Human cytomegalovirus UL82 promotes colorectal cancer cell proliferation through inhibiting the ubiquitination of OGDH via ANGPT2.

1
Introduction
Colorectal cancer (CRC) is the third most common cancer worldwide, accounting for approximately 10 % of all cancer cases, and it is the second leading cause of cancer-related deaths globally [1]. In recent years, an increasing number of studies have shown the presence of human cytomegalovirus (HCMV) in CRC tissues, which is not detected in adjacent normal colon biopsy samples [2]. Our previous research reported that the HCMV UL82 is most abundantly expressed in tumor tissues of HCMV-positive patients and is associated with poor patient prognosis [3]. The UL82 encodes a phosphoprotein with an apparent molecular weight of 71 kDa, pp71, which localizes to the nucleus immediately after infection and initiates viral gene expression [4]. UL82 can achieve its immune evasion function by suppressing endogenous, innate, and adaptive immune response molecules, and it can also control cell cycle progression by inhibiting the Rb family of tumor suppressor proteins and the oncomiR miR 21 [5]. Furthermore, pp71 can degrade the hypophosphorylated members of the retinoblastoma family in a proteasome-dependent manner, thereby stimulating DNA synthesis in quiescent cells and promoting the progression of the cell cycle [6,7]. Therefore, we speculate that UL82 plays a significant role in the progression of CRC.
Oxoglutarate dehydrogenase (OGDH) is primarily localized in the mitochondrial matrix, where it forms the rate-limiting enzyme 2-ketoglutarate dehydrogenase complex (OGDHc) together with dihydrolipoyl transferase (DLST) and dihydrolipoyl dehydrogenase (DLD) in the tricarboxylic acid (TCA) cycle [8]. The TCA cycle is a central pathway for generating cellular energy and precursors for biosynthetic pathways, and recent studies have indicated that metabolic enzyme aberrations affecting the integrity of the TCA cycle are associated with various tumor pathological processes [9]. In gastric cancer (GC), OGDH interacts with SIRT5, and SIRT5's desuccinylation of OGDH inhibits the activity of the OGDH complex, thereby disrupting mitochondrial function and redox status, and subsequently inhibiting the growth and migration of GC cells [10]. Another study found that the mRNA and protein levels of OGDH are significantly elevated in GC tissues and positively correlate with the clinical pathological parameters of GC patients. OGDH promotes the proliferation and migration of GC cells and the tumorigenesis in nude mice by enhancing mitochondrial function and activating the Wnt/β-catenin signaling pathway [11]. However, the impact of OGDH on the proliferation of CRC cells has not yet been reported.
Angiopoietin-2 (ANGPT2) is located on chromosome 8p23 and encodes a long protein consisting of 496 amino acids, belonging to the family of secreted glycoproteins [12]. It is overexpressed in a variety of malignant tumors, such as colorectal cancer [13], gastric cancer [14], liver cancer [15], and breast cancer [16]. ANGPT2 can mediate vascular leakage and increased permeability, allowing matrix metalloproteinases (MMPs) to seep into the endothelial cell gaps, promoting the intravasation of tumor cells, and thereby promoting tumor progression [17]. ANGPT2 is also associated with mitochondrial function. In melanoma, knockdown of ANGPT2 can disrupt cellular redox homeostasis, such as increasing the biosynthesis of reactive oxygen species (ROS) and causing the collapse of cellular antioxidant defense mechanisms, leading to elevated levels of oxidative stress within the cell, resulting in cell necrosis [18].
In this study, we reported that UL82 promotes the proliferation of CRC cells by upregulating ANGPT2 expression and inhibiting the ubiquitination of OGDH. Our findings reveal a novel mechanism by which UL82 enhances CRC cell proliferation, highlighting the significant role of UL82 in CRC progression.

2
Materials and methods
2.1
Data source
GSE32323 (n = 34) and GSE39582 (n = 585) datasets were retrieved from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/gds/) for further analysis.

2.2
Cell culture
HEK293T, SW620, and HCT116 cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) (https://www.cellbank.org.cn/). All cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10 % fetal bovine serum (10099141, Gibco) and 1 % antibiotics (100U/ml penicillin and 100 μg/mL streptomycin, C0222, Beyotime). The incubator was set at 37 °C with 5 % CO2.

2.3
Cell transfection
Cultivate HEK-293T cells to a confluence of 70 %–80 %. Prepare two tubes, A and B. For tube A, mix 10 μL of Lipofectamine 3000 transfection reagent (Invitrogen, USA) with 250 μL of serum-free Opti-MEM medium (11058021,Gibco) and incubate at room temperature for 5 min. For tube B, mix 0.625 μg of pMD2. G, 1.875 μg of psPAX2 (Biovector Science Lab), 2.5 μg of the target plasmid, and 10 μL of P3000 reagent with 250 μL of serum-free Opti-MEM medium. Combine the contents of tubes A and B, and let the mixture stand at room temperature for 20 min. Add the mixture dropwise to the culture dish containing HEK-293T cells, and supplement with Opti-MEM to a total volume of 2 mL. Incubate in a cell culture incubator for 6–8 h, then replace with complete culture medium containing 10 % fetal bovine serum. Collect the viral supernatant at 48 h and 72 h post-transfection.

2.4
Western blot analysis
Cells were collected during the logarithmic growth phase and lysed with RIPA buffer (Beyotime, shanghai, China) containing a mixture of protease inhibitors (1:100, Beyotime, China) to extract total proteins. The samples were then adjusted to the appropriate concentration with loading buffer and PBS, and heated to denature. Equal amounts of protein samples were loaded into the wells of an SDS-PAGE gel for electrophoresis, followed by wet transfer of the proteins onto a PVDF membrane (Millipore, USA) with a pore size of 0.45 μm. The membrane was blocked with 5 % skim milk for 2 h and washed with TBST. Based on the molecular weight markers, the membrane containing specific protein bands was excised. The membrane was then incubated with the corresponding primary antibodies overnight at 4 °C. The primary antibodies are indicated in supplementary Table. After washing away the primary antibody, the membrane was incubated with HRP-conjugated secondary antibodies. Immunoreactive proteins were detected using an ECL reagent (Bio-Rad,USA) according to the manufacturer's protocol.

2.5
Immunoprecipitation (IP) assay
Equal numbers of cells were collected and total proteins were extracted using IP lysis buffer. 2 μL of anti-OGDH antibodies (15212-1-AP, Proteintech) were incubated with the protein samples at 4 °C for 12 h, followed by the addition of A/G PLUS-agarose (Beyotime, China) and further incubation at 4 °C for 1–3 h. After washing with wash buffer, the antibody-agarose-protein complexes were obtained and mixed with SDS-PAGE sample loading buffer (Beyotime, China). The mixture was then heated at 95 °C in a dry incubator for 10 min. The protein solution was further evaluated by Western blot analysis.

2.6
RNA extraction, reverse transcription, and real-time RT-PCR
Total RNA was extracted using the RNAiso Plus kit (Takara, Japan) following the manufacturer's instructions. RNA was reverse transcribed into cDNA using the PrimeScript™ RT Reagent Kit (Takara, Japan). Real-time PCR was performed with the TB Green® Premix Ex Taq™ II FAST qPCR Kit (Takara, Japan) and the following conditions: denaturation at 95 °C for 3 min, and 40 cycles of 95 °C for 10 s and 60 °C for 30s, with a final extension for 30s at 72 °C. The following primers were used for PCR: forward primer for OGDH 5′-TTGGCTGGAAAACCCCAAAAG-3′, reverse primer 5′-TGTGCTTCTACCAGGGACTGT-3′, forward primer for ANGPT2 5′-AACTTTCGGAAGAGCATGGAC-3′, reverse primer 5′-CGAGTCATCGTATTCGAGCGG-3’.

2.7
Cell counting kit-8 (CCK-8) assay
Prepare cell suspensions at a density of 5 × 10^5 cells/mL for SW620 or 4 × 10^5 cells/mL for HCT116. Add 100 μL of the cell suspension to each well of a 96-well plate. At 0, 24, 48, and 72 h, add 10 μL of Cell Counting Kit-8 (CCK-8, Beyotime, China) to each well and incubate at 37 °C for 2 h. Measure the absorbance at 450 nm. The experiment was repeated three times.

2.8
Colony formation assay
Digest cells in logarithmic growth phase with trypsin and seed them into six-well plates at a density of 1000 cells per well for SW620 or 800 cells per well for HCT116. Culture the cells for three weeks. After washing with PBS, fix the cells with 4 % paraformaldehyde for 20 min, followed by staining with crystal violet for 20 min. After washing, photograph the cell colonies and count the number of colonies using ImageJ software. Each experiment was repeated at least three times.

2.9
Ethynyl-2′-deoxyuridine (EdU) incorporation assay
When the cells in a 12-well plate reach approximately 50 % confluence, add EdU (Beyotime, China) at a concentration of 10 μM and incubate at 37 °C for 2 h. After fixation with 4 % paraformaldehyde and permeabilization with 0.3 % Triton X-100, prepare the Click reaction mixture according to the manufacturer's instructions and use it within 15 min. Counterstain the cell nuclei with Hoechst 33342, and after washing, photograph the cells under a fluorescence microscope.

2.10
Xenograft models
The animal experiments were approved by the Institutional Animal Care and Use Committee of Wenzhou Medical University (Ethics Approval Number: wydw2024-0536). Female 4-week-old BALB/c nude mice were purchased from Vital River Laboratories (Beijing, China) or GemPharmatech Co., Ltd and housed under specific pathogen-free (SPF) conditions. Mice were double-blinded and randomly divided into eight groups (five mice per group). Control or experimental group SW620 cells (2 × 10^6 cells) were suspended in 100 μl PBS and injected subcutaneously into the mice. Tumor volume was measured daily with a caliper. Tumor volume was calculated using the following formula: V = (width^2 × length) × 0.5. The nude mice were then euthanized by spinal dislocation, and the xenografted tumors were excised for photography and weighing.

2.11
RNA sequencing
RNA sequencing was performed on SW620 cells transfected with either pCMV-UL82 or pCMV plasmids using the Illumina HiSeq 2500 platform by Novogene (Beijing, China). The differential expression analysis of RNA sequencing data was conducted using the SangerBox platform (http://www.sangerbox.com/tool), with the screening criteria set as |log 2 fold change| > 1 and p-value <0.05 [19]. Enrichment analysis of the RNA sequencing data was performed using GSEA_4.3.3 software, with a p-value <0.05 considered statistically significant [20]. Sequencing of SW620 cells transfected with pLVX-shANGPT2 and pLVX-shNC was carried out by Shanghai Personalbio Biotechnology Co., Ltd. (Shanghai, China), and the sequencing data were subjected to transcriptome bioinformatics analysis using the Personalbio Cloud platform (https://www.genescloud.cn).

2.12
Metabolomics analysis
Metabolites in SW620 cells transfected with pCMV-UL82 or pCMV were identified using LC-MS methods by Personalbio, and the data were also analyzed on the Personalbio Cloud platform.

2.13
Statistical analysis
Survival analyses were performed utilizing the Kaplan-Meier technique, with comparisons made via the log-rank test. For the statistical evaluation of differences in variables between two groups, unpaired Student's t-tests and ANOVA were employed. The experimental data presented in the manuscript were processed and analyzed employing GraphPad Prism version 8.0.2 software. Significance levels ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

3
Results
3.1
UL82 promotes the proliferation of CRC cells
Previous studies have demonstrated that HCMV can modulate cell growth and proliferation through diverse mechanisms. Therefore, we hypothesized that HCMV UL82 may influence cell proliferation [21]. To investigate the impact of UL82 on CRC progression, CRC cell lines (SW620, HCT116) overexpressing UL82 were constructed via a lentiviral expression vector (Fig. 1A). Cell proliferation was assessed using the CCK-8 assay, and the results demonstrated that overexpression of UL82 significantly enhanced the proliferative capacity of CRC cells (Fig. 1B). Colony formation assays revealed that UL82 promoted colony formation (Fig. 1C). Additionally, the increased rate of EdU-positive cells demonstrated that UL82 can enhance DNA synthesis, thereby promoting proliferation (Fig. 1D). To investigate the in vivo growth of CRC cells, SW620 cells transfected with either UL82 or vector were inoculated into the flanks of nude mice. The results indicated that compared with the control group transfected with the vector, the UL82-transfected group exhibited significantly faster tumor growth and larger tumor volumes and weights (Fig. 1E).

3.2
UL82 enhances the expression of key TCA cycle enzyme OGDH in CRC cells
Metabolic regulation is involved in various cellular processes, including cell proliferation [22]. To investigate whether UL82 impacts the energy metabolism of CRC cells, transcriptome sequencing was performed on SW620 cells overexpressing UL82 and their control counterparts. Differential expression analysis identified 285 differentially expressed genes (Fig. 2A), and GSEA enrichment analysis indicated that UL82 is associated with oxidative phosphorylation pathways (Fig. 2B). Oxidative phosphorylation is a crucial component of energy metabolism in the body [23]. Therefore, we conducted metabolomic analysis on SW620 cells overexpressing UL82 and their controls, and the metabolite difference analysis showed that Isocitric acid and L-Rhamnono-1,4-lactone were increased in the UL82 group, with Isocitric acid showing the most significant increase (Fig. 2C). Isocitric acid is a key product in the TCA cycle [24], and its elevation suggests that UL82 may influence the TCA cycle in CRC cells. Western blot was used to detect the expression levels of TCA cycle-related enzymes such as CS and DLD, and the band intensities were analyzed using Image-J software. The results showed that the protein expression levels of DLST and OGDH were higher in the UL82 overexpression group than in the control group (Fig. 2D).
Given that DLST and OGDH are two subunits comprising the 2-ketoglutarate dehydrogenase complex, to investigate which protein enhances the stability of this complex, DLST and OGDH were separately knocked down in SW620 cells overexpressing UL82. The results showed that knocking down DLST had no effect on OGDH, while knocking down OGDH significantly reduced the expression of DLST (Fig. 2E and F). Therefore, we speculate that OGDH is the key protein that stabilizes the 2-ketoglutarate dehydrogenase complex. Additionally, OGDH expression modulates the proliferative capacity of colorectal cancer (CRC) cells. CCK-8 assay demonstrated that overexpression of OGDH significantly promoted cell proliferation in SW620 cells, whereas knockdown of OGDH suppressed cellular proliferative ability (Fig. S1).

3.3
OGDH knockdown inhibits the proliferation of CRC cells overexpressing UL82
To explore the mechanisms underlying UL82-mediated promotion of CRC cell proliferation, we performed knockdown of OGDH in CRC cells overexpressing UL82, and used qRT-PCR and Western blot experiments to detect the knockdown cell lines. The results showed that OGDH was significantly reduced at both the mRNA and protein levels, indicating effective knockdown of OGDH (Fig. 3A and B).
CCK-8 and colony formation assays were utilized to assess the effect of decreased OGDH expression on the proliferative capacity of CRC cells. The results demonstrated that compared to the shNC group, the knockdown of OGDH inhibited the proliferation of CRC cells (Fig. 3C and D). The EdU assay further confirmed that knocking down OGDH could suppress DNA synthesis in cells (Fig. 3E), also indicating that the knockdown of OGDH inhibits cell proliferation. To investigate the impact of OGDH on tumorigenesis in vivo, we established a xenograft tumor model. SW620-UL82 OE cells transfected with sh-OGDH or empty vector were inoculated into nude mice. After 21 days, the xenograft tumors in mice transfected with sh-OGDH were found to be smaller than those in the control mice (Fig. 3F).

3.4
UL82 promotes OGDH expression through upregulation of ANGPT2
To elucidate the mechanisms by which UL82 regulates OGDH, we analyzed the transcriptomic data from SW620 cells overexpressing UL82 and discovered that UL82 significantly upregulated genes such as EDARADD, GZMB, GPR137B, TCF4, and ANGPT2 (Fig. 4A). Among them, GZMB, GPR137B, and ANGPT2 were highly expressed in tumor tissues in the GSE32323 dataset (Fig. 4B). Studies have indicated that GZMB plays a negative regulatory role in CRC progression [25]. Meanwhile, GPR137B is suggested to be involved in M2 macrophage polarization and is not associated with tumor mitochondrial metabolism [26]. Therefore, they are not within the scope of our current investigation. Additionally, we indicated that in CRC patients, the high expression group of ANGPT2 had a poorer survival rate than the low expression group (Fig. 4C). ANGPT2 is also associated with cell proliferation. Overexpression of ANGPT2 in SW620 cells enhances cellular proliferative capacity, while knockdown of ANGPT2 inhibits cell proliferation (Fig. S2). Thus, we focused on ANGPT2. We verified the transcriptome sequencing data using qRT-PCR and Western blot, and the results demonstrated that both the mRNA and protein levels of ANGPT2 were significantly upregulated (Fig. 4D and E).
Given that UL82 upregulates both ANGPT2 and OGDH (Fig. 4F), to investigate the relationship between ANGPT2 and OGDH, OGDH was knocked down in SW620 cells overexpressing UL82, and subsequent Western blot analysis was performed to assess ANGPT2 expression levels. Results demonstrated that OGDH knockdown did not affect ANGPT2 expression (Fig. 4G). Subsequently, we performed knockdown of ANGPT2 in CRC cells that were overexpressing UL82. Western blot experiments confirmed that the protein expression levels of ANGPT2 were significantly suppressed, and the protein expression levels of OGDH decreased (Fig. 4H). The results indicate that UL82 regulates OGDH in an ANGPT2-dependent manner.

3.5
ANGPT2 inhibits the ubiquitination and degradation of OGDH
To elucidate the mechanism by which ANGPT2 regulates OGDH, transcriptome sequencing was performed on SW620 UL82 OE cells with ANGPT2 knockdown, but the results showed that the transcriptional level of OGDH remained unchanged following ANGPT2 knockdown (Fig. 5A). Therefore, we speculated that ANGPT2 might regulate the expression of OGDH protein through post-translational modifications. Data of CHX chase assay showed knockdown of ANGPT2 accelerated the degradation of OGDH, suggesting that ANGPT2 may be involved in the degradation process of OGDH (Fig. 5B). It is reported that proteins can be degraded through the ubiquitin-proteasome pathway. OGDH was immunoprecipitated from cells, and ubiquitin conjugation levels were detected. Results revealed increased ubiquitination of OGDH in SW620 UL82 OE cells with ANGPT2 knockdown (Fig. 5C). Treatment of these cells with the proteasome inhibitor MG132 blocked OGDH protein degradation (Fig. 5D). These results indicate that ANGPT2 prevents the degradation of OGDH through the ubiquitin-proteasome system (UPS).

3.6
UL82 promotes cell proliferation via the ANGPT2/OGDH axis
To investigate the role of ANGPT2 in CRC cells, we performed ANGPT2 knockdown in CRC cells overexpressing UL82. The results demonstrated that compared with the control group, cells with ANGPT2 knockdown exhibited a significant reduction in absorbance values in the CCK-8 assay, indicating that their proliferative capacity was attenuated (Fig. 6A). Consistently, the colony formation assay revealed that the number and size of colonies formed by ANGPT2-knockdown cells were significantly decreased (Fig. 6B). SW620 cells overexpressing UL82 and with ANGPT2 knockdown were implanted into nude mice, with the vector and shNC groups serving as controls to monitor tumor growth. The results indicated that compared with the shNC group, nude mice with ANGPT2 knockdown exhibited a significantly slower tumor growth rate and a marked reduction in tumor volume. Conversely, both the tumor growth rate and volume in the shNC group were larger than those in the vector group. Consistent trends were also observed in tumor weight measurements. These findings suggest that ANGPT2 knockdown reversed the proliferative effects of UL82 on CRC cells (Fig. 6C).

4
Discussion
The metabolic reprogramming of tumor cells, characterized by enhanced glycolysis, oxidative phosphorylation, glutaminolysis, and lipid metabolism, facilitates their proliferative demands [27]. In recent years, there has been a growing interest in the study of the UL82, but few reports on its role in tumor cell metabolism. Our research indicated that UL82 can promote oxidative phosphorylation in CRC cells, which is confirmed by both transcriptomic and metabolomic analyses. Furthermore, UL82 can upregulate the expression of ANGPT2, which can inhibit the degradation of OGDH through deubiquitination. OGDH and ANGPT2 play important roles in the proliferation of CRC cells, and knockdown of either OGDH or ANGPT2 can reverse the proliferative effects of UL82 on CRC cells.
Through transcriptome sequencing analysis and metabolomic analysis, this study suggested that UL82 plays a role in the oxidative phosphorylation pathway of CRC cells and promotes the elevation of the important intermediate metabolite Isocitric acid in the TCA cycle. Additionally, the key enzyme of the TCA cycle, OGDH, is abnormally overexpressed in CRC cells, which in turn affects their proliferative capacity. Previous studies had indicated that TCA cycle metabolites can act as signaling molecules, controlling cellular processes such as DNA methylation, chromatin modification, and hypoxia response [28]. The TCA cycle is also a central hub for cellular energy and biosynthetic precursors, playing a significant role in the development and progression of cancer [24]. The involvement of OGDH in the TCA cycle and its capacity to promote the proliferation of CRC cells lend substantial support to our research findings [29].
In previous studies, OGDH has been shown to be subject to post-translational modifications. OGDH can be desuccinylated by SIRT5, which affects the progression of gastric cancer [8]. Ubiquitination is a key regulator of protein degradation, especially in the ubiquitin-proteasome pathway [30]. In the present study, it was observed that in SW620 cells overexpressing UL82, the ubiquitin abundance associated with OGDH was significantly increased following ANGPT2 knockdown. Additionally, treatment with MG132 effectively blocked the degradation of OGDH. These findings indicate that ANGPT2 knockdown accelerates the degradation of OGDH via the ubiquitin-proteasome pathway.
ANGPT2 can affect mitochondrial function, and in hepatocellular carcinoma cells, exogenous ANGPT2 can antagonize doxorubicin-induced oxidative stress and mitochondrial dysfunction, maintaining mitochondrial integrity [31]. Therefore, we speculate whether ANGPT2 has an effect on proteins within the mitochondria. This study indicates that following the expression of UL82 in CRC cells, the protein levels of both ANGPT2 and OGDH are upregulated. Furthermore, knockdown of ANGPT2 leads to a reduction in OGDH expression levels and suppresses the proliferative capacity of CRC cells.
In addition to Isocitric acid, UL82 can also upregulate other metabolites, such as uracil and L-threonine. Uracil is a specific base of ribonucleic acid (RNA) and corresponds to thymine in deoxyribonucleic acid (DNA), being a raw material for the synthesis of nucleic acids [32], and is closely related to cell division and proliferation. L-threonine accumulates in glioblastoma stem cells, providing fuel for glioblastoma through YRDC-mediated codon-biased translational reprogramming, playing an important role in cell growth and tumor progression [33]. However, we have not focused on the impact of metabolites on the progression of CRC, which can be a direction for future research.
In summary, this study elucidated the molecular mechanisms by which the HCMV tegument protein UL82 promotes the progression of CRC. Integrated analyses of transcriptomics and metabolomics indicate that UL82 significantly enhances tumor growth by upregulating the expression of OGDH. Mechanistic investigations revealed that UL82 inhibits the ubiquitination and degradation of OGDH by upregulating ANGPT2, thereby maintaining OGDH protein stability and promoting CRC cell proliferation. These findings not only provided novel insights into the mechanisms of tumor cell metabolic reprogramming but also laid the foundation for the potential diagnostic and therapeutic applications of UL82 in CRC.

CRediT authorship contribution statement
Lanni Wang: Writing – original draft, Data curation, Conceptualization. Ruini Li: Writing – original draft, Data curation, Conceptualization. Haitao Ren: Writing – original draft, Data curation, Conceptualization. Chaoyi Jiang: Data curation. Ye Shi: Data curation. Bing Wang: Data curation. Wenyu Jin: Data curation. Jiaxue Wang: Data curation. Linhua Lan: Writing – review & editing. Feng Xu: Writing – review & editing. Guangxin Xiang: Writing – review & editing. Xiaoqun Zheng: Writing – review & editing.

Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.