ESM1 and protein lysine L-lactylation modification (Pan-kla) in ovarian cancer: coexpression, clinical significance, and prognostic value

Abstract

Background

Ovarian cancer (OC) remains a highly lethal gynaecologic malignancy. Endothelial cell-specific molecule 1 (ESM1) and protein lysine L-lactylation modification (Pan-kla) are key players in tumour microenvironment regulation, which involves metabolic reprogramming, angiogenesis, and immune modulation. However, their coexpression patterns, clinical relevance, and synergistic prognostic impact in OC patients have not been fully elucidated.

Methods

In this study, immunohistochemistry (IHC) was used to analyse ESM1 and Pan-kla expression in 131 ovarian cancer tissue samples from patients diagnosed between 2014 and 2024. Clinical parameters (e.g., FIGO stage, CA125 level, and ascites) and survival data were collected. Statistical analyses, including Kaplan–Meier survival and risk stratification, were performed using R software.

Results

ESM1 and Pan-kla were significantly overexpressed in OC tissues, with a strong positive correlation (P < 0.0001). High expression of both biomarkers was associated with adverse clinical features: advanced FIGO stage, elevated CA125 levels, and malignant ascites. Survival analysis revealed that high ESM1 expression increased the risk of death, whereas high Pan-kla expression increased the risk. Patients exhibiting combined ESM1/Pan-kla expression were stratified into four prognostic subgroups, with the dual-high group exhibiting the worst survival (P < 0.001).

Conclusion

ESM1 and Pan-kla synergistically promote OC progression through metabolic–epigenetic cross-regulatory mechanisms. Their combined assessment provides robust prognostic stratification and reveals potential targets for overcoming therapeutic resistance in ovarian cancer.

Graphical Abstract

Supplementary Information

The online version contains supplementary material available at 10.1186/s13000-026-01748-0.

Introduction

Ovarian cancer remains among the most lethal gynaecologic malignancies, with epithelial ovarian cancer (EOC) accounting for approximately 90% of cases [1]. Despite advancements in surgical cytoreduction and platinum-based chemotherapy, the prognosis of OC remains poor and OC is characterized by a high recurrence rate and chemoresistance, leading to a five-year survival rate of less than 45% [2]. Tumour heterogeneity, late diagnosis, and the immunosuppressive tumour microenvironment (TME) further complicate therapeutic outcomes [3]. Recent molecular classifications have refined tumour stratification into histological subtypes (e.g., high-grade serous, endometrioid, and clear cell), yet targeted therapies, including PARP inhibitors and antiangiogenic agents, have shown limited efficacy in unselected populations, underscoring the need for novel biomarkers and mechanistic insights [2].

Endothelial cell-specific molecule 1 (ESM1), a secreted protein, plays a critical role in the tumour microenvironment, metabolic reprogramming, angiogenesis, and immune regulation [4, 5]. Our team has conducted a comprehensive and in-depth exploration of ESM1, covering various cancer types and disease models.

In lung cancer, our findings indicate that ESM1 promotes lipid metabolic reprogramming, proliferation, migration, and angiogenesis by activating the AKT signalling pathway and upregulating the expression of SCD1 and FASN [6]. In clear cell renal cell carcinoma, we were the first to report the overexpression of ESM1 and its role in promoting the proliferation, invasion, and angiogenesis of renal cancer cells via activation of Akt/mTOR and Ras pathways [7]. In cardiovascular diseases, ESM1 plays a protective role in endothelial cells by upregulating autophagy to counteract palmitic acid (PA)-induced damage, thereby slowing the progression of atherosclerosis (AS) [8].

Most importantly, in OC, we systematically validated for the first time the high expression of EM1 and its association with poor prognosis, revealing its ability to promote OC progression through multiple mechanisms [4, 5, 9–12]. On the one hand, within the hypoxic microenvironment, ESM1 is transcriptionally upregulated by HIF-1α, promotes PKM2 SUMOylation, enhances the Warburg effect (glycolysis), and drives fatty acid synthesis and vascular mimicry (VM) [9]. On the other hand, lactate upregulates ESM1 and SCD1 expression, inhibits the antitumour response of CD8 + T cells, and activates the Wnt/β-catenin pathway to exacerbate drug resistance [11]. Moreover, ESM1 suppresses PTEN expression by inhibiting the long noncoding RNA GAS5 and upregulating the expression of miR-23a-3p, thereby activating the PI3K/Akt pathway and leading to cisplatin resistance [12]. Furthermore, ESM1 interacts with ANGPTL4, promoting lipid metabolic reprogramming and angiogenesis through the JAK2/STAT3 pathway, thereby accelerating ovarian cancer progression [10]. Although our research has demonstrated the role of the association between ESM1 and lactate in ovarian cancer progression, the precise regulatory mechanism by which lactate controls ESM1 remains to be elucidated.

Lysine L-lactylation modification (Pan-kla) involves the addition of L-lactate molecules to the lysine residues of proteins and was discovered by the team of Professor Yingming Zhao at the University of Chicago in 2019 [13, 14]. Lactate, an important metabolic substance in the body, plays crucial roles in both physiological and pathological processes, and the level of lactylation is closely related to the abundance of lactate [15–17]. The Warburg effect was initially used to describe the phenomenon of increased lactate production in tumours and is involved in the regulation of various cellular processes, such as angiogenesis, hypoxia, macrophage polarization, and T-cell activation [18, 19]. Advances in research on lysine L-lactylation modification in cancer highlight its role as a key node in the cross-regulation of metabolism and epigenetics, driving cancer progression by influencing tumour proliferation, drug resistance, and immune microenvironment remodelling [20]. Specifically, studies have revealed the molecular mechanisms underlying lactylation in various cancers; for example, in liver cancer, lactylation at the K28 site of adenylate kinase 2 can promote glycolysis and nucleotide metabolism, thereby accelerating tumour growth and metastasis [21]; in melanoma, lactate-induced lactylation of the LSD1 enzyme leads to targeted therapeutic resistance by inhibiting the ferroptosis pathway [22]; and in colorectal cancer, histone H4K12la modification enhances the resistance of cancer stem cells to chemotherapy by upregulating the expression of the glutathione synthetase GCLC [23]. These findings not only deepen the understanding of the carcinogenic mechanism of the Warburg effect but also promote the exploration of therapeutic strategies targeting lactylation; for instance, inhibiting the lactate-producing enzyme LDHA or the lactyltransferase AARS1 can reverse the inactivation of tumour suppressor function mediated by p53 protein lactylation, providing new directions for overcoming drug resistance [24]. Notably, lactate accumulation in the hypoxic ovarian TME may synergize with ESM1 to sustain angiogenesis and immunosuppression [11], but this interplay remains unexplored.

In summary, the functional role of ESM1 in ovarian cancer and its regulatory network have not been fully elucidated, but research on how lactylation influences ovarian cancer progression remains in its early stages. Notably, elevated levels of lactate in the ovarian cancer TME may involve dual mechanisms; lactylation might directly regulate the expression or function of ESM1, thereby affecting angiogenesis and metastasis [11]. ESM1 could also mediate interactions between endothelial cells and tumour cells, further exacerbating lactate accumulation and epigenetic remodelling [9]. However, the specific molecular association between ESM1 and lactylation, their synergistic mechanisms in chemotherapy resistance, and the therapeutic potential of targeting this pathway still lack systematic investigation. In our study, we found that ESM1 and lactic acid levels were significantly increased in different ovarian cancer patients and that their expression levels were closely related to prognosis and survival in these patients. These findings provide a new direction for further exploration of the pathogenesis and prevention and treatment mechanism of ovarian cancer.

Methods and materials

Collection of patient tissue

In our study, 131 tissue samples from ovarian cancer patients who received a pathological diagnosis at Zhuzhou Central Hospital, Xiangya School of Medicine, Central South University, between June 2014 and September 2024 were taken as experimental subjects. The flowchart of the study design is shown in Fig. 1. We summarized the main clinical and pathological data of the patients, including tumour site, tumour size, metastasis status, and commonly used immunohistochemical indicators, including ESM1 and Pan-kla. The collected tissue samples were first used to determine the pathological type of ovarian cancer using HE staining, after which immunohistochemical staining was performed.

Fig. 1.

Flowchart of the research design. All patients were followed up until death or until February 1, 2025. OC, ovarian cancer

Immunohistochemistry (IHC)

The prepared tissue sections were placed in environmentally friendly dewaxing reagent (G1128; Servicebio) and dewaxed at room temperature for 20–30 min according to the thickness of the sections. The sections were then rehydrated in sequence with anhydrous ethanol (100092683, Servicebio) and gradient ethanol. Afterwards, the slices were washed in running water for 10 min to ensure thorough dewaxing and rehydration. The slices were then placed into a citric acid repair solution (G1202, Servicebio) and repaired at high pressure and high temperature for 15 min. After the sections were cooled to room temperature, an immunohistochemistry pen (G6100, Servicebio) was used to draw circles around the tissue area. Aftewards, the cells were washed in PBS (G0002, Servicebio) 3 times for 3 min each time. The washed sections were placed in an endogenous peroxidase blocker and incubated at room temperature for 30 min to block endogenous peroxidase activity. Subsequently, blocking solution (AWB0214b; Abiowell) was added dropwise to the plate, which was incubated at room temperature for 30 min and then the sections were washed three times with PBS for 3 min each time. Then, the corresponding primary antibodies were added dropwise to the circle (ESM1 (bs-3615R, Bioss) and Pan-kla (PTM-1401RM, PTMBio)) and incubated at 4 °C for 14 h. The slices were then washed 5 times in PBS for 5 min each time. Afterwards, secondary antibody (GB23204; Servicebio) was added dropwise to the sections, which were incubated at room temperature for 50 min, after which the sections were placed in PBS for 5 min each time. Subsequently, DAB (G1212; Servicebio) was used to develop colour for approximately 5 min, and the colour rendering time was controlled under a microscope. Afterwards, the cells were rinsed with tap water to stop colour development, and the nuclei were counterstained with haematoxylin (G1004; Servicebio) for 3 min and then rinsed with tap water. After the cells were differentiated with haematoxylin differentiation solution (G1039; Servicebio), the blue colour of the tap water was reversed for 10 min. Finally, the sections were dehydrated with gradient alcohol, dried and sealed with mounting medium (G1404; Servicebio).

The immunohistochemical staining results were independently evaluated by two pathologists who were blinded to the clinicopathological data. The widely used H-score system was employed for analysing ESM1 and Pan-kla staining [25]. This score integrates staining intensity and the percentage of positive cells: staining intensity was graded as 0 (negative), 1 (weakly positive, light yellow), 2 (moderately positive, brownish yellow), or 3 (strongly positive, brown); the percentage of positive cells refers to the proportion of positive cells at each intensity level relative to the total number of tumour cells in the field of view. The H score was calculated using the formula ∑(intensity score × percentage of cells at that intensity), yielding a theoretical range of 0 to 300. To convert the continuous variable into a binary categorical variable for survival analysis, on the basis of previous studies and the receiver operating characteristic (ROC) curve analysis of our cohort, an H score > 150 was ultimately defined as the cut-off for “high expression”, whereas an H score ≤ 150 was defined as “low expression”. The results from the two scorers showed good agreement (intraclass correlation coefficient > 0.85).

All sample collection and utilization protocols were reviewed and approved by the Medical Ethics Committee of Zhuzhou Central Hospital, Xiangya Medical College, Central South University (#LLYPJ2025004-01), which strictly adhered to the Helsinki Declaration. Enrolled patients provided written informed consent, and the specimen handling procedures complied with the Human Genetic Resources Management Regulations for biobank construction standards.

Statistical analysis

All the statistical analyses were performed using two-tailed tests, with a significance threshold of P < 0.05 applied to all the assessments. The R programming language was used for all the statistical calculations.

Results

Baseline characteristics of the study population

Our study conducted follow-up and analysis on 131 ovarian cancer patients (Table 1). The mean age of the cohort was 57.8 ± 10.2 years, with 57.52% being postmenopausal women aged more than 50 years, aligning with the disease’s high-risk demographic characteristics. Pathologically, epithelial carcinoma (89.3%, 117 cases) predominated, primarily high-grade serous carcinoma, along with germ cell tumours (4.6%, 6 cases) and metastatic tumours (6.1%, 8 cases); the latter frequently originated from gastrointestinal or primary breast sites. The average tumour diameter was 5.2 ± 2.8 cm, with 68.7% of the patients exhibiting unilateral involvement. Clinically, 62.5% (82 patients) presented with advanced-stage disease (FIGO III-IV) at initial diagnosis and 36.64% (48 patients) had malignant ascites. Tumour marker testing revealed a median CA125 concentration of 284.6 U/mL (range: 38–1520) and a median HE4 concentration of 315.8 pmol/L.

Table 1.

Baseline characteristics of the ovarian cancer

Characteristic	OC(n = 131)
Age(years)	57.8 ± 10.2
< 50	56(42.74%)
> 50	75(57.52%)
Diagnostic category	3
Epithelial OC	117(89.31%)
Germ Cell Tumors	6(4.6%)
Metastatic Tumors	8(6.1%)
FIGO staging, Ⅰ-Ⅱ/Ⅲ-Ⅳ	29(37.5%)/82(62.5%)
Tumor size(cm)	5.2 ± 2.8
Ovarian involvement, unilateral/bilateral	90(68.7%)/41(31.3%)
Median value of CA125(U/mL)	284.6
Median value of HE4(pmol/L)	315.8

Relationships between the expression of ESM1 and Pan-kla in patients with OC and clinical pathological factors

We evaluated the expression of ESM1 in OC patients in relation to various clinical parameters, including age, CA125 level, Pan-kla expression, and the presence of malignant ascites, and significant correlations were detected (Table 2). A marked difference was found between patients under and over 50 years of age (X² = 3.92, P = 0.0477), with a greater proportion of patients over 50 years of age showing high ESM1 expression. Additionally, CA125 levels strongly correlated with ESM1 expression (X² = 46.72; P < 0.0001). Notably, patients with high CA125 levels exhibited significantly higher ESM1 expression, further underscoring the relevance of ESM1 in the pathophysiology of OC. A similar strong association was observed between Pan-kla expression and ESM1 levels (X² = 85.41, P < 0.0001), where high Pan-kla expression was significantly linked to high ESM1 expression in tumour samples. Furthermore, the presence of malignant ascites was significantly correlated with ESM1 expression (X² = 12.45, P < 0.0001), with a higher proportion of patients with ascites showing elevated ESM1 expression. These findings suggest that ESM1 is closely associated with the key clinical features of OC, indicating its potential as a prognostic biomarker in ovarian cancer.

Table 2.

The expression of ESM1 in OC based on IHC staining

Pathological parameters	Patient number	ESM1 level		X²	P value
		Low	High
Age				4.10	0.043
< 50	56	23	33
> 50	75	18	57
CA125				46.72	< 0.0001
Low	23	21	2
High	108	20	88
Pankla				85.41	< 0.0001
Low	30	30	0
High	101	11	90
Malignant ascites				12.45	< 0.0001
Yes	48	6	42
No	83	35	48

Similarly, Pan-kla expression strongly correlated with advanced disease features (Table 3). High Pan-kla expression was present in 47 of 48 patients with malignant ascites, which was significantly higher than that in patients without ascites (54/83) (X² = 18.59; P < 0.0001). Furthermore, high CA125 levels were strongly associated with increased Pan-kla expression (96/108 vs. 5/23; P < 0.0001), and a robust correlation was noted between Pan-kla and ESM1 levels (X² = 85.41; P < 0.0001). Pan-kla expression also exhibited a modest but significant association with age, with a higher proportion of patients older than 50 years showing elevated expression (53/75 vs. 48/56; X² = 4.12; P = 0.0426). We evaluated ESM1 and Pan-kla expression in 131 °C tissue samples through immunohistochemistry. Both proteins were predominantly localized in the cytoplasm of OC cells. High ESM1 expression was observed in 96 patients, whereas Pan-kla expression was high in 101 patients. Representative immunohistochemical staining images are shown in Fig. 2.

Table 3.

The expression of Pankla in OC based on IHC staining

Pathological parameters	Patient number	Pankla level		X²	P value
		Low	High
Age				4.12	0.0426
< 50	56	8	48
> 50	75	22	53
CA125				48.42	< 0.0001
Low	23	18	5
High	108	12	96
ESM1				85.41	< 0.0001
Low	41	30	11
High	90	0	90
Malignant ascites				18.59	< 0.0001
Yes	48	1	47
No	83	29	54

Fig. 2.

Immunohistochemical staining images. Representative immunohistochemical staining images of ESM1 and Pan-kla expression. The distribution of the staining intensity in 131 samples is shown

Collectively, these findings demonstrate that increased ESM1 and Pan-kla expression is closely associated with adverse clinicopathological features, including elevated CA125 levels and malignant ascites, suggesting their potential utility as biomarkers of disease progression in OC.

Relationships between ESM1 and Pan-kla expression and clinicopathological factors and patient survival time

To evaluate the impact of ESM1 and Pan-kla expression on patient prognosis, we grouped patients on the basis of their IHC scores and compared survival differences among the groups through survival analysis. At the individual gene level, high ESM1 expression was identified as a significant unfavourable prognostic factor in ovarian cancer (HR = 3.15, 95% CI: 1.70–5.84, P < 0.001). These findings indicate that assessing ESM1 expression levels in ovarian cancer patients can effectively distinguish high-risk individuals (Fig. 3A). Concurrently, high expression of Pan-kla served as a significant marker of poor prognosis in ovarian cancer patients (HR = 0.27, 95% CI: 0.12–0.59, P = 0.001). Therefore, detecting Pan-kla protein expression levels is highly valuable for evaluating the prognosis of patients with ovarian cancer (Fig. 3B).

Fig. 3.

Prognostic impact of ESM1 and Pan-kla expression in ovarian cancer patients. A Kaplan–Meier survival curve analysis based on ESM1 expression. B Kaplan–Meier survival curve analysis based on Pan-kla expression. C Combined analysis of ESM1 and Pan-kla expression stratified patients into four distinct risk groups

Furthermore, combining the expression levels of ESM1 and Pan-kla allowed for highly significant stratification of ovarian cancer patients into four distinct risk strata with markedly different prognoses (overall P < 0.001, Fig. 3C). Specifically, the “dual-high” expression group (high ESM1 + high Pan-kla) had the worst prognosis and shortest survival time (HR = 5.075, 95% CI: 2.116–12.174), whereas the “dual-low” expression group (low ESM1 + low Pan-kla) had the most favourable outcome (HR = 0.650, 95% CI: 0.620–0.679). The groups with single high expression showed intermediate risk, with the ESM1 high + Pan-kla low group having an HR of 2.0534 (95% CI: 0.738–5.711) and the ESM1 low + Pan-kla high group having an HR of 1.1288 (95% CI: 0.136–9.396). This synergistic effect was highly statistically significant (P < 0.001), confirming that the “ESM1 + Pan-kla” combination constitutes a powerful prognostic model capable of effectively identifying high-risk patients and providing critical insights for individualized treatment strategies.

Discussion

Our study systematically analysed 131 ovarian cancer tissue samples to thoroughly investigate the expression characteristics, clinical significance, and molecular mechanisms of ESM1 and Pan-kla in ovarian cancer. The results demonstrated that both ESM1 and Pan-kla were significantly overexpressed in ovarian cancer tissues, with a strong positive correlation noted between their expression levels. Clinical pathological analysis revealed that high expression of ESM1 and Pan-kla was significantly associated with adverse clinical features, including advanced FIGO stage, elevated CA125 levels, and malignant ascites, suggesting their potential synergistic role in ovarian cancer progression.

Furthermore, survival analysis revealed that high ESM1 expression increased the risk of patient death, whereas high Pan-kla expression increased the risk. More importantly, the combined ESM1/Pan-kla risk stratification model effectively categorized patients into four distinct prognostic subgroups. The “dual-high” expression group had the worst prognosis, whereas the “dual-low” expression group had the most favourable outcome. This stratification capability was significantly superior to that of single biomarkers or traditional clinical indicators, highlighting the value of multidimensional biomarker assessment in precision medicine.

From a mechanistic perspective, the results of this study suggest that ESM1 and Pan-kla may promote ovarian cancer malignancy through multiple pathways. Our previous research indicated that ESM1 promotes cholesterol synthesis and lipid metabolism reprogramming via the IGF2BP3/ESM1/KLF10/BECN1 axis [5] while also activating the Akt/mTOR/VEGF-A signalling pathway to mediate angiogenesis [10, 12]. Additionally, Pan-kla, as a novel metabolic-epigenetic modification, is produced via the glyoxalase pathway (GLO1/GLO2) from the glycolytic byproduct methylglyoxal, leading to D-lactate modification of key immune signalling proteins. This inhibits the production of proinflammatory cytokines and negatively regulates innate immune responses. Our previous research indicated that the accumulation of lactate in the ovarian cancer tumour microenvironment may promote the expression of ESM1, which in turn further suppresses the activity of CD8 + T cells [11]. Concurrently, targeting the ESM1-PKM2 interaction can reverse the Warburg effect and inhibit vasculogenic mimicry [9]. Furthermore, intervention in the lactate-ESM1-lipid metabolism axis may restore the antitumour immune response [11]. This study is the first to focus on the use of ESM1/Pan-kla as potential therapeutic targets, providing novel insights for overcoming resistance to chemotherapy and immunotherapy in ovarian cancer.

However, this study has several limitations, including its single-centre retrospective design and relatively limited sample size. Future multicentre prospective studies are needed to validate its clinical applicability and to further elucidate the role of the interaction between ESM1 and Pan-kla in ovarian cancer progression. Furthermore, exploring small-molecule inhibitors targeting this pathway or combination therapy strategies will be important for translational research.

In summary, this study not only confirms the value of ESM1 and Pan-kla as prognostic biomarkers for ovarian cancer but also reveals the central role of metabolic–epigenetic cross-regulation in tumour progression, providing a theoretical and experimental basis for the development of novel therapeutic strategies. Further exploration in this research direction is expected to open new avenues for the precise treatment of ovarian cancer.

Conclusion

This study confirms that ESM1 and Pan-kla are significantly coexpressed and positively correlated in ovarian cancer tissues and that their high expression is closely associated with adverse prognostic features such as advanced clinical stage, malignant ascites, and elevated CA125 levels. This research not only reveals the critical role of metabolic–epigenetic cross-regulation in ovarian cancer progression but also provides a novel biomarker combination for prognostic stratification, with significant clinical implications for improving individualized treatment and overcoming drug resistance strategies in ovarian cancer.

Abbreviations

OC

Ovarian cancer

Pan-kla

Protein lysine L-lactylation modification

IHC

Immunohistochemistry

EOC

Epithelial ovarian cancer

TME

Tumour microenvironment

ESM1

Endothelial cell-specific molecule 1

PA

Palmitic acid

AS

Atherosclerosis

VM

Vascular mimicry

Authors’ contributions

Xiyun Quan and Yukun Li: conceptualization, methodology, software. Wenchao Zhou, Yang Zhou and Tian Zeng: data curation, writing- original draft preparation. Zhenqin Guo and Yi Deng: visualization, investigation. Yukun Li: supervision. Xiyun Quan: validation. Yukun Li and Xiyun Quan: writing- reviewing and editing.

Funding

The present study was supported by the Natural Science Foundation of China (82303246), the Natural Science Foundation of Hunan Province (2025JJ50493) and the Health Research Project of the Hunan Provincial Health Commission (W20243173).

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

This study included 131 °C tissue samples from patients with ovarian cancer collected by the Department of Pathology at Zhuzhou Hospital Affiliated with the Xiangya School of Medicine between 2014 and 2024. All sample collection and utilization protocols were reviewed and approved by the Hospital Ethics Committee (#LLYPJ2025004-01), which strictly adhered to the Helsinki Declaration. Enrolled patients provided written informed consent, and the specimen handling procedures complied with the Human Genetic Resources Management Regulations for biobank construction standards.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.