Identification of USP29 as a key regulator of nucleotide biosynthesis in neuroblastoma through integrative analysis of multi-omics data

ABSTRACT

Cancer cells show enhanced nucleotide biosynthesis, which is essential for their unlimited proliferation, but the underlying mechanisms are not entirely clear. Ubiquitin specific peptidase 29 (USP29) was reported to sustain neuroblastoma progression by promoting glycolysis and glutamine catabolism; however, its potential role in regulating nucleotide biosynthesis in tumor cells remains unknown. In this study, we depleted endogenous USP29 in MYCN-amplified neuroblastoma SK-N-BE2 cells by sgRNAs and conducted metabolomic analysis in cells with or without USP29 depletion, we found that USP29 deficiency caused a disorder of intermediates involved in glycolysis and nucleotide biosynthesis. De novo nucleotide biosynthesis was analyzed using ¹³C₆ glucose as a tracer under normoxia and hypoxia. The results indicated that USP29-depleted cells showed inhibition of nucleotide anabolic intermediates derived from glucose, and this inhibition was more significant under hypoxic conditions. Analysis of RNA sequencing data in SK-N-BE2 cells demonstrated that USP29 promoted the gene expression of metabolic enzymes involved in nucleotide anabolism, probably by regulating MYC and E2F downstream pathways. These findings indicated that USP29 is a key regulator of nucleotide biosynthesis in tumor cells.

KEYWORDS:

• USP29
• cancer metabolism
• nucleotide biosynthesis
• neuroblastoma

Introduction

Nucleotides are essential to many cellular processes including DNA replication, gene transcription, and ribosome biogenesis, although the uptake of nucleotides from extracellular sources is negligible.¹ In mammalian cells, nucleotides are synthesized through either the de novo pathway or the salvage pathway. In the de novo pathway, which acts as the main pathway, the cells utilize glucose and amino acids to synthesize both purine and pyrimidine, with glucose and amino acids providing carbon and nitrogen respectively; for the salvage pathway, the nucleoside backbones and free bases derived from the hydrolysis of nucleic acids are recycled for nucleic acid synthesis.^2,3 It has been well characterized that cancer cells often showed enhanced nucleotide biosynthesis, thereby sustaining unlimited cell proliferation.² Cancer cell nucleotide synthesis is regulated by several critical transcription factors, MYC and Rb/E2F in particular, which promote nucleotide synthesis by inducing a series of genes that encode the enzymes in the nucleotide biosynthetic pathways.^4,5

Neuroblastoma, the most common extracranial solid tumor of childhood, is thought to originate from undifferentiated neural crest cells.^6,7 Several genetic alterations have been shown in neuroblastoma, including amplification of MYCN, a member of the oncogenic MYC family, which is found in approximately 20% of cases and correlates with high-risk disease and poor patient survival; to date, it remains the best-characterized genetic marker of high-risk neuroblastoma.^6,7 Although the outcome for patients with high‑risk neuroblastoma has improved, the 5-year overall survival probability for these patients is only approximately 50%.⁸

USP29 has been reported to play an emerging role in several cancers. In our previous study, we revealed that USP29 promoted tumor cell aerobic glycolysis and glutamine anaplerosis by deubiquitinating and stabilizing the oncogenic MYC family and HIF1α, and systemic knockout of USP29 significantly suppressed tumor progression and prolonged the survival of tumor-bearing mice.⁹ Consistently, USP29 was also reported to sustain glycolysis and sorafenib resistance in hepatocellular carcinoma by stabilizing HIF1α, indicating the important role of USP29 in regulating tumor cell metabolic reprogramming in various types of cancer.¹⁰ Moreover, USP29 was reported to stabilize p53,¹¹ Claspin,¹² Cdc25A,¹³ VIRMA¹⁴ and Snail1^15,16 in tumor cells. However, the potential role of USP29 in regulating nucleotide metabolism remains unclear.

In this study, through an integrated analysis of metabolomics, metabolic flux, and transcriptomics data in neuroblastoma SK-N-BE2 cells with or without USP29 depletion, we revealed that USP29 plays a key role in promoting tumor cell nucleotide synthesis potentially by regulating the MYC and E2F pathways.

Methods

Cell culture

Neuroblastoma SK-N-BE2, BE-2C and SK-N-AS cells were kindly provided by Professor Guoliang Qing (Medical Research Institute, Wuhan University, Wuhan, China), and 293T cells were purchased from Procell Company (Wuhan, China). Neuroblastoma cells were cultured in RPMI-1640 (HyClone) medium with 10% FBS (Gibco) and 1% penicillin/streptomycin (HyClone), and 293T cells were maintained in DMEM (HyClone) containing 10% FBS and 1% penicillin/streptomycin.

Lentiviral transduction

The lentiCRISPRv2 was used for plasmid construction and transfected into 293T cells together with the helper plasmids pMD2.G and psPAX2 using Lipo2000 (Thermo Fisher Scientific). Viral supernatant was collected at 48 h post transfection and filtered using a 0.22 μm sterile membrane. For infection of SK-N-BE2 cells, cells grown to 60–80% confluence were incubated with an appropriate amount of viral supernatant and 4 μg/mL polybrene in a final volume of 2 mL for 24 h. The infected cells were selected with 2 μg/mL puromycin for at least 36 h before additional experiments were performed. The USP29 sgRNA sequence was 5’-TTGAGCAGTAGTGCACCTGT-3’, and the control sgRNA was 5’-GGGCGAGGAGCTGTTCACCG-3’.

Immunoblot

Cells were collected using PBS, and total proteins were extracted with lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 1% SDS, 1 mM EDTA). Proteins were separated by SDS‒PAGE and transferred to PVDF membranes (Bio-Rad). After blocking with 5% nonfat dry milk in TBST, membranes were incubated with the primary antibody overnight at 4°C and then a horseradish peroxidase-conjugated secondary antibody. Protein bands were visualized using the SuperSignal chemiluminescence kit (Merck Millipore) and the Bio-Rad ChemiDoc XRS+ imaging system. The antibodies used in this study were actin (ABclonal, AC026) and USP29 antibodies (ABGENT, AP2153C).

Metabolomic analysis

The metabolomic analysis was performed as previously described.¹⁷ In brief, ten million SK-N-BE2 cells with or without USP29 knockout were collected and quenched in liquid nitrogen. Metabolite samples were prepared for analysis using standard solvent extraction methods and then subjected to the gas chromatograph system coupled with a Pegasus HT time-of-flight mass spectrometer. The resulting three-dimensional data involving the peak number, sample name, and normalized peak area were fed to the SIMCA14.1 software package (V14.1, Sartorius Stedim Data Analytics AB, Umea, Sweden) for principal component analysis (PCA) and orthogonal projections to latent structures-discriminate analysis (OPLS-DA). PCA showed the distribution of the original data. The first principal component of variable importance in the projection (VIP) was obtained to refine this analysis. VIP values exceeding 1 were first selected as changed metabolites. In step 2, the remaining variables were then assessed by Student’s t test (P value > 0.05), and variables were discarded between two comparison groups.

Stable isotope tracing

¹³C₆-glucose (Cat#110187-42-3) was purchased from the Cambridge Isotope Laboratories. SK-N-BE2 cells were cultured under normoxia or hypoxia (1% O₂) for 12 h before the medium was replaced with RPMI-1640 containing the labeled nutrients. Media were replaced with glucose-free RPMI-1640 (Gibco) with 2 mg/mL ¹³C₆-glucose and 10% FBS for 1 or 24 h after cells were washed twice with PBS; for hypoxic tracing, the labeled medium was pretreated in hypoxia for 3 h. Cellular metabolite extraction was performed as described previously.⁹ Briefly, cells were treated with 80% methanol (prechilled to −80°C) and centrifuged (14,000×g) at 4°C for 15 min. The supernatant was then collected and evaporated. The metabolite analysis was conducted at the Metabolomics Facility Center in the National Protein Science Technology Center of Tsinghua University.

RNA sequencing and gene set enrichment analysis

RNA sequencing was performed as previously described,⁹ and RNA-Seq data in SK-N-BE2 cells have been deposited in Gene Expression Omnibus with the accession code GSE180797. Gene set enrichment analysis (GSEA) was used to identify pathways and gene clusters.¹⁸

RNA isolation and qRT‒PCR

RNA isolation and qRT‒PCR were performed as previously described.⁹ Briefly, total cellular RNA was extracted with TRIzol reagent (Vazyme, Nanjing, China) according to the manufacturer’s protocol, complementary DNA was prepared using a Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme, Nanjing, China), and quantitative PCR was conducted using FAST SYBR Green Master Mix (Vazyme, Nanjing, China) on a CFX Connect Real-Time PCR System (Bio-Rad). Relative expression of the mRNA was calculated by the 2^−ΔΔCt method and normalized to actin expression using specific primers as shown in Supplementary Table S1.

Statistical analysis

Data analysis was carried out using GraphPad Prism 6. Statistical significance was calculated by unpaired two-tailed Student’s t test between two groups or by one-way ANOVA with Tukey’s corrections when comparing three or more groups. Differences were considered significant when p <  0.05.

Results

Metabolomic analysis of USP29-depleted SK-N-BE2 cells

To explore the effects of USP29 on tumor metabolism at the global level, we depleted endogenous USP29 in neuroblastoma SK-N-BE2 cells by validated sgRNA (Supplementary Figure S1a). The total metabolites were extracted from cells with or without USP29 depletion and subjected to metabolomics analysis. As shown in Figure 1a, 56 up-regulated metabolites and 22 down-regulated metabolites were found, indicating that USP29 deficiency may cause a major effect on cellular metabolite accumulation. In a previous study, we reported that the depletion of USP29 in tumor cells significantly inhibited glucose consumption and lactate production.⁹ Interestingly, the metabolomics data showed that representative glycolytic intermediates, including glucose (Glc), fructose 6 phosphate (F6P), pyruvate (Pyr) and lactate (Lac), were markedly accumulated along with USP29 deficiency (Figure 1b). Although the underlying mechanisms are not clear, these results may explain why USP29-depleted tumor cells show less glucose absorption and lactate secretion.

Figure 1. Metabolomic analysis of USP29-depleted SK-N-BE2 cells.

(a) Heatmap presentation of 78 metabolites showing significant differences in cells with or without USP29 knockout, each group contains seven replicates; (b). Analysis of metabolites associated with aerobic glycolysis; Glc (glucose), F6P (fructose 6 phosphate), Pyr (pyruvate), Lac (lactate); (c) Intracellular purine base and pyrimidine base; (d) Intracellular purine nucleotide, AMP (adenosine 5-monophosphate), GMP (guanosine 5-monophosphate).*p < .05, **p < .01.

In addition to the alteration of glycolytic intermediates, there was a significant increase in intracellular purine and pyrimidine bases, including hypoxanthine, adenosine, guanosine, and thymine (Figure 1c), which are essential precursors of nucleotide synthesis,¹⁹ indicating that the cellular nucleotide synthesis might be compromised. Consistent with this hypothesis, the downstream metabolites adenosine 5-monophosphate (AMP) and guanosine 5-monophosphate (GMP) were down-regulated (Figure 1d). These findings suggested that USP29 may play an important role in regulating cancer cell metabolism, especially in nucleotide biosynthesis.

USP29 deficiency decreases nucleotide biosynthesis

In addition to the use of glucose as an energy source for ATP generation, cancer cells display enhanced uptake of glucose, a major nutrient of mammalian cells, providing an important source of carbon to support the biosynthesis of nucleotides (Figure 2a), lipid production and non-essential amino acids (NEAAs).^1,20 We then assessed nucleotide synthesis flux in SK-N-BE2 cells using ¹³C₆ glucose (all six carbons are ¹³C-labeled, M6) as the tracer. Inosine monophosphate (IMP) is an important metabolic intermediate of purine synthesis, and we observed elevated M5 IMP enrichment when the cells were labeled for 1 h but decreased enrichment for cells labeled for 24 h (Figure 2b). Despite this, USP29 depletion significantly reduced the enrichment of M5 purine intermediates, including AMP, ADP/dGDP, ATP, GDP and GTP, either labeled for 1 or 24 h (Figure 2c). As expected, the enrichment of representative M5 pyrimidine intermediates (including UMP, UDP and UTP) was down-regulated (Figure 2d). These findings suggested that USP29 may play a positive role in de novo nucleotide synthesis.

Figure 2. USP29 deficiency decreases nucleotide biosynthesis.

(a) Carbon fate map showing the isotopomer distribution of indicated nucleotides derived from ¹³C₆ glucose. Glc (Glucose), R-5-P (Ribose 5-phosphate), IMP (hypoxanthine nucleotide), AMP (adenosine 5-monophosphate), ADP (adenosine diphosphate), ATP (adenosine triphosphate), GMP (guanine 5-monophosphate), GDP (guanine diphosphate), GTP (guanine triphosphate), UMP (uridine monophosphate), UDP (uridine diphosphate), TMP (thymine monophosphate), UTP (uridine triphosphate), CTP (cytosine triphosphate). (b) SK-N-BE2 cells with or without USP29 knockout were labeled with ¹³C₆ glucose for 1 h or 24 h, and isotope-labeled IMP was analyzed. (c) Isotope-labeled purine nucleotides were analyzed. (d) Isotope-labeled pyrimidine nucleotides were analyzed. Each group contains three replicates, NS (no significance), *p <0 .05, **p <0 .01.

USP29 promotes nucleotide biosynthesis under hypoxia

One of the best-known reasons for altered tumor metabolism is the unique hypoxic stresses that exist within the tumor, and perhaps the most important aspect of how cells respond to this unique microenvironment is the activity of the HIF1α transcription factor.²¹ USP29 was reported to promote tumor cell glycolysis in normoxia or hypoxia by stabilizing HIF1α,^9,10 and thus, we proposed that USP29 may also regulate de novo nucleotide synthesis under hypoxia.

To assess this hypothesis, we depleted endogenous USP29 in SK-N-BE2 cells and then analyzed nucleotide synthesis in cells cultured under hypoxic conditions (1% O₂) using ¹³C₆ glucose as a tracer. As expected, we observed a decreased enrichment of ¹³C₆ glucose-derived M5 purine and pyrimidine intermediates (Figure 3a,b); interestingly, these down-regulations seemed to be more significant when compared to normoxia (Figure 2). These results showed that USP29 may sustain de novo nucleotide biosynthesis under hypoxia.

Figure 3. USP29 deficiency decreases nucleotide biosynthesis under hypoxia

(a) SK-N-BE2 cells were cultured in 1% O₂ condition and labeled with ¹³C₆ glucose for 1 or 24 h. The isotope-labeled purine nucleotides were analyzed. (b) Isotope-labeled pyrimidine nucleotides were analyzed. NS (no significance). Each group contains three replicates, NS (no significance), **p <0 .01.

USP29 regulates nucleotide biosynthesis by promoting the MYC and E2F pathways

To explore the potential mechanisms underlying USP29 regulation of nucleotide biosynthesis, we analyzed the transcriptome data of SK-N-BE2 cells with or without depletion (GSE180797) and revealed 18 metabolic enzyme genes that were deeply involved in nucleotide synthesis⁵ and significantly suppressed (Figure 4a). These genes encode proteins including 7 enzymes (PRPS1, PPAT, GART, PFAS, PAICS, ATIC, and GMPS) that directly participate in the biosynthesis of purine nucleotides from R-5-P (Figure 4b). Nucleotide biosynthesis has feeder pathways, including the serine-glycine pathways and amidotransferase reactions, that provide carbon and nitrogen precursors;⁵ the expression of six enzymes (GLUD1, GOT2, PHGDH, PSAT1, SHMT1, and SHMT2) involved in these pathways was also depleted (Figure 4c). We confirmed the downregulation of these genes by RT‒qPCR in neuroblastoma SK-N-BE2, BE-2C, and SK-N-AS cells (Figures 4d and S1). Oncogenic MYC and E2F are two critical transcription factor families that are often overexpressed in cancers and directly transcribe many enzymes involved in nucleotide biosynthesis.⁵ Our previous study showed that USP29 positively regulates the MYC downstream pathway.⁹ Interestingly, we also revealed that USP29 depletion markedly suppressed the gene signatures of E2F (Figure 4e) by gene set enrichment analysis (GSEA).¹⁸ These results indicated that USP29 may promote nucleotide biosynthesis by regulating the MYC and E2F downstream pathways.

Figure 4. Transcriptomic analysis of USP29-depleted SK-N-BE2 cells.

(a) Heatmap presentation of 18 down-regulated genes that participated in nucleotide synthesis. (b) A simplified schematic diagram regarding the GMP synthesis from R-5-P; the red font enzymes were included in A; PRPS1 (Phosphoribosyl Pyrophosphate Synthetase 1), PPAT (Phosphoribosyl Pyrophosphate Amidotransferase), GART (Phosphoribosylglycinamide Formyltransferase), PFAS (Per- and Polyfluoroalkyl Substances), PAICS (phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccinocarboxamide synthase), ATIC (5-Aminoimidazole-4-Carboxamide Ribonucleotide Formyltransferase/IMP Cyclohydrolase), GMPS (Guanine Monophosphate Synthase). (c) A simplified schematic diagram regarding amino acids metabolism supported nucleotide biosynthesis, the red font enzymes were included in A; GLUD1 (Glutamate Dehydrogenase 1), GOT2 (Glutamic-Oxaloacetic Transaminase 2), PHGDH (Phosphoglycerate Dehydrogenase), PSAT1 (Phosphoserine Aminotransferase 1), SHMT1/2 (Serine Hydroxymethyltransferase 1/2), Glu (glutamic acid), 3PG (3-phosphoglycerate), Asp (aspartic acid). (d) RT‒qPCR analysis of genes expression in SK-N-BE2 cells with or without USP29 depletion (e) Gene set enrichment analysis (GSEA) of E2F target gene sets in the differential expression profile of SK-N-BE2 cells upon USP29 depletion (https://www.gsea-msigdb.org/gsea/index.jsp). **p <0 .01.

Discussion

Deubiquitinases were reported to be involved in many aspects of metabolic processes by regulating metabolic enzymes, transporters, transcription factors, and/or signaling pathways.²² In neuroblastoma, we previously revealed that USP29 promotes glycolysis and glutamine catabolism in normoxia and/or hypoxia.⁹ Considering that glucose and glutamine are two major nutrients of tumor cells, it is conceivable that USP29 may also influence nucleotide synthesis by regulating the feeder pathways; however, the specific role and underlying mechanisms of USP29 in de novo nucleotide biosynthesis remain unclear. In this study, we found that USP29-depleted SK-N-BE2 cells showed significant suppression of glucose-derived nucleotide biosynthesis and gene expression of metabolic enzymes, which were involved in both de novo nucleotide biosynthesis processes and feeder pathways, in both normoxia and hypoxia. Analysis of the USP29-mediated transcriptome suggested that it may function by regulating the MYC and E2F pathways, which play a critical role in regulating de novo nucleotide biosynthesis.⁵ Together with previous studies,^9,10 this study revealed that USP29 acts as a key regulator of tumor cell metabolic reprogramming by affecting several oncogenic pathways.

Dysregulated cancer cell metabolism provides not only proliferative advantages but also new opportunities for cancer therapy, and targeting tumor cell nucleotide biosynthesis has been successfully used in modern chemotherapy.²³ Unfortunately, inhibitors of nucleotide metabolism (also known as antimetabolites), including methotrexate, 5-fluorouracil, 6-mercaptopurine and pemetrexed, which antagonize the activity of enzymes involved in nucleotide biosynthesis, frequently cause severe side effects due to on-target inhibition of the same enzymes in normal cells. Deubiquitinases have been regarded as potential therapeutic targets in cancer metabolism and treatment.^22,24,25 In this study, we revealed the key role of USP29 in promoting nucleotide biosynthesis, given that USP29 may be an alternative target for targeting cancer cell nucleotide metabolism. Indeed, USP29 depletion has been reported to suppress malignant phenotypes in several other cancers, including gastric cancer,^15,16 colorectal cancer^14,26 and hepatocellular carcinoma cells.¹⁰

In summary, we revealed a key role of USP29 in regulating cancer cell nucleotide biosynthesis, and our findings highlight that USP29 may become a potential therapeutic target for neuroblastoma.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Disclosure statement

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

Data availability statement

All data generated or analyzed during this study are included in this article. The datasets used and/or analyzed during the current study are available from the corresponding authors on reasonable requests.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/15384047.2023.2237200

Additional information

Funding

This study is supported in part by the National Natural Science Foundation of China (82203282).

Notes on contributors

Ye Kang

Ye Kang Assistant research fellow, Department of Cancer Precision Medicine, The MED-X Institute, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, China; Email: [email protected]

Yahuan Yu

Yahuan Yu Graduate student, Department of Nephrology, The Affiliated Hospital of Qingdao University, Qingdao, China; Email: [email protected]

Yijia Liu

Yijia Liu Technician, Precision Medicine Center, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, China; Email: [email protected]

Yiwen Pan

Yiwen Pan Technician, Precision Medicine Center, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, China; Email: [email protected]

Ru Zhang

Ru Zhang Technician, Department of Cancer Precision Medicine, The MED-X Institute, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, China; Email: [email protected]

Doudou Ren

Doudou Ren Technician, Department of Cancer Precision Medicine, The MED-X Institute, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, China; Email: [email protected]

Zeqiong Cai

Zeqiong Cai Technician, Department of Cancer Precision Medicine, The MED-X Institute, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, China; Email: [email protected]

Junpeng Ma

Junpeng Ma Technician, Center for Molecular Diagnosis and Precision Medicine, Department of Clinical Laboratory, The First Affiliated Hospital of Nanchang University, Nanchang, China; Email: [email protected]

Xiaofan Xiong

Xiaofan Xiong Assistant research fellow, Department of Tumor and Immunology, Precision Medicine Institute, Building 21, Western China Science and Technology Innovation Harbor, Xi’an, China; Email: [email protected]

Qi Zhang

Qi Zhang Assistant research fellow, Department of Emergency Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; Email: [email protected]

Chengsheng Zhang

Chengsheng Zhang Director, Center for Molecular Diagnosis and Precision Medicine, Department of Clinical Laboratory, The First Affiliated Hospital of Nanchang University, Nanchang, China; Email: [email protected]

Rongfu Tu

Rongfu Tu Assistant research fellow, Department of Cancer Precision Medicine, The MED-X Institute, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, China; Email: [email protected]