EPDR1 promotes gastric cancer progression via STAT3-mediated fatty acid metabolic reprogramming.

Introduction
Gastric cancer (GC) is a heterogeneous malignant tumor occurring in any part of the stomach, the most common of which is adenocarcinoma. It originates from the innermost glands of the stomach [1]. GC pathogenesis is complex, involving abnormal regulation of various genes and pathways. Surgical resection is an effective treatment, and advancements in targeted therapy and immunotherapy have improved survival rates [2]. However, the prognosis for GC patients remains poor, with a low overall 5-year survival rate [3]. Due to the unusually active proliferation, extensive metastasis, therapeutic resistance of GC cells, and the lack of typical symptoms and effective early diagnostic biomarkers, GC patients are often diagnosed in advanced or late stages [4]. Therefore, exploring the molecular mechanisms of GC pathogenesis and progression, and identifying biomarkers for diagnosis and prognosis monitoring, could enhance early diagnosis rates, facilitate targeted drug development, and improve GC treatment.
The EPDR1 gene, also known as MERP1 and UCC1, encodes a protein related to ependymal proteins and is a member of the ependymal protein family [5]. Ependyma is a type II transmembrane protein and a glycoprotein family involved in cell–cell contact [6]. Recent studies have suggested that EPDR1 is closely linked to the pathological or developmental processes of various tumors. EPDR1 expression is significantly increased in tumor tissues, promoting cell proliferation, migration, invasiveness, and adhesion to type I collagen fibers [7, 8]. In obese individuals, the upregulation of EPDR1 may enhance β-cell function, helping to maintain glucose homeostasis [6]. Notably, there are significant differences in EPDR1 expression between BLCA and adjacent normal bladder tissues, with EPDR1 levels increasing as BLCA tumors progress and metastasize [9]. EPDR1 protein has been detected in human plasma samples, indicating its involved in intertissue metabolic crosstalk. Silencing human brown fat cells EPDR1 reduces norepinephrine (NE)-induced mitochondrial proton leak respiration and affects mitochondrial protein expression [10]. These findings suggest that EPDR1 regulates systemic energy metabolism through regulating mitochondrial metabolism in target cells. EPDR1 has potential as a novel prognostic biomarker and may become an effective target for the diagnosis and treatment of certain cancers [9, 11]. Given that EPDR1 is expressed differently in various cancers, it is believed to have complex roles in carcinogenesis and metastasis. However, EPDR1 has not been studied in GC. Its mechanism of action on the progression of GC remains unclear.
The JAK/STAT signaling pathway is a widely expressed signal transduction pathway in cells, involved in cell proliferation, differentiation, apoptosis, and immune regulation [12]. The STAT family consists of seven proteins, with STAT3 being one of them [13]. Multiple studies have shown that STAT3 is abnormally expressed in various cancers and plays a significant role [14, 15]. Specifically, abnormal STAT3 expression is associated with GC patients, make it a promising candidate as a prognostic biomarker for GC [14, 16]. Abnormal expression of STAT3 contributes to the proliferation of cancer cells, promotes inflammation, and facilitates EMT metastasis [17, 18]. Immunohistochemical staining of tissues from patients with gastric adenocarcinoma revealed a high correlation between STAT3 expression and TNM stage and survival rate [19]. These findings suggest that STAT3 can serve as a diagnostic biomarker, and limiting its activity may help prevent malignancies. Overall, these findings suggest that targeting abnormal JAK/STAT signaling in GC may hold great potential as a new therapeutic intervention for cancer patients.
Metabolic reprogramming is recognized as a common hallmark of cancer. It supports the prolonged energy requirements of tumor cells due to rapid growth and proliferation [20]. Fatty acid oxidation (FAO), an important energy metabolism pathway, plays a crucial role in tumor energy metabolism reprogramming [21, 22]. FAO consists of a series of cyclic reactions that lead to fatty acid shortening and the production of important metabolites [23, 24]. Fatty acid oxidation can provide higher energy than carbohydrate, and tumor cells can improve the efficiency of energy supply by increasing the level of FAO [25]. Recently, increasing evidence has shown that tumor cells need to provide a metabolic advantage to survive through metabolic reprogramming, which includes changes in fatty acid (FA) metabolism [26]. providing a selective advantage for cancer cells during metastasis [27]. Rapidly proliferating tumor cells require large amounts of cholesterol for membrane organelle and FA synthesis. Therefore, targeting key enzymes that mediate fatty acid metabolism, such as CD36 and CPT1A [27], has been shown to be effective in inhibiting cancer metastasis. But it is still unclear how GC cells reprogram the regulatory mechanism of FAO during metastasis.
This study seeks to uncover the mechanism by which EPDR1 regulates FAO reprogramming in GC through modulation of CPT1A activity. The findings indicate that the expression level of EPDR1 has increased in GC, and it may via STAT3 bind CPT1A and activated it, thereby influencing fatty acid oxidation in GC. Furthermore, high EPDR1 expression was identified as an independent prognostic factor for predicting cancer patient outcomes. These results highlight the crucial role of fatty acid oxidative reprogramming in the biological characteristics of cancer malignancy and offer novel insights for selecting therapeutic targets and prognostic markers, with the potential to contribute to early cancer detection and treatment.

Materials and methods

Collection of clinical samples
Primary tumor tissues and paired normal gastric tissues (5 cm above tumor margins) were collected from 30 GC patients during surgery. Samples were divided into aliquots for immediate liquid nitrogen freezing (−196 °C) or 4% formaldehyde fixation/paraffin embedding. Ethical approval was obtained from Zhongshan Hospital (Xiamen) Ethics Committee (No. B2023-133).

Cell culture
GC cell lines (MGC803, AGS, NCI-N87, HGC-27, BGC823) and normal gastric epithelial cells (GES-1) were acquired from Shanghai Cell Bank of Chinese Academy of Sciences. Cells were maintained in DMEM/RPMI-1640 (Gibco, Grand Island, NY, USA) supplemented with 10% FBS under 37 °C/5% CO₂, with medium replacement every 48 h.

Other methods
Additional methods are described in Data S1. The details of the primer sequences are provided in Suppl. Table I.

Results

EPDR1 is upregulated in STAD and associated with poor prognosis in GC patients
EPDR1 has shown both oncogenic and tumor-suppressive properties [7], but its specific role in STAD remains unclear. To explore this, we analyzed the TCGA database and GES27342 database and found that EPDR1 expression is significantly upregulated in STAD (Figs. 1A and S1A), suggesting a potential link between EPDR1 and STAD. In order to verify the relationship between EPDR1 and GC, we measured EPDR1 mRNA levels in 54 pairs of GC tissues and matched normal gastric mucosa tissues using qRT-PCR, revealing increased EPDR1 levels in GC tissues (Fig. 1B). Western blot analysis of 10 pairs of GC tissues and normal gastric mucosa tissues further confirmed elevated EPDR1 protein levels in GC tissues (Fig. 1C). Additionally, qRT-PCR and Western blot were used to assess EPDR1 mRNA and protein levels in normal gastric epithelial cell lines (GSE-1) and GC cell lines (MGC-803, AGS, NCI-N87, HGC-27, and BGC-823), demonstrating high EPDR1 expression in GC cell lines (Fig. S1D, E). Immunohistochemical analysis of a larger sample size further confirmed EPDR1 overexpression in GC tissues compared to normal tissues (Fig. 1D). Quantitative PCR analysis provided specific differential data on EPDR1 expression between GC tissues and normal gastric mucosa tissues (Fig. 1E). These results indicate that EPDR1 is upregulated in STAD, suggesting a potential relationship between EPDR1 and GC.
The elevated EPDR1 expression in STAD tissue prompted us to explore its clinical correlation with GC progression, prognosis, and recurrence. Analysis of clinical data from 192 GC patients revealed that high EPDR1 expression was associated with poorer OS and DFS compared to low expression (Fig. 1F, G), indicating that high EPDR1 expression may predict a worse treatment response and shorter survival. To assess the effect of EPDR1 expression on prognosis, Cox proportional hazards model was used for independent prognostic regression analysis revealed that EPDR1 (HR = 1.168; P = 0.043) may serve as an independent diagnostic indicator for the overall survival of GC patients (Figs. 1H, S1B). A nomogram predicting 1-, 2-, 3-, and 5-year overall survival of GC patients showed that EPDR1 accounted for a large proportion of the total score (Fig. S1C), indicating its critical role as a prognostic factor. These results confirm that high EPDR1 expression is an independent prognostic indicator of poor overall survival in GC patients. Overall, these findings suggest that EPDR1 upregulation in STAD and its high expression in GC are closely linked to poor prognosis, providing new insights and a potential target for GC prognosis evaluation and treatment.

EPDR1 functions as an oncogene and accelerates the progression of GC
Given the elevated expression of EPDR1 in STAD tissues, we hypothesized that EPDR1 might act as a tumor-promoting factor in STAD. To validate this hypothesis, we used shRNA to establish EPDR1 knockdown cell models, sh-EPDR1#1 and sh-EPDR1#2, in HGC-27 and NCI-N87 cell lines with high EPDR1 expression. qRT-PCR and Western blot analysis confirmed the accurate and effective knockdown of EPDR1 in GC cells (Fig. 2A, B). Subsequently, we conducted a series of in vitro cell function experiments to evaluate the role of EPDR1 in GC cell proliferation, invasion, and migration. CCK-8 and Colony formation assays demonstrated inhibited cell proliferation upon EPDR1 knockdown (Fig. 2C, D). Consisted these results, the EDU assay also revealed a decrease in proliferation ability upon EPDR1 knockdown. (Fig. S2A). Transwell assays demonstrated reduced invasion abilities (Fig. 2E), and wound healing assays showed decreased migration abilities upon EPDR1 knockdown (Fig. S2B). These findings suggest EPDR1's crucial role in GC proliferation, metastasis, and invasion.
Explore the function of EPDR1 in vivo, we conducted subcutaneous tumorigenesis experiments in nude mice. The results demonstrated that EPDR1 knockdown effectively inhibited the growth of subcutaneous GC tumors (Fig. 2F), confirming its role in promoting GC cell proliferation. Tumor growth curves showed a significant reduction in tumor formation speed in the EPDR1 knockdown group (Fig. 2G). After 20 days, tumor volume and weight were reduced in the knockdown group compared to the normal expression group (Fig. 2H), suggesting that EPDR1 can promote the tumor formation of GC cells in vivo.
To further understand the effects of EPDR1 on biological processes such as GC cell proliferation and epithelial mesenchymal transition (EMT), we performed immunohistochemical (IHC) staining on tumor slices. The results revealed high EPDR1 protein expression in GC tissues, with decreased Ki-67 and N-cadherin expression and increased E-cadherin expression upon EPDR1 knockdown (Fig. 2I). Western blot analysis showed reduced N-cadherin and vimentin, and increased E-cadherin in EPDR1 KD cells (Fig. S2C), indicating EPDR1 promotes GC cell proliferation and metastasis by influencing EMT. Overall, our findings provide compelling evidence that EPDR1 acts as an oncogene and accelerates GC progression.

Knockdown of EPDR1 expression negatively regulates fatty acid metabolism in GC
To elucidate the specific mechanism underlying pro-oncogenic role of EPDR1, we assessed mRNA expression levels in GC cells after EPDR1 knockdown using transcriptome sequencing technology, providing extensive gene expression data to analyze EPDR1's impact on GC cell metabolism (Fig. 3A). We conducted enrichment analysis using KEGG and GSEA. The results indicated the abnormal expression of EPDR1 in GC cells is closely related to fatty acid metabolism (Fig. 3B, C). It has been reported that alterations in fatty acid metabolism can confer metabolic advantages to tumor cells [27]. This suggests that EPDR1 may influence the biological behavior of GC cells by regulating fatty acid metabolism, particularly through CPT1A, the key rate-limiting enzyme of FAO (Fig. 3D). To further investigate the connection between EPDR1 and fatty acid metabolism, we also performed targeted metabolomics analysis of acylcarnitines and acyl-CoA to confirm the utilization of fatty acids through the carnitine shuttle system. The results showed that the levels of acylcarnitines and acyl-CoA were significantly altered in shEPDR1 cells (Fig. 3E). Therefore, we investigated whether EPDR1 contributes to metabolic reprogramming in GC. We performed Seahorse-based metabolic flux analysis to assess the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in EPDR1 knockdown (KD) cell lines. The absence of EPDR1 KD cells resulted in decreased basal and FCCP-induced maximal respiration, ATP production, and spare respiratory capacity (Figs. 3F, G and S3A, B). This indicates that knocking down EPDR1 expression affects the biological behavior of GC cells by reducing fatty acid metabolism. Overall, these results suggest that EPDR1 positively regulates fatty acid metabolism in GC, and knockdown of EPDR1 expression inhibits fatty acid metabolism in GC cells.

EPDR1 reprograms FAO pathway in GC
Considering that EPDR1 regulates lipid changes in cells, we examined whether deletion of EPDR1 could inhibit FAO to meet bioenergetic requirements in GC cells. We initially observed a positive correlation between EPDR1 and CPT1A expression in GC through Spearman analysis (Fig. 4A). Given CPT1A's role as a key enzyme in the FAO pathway, we hypothesized that EPDR1 might influence the FAO pathway via CPT1A regulation. Subsequent analysis revealed that EPDR1 silencing decreased CPT1A mRNA and protein levels in EPDR1 knockdown (KD) cells (Fig. 4B, C), along with a reduction in CPT1A enzyme activity (Fig. S4A). Conversely, EPDR1 overexpression (OE) induced CPT1A mRNA and protein expression (Fig. S4B, C). Western blot analysis indicated that knocking down EPDR1 reduced the expression of CPT1A in GC cells; however, overexpression of CPT1A did not increase the expression of EPDR1. This suggested that CPT1A was a downstream molecule of EPDR1 (Fig. S4D). Additionally, we confirmed through the colony formation assay that knocking down EPDR1 could inhibit the proliferative capacity of CPT1A OE cells (Fig. S4E). The next, intracellular lipid staining using Nile Red showed increased lipid content in EPDR1 KD cells (Fig. 4D). When treating HGC-27 and NCI-N87 cells with BSA-conjugated Oleic acid, we found that EPDR1 KD cells exhibited significantly enhanced proliferation and invasion abilities (Fig. S4F, G). Mass spectrometry-based carnitine metabolomics analysis further demonstrated reduced levels of several carnitine intermediates in cells after EPDR1 KD (Fig. 4E). To assess the therapeutic implications of EPDR1-mediated FAO regulation, we treated cells with the CPT1A inhibitor etomoxir, which abolished the increased clonal potential of EPDR1 OE cells (Fig. 4F). Additionally, etomoxir treatment abrogated the enhanced wound healing potential of EPDR1 OE cells in a wound healing assay (Fig. 4G). Overall, our findings suggest that EPDR1 contributes to FAO regulation, and its regulation of FAO activity presents a potential metabolic vulnerability in GC.

EPDR1 regulates the JAK/STAT pathway in GC
To delve into the signaling pathways underlying EPDR1-mediated FAO regulation, we conducted gene set enrichment analysis (GSEA). Our results revealed that the JAK/STAT pathway was significantly inhibited upon EPDR1 knockdown (Fig. 5A), implying EPDR1's involvement in activating this pathway. Considering STAT3 as a pivotal transcription factor in the JAK/STAT signaling pathway, we analyzed its levels in the EPDR1 KD cell line. Western blot analysis demonstrated that silencing EPDR1 suppressed the expression of p-STAT3, STAT3, and CPT1A (Fig. 5B). These findings confirm EPDR1's role in activating the JAK/STAT signaling in GC.
Then explored whether STAT3 activation modifies the FAO pathway in GC cells. STAT3 knockdown inhibited the promoter of CPT1A and decreased CPT1A mRNA expression in GC cells (Figs. 5C and S5A). ChIP-PCR experiments further showed that the CPT1A promoter binds to STAT3 in GC cells (Fig. 5D), providing direct molecular evidence of the interaction between CPT1A and STAT3. Next, we assessed the changes in GC cell energy metabolism after treatment with the STAT3 inhibitor Stattic. The results indicated that Stattic inhibited lipid metabolism (Fig. S5B) and reduced OCR and ATP production in GC cells (Fig. S5C). These observations suggest that STAT3 activation is crucial in the EPDR1-regulated FAO pathway in GC.
Spearman analysis revealed a positive correlation between CPT1A and STAT3 expressions, as well as between EPDR1 and STAT3 expressions in GC (Fig. 5E). Furthermore, we investigated whether JAK/STAT signaling contributes to EPDR1-mediated regulation of the FAO pathway in GC cells. As anticipated, Stattic treatment abrogated the proliferative and invasive effects of EPDR1 OE cells (Figs. 5F and S5D). Collectively, our data suggest that EPDR1 regulates the FAO pathway by activating JAK/STAT signaling, which subsequently promotes GC metastasis.

Targeting EPDR1 can improve chemotherapy sensitivity in GC
Colony formation assays revealed that both Stattic and 5-FU significantly inhibited the proliferation of GC cells, with a further strengthened inhibitory effect when the two drugs were combined (Fig. 6A), indicating potential synergistic effects. This was corroborated by apoptosis experiments, where both drugs promoted apoptosis in these cells, with a significantly increased level when used together (Fig. 6B). In vivo assays in nude mice further verified the effectiveness of Stattic and 5-FU in GC treatment, with both drugs significantly inhibiting subcutaneous tumor growth and enhanced inhibitory ability when combined (Fig. 6C). Tumor volume monitoring showed a time-dependent decrease in tumor size for both drugs, with the combination resulting in smaller tumors (Fig. 6D). On the 25th day, tumor weights were significantly reduced by both drugs (Fig. 6E). In summary, our experimental results demonstrate that targeting EPDR1 enhances chemosensitivity in GC by inhibiting cell proliferation and promoting apoptosis, offering a novel strategy with potential clinical application for GC treatment. Additionally, this study provides a foundation for further research on the mechanism of targeting EPDR1 in GC therapy.

Discussion
Accumulating evidence suggests that metabolic reprogramming in cancer cells supports their energy and metabolic demands during tumor metastasis. Reprogramming lipid metabolism acts a pivotal role in tumorigenesis [28]. EPDR1 has currently been identified in the secretome of adipocytes. And it plays an important role in the process of adipogenesis [10]. Utilizing KEGG and GSEA, we analyzed the mRNA expression levels following EPDR1 knockdown in GC cells and found that fatty acid metabolic pathways were notably enriched in GC and the expression of CPT1A was significantly decreased. Preliminary results indicated that EPDR1 might directly influence and activate CPT1A by modulating STAT3 phosphorylation in the JAK/STAT signaling pathway, thereby promoting fatty acid oxidation and GC progression. Therefore, we emphasized the significant value and potential of the EPDR1–CPT1A–STAT3 axis in the treatment of GC.
EPDR1 possesses lipid-binding molecular function, participates in the biological process of cell matrix adhesion, and its cellular components are localized to the extracellular region and lysosome [29]. Additionally, the absence of EPDR1 in β-cells impairs mitochondrial respiration and increases the ATP/ADP ratio. This suggests that EPDR1 can regulate and influence cellular metabolic processes [30]. While its role in various cancer progressions has been established [6, 7, 9], its function and mechanism of action in GC remain unreported. Our study revealed that EPDR1 played a positive regulatory role in GC, and high EPDR1 expression is an independent prognostic indicator of poor overall survival in GC patients. We performed sequencing and analysis of the transcriptome of GC cells with EPDR1 knockdown and observed a remarkable correlation between aberrant EPDR1 expression in GC cells and fatty acid metabolism. Additional experiments further confirmed that knockdown of EPDR1 suppressed fatty acid metabolism in GC. These findings indicate that upregulated EPDR1 in GC cells elevates the level of fatty acid metabolism, conferring a metabolic advantage for tumor development and subsequently promoting GC progression. Overall, we proposed for the first time that EPDR1 plays a crucial role in the reprogramming of fatty acid metabolism in cancer cells.
Studies in ovarian cancer models have shown that lipids produced by adipocytes can be transferred to cancer cells, promoting tumor growth, indicating that local adipose tissue may directly support cancer cells [31]. Furthermore, the absorption of fatty acids by adipocytes enables cancer cells to survive under nutrient-deprived conditions by enhancing mitochondrial fatty acid oxidation (FAO) [32]. MSC-NPRA loop drives fatty acid oxidation to promote stemness and chemoresistance of gastric cancer [33]. LINC00924 is overexpressed in GC and promotes FAO and fatty acid uptake, which are crucial for the survival and sphere formation of matrix-isolated GC cells [34]. CPT1A, a crucial rate-limiting enzyme in the FAO pathway, governs the influx of fatty acids into mitochondria for oxidation [35]. Its expression has been found to be elevated in various tumors and is linked to cancer exacerbation and fatty acid oxidative reprogramming [36]. Targeting CPT1A in the mitochondrial FAO pathway has demonstrated clinical benefits in radiotherapy for nasopharyngeal carcinoma (NPC) and cancer patients [36]. pep-AKR1C2 promotes CPT1A expression by modulating YAP phosphorylation, thereby enhancing FAO and ATP production [37]. Inhibition of lipid metabolism and FAO key genes has been shown to hinder the progression of solid tumors [38]. In this study, our findings aligned with these observations. Upon knockdown of EPDR1, the enzyme activity and expression level of CPT1A in GC cells were significantly reduced, cellular fat content increased, and the level of intracellular carnitine metabolites decreased notably. Etomoxir was able to block the proliferative, migratory, and invasive effects of EPDR1. However, we have yet to elucidate the specific molecular mechanisms by which EPDR1 regulates the expression of CPT1A. Overall, our study indicated that EPDR1 influences the FAO pathway by positively regulating CPT1A, which subsequently impacts the development of GC.
JAK/STAT signaling pathway is commonly observed to be aberrantly activated in cancer [39]. Our findings through GSEA revealed that the JAK/STAT pathway was significantly inhibited after knockdown of EPDR1. Further western blot experiments confirmed a decrease in the protein expression levels of p-STAT3 and STAT3 in GC cells upon EPDR1 knockdown. The use of Stattic blocked the proliferative effects of EPDR1, supporting the notion that EPDR1 influences cell proliferation via the JAK/STAT pathway. STAT3, a transcriptional activator and member of the STAT protein family, STAT3 acts as a transcription factor due to its SH2 domain [40] However, the SH2 domain's role in the activation and signaling of other pathways, such as the PI3K pathway, suggests it may be involved in a broader range of pathways. In summary, our study elucidated the potential of targeting EPDR1 in conjunction with CPT1A inhibitors and STAT3 inhibitors for the treatment of GC. However, this still needs to be confirmed in further in vivo studies.
In brief, our study has uniquely demonstrated the elevated expression of EPDR1 in GC and established a positive correlation between the expression of CPT1A and STAT3 with EPDR1 in this context. This is the first instance where the JAK/STAT signaling pathway and the FAO pathway have been jointly examined and analyzed. Our findings suggest that EPDR1 may instigate FAO reprogramming via STAT3-mediated transcriptional activation of CPT1A, thereby augmenting the malignant biological traits of GC and ultimately facilitating its progression. Moreover, given the concurrent expression of these genes in GC, they may present themselves as viable targets for GC treatment. The simultaneous targeting of these genes or their respective signaling pathways could potentially offer a more efficacious strategy to impede the proliferation of GC cells. Nevertheless, it is important to note that this study is based on cell experiments with a restricted clinical sample size. Hence, further corroboration of our findings may necessitate a larger clinical sample size or multicenter data in the future. Furthermore, while this study has specifically focused on the interplay between EPDR1 and the JAK/STAT signaling pathway and the FAO pathway, it has not delved into other factors and pathways that may also play a role in cancer progression.

Supplementary Information