NCEH1 promotes breast cancer progression by regulating NRP1 and activating the TNF-α/NF-κB signalling pathway.

Introduction
Breast cancer is a highly prevalent malignant tumour worldwide, characterized by high heterogeneity. Its incidence rate reaches 11.5%, ranking second only to lung cancer [1,2]. Although current treatments have expanded from traditional approaches such as local excision, chemotherapy, radiotherapy, and endocrine therapy to the field of targeted therapies, patients still face the challenge of tumour recurrence and metastasis [3]. Therefore, identifying suitable biomarkers for breast cancer is critical for elucidating its pathogenesis and improving survival rates.
Neutral cholesterol ester hydrolase 1 (NCEH1) is one of the key hydrolases in the lipid metabolic pathway in cells. NCEH1 is a serine hydrolase. The gene encoding NCEH1 is located on human chromosome 3. Structurally, its N-terminus is connected to the endoplasmic reticulum (ER) membrane, while the catalytic domain resides within the ER lumen. This structural characteristic allows it to play a catalytic role at specific locations within cells and participate in lipid metabolism processes [4].
According to a previous study, it has been found that NCEH1 can promote the hydrolysis of 2-acetylmonoalkyl glycerol ether to produce monoacylglyceride enzyme. Both are overexpressed in various cancer cells such as melanoma, pancreatic cancer, and ovarian cancer. The high expression is closely related to cancer cell proliferation, invasion, and other aggressive behaviours [5]. Recent studies have shown that NCEH1 is highly expressed in various malignant tumours. Single-cell RNA sequencing analyses demonstrate high NCEH1 expression in pancreatic cancer samples. This genetic marker serves as an independent prognostic factor and a biomarker for predicting chemoresistance and immune cell infiltration [6]. In addition, Tang et al. further identified NCEH1 as a prognostic predictor for gastric cancer patients and
a marker of response to chemotherapy combined with immune checkpoint inhibitors through transcriptomic analysis of cholesterol metabolism genes [7]. Similarly, RNA sequencing in head and neck cancer epithelial-mesenchymal transition (EMT) revealed that an NCEH1-based gene risk score predicts overall survival (OS) [8].
Neuropilin-1 (NRP1) is a glycoprotein that plays critical roles in various processes, including angiogenesis, tumour malignant progression, and the regulation of immune system homeostasis [9,10]. Previous studies have demonstrated that NRP1 acts as a co-receptor binding multiple extracellular ligands. It not only promotes tumour angiogenesis by enhancing the vascular endothelial growth factor (VEGF) signalling pathway but also interacts with various ligands such as semaphorins and transforming growth factor β), thereby activating multiple downstream oncogenic signalling pathways and regulating tumour cell migration and invasion [11,12]. Multiple evidences indicate abnormal NRP1 expression and its carcinogenic effects in various malignancies [13–15]. For instance, overexpression of NRP1 in breast cancer accelerates tumour progression [16]. In non-small cell lung cancer cells, the expression level of NRP1 shows a synchronized increase with the expression of EMT-related proteins [17]. Furthermore, NRP1 influences the stability of regulatory T cells within the tumour microenvironment, participating in immune regulation [18].
It is well-known that TNF-α can accelerate malignant cancer progression by promoting cell proliferation, inhibiting apoptosis, and inducing angiogenesis [19]. NF-κB is a key regulatory molecule in the TNF-α signalling pathway [20,21]. Studies demonstrate that NF-κB mediates multiple processes such as angiogenesis, inflammatory responses, cell proliferation, metastasis, and tumour development in various eukaryotic cells [22]. In glioblastoma cells, inhibition of NF-κB activity significantly downregulates the expression of the pro-angiogenic factor VEGF [23]. The NF-κB signalling pathway plays a significant role in the development and metastasis of breast cancer [24]. Frequent overactivation of this pathway creates a favourable microenvironment for tumour growth [25]. It regulates the expression of various molecules associated with cell invasion, promotes EMT, and accelerates the metastatic process [26].
Given its overexpression in multiple cancers and strong association with tumour progression, NCEH1 may represent a potential therapeutic target. We hypothesize that NCEH1 may play a critical role in breast cancer. This study aims to elucidate its biological functions and mechanisms in breast cancer. Our findings demonstrate that the expression level of NCEH1 in breast cancer is significantly increased. NCEH1 can promote the proliferation and migration of breast cancer cells and participate in the regulation of EMT. We further demonstrated that NCEH1 can affect NRP1 expression and promote breast cancer progression through the TNF-α/NF-κB signalling pathway.

Methods

Bioinformatics analysis
The expression of NCEH1 in tumour and normal tissues was analysed using the GEPIA2 platform (http://gepia2.cancer-pku.cn/) [27] based on the Genotype-Tissue Expression (GTEx) and the Cancer Genome Atlas (TCGA) databases. The criteria are as follows: log2 Fold change = 1 and p value ≤0.01. The Kaplan–Meier Plotter database (https://kmplot.com/analysis/) [28] was employed to evaluate the correlation between NCEH1 expression and relapse-free survival (RFS) or distant metastasis-free survival (DMFS) in breast cancer patients. The UALCAN database (http://ualcan.path.uab.edu) [29] assessed the association of NCEH1 with axillary lymph node metastasis. TIMER 2.0 (https://cistrome.shinyapps.io/timer/) [30] was used to analyse the correlation between NCEH1 and NRP1 expression, visualized as scatter plots with a significance threshold of p valve set to less than 0.05.

Cell lines
Breast cancer cell lines (MDA-MB-231, MDA-MB-453, HCC1806, HCC1954, MCF-7, SK-BR3), human mammary epithelial cells (MCF-10A), and HEK293T cells were obtained from the American Type Culture Collection (ATCC). MCF-10A cells were cultured in mammary epithelial cell-specific medium (LONZA, CC-3151). MDA-MB-231, MCF-7, SK-BR3, and HEK293T cells were cultured in DMEM (GIBCO,
C11995500BT), HCC1806 and HCC1954 cells were cultured in RPMI 1640 (GIBCO, C11875500BT), and MDA-MB-453 cell was cultured in L-15 (Solarbio, LA9510), all supplemented with 10% fetal bovine serum (FBS) (VivaCell, C04001-500), 100 μg/mL streptomycin, and 100 U/mL penicillin (NCM Biotech, C100c5). All cells were cultured at 37°C in a humidified incubator with 5% CO2.

siRNA transfection
MDA-MB-231 and MCF-7 cells at 50% confluence in 6-well plates were transfected with specific small interfering RNA (siRNAs) targeting NCEH1 (si-NCEH1-1, 5’-GGCUAGUUCCAAAGGUUUATT-3′, 5′-UAAACCUUUGGAACUAGCCTT-3′. si-NCEH1-2, 5′-CCGGACUAGGAAUAGUUACAUTT-3′, 5′-AUGUAACUAUUCCUAGUCCGGTT-3’.) or nonspecific siRNA (Sangon Biotech (Shanghai) Co., Ltd.) using Lipofectamine 2000 (Invitrogen, 11,668,019). Each siRNA and transfection reagent were diluted in serum-free medium (GIBCO, 31,985,070), mixed and incubated for 20 min before adding to cells. Forty-eight hours after transfection, knockdown efficiency was validated via qPCR and Western blotting.

Cell viability and colony formation assays
Cell viability and colony formation assays used specific siRNA or overexpressing vectors to transfect breast cancer cells. For viability assays, transfected MDA-MB-231 and MCF-7 cells were seeded in 96-well plates. At indicated time points, 10 μL of Cell Counting Kit-8 (CCK8) (NCM Biotech, C6005) reagent was added per well, incubated for 2–4 h, and the optical density (OD) at 450 nm was measured using a Bio-Rad microplate reader. For colony formation assays, transfected cells were harvested and re-seeded into 6-well plates and cultured for 7–14 days until cell colonies appeared. Cell colonies were fixed in methanol for 10 min, stained with 0.1% crystal violet (Solarbio, C8470) at room temperature, protected from light for 15 min, photographed and counted.

Scratch wound healing and Transwell migration assays
Scratch wound healing and Transwell migration assays used specific siRNA or overexpressing vectors to infect breast cancer cells. For scratch assays, transfected MDA-MB-231 and MCF-7 cells were scratched by using a sterile 200 μL pipette tip in 6-well plates until cell fusion reached 80–90%. Cells were washed and cultured in serum-free medium. The scratched areas were photographed using a microscope at 0 and 24 h, respectively. For the Transwell migration assay, the transfected cells were resuspended in 100 μL of serum-free medium and added to the upper chamber, while the lower chamber contained 600 μL of complete medium with 10% FBS in a 24-well plate. After 24 h, cells were fixed in methanol for 10 min and stained with 0.1% crystal violet for 15 min, protected from light. Wipe it clean and use a microscope (Leica) to image.

Clinical samples
Samples of breast tumours and adjacent tissues were collected from patients diagnosed with breast cancer at the Second Affiliated Hospital of Dalian Medical University. Tissues were collected and stored in a refrigerator at −80°C.
All operations in this study were carried out in accordance with the Declaration of Helsinki. All participating patients have signed informed consent forms. The study protocol was approved by the Institutional Ethics Committee (Project Registration Number: KY2025-114–01).

Western blotting
Cells were lysed in Radio Immunoprecipitation Assay (RIPA) (Beyotime, P0013B) buffer with protease and phosphatase inhibitors. After thorough lysis and centrifugation, the protein concentration of the supernatant was measured using the bicinchoninic acid (BCA) protein detection kit (TIANGEN, PA115-02). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE),
transferred to nitrocellulose (NC) membranes (Milipore, HATF08130), and blocked with 5% skim milk 1 h. Then the primary antibody was incubated overnight at 4°C. The main antibodies used in this study include NCEH1 (1:1000, Solarbio), NRP1 (1:1000, Proteintech)、E-cadherin (1:1000, Cell Signaling Technology), N-cadherin (1:1000, Cell Signaling Technology), Vimentin (1:1000, Cell Signaling Technology), β-catenin (1:1000, Cell Signaling Technology), Slug (1:1000, Cell Signaling Technology), Snail (1:1000, Cell Signaling Technology), TNF-α (1:1000, Wanleibio), NF-κB (1:1000, Wanleibio), p-NF-κB (1:1000, Wanleibio), IκBα (1:1000, Wanleibio), p-IκBα (1:1000, Wanleibio), GAPDH (1:5000, Wanleibio). After washing, the NC membrane was incubated with the corresponding secondary antibody at room temperature. Western blotting was detected using enhanced chemiluminescence.

RNA extraction and qPCR
Total RNA was extracted with TRIzol reagent (Takara, RNAiso Plus T9108) according to the manufacturer’s instructions and reverse-transcribed using MonScript™ RTIII All-in One Mix (Monad, MR05101M). qPCR was performed with Hieff UNICON SYBR Green Master Mix (Yeasen, 11184ES08). The primers used in this experiment included NCEH1 (forward 5′-CCATTGAATACAGGCTAGTTCC-3′, reverse 5′-CAAATTCTGCCTGGATCAACC-3′), NRP1 (forward 5′-CCCAACAGCCTTGAATGCAC-3′, reverse 5′-ATTTCTAGCCGGTCGTAGCG-3′), and GAPDH (forward 5′-CTGGGCTACACTGAGCACC-3′, reverse 5′-AAGTGGTCGTTGAGGGCAATG-3′).

Lentiviral transfection
The target plasmids NCEH1 shRNA, NCEH1 overexpression (OE), NRP1 OE and packaging plasmids ps.PAX2 and pMD2.G were mixed with Polyethylenimine (PEI) (Yeasen, 40816ES01) and allowed to stand at room temperature for 20 min. The mixture was added to a HEK293T petri dish to prepare virus fluid. The obtained virus solution was mixed with the culture medium, and 8 μg/mL polybrene (Yeasen, 40804ES76) was added to infect MB-MDA-231 and MCF-7 cells. After screening with puromycin (MedChemExpress, HY-B1743), stably transfected cells were obtained.

Immunohistochemistry (IHC)
Tumour tissue was fixed overnight in 10% formalin for IHC. Embed in wax and cut into 4 µm slices. Tissue sections were deparaffinized, rehydrated, and stained with antibodies against NCEH1 (1:100, Solarbio), NRP1 (1:100, Proteintech), or Ki67 (1:100, Abcam) using standard protocols (ZSGB-BIO, PV-9000).

Immunofluorescence (IF)
MDA-MB-231 and MCF-7 cells were seeded on cell slides. Fixed with 4% paraformaldehyde. Cells were permeabilized with 0.5% TritonX-100 (Solarbio, IT9100). After that, NCEH1 antibody (1:50, Solarbio) was used overnight at 4°C, and after the incubation with fluorescent secondary antibody (1:500, Proteintech), the coverslip was stained with DAPI (Solarbio, S2110) to counterstain the cell nucleus. The location of NCEH1 protein in MDA-MB-231 and MCF-7 cells was observed using a microscope.

Cell cycle, apoptosis, and senescence assays
All experiments were performed 48 h after cell transfection. For cell cycle experiment, transfected cells were fixed overnight at 4°C using 70% ethanol, stained with propidium iodide (KGI Biosciences, KGA9101-100), and detected by flow cytometry. For the apoptosis experiment, cell precipitates were collected after digestion with EDTA free of trypsin, resuspended and stained using Annexin V-FITC/PI staining (KGI Biosciences, KGA1101-100). The apoptosis results were detected by flow cytometry. For the senescence assays, the cell aging kit (Beyotime, C0602) was used to stain the cells according to the instructions, and then the staining was observed using a microscope.

Hematoxylin-eosin staining (H&E staining)
Paraffin sections of mouse tissues were first baked at 60°C for 2 h. After dewaxing and rehydration of the paraffin sections, the cell nuclei were stained with hematoxylin staining solution, followed by staining of the cytoplasm with eosin staining solution (Solarbio, G1120). After the sections were dried, they were mounted and preserved with neutral balsam.

Animal studies
The animal experimental protocol was conducted in accordance with the animal license agreement approved by the Research Committee of Dalian Medical University (Registration Number: AEE24134). Healthy 4-week-old female BALB/c nude mice were selected and purchased from SPF (Beijing) Biotechnology Co., Ltd. Mice were numbered and randomly assigned to groups using a randomization tool. For the in vivo proliferation assay, female BALB/c nude mice (n = 20) were randomly divided into four groups (sh-NC, NRP1-OE, sh-NCEH1, sh-NCEH1 + NRP1-OE, with five mice in each group). MDA-MB-231 cells were injected subcutaneously into the axilla of mice at a dose of 5 × 106 cells. Five days after injection, the tumour size was recorded every 5 days using a vernier caliper, and the tumour volume was calculated as V=(length × width2)/2. When tumours in the control group reached approximately 1 cm, the animals were euthanized, the tumour was excised and photographed. For the in vivo metastasis assay, female BALB/c nude mice (n = 20) were randomly divided into four groups (sh-NC, NRP1-OE, sh-NCEH1, sh-NCEH1 + NRP1-OE, with five mice in each group). After tail vein injection of MDA-MB-231 cells at a dose of 5 × 106 cells into the mice, the animals were euthanized 3 months post-injection. The lung tissues were harvested for paraffin-embedded for subsequent analysis.

Statistical analysis
Data were analysed and charted using GraphPad Prism 9.0. Experimental data are expressed as mean ± standard deviation (SD). Two group comparisons were analysed by Student’s t-test, and multiple group comparisons were analysed by analysis of variance (ANOVA). p < .05 was considered statistically significant.

Results

Differential expression of NCEH1 in tumours and its clinical prognostic relevance
Analysis of public databases revealed significantly elevated NCEH1 expression in breast cancer tissues compared to normal tissues (Figure 1(A)). In different types of breast cancer, the expression level of NCEH1 in tumour tissues was higher than that in normal tissues (Figure 1(B)). UALCAN database analysis demonstrated a significant association between high NCEH1 expression and lymph node metastasis (Figure 1(C)). In addition, survival analysis from the Kaplan–Meier Plotter database showed that NCEH1 expression levels were significantly correlated with patient prognosis, and breast cancer patients with high NCEH1 expression had shorter RFS and DMFS (Figure 1(D)). Bioinformatics analysis showed that the high expression of NCEH1 is linked to malignant progression and poor prognosis in breast cancer.

In order to verify the clinical applicability of bioinformatics analysis results, we detected and localized the expression of NCEH1 in clinical breast cancer tissue samples. IHC showed prominent NCEH1 expression in Luminal A, Luminal B, triple-negative breast cancer (TNBC), and HER2+ subtypes, with predominant cytoplasmic localization (Figure 1(E)). Western blotting results showed that in breast cancer tissues, the expression of NCEH1 increased significantly compared to the adjacent tissues (Figure 1(F)). Additionally, NCEH1 was overexpressed in breast cancer cell lines (MDA-MB-231, MDA-MB-453, HCC1806, HCC1954, SK-BR3, MCF-7) relative to normal mammary epithelial cells (MCF-10A) (Figure 1(G)). We selected triple negative breast cancer cells MDA-MB-231 and Luminal breast cancer cells MCF-7 with relatively high expression levels as the next experimental cells. To further understand the distribution of NCEH1, we conducted immunofluorescence experiments in MDA-MD-231 and MCF-7 cells, and we found that
NCEH1 was mainly localized in the cytoplasm of tumour cells (Figure 1(H)). Collectively, these results suggest that NCEH1 is highly expressed in breast cancer and may be involved in the occurrence and development of breast cancer.

NCEH1 promotes breast cancer cell growth and migration
To investigate the functional role of NCEH1, we silenced its expression using NCEH1-specific siRNAs. qPCR (Figure 2(A)) and Western blotting (Figure 2(B)) demonstrated successful transfection of NCEH1 siRNA and significant knockdown of NCEH1 expression. NCEH1 silencing significantly inhibited the proliferation ability (Figure 2(C)) and colony formation ability (Figure 2(D)) in MDA-MB-231 and MCF-7 cells. It shows that it has a promoting effect on the growth of breast cancer.

In order to explore the migration ability of NCEH1 in breast cancer, we conducted scratch wound healing assays. After silencing NCEH1, MDA-MB-231 and MCF-7 cells inhibited scratch healing for a certain period of time and slowed down the speed (Figure 2(E)). Transwell migration assay showed that silencing NCEH1 significantly reduced the migration ability of MDA-MB-231 and MCF-7 cells (Figure 2(F)). The above results suggest that NCEH1 can accelerate the progression of breast cancer and promote tumour migration.
In order to demonstrate the carcinogenic effect of NCEH1 in breast cancer cell lines, we transfected lentivirus fluids overexpressing NCEH1 into MDA-MB-231 and MCF-7 cells. NCEH1 overexpression efficiency was demonstrated by qPCR (Figure 2(G)) and Western blotting (Figure 2(H)). Our results demonstrated that overexpression of NCEH1 significantly increased the viability (Figure 2(I)) and colony forming ability (Figure 2(J)) of MDA-MB-231 and MCF-7 cells and promoted the migration ability of cells (Figures 2K and 2L). The above results further support that NCEH1 promotes the proliferation and migration capabilities of breast cancer cells and accelerates the progression of breast cancer.

NCEH1 modulates cell cycle, apoptosis, and senescence
Based on the mentioned effect of NCEH1 on the proliferation of breast cancer cells, we conducted cell cycle assays and found that NCEH1 silencing induced G2/M phase arrest in MDA-MB-231 and MCF-7 cells (Figure 3(A)). When cells enter the G2/M arrest state, it will inhibit cell mitosis, delay cell proliferation, and is not conducive to DNA damage repair. Apoptosis assays demonstrated that NCEH1 silencing increased
the number of early and late apoptosis of tumour cells, promoting apoptosis of breast cancer cells (Figure 3(B)). Furthermore, senescence-associated β-galactosidase activity was elevated in silenced cells (Figure 3(C)), indicating enhanced cellular senescence.

NCEH1 regulates epithelial-mesenchymal transition (EMT)
EMT refers to the process in which epithelial cells undergo a series of changes and eventually transform into cells with mesenchymal characteristics [31]. Abnormal activation of EMT changes cell characteristics, enhances migration and invasion capabilities, anti-apoptotic properties, and promotes extracellular matrix degradation [32]. Based on the results of phenotyping experiments of NCEH1 in breast cancer cells, high expression of NCEH1 can promote the proliferation and migration of cells, and knockdown of NCEH1 can promote the apoptosis of breast cancer cells. We speculate that NCEH1 may promote tumour progression through EMT. We found that after NCEH1 knockdown, the expression of E-cadherin increased in MDA-MB-231 and MCF-7 cells, while the expression levels of N-cadherin, β-catenin, and Vimentin all decreased, and the mesenchymal characteristics of the cells were weakened. In addition, the expression levels of Slug and Snail proteins, key transcription factors promoting EMT, were also significantly reduced (Figure 4(A)). These results demonstrated that NCEH1 knockdown can inhibit EMT and help inhibit cell metastasis and invasion capabilities. This implies that NCEH1 drives EMT to enhance metastatic potential.

NCEH1 promotes tumour growth via NRP1 regulation
To further analyse the potential molecular mechanism by NCEH1 promotes breast cancer progression, we analysed the transcriptome changes after silencing NCEH1 expression in MDA-MB-231 (Figure 4(B)). In addition, KEGG enrichment analysis of differentially expressed genes showed that NCEH1 silencing affects a range of signalling pathways in breast cancer cells (Figure 4(C)). Because in the early stage, we verified that NCEH1 can promote the occurrence and development of breast cancer through EMT. Through a large number of literature review, neuropilin 1 (NRP1) was screened among differential genes. NRP1 is a unique transmembrane glycoprotein that plays a role in promoting tumour progression, accelerating angiogenesis, and regulating immune responses. NRP1 has been shown to be an effector molecule that mediates EMT and accelerates tumour progression through EMT in a variety of tumours [33–35]. Notably, NCEH1 expression positively correlated with NRP1 levels in breast cancer (Figure 4(D)). Therefore, we speculate that NCEH1 may promote the malignant progression of breast cancer by affecting the expression of NRP1. It was determined by qPCR and Western blotting analysis that silencing of NCEH1 reduced NRP1 mRNA and protein levels (Figures 4E and 4F). We simultaneously silenced NCEH1 and overexpressed NRP1 in breast cancer cells to analyze their impact on the malignant phenotype of the cells. Our results showed that NCEH1 silencing significantly inhibited cell proliferation and migration compared to control groups, while these effects were partially reversed after NRP1 overexpression (Figures 5A–5C).

Abnormal activation of TNF signalling pathway is closely related to tumour growth, invasion, metastasis and angiogenesis [36]. TNF can promote the proliferation and survival of tumour cells by activating transcription factors such as NF-κB, and inhibit apoptosis of tumour cells [22]. In addition, studies have shown that NRP1, as a co-receptor for TNF-α, can promote the expression of NF-κB under stimulation from TNF-α [37]. RenD, XuJ and Kim et al. have reported that in different malignant tumours, the NF-κB pathway can regulate EMT and promote cancer progression [38–40]. Mechanistically, we found that NCEH1 knockdown suppressed TNF-α/NF-κB signalling, while NRP1 overexpression restored pathway activation (Figure 5(D)). In addition, inhibition of NCEH1 and overexpression of NRP1 reversed the inhibitory effect of knockdown of NCEH1 on EMT (Figure 5(D)). Similarly, in vivo experiments with breast cancer cells with stable knockdown NCEH1 and NRP1 overexpression showed that NRP1 overexpression partially alleviated the tumour growth inhibition caused by NCEH1 knockdown (Figures 5E–5G). IHC staining of the formed xenografts further demonstrated that overexpression of NRP1 salvaged the tumour growth inhibition caused by NCEH1 (Figure 5(H)). In vivo experiments on lung metastasis of breast cancer cells demonstrated that breast cancer cells with stable NCEH1 knockdown and NRP1 overexpression also showed that NRP1 overexpression partially alleviated the NCEH1-mediated inhibitory effect on tumour metastasis caused by NCEH1 knockdown (Figure 5(I)). These results support the hypothesis that NCEH1 promotes breast cancer growth and metastasis through NRP1 expression.

Discussion
Breast cancer is the most common female malignant tumour worldwide, seriously endangering women’s physical and mental health [41]. While advancements in early detection and treatment have reduced mortality rates, the heterogeneity and complexity of breast cancer often lead to poor prognosis and diminished quality of life, presenting ongoing challenges for patients [42,43]. Overall, the epidemiological profile of breast cancer reflects at least in part our limitations in understanding the molecular signalling pathways of tumourigenesis and the functions of many genes in cancer [44,45]. Therefore, elucidating the signalling mechanisms underlying breast cancer initiation and progression may reveal novel therapeutic strategies and targets.
NCEH1 is one of the important hydrolases in the lipid metabolic pathway in cells. The gene encoding NCEH1 is located on the q arm of chromosome 3, a chromosomal region that is highly amplified in various malignant tumours [46]. Notably, the reliability of NCEH1 as a therapeutic target is supported by its consistent carcinogenic effects observed in multiple malignant tumours. Growing evidence indicates that NCEH1 is abnormally overexpressed in various cancer type and is closely associated with poor prognosis. Jessani N et al. first found that NCEH1 is positively correlated with the proliferation ability and invasiveness of malignant tumour cells [5]. In addition, literature has shown that the expression of NCEH1 is significantly increased in various cancers such as pancreatic cancer, gastric cancer and prostate cancer and is closely related to the degree of local invasion and lymph node metastasis of the tumour [47–49]. NCEH1 is highly expressed in pancreatic cancer samples, participating in cancer progression and chemoresistance to gemcitabine, suggesting its potential as a target for efficacy evaluation and resistance reversal in pancreatic cancer [6]. In cholangiocarcinoma, serum and tissue cholesterol levels are significantly elevated, promoting tumour development. Aurora kinase B (AURKB) can increase intratumoral cholesterol levels by regulating NCEH1, modulating lipid metabolism to drive tumour growth [50]. Furthermore, analysis of public datasets for breast cancer and major depressive disorder (MDD) showed significant upregulation of
NCEH1, correlating with poor prognosis in breast cancer patients and the progression of MDD [51]. These studies collectively demonstrate that NCEH1 maintains a oncogenic function across multiple tumour types, providing strong evidence for its reliability as a therapeutic target in breast cancer treatment.
We have identified NCEH1 as an important biomarker and therapeutic target in breast cancer and have described a new mechanism for NCEH1 to promote breast cancer progression by affecting NRP1 expression and mediating the TNF-α/NF-κB signalling pathway. This may be the first report on the carcinogenic ability and possible molecular mechanisms of NCEH1 in the development of breast cancer.
Through bioinformatics analysis, we determined the expression profile of NCEH1 in breast cancer and found that high expression of NCEH1 is associated with poor prognosis. Clinical validation confirmed these findings, with immunohistochemistry and Western blotting demonstrating upregulated NCEH1 protein levels in tumours. In order to further explore the potential mechanism of NCEH1 in breast cancer, we conducted experiments and found that knockdown of NCEH1 significantly inhibited the proliferation and migration of breast cancer cells, indicating that NCEH1 plays a key role in regulating the malignant phenotype of breast cancer.
In terms of molecular mechanisms, we found that NCEH1 knockdown induced G2/M phase arrest and apoptosis while promoting cellular senescence. Cell cycle disorder is one of the hallmarks of cancer. Since the G2/M phase is the late stage of DNA synthesis and the G2/M checkpoint is the last checkpoint of cell mitosis, most tumour cells have defects in the G1/S checkpoint [52]. The cell cycle is arrested in the G2/M phase, which is not conducive to the repair of DNA damage, thereby passing the damaged DNA to daughter cells, which in turn causes apoptosis [53]. Cell aging is characterized by decreased cell proliferation ability and decline in physiological functions. After knocking down NCEH1, the proliferation rate of cancer cells slows down and may enter a stable non-dividing state, thereby inhibiting tumour growth and metastasis.
EMT refers to the biological process of transition from an epithelial to a mesenchymal phenotype [54]. Abnormal activation of EMT by tumour cells leads to increased invasiveness and mobility, which is considered a key event in tumour progression [55]. Our research shows that NCEH1 can mediate EMT, thereby participating in tumour adhesion and invasion.
Considering the important role of NCEH1 in EMT and the differentially expressed genes and signal transduction pathways based on RNA-Seq results. We explored the effect of NCEH1 on NRP1 regulation and its relationship with the TNF signalling pathway. NRP1 is widely expressed in tumour blood vessels and plays a role in promoting tumour progression, accelerating angiogenesis, and regulating immune responses through interaction with multiple regulatory factors [56]. NRP1 has been shown to be an effector molecule mediating EMT [57]. Studies have shown that NRP1 is up-regulated in cervical cancer, is related to disease progression and affects EMT [33]. NRP1 promotes the migration of liver cancer cells through EMT [34] and is also involved in TGF-β1-induced EMT in non-small cell lung cancer [35]. Abnormal activation of TNF signalling pathway is closely related to tumour growth, invasion, metastasis and angiogenesis. TNF mainly includes TNF-α and TNF-β, of which TNF-α is deeply researched and closely related to many diseases. TNF-α binds to TNF receptors (TNFR) on the cell surface, which in turn activates NF-κB [58]. Thereby promoting the proliferation and survival of tumour cells and inhibiting tumour cell apoptosis. It can also accelerate angiogenesis and promote the migration of tumour cells. TNF-α
can promote EMT-related tumour invasion [59]. In different malignant tumours, the NF-κB pathway can regulate EMT and promote cancer progression [38–40,60]. NRP1 is a key factor in the TNF-α pathway and promotes the expression of NF-κB [37]. Combined with our findings that NCEH1 positively regulates NRP1 at both the mRNA and protein levels in breast cancer cells, and NRP1 overexpression significantly reverses the inhibitory effect caused by NCEH1 silencing. We fully believe that NCEH1 activates NRP1 to regulate the TNF-α/NF-κB signalling pathway and promotes breast cancer progression.
As a key downstream target of NCEH1, NRP1 plays an important role in promoting the progress of breast cancer. Based on our existing experimental evidence and relevant published studies, we have conducted an in-depth analysis of the potential mechanism by which NCEH1 regulates NRP1 transcription. As a neutral cholesterol ester hydrolase, NCEH1 primarily hydrolyses intracellular neutral cholesterol esters to release free cholesterol (FC). Cholesterol is a key component of the cell membrane, directly influences membrane fluidity and the structure and function of lipid rafts [61]. The signal transduction of many receptor tyrosine kinases, such as Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), Epidermal Growth Factor Receptor (EGFR), relies on lipid rafts [62]. NRP1, as a co-receptor for VEGFR2, enhances complex stability through interaction [63]. Altered NCEH1 activity might affect the composition and function of lipid rafts, indirectly influencing the stability or signalling of NRP1, thereby providing feedback regulation on its transcriptional control.
Research has found that intracellular cholesterol can directly bind to Hypoxia Inducible Factor-1α (HIF-1α), enhancing its stability. Elevated intracellular cholesterol levels inhibit HIF-1α ubiquitination and degradation, thereby increasing its activity and transcriptional function [64]. Furthermore, changes in NCEH1 can lead to metabolic reprogramming, such as endoplasmic reticulum stress and reactive oxygen species level alterations, indirectly affecting HIF-1α regulation. HIF-1α has been confirmed to directly bind to the NRP1 promoter region located between positions −2009 and −2017. Studies have shown that HIF-1α upregulates NRP1 expression, promoting vasculogenic mimicry formation in lung adenocarcinoma and contributing to malignant progression [65]. Additionally, research in prostate cancer cells indicates that HIF1α binds to a specific region of the NRP1 promoter, regulating its transcriptional activation. NRP1 also interacts with EGFR, further activating the AKT signalling pathway, thereby promoting malignant progression in prostate cancer [18]. Therefore, we propose that NCEH1 may indirectly affect NRP1 transcription by regulating intracellular lipid homeostasis and signalling pathways.
Our current research suggests that NCEH1 could serve as a potential biomarker and therapeutic target for breast cancer. Of course, our research also has some limitations. In order to deeply explore the cancer-promoting role of NCEH1 in breast cancer, more research is needed in larger, multi-center breast cancer patients in the future. We expect that NCEH1 will become a new clinical prognostic and treatment indicator. More importantly, the regulatory relationship between NCEH1 and NRP1 still needs further experiments to verify. Given the complexity of tumour transcription, we need to further study whether NCEH1 cooperates with other factors or opposes the regulation of other breast cancer-related genes. Furthermore, this study has only validated the anti-tumour effects of NCEH1 intervention at cellular and animal levels, without initiating research on NCEH1-targeted drug development or combination therapies. Future work could focus on screening NCEH1-targeted inhibitors and exploring combination treatment strategies, thereby providing more comprehensive evidence to support the clinical translation of this therapeutic target.
In summary, this study demonstrates that NCEH1 can affect the transcription of NRP1 and thus upregulate its expression level. Both of them promote breast cancer proliferation and migration and other malignant phenotypes by activating the TNF-α/NF-κB signalling pathway and promote the progress of breast cancer.

Conclusion
In conclusion, this study proves that NCEH1 is a key gene in breast cancer and it can affect NRP1 expression and promote breast cancer progression through activation of the TNF-α/NF-κB signalling pathway. This discovery marks the possibility that NCEH1 may become a new biomarker for breast cancer and a key target for therapeutic intervention.