 3, t-test). F: Western blotting analysis of the protein levels of UCK2 in U87 cells, GBM-1 cells, and GSCs (biological replicates, n = 3, t-test). G: Microsphere-forming capacity of U87, GBM-1 and GSC cells. H: Western blotting analysis of the protein levels of nestin in U87, GBM-1 and GSC cells. Data are shown as the mean ± SEM. **p < 0.01 versus the peripheral group.

Microsphere formation assays and ALDH+ cell sorting were used to enrich GSCs. Western blot analysis revealed a significant increase in the expression of the stem cell marker nestin in U87 and GBM-1-derived GSCs compared to their parental cells (Figure 1(B)). Additionally, these GSCs exhibited a markedly enhanced sphere formation capacity (Figure 1(C)).

Figures 1(D,E) show that HES5, OPRD1, EPB41, FOXN2, UCK2, MAGI2, and MAST1 were significantly upregulated in GSCs compared to neural stem cells, with UCK2 being the most prominent DEG. Figure 1(F) illustrates UCK2 expression across three GSC and three neural stem cell samples in the GSE157506 dataset.

Western blotting confirmed that UCK2 expression was notably higher in U87 and GBM-1 cells than in normal cells, with an even greater increase in GSCs (Figures 1(G,H)). These results highlight UCK2’s potential role in GSC stemness and tumourigenesis, suggesting it as a promising target for further mechanistic and therapeutic investigation.

3.2. Knockdown and overexpression of UCK2 significantly affect the stemness characteristics of GSCs

This study then focused on stably knocking down or overexpressing the UCK2 gene in GSCs. GBM-1 and U87 cells were transfected with pcDNA-UCK2 and UCK2 shRNA#1/2. Western blotting confirmed the successful creation of UCK2-overexpressing and knockdown cell lines. Notably, UCK2 was stably downregulated in U87-GSCs and upregulated in GBM-1-GSCs (Figure 2(A)). Moreover, UCK2 modulation directly affected the protein levels of stemness factors: knockdown decreased, while overexpression increased the levels of C-myc, OCT4, and LIN28 (Figure 2(A)).

Figure 2.

Knockdown and overexpression of UCK2 significantly affect the stemness characteristics of GSCs.

A: Western blot analysis of the protein levels of UCK2 and stemness-related transcription factors C-MYC, OCT4 and LIN28 in U87-GSCs and GBM-1-GSCs with UCK2 knockdown or overexpression. B: Representative images of mouse tumors, tumor volume and tumor weight. C: Colony formation ability of U87-GSCs and GBM-1-GSCs with UCK2 knockdown or overexpression, number of spheres formed and images taken, scale bar: 200 μm (biological replicates, n = 5, Independent Samples t-test). D: Colony formation assay showing the proliferation ability of U87- GSCs and GBM-1-GSCs with UCK2 knockdown or overexpression (biological replicates, n = 3, t-test). E: Transwell assay showing the migration and invasion abilities of U87-GSCs and GBM-1-GSCs with UCK2 knockdown or overexpression; scale bar: 50 μm (biological replicates, n = 3, t-test). Data are shown as the mean ± SEM. **p < 0.01 versus the NC shRNA group.

In vivo tumorigenicity assays in nude mice provided strong evidence of UCK2’s role in glioma growth. Tumors formed by sh-UCK2 cells were significantly smaller than those by sh-NC cells, highlighting UCK2’s crucial role in glioma development (Figure 2(B)). Additionally, UCK2 knockdown or overexpression respectively reduced or enhanced the size and diameter of microspheres, indicative of the cells’ stem-like properties (Figure 2(C)).

Colony formation and Transwell assays further revealed UCK2’s effects on GSCs. UCK2 knockdown significantly suppressed cell proliferation (Figure 2(D)), migration, and invasion (Figure 2(E)), while its overexpression promoted these malignant behaviors.

These results highlight UCK2 as an oncogenic driver in GSCs, accelerating stemness and fostering a malignant phenotype, thereby contributing to glioma’s aggressiveness.

3.3. UCK2 gene activates glycolysis to promote the characteristics of CSCs under hypoxia

To evaluate tumors with high UCK2 expression, a mouse xenograft tumor model was used. Given that hypoxia is a hallmark of the tumor microenvironment and prevalent in solid tumors, U87 cells were subcutaneously injected into nude mice. Immunofluorescence staining on frozen sections of the resulting tumors was then performed to examine the localization and expression levels of UCK2. Subsequently, HIF-1α immunofluorescence staining and HE staining were carried out to further elucidate the microenvironmental context.

The results show that HIF-1α, a key regulator under hypoxic conditions, accumulated predominantly in the tumor’s central region compared to the peripheral region (Figure 3(A)). Additionally, the central tumor regions exhibited elevated expression of stemness-related factors C-MYC, OCT4 and LIN28 (Figure 3(A)), suggesting a link between UCK2 and the stem-like properties of tumor cells.

Figure 3.

The UCK2 gene activates glycolysis to promote the characteristics of cancer stem cells under hypoxia.

A: Central and peripheral regions of transplanted tumors co-stained with fluorescent markers of stemness (HIF-1α, C-MYC, OCT4 and LIN28), which were stained blue with DAPI, scale bar: 50 μm, (biological replicates, n = 5). B: Transplanted tumors divided into central and peripheral regions and analyzed by HE staining, scale bar: 50 μm. C: RT–qPCR analysis of UCK2 mRNA expression in central and peripheral cancer cells (biological replicates, n = 3, t-test). D: Stemness of cancer cells in the central and peripheral regions examined by microsphere formation assay, scale bar: 200 μm (biological replicates, n = 3, t-test). E: Glucose uptake, lactate production, cellular ATP levels and PDH activity after central and peripheral zone cells were isolated from transplanted tumors (biological replicates, n = 3, t-test). F: Volcano plot of DEGs in GSE45117 dataset, which contains four glioma primary tumor cell lines exposed to hypoxia for 48 h and four glioma cell lines exposed to normoxia for 48 h. Data are shown as the mean ± SEM. **p < 0.01 versus the peripheral group.

Primary cultures were conducted on cells isolated from the central and peripheral zones of the hypoxic tumor tissue (Figure 3(B)). RT-qPCR analysis confirmed significantly higher UCK2 mRNA levels in the central region than in the peripheral region (Figure 3(C)). Tumor xenograft and sphere-forming assays using these primary cultures demonstrated a marked enhancement in the sphere-forming capacity of cells from the hypoxic central zone (Figure 3(D)), indicative of their stem-like nature.

Furthermore, glycolytic activity was investigated, with results showing that central zone cells exhibited higher glucose uptake, lactate production and ATP levels than peripheral zone cells (Figure 3(E)). This indicates that cells in the central region were more reliant on glycolysis, a metabolic adaptation often seen in hypoxic tumor conditions.

The investigation was further supported by obtaining the GSE45117 dataset, which includes four glioma primary tumor cell lines subjected to hypoxic conditions for 48 h and four cell lines under normoxic conditions for the same duration. Analysis of DEGs identified 280 upregulated genes in hypoxic cell lines, with UCK2 prominently detected among them (Figure 3(F)). This finding serves as additional validation of the experimental outcomes and strengthens the association among UCK2, hypoxia and the stem-like characteristics of glioma cells.

3.4. UCK2 knockdown effectively inhibits glycolysis and promotes the stemness of cells in the central hypoxic zone

Experiments were conducted in primary glioma cells from the tumor’s central region, involving UCK2 knockdown and treatment with the glycolysis inhibitor 2-DG. The results showed striking similarities between UCK2 knockdown and 2-DG treatment. Both led to reduced UCK2 expression (Figure 4(A)), decreased glucose uptake (Figure 4(A)), lower lactate production (Figure 4(C)), and diminished ATP levels (Figure 4(D)). Additionally, PDH activity increased (Figure 4(E)), and there was a reduction in sphere formation (Figure 4(F)), cell proliferation (Figure 4(G)), and migration and invasion capabilities (Figure 4(H)).

Figure 4.

UCK2 knockdown effectively inhibits glycolysis and promotes the stemness of cells in the central hypoxic zone.

A: RT–qPCR analysis of UCK2 expression after UCK2 knockdown or 2-DG treatment in primary cells isolated from the central region of mouse transplanted tumors (biological replicates, n = 3, t-test). B: ELISA analysis of glucose uptake in primary cells isolated from the central region of mouse transplanted with tumors after UCK2 knockdown or 2-DG treatment (biological replicates, n = 3, t-test). C: ELISA analysis of lactate production in primary cells isolated from the central region of mouse transplanted tumors after UCK2 knockdown or 2-DG treatment (biological replicates, n = 3, t-test). D: ELISA analysis of ATP levels in primary cells isolated from the central region of mouse transplanted tumors after UCK2 knockdown or 2-DG treatment (biological replicates, n = 3, t-test). E: ELISA analysis of PDH activity in primary cells isolated from the central region of mouse transplanted tumors after UCK2 knockdown or 2-DG treatment (biological replicates, n = 3, t-test). F: Stemness of cancer cells examined by microsphere formation assay after UCK2 knockdown or 2-DG treatment in primary cells isolated from the central region of mouse transplanted tumors, scale bar: 200 μm (biological replicates, n = 3, t-test). G: Colony formation analysis of primary UCK2 knockdown or 2-DG-treated cells to determine the proliferation of cells isolated from the central region of mouse transplanted tumors (biological replicates, n = 3, t-test). H: Transwell assay analysis of the migration and invasion of primary cells isolated from the central region of mouse transplanted tumors after UCK2 knockdown or 2-DG treatment, scale bar: 50 μm (biological replicates, n = 3, t-test). Data are shown as the mean ± SEM. **p < 0.01 versus the sh-NC group. ##p < 0.01 versus the vehicle group.

These results highlight UCK2’s crucial role in glycolysis and cancer stem cell maintenance under hypoxia. UCK2 knockdown significantly curtailed glycolysis and the stem-like traits of glioma cells, indicating that UCK2 is a key regulator of these cells’ adaptability and malignancy in the hypoxic tumor microenvironment.

3.5. UCK2 activates glycolytic levels and stemness traits through the PI3K/Akt/HIF-1α pathway

The PI3K/Akt/HIF-1α pathway is crucial in glioma pathophysiology. To explore UCK2’s impact on this pathway, we used LY2, a specific PI3K/Akt/HIF-1α inhibitor. LY2 treatment did not affect UCK2 protein expression (Figure 5(A)) but significantly reduced the expression of PI3K/Akt/HIF-1α-related proteins (Figure 5(A)), glucose uptake (Figure 5(B)), lactate production (Figure 5(C)), and ATP levels (Figure 5(D)). It also increased PDH activity (Figure 5(E)) and decreased glioma cells’ sphere-forming ability (Figure 5(F)), proliferation (Figure 5(G)), and migration and invasiveness (Figure 5(H)).

Figure 5.

UCK2 activates glycolytic levels and stemness traits through the PI3K/Akt/HIF-1α pathway.

A: Western blot analysis of the protein levels of UCK2, HIF-1α, PI3K, p-AKT and AKT in primary cells isolated from the central region of mouse transplanted tumors after UCK2 knockdown or/and LY2 treatment (biological replicates, n = 3, t-test and one-way ANOVA). B: ELISA analysis of glucose uptake in primary cells isolated from the central region of mouse transplanted tumors after UCK2 knockdown or LY2 treatment (biological replicates, n = 3, t-test and one-way ANOVA). C: ELISA analysis of lactate production in primary cells isolated from the central region of mouse transplanted tumors after UCK2 knockdown or LY2 treatment (biological replicates, n = 3, t-test and one-way ANOVA). D: ELISA analysis of cellular ATP levels in primary cells isolated from the central region of mouse transplanted tumors after UCK2 knockdown or LY2 treatment (biological replicates, n = 3, t-test and one-way ANOVA). E: ELISA analysis of PDH activity in primary cells isolated from the central region of mouse transplanted tumors after UCK2 knockdown or LY2 treatment (biological replicates, n = 3, t-test and one-way ANOVA). F: Stemness of cancer cells checked by microsphere formation assay after UCK2 knockdown or LY2 treatment in primary cells isolated from the central region of mouse transplanted tumors, scale bar: 200 μm (biological replicates, n = 3, t-test and one-way ANOVA). G: Colony formation analysis of primary cells isolated from the central region of mouse transplanted tumors showing proliferation after UCK2 knockdown or LY2 treatment (biological replicates, n = 3, t-test and one-way ANOVA). H: Transwell assay analysis of the migration and invasion of primary cells isolated from the central region of mouse transplanted tumors after UCK2 knockdown or LY2 treatment, scale bar: 50 μm (biological replicates, n = 3, t-test and one-way ANOVA). I: ELISA analysis of glucose uptake in primary cells isolated from the central region of mouse transplanted tumors after UCK2 knockdown or/and DA treatment (biological replicates, n = 3, t-test and one-way ANOVA). J: ELISA analysis of lactate production in primary cells isolated from the central region of mouse transplanted tumors after UCK2 knockdown or/and DA treatment (biological replicates, n = 3, t-test and one-way ANOVA). K: ELISA analysis of cellular ATP levels in primary cells isolated from the central region of mouse transplanted tumors after UCK2 knockdown or/and DA treatment (biological replicates, n = 3, t-test and one-way ANOVA). L: ELISA analysis of PDH activity in primary cells isolated from the central region of mouse transplanted tumors after UCK2 knockdown or/and DA treatment (biological replicates, n = 3, t-test and one-way ANOVA). M: Stemness of cancer cells checked by microsphere formation assay after UCK2 knockdown or/and DA treatment in primary cells isolated from the central region of mouse transplanted tumors, scale bar: 200 μm (biological replicates, n = 3, t-test and one-way ANOVA). N: Colony formation analysis of primary cells isolated from the central region of mouse transplanted tumors showing proliferation after UCK2 knockdown or/and DA treatment (biological replicates, n = 3, t-test and one-way ANOVA). O: Transwell assay analysis of the migration and invasion of primary cells isolated from the central region of mouse transplanted tumors after UCK2 knockdown or/and DA treatment, scale bar: 50 μm (biological replicates, n = 3, t-test and one-way ANOVA). Data are shown as the mean ± SEM. n = 3. **p < 0.01. nsP > 0.05.

Notably, after LY2 treatment, UCK2 knockdown did not significantly alter these cellular behaviors, suggesting that inhibiting the PI3K/Akt/HIF-1α pathway post-UCK2 suppression may be ineffective due to a lack of available targets. This highlights a complex interplay between UCK2 and this pathway, warranting further investigation.

To further elucidate this relationship, we introduced DA, a PI3K/Akt/HIF-1α pathway activator. DA treatment counteracted the suppressive effects of UCK2 knockdown on various cellular activities, including glucose uptake (Figure 5(I)), lactate generation (Figure 5(J)), ATP levels (Figure 5(K)), sphere-forming ability (Figure 5(M)), proliferation (Figure 5(N)), and migration and invasion (Figure 5(O)). Additionally, DA reversed the increased PDH activity observed after UCK2 reduction (Figure 5(L)). This suggests that DA may modulate glioma cell metabolism and behavior through the PI3K/Akt/HIF-1α pathway, which is intricately linked with UCK2’s regulatory role.

3.6. UCK2 stabilizes PI3K protein expression by deubiquitination modification

This study meticulously examined how UCK2 affects PI3K ubiquitination to elucidate the mechanisms underlying UCK2-mediated activation of the PI3K/Akt/HIF-1α pathway. The data showed that UCK2 overexpression significantly reduced PI3K ubiquitination while increasing PI3K protein expression (Figure 6A).

Figure 6.

UCK2 stabilizes PI3K protein expression by deubiquitination modification.

A: Ubiquitination level of PI3K protein after overexpression of UCK2 in primary cells isolated from the central region of mouse transplanted tumors by using immunoprecipitation assay (biological replicates, n = 3). B, C: Co-IP detection of PI3K and UCK2 protein interactions (biological replicates, n = 3).

Co-IP assays provided strong evidence of a robust interaction between UCK2 and PI3K, with notable enrichment of PI3K in UCK2 antibody pull-downs and vice versa (Figures 6(B,C)).

Collectively, these findings suggest that UCK2 enhances PI3K stability and expression via deubiquitination, thereby activating downstream Akt/HIF-1α signaling. This interplay offers new insights into the signaling networks governing cellular metabolism and hypoxia response, potentially revealing new therapeutic targets in glioma and other hypoxia-driven cancers.

4. Discussion

The transformation of tumor cells, known as reprogramming, facilitates a transition between differentiated cancer cells and stem-like CSCs. In the laboratory, the overexpression of four key transcription factors – OCT4, KLF4, SOX2 and C-MYC – has been shown to convert various somatic cells into pluripotent stem cells. Notably, a subset of CSCs exhibits increased expression levels of genes pivotal for self-renewal, such as MYC and SOX2 [13]. The present research shed light on the role of UCK2, a gene whose knockdown or overexpression significantly modulates the expression levels of stemness-associated transcription factors, including C-MYC, OCT4 and LIN28. This finding suggests that UCK2 is a critical mediator in the reprogramming of glioma cells. UCKs are integral to the nucleotide salvage pathway, with UCK2 being a pyrimidine ribonucleoside kinase. It is encoded on the long arm of chromosome 1, specifically at locus 22–23.2 and closely related to UCK1. Both enzymes phosphorylate uridine and cytidine into their monophosphate forms, which are essential for the synthesis of pyrimidine nucleotides that constitute DNA and RNA. However, UCK2 demonstrates a substantially higher catalytic efficiency for these substrates, approximately 15–20 times that of UCK1. Beyond its metabolic role, UCK2 functions as an activator for nucleotide analogues, playing a significant part in the activation of drugs used in antiviral and antitumor therapies. Through weighted gene co-expression network analysis, Chen et al. [14] identified gene modules linked to glycolysis in hepatocellular carcinoma and explored their functions. The study revealed an upregulation of the glycolytic pathway in hepatocellular carcinoma, and prognostic signatures were developed for genes such as CDCA8, RAB5IF, SAP30 and UCK2. Similarly, Jiang et al. [15] identified 47 metabolism-related DEGs associated with the prognosis of patients with endometrial cancer, many of which were enriched in amino acid, glycolysis and glycerophospholipid metabolism pathways, including UCK2. This study pioneers the proposition that UCK2 is a central gene in the regulation of stemness and glycolytic reprogramming in cancer. The metabolic characteristics of CSCs have been a focus of intensive research, with the energy acquisition of these cells predominantly relying on glycolysis rather than oxidative phosphorylation. For instance, primary tumor cells derived from MMTV-Wnt-1-induced breast tumors predominantly use glycolysis for energy production, in contrast with non-tumourigenic mammary cells [16]. Additionally, glycolysis is the primary pathway for glucose metabolism in radioresistant nasopharyngeal carcinoma cells and CD133+CD49+ hepatocellular carcinoma tumor-initiating cells [17]. The deficiency of FBP1 leads to increased glycolysis and enhanced stemness in glioma cells by promoting β-catenin interactions, thus fostering tumor progression [18]. The findings indicate that UCK2 is abnormally overexpressed in GSC populations, including those that form spheres and are ALDH+. The knockdown of UCK2 significantly reduced glycolysis in glioma cells and impeded the maintenance of stemness. Collectively, these results suggest that the reprogramming of glioma cells is governed by the UCK2 gene and is primarily associated with the tumors’ self-renewal capacity.

Recent years have seen a paradigm shift in the development of cancer stem cell models. The focus has transitioned from culturing established tumor cell lines in vitro to utilizing fresh tumor specimens and early passages of primary cells derived from xenograft tumors. A growing consensus amongst researchers emphasizes the importance of employing xenograft assays to assess the presence and activity of CSCs. In line with this approach, a nude mouse xenograft model was utilized in the present study to investigate the microenvironment and characteristics of UCK2-expressing tumor cells. The immunofluorescence staining of the xenograft tumors revealed a notable concentration of UCK2 in the central regions of the tumor mass. This distribution pattern suggests a correlation between the function of the UCK2 protein and the hypoxic conditions typically found in solid tumors. Zhang et al. [19] reported a significant upregulation of UCK2 in the hypoxic microenvironment of hepatocellular carcinoma. These corroborating results lend further credence to the present study’s conclusions, highlighting the potential role of UCK2 in the context of CSCs and the hypoxic tumor microenvironment.

The PI3K/AKT signaling pathway plays a pivotal role in the regulation of cancer cell growth [20]. Its abnormal activation is implicated not only in the promotion of aberrant cell growth, proliferation and differentiation but also in the mediation of glycolysis, a critical metabolic process in cancer cells [21]. This pathway has the capacity to augment the expression and membrane transport of glucose transporters, thereby enhancing glucose uptake and activating key glycolytic enzymes, which, in turn, boost the efficiency of glycolysis [22]. Hypoxia, a common feature in the tumor microenvironment, is known to activate the PI3K/Akt signaling pathway, leading to the upregulation of HIF-α expression in cells. The expression of HIF-α is closely associated with the pathogenesis of a myriad of diseases [23]. The phosphorylation of AKT by this pathway further propels glucose uptake and stimulates glycolysis in tumor cells. Additionally, hypoxia-induced upregulation of SLC7A11 via the PI3K/AKT/HIF-1 axis has been shown to enhance the resistance of glioma to sulfamethoxazole-induced ferroptosis [24]. Hou et al. [25] discovered that glioma stem-like cells can be targeted by the benzene ester derivative ACT001, which inhibits the AEBP1/PI3K/AKT signal transduction pathway. The findings of the present research underscore the ability of UCK2 to enhance glycolysis and augment stemness characteristics via the PI3K/Akt/HIF-1α pathway. Mechanistic exploration revealed that UCK2 increases the stability and expression of PI3K protein through a deubiquitination process, subsequently initiating downstream Akt/HIF-1α signaling events. Previous studies have established that UCK2 interacts with the epidermal growth factor receptor (EGFR), impeding EGFR-induced ubiquitination and degradation. This interaction results in heightened activation of the EGFR-AKT pathway, contributing to the enhanced metastatic potential of hepatocellular carcinoma [26]. The deubiquitination of PI3K proteins by UCK2 is a key regulatory mechanism. This process of deubiquitination is not just a simple protein modification, but a precise regulation of PI3K protein stability and expression, which leads to a series of profound biological effects. At the molecular level, deubiquitination prevents the PI3K protein from being degraded by the proteasome, allowing it to accumulate in the cell at a concentration that allows it to transmit signals more efficiently. This process is analogous to opening an unobstructed gateway to signaling pathways, allowing downstream Akt/HIF-1α signaling events to be initiated. The specificity of UCK2’s action on PI3K proteins compared to other known deubiquitinating enzymes may stem from its unique structure and catalytic mechanism. An in-depth study of the sites where UCK2 interacts with PI3K and the specific chemical process of deubiquitination will help us to understand this regulatory mechanism more comprehensively. In addition, considering the widespread aberrant activation of the PI3K/AKT signaling pathway in many cancers, UCK2-mediated deubiquitination of PI3K may become a new target for cancer therapy. By inhibiting the activity of UCK2 or interfering with its interaction with PI3K, it is possible to block the overactivation of this signaling pathway, thereby inhibiting the malignant phenotypes of cancer cell growth, proliferation, and glycolysis. This study corroborates the conclusion of the present study by demonstrating UCK2’s capability to deubiquitinate and stabilize target proteins, thereby modulating critical signaling pathways implicated in cancer progression.

In conclusion, the findings indicate that UCK2 plays a pivotal role as a metabolic regulator in glucose metabolism by stabilizing the expression of the PI3K protein through deubiquitination, which, in turn, activates the Akt/HIF-1α signaling pathway. This intricate regulatory mechanism effectively modulates the glycolytic activity within tumor cells and supports the preservation of stem-like properties in glioma cells, even under the harsh conditions of hypoxia (Figure 7). The discovery of UCK2’s role in these processes could potentially usher in novel therapeutic strategies and offer renewed optimism for patients battling malignant gliomas. On the basis of elaborating the research findings and their significance, this study has added descriptions of the research methods and the advantages of the models employed. For instance, it highlights the use of bioinformatics analysis to screen for key genes from vast amounts of data, thereby providing comprehensive information on gene expression for the study. The combination of various experimental techniques, such as cell experiments, animal experiments, and protein interaction experiments, has been utilized to verify the research hypotheses from different perspectives, thus lending greater persuasiveness to the research results. The application of the nude mouse xenograft model allows for a closer approximation to in vivo conditions, enabling the study of the growth and metabolic characteristics of tumor cells within the body and providing an important reference for preclinical research. Whilst the study on UCK2’s role as a metabolic regulator in glucose metabolism presents significant insights, several limitations must be consider. Firstly, although the study has found that UCK2 activates the Akt/HIF-1α pathway by stabilizing PI3K protein, the molecular mechanism by which UCK2 precisely recognizes the PI3K protein and performs deubiquitination modification remains unclear. At present, it is only known that there is an interaction between the two and the effect of deubiquitination on PI3K protein expression, but the specific action sites and other molecules involved need further research. Secondly, this study was mainly conducted in glioma cell lines and nude mouse models, with relatively simple experimental conditions. Different types of glioma cells and other cancer cells may have different responses to the expression and function of UCK2, and the in vivo tumor microenvironment is more complex, containing various cell types and extracellular matrix components. The universality of the study results in other cancer types and more complex in vivo environments needs further verification. In addition, the study mainly focuses on the effects of UCK2 on glycolysis and cancer stemness, although it involves the PI3K/Akt/HIF-1α pathway, but less discussion on the changes of other related metabolic pathways, such as fatty acid metabolism, amino acid metabolism, etc., in this process and their interrelationships with glycolysis. The metabolism of tumor cells is a complex network, and these less studied metabolic pathways may be closely related to the function of UCK2, affecting the comprehensive understanding of the regulation mechanism of tumor metabolism. In the experimental process, there may be bias in the selection of cell lines. The U87 and GBM-1 cell lines used in this study may not fully represent the characteristics of all glioma cells, and the differences between different cell lines may affect the universality of the experimental results. In addition, the nude mouse model used in animal experiments has immune deficiencies, which is different from the human body with normal immune function, and this may lead to deviations between the experimental results and the actual clinical situation. In terms of data analysis, although various statistical methods were used, the sample size was relatively small, which may not accurately reflect the overall situation and increase the uncertainty of the results.

Figure 7.

Mechanism diagram.

Knockdown of UCK2 acts as a switch for glucose metabolism by inhibiting the PI3K/Akt/HIF-1α signaling axis, thereby preventing glycolysis in glioma cells, which in turn promotes the maintenance of glioma cell stemness under hypoxic conditions. Subsequently, this led to the inhibition of cell migration and invasion ability, which in turn exerted anti-tumor effects.

Funding Statement

The study was supported by the Project of Jiangsu Health Vocational College University-Level Scientific Research [JKC201971]; the Key Program of Kangda College of Nanjing Medical University Science and Technique Development Foundation Project [KD2022KYJJZD059]; the Fengcheng Talent Support Project of Taizhou.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Authors’ Contributions

Xiaopeng Ding and Jingying Wang were responsible for research design. Peng Yu and Jia Yin were responsible for conducting the experiments. Jingying Wang and Peng Yu were responsible for data acquisition and data analysis. Xiaopeng Ding and Jia Yin were responsible for writing the manuscript. All the authors have contributed to the completion of this paper.

Data availability statement

The datasets used during the present study are available from the corresponding author upon reasonable request.

Ethical approval

Animal experiments were approved and supervised by the Animal Ethics Committee of the Taixing People’s Hospital. All methods were carried out in accordance with relevant guidelines and regulations.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/15384101.2025.2539643