NUF2 regulated the progression of hepatocellular carcinoma through modulating the PI3K/AKT pathway via stabilizing ERBB3

Highlights

• •Overexpression of NUF2 was associated with poor prognosis in hepatocellular carcinoma.
• •NUF2 interacts with ERBB3 to stabilize its expression.
• •NUF2 activates the PI3K/AKT pathway through its interaction with ERBB3.

Abstract

Hepatocellular carcinoma (HCC) is among the most prevalent and lethal cancers worldwide. The NDC80 kinetochore complex component NUF2 has been previously identified as up-regulating in HCC and associated with patient prognosis. However, the pathophysiological effects and molecular mechanisms of NUF2 in tumorigenesis remain unclear. In this study, we confirmed a significant increase in NUF2 expression in HCC tissues and established a correlation between high NUF2 expression and adverse outcomes in HCC patients. Through in vitro and in vivo experiments, we demonstrated that genetic inhibition of NUF2 suppressed the proliferation of HCC cells and disrupted the cell cycle. Further investigation into the molecular mechanisms revealed that NUF2 interacted with ERBB3, inhibiting its ubiquitination degradation, thus activating the PI3K/AKT signaling pathway and influencing cell cycle regulation. Overall, this study revealed the crucial role of NUF2 in promoting the malignant progression of HCC, suggesting its potential as both a prognostic biomarker and a therapeutic target for HCC.

Graphical abstract

Introduction

According to the Global Cancer Statistics of 2020, liver cancer accounted for approximately 906,000 new cases and 830,000 deaths worldwide [1]. Among primary liver cancers, hepatocellular carcinoma (HCC) is the most prevalent type, comprising about 90 % of cases [2]. However, despite the presence of multiple treatment methods for combating HCC, such as surgery, local ablation, targeted therapy, and immunotherapy, patients' prognosis remains unsatisfactory and prone to recurrence and metastasis [2], [3], [4]. Therefore, gaining a deeper understanding of the underlying mechanisms of HCC is crucial for improving patient outcomes.

NUF2, also known as CDCA1, is an essential component of the Ndc80/Nuf2 complex, which plays a critical role in ensuring proper chromosomal segregation during mitosis [5,6]. Previous studies have indicated that NUF2 is overexpressed in HCC and other cancers and is associated with immune infiltrates, making it a valuable prognostic biomarker for predicting early recurrence after surgical resection in HCC [5,[7], [8], [9]]. Additionally, our prior research has demonstrated that NUF2 is overexpressed in cholangiocarcinoma (CCA) and promotes CCA progression by regulating the degradation of the TFR1 protein [10]. These findings suggest that NUF2 may have a broader impact in HCC.

In this study, our aim was to unravel the molecular mechanisms and biological functions of NUF2 in HCC. Our findings confirmed the elevated expression of NUF2 in HCC and its significant correlation with the prognosis of HCC patients. Importantly, we discovered that NUF2 interacted with ERBB3, leading to the stabilization of ERBB3 and inhibiting its ubiquitinated degradation. This interaction further activated the PI3K/AKT signaling pathway, contributing to the progression of HCC.

Based on our results, NUF2 emerged as a potential therapeutic target for HCC and a prognostic biomarker. By understanding the underlying mechanisms of NUF2 in HCC, we can explore new therapeutic strategies to improve patient outcomes.

Materials and methods

Cell culture

The human HCC cell lines used in this study, namely Hep-3B, Hep-1, Huh7, HCCLM3, and Hep-G2, were obtained from the Chinese National Human Genome Center in Shanghai, China. All cell lines were maintained in a viable state. The cells were cultured in DMEM culture media supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin/streptomycin at 37 °C in a 5 % CO₂ atmosphere. Additionally, we performed short tandem repeat (STR) analysis to authenticate each of the cell lines.

Transfection of small interfering RNA (siRNA)

Small interfering RNA (siRNA) specifically targeting NUF2, ERBB3, and a non-specific control (si-Ctrl) were acquired from GenePharma (Shanghai, China) for transfection. Lipofectamine 3000 (Thermo Fisher Scientific, MA, USA) was employed as the transfection reagent following the manufacturer's protocol. The cells were cultured until they reached a confluency of 30–40 %. After a six-hour transfection period, the medium containing the transfection reagents was replaced with fresh medium. Total proteins and RNA were collected 48 h after transfection. The siRNA sequences were as follows:

• si-NUF2:

• 5′-UCAAAAAUCAGCAAACGUCAA-3′

• 5′-AUAUGAAUCACAAUCUCAGCU-3′

• 5′-AGAUCUUAUUGCGAAUAUGAA-3′

• si-ERBB3:

• 5′-ACGAAGAUGGCAAACUUCCCA-3′

• 5′-UGUUAUAGUUCAACAUGACGA-3′

• 5′-UUAGAAAAACGGUUAACAGAU-3′

Western blot

Total protein extraction was performed using the RIPA kit (Beyotime, Shanghai, China). The extracted proteins were then subjected to SDS-PAGE for separation, followed by transfer onto PVDF membranes. Subsequently, the membranes were incubated overnight at 4 °C with specific antibodies. β-actin was utilized as a loading control. ECL Plus (EMD Millipore, Billarica, MA, USA) was employed to visualize the ECL signals.

RNA extraction and quantitative real-time PCR (qRT-PCR)

According to the manufacturer's instructions, total RNA was obtained using the RNA Quick Purification Kit (YiShanbio, Shanghai, China). The PrimeScript RT Reagent Kit was used to perform reverse transcription. An endogenous control gene was thought to β-actin.

CCK-8

The Cell Counting Kit-8 (CCK-8) was purchased from MCE in Shanghai, China. A total of 13,000 cells per well were seeded in 96-well plates and incubated overnight in 100 µl of complete culture media. At 0, 24, 48, 72, 96, and 120 h, 10 µl of CCK-8 solution was added to each well. After a 2-hour incubation, the absorbance at 450 nm was measured using a spectrophotometer (Thermo Scientific, Pittsburgh, PA, USA).

5ethynyl-2′-deoxyuridine (EdU) incorporation assay

A total of 25,000 cells per well and 100 µl of medium containing 10 % FBS were added to each well of a 96-well plate. After an initial 24-hour incubation, 1 µl of EdU was added to each well, and the cells were further incubated for 2 h at 37 °C. Following this, the cells were fixed with 4 % paraformaldehyde for 30 min at 37 °C and permeabilized with 0.5 % Triton X-100 for 10 min. The cells were then stained with EdU using the Apollo dyeing procedure for 30 min. To visualize the nuclei, Hoechst stain was added and incubated for an additional 30 min. Finally, a fluorescence microscope was utilized to determine the percentage of nucleated cells that were stained with EdU.

Clone formation

Approximately 800 cells were evenly distributed and plated into each well of 6-well plates containing 3 ml of complete culture media per well. For the first ten days, the cells were treated with phosphate-buffered saline (PBS). After this period, the cells were fixed with ethyl alcohol for one minute. Following the fixation process, the cells were stained with a 1 % crystal violet solution for 20 min. The colonies formed by the cells were then visually counted.

Cycloheximide chase assay

After exposure to the protein synthesis inhibitor CHX (50 µM) for different durations (0, 2, 4, and 8 h), cells were harvested and placed in RIPA buffer supplemented with proteinase inhibitors. The cell lysate was centrifuged and sonicated, and the resulting supernatants were mixed with SDS-PAGE loading buffer. Immunoblotting was performed to analyze the protein samples and investigate their expression levels and changes over time.

Immunofluorescence

To preserve cellular architecture, the fixed cells were treated with 4 % paraformaldehyde at 37 °C for 45 min. Permeabilization was achieved by applying 0.3 % Triton X-100 for 15 min. To prevent nonspecific binding, the cells were then blocked with 5 % donkey serum for 30 min. Specific primary antibodies were added and allowed to incubate with the cells overnight at 4 °C. For visualization of the target proteins, fluorescent secondary antibodies labeled with 594, 488, or FITC were applied to the cells and incubated for 2 h at room temperature. DAPI staining was used to highlight the DNA in the nucleus. Finally, fluorescence images capturing the labeled proteins and cellular structures were acquired using a confocal fluorescence microscope.

Immunoprecipitation

The cells were lysed using Nonidet P-40 Substitute. Specific antibodies and IgG were added to the lysate, and the mixture was incubated overnight at 4 °C to capture protein complexes. The following day, Protein A + G magnetic beads were employed to collect the antigen-antibody complexes. After two hours of mixing at room temperature, the supernatant was removed to extract the antigen-antibody complexes for subsequent analysis. The protein complexes were separated by SDS-PAGE and identified using western blot assays with corresponding antibodies.

Ubiquitination assay

Cells that had been transfected with specific plasmids or siRNAs were pre-treated with the proteasome inhibitor MG132 (Beyotime, Shanghai, China) for 8 h prior to collection.

Tumor xenograft transplantation experiments

The animal studies conducted in this research were approved by the Nanjing Medical University (NJMU) Institutional Animal Care and Use Committee. BALB/c nude mice were obtained from the Model Animal Research Centre of Nanjing University. Five-week-old mice were used to establish the subcutaneous tumor model. For the xenograft model, 1 × 10^6 cells were individually injected into the left side. Tumor weights were measured every five days. After 25 days of subcutaneous injection, the mice were euthanized, and the xenograft tumors were excised. The dissected tumors underwent H&E (Hematoxylin and Eosin) and IHC (Immunohistochemistry) staining for further examination.

Immunohistochemical staining and tissue microarrays (TMA)

The immunohistochemical staining and TMA are described in detail in our previous research reports [11].

Statistical analysis

The statistical analyses were performed using GraphPad Prism version 7. The specific tests used depended on the type of data and included log-rank tests, unpaired two-tailed t-tests, one-way ANOVA, and Tukey's multiple comparison tests. A p-value less than 0.05 (*P < 0.05, **P < 0.01, ***P < 0.001) was considered statistically significant.

Results

NUF2 expression was found to be upregulated in HCC and exhibited a significant correlation with a poorer prognosis

To explore the differential genes associated with the onset and progression of HCC, a comprehensive analysis of gene expression using the TCGA database and other datasets was conducted. The analysis identified NUF2, a component of the Ndc80 kinetochore complex, as a potential candidate of interest (Fig. 1A). Previous studies have indicated the potential of NUF2 as a prognostic indicator for HCC. Additionally, our own research had demonstrated the involvement of NUF2 in regulating the progression of cholangiocarcinoma through autophagy modulation. Therefore, NUF2 was selected as the focus of this study to further investigate its role in the occurrence and development of HCC. Initial analysis of the TCGA database revealed high expression levels of NUF2 in HCC (Fig. 1B–C). This finding was further validated by analyzing additional datasets obtained from the Gene Expression Omnibus (GSE36376, GSE54236, and GSE63898). Consistently, the results showed increased expression of NUF2 in HCC tumors compared to adjacent tissues (Fig. 1D, Figure S1A–B). To validate the differential expression of NUF2 in HCC, we performed detection on 30 pairs of HCC and adjacent tissues in our center. The analysis revealed upregulation of NUF2 mRNA expression in HCC tissues compared to matched normal tissue samples (Fig. 1E). This finding was further supported by Western blot analysis, which demonstrated a significant increase in NUF2 protein levels in HCC tissues (Fig. 1F). Immunohistochemical staining of HCC tissues provided additional confirmation of NUF2 overexpression in HCC (Fig. 1G, Figure S1C).

Fig. 1.

NUF2 upregulation in HCC and its association with poor prognosis.

(A) HCC differential gene analysis in the TCGA dataset.

(B-C) Analysis of NUF2 expression and mRNA levels in tumor and normal samples using the TCGA database.

(D) Analysis of NUF2 expression in tumor and normal samples using the GEO database.

(E) qRT-PCR analysis of NUF2 expression in HCC tumor and normal tissue (n = 30).

(F) Western blot analysis of NUF2 expression in HCC tumor and normal tissue.

(G-H) Immunohistochemistry (IHC) staining images of NUF2 in HCC tumor and normal tissue.

(I-J) Survival analysis of NUF2 in HCC based on the TMA data. *P < 0.05, **P < 0.01, ***P < 0.001.

To investigate the relationship between NUF2 expression and prognosis, the association between NUF2 expression and patient outcomes was explored using the TMA dataset. Kaplan-Meier survival analysis demonstrated that patients with high NUF2 expression had significantly shorter overall survival (OS) and disease-free survival (DFS) compared to HCC patients with low NUF2 expression (Fig. 1H–I).

NUF2 promoted HCC cell proliferation and metastasis in vitro

To gain further insights into the function of NUF2 in HCC, we assessed its expression in five HCC cell lines (Fig. 2A–B). Based on the expression levels observed, we selected Hep-3b, which exhibited the highest NUF2 expression, for the knockdown experiment. Conversely, we chose Hep-1, which displayed relatively low NUF2 expression, for the overexpression experiment. The efficiency of NUF2 knockdown or overexpression was confirmed using Western blotting and qRT-PCR (Fig. 2C–F).

Fig. 2.

Exploring NUF2 Expression and Functional Analysis in HCC Cells.

(A-B) Expression of NUF2 in five hepatocellular carcinoma (HCC) cell lines.

(C-F) Validation of knockdown and overexpression efficiency of NUF2 using Western blot (WB) and reverse transcription polymerase chain reaction (RT-PCR).

(G-H) Detection of EdU incorporation in PIN1 stable knockdown and overexpression cells, represented by a scale bar (50 μm). *P < 0.05, **P < 0.01, ***P < 0.001.

We examined the cellular phenotypes following NUF2 knockdown or overexpression to gain a better understanding of how NUF2 levels influenced the biological properties of HCC cells. EdU experiments revealed that NUF2 deletion significantly decreased the proportion of red cells (indicating cells in the proliferative phase), whereas NUF2 overexpression significantly increased the percentage of red cells (Fig. 2G–H). CCK-8 experiments demonstrated that NUF2 overexpression enhanced cell proliferation, whereas NUF2 knockdown significantly decreased cell proliferation in HCC (Figure S1D–E). Furthermore, colony formation experiments indicated that NUF2 knockdown decreased colony formation, whereas NUF2 overexpression had the opposite effect (Fig. 3A).

Fig. 3.

Investigating the Impact of NUF2 on Proliferation and Cell Cycle Regulation in HCC.

(A) Proliferation Ability Analysis Following NUF2 Knockdown or Overexpression Through Clone Formations.

(B) KEGG Enrichment Analysis of NUF2 Using the TCGA Database.

(C-D) Levels of Cyclin D1 in Hep-1 and Hep-3B cells as Determined by Western blot Analysis.

(E-F) Cell Cycle Analysis of HCC Cells Using Flow Cytometry. *P < 0.05, **P < 0.01, ***P < 0.001.

Subsequently, we investigated the metastatic role of NUF2 in HCC cell lines through wound healing and transwell assays. The results showed that NUF2 knockdown impaired the migration capacity of HCC cells, whereas NUF2 overexpression promoted HCC cell migration (Figure S1F–G).

NUF2 regulated HCC proliferation and cell cycle via the PI3K/AKT signaling pathway

We conducted a functional annotation study of NUF2-associated differentially expressed genes (DEGs) in HCC patients using TCGA datasets to uncover the underlying mechanism by which NUF2 influences HCC development. According to the results of the KEGG enrichment analysis, NUF2 may enhance HCC progression by regulating the cell cycle (Fig. 3B). Subsequently, we observed that NUF2 knockdown led to a decrease in Cyclin-D1 expression, while NUF2 overexpression had the opposite effect (Fig. 3C–D). Flow cytometry analysis further revealed that NUF2 knockdown led to an accumulation of cells in the G0/G1 phase, accompanied by a decrease in the proportion of cells in the S and G2/M phases. Conversely, NUF2 overexpression resulted in a significant increase in the percentage of cells in the S and G2/M phases, along with a reduction in the G0/G1 phase population (Fig. 2E–F).

To investigate the potential mechanisms through which NUF2 regulates HCC, we performed Western blot on several classic cancer-related signaling pathways, including phosphatidylinositol-3-kinase (PI3K)/AKT, JAK/STAT3, WNT/β-Catenin, and JNK/P38, following NUF2 knockdown and overexpression. The results indicated that NUF2 knockdown resulted in downregulation of P-AKT and P-PI3K compared to control cells (Fig. 4A). Conversely, NUF2 overexpression promoted the activation of P-AKT and P-PI3K (Fig. 4B). However, no significant differences were observed in JAK/STAT3, WNT/β-Catenin, RAS/RAF, and JNK/P38 (Figure S2A–D).

Fig. 4.

Compound 7 reverses the promotional effect of NUF2 overexpression on HCC cells.

(A-B) Western blot analysis of NUF2, PI3K, P-PI3K, AKT, and P-AKT protein levels.

(C) CCK8 assays.

(D) EdU assays.

(E) Clone formation.

(F) Flow cytometry was showed compound 7 reversed the promotional effect of NUF2 overexpression on the proliferation of HCC cells. *P < 0.05, **P < 0.01, ***P < 0.001.

To understand the role of P-PI3K or P-AKT in NUF2-mediated HCC proliferation and cell cycle regulation, we conducted rescue experiments using an AKT inhibitor (Compound 7) (Figure S2E). The results showed that the addition of the AKT inhibitor significantly restored HCC cell proliferation and cell cycle alterations caused by NUF2 overexpression. CCK-8, EdU assays, and colony formation experiments demonstrated that the AKT inhibitor markedly reduced HCC cell proliferation compared to the vector groups (Fig. 4C–E). Flow cytometry analysis revealed that the AKT inhibitor rescued the decrease in the fraction of cells in the G0/G1 phase and increased the proportion of cells in the S and G2/M phases induced by NUF2 overexpression (Fig. 4F). These findings suggested that NUF2 may promote HCC progression through the regulation of the PI3K/AKT signaling pathway.

NUF2 interacted and co-localized with ERBB3

Previous studies have indicated that NUF2 can regulate ERBB3 expression in the context of epithelial ovarian cancer progression via the PI3K-AKT and MAPK signaling axes [12]. However, the specific mechanism by which NUF2 regulates ERBB3 expression has not been extensively investigated in HCC. To confirm the relationship between NUF2 and ERBB3, a co-immunoprecipitation (Co-IP) assay was performed to verify the binding between these two proteins (Fig. 5A–B). Additionally, co-immunofluorescence (Co-IF) analysis revealed a high degree of spatial concordance between endogenous NUF2 and ERBB3 in HCC cells (Fig. 5C–D), further supporting their interaction.

Fig. 5.

NUF2 interacted and co-localized with ERBB3.

(A-B) Co-IP analysis of NUF2 and ERBB3.

(C-D) Confocal immunofluorescence analysis of NUF2 and ERBB3 expression.

(E-F) Changes in ERBB3 protein level after NUF2 knockdown or overexpression.

(G-H) The knockdown efficiency of ERBB3.

We investigated whether changes in NUF2 protein expression correlated with changes in ERBB3 expression. Fortunately, our results demonstrated that the protein level of ERBB3 was upregulated in the NUF2 overexpression group and downregulated in the NUF2 knockout group (Fig. 5E–F). However, no significant changes were observed at the mRNA level (Figure S2F–G). To explore the function of ERBB3 in HCC, knockdown and overexpression experiments of ERBB3 were performed in HCC cells (Fig. 5G–H). Western blot showed that the expression of NUF2 was not altered in ERBB3 overexpression or knockdown scenarios (Figure S2H–I). Proliferation assays, including EdU assays, CCK8, and colony formation assays, were conducted to assess the effect of ERBB3 on cell proliferation. The results demonstrated that ERBB3 knockdown impaired the capacity for proliferation in HCC cells, while ERBB3 overexpression promoted proliferation of HCC cells (Fig. 6A–D). Furthermore, we examined the changes in PI3K/AKT signaling following ERBB3 knockdown or overexpression. The results revealed that P-PI3K and P-AKT were inhibited or activated following ERBB3 knockdown or overexpression (Fig. 6E–F).

Fig. 6.

ERBB3 promoted HCC cell proliferation in vitro.

(A-B) EdU assays.

(C) Clone formation.

(D) CCK8 assays.

(E-F) Western blot analysis of ERBB3, AKT, PI3K, P-AKT, and P-PI3K protein levels.

(G-H) The control and NUF2 knockdown and overexpression in Hep-3B and Hep-1 cells were treated with CHX at different time points.

These findings suggested that NUF2 may regulate the expression of ERBB3 through post-translational modifications, and in turn, ERBB3 promotes HCC cell proliferation.

NUF2 inhibited the ubiquitination-mediated degradation of ERBB3 in HCC

Previous studies have documented that ERBB3 undergoes constitutive ubiquitination. The incubation with heregulin further amplifies the ubiquitination and subsequent degradation of ERBB3 [13,14]. The degradation of endogenous proteins primarily occurs through proteasomes or lysosomes, namely the Endoplasmic Reticulum-Associated Protein Degradation (ERAD) pathway and lysosomal degradation. To evaluate this phenomenon, we performed a cycloheximide (CHX) pulse-chase assay and observed that manipulating NUF2 expression either by knockdown or overexpression led to an augmented or restricted degradation of ERBB3 protein in HCC cells (Fig. 6G–H). To understand the mechanism underlying ERBB3 degradation, we treated HCC cells with the proteasome inhibitor MG132 and the lysosomal inhibitor chloroquine (CQ). As anticipated, treatment with MG132, but not CQ, rescued the NUF2-mediated downregulation of ERBB3 (Fig. 7A–D). Furthermore, we conducted in vivo ubiquitination assays to investigate the involvement of NUF2 in the regulation of ERBB3 ubiquitination levels. The results demonstrated that manipulating NUF2 expression significantly affected the ubiquitination level of ERBB3 in HCC cells (Fig. 7E–F).

Fig. 7.

NUF2 inhibited the ubiquitination-mediated degradation of ERBB3 in HCC cells.

(A-B) Cells with NUF2 knockdown or overexpression were treated with MG132 (20 μM) for 10 h, and then ERBB3 protein level was determined by western blotting.

(C-D) Cells with NUF2 knockdown or overexpression were treated with CQ (50 μM) in Hep-3B and Hep-1.

(E-F) The NUF2 knockdown or overexpression Hep-3B or Hep-1 cells were incubated with MG132 at 10 μM for 10 h. Then cell lysates were prepared and immunoprecipitated with an anti-ERBB3 antibody and then immunoblotted with the indicated antibodies.

(G-H) Clone formation. *P < 0.05, **P < 0.01, ***P < 0.001.

These findings illustrate that deletion of NUF2 enhances the degradation of ERBB3 through the ubiquitin-proteasome pathway in HCC cells.

NUF2 promoted HCC progression via the ERBB3/PI3K/AKT signaling pathway

We further investigated the role of ERBB3 in mediating the effects of NUF2 on the progression of HCC. To assess the proliferation ability of HCC cells, we conducted clone formation, CCK8, and EdU assays, while flow cytometry was employed to evaluate changes in the cell cycle. As expected, overexpressing ERBB3 significantly reversed the tumor-inhibitory effect caused by NUF2 knockdown, indicating the involvement of ERBB3 in NUF2-induced malignancy of HCC (Fig. 8A–B, Figure S2J–K). Furthermore, we utilized a xenograft model to explore the role of the NUF2/ERBB3/PI3K/AKT pathway in HCC in vivo. The results demonstrated that NUF2 overexpression promoted tumor growth. However, inhibition of ERBB3 and AKT mitigated the HCC growth induced by NUF2 overexpression (Fig. 8C). Immunohistochemical staining of xenograft tissues revealed an upregulation of Ki67 expression in the NUF2 overexpression group (Fig. 8D).

Fig. 8.

ERBB3 Overexpression and NUF2 Knockdown on Proliferation and Tumorigenicity in HCC.

(A) Flow cytometry showed ERBB3 overexpression reversed the inhibited effect of NUF2 knockdown on the proliferation and cell cycle of HCC cells.

(B) EdU assays.

(C) Typical images of subcutaneous xenograft tumor. Weight of the xenograft tumor (n = 5).

(D) Ki67, NUF2, and P-AKT expression in different groups of xenografts. *P < 0.05, **P < 0.01, ***P < 0.001.

Collectively, these findings indicate that the NUF2-ERBB3/PI3K/AKT axis plays a critical role in the progression of HCC, suggesting its potential as a therapeutic target.

Discussion

HCC is a highly prevalent and lethal tumor, with Chinese HCC patients accounting for 50 % of global cases [15,16]. Despite advancements in systemic therapy, the heterogeneity of HCC and its limited treatment efficacy highlight the urgent need for improved diagnostic and prognostic biomarkers [17].

NUF2, a component of the NDC80 complex, plays a critical role in the spindle checkpoint and chromosomal segregation during mitosis [6]. Considerable evidence suggests that NUF2 plays a pivotal role in the development of various cancers. In this study, we utilized the TCGA database to identify key genes associated with HCC progression, leading to the recognition of NUF2 as a potential candidate. Previous investigations have reported increased expression of NUF2 in several human malignancies, including colorectal, gastric, oral, breast, osteosarcoma, and cholangiocarcinoma cancers [10,[18], [19], [20], [21]]. Additionally, NUF2 peptide vaccination has shown promising outcomes in a Phase I clinical study targeting castration-resistant prostate cancer (CRPC) [22]. Notably, previous studies have consistently highlighted the importance of NUF2 in HCC. However, the specific mechanisms underlying the role of NUF2 in HCC have remained unclear until now [5,[22], [23], [24]]. Therefore, this study aimed to investigate the involvement of NUF2 in the pathogenesis of HCC. We confirmed the overexpression of NUF2 in HCC and explored its functional role. Through an array of assays and animal models, we demonstrated that the suppression of NUF2 significantly impedes cell proliferation both in vitro and in vivo. Furthermore, the Gene Set Enrichment Analysis (GSEA) shed light on the potential involvement of NUF2 in regulating the cell cycle, a finding consistent with prior research [24].

Increasing evidence indicates that dysregulation of the cell cycle can contribute to enhanced cell proliferation [25]. In addition to its role in inhibiting HCC cell proliferation, our study demonstrated that downregulation of NUF2 can induce cell cycle arrest in the G0/G1 phase. Any disruption in the regulatory program of the cell cycle can result in abnormal cell proliferation [26]. In our study, we investigated whether NUF2 influences classical signaling pathways involved in the cell cycle, such as PI3K/AKT, JAK/STAT3, and JNK/p38 [27], [28], [29]. Our study revealed that NUF2 promotes the progression of HCC by regulating the PI3K/AKT signaling pathway. The PI3K/Akt signaling pathways exert substantial influence on multiple facets of cell growth and survival, both in physiological and pathological contexts. Notably, inhibitors of the PI3K/AKT signaling pathway hold a vital role in anti-tumor strategies, with various PI3K/AKT inhibitors under development. However, each generation of inhibitors is prone to engendering drug resistance [30]. Consequently, it becomes particularly imperative to scrutinize the mechanisms governing the regulation of PI3K/AKT.

According to reports, ERBB3 is identified as the sole member of the ErbB family, possessing multiple binding sites for PI3K and acting as the primary recruiter of PI3K. ERBB3 belongs to the epidermal growth factor receptor (EGFR) family and possesses a functionally impaired tyrosine kinase domain, primarily engaging in the PI3K/AKT survival/mitogenic pathway. Additionally, it interacts with various proteins such as GRB, SHC, SRC, ABL, rasGAP, SYK, and the transcription regulator EBP1 [31]. While previous studies have indicated the potential role of NUF2 in regulating the expression of ERBB3, the specific regulatory mechanisms remain unclear. In our investigation, we aimed to explore the molecular mechanisms underlying NUF2′s involvement in HCC. Through Co-IP, we identified ERBB3 as a molecule that interacts with NUF2 and acts as a cancer initiator, promoting the formation and progression of HCC. In our study, we observed that the expression level of ERBB3 protein was influenced by the expression of NUF2. However, no significant changes were observed at the mRNA level. Based on these findings, we speculate that NUF2 may affect ERBB3 expression through post-translational modifications (PTM). Ubiquitination, as a PTM of proteins, serves crucial functions in addition to protein degradation. It plays an important role in regulating various cellular processes and functions [32]. Previous studies have reported that ERBB3 undergoes constitutive ubiquitination. Additionally, the incubation with heregulin further enhances the ubiquitination process, leading to the subsequent degradation of ERBB3 [13,14]. Our results suggest that the absence of NUF2 leads to increased degradation of ERBB3 through the ubiquitin-proteasome pathway in HCC cells.

Certainly, it is crucial to emphasize that although we have conducted preliminary investigations on the molecular mechanisms of NUF2 in HCC, the heterogeneity of tumors and the multifunctionality of genes require more focused research. Our discussion has primarily focused on the role of NUF2 in promoting the proliferation of HCC cells through the regulation of the PI3K/AKT pathway. However, there is still much uncertainty regarding whether NUF2 modulates proliferation through alternative pathways or mechanisms, as well as its involvement in other biological behaviors. Therefore, further investigation is needed to address these questions and gain a better understanding of the full scope of NUF2′s functions in HCC.

In conclusion, our investigation highlights the importance of NUF2 in HCC, providing insights into its role in cell proliferation and the cell cycle by activating the PI3K/AKT signaling pathway through its interaction with ERBB3. Targeting NUF2 or the NUF2-ERBB3 interaction emerges as a promising therapeutic strategy for HCC.

Conclusion

In summary, our investigation has provided compelling evidence of the significant upregulation of NUF2 expression in HCC, which is associated with an adverse prognosis. Importantly, NUF2 has emerged as a key regulator of cell proliferation while concurrently affecting cell cycle progression, acting through the modulation of the ERBB3/PI3K/AKT signaling pathway. These findings have advanced our understanding of NUF2′s functional role in HCC and highlighted its potential as a therapeutic target for intervention. Future research should further explore the therapeutic implications of targeting NUF2 in the context of HCC therapy.

Declarations

Ethics approval and consent to participate

This study was performed in compliance with the Declaration of Helsinki, and it was approved by the Ethics Committee of The First Affiliated Hospital of Nanjing Medical University.

Funding statement

We gratefully acknowledge the support of the following grants for this work: National Natural Science Foundation of China (81472306); Special Fund for talents of the Jiangsu Province Hospital (YNRCZN015).

CRediT authorship contribution statement

Yiwei Liu: Conceptualization, Methodology, Writing – review & editing. Yuming Wang: Conceptualization, Writing – original draft. Jifei Wang: Data curation. Wangjie Jiang: Investigation. Yananlan Chen: Software, Supervision. Jijun Shan: Methodology, Resources. Xiao Li: Project administration. Xiaofeng Wu: Validation, Visualization.

Declaration of competing interest

The authors declare that they have no competing interests.