Regulation of Histone Emulsification by HPDL via LDHA/LDHB Promotes EC Cell Proliferation

1  Introduction

Endometrial cancer (EC), the third leading cause of gynecological cancer-related deaths in women [1]. The poor prognosis of advanced EC is primarily driven by late diagnosis and acquired therapy resistance, with 5-year survival dropping from 95% in Stage I to 17% in Stage IV [2]. In 2022 alone, the American Cancer Society reported approximately 66,000 new EC cases and 13,000 deaths in the United States, underscoring its growing clinical burden [3]. Emerging evidence highlights lactate as a dual-function metabolite in tumor biology, serving not only as a key carbon source for cellular metabolism but also as a signaling molecule within the tumor microenvironment (TME). Under hypoxic or semi-hypoxic TME conditions, tumor cells secrete excess lactate, fostering a protumoral niche linked to metastasis and poor clinical outcomes [4,5]. Notably, lactate also acts as a substrate for histone lactylation—a novel posttranslational modification implicated in epigenetic regulation [6].

In our prior work, we leveraged The Cancer Genome Atlas (TCGA) database to construct a lactate-related prognostic model for EC, identifying 4-hydroxyphenylpyruvate dioxygenase-like (HPDL) as a key candidate gene. HPDL is an intronless protein-coding gene encoding a mitochondrial enzyme with putative 4-hydroxyphenylpyruvate dioxygenase activity, as annotated in Gene Ontology (GO) databases [7,8]. While HPDL mutations are associated with autosomal recessive neurodevelopmental disorders [9], recent studies reveal their oncogenic role in metabolic reprogramming. For instance, HPDL promotes glutamine metabolism to mitigate oxidative stress in pancreatic ductal adenocarcinoma [10].

To elucidate HPDL’s mechanism in EC, we performed GO/Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses on HPDL and its positively correlated genes (correlation coefficient >0.3). These genes were enriched in pathways such as fatty acid metabolism, mitochondrial metabolism, and central carbon metabolism in cancer, a pathway directly linked to lactate dehydrogenase A (LDHA) and B (LDHB). Both LDHA and LDHB, established oncogenes, are overexpressed in EC and exhibit a positive correlation with HPDL expression. We hypothesize that HPDL drives EC progression by upregulating LDHA/LDHB, potentially through histone lactylation-mediated epigenetic regulation.

This study aims to investigate whether HPDL enhances EC cell proliferation and worsens patient prognosis by upregulating LDHA/LDHB via histone lactylation. Through cellular and animal-level experiments, we seek to validate this hypothesis, thereby clarifying the roles of HPDL and histone lactylation in EC pathogenesis. Our findings may provide a foundation for novel therapeutic strategies targeting lactate metabolism in endometrial cancer.

2  Materials and Methods

2.1 Ethics Approval and Compliance Statement

This study was approved by the Ethics Committee of the First Affiliated Hospital of Bengbu Medical College (Approval No. 2023432). The experimental animal protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Jinan University (ethics approval number: IACUC-20230106-01). All methods were carried out in accordance with relevant guidelines and regulations, including the Declaration of Helsinki for human studies, ARRIVE guidelines for animal research, etc. Written informed consent was obtained from all participants. A total of 160 tissue samples were collected, including 80 EC tissue samples from patients diagnosed with EC and 80 normal endometrial tissue samples from control patients. All samples were obtained from individuals who underwent surgical procedures at the First Affiliated Hospital of Bengbu Medical College between January 2021 and December 2022.

2.2 Cell Line Acquisition and Culture

HEC-1-B and AN3CA EC cell lines were authenticated by STR profiling (American Type Culture Collection (ATCC), Manassas, VA, USA, Cat. No. CL-0100) and consistently tested negative for mycoplasma contamination using the MycoFluor™ Mycoplasma Detection Kit (Thermo Fisher, Waltham, MA, USA, Cat. No. M-7006) in monthly screenings. Cells were cultured in Minimum Essential Medium (MEM; Wuhan Procell Life Technology Co., Ltd., Cat. No. PM150410) under standardized conditions: a humidified atmosphere containing 5% CO2 at 37°C. The culture medium was replaced every 24–48 h, and cell growth status was regularly monitored. Subculturing was performed when cells reached 90% confluence to maintain optimal proliferation.

2.3 Immunofluorescence Analysis

After 48 h of lentiviral infection (for knockdown) or plasmid transfection, three distinct plasmids (SiHPDL1178181, SiHPDL1178182, SiHPDL1178183 (GeneChem, Shanghai, China)) were used to transiently knock down HPDL expression in EC cell lines (AN3CA and HEC-1-B cells). Forty-eight hours post-transfection, the transfection efficiency of different plasmids was compared by measuring fluorescence intensity. Results indicated that SiHPDL1178181 exhibited the highest transfection efficiency. This plasmid was packaged into lentivirus (SiHPDL1178181, Jike Gene) and lentivirus (LV) (LV-HPDL (28880-1; GeneChem)). AN3CA and HEC-1-B cells were washed three times with 1× PBS (pH 7.4), detached with 0.25% trypsin, and seeded into 2 cm diameter confocal imaging dishes at a density of 1 × 105 cells per dish. Following a 12–24 h incubation period to ensure complete adherence, cells were removed from the incubator and subjected to three washes with room temperature (RT) PBS. Subsequent steps included fixation, permeabilization, and blocking, followed by overnight incubation at 4°C with shaking in primary antibody solutions. Antibody dilutions were as follows: anti-H3K18lac (PTM BioLab, Inc, Hangzhou, China, Cat. No. 1406, dilution 1:50), anti-pan kla (PTMBIO, Hangzhou, China; Cat. No. 1401, dilution 1:50), anti-HPDL (Proteintech, Wuhan, China, Cat. No. 20777-1-AP, dilution 1:20), anti-H3 (Abbkine Scientific Co. Ltd., Wuhan, China, Cat. No. A01070, dilution 1:100), and anti-LDHA/LDHB (Proteintech, Cat. No. 19987-1-AP, dilution 1:50). Cells were then incubated with Goat Anti-Rabbit IgG H&L/FITC antibody (BIOS Technology Co., Ltd., Taichung, China, Cat. No. bs-0295G-FITC, dilution 1:200) for 2 h at RT in the dark. After completing the immunostaining protocol, samples were imaged using a confocal microscope (Leica Microsystems, Ernst-Leitz-Str. 17-37, 35578 Wetzlar, Germany, STELLARIS 5/STELLARIS 8).

2.4 Scratch Assay Protocol

A 6-well plate was pre-marked on the underside with three parallel horizontal lines spaced 1 cm apart, spanning the diameter of each well. AN3CA and HEC-1-B Cells (5 × 105 per well) were seeded and incubated for 24 h to achieve >90% confluency. At 24 h post-seeding, the plate was removed from the incubator, and uniform longitudinal scratches were created perpendicular to the pre-drawn horizontal lines using a vertically held 1 mL pipette tip. Detached cells were removed by washing three times with 1× PBS (pH 7.4) followed by the addition of fresh serum- and antibiotic-free MEM medium. Initial scratch widths were documented via imaging (timepoint = 0 h). Post-treatment, the plate was re-incubated, with medium refreshed at designated intervals. Migration progress was monitored by imaging scratches at 6, 12, 24, and 48 h. Scratch closure was quantified using ImageJ software (National Institutes of Health (NIH), Bethesda, MD, USA, Version: 1.53t-1) to measure intercellular distances at each time point.

2.5 Plate Colony Formation Assay

The AN3CA and HEC-1-B HPDL overexpression cell lines, along with their respective knockdown cell lines and parental EC cell lines, all exhibiting good growth status, were detached and counted after being washed three times with 1× PBS (pH 7.4). Each cell line was seeded in 6-well plates at a density of either 500 or 1000 cells per well. The medium was replaced every 3–4 days, and colony formation was monitored. After approximately 10–14 days, colony formation was visible under the microscope (Leica Microsystems, STELLARIS 5/STELLARIS 8). Subsequently, the cells were washed three times with PBS, and 1 mL of 4% paraformaldehyde was added to each of the six wells for fixation, which lasted for 30 min. The cells were then washed three times with PBS, and 1 mL of methanol was added to each of the six wells for permeabilization, which took 20 min. Finally, the cells were washed three times with PBS, 1 mL of a 0.1% crystal violet solution was added to each of the six wells for staining, which lasted for 30 min, and after being washed three times with PBS, the cells were dried and photographed.

2.6 Cell Viability Assay (CCK-8 Assay)

The AN3CA and HEC-1-B HPDL overexpression cell lines, along with the knockdown cell lines and the parental EC cell lines, all exhibiting good growth status, were washed three times with 1× PBS (pH 7.4), detached, counted, and seeded into 96-well plates at a density of 500 cells per well (with 5 replicate wells per group). After the cells had attached to the plates, the culture was continued for 24, 48, 72, 96, and 120 h. At each of these time points, the cells were transferred to new 96-well plates, 100 μl of medium and 10 μl of Cell Counting Kit-8 (CCK-8) (Beyotime, Shanghai, China, Cat. No. C0038) were added to each well, and the plates were incubated for 0.5 to 2 h. The optical density (OD) was measured at 450 nm using a microplate reader (Nikon Eclipse, Tokyo, Japan, TE 300) during this incubation period.

2.7 Transwell Migration Assay

AN3CA and HEC-1-B cells (including HPDL-overexpressing, knockdown, and parental EC cell lines) in the logarithmic growth phase were washed three times with 1× PBS (pH 7.4), detached, and counted. A 200 μl suspension of serum-free MEM containing 1 × 105 cells was added to the upper chamber of the Transwell apparatus (Thermo Fisher, Cat. No. 141002) while the lower chamber was filled with 500 μl of medium containing 20% fetal bovine serum (FBS; Biosharp, Hefei, China, Cat. No. BL302A). After 24 h of incubation, cells were washed three times with PBS, fixed with 4% paraformaldehyde (30 min), permeabilized with methanol (Chemical Book, 67-56-1) (20 min), and stained with 0.1% crystal violet (30 min at room temperature). Following two additional PBS washes and air-drying, non-migrated cells on the upper chamber surface were gently removed using cotton swabs. Migrated cells on the lower membrane surface were quantified under a fluorescence microscope (Nikon Elipse TE 300, Tokyo, Japan). We added the histone lactylation inhibitor sodium oxamate (Absin, Shanghai, China, CAS 811929) to the HPDL overexpression group and observed the change in cell invasion in the HPDL overexpression group after lactate inhibition. We added the histone lactation enhancer Sodium L-lactate (L-NaLa; Sigma, Taufkirchen, Germany, Cat. No. 71718) to the HPDL knockdown group to observe the alteration of cell invasion in the HPDL knockdown group after histone lactate inhibition.

2.8 Flow Cytometric Analysis of the Cell Cycle, Histone

Logarithmic-phase AN3CA and HEC-1-B HPDL-overexpressing cells, knockdown cells, and parental EC cell lines were washed three times with 1× PBS (pH 7.4) and detached for counting. The cells were then seeded in 6-well plates (2 × 105 cells per well), and the plates were incubated for 24 h. One milliliter of the prepared cell suspension was centrifuged, and the supernatant was removed. Next, 500 μl of 70% cold ethanol was added, and the cells were fixed at 4°C for 2 h to overnight. After washing the cells with PBS to remove the fixative, 100 μl of RNase A was added, and the cells were incubated in a 37°C water bath for 30 min. Then, 400 μl of BeyoFC™ PI Staining Solution (Beyotime, Cat. No. C1734) was added to each well. After staining for 15 min in the dark, the cells were injected into a flow cytometer (BD Biosciences, BD FACSymphony™, Milpitas, CA, USA) for detection, and red fluorescence at an excitation wavelength of 488 nm was recorded.

2.9 Chromatin Immunoprecipitation (ChIP)-Quantitative Polymerase Chain Reaction (qPCR) Assay

AN3CA and HEC-1-B HPDL overexpression cell lines, along with their corresponding knockdown cell lines and parental EC cell lines, were cultured in 10 cm dishes containing 10 mL of complete medium. Once they reached over 90% confluence, the cells were harvested and stored. For crosslinking, 270 μl of 37% formaldehyde was added directly to the culture medium and gently mixed to achieve a final formaldehyde concentration of 1%. Chromatin immunoprecipitation (ChIP) was performed using a commercial ChIP Assay Kit (Beyotime, Shanghai, China, Cat. No. P2078) according to the manufacturer’s instructions. The immunoprecipitated chromatin was then purified and subjected to qPCR analysis. RNA reverse transcription: RNA sample mixed and centrifuged, and placed on ice for later use. The TAKARA (Suzhou Novoprotein Scientific Inc, Suzhou, China, Cat. No. E096) reagent kit is melted on ice, shaken first, and then centrifuged. Reverse transcription system 20 μl system: Reagent 14 μl + (2, 3, 4) each 1 μl + 5 (the remaining liquid is replenished to 20 μl) + total RNA sample 1 μg. Mix and centrifuge after adding the sample. Place it on the reverse transcriptase, adjust the parameters: 37°C, 15 min; 85°C, 5 s; ends at 4°C. The cDNA obtained by reversing the qPCR amplification step is used as the sample. The reaction system is configured with 10 μl of system per well, and 3 sub-wells are set for each sample. The total amount of the 3 wells is prepared together. After adding the sample, mix well, exhaust and put it into an 8-tube, and wait for detection in the dark. PCR amplification instrument (Bio-Rad, Shanghai, China, Cat. No. S1000) setting parameters: preheat at 95°C for 20 s; 40 cycles: 15 s at 95°C, 60 s at 60°C. Record the cycle threshold (Ct) of each gene in the experiment and use the 2−ΔΔCt method to calculate the mRNA expression of the target gene. The PCR primers are shown in Table A2.

2.10 Western Blotting (WB)

Nuclear proteins were extracted from 50 mg of endometrial tissue or cultured cells using a nuclear and cytoplasmic protein extraction kit. The extracted nuclear proteins were aliquoted and stored at –80°C for subsequent use. The Western blotting procedure included gel preparation, electrophoresis, membrane transfer, membrane washing, and antibody incubation. Primary antibodies were diluted as follows: anti-H3K18lac (PTMBIO, Cat. No. 1406, Dilution 1:2000), anti-pantothenic acid anti-pan Kla (PTMBIO, Cat. No. 1401, Dilution 1:1000) (1:50 is redundant here and should be removed), anti-HPDL (Proteintech, Cat. No. 20777-1-AP, Dilution 1:1000), anti-H3 (Abbkine, Cat. No. A01070, Dilution 1:3000), and anti-LDHA/LDHB (Proteintech, Cat. No. 19987-1-AP, Dilution 1:2000). Membranes were incubated with the primary antibodies at 4°C overnight. Subsequently, membranes were incubated with a horseradish peroxidase (HRP)-conjugated goat anti-rabbit (Abbkine, Cat. No. BMS005, Dilution 1:7000) for 2 h at room temperature. After further washing, the blots were subjected to chemiluminescent detection. The histone lactylation inhibitor sodium oxamate (MedChemExpress, Monmouth Junction, NJ, USA, CAS No. 565-73-1) was added to HPDL-overexpressing HEC-1-B cells at concentrations of 0, 10, 20, and 30 mmol. WB analysis showed that the signals of the anti-Pan Kla and anti-H3K18lac antibodies were significantly reduced in these cells. Antibodies & Detection Reagents in Table A1.

2.11 Plasmid Transfection and Lentiviral Transfection

In cell culture, when cells grow to approximately 90% confluence, they are seeded into 6-well plates (e.g., for AN3CA cells: T25 flasks are seeded the previous morning, and after 24 h, the cell density reaches 80%–90%, ready for transfection. HEC-1-B cells grow slightly slower, typically reaching over 80% confluence after 48 h. HPDL knockdown plasmids (with catalog numbers SiHPDL11781181, SiHPDL11781182, and SiHPDL11781183) and an overexpression plasmid, both carrying green fluorescent protein (LV-HPDL (28880-1), are added. Transfection is carried out for 48–72 h, after which RNA and protein are harvested in advance for subsequent experiments. For lentiviral transfection, the viral solution is diluted. One aliquot of lentivirus and transfection reagent A (HitransG A, Cat. No. REV005) is taken out. The viral titer for the HPDL knockdown strain is 1 × 109, and for the HPDL overexpression virus, it is 3 × 109. The viral solution is prepared at a 1:50 ratio: For the HPDL knockdown strain: 4 μl of virus + 196 μl of MEM medium. For the HPDL overexpression virus: 2 μl of virus + 198 μl of MEM medium. For the control (con): 3 μl of virus + 197 μl of MEM medium.

2.12 Laboratory Animals

Forty female BALB/c Anu nude mice (4 weeks old; 14–16 g; Beijing Huafu Kang Co., Ltd., strain code 490; RRID: IMSR_APC:001) were housed under specific pathogen-free (SPF) barrier conditions. Randomly divided into 4 groups, with 5 mice in each group, representing variants of the AN3CA cell line—including HPDL overexpression group, HPDL knockout group, siNC transfection group, and parental AN3CA group. Cells from each group were amplified in 10-cm culture dishes. Cells were harvested at >90% confluence during logarithmic growth phase, following confirmation of mycoplasma—and bacterial—free status. Subsequently, 5 × 106 cells of each variant were subcutaneously inoculated into each mouse. Tumor dimensions were measured blindly by two independent investigators using digital calipers, with volume calculated as (V = π/6 × L × W2), L represents Length (the longest dimension of the tumor), while W represents Width (the shorter dimension perpendicular to the length). Humane endpoints were strictly enforced: tumor volume >1500 mm3 or >20% body weight loss. All animal procedures were approved by the Jinan University Institutional Animal Care and Use Committee (IACUC Approval #: IACUC-20230106-01) in accordance with institutional guidelines.

2.13 Data Analysis Methods

The Cancer Genome Atlas (TCGA; https://portal.gdc.cancer.gov/, V23.0), Genotype-Tissue Expression project (GTEx) database (https://www.genome.gov/Funded-Programs-Projects/Genotype-Tissue-Expression-Project). GO/KEGG enrichment analyses: Gene Ontology (GO) and KEGG pathway enrichment analyses were performed on differentially expressed genes (DEGs) using the ‘clusterProfiler’ R package (version 4.16.0), R Studio (version 4.2.0) with the top five enriched pathways presented in this study. Gene Set Enrichment Analysis (GSEA) analysis was conducted using ‘ggplot2’ (version 3.3.3), filtering results with a False Discovery Rate (FDR) < 0.25 and p.adjust < 0.05.

2.14 Statistical Analysis

Data from western blot (WB), cellular immunofluorescence staining, and functional assays—including scratch wound healing, plate colony formation, and Transwell migration/invasion experiments—were analyzed using ImageJ2 Core framework for multidimensional imaging (Bethesda, MD, USA, https://imagej.net/) and GraphPad Prism Version 10.5 (San Diego, CA, USA, https://www.graphpad.com/). Non-parametric statistical tests (Wilcoxon rank-sum test for two-group comparisons or Kruskal-Wallis test for multi-group comparisons) were applied where appropriate. Parametric t-tests or chi-square tests were used for pairwise comparisons of normally distributed or categorical data, respectively. A p-value < 0.05 was deemed statistically significant.

3  Results

3.1 The Level of Histone Lactylation Is Elevated in EC Tissues

We detected significantly higher levels of pan-histone lactylation (Pan Kla) in endometrial carcinoma tissues compared to normal endometrial tissues via Western blotting (WB) (p < 0.05, Fig. 1A,B). Simultaneously, The results of WB showed that the Pan Kla bands in the two EC groups near histone H3, with a molecular weight of 17 kDa, changed significantly. Thus, we hypothesized that lactylation of histone H3 (with a molecular weight of approximately 17 kDa for the modified protein) might be the main factor contributing to the increase in the overall lactylation level in EC patients. Further validation assays revealed that the level of H3K18 lactylation (H3K18la) was significantly increased in EC tissues compared with normal tissues (p < 0.001, Fig. 1C,D), consistent with the increased Pan Kla level in EC patients. lactate concentrations were measured in both endometrial carcinoma and normal endometrial tissues, revealing markedly higher lactate levels in the cancerous tissues (p < 0.001, Fig. 1E). We used immunohistochemistry to detect Pan Kla and H3K18la in EC tissues and normal endometrial tissues, and the results revealed Pan Kla and H3K18la staining in the nucleus and cytoplasm of EC tissues; Pan Kla was localized predominantly in the cytoplasm, H3K18la was localized predominantly in the nucleus, and the difference was significant (p < 0.001). The H3K18la and Pan Kla levels in EC tissues were also significantly higher than those in paracancerous tissues (p < 0.001) (Fig. 1F–H). The above results further confirmed that the level of histone lactylation was elevated in EC tissues. The list of clinical cases of EC is provided in (Table A3).

Figure 1: Histone lactylation modification levels in EC tissues. (A) Expression of Pan Kla and H3K18la in EC tissues; (B) Western blot (WB) quantification results of Pan Kla expression in EC versus normal endometrial tissues; (C) WB quantification results of H3K18la expression in EC versus normal endometrial tissues; (D) Detection of lactate levels in EC tissues; (E) Pan Kla expression in cancer tissues, paracancerous tissues, and normal endometrial tissues with immunohistochemical quantification results; (F) H3K18la expression in cancer tissues, paracancerous tissues, and normal endometrial tissues with immunohistochemical quantification results. Lactic acid levels in EC tissues were higher than those in normal endometrial tissues. Scale bar is 100 μm. (G) Expression of Pan Kla in EC tissues, normal endometrial tissues, and paracancerous tissues. (H) Expression of H3K18la in EC tissues, normal endometrial tissues and paracancerous tissues. *denotes p < 0.05, and ***denotes p < 0.001. ns denotes no statistical significance

3.2 The Expression of HPDL Was Significantly Increased in EC Tissues

The analysis of the TCGA + GTEx database revealed that HPDL gene expression levels were significantly higher in endometrial carcinoma (EC) patient tissues compared to both normal and endometrial carcinoma-adjacent non-cancerous tissues (Fig. 2A). The HPDL gene correlates with poor prognosis in endometrial carcinoma (EC) patients (Fig. 2B). We subsequently validated HPDL expression in EC development. Compared to normal tissue and adjacent non-cancerous tissue, qPCR results confirmed significant overexpression of the HPDL gene in EC tissue (Fig. 2C). Immunohistochemistry revealed high HPDL expression in EC patient tissues (Fig. 2D). Furthermore, HPDL expression was higher in poorly differentiated endometrioid carcinomas than in well-differentiated endometrioid carcinomas. Both clear cell carcinomas and serous carcinomas exhibited significantly higher HPDL expression than normal endometrium and well-differentiated endometrioid carcinomas (Fig. 2E).

Figure 2: HPDL was significantly highly expressed in EC tissues. (A) Expression of HPDL in cancerous and normal tissues within the TCGA database; (B) Prognostic analysis of HPDL within the TCGA database. (C) Expression of HPDL mRNA in cancerous tissues and normal endometrium; (D) Expression of HPDL in EC tissues, normal endometrial tissues, and paracarcinomatous tissues. Scale bar is 100 μm; (E) Expression of HPDL in endometrial cancers of different pathologic types: Normal endometrium; Hyperfractionated endometrioid carcinoma; High-grade plasmacytoid carcinoma; Clear cell carcinoma; Pellucidar carcinoma; Hypofractionated endometrioid carcinoma. The scale bar represents 100 μm, and the black arrows in the figure indicate sites of positive expression of HPDL in the tissues, **signifying p < 0.01, and ***signifying p < 0.001

3.3 Knockdown of HPDL Reduces the Level of Histone Lactylation in EC Cells

Western blot analysis confirmed that SiHPDL11781181 (We constructed three HPDL gene knockdown plasmids, designated SiHPDL11781181, SiHPDL11781182, and SiHPDL11781183, to determine which sequence yields the most effective knockdown) resulted in the lowest HPDL protein expression level after transient transfection in AN3CA and HEC-1-B cells, and SiHPDL11781181 also exhibited the highest knockdown efficiency (Fig. 3A,B). Thus, we selected SiHPDL11781181 for lentiviral packaging to establish EC cell lines with stable knockdown. Furthermore, qPCR revealed that HPDL mRNA expression was lowest in the SiHPDL groups of AN3CA and HEC-1-B cells (Fig. 3C,D), and we observed a significant reduction in the lactate content in AN3CA and HEC-1-B cell lines with transient HPDL knockdown (Fig. 3E,F). Western blot analysis of the Pan Kla and H3K18la levels in HPDL-knockdown AN3CA and HEC-1-B cells indicated that the levels of Pan Kla and H3K18la were significantly reduced by HPDL knockdown (Fig. 3A,B). suggesting that knockdown of HPDL significantly inhibited histone lactylation. After L-NaLa treatment of normal and HPDL knockdown AN3CA cell lines, the expression of H3K18la was detected by immunofluorescence staining, with green fluorescence indicating H3K18la and blue fluorescence indicating the nuclear marker DAPI. The level of H3K18la was significantly reduced in the HPDL knockdown group compared with the NC group, and the level of H3K18la was significantly elevated after the addition of 30 mmol/L lactate (Fig. 3G). Different concentrations (0, 10, 20, and 30 mmol/L) of sodium lactate were added to NC and SiHPDL11781181 AN3CA cells, and measurement of the Pan Kla and H3K18la levels in the cells in both groups confirmed that the histone lactylation level in endometrial carcinoma cells was elevated, but there was no significant increase in HPDL expression (Fig. 3H,I). The above results indicated that sodium lactate supplementation could significantly increase the histone lactylation level and that the lactate-induced increase in the histone lactylation level did not affect the expression of HPDL.

Figure 3: Knockdown of HPDL decreased the level of histone lactonization modification in EC cells. (A) Western blot (WB) analysis confirming the expression of histone lactate dehydrogenase (HLD) antibodies in AN3CA cells with HPDL knockdown; (B) WB analysis confirming the expression of HLD antibodies in HEC-1-B cells with HPDL knockdown; (C) Quantitative polymerase chain reaction (qPCR) analysis of HPDL expression following triple-sequence plasmid knockdown in AN3CA cells; (D) qPCR analysis of HPDL expression following triple-sequence plasmid knockdown in HEC-1-B cells; (E) Measurement of lactate content in AN3CA cells subsequent to HPDL knockdown; (F) Measurement of lactate content in HEC-1-B cells subsequent to HPDL knockdown. (G) Green fluorescence signifies HPDL protein expression, while blue fluorescence indicates immunofluorescence staining of cell nuclei; scale bar is 50 μm; (H) Expression levels of HPDL, Pan Kla, and H3K18la after the addition of varying concentrations of sodium lactate to AN3CA Si HPDL knockdown cell lines; scale bar represents 50 μm. Expression levels of HPDL, Pan Kla, and H3K18la in AN3CA Si HPDL knockdown cell lines with the addition of varying concentrations of sodium lactate; scale bar represents 50 μm; (I) Expression levels of HPDL, Pan Kla, and H3K18la after the addition of varying concentrations of sodium lactate to AN3CA normal cell lines. *indicates p < 0.05, **indicates p < 0.01, ***indicates p < 0.001

3.4 The Histone Lactylation Level Is Elevated in EC Cells after the Overexpression of HPDL

We transfected AN3CA and HEC-1-B cells with the lentiviral overexpression vector LV-HPDL (28880-1) to construct a cell line with stable HPDL overexpression. Following puromycin screening, once the cells had stabilized, Western Blot (WB) analysis revealed a significant increase in HPDL expression in the HPDL-overexpressing HEC-1-B cell line, as well as a notable increase in the signal of the anti-Pan Kla antibody in the HPDL-overexpressing group. The signal of the anti-H3K18la antibody was also elevated (Fig. 4A). The histone lactylation inhibitor sodium oxamate was added to HPDL-overexpressing HEC-1-B cells at concentrations of 0, 10, 20, and 30 mmol. WB analysis showed that the signals of the anti-Pan Kla and anti-H3K18la antibodies were significantly reduced in these cells, although HPDL expression remained unchanged (Fig. 4B). Lactate content assays indicated that lactate levels were significantly higher in HPDL-overexpressing cells but markedly lower in HPDL-knockdown cells (Fig. 4C,D). Immunofluorescence analysis of AN3CA cells demonstrated that H3K18la and Pan Kla levels were elevated in the HPDL overexpression group, and the addition of oxamate to the HPDL-overexpressing cells significantly decreased the H3K18la and DAPI signals (Fig. 4E–G). This confirmed that oxamate could inhibit histone lactylation in endometrial carcinoma cells and that the addition of oxamate did not inhibit HPDL expression after histone lactylation was suppressed.

Figure 4: The level of histone lactylation was elevated in EC cells overexpressing HPDL (A) Expression levels of HPDL, Pan Kla, and H3K18la following the overexpression of HPDL in the HEC-1-B cell line; (B) Expression levels of HPDL, Pan Kla, and H3K18la after treating the HEC-1-B cell LV-HPDL with varying concentrations of sodium oxalate; (C) Overexpression and knockdown strains of AN3CA; (D) Lactate levels in HEC-1-B overexpression and knockdown strains; (E) Immunofluorescence of AN3CA cells for quantifying H3K18la immunofluorescence across different groups; (F) Immunofluorescence of AN3CA cells for quantifying DAPI immunofluorescence across different groups; (G) Immunofluorescence of AN3CA cells for quantifying the expression of H3K18la and DAPI, scale bar is 50 μm; *indicating p < 0.05, **indicating p < 0.01, ***indicating p < 0.001, and ns indicating no statistical significance

3.5 HPDL Promotes the Proliferation of EC Cells

To verify the effect of HPDL expression on cell proliferation, we laid 96-well plates in the SiNC, SiHPDL, LVHPDL groups of AN3CA and HEC-1-B cells, with 5 wells in each group, and detected the absorbance of CCK-8 at different times to plot the curves as shown in the graphs (Fig. 5A,B), which showed that the cell proliferation rate of the LVHPDL group was significantly increased compared to that of the SiNC group. The cell proliferation of SiHPDL group was significantly decreased. The SiNC, SiHPDL, and LVHPDL groups of AN3CA and HEC-1-B were laid in 6-well plates, and plate cloning experiments were performed, and the number of cell clones was detected after 2 weeks of incubation, and the results showed that the number of cell clones of the LVHPDL group > SiNC group > SiHPDL group, and the difference was statistically significant (Fig. 5C–E), which showed that the cell proliferation ability of cells in the HPDL overexpression group was enhanced, and the cell proliferation ability of cells in the knockdown group was decreased, and the expression of HPDL could promote the proliferation of endometrial cancer cells. The effect of HPDL expression on the cell cycle was detected by flow cytometry, and the results showed that the proportion of S phase, which reflects the proliferation ability of cells, increased in the LVHPDL group, and the proportion of G0/G1 decreased; whereas, the proportion of S phase decreased and the proportion of G0/G1 increased after the knockdown of HPDL (Fig. 5F), suggesting that the expression of HPDL may regulate the cycle.

Figure 5: The expression of HPDL promotes the proliferation of EC cells and affects the cell cycle. (A) CCK-8 profiles in AN3CA cells; (B) CCK-8 profiles in HEC-1-B cells; (C) Quantitative results of plate cloning assay of HEC-1-B cells; (D) Plate cloning assay of AN3CA and HEC-1-B cells; (E) Quantification results of plate cloning of AN3CA cells; (F) AN3CA cell cycle assay, *indicating p < 0.05 and ***indicating p < 0.001

3.6 HPDL Promotes the Migration and Invasion of EC Cells

For further elucidation of the effect of HPDL on the biological phenotype of endometrial cancer (EC) cells, this study examined the migratory ability of AN3CA and HEC-1-B cells after HPDL overexpression and knockdown by scratch assay. The results showed that the scratch healing rate was significantly higher in the LV-HPDL group compared with the SiNC group, while it was significantly lower in the SiHPDL group (Fig. 6A–C). Transwell migration assay showed that the number of membrane-penetrating cells was significantly higher in the LV-HPDL group compared with the SiNC group in both AN3CA and HEC-1-B cells, while it was significantly lower in the SiHPDL group (Fig. 6D,F), confirming that HPDL significantly enhanced the migration ability of EC cells. Transwell invasion assay showed that the number of membrane-penetrating cells was significantly increased in the LV-HPDL group compared to the SiNC group, whereas it was significantly decreased in the SiHPDL group (Fig. 6E,G), confirming that HPDL promotes the invasive ability of EC cells.

Figure 6: HPDL promotes the migration and invasion of EC cells. (A) Cell scratch assay of the normal, overexpression and knockdown groups of the HPDL gene in AN3CA cells, scale bar is 50 μm; (B) Cell scratch assay of the normal, overexpression and knockdown groups of the HPDL gene in HEC-1-B cells; (C) AN3CA and HEC-1-B cells with the normal, overexpression and knockdown groups of the HPDL gene in the quantitative histograms of cell scratch assay cells; (D) quantitative histograms of Transwell migration assay of AN3CA cells; (E) quantitative histograms of Transwell migration assay of HEC-1-B cells; (F) Transwell migration assay of AN3CA and HEC-1-B cells, scale bar is 50 μm; (G) AN3CA and HEC-1-B cells Transwell invasion assay; (H) AN3CA and HEC-1-B cell HPDL overexpression Transwell invasion assay; (I) Transwell invasion assay with HPDL knockdown in AN3CA and HEC-1-B cells, ***indicating p < 0.001

We added the histone lactylation inhibitor sodium oxalate to the HPDL overexpression group and observed the change in cell invasion in the HPDL overexpression group after histone lactylation inhibition. 6-well plate Transwell invasion experiments were performed by establishing the SiNC group, LV-HPDL group and LV-HPDL+oxalate group of AN3CA and HEC-1-B cells, and found that the LV-HPDL group had a significant increase in the number of membrane-penetrating cells compared with the SiNC group, while the LV-HPDL+oxalic acid group had a significant decrease compared with the LV-HPDL group (Fig. 6H). The results confirmed that histone lactylation modification inhibited cell migration and invasion induced by HPDL overexpression, suggesting that HPDL overexpression may promote migration and invasion of EC cells by regulating histone lactylation.

We added the histone lactylation enhancer sodium lactate to the HPDL knockdown group to observe the alteration of cell invasion in the HPDL overexpression group after histone lactylation inhibition. 6-well plate Transwell invasion experiments were performed by establishing the SiNC group, the SiHPDL group and the SiHPDL+sodium lactate group of AN3CA and HEC-1-B cells, and the results showed that the number of membrane-penetrating cells in the SiHPDL group was significantly fewer than the SiNC group, whereas the SiHPDL+sodium lactate group had significantly more than the SiHPDL group (Fig. 6I). These confirmed that promotion of histone lactylation reversed the decrease in cell migration and invasion ability caused by HPDL knockdown, further suggesting that HPDL knockdown may inhibit the migration and invasion of EC cells by regulating histone lactylation.

3.7 HPDL Promotes Histone Lactylation in EC Cells by Upregulating LDHA/LDHB Expression

We downloaded genes that were positively correlated with HPDL in EC from the TCGA database. A total of 544 genes had correlation coefficients greater than 0.3, and 466 of these genes were found to be differentially expressed in EC through expression analysis. The differentially expressed genes underwent GO/KEGG enrichment analyses, which indicated that these genes were primarily enriched in fatty acid metabolism, mitochondrial metabolism, carbon metabolism, and central carbon metabolism in cancer (Table 1).

Within these processes, the downstream genes of HPDL in the carbon metabolism pathway of cancer cells are LDHA and LDHB. LDHA and LDHB participate in glycolytic metabolism, and their metabolite is lactate; therefore, since lactate serves as a substrate for histone lactylation, LDHA and LDHB may play a crucial role in histone lactylation. Consequently, we concentrated on this pathway and investigated the relationship between HPDL and its associated genes, LDHA/LDHB, as well as their regulatory roles in histone lactylation within endometrial cancer. The pathway map associated with the GO/KEGG analyses is depicted in (Fig. 7A,B. Western blot (WB) detection revealed that after knocking down LDHA and LDHB, quantitative PCR results showed no significant changes in HPDL mRNA expression levels (Fig. 7C), and HPDL protein expression did not exhibit significant differences in Parazacco spilurus subsp. spilurus (Fig. 7D), suggesting that there was no significant alteration in HPDL expression subsequent to substantial inhibition of LDHA/LDHB expression. The mRNA and protein levels of LDHA and LDHB in AN3CA cells were examined across three groups: the HPDL overexpression group, the HPDL knockdown group, and the SiNC group. Western blot (WB) analysis revealed that the expression of LDHA/LDHB was significantly downregulated in the SiHPDL group and significantly upregulated in the LV-HPDL group (Fig. 7D). Quantitative polymerase chain reaction (qPCR) analysis indicated that LDHA/LDHB mRNA expression was decreased in the SiHPDL group and significantly increased in the LV-HPDL group (Fig. 7E). These results suggest that knockdown of HPDL significantly downregulates LDHA/LDHB expression, while overexpression of HPDL significantly upregulates it.

Figure 7: HPDL promotes histone lactylation levels in EC cells by upregulating LDHA/LDHB expression. (A) Chordal graph of GO/KEGG analysis; (B) Western blot (WB) demonstrates detection of Pan Kla, H3K18la antibody expression after LDHA and LDHB inhibition; (C) Reverse transcription-quantitative polymerase chain reaction (qPCR) showing the expression of HPDL in AN3CA cells with SiNC, SiLDHA, and SiLDHB; (D) WB demonstration of fluorescence transfection efficiency and LDHA/LDHB and HPDL expression in AN3CA cells with SiNC, SiLDHA, and SiLDHB; (E) qPCR of AN3CA cells with decreased LDHA/LDHB expression in the SiHPDL group, and decreased LDHA/LDHB expression in the LVHPDL group, where LDHA/LDHB expression increased; (F) H3K18la expression in the LDHA promoter region in AN3CA cells with SiNC, LV-HPDL, and LV-HPDL+Oxmate; (G) H3K18la expression in the LDHB promoter region in AN3CA cells with SiNC, LV-HPDL, and LV-HPDL+Oxmate; (H) H3K18la expression in the LDHA promoter region in AN3CA cells with SiNC, SiHPDL, and SiHPDL+L-NaLa; (I) H3K18la expression in the LDHB promoter region in AN3CA cells with SiNC, SiHPDL, and SiHPDL+L-NaLa; *indicates p < 0.05, **indicates p < 0.01, ***indicates p < 0.001

Following the knockdown of LDHA and LDHB, Western blot analysis revealed that the signals from the anti-Pan Kla and anti-H3K18la antibodies were significantly suppressed. This indicates that the knockdown of LDHA/LDHB markedly inhibited histone lactylation in EC cells (Fig. 7B). The levels of H3K18la in the promoter regions of LDHA and LDHB were elevated in the HPDL-overexpressing AN3CA cell line and subsequently decreased upon the addition of oxalate (Fig. 7F,G). Supplementation with sodium oxamate led to a reduction in the H3K18la level in the LDHA promoter region (Fig. 7F,G). The levels of H3K18la in the LDHA and LDHB promoter regions were diminished in the AN3CA HPDL-knockdown cell line, and the introduction of sodium lactate increased the levels of H3K18la in the promoter regions of both LDHA and LDHB (Fig. 7H,I). These results confirm that HPDL regulates the expression of LDHA and LDHB through histone lactylation at H3K18. In summary, these findings indicated that HPDL regulated the expression of LDHA/LDHB; however, knockdown of LDHA/LDHB had no significant effect on the expression of HPDL. Additionally, knockdown of LDHA/LDHB significantly inhibited histone lactylation in AN3CA cells.

3.8 In Vivo Experiments Confirmed That HPDL Promotes Endometrial Cell Proliferation and Growth

We randomly divided 4-week-old BALB/c nude mice into four groups of five mice each. Fig. 8A,B demonstrated the macroscopic morphology of mice and tumors after euthanasia, and there was no significant difference in the initial body weights of the mice (Fig. 8C). After the isolation and quarantine period, we established SiNC group, AN3CA control group, HPDL knockdown group, and HPDL overexpression group. After culturing the cells by passaging to an optimal growth state, the mice were inoculated at a density of 5 × 106 cells per mouse. The tumor volume of the LV-HPDL overexpression group was significantly larger than that of the SiNC group and AN3CA control group, whereas the tumor volume of the HPDL knockdown (SiHPDL) group was significantly smaller than that of the SiNC group, and the difference in tumor weight was statistically significant (Fig. 8D). The tumor growth rates and tumor sizes in the HPDL overexpression group were significantly higher than those of the SiNC group (Fig. 8D). The tumor growth rates and tumor sizes in the HPDL overexpression group were statistically significant. It had a significantly higher tumor growth rate and tumor size than the SiNC group and AN3CA control group, while the HPDL knockdown (SiHPDL) group was significantly lower than these two control groups (Fig. 8E). These findings indicated that HPDL overexpression significantly promoted, whereas HPDL knockdown significantly inhibited, tumor growth in nude mice.

Figure 8: The expression of HPDL promotes tumor growth. (A,B) Mouse macroscopic and tumor phenotypes post-euthanasia; (C) Initial body weight of mice; (D) Tumor body weight; (E) Tumor volume growth curves. ns denotes p > 0.05, *denotes p < 0.05, **denotes p < 0.01, and ***denotes p < 0.001

4  Discussion

4.1 HPDL Has the Potential to Predict the Prognosis of EC Patients

Endometrial carcinomas (ECs) represent a group of malignant epithelial tumors arising from the endometrium and rank among the most prevalent gynecological malignancies [11]. While early-stage EC patients generally exhibit favorable prognoses, those with advanced, recurrent, or metastatic disease face significantly poorer outcomes. Given the limited efficacy of radical surgery in such cases [12], there is an urgent clinical need to identify novel molecular biomarkers for diagnosis, prognostication, and targeted therapy.

The HPDL gene, a recently characterized single-exon gene encoding a mitochondrial-localized protein [13], has emerged as a candidate of interest. Although initially implicated in tyrosine metabolism, metabolic profiling of HPDL-mutant cells and murine models revealed no tyrosine pathway alterations but significant dysregulation in other metabolic compartments. This phenotype aligns with HPDL loss-of-function causing a distinct pediatric neurometabolic mitochondrial disorder [14]. Critically, oncogenic roles for HPDL have been reported: Ye et al. demonstrated that HPDL overexpression promotes pancreatic ductal adenocarcinoma (PDAC) tumorigenesis in vitro, whereas its suppression inhibits proliferation and clonogenicity [10].

Extending these findings to EC: Through constructing a lactate-related prognostic model for endometrial cancer, our prior work identified HPDL as a high-risk gene. Subsequent validation via Western blot and quantitative PCR analyses of clinical specimens confirmed significantly elevated HPDL expression in EC tissues compared to normal endometrium. Critically, this overexpression correlated with adverse clinical outcomes, underscoring HPDL’s potential as both a prognostic biomarker and therapeutic target in EC.

4.2 Elevated Levels of Histone Lactylation in Endometrial Cancer

Histones are proteins that consist of a nucleosome core wrapped in DNA and contain numerous posttranslational modifications in the N-terminal tail. These modifications can regulate physiological processes closely related to gene expression, such as RNA transcription, DNA replication, and DNA damage repair, either by altering the structure of the nucleosome or by recruiting nonhistone proteins [15]. Many of the canonical types of histone posttranslational modifications, such as methylation and acetylation, have long been recognized as important in the pathogenesis of a range of other diseases, including cancer [16]. Researchers have identified lactylation as a novel epigenetic modification of histone lysine residues, and detected 28 lactylation sites in core histones across human and mouse cells [17]. Hypoxia and bacterial stress induce lactate production via glycolysis, and lactate in turn functions as a precursor to stimulate histone lactylation [18]. Subsequently, an increasing number of studies have shown that tumours, inflammation, metabolism, and hypoxia are associated with the regulation of histone lactylation, and Feng et al. [19] reported that lactate induced histone lactylation at the promoters of profibrotic genes in macrophages, consistent with the increase in this epigenetic modification in fibrotic lungs. This lactate-induced histone lactylation and fibrotic gene expression were mediated by p300, as evidenced by their reductions in p300-deactivated macrophages [20]. Yang et al. [21] demonstrated that increased glycolytic activity could induce endometrial histone lactylation, a newly identified histone modification, remodelling a novel function in uterine tolerance. The results from in vitro and in vivo models support an important role for lactate in inducing endometrial H3K18la and regulating redox homeostasis and the apoptotic balance to ensure successful embryo implantation. Our group previously confirmed that the level of histone lactylation is elevated in EC tissues.

4.3 HPDL Expression Drives Proliferation and Cell Cycle Progression in EC

Dysregulation of cell cycle machinery is a hallmark of tumorigenesis and represents a rational therapeutic target [22]. Notably, cell cycle pathways intersect with cancer metabolism and immune evasion mechanisms [23], suggesting that targeting these pathways may simultaneously inhibit proliferation, reverse metabolic reprogramming, and restore immune surveillance. While small-molecule inhibitors (SMIs) and proteolysis-targeting chimeras (PROTACs) have shown promise in modulating cell cycle proteins [24], recent work also implicates cytoplasmic DNA sensors (e.g., cGAS-STING) in cell cycle control and tumor suppression [25].

In this study, we examined the migration ability of AN3CA and HEC-1-B cells after HPDL overexpression and knockdown by scratch assay, and confirmed that HPDL could promote the invasion ability of EC cells. The histone lactylation inhibitor sodium oxalate was added to the HPDL overexpression group to observe the alteration of cell invasion in the HPDL overexpression group after the inhibition of histone lactylation. The results confirmed that histone lactylation modification could inhibit HPDL overexpression-induced cell migration and invasion, suggesting that HPDL overexpression might promote the migration and invasion of EC cells through the regulation of histone lactylation. The histone lactylation enhancer sodium lactate was added to the HPDL knockdown group to observe the alteration of cell invasion in the HPDL knockdown group after the inhibition of histone lactylation. These confirmed that the promotion of histone lactylation reversed the decrease of cell migration and invasion caused by HPDL knockdown, which further suggests that HPDL knockdown may inhibit the migration and invasion of EC cells through the modulation of histone lactylation.

HPDL drives EC progression by accelerating cell cycle transition and promoting metastasis via histone lactylation. Targeted inhibition of HPDL represents a promising therapeutic strategy for endometrial cancer.

4.4 HPDL Upregulates LDHA/LDHB Expression and Promotes Histone Lactylation in EC Cells

Tumor cells exhibit high metabolic plasticity and can engage in anaerobic glycolysis and lactate production under hypoxic conditions [26]. The enzymes responsible for the reversible conversion of pyruvate to lactate include LDHA and LDHB [27]. LDHA has a high affinity for pyruvate and preferentially converts it to lactate and NADH to NAD+ under anaerobic conditions, while LDHB has a high affinity for lactate and preferentially converts it back to pyruvate in the presence of sufficient oxygen, also converting NAD+ to NADH. In addition to their undisputed roles in tumor cell metabolism and adaptation to adverse environmental or cellular conditions, LDHA and LDHB are also involved in histone lactylation [28]. Nian et al. [29] reported that LDHA expression is upregulated in human non-small cell lung cancer (NSCLC) tissues and correlates with poor disease prognosis. LDHA further increases histone lactylation and activates the expression of HK-1 and IDH3G, leading to metabolic disorders and tumorigenesis in lung cancer cells. Tang et al. [30] reported that the levels of LDHA, intracellular lactate, and histone lactylation progressively increase during osteogenic differentiation. Downregulation of LDHA affects the formation of mineralized nodules and ALP activity; knockdown of LDHA decreases the enrichment of histone lactylation at the JunB promoter, and exogenous lactate treatment reverses this effect [31].

4.5 HPDL Expression Regulates Histone Lactylation in Endometrial Cancer

In this study, we revealed for the first time the critical regulatory role of hydroxy acid dehydrogenase (HPDL) on histone lactate modification in endometrial cancer (EC) by functional experiments. Our results show that transient knockdown of HPDL expression in two endometrial cancer cell lines, AN3CA and HEC-1-B, resulted in a significant decrease in intracellular lactate content, overall protein lactate level (Pan Kla), and specific histone H3K18 lactate (H3K18la) modification. This finding directly links HPDL, a metabolic enzyme, to the emerging epigenetic modification of histone lactylation, providing a novel “metabolic-epigenetic” perspective for understanding the mechanisms of endometrial cancer progression.

At the core of our findings is the demonstration that HPDL is a key upstream factor in maintaining high lactate levels in cancer cells, and is thought to catalyze the oxidative decarboxylation of 2-hydroxy acids (e.g., 2-hydroxyglutaric acid) in the mitochondria, a process that is potentially linked to energy metabolism and lactate metabolism networks [32]. Although its exact function is still being explored, our data clearly show that knockdown of HPDL directly contributes to the depletion of the intracellular lactate pool. This strongly suggests that in EC cells, HPDL may act as a “gatekeeper” of intracellular lactate levels through its metabolic function by influencing a key aspect of the glycolytic process: lactate production or lactate conversion. This finding provides a new example of how tumor cells finely regulate their metabolite concentrations to drive downstream signaling pathways.

The decrease in intracellular lactate levels directly led to a widespread inhibition of histone lactate modification. This causal relationship perfectly corroborates the groundbreaking study by Prof. Yi Zhang’s team in 2019, which for the first time suggested that lactate could serve as a precursor substrate for histone lactonization, thereby directly coupling cellular metabolism to epigenetic regulation [17]. As a novel epigenetic modification, histone lactylation has been shown to activate the expression of genes involved in tumorigenesis, Warburg effect, and immune regulation, among other related genes, by altering chromatin structure and recruiting transcriptional regulatory complexes. Thus, the down-regulation of Pan Kla and H3K18la that we observed is not an isolated biochemical phenomenon, but rather implies that HPDL knockdown may have triggered the reprogramming of a series of pro-oncogenic transcriptional programs by decreasing the level of lactonization.

Of particular interest is the downregulation of H3K18la. It has been shown that H3K18la is an active transcriptional activation marker on gene promoters. In macrophages, it was shown to be involved in the activation of M2-type polarization-related genes. In the context of cancer, high levels of H3K18la are strongly associated with malignant progression of several tumors [33]. Our results suggest that HPDL-driven lactate production is required to maintain high levels of H3K18la in endometrial cancer. This implies that HPDL may specifically activate a critical set of genes that drive EC cell proliferation, migration, or stemness maintenance through upregulation of H3K18la, thereby exerting its oncogenic function. Future combined ChIP-seq (chromatin immunoprecipitation sequencing) and RNA-seq (transcriptome sequencing) analyses will hopefully reveal precisely which oncogenes are dependent on the HPDL-H3K18la axis for their activation.

From a clinical translational perspective, our study has dual implications. First, HPDL and its regulated histone lactylation levels might serve as novel prognostic biomarkers for EC patients. High levels of HPDL/H3K18la may portend greater metabolic activity and poorer clinical prognosis. Second, and more importantly, HPDL shows great promise as a potential therapeutic target. Compared with the traditional strategy of targeting key enzymes of glycolysis (e.g., LDHA), targeting upstream HPDL may be more specific and effective in cutting off the source of lactate, thus not only inhibiting the energy supply of tumor cells, but also reversing their pro-cancer epigenetic status, thus achieving the therapeutic effect of “killing two birds with one stone”. In addition, in view of the inhibitory effect of lactate on immune cells in the tumor microenvironment [34], targeting HPDL may also enhance the efficacy of existing immunotherapies by decreasing lactate levels and remodeling the immune microenvironment.

Of course, this study still has some limitations. The current data were mainly derived from in vitro cell line models, and further validation in in vivo animal models (e.g., transplantation tumor models) and a wider range of EC clinical samples is needed. In addition, the specific molecular mechanisms by which HPDL regulates lactate levels (e.g., what are its most critical substrates and how does it cross-talk with the glycolytic pathway) remain to be elucidated in depth.

5  Limitations

The limitation of this study is that our team focused on an in vitro EC cell model (AN3CA/HEC-1-B) and in vitro cellular experiments, as well as a clinical case study, which would have been better with more cases. Limitations of the evidence for the mechanism of the direct interaction of HPDL-LDHA/LDHB. “Although our in vivo data establish HPDL as a viable therapeutic target, they do not resolve whether the mechanisms identified in vitro (e.g., mechanism) underpin these phenotypes. Future studies using [specific tools] will bridge this gap”.

6  Conclusion

This study found that inhibiting the expression of LDHA or LDHB significantly suppressed histone lactylation in endometrial cancer. However, such inhibition did not notably affect HPDL expression. Conversely, suppressing HPDL expression led to a significant decrease in LDHA/LDHB expression, and overexpression of HPDL markedly downregulated LDHA/LDHB expression. Consequently, we concluded that HPDL can facilitate histone lactylation in EC by upregulating LDHA/LDHB.

Acknowledgement: We would like to acknowledge the financial support from the Anhui Provincial Research Preparation Plan Project [No. 2022AH051536].

Funding Statement: This work was supported by the Anhui Provincial Research Preparation Plan Project [No. 2022AH051536] and the Science and Technology Projects in Guangzhou [No. 2025A03J4177].

Author Contributions: Yan Wang and Jialei Zhu designed the study, wrote the first draft, submitted, revised, and embellished the manuscript. Lingling Wang and Caizhi Wang guided topic selection and experimental design, revised the manuscript, and supervised the submission process. Shiyang Wei, Lijie Jin, Zhanqiu Liu, Jie Xu, Nana Yang and Xuefeng Jiang revised the first draft of the manuscript and approved the final version after incorporating feedback from all authors. All authors reviewed and approved the final version of the manuscript.

Availability of Data and Materials: All data generated or analyzed during this study are included in this published article.

Ethics Approval: This study was approved by the Ethics Committee of the First Affiliated Hospital of Bengbu Medical College (Approval No. 2023432). We obtained informed consent from all patients. The experimental animal protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Jinan University (ethics approval number: IACUC-20230106-01).

Conflicts of Interest: The authors state that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper. The authors disclose the following financial interests/personal relationships, which may be considered as potential competing interests: Yan Wang reports receiving financial support from The First Affiliated Hospital of Bengbu Medical University. Yan Wang has a relationship with The First Affiliated Hospital of Bengbu Medical University, encompassing employment and funding grants. Should there be additional authors, they too declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper.

Appendix A

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