NUP205 Stabilized YAP1 Protein to Stimulate Growth of Hepatocellular Carcinoma Cells in vitro and in vivo

Introduction

Liver cancer is a heterogeneous malignancy which has the fifth highest incidence and the second highest mortality of cancers in China. Five year survival rates are below 10%.^1,2 Hepatocellular carcinoma (HCC) is the most prevalent type of primary liver cancer, accounting for approximately 80–85% of all liver cancers.³ HCC shows rising worldwide incidence and mortality with 906,000 new cases in 2020, 830,000 deaths and a five-year survival rate of 22%.^3,4 Many patients receive a diagnosis at a locally advanced or late stage owing to the paucity of sensitive diagnostic methods or biomarkers. Targeted molecular drugs, such as sorafenib and lenvatinib, and immune checkpoint inhibitors have extended overall survival but prognoses remain poor and drug resistance is a challenge for systemic therapy.^5–7 There is, therefore a pressing need to identify therapeutic targets and develop anti-HCC drugs.

Yes-associated protein (YAP1) is involved in Hippo signaling with effects on cell proliferation, survival and tissue regeneration. YAP1 overexpression promoted proliferation, invasion and metastasis of various cancer cells and has been implicated in resistance to antitumor therapies.^8,9 YAP1 has been shown to influence the cell cycle, apoptosis, invasion, migration and glycolysis in liver cancer cells.^10,11

Nuclear pore protein 205 (NUP205) mediates transport between cytoplasm and nucleus¹² and its expression has been correlated with steroid-resistant nephrotic syndrome (SRNS), influencing the pathogenesis of focal segmental glomerulosclerosis (FSGS).^13,14 Previous studies have implicated NUP205 in tumorigenesis and elevated NUP205 protein has been observed in low-grade gliomas in which it is linked to poor prognosis and lymphocytic immune infiltration.¹⁵ Indeed, NUP205 may be a cancer driver for ovarian yolk sac tumors.¹⁶ NUP205 has been previously implicated in HCC in which it is overexpressed and expression correlated negatively with sensitivity to the anti-cancer drug, 5-fluorouracil (5-FU).¹⁷ It has also been shown to be a downstream target regulated by IncRNAs and microRNAs.^18,19

Previous work on NUP205 and its role in cancer has consisted largely of preliminary bioinformatics analysis, particularly regarding the biological behavior of tumor cells, an area which remains unclear. The current study extends such observations by investigating the impact of NUP205 knockdown and overexpression on proliferation and apoptosis of HCC cells in vitro and on tumor growth in vivo. Interactions of NUP205 with YAP1 and the effect on YAP1 expression and stability have been explored.

Materials and Methods

Ethics Statement

Ethical approval was granted by the Ethics Review Board of Jiangxi Cancer Hospital (approval number: 2024ky095). Experiments were performed in accordance with the Declaration of Helsinki (amended 2024) and written informed consent was obtained from all participants for analysis and publication of data.

Patients

Sixteen patients who received a diagnosis of HCC at Jiangxi Cancer hospital between January 2016 and December 2024 were enrolled. Inclusion Criteria were patients aged 18–65 years who underwent HCC resection and had cytological or histopathological confirmation of HCC. Exclusion Criteria were patients with mixed-type HCC or cholangiocarcinoma. No patient had received radiotherapy or chemotherapy and all were treated by liver resection. Sixteen pairs of HCC and adjacent non-tumor tissues (ANT, at least 2 cm away from the resection margin) were randomly selected and frozen at −80 °C for immunohistochemical (IHC) staining of NUP205.

Cell Culture

Human HCC cell lines, SMMC-7721 and BEL-7402, were obtained from the Beijing BeNa Culture Collection and Huh-7, SNU387, HCCLM3, Hep3B and SK-Hep-1 from the National Infrastructure of Cell Line Resource (Shanghai, China). SK-Hep1 cells were cultured in MEM with 1% NEAA, 1 mM sodium pyruvate and 10% FBS; Huh7 and HCC LM3 cells in DMEM with 10% FBS and Bel-7402, SMMC 7721 and SNU387 cells in RPMI-1640 medium with 10% FBS at 37 °C in 5% CO₂.

siRNA for NUP205 Knockdown and Transfection

siRNA oligonucleotides targeting human NUP205 mRNA were synthesized by Shanghai Jierui Biotechnology Co., Ltd. SK-HEP1 cells were seeded into 6-well plates at a density of 12×10⁶ cells/well and HCC-LM3 cells at 18×10⁶ cells/well, cultured to 80% confluence and transfected with siRNAs using Lipofectamine TM 3000 reagent (Thermo Fisher Scientific, USA), according to the manufacturer’s instructions. Negative control (NC) sense sequence was: 5′-UUCUCCGAACGUGUCACGUTT-3′; NUP205 sense sequences were: NUP205-siRNA#1: 5′-GCCAGUCACCUUUAGGCAATT-3′; NUP205-siRNA#2: 5′-CCGACUAUCAUGGGCUCUUTT-3′.

Plasmids for NUP205 Overexpression and Transfection

Cells were seeded into 6-well plates at a density of 8×10⁵ cells/well. 3 μg ANUP205 overexpression plasmid and negative control plasmid (PPL, USA) were mixed with LipofectamineTM 3000 transfection reagent to prepare NUP205 overexpression complexes which were incubated with SK-HEP1 and HCC-LM3 cells for 10 hours. mRNA was isolated 48 hours and proteins 72 hours post-transfection.

Quantitative Real-Time PCR (RT-qPCR)

Cell and xenograft tissue samples were lysed with TRIzol reagent (Thermo Fisher Scientific, USA) and RNA isolated by FreeZol Reagent kit (Vazyme, China). Total RNA was reverse transcribed into cDNA by qRT SuperMixII kit (Vazyme, China) and quantified by Nanodrop 2000. qPCR was performed using AceQ qPCR SYBR Green Master Mix (Vazyme, China) on a real-time PCR system (Thermo Scientific, USA). The thermocycler was programmed to cycle through the following phases: 95 °C for 5 mins, 40 cycles of 95 °C for 10s, 60 °C for 30s, melting at 95 °C for 15s, 60s at 60 °C and 15s at 95 °C. Primer sequences were: NUP205: 5′-GATCCAGGAGTGTTAGGTTGCC-3′; 18S: 5′-AGGTCTGTGATGCCCTTAGATGTC-3′; YAP1: 5′-GGAGAGGAGCTGATGCCAAG-3′.

Western Blotting

Cells were lysed on ice in 2% SDS buffer with phosphatase and protease inhibitors for 10 minutes, protein extracted and concentration measured using a BCA kit (Biyun Tian Biotechnology Co., Ltd). Equal quantities of protein were loaded into the wells of a 10% gel, proteins separated by PAGE and transferred to PVDF membrane, followed by blocking with 5% skimmed milk. The membrane was incubated overnight at 4 °C with primary antibodies raised against NUP205, YAP1 (Proteintech, China), Bcl-2, BAX, caspase-9, Cleaved PARP, Cleaved caspase-3, Cleaved caspase-7 and Cleaved caspase-9 (Cell Signaling Technology, USA) and with secondary antibodies (HRP-conjugated goat polyclonal anti-rabbit and HRP-conjugated goat polyclonal anti-mouse IgG, Cell Signaling Technology, MA, USA, 1:5000 in TBST) for 2h at room temperature. Enhanced chemiluminescence reagents (Millipore, MA, USA) were used for detection by Gel (2000) image analyzer (Bio-Rad, CA, USA).

Cell Viability

Cell viability was assessed using a CCK-8 kit (Cellcook Biotech Co., Ltd., China), according to the manufacturer’s instructions. In brief, cells were seeded into a 96-well plate at a density of 1500–2000 cells/well, cultured overnight and transfected with siNC or siNUP205 siRNAs for 10 hours. 10 μL/well CCK-8 solution was added, plates incubated at 37 °C for 1.5 hours and absorbance at 450 nm measured using a microplate reader (Varioskan LUX, USA).

Colony Formation

Cells were seeded into a 6-well plate at a density of 1500 cells/well, cultured overnight and transfected with siNC or siNUP205 siRNAs for 10 hours. Plates were cultured for 10–12 days, fixed with methanol and stained with 0.5% crystal violet before colonies were counted using ImageJ software.

Cell Proliferation

Cells were seeded into a 96-well plate at a density of 1500 cells/well, cultured for 24 h and transfected with siNC or siNUP205 siRNAs for 10 hours. Proliferation was measured by EDU kit (Guangzhou Ribo Bio-Technology Co., Ltd.), according to the manufacturer’s instructions.

Apoptosis Assay

Apoptosis was measured in cultured cells by Annexin V-FITC Apoptosis Detection Kit (Vazyme, China), according to the manufacturer’s guidelines. In brief, cells were transfected with siNC or siNUP205 siRNAs for 72 hours, stained with Annexin V-FITC and propidium iodide (PI) and analyzed by FACSCaliburTM flow cytometer (BD Biosciences, USA). Mitochondrial membrane potential was measured by JC-1 Mitochondrial Membrane Potential Detection Kit (Beyotime, China). In brief, cells were transfected with siNC or siNUP205 siRNAs for 10 hours, incubated in complete culture medium for 72 hours, harvested, washed twice with cold PBS and resuspended in 1 mL JC-1 dye for 30 min. incubation at 37 °C. Samples were analyzed by FACSCaliburTM flow cytometer. Apoptosis was measured in xenograft tumors by terminal deoxynucleotidyl transferase (TdT) biotin-dUTP nick-end labeling (TUNEL) assay using a TUNELApoptosis Detection Kit (KeyGen BIOTECH, China), according to the manufacturer’s instructions.

Xenograft Tumors in Nude Mice

Female Balb/c nude mice, aged 3–4 weeks, mass 18–22 grams were purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd. and housed under SPF conditions with a 12 h light/12 h dark cycle and access to food and water ad libitum. 7×10⁶ HCC-LM3 cells were subcutaneously injected into the axillary region, tumors allowed to grow to 100 mm³ and mice randomly assigned to two groups (n = 6).²⁰ Animals were treated with either 10 μg control siRNA or 10 μg NUP205 siRNA with 7.5 μL Entranster^TM in vivo transfection reagent (Yingrun Bio-System, Beijing) via intra-tumoral injection every two days.

Tumor volume (TV) and body weight were monitored. TV was calculated as TV (mm³) = 1/2 × A × B², where A was the longest and B the shortest diameter of the tumor. Mice were anesthetized with gradually increasing concentrations of carbon dioxide (CO₂, approximately 40% of the chamber volume per minute) until loss of consciousness was confirmed.

Cervical dislocation was subsequently performed as a secondary method of euthanasia to ensure death, in strict accordance with the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals (2020 Edition) and the National Guidelines for the Care and Use of Laboratory Animals in China.

After 33 days of treatment, the mice were humanely euthanized, and the tumors were excised for hematoxylin–eosin (H&E), immunohistochemistry (IHC), and TUNEL staining, as well as qRT-PCR and Western blot analyses.

All animal care and experimental procedures were conducted in accordance with institutional guidelines and ethical approval was obtained from the Animal Care and Use Committee of China Pharmaceutical University (Approval No. 2023-10-018).

Immunohistochemistry Staining

NUP205 expression in 4μm sections of liver cancer and paired non-cancerous tissue was assessed by IHC with a primary anti-NUP205 antibody (slices thickness: 4 μm). NUP205 expression and the degree of cell proliferation was assessed in xenograft tumor tissues using anti-NUP205 and anti-Ki67 (1:100, Abcam, UK) antibodies.

Co-Immunoprecipitation (Co-IP)

Cells were seeded into 6 well plates at a density of 1×10⁶ cells/well, cultured for 72h, transfected with siNC and siNUP205 siRNA for 10 h, medium changed and cultured for 72 h. Proteins were extracted, quantified and incubated overnight at 4 °C with a magnetic bead-antibody conjugate.

Cell Immunofluorescence Assay

Cells were seeded into 12 well plates at a density of 1×10⁵ cells/well, cultured for 48h, transfected with siNC and siNUP205 siRNA for 10 h, medium changed and cultured for 48 h. Cells were fixed with 4% formaldehyde solution pre-chilled to 4 °C and fixed at room temperature for 30 minutes before 3% BSA and 0.1% Triton X-100 in PBS was added for blocking at room temperature for 50 minutes. Cells were washed twice with PBS at 120 rpm for 5 min. 40 μL/well primary antibody dilution solution was added to clean slides, cell slides placed on top and incubated overnight at 4 °C. Hoechst cell nuclear stain was added to the secondary antibody dilution solution at a ratio of 1:2000, cell coverslip inverted over slide with the secondary antibody dilution solution and incubated at room temperature in the dark for 1 hour. Coverslips were removed, slides washed with PBS, dried and 8–10 μL anti-fluorescence quencher added for storage at 4 °C in the dark. Slides were analyzed under a confocal microscope.

Statistical Analysis

Statistical analyses were performed using GraphPad Prism 9 software. Data are presented as mean ± Standard Deviation (SD). Inter-group differences were assessed by Student’s t-test and one-way Analysis of Variance (ANOVA) with post-hoc multiple comparisons. Statistical significance is indicated as follows from a threshold of p < 0.05 which was considered to indicate significance: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Results

NUP205 Was Overexpressed in Hepatocellular Carcinoma Tissues and Cells

Data reflecting NUP205 mRNA expression in cancer and adjacent normal tissues was accessed from The Cancer Genome Atlas (TCGA) database through the University of Alabama Cancer database (UALCAN) website. NUP205 was overexpressed in many cancer tissues, including HCC (Figure 1A and B). NUP205 mRNA expression also generally increased with HCC clinicopathological grade in TCGA tissues, although the small grade 4 sample size (n = 12) should be interpreted with caution (Figure 1C). IHC staining of tissues collated from patients (n = 16) recruited by the current study indicated higher levels of NUP205 protein in HCC than in ANT (Figure 1D). The data demonstrate overexpression of NUP205 in HCC tissues.

Figure 1 NUP205 expression in cancer and non-tumor adjacent tissues from TCGA database. (A) NUP205 mRNA expression in tumor (red bars) and non-tumor (blue bars) samples; (B) NUP205 mRNA expression in HCC (n = 371) and normal tissues (n = 50) from TCGA database; (C) NUP205 mRNA expression in HCC samples from TCGA by grade; (D) IHC detection of NUP205 expression in 16 paired patient samples of HCC and adjacent normal tissues. Representative images are shown to the left with quantitative analysis to the right. Scale bars: 100 μm. Bars represent mean ± SD. ****p < 0.0001; ns = not significant (Log rank test). Abbreviations: NUP205, Nuclear pore protein 205; HCC, Hepatocellular carcinoma; IHC, immunohistochemical; SD, Standard Deviation.

High Expression of NUP205 Was Associated with Poor HCC Prognosis

Kaplan-Meier analysis was performed on TCGA HCC data using the Tumor Gene Prognosis Prediction Database (http://ualcan.path.uab.edu/analysis.html). NUP205 overexpression was associated with reduced overall survival (OS) and disease-specific survival (DSS) in HCC patients (Figure 2A–C). Similar associations of NUP205 expression with OS and DSS were seen for both genders (Figure 2D and E). OS and DSS indicate that NUP205 is clinically associated with poorer prognosis in HCC patients.

Figure 2 NUP205 expression and HCC prognosis. (A) Kaplan-Meier OS curves for HCC patients from TCGA database with high or low NUP205 expression; (B) Kaplan-Meier DSS curves for HCC patients from TCGA database with high or low NUP205 expression; (C) Effect of NUP205 expression on survival of HCC patients from TCGA database; (D and E) Kaplan-Meier OS curves for male and female HCC patients from TCGA database with low or high NUP205. p values calculated by Log rank test. Abbreviations: NUP205, Nuclear pore protein 205; HCC, Hepatocellular carcinoma; OS, overall survival.

NUP205 Silencing Inhibited HCC Cell Proliferation

NUP205 mRNA and protein expression were evaluated in six liver cancer cell lines: SMCC 7721, Hep3B, HCC LM3, SK-Hep1, Huh7 and Bel 7402 and NUP205 mRNA expression found to be highest in HCC LM3 and SK-Hepl cells (Figure 3A and B) which were used for further experiments. Transfection of these cell-lines with siNUP205 resulted in an 82.55% (100% − (14% + 20.9) / 2) reduction in levels of NUP205 mRNA in HCCLM3 and 71.05% (100% − (29.5% + 28.4) / 2) in SK-Hep1. NUP205 protein was reduced by 56% in HCC LM3 and by 60% in SK-Hep1 (Figure 3C and D). Transfection with siNUP205 was considered to produce a satisfactory knockdown of expression in HCC LM3 and SK-Hep1 cells.

Figure 3 NUP205 expression in HCC cell lines and effect of NUP205 silencing on proliferation. (A and B) NUP205 mRNA and protein expression in HCC cells; (C and D) Silencing of NUP205 by transfection with siNUP205 siRNAs. Effects on NUP205 protein (C) and mRNA (D) in HCC LM3 and SK-Hep1 cells are shown; (E and F) Viability of HCC LM3 and SK-Hep1 cells transfected with siNUP205 measured by CCK-8 assay; (G and H) Colony formation by HCC LM3 and SK-Hep1 cells transfected with siNUP205. Representative images are shown on the left with quantitative analysis on the right; (I and J) Proliferation of HCC LM3 and SK-Hep1 cells transfected with siNUP205 measured by EDU assay. Representative images are shown on the left with quantitative analysis on the right. Bars represent mean ± SD. #Indicates a separator and has no practical meaning. *p < 0.05, **p < 0.01, ****p < 0.0001 (one-way ANOVA multiple comparison). Scale bars: 100 μm. Abbreviations: NUP205, Nuclear pore protein 205; HCC, Hepatocellular carcinoma; SD, Standard Deviation; ANOVA, Analysis of Variance.

Growth curves were constructed for these two cell types using a CCK-8 cell viability assay. Knockdown of NUP205 by siNUP205 transfection reduced cell growth relative to cells transfected with siNC (Figure 3E and F). Colony formation was also decreased (Figure 3G and H). Cell proliferation was measured by EDU assay and blue fluorescence indicates nuclear labelling with green fluorescence indicating nuclear incorporation of EDU in the images. NUP205 knockdown reduced the ratio of green to blue fluorescence, indicating reduced rates of proliferation (Figure 3I and J). Thus, the knockdown of NUP205 inhibited HCC LM3 and SK-Hepl cell proliferation implying that NUP205 may have a role in the promotion of HCC cell growth.

NUP205 Overexpression Promoted HCC Cell Proliferation

NUP205 overexpression was achieved by the transient transfection of HCC LM3 and SK-Hep1 cells with ANUP205 plasmid and analysis by qRT-PCR and Western blotting showed upregulation of NUP205 mRNA and protein (Figure 4A and B). Growth curves showed increased rates in both cell types when NUP205 was overexpressed (Figure 4C and D). Colony formation was reduced in the HCC LM3 cell-line but not in the SK-Hep1 cell-line (Figure 4E). It may be concluded that higher rates of NUP205 expression had a generally stimulatory effect on the growth of HCC cells, supporting the view of the involvement of this protein in the growth of HCC tumors.

Figure 4 Effects of NUP205 overexpression on HCC cell proliferation. (A and B) Expression of NUP205 mRNA assessed by RT-qPCR and of NUP205 protein assessed by Western blotting following transfection of HCC LM3 and SK-Hep1 cells with NUP205 overexpressing plasmid; (C and D) Growth curves constructed from CCK-8 assays of HCC LM3 and SK-Hep1 cell viability following transfection with NUP205 overexpressing plasmid; (E) Colony formation by HCC LM3 and SK-Hep1 cells following transfection with NUP205 overexpressing plasmid. Bars represent mean ± SD. #Indicates a separator and has no practical meaning. *p < 0.05, **p < 0.01, ****p < 0.0001, ns = not significant by two-tailed Student’s t-test and one-way ANOVA multiple comparison. Abbreviations: NUP205, Nuclear pore protein 205; HCC, Hepatocellular carcinoma; SD, Standard Deviation; ANOVA, Analysis of Variance.

NUP205 Silencing Induced Apoptosis in HCC Cells

Measurements of mitochondrial membrane potential (MMP) were made by JC-1 assay on the grounds that decreased potential is considered to be a signal for the release of apoptotic factors and the onset of apoptosis. Silencing of NUP205 produced a decrease in MMP in HCC LM3 and SK-Hepl cells which is represented by decreased red and increased green fluorescence in the images shown in Figure 5A. The data indicated that NUP205 knockdown increased pro-apoptotic changes in these cells. Annexin V/PI assays showed that 50% HCC LM3 cells were apoptotic after NUP205 silencing compared with 10% control cells (Figure 5B). Similarly, 30% SK-Hep1 cells were apoptotic after transfection with siNUP205, compared with 8% control cells (Figure 5B). Hoechst staining was used to assess nuclear morphology and indicated nuclear condensation and darker staining following transfection with siNUP205 in both HCC LM3 and SK- Hep1 cells (Figure 5C). Measurements were made of the expression of apoptosis effector proteins, Cleaved-caspase 7, Cleaved-caspase 3, Cleaved PARP, BAX and Bcl-2. Pro-apoptotic factors, Cleaved PARP, BAX, Cleaved-caspase 7 and Cleaved-caspase 3 were found to be increased in both cell types and the anti-apoptotic factor, Bcl-2, was reduced in SK-Hep1 cells (Figure 5D). The data described above indicate that silencing of NUP205 resulted in a stimulatory effect on apoptosis in the HCC cell-lines studied.

Figure 5 Effects of NUP205 silencing on apoptosis in HCC LM3 and SK-Hep1 cells. (A) Measurements of MMP by JC-1 assay in HCC LM3 and SK-Hep1 cells after transfection with siNUP205. Representative images are shown on the left with quantitative analysis of green/red fluorescence on the right; (B) Measurements of apoptosis by Annexin V/PI assay in HCC LM3 and SK-Hep1 cells after transfection with siNUP205. Representative flow cytometry images are shown on the left with quantitative analysis of apoptotic cells on the right; (C) Hoechst staining to show nuclear changes in HCC LM3 and SK-Hep1 cells after transfection with siNUP205. Representative images are shown with white arrows indicating apoptotic nuclei. Scale bar: 100 μm; (D) Expression of effectors involved in apoptosis in HCC LM3 and SK-Hep1 cells after transfection with siNUP205. Protein expression is shown in the gel (left) and mRNA expression in the plots (right). Expression was normalized to β-actin. Bars represent mean ± SD. #Indicates a separator and has no practical meaning. *p < 0.05, ***p < 0.001, ****p < 0.0001 by one-way ANOVA with multiple comparison. Abbreviations: NUP205, Nuclear pore protein 205; HCC, Hepatocellular carcinoma; SD, Standard Deviation; ANOVA, Analysis of Variance.

NUP205 Silencing Inhibited HCC Tumor Growth and Promoted Apoptosis in vivo

A xenograft HCC tumor model was established by subcutaneous injection of HCC LM3 cells into the flanks of nude mice. siNUP205 and transfection reagent were administered by intra-tumoral injection every two days after the tumor had achieved a 100mm³ volume until a 1000–1500 mm³ was achieved, at which point, mice were sacrificed.

Differences in tumor volume and mass between siNC and siNUP205 groups could be seen by day 33, indicating an impact of NUP205 knockdown on tumor growth in vivo (Figure 6A–F). Histological analysis was performed on tumor tissues by H&E, Ki67 and TUNEL staining (Figure 6G–I). H&E analysis showed changes in tumor cell morphology indicative of increased rates of apoptosis. siNC control tumors had tightly packed cells with normal morphology but siNUP205 tumors had significant necrosis with cytoplasmic and nuclear shrinkage and increased intercellular spaces. Decreased levels of actively proliferating cells (Ki67) and increased numbers of apoptotic cells (TUNEL) in tumors treated with siNUP205 compared with control tumors treated with siNC were also seen. Consistent with the histological and TUNEL findings, Western blot analysis showed an increase in the expression of pro-apoptotic proteins, including Cleaved PARP, BAX, and Cleaved caspase-3/7, in the siNUP205 group. Notably, the levels of Cleaved caspase-3/7 were significantly higher than those in the control group (P < 0.001) (Figure 6J). It appears that the silencing of NUP205 in xenograft tumors in vivo resulted in reduced rates of tumor growth which were reflected in reduced levels of proliferating tumor cells and increased rates of apoptosis.

Figure 6 Effects of NUP205 silencing on growth of human HCC xenograft tumors in nude mice. (A and B) Photographic images of mice and excised tumors from control (siNC injected) and NUP205 knockdown (siNUP205 transfected) animals; (C) Tumor mass at 33 days from control and NUP205 silenced tumors; (D) Time course of tumor volume in siNC and siNUP205 treated mice; (E) Time course of relative tumor volume in siNC and siNUP205 treated mice. Relative tumor volume was calculated as (tumor volume siNUP205)/ (tumor volume siNC) × 100; (F) Time course of mouse body weight during treatment; (G) Ki67 expression by siNC and siNUP205 treated tumor tissues detected by IHC. Representative images are shown on the left with quantitative analysis on the right; (H) Apoptotic cells in tumors from siNC and siNUP205 treated animals assessed by TUNEL staining. Representative images are shown on the left with quantitative analysis on the right; (I) Tumor tissue morphology assessed by H&E staining; (J) Representative Western blotting images showing expression of Cleaved PARP, Caspase 9, Cleaved caspase 9, Bcl-2, BAX, Caspase7 and Cleaved caspase 3/7 proteins in tumors treated with siNC or siNUP205 (n = 6). Image scale bars: 50 μm, 20 μm. Bar charts represent mean ± SD. #Indicates a separator and has no practical meaning. *p < 0.05, **p < 0.01, ***p < 0.001 by two- tailed Student’s t-test. Abbreviations: NUP205, Nuclear pore protein 205; HCC, Hepatocellular carcinoma; SD, Standard Deviation.

NUP205 Silencing Inhibited YAP1 Expression and Stability in HCC Cells

NUP205 silencing has been reported to inhibit the nuclear transport of YAP1 protein in podocytes.¹⁴ The expression of YAP1 mRNA was assessed by RT-qPCR in HCC LM3 and SK-Hepl cells with NUP205 knockdown or overexpression and no significant differences were found (Figure 7A and B). Western blotting analysis showed lower expression of YAP1 protein in siNUP205 transfected cells and higher levels in NUP205 overexpressing cells (Figure 7C and D). Results obtained from examination of tumor tissues from the in vivo mouse model produced results consistent with those from the cells growing in vitro. No differences were found in YAP1 mRNA after treatment of tumors with siNUP205 but levels of YAP1 protein were decreased (Figure 7E and F). NUP205 silencing led to the downregulation of YAP1 protein both in vitro and in vivo.

Figure 7 Effects of NUP205 on YAP1 expression. (A and B) YAP1 mRNA expression assessed by RT-qPCR in HCC LM3 and SK-Hep1cells with NUP205 knockdown (A) or overexpression (B); (C) YAP1 protein expression assessed by Western blotting in HCC LM3 and SK-Hep1 cells with NUP205 overexpression. Representative image shown in upper panel with quantitative analysis in lower panel; (D) YAP1 protein expression assessed by Western blotting in HCC LM3 and SK-Hep1 cells with NUP205 silencing. Representative image on the left with quantitative analysis on the right; (E and F) Expression of YAP1 mRNA and protein in human xenograft HCC tumors in a mouse model treated with siNC (control) or siNUP205 (NUP205 silencing); (G and H) Representative immunofluorescence staining images to show the localization of YAP1 in HCC LM3 and SK-Hep1 transfected with siNC (control) or siNUP205 (NUP205 knockdown, n = 3). Scale bars: 20 μm. Bar charts represent mean ± SD. #Indicates a separator and has no practical meaning. *p < 0.05, **p < 0.01, ****p < 0.0001. Abbreviations: ns, not significant; NUP205, Nuclear pore protein 205; HCC, Hepatocellular carcinoma; RT-qPCR, Quantitative Real-Time PCR; YAP1, Yes-associated protein; SD, Standard Deviation.

Immunofluorescence assays of HCC LM3 and SK-Hepl cells were conducted to assess the subcellular localization of YAP1 protein following manipulation of NUP205. NUP205 silencing did not change YAP1 localization (Figure 7G and H). These findings are consistent with the view that NUP205 knockdown decreased the expression of YAP1 protein but not YAP1 mRNA and did not affect subcellular localization of YAP1. The data suggest that NUP205 may affect the stability of YAP1 protein in hepatocellular carcinoma.

HCC LM3 and SK-Hepl cells that had been transfected with siNUP205 were treated with the protein synthesis inhibitor, cycloheximide (CHX), for 6, 12 and 24 hours. Treatment of NUP205 silenced cells with CHX produced a greater downregulation of YAP1 protein, suggesting decreased stability of YAP1 under these conditions (Figure 8A and B). Treatment with the proteasomal inhibitor, MG132, reversed the downregulation of YAP1 in NUP205 knockdown cells but treatment with the autophagolysosomal inhibitor, chloroquine (CQ), did not, indicating that the proteasomal pathway is likely to be involved in YAP1 degradation in NUP205 silenced cells (Figure 8C and D). Immunoprecipitation (IP) experiments were performed to indicate the ubiquitination status of YAP1 protein in SK-Hep1 cells. NUP205 silencing increased YAP1 ubiquitination (Figure 8E). The data presented are consistent with the view that NUP205 silencing reduced the stability of YAP1 protein and facilitated its degradation via the ubiquitin-proteasome pathway. The implication is that, under conditions of high NUP205 expression in HCC cells, YAP1 will be present at high levels.

Figure 8 Levels of YAP1 protein in NUP205 knockdown HCC cells. (A and B) HCCLM3 (A) and SK-Hep1 (B) cells were transfected with siNUP205 and treated with 30 μg/mL CHX for 0, 6, 12 and 24 h. Western blotting images showing levels of YAP1 protein are shown on the left with quantitative analysis on the right; (C and D) HCC LM3 (C) and SK-Hep1 (D) cells were transfected with siNUP205 and treated with 10 μM MG132 for 12 h or 25 μM CQ for 12 h (n = 3). Western blotting images with levels of NUP205 and YAP1 are shown in the upper panels with quantitative analysis in the lower panels; (E) Immunoprecipitation assays to show levels of ubiquitylated YAP1 protein in NUP205 silenced SK-Hep1 cells. Bars represent mean ± SD. #Indicates a separator and has no practical meaning. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant by one-way ANOVA with multiple comparison. Abbreviations: NUP205, Nuclear pore protein 205; HCC, Hepatocellular carcinoma; CHX, Cycloheximide; YAP1, Yes-associated protein; SD, Standard Deviation; ANOVA, Analysis of Variance.

Discussion

Primary HCC is a common malignant tumor with high worldwide mortality and increasing incidence¹ and survival is adversely affected by low rates of early diagnosis and high rates of recurrence.²¹ Primary liver cancer has three main pathological types, HCC, intrahepatic cholangiocarcinoma and combined HCC-cholangiocarcinoma, with HCC being the most common and frequently fatal, accounting for approximately 80–85% of primary liver cancers.²² Systemic therapy with first line drugs, such as receptor tyrosine kinase (RTK) inhibitors, sorafenib and lenvatinib, is a treatment option²³ but the potential of immune checkpoint inhibitors (ICIs) has been demonstrated by the success of the IMbrave150 trial in 2020. The combination of the anti-PD-L1 monoclonal antibody (mAb), atezolizumab, and anti- vascular endothelial growth factor (VEGF) mAb, bevacizumab, gave significant benefits for OS and progression-free survival compared with sorafenib.²⁴ HCC is an innately drug-resistant tumor and HCC patients are generally insensitive to chemotherapy drugs.²⁵ The high phenotypic and molecular heterogeneity of liver cancer cells facilitates resistance to conventional chemotherapy or targeted therapy following initial improvement and leads to multidrug resistance.²⁶ Treatment resistance and disease progression remain as challenges for HCC,²³ illustrating the urgency for exploration of HCC disease mechanisms to identify therapeutic targets.

The nuclear pore complex (NPC) is a highly conserved channel in the nuclear envelope that mediates mRNA export to the cytosol and bidirectional protein transport. Many chromosomal loci physically interact with nuclear pore proteins (Nups) to promote transcriptional repression, transcriptional activation or transcriptional localization. Interaction with the NPC also affects the spatial arrangement of genes, interchromosomal clustering and folding of topologically associated domains. Thus, the NPC is a spatial organizer of the genome and regulator of genome function.²⁷ The NPC is a large 110 megadalton structure, composed of around 1000 proteins, including the nucleoporin, NUP205.²⁸ The complex transports macromolecules across the nuclear membrane, regulating the flow of genetic information from transcription to translation.²⁹ Mutations in nucleoporin genes, NUP93 and NUP205, contribute to steroid-resistant nephrotic syndrome and focal segmental glomerulosclerosis (FSGS) but mechanisms by which NUP205 dysfunction leads to podocyte injury in FSGS remain unclear.¹³ Pathogenic variants in six genes encoding Nups, NUP85, NUP93, NUP107, NUP133, NUP160 and NUP205, cause monogenic steroid-resistant nephrotic syndrome (SRNS), referred to as nucleoporin-associated SRNS.³⁰ NUP205 is required for the nuclear import of the cell growth-associated transcription factors, YAP and TAZ, and loss of NUP205 reduced interactions between the YAP/TAZ complex and TEAD1, decreasing the expression of target genes.¹⁴ NUP205 downregulation has been shown to impair podocyte proliferation and increase cell death¹⁴ and bioinformatics studies have suggested that NUP205 is a driver for thyroid, bladder and lung cancers.^31–33 However, little is known about roles of NUP205 in HCC and previous work has been limited to bioinformatics analyses showing overexpression in HCC and association with lower survival rates, immune infiltration and poor prognosis.¹⁸ To the best of our knowledge, the current work is the first to examine the role of NUP205 in HCC cell proliferation and apoptosis and investigate underlying mechanisms.

The Hippo pathway is an evolutionarily conserved signaling cascade involved in organ development, epithelial cell homeostasis, tissue regeneration, wound healing and immune regulation. Many of these functions are mediated through downstream transcriptional effectors, YAP and TAZ, which control TEAD transcription factors. Dysregulated Hippo signaling and YAP/TAZ-TEAD activity have been linked to various diseases, particularly cancer, making this an attractive therapeutic target.³⁴ YAP1 is a transcriptional co-activator that is negatively regulated by Hippo signaling and is involved in cell growth, tissue homeostasis and organ size.³⁵ Hippo signaling normally suppresses YAP1 function, maintaining stem cells in a quiescent state but tissue damage leads to YAP1 activation and the generation of new cells for stem cell self-renewal and tissue repair.³⁶ YAP1 has also been linked to the proliferation, invasion and metastasis of various tumor cells³⁷ and may be activated in tumors by loss-of-function mutations in upstream regulatory factors of the Hippo pathway, such as LATS1/2, MST1/2 or NF2, or mutations in genes, such as G protein-coupled receptors or viral proteins, that influence Hippo.⁸ Hyperactivated YAP1 has been shown to promote HCC cell proliferation and glycolysis, correlating with poor prognosis.³⁸ The current work illustrates the elevated NUP205 expression that characterizes HCC tissues compared with adjacent non-cancerous tissues and supports the association with poorer prognosis. NUP205 also affected proliferation and apoptosis in the HCC cell-lines of the present study.

The current findings indicate a novel regulatory axis involving NUP205 and YAP1 in HCC. Impaired degradation of YAP1 and the interleukin-6 family signaling protein, IL6ST, has previously been demonstrated in HCC.¹⁰ The current work extends such findings by demonstrating that NUP205 knockdown did not affect YAP1 mRNA but promoted the ubiquitination and degradation of YAP1 protein via a proteasomal pathway. We suggest that the role of NUP205, which is known to be overexpressed in many cancer types, is to stabilize YAP1 protein, allowing the continued stimulation of gene transcription concerned with cell growth. Thus, NUP205 overexpression is conducive to the unregulated cell proliferation characteristic of tumor cells.

Conclusion

The current findings demonstrate a role of NUP205 in proliferation and apoptosis of HCC cells, explaining its link with poor prognosis. NUP205 may facilitate ubiquitination of YAP1 protein. The NUP205/YAP1 axis may be a therapeutic target for HCC treatment. The current work gives mechanistic insights into NUP205 regulation of HCC proliferation and apoptosis and identifies NUP205 as a promising therapeutic target for HCC.