CPT1A mediates succinylation of LDHA at K318 site promoteing metabolic reprogramming in NK/T-cell lymphoma nasal type

Abstract

Carnitine palmitoyltransferase 1A (CPT1A), a succinylating enzyme, is highly expressed in various malignant tumors and promotes tumor progression. Succinylation is a posttranslational modification that has been reported in various diseases, but its role in NK/T-Cell lymphoma nasal type (ENKTL-NT) remains underexplored. In this study, bioinformatics analysis showed that glycolytic is a major metabolic pathway in ENKTL-NT as the expression of many glycolytic related kinases are increased. CPT1A probably mediates glycolytic process, as indicated by GO-enrichment analysis. Studies showed that CPT1A was upregulated in ENKTL-NT tissues, and that high CPT1A expression was associated with poor prognosis of ENKTL-NT. CPT1A promoted the proliferation, colony formation, invasion and glycolytic process of ENKTL-NT cells and suppresses apoptosis. Mechanistically, CPT1A promotes succinylation of LDHA at lysine 318 (K318), which increase the protein stability and the final protein level of LDHA. Both knockdown and mutation (K318R) of LDHA abolished the cancer-promoting effects of CPT1A in ENKTL-NT. In all, this study reveals the mechanism underlying the cancer-promoting effects of CPT1A via inducing LDHA succinylation and metabolic reprogramming in ENKTL-NT. These findings might provide potential targets for the diagnosis or therapy of ENKTL-NT.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10565-025-09994-6.

Introduction

NK/T-Cell lymphoma is a rare and aggressive non-Hodgkin's lymphoma. Most of the malignant cells originate from mature nature killer (NK) cells, with a small portion coming from NK-like T cells. The NK/T-Cell lymphoma originating from the nasal is termed as NK/T-Cell lymphoma (NKTL) nasal type (ENKTL-NT) (Chen et al. 2022; Harabuchi et al. 2019; Kandel et al. 2023). Oncogenic virus infection is an important factor promoting the occurrence and development of ENKTL-NT, such as Epstein-Barr virus (EBV) infection (Feng et al. 2023). Mutations in EBV and antigen 4 can make cytotoxic T cells unable to recognize and clear EBV, which may be an important factor in inducing ENKTL-NT.Ethnic factors are also the main cause, which is relatively rare in Europe and the United States, but more common in Asia (Asano et al. 2013; Santos et al. 2021; Doesum et al. 2021). ENKTL-NT is mainly manifested as progressive and destructive lesions in the nasal or facial midline area (Tse et al. 2022). Early symptoms include local swelling, nasal congestion, epistaxis, as well as local erosion, necrosis and ulceration. In the late stage, perforation of the upper palate and nasal septum may occur, accompanied by a foul odor, granulation like new organisms, and bleeding (Chang and Kim 2022; Obara and Amoh 2020; Geller et al. 2018). ENKTL-NT is a highly invasive malignant lymphoma that progresses rapidly and is insensitive to chemotherapy. To date, treatments for ENKTL-NT are still completely reliant on radiotherapy, chemotherapy or a combination of both, but these have been met with limited success with low complete response rates (Tian et al. 2023; Tse et al. 2019; Xiong et al. 2021). There is thus a compelling need to develop alternative strategies for ENKTL-NT.

CPT1A has been associated with tumor progression in various cancers, including breast, gastric, lung, and prostate cancer (Xiong et al. 2018; Wang et al. 2022; Ma and Chen 2024; Joshi et al. 2020). The inhibition or depletion of CPT1A can lead to apoptosis, thereby curbing cancer cell proliferation and chemoresistance (Hu et al. 2023). Recently, CPT1A has been confirmed to have lysine succinyltransferase activities (Kurmi et al. 2018). Succinylation is a newly discovered and multi-enzyme-regulated post-translational modification (PTM) that is associated with the initiation and progression of cancer (Lu et al. 2021). For example, succinylation of S100A10 by CPT1A promotes the invasion of human gastric cancer cells (Wang et al. 2019). CPT1A can interact with the autophagy-related protein ATG16L1 and stimulate the succinylation of ATG16L1, which in turn drives autophagosome formation and autophagy, thus curbing cancer cell proliferation and chemoresistance (Sun et al. 2023).

Changes in energy metabolism are one of the characteristics of tumor cells. Malignant cells are inclined to obtain energy for rapid growth and reproduction through glycolysis, lactate as a byproduct of glycolysis, plays a regulatory role in different aspects of energy metabolism and signal transduction, and then regulates tumor metabolism (Li et al. 2022). Lactate dehydrogenase A (LDHA) is a key enzyme in aerobic glycolysis, and is mainly expressed in skeletal muscle, where it preferentially converts pyruvate to lactate and NADH to NAD⁺ (Ždralević et al. 2018). High expression of LDHA is often associated with poor prognosis and high metastasis rate (Dorneburg et al. 2018). The activating transcription factor 3 (ATF3) protein is specifically bound to LDHA to effectively suppress LDHA-mediated metabolic reprogramming, thus affecting tumor cells proliferation and apoptosis (Chen et al. 2024a). LDHA is post-translationally palmitoylated by ZDHHC9 at cysteine 163, which promotes its enzyme activity, lactate production, and reduces reactive oxygen species (ROS) generation, and then alters pancreatic cancer response to chemotherapy (Chen et al. 2024b). Therefore, LDHA is considered a promising new target in the prevention and treatment of tumors (Han et al. 2023). In this study, we aimed to explore the role of LDHA and succinylation in ENKTL-NT and identify succinylated targets that could be associated with cancer progression and prognosis. To this aim, we performed quantitative lysine succinylome analysis in human ENKTL-NT tissues and adjacent normal tissues using bioinformatics, gene ontology (GO) functional enrichment and gene set enrichment analysis (GSEA). Moreover, we investigated the mechanism underlying high expression of LDHA in ENKTL-NT.

Materials & methods.

Reagents

RPMI medium (Cat. No. R6504-10 L) was purchased from Sigma, DMEM (Cat. No. D5648-10 L) was purchased from Sigma, and FBS (Cat. No.) was obtained from Sigma. ShRNAs specific to CPT1A was obtained from the Cenepharma company (shanghai, China), LDHA-specific polyclonal antibody (Cat. No. 19987–1-AP), CPT1A monoclonal antibody (Cat. No. 66039–1-Ig) and anti-succinyllysine rabbit pAb (Cat. No.PTM-401) were purchased from Proteintech Group, Inc (shanghai, China). CPT1 inhibitor Teglicar (Cat. No. ST1326) and glycolytic inhibitor 2-deoxyglucose (2-DG) (Cat. No. HY-13966) were obtained from MCE company (shanghai, China). Protein synthesis inhibitor cycloheximide (CHX) was obtained Selleck Chemicals, Houston (TX, USA). Cell Counting Kit-8 (Cat. No. C0038) was obtained from Beyotime. Lactate assay kit (Cat. No. K607-100) and pyruvate assay kit (Cat. No. K679-100) were obtained from Biovision.

Bioinformatic analysis

GEO database with the GSE90597 dataset, a GPL10739 detection platform, was used for Bioinformatic analysis. GSE90597 dataset consists of 66 cases of NK/T cell lymphoma, including 19 intranodal lymphoma and 47 cases of extranodal lymphoma (including 20 nasal type NK/T lymphoma, 15 non-nasopharyngeal type, and 12 unknown type). As extranodal NK/T cell lymphoma mostly occurs in the nasal cavity, also known as nasal type NK/T cell lymphoma (ENKTL-NT), we prioritized ENKTL-NT as the research object. In GSE90597 dataset, 20 cases of ENKTL-NT and 19 cases of intranodal NK/T lymphoma were selected for comparative analysis. The analysis used the RMA function of the R language oligo software package for data normalization, and then use the R language limma software package to analyze the differentially expressed genes (DEG) between groups. The screening threshold for DEG was p value < 0.05 and log₂(fold changes) > 1.5.

Tissue specimens

This study collected 10 ENKTL-NT tissue samples from the Hunan Cancer Hospital, The Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University (Changsha, China). In addition, this study collected 10 lower turbinate mucosa tissue samples from patients with chronic rhinitis. The lower turbinate tissues ware regarded as the normal tissues compared to ENKTL-NT tissues. All patients were informed of the investigational nature of the study. Written informed consent was obtained from them before the experiment. This study was reviewed and approved by the Ethics Committee of Hunan Cancer Hospital. The tissues were washed with physiological saline to remove blood stains and then placed in formalin solution for pathological testing, or in liquid nitrogen for subsequent molecular biology testing.

Cell lines and culture

The cell lines NK-92, NK-92MI and Hut-102 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). SNK-6 cells were obtained from Cmbio company (#CM-1818S, Shanghai, China). YTS cells were purchased from the CHI Scientific, Inc. (Beijing, China). The cell lines NK and HEK293T cells were obtained from the Shanghai Institute of Life Sciences (Shanghai, China). SNK-6, YTS, NK-92, NK-92MI, Hut-102 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), and NK, HEK293T cells were grown in DMEM (high glucose) supplemented with heating-activated 10% FBS.

Site-directed mutagenesis and cell transfection

Succinylation mutants of LDHA were generated by the Cenepharma company (shanghai, China) to convert each lysine (K318, K232) to glutamic acid (K318R, K232R). The mutant vectors were conducted at Sangon Biotech. The mutation was confirmed by DNA sequencing. Lipofectamine 3000 (Invitrogen) was used for cell transfection followed by the manual.

Realtime-PCR

The tissue or cells of each experimental group were lysed and total RNA was isolated using the RNeasy mini kit (Qiagen). The extracted RNA was then converted to cDNA with RevertAidTM M-MulV Reverse Transcriptase (Bio-Rad). The mRNA levels of PGD, PFKP, HK1, HK2, LDHA, LDHB, PKM2, GP1 and CPT1A were then quantitated using SYBR Green PCR Master mix (Bio-Rad) according to manufacturer’s recommendations.

Western blotting assay

Total protein was prepared from gastric tissues or cultured cell samples using RIPA lysis buffer containing protease inhibitor cocktail and centrifuged at 10000 g at 4 °C for 15 min. Supernatants were mixed with SDS‐PAGE sample‐loading buffer, boiled for 5 min, and then subjected to SDS‐PAGE. After being transferred onto polyvinylidene fluoride membranes, non‐specific binding was blocked with 5% nonfat milk. The blots were probed with the following primary antibodies: LDHA-specific polyclonal antibody, CPT1A monoclonal antibody, anti-succinyllysine rabbit pAb or β‐actin antibody.

Co-immunoprecipitation assay

The cells of each experimental group were lysed in Co-IP buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, and 1 mM EDTA) containing protease inhibitors (Roche Applied Science, Mannheim, Germany) on ice for 30 min. Cells were centrifuged and supernatant was collected, and then IP was pulled down by LDHA-specific polyclonal antibody, IB was detected by anti-succinyllysine rabbit pAb or CPT1A monoclonal antibody. IgG as control, and input by CPT1A antibody, LDHA antibody and β-actin detection, and then analyzed by western blotting.

Cell proliferation assay

Cells (1 × 10⁵ per well) were cultured in a 96-well plate in each experimental group, cell proliferation was determined using Cell Counting Kit-8 (CCK-8). Adding with 10 μl CCK-8 solution in the plate, and incubating for 1 h in a humidified incubator, then measuring absorbance at 450nm after the appropriate color change was observed.

Colony formation assay

500 SNK-6 and YTS cells of each experimental group were seeded separately into each well of a 6-well plate in RPMI-1640 medium supplemented with heatinactivated 10% FBS. Appropriate concentration of 2-DG or ST1326 was also added into each well in some experiments. The plates were placed in humidified incubators at 37 °C with 5% CO₂ for 2–3 weeks, after which colony formation was scored with an inverted microscope.

Flow cytometry

Cells from each experimental group were collected and stained with 5 μl of fluorochrome conjugated Annexin V-PE and 7-AAD with AnnexinV-PE + 7-AAD apoptosis kit for 30 min at 4 ℃. Data acquisition used a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA) and analysis was carried out using FlowJo (v 0.10.5.3, TreeStar, Woodburn, OR, USA).

Lactate assay

The 46 µl experimental sample, 2 µl lactate probe and 2 µl lactate enzyme mix were evenly mixed and added to each well of a 96-well plate. Meanwhile, a standard curve was established using L ( +) -lactate standard. The reactants were incubated at room temperature and away from light for 30 min, then measuring absorbance at 570nm to calculate lactate level.

Pyruvate assay

The cells in each experimental group were lysed and centrifuged to collect supernatant for standby. The supernatant sample, pyruvate probe and pyruvate enzyme mix were evenly mixed and added to each well of a 96-well plate. Meanwhile, a standard curve was established using pyruvate standard. The reactants were incubated at room temperature and away from light for 30 min, then measuring absorbance at 570nm to calculate pyruvate level.

Mitochondrial oxygen consumption rate assay

The cells in each experimental group were collected at different time points, oxygen consumption rate (OCR) using Seahorse assay.

LDH activity assay

The experimental samples were added 0.2 ml of pre-cooled extracting solution, which were homogenized for 10 min on ice by homogenizer, and then centrifuged 10000g at 4 ℃ for 15 min, and collected supernatant for standby. After supernatant was treated with LDH activity assay kit, the absorbance was determined at 450nm to calculate LDH activity level.

LDH protein stability assay

After transfection of CPT1A (KD), HA-LDHA (WT), HA-LDHA (K232R) or HA-LDHA (K318R) plasmid into SNK-6 cells for 0、2、4、6、10h, adding protein synthesis inhibitor cycloheximide (CHX, 10μg/ml), endogenous and exogenous LDHA levels through WB detection.

4D Label-free succinylation modification quantitative proteome

This study detected succinylation in SNK-6 cells using the 4D Label-Free Succinylation Modification Quantitative Proteome from Hangzhou Jingjie Biotechnology Co.,Ltd.. SNK-6 cells from CPT1A SiRNA knockdown (n = 3) and SiRNA-normal control (NC) (n = 3) groups were collected. Under sonication, cells were lysed and transferred to succinylated resin (PTM Bio, China, cat: PTM-402) for overnight incubation at 4 °C with. The peptides was analyzed by liquid chromatography-mass spectrometry (LC–MS) following evacuation and desalting in accordance with the C18 ZipTips instructions.

Nude mice tumor model

We utilized 6 to 8 week old nude mice, which were housed in a temperature‐controlled environment with a 12 h light‐dark cycle and were allowed free access to water and food. All animal procedures were approved by the Ethics Committee of Hunan Cancer Hospital. Approximately 1 × 10⁶ treated SNK-6 cells injected into nude mice to construct nude mice tumor model. The mice were euthanized and umor tissues were removed after 4 weeks. Tumor volume was calculated as width × length × (width + length)/2. CPT1A and LDHA levels were examined by WB, cell proliferation and apoptosis were measured by IHC and DAPI + TUNEL fluorescent staining.

Statistical analysis

All experiments were repeated at least three times. All values in the figures and text are expressed as mean ± SEM. Two‐tailed Student's t‐tests were used to compare two groups. Comparisons between three or more groups were analyzed using one-way ANOVA. All statistical analyses were performed with GraphPad Prism (v6.0, GraphPad Software, San Diego, CA, USA). Differences were considered significant at P < 0.05 and expressed by “*”.

Results

Glycolysis is an important metabolic pathway in ENKTL-NT progression

Using GSE90597 dataset, bioinformatics analysis was conducted to screen out DEGs between 20 cases of ENKTL-NT and 19 cases of nodal NKTL in the database. Here, using p-value < 0.05 and log₂FC > 1.5 as screening standard for DEGs, we identified 4799 DEGs, of which 1468 genes were upregulated and 3331 genes were downregulated in ENKTL-NT (Fig. 1A shows the top 1000 DEGs in a heatmap). We continued to perform gene ontology (GO) functional enrichment analysis using the DEGs (Fig. 1B and Table 1). The results showed significant gene enrichment in metabolic pathways such as glycolytic process, glycogen metabolic process, pyruvate metabolic process, glycoprotein catabolic process, fatty acid oxidation and metabolic process, as well as NK cell mediated cytotoxicity regulation and tumor specific NK/T cell differentiation. Among the glycolytic process, glycolytic related hexokinase (HK), 6-phosphofructokinase (PFK), pyruvate kinase (PK), lactate dehydrogenase (LDH), etc. were all upregulated in ENKTL-NT (Fig. 1C). Therefore, we speculated that glycolysis may be a typical metabolic process in ENKTL-NT.

Fig. 1.

Glycolysis process mediates ENKTL-NT. (A) Heat map of DEGs between ENKTL and nodal NKTL (Red indicates high gene expression, blue indicates low gene expression). (B) GO enrichment analysis results of DEGs between ENKTL and nodal NKTL. (C) MRNA expression levels of glycolytic-related enzymes

Table 1.

GO enrichment analysis results of DEGs between ENKTL and nodal NKTL

ID	Description	GeneRatio	p-value	Adjust p-value
GO:0006096	glycolytic process	0.2639	0.00084	0.00089
GO:0006110	regulation of glycolytic process	0.2277	0.004203	0.004211
GO:0042269	regulation of natural killer cell mediated cytotoxicity	0.2128	0.000188	0.000194
GO:0001865	NK T cell differentiation	0.17	0.00147	0.00155
GO:0006090	pyruvate metabolic process	0.0833	0.000374	0.00039
GO:0051258	protein polymerization	0.0854	0.002095	0.002099
GO:2,000,377	regulation of reactive oxygen species metabolic process	0.0805	0.000117	0.000127
GO:1,903,299	regulation of hexokinase activity	0.1818	0.011872	0.011883
GO:0006516	glycoprotein catabolic process	0.1983	0.028667	0.028689
GO:0005977	glycogen metabolic process	0.2333	0.001157	0.001168
GO:2,001,237	negative regulation of extrinsic apoptotic signaling pathway	0.101	0.004442	0.004463
GO:0006631	fatty acid metabolic process	0.0725	0.000756	0.000769
GO:0019395	fatty acid oxidation	0.1273	0.002709	0.002717

CPT1A mediates glycolytic process in ENKTL-NT

Succinylation of proteins has been proven to promote tumor cell metastasis and proliferation in various cancers. This study conducted bioinformatics analysis using GSE90597 data to investigate the mechanism of lysine succinylation in ENKTL-NT and identify succinylation target molecules that may be related to cancer progression and prognosis It showed that succinylating enzymes CPT1A was upregulated in ENKTL-NT (Fig. 2A). CPT1A mainly mediates non-histone succinylation modification compared to EP300 that mediates succinylation modification of histone H3. According to CPT1A gene expression level, samples were divided into CPT1A-up and CPT1A-down groups, and used to find the biological processes related to the DEGs via GO-enrichment analysis (Fig. 2B and Table 2). The results showed that CPT1A-associated genes were significantly enrichment in glycolytic process, carbohydrate catabolic process, cell migration and proliferation, intercellular adhesion, etc., indicating that CPT1A may affect glycolytic or pyruvate metabolic process of ENKTL-NT. Subsequently, we performed gene set enrichment analysis (GSEA) of CPT1A-associated DEGs (Fig. 2C), and the results showed that glycolytic and pyruvate metabolic process pathways were related to CPT1A upregulation.

Fig. 2.

CPT1A mediates glycolytic process in ENKTL-NT. (A) Expression boxplot and overall survival probability of CPT1A in ENKTL and nodal NKTL. (B) GO enrichment analysis results of CPT1A-associated DEGs between ENKTL and nodal NKTL. (C) GSEA of CPT1A-associated DEGs

Table 2.

GO enrichment analysis results of CPT1A-associated DEGs between ENKTL and nodal NKTL

ID	Description	GeneRatio	pvalue
GO:0006110	regulation of glycolytic process	0.1277	0.002373
GO:0006096	glycolytic process	0.2639	0.000461
GO:0016052	carbohydrate catabolic process	0.1795	0.000041
GO:0070206	protein trimerization	0.3400	0.002740
GO:0030335	positive regulation of cell migration	0.2200	0.032890
GO:0006541	glutamine metabolic process	0.1875	0.004727
GO:2001237	negative regulation of extrinsic apoptotic signaling pathway	0.1525	0.018124
GO:0002370	natural killer cell cytokine production	0.1340	0.015118
GO:0043506	regulation of JUN kinase activity	0.2100	0.064210
GO:0051258	protein polymerization	0.1429	0.001478
GO:0007043	cell–cell junction assembly	0.0910	0.001188
GO:0043406	positive regulation of MAP kinase activity	0.1340	0.000407
GO:0008284	positive regulation of cell population proliferation	0.1890	0.033487

CPT1A promotes cell proliferation and glycolytic process in ENKTL-NT

We collected ENKTL-NT and normal nasal mucosa tissues (N = 10). CPT1A was highly expressed in ENKTL-NT tissue (p < 0.05; Fig. 3A,B). In addition, CPT1A had high expression in ENKTL-NT cell lines SNK-6 and YTS than in NK cells (p < 0.001; Fig. 3C). To investigate the mechanism of CPT1A in ENKTL-NT, we respectively transfected CPT1A shRNAs or CPT1A expression vector into SNK-6 and YTS cells. CPT1A expression significantly decreased in after the transfection with shRNA1 and shRNA2 targeting to CPT1A (p < 0.001; Fig. 3D and Supplementary Fig. 1A), while the CPT1A expression was increased after the transfection with the expression vector (p < 0.001). CPT1A knockdown inhibited cell proliferation and cloning formation rate, but CPT1A overexpression increased cell proliferation and cloning formation rate (p < 0.05, p < 0.01 or p < 0.001; Supplementary Fig. 1B, C). The results indicates that CPT1A regulate cell proliferation and invasion of ENKTL-NT. Furthermore, we found that the content of glycolytic products pyruvate and lactate were lower in CPT1A knockdown group than in NC group (p < 0.001; Supplementary Fig. 1D,E), while mitochondrial oxygen consumption rate (OCR) was higher in CPT1A knockdown group than in NC group (p < 0.05 or p < 0.01; Supplementary Fig. 1F). The content of glycolytic products pyruvate and lactate were higher in CPT1A overexpression group than in NC group (p < 0.001; Supplementary Fig. 1D, E), while mitochondrial oxygen consumption rate (OCR) was lower in CPT1A overexpression group than in NC group (p < 0.05, Supplementary Fig. 1F). The results suggests that CPT1A also regulates glycolytic process.

Fig. 3.

CPT1A facilitates cell proliferation, colony formation and glycolytic process in ENKTL-NT. CPT1A expression in ENKTL-NT and normal nasal mucosa tissue by WB (A) and IHC (B) detection. (C) CPT1A expression in ENKTL-NT cell lines SNK-6 and YTS, NKTL non-nasal type cell lines NK-92 and NK-92MI, and T lymphoma cell line Hut-102 through WB detection. NK cells as NC group. Using CPT1A-SnRNA1(KD) and CPT1A-SnRNA2(KD) to knockdown (KD) CPT1A expression or CPT1A(OE) to overexpress (OE) CPT1A, and respectively transfected them into SNK-6 and YTS cells, then CPT1A expression by WB (D) detection.P-value < 0.05 was defined as specific statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001)

CPT1A induces LDHA succinylation via directly interacting with LDHA

Using GSE90597 dataset, bioinformatics analysis showed that LDHA is highly expressed in ENKTL-NT compared to nodal NKTL. High expression of LDHA is associated with poor survival rate in both ENKTL-NT and nodal NKTL (Fig. 4A). LDHA is a key protein in glycolysis process. Given CPT1A also regulates the glycolysis process, we speculated that there are some relationship between CPT1A and LDHA in ENKTL-NT. 4D Label-Free Succinylation Modification Quantitative Proteome showed that CPT1A knockdown led to the change in 212 lysine sites, including 173 downregulated sites (corresponding 71 proteins), 39 upregulated sites (corresponding 28 proteins) (Result was not shown). Two LDHA succinylation sites (K318, K232) were downregulated in CPT1A (KD) group (Fig. 4B and Table 3). As indicated by western blot and Co-IP studies, CPT1A knockdown reduced the overall succinylation in SNK-6 and YTS cells as well as succinylation in LDHA protein (p < 0.001; Fig. 4C, E). In addition, CPT1A knockdown was associated with a reduced LDHA protein level (Fig. 4E). To further confirm that CPT1A may mediate LDHA-succinylation, FLAG-CPT1A and HA-LDHA vectors were co-transfected into cells with endogenous CPT1A knockdown. Compared to transfection with HA-LDHA vectors alone, the co-transfection with FLAG-CPT1A and HA-LDHA vectors increased the succinylation of the exogenous LDHA (Fig. 4D). The direct interaction between CPT1A and LDHA was also confirmed (Fig. 4D). Transfection with HA-LDHA and CPT1A shRNA decreased the succinylation of the exogenous LDHA (Fig. 4F). The results indicated that CPT1A interacts with LDHA, and then induce LDHA succinylation.

Fig. 4.

CPT1A mediates LDHA-succinylation and interacts with LDHA. (A) LDHA expression in ENKTL-NT through GSE90597 database analysis. (B) Protein secondary mass spectrometry for K232 and K318. The the overall (C),exogenous (D), endogenous (E) and CPT1A (KD) (F) LDHA-succinylation expression in cells by WB and Co-IP detection. P-value < 0.05 was defined as specific statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001)

Table 3.

Lysine sites in CPT1A (KD) group regulation type of LDHA

Protein accession	Position	Amino acid	B/A Ratio	Regulated Type	Gene name	Score	Charge	Modified sequence
P00338	318	K	0.48	down	LDHA	134.20	2.00	K(1)SADTLWGIQK
P00338	232	K	0.54	down	LDHA	132.93	2.00	EVHK(1)QVVESAYEVIK
P00338	81	K	0.92	None	LDHA	110.91	2.00	IVSGK(1)DYNVTANSK

LDHA promotes ENKTL-NT proliferation through inducing glycolytic process

After transfecting LDHA shRNA1 and LDHA expression plasmids into SNK-6 cell, LDHA protein level was reduced and increased, respectively (Fig. 5A). LDHA knockdown reduced the cell proliferation, colonly formation rate, and the levels of glycolytic products pyruvate and lactate, but increased apoptosis and OCR (p < 0.05, p < 0.01 or p < 0.001, Fig. 5B-G). However, LDHA overexpression increased the cell proliferation, colonly formation rate, and the levels of glycolytic products pyruvate and lactate, but reduced apoptosis and OCR (p < 0.05, p < 0.01 or p < 0.001; Fig. 5B-G). 2-deoxyglucose (2-DG), a glycolytic inhibitor, was added to determine that the regulatory effect of LDHA on the cell phenotypes is related to the glycolytic process. Treatment with 2-DG abolished the effect of LDHA overexpression in the cell proliferation, colonly formation rate, the levels of glycolytic products pyruvate and lactate, apoptosis and OCR (Supplementary Fig. 2A-F). The results suggests that LDHA promotes ENKTL-NT proliferation through inducing glycolytic process.

Fig. 5.

CPT1A promots glycolytic process by mediating LDHA-succinylation. (A) LDHA expression levels in SNK-6 cell of transfecting LDHA (KD) or LDHA (OE) by WB detection. Cell proliferation and invasion ability in SNK-6 by CCK-8 (B), soft agar colony formation assay (C) and flow cytometry (D) detection. The levels of pyruvate (E), lactate (F) and OCR (G) for glycolysis process. P-value < 0.05 was defined as specific statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001)

Suppression of CPT1A reduces LDHA expression, ENKTL-NT proliferation and glycolytic process

To further study the relationship between CPT1A, LDHA-succinylation and glycolysis, we treated SNK-6 and YTS cells with CPT1 inhibitor Teglicar (ST1326) (1μM, 5μM, 10μM) for 48h, the results showed that ST1326 dose-dependently reduced LDHA succinylation and the total LDHA protein levels(Supplementary Fig. 3A). In addition, ST1326 suppressed the cell proliferation, colony formation and levels of pyruvate and lactate, but increased apoptosis and OCR (Supplementary p < 0.05, p < 0.01 or p < 0.001; Fig. 3B-H). In summary, CPT1A facilitates ENKTL-NT proliferation and glycolytic process by inducing LDHA succinylation.

CPT1A induces LDHA succinylation at the site K318 to improve the protein stability

Previous studies have found that, LDHA-succinylation level in K318 and K232 are downregulated after CPT1A knockdown. Therefore, we speculated that the regulatory sites of CPT1A on LDHA-succinylation may be K318/ K232. We constructed HA-LDHA (K232R) and HA-LDHA (K318R) plasmids (K → R represents de-succinylation mutant), and respectively co-transfected them with FLAG-CPT1A into cells. We found that FLAG-CPT1A increased LDHA-succinylation level of HA-LDHA (WT) and HA-LDHA (K232R), but had minor effect on LDHA (K318R) (Fig. 6A), indicating that CPT1A induces LDHA-succinylation in K318, but not in K232. Adding 10μg/ml CHX led to the gradual reduction of LDHA. Transfecting CPT1A shRNA into cells accelerated the reduction of LDHA (Fig. 6B).HA-LDHA (WT) was gradually reduced after the treatment with CHX. HA-LDHA (K318R), but not HA-LDHA (K232R), showed accelerated the reduction compared to HA-LDHA (WT) (Fig. 6C), which suggested that LDHA-succinylation at K318 increases the protein stability. LDHA knockdown suppressed cell proliferation and colony formation, but it was reversed by transfecting with HA-LDHA (WT) and HA-LDHA (K232R). Transfecting with HA-LDHA (K318R) failed to impact the effect of LDHA knockdown (Fig. 6D-E). Transfecting with HA-LDHA (WT), but not HA-LDHA (K318R), reversed the OCR, lactate acid and pyruvate levels after LDHA knockdownsuggesting that K318 may be the action site of LDHA-succinylation regulating glycolysis (Fig. 6F-H). Knockdown of CPT1A down-regulated LDHA protein level, suppressed cell proliferation and colony formation and reduced LDHA activity, lactate acid and pyruvate levels, but increased OCR. Overexpression of LDHA reversed all the effects by CPT1A knockdown (Fig. 7A-F). These results suggested that LDHA mediates most of the effect of CPT1A in the regulation of cell proliferation and glycolysis.

Fig. 6.

K318 is the site of LDHA-succinylation and glycolysis mediated by CPT1A. Interaction between LDHA and CPT1A, LDHA-succinylation level of HA-LDHA (K318R) and FLAG-CPT1A co-transfected into SNK-6 or 293T cells by Co-IP A detection. CPT1A (KD) transfected into SNK-6 cells were treated with CHX, LDHA-succinylation level of through WB B detection, HA-LDHA (WT), HA-LDHA (K232R) or HA-LDHA (K318R) transfected into SNK-6 cells were treated with CHX through WB C detection, and the detection time was 0, 2, 4, 6, 10 h after adding CHX. LDHA(KD) was co-transfected into SNK-6 cells with LDHA(WT), LDHA(K232R) or LDHA(K318R), cell proliferation and invasion ability in SNK-6 by CCK-8 D and soft agar colony formation assay E detection. The levels of OCR F, pyruvate G and lactate H for glycolysis process. P-value < 0.05 was defined as specific statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001)

Fig.7.

LDHA and CPT1A expression by WB (A) detection, cell proliferation and invasion ability in SNK-6/YTS cells by CCK-8 (B) and soft agar colony formation assay (C) detection, the levels of pyruvate (D), lactate (E), LDHA activity (F) and OCR (G) by biochemical method detection in LDHA (OE) or CPT1A (KD) + LDHA (OE). P-value < 0.05 was defined as specific statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001)

LDHA and CPT1A mediate ENKTL-NT growth process in vivo

SNK-6 cells were subjected to CPT1A(OE) and CPT1A(OE) + LDHA(KD). The treated cells were subcutaneously injected into the hind limb of nude mice (N = 4), and tumor volume was observed after 7 days of feeding. Tumor growth rate in CPT1A(OE) group was significantly higher than that in NC group, while the tumor growth rate in CPT1A(OE) + LDHA(KD) group was lower (Fig. 8A,B). The expression levels of CPT1A and LDHA in CPT1A(OE) group were higher than those in NC group, while LDHA expression in CPT1A(OE) + LDHA(KD) group was lower (Fig. 8C). In addition, CPT1A (OE) group promoted cell proliferation and inhibited apoptosis, while CPT1A(OE) + LDHA(KD) group showed the opposite effects (Fig. 8D, E). CPT1A (OE) group showed increased lactate acid concentrations in the tumor tissues and peripheral blood, but the increased concentration was reversed after LDHA knockdown. These results suggested that CPT1A induces ENKTL-NT growth proformance through LDHA.

Fig. 8.

ENKTL-NT tumor growth in vivo. (A) After treatment, the tumor volume of nude mice in each experimental group was measured every 3 d after feeding for 7 d, and drawing tumor growth curve. (B) After 25 d, nude mice were euthanized and tumor tissues were collected. (C) The expression levels of CPT1A and LDHA in nude mice were detected by WB. (D) The level of cell proliferation-associated antigen KI67 by IHC and index of distribution (IOD) detection. (E) The apoptosis of tumor cells was detected by DAPI + TUNEL fluorescent staining (F) The lactate acid concentrations in the tumor tissues and peripheral blood. P-value < 0.05 was defined as specific statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001)

Discussion

The low success rates that current treatments on ENKTL-NT are showing has urged us to discover better treatment alternatives (Moon et al. 2016). Several key findings in recent years have helped to shed light on the pathogenesis of ENKTL-NT. Recently, many novel PTMs have been identified, which can mediate tumourigenesis and tumour progression as initiator, including acetylation, methylation, ubiquitylation and succinylation, etc. (He et al. 2023; Fernandez and Patnaik 2022; Yang et al. 2019). Metabolic reprogramming is a common hallmark of cancers, and study has found that succinylation can mediate cancer cell metabolism by altering the structure or activity of metabolism-related proteins and plays vital roles in metabolic reprogramming (Liu et al. 2023). Our results definitely shown that significant gene enrichment in metabolic pathways of ENKTL-NT tissue, such as glycolytic process, glycogen metabolic process, pyruvate metabolic process, glycoprotein catabolic process, fatty acid oxidation and metabolic process.

In particular, CPT1A is found that has lysine succinyltransferase activity (Kurmi et al. 2018). Succinylation of lysine residues changes the charge status from + 1 to −1 under physiological conditions, and adds a four‐carbon acyl group to lysine whereby succinyl groups are transferred from succinyl-CoA to the specific alpha-amino of lysine (Shen et al. 2023). CPT1A promotes succinylation of mitochondrial fission factor (MFF) at lysine 302 (K302), which protects against Parkin-mediated ubiquitin-proteasomal degradation of MFF, and promote the growth and proliferation of ovarian cancer cells (Zhu et al. 2023). CPT1A promots the succinylation of SP5, which strengthenes the binding between SP5 and the promoter of PDPK1, SP5 activated PDPK1 transcription and PDPK1 activated the AKT/mTOR signal pathway, and then promots the viability and glycolysis of prostate cancer cells (Liu et al. 2024). However, the mechanism of CPT1A on protein succinylation modification in ENKTL-NT is rarely studied. In this study, we discovered that CPT1A and glycolytic related kinases (LDHA and LDHB) have high expression in ENKTL-NT tissue. LDHA is the key enzyme in aerobic glycolysis and preferentially converts pyruvate to lactate, high expression is often associated with poor prognosis and high metastasis rate (Sharma et al. 2022). Therefore, we speculate CPT1A maybe mediate metabolic reprogramming of ENKTL-NT.

In contrast to normal cells, increased glycolytic flux is a distinguishing feature of the highly proliferative cancer cells, which supports them to adapt to a hypoxic environment and also protects them from oxidative stress. We next sought to determine the specific molecular mechanism by which metabolic reprogramming is regulated. We found that CPT1A promotes proliferation and invasion of ENKTL-NT cells, and then glycolytic products pyruvate and lactate also have high expression in cells. We also found that CPT1A mediates LDHA-succinylation and interacts with LDHA, and then increase LDHA activity, thereby affecting metabolic reprogramming and tumour cells progression. Therefore, we speculate that the increased succinylation of LDHA resulting from CPT1A highly expression regulates the aggression of ENKTL-NT cells by increasing LDHA activity.

CPT1A succinylates LDHA on K222, which reduces the binding and inhibits the degradation of LDHA, as well as promotes GC invasion and proliferation (Li et al. 2020). LDHA plays multiple roles in B-cell lymphoma pathogenesis via FER pathways, silencing LDHA led to reduced mitochondrial membrane integrity, adenosine triphosphate (ATP) production, glycolytic activity, cell viability and invasion (Feng et al. 2024). lncRNA GLTC is a binding partner of LDHA, can target LDHA-succinylation at lysine 155 (K155) via competitive inhibition of the interaction between SIRT5 and LDHA, thereby enhancing LDHA enzymatic activity, and then promote progression and radioiodine resistance in papillary thyroid cancer (Shi et al. 2023). A significant increase in the succinylation at lysine 118 (K118su) of LDHA can promote LDH activity and migration and invasion of prostate cancer cells, and Sirtuin 5 (SIRT5) is a NAD⁺-dependent desuccinylase can reduce LDHA-K118su expression (Kwon et al. 2023). Based on these, we speculate whether there are also specific sites of succinylation modification in LDHA. Our studies have found that knockdown CPT1A, LDHA sites in K318 and K232 are also downregulated. Moreover, the K318R (a de-succinylation mutant) but not K232R appears to interfere with the succinylation of LDHA and combination of LDHA and CPT1A, and inhibites cell proliferation, LDHA activity and glycolysis level. These also further suggested that LDHA mediates ENKTL-NT growth process through CPT1A in vivo.

In conclusion, our study highlights a novel molecular mechanism of the regulation of metabolic reprogramming via CPT1A interactes with LDHA (K318) and regulates LDHA succinylation. The mechanism diagram was shown in Fig. 9. Our results may expedite the recognition of K318‐succinylation of LDHA as a novel biomarker and therapeutic target for ENKTL-NT.

Fig. 9.

The mechanism diagram of the effect of CPT1A in ENKTL-NT tumor CPT1A promotes succinylation of LDHA at lysine 318 (K318), which increase the protein stability and the final protein level of LDHA. LDHA exerts the cancer-promoting effects via the metabolic reprogramming in ENKTL-NT

Supplementary Information

Below is the link to the electronic supplementary material.

Authors’ contribution

Authors’ contribution Study concept and design: Hao Tian,Yi Ge, Jianjun Yu, Xing Chen; data acquisition: Honghan Wang, Xu Cai, Zhenfeng Shan; data analysis and interpretation: Hao Tian, Yi Ge, Jianjun Yu; drafting of the manuscript: Xing Chen, Honghan Wang, Xu Cai, Zhenfeng Shan, Liang Zuo; statistical analysis: Hao Tian,Yi Ge, Yan Liu; study supervision: Hao Tian,Yi Ge, Liang Zuo,Yan Liu.

Funding

This study was supported by the natural sciences funding project of Hunan Province(2023JJ40411),the natural sciences funding project of Changsha City(kq2208149),the Crucial Science and Technology Project of Hunan Province(2023ZJ1124),the Supported By Hunan Cancer Hospital Climb Plan(ZX2021002-5).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethical approval

The clinical and animal study has been approved by the Ethics Committee of Hunan Cancer Hospital & The Affiliated Cancer Hospital of Xiangya School of Medicine(2024110).

Conflict of interest

The authors declare no competing interests.

Consent for publication

Informed consent was obtained from the patient for publication of the pathological images.