We collected the formalin-fixed paraffin-embedded (FFPE) tissue blocks of patients who underwent radical hysterectomy as primary treatment of cervical cancer in a tertiary referral hospital, Gil Medical Center, from January 2007 to December 2008. To reduce the errors during histologic interpretation of Nedd4 expression, we included FFPE blocks that contained normal epithelium, cervical intraepithelial neoplasia (CIN) III, and cancer in the same slide simultaneously. Patients were also eligible if the block had either normal epithelium or CIN III, along with cancer at the same time. The Gil Medical Center Institutional Review Board has approved the study (GAIRB2022-359). The informed consent was exempted due to the retrospective nature of this study.

The medical record of eligible patients was reviewed, and following items were analyzed: age; International Federation of Gynecology and Obstetrics (FIGO) stage; type of operation; involvement of lymph node, parametrium, and resection margin; tumor size; lymphovascular space invasion; adjuvant treatment; date of operation, recurrence, death, and last visit. FIGO stage was revised in 2009 and 2018, and the stage was determined by both staging systems [14] [15] . Progression-free survival (PFS) and overall survival (OS) were defined as the interval from the date of radical hysterectomy to the date of recurrence, death, or last follow-up.

Immunohistochemical staining for Nedd4-1 was performed using streptavidin-peroxidase assay according to the manufacturer’s instructions. The paraffin tissue sections (4-μm) were deparaffinized, and endogenous peroxidase was inactivated with 3% H₂O₂. For antigen retrieval, deparaffinized sections were immersed in 10 mmol/L citrate buffer and heated at 99˚C - 100˚C for 30 minutes. Then, the sections were incubated sequentially with primary anti-Nedd4-1 antibody (Millipore, Billerica, MA) at 1:100 dilution for 60 minutes, second antibody for 10 minutes, and horseradish peroxidase-streptavidin conjugate for 30 minutes. The slides were stained with 3,3’-diaminobenzidine staining and counterstained with hematoxylin and mounted.

A specialized gynecologic pathologist evaluated Nedd4-1 expression by grading the intensity of cytoplasmic staining. Immunostaining intensity was scored as 0 (negative), 1 (weak), or 2 (moderate/strong), and overexpression was defined according to both intensity and proportion of stained cells, as summarized in Table 1 . All immunostaining scores were evaluated by this single experienced gynecologic pathologist, thereby minimizing inter-observer variability.

The human cervical cancer cell lines, HeLa and CaSki were purchased from Korean Cell Line Bank and maintained in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% (v/v) fetal bovine serum (FBS, Welgene, Inc.), 1% (v/v) penicillin and streptomycin at 37˚C in a humidified incubator with 5% CO₂.

HeLa and CaSki cells were transfected using the Lipofectamine RNAiMAX (Invitrogen) transfection reagent as indicated in the manufacturer’s protocol. Two different siRNAs for Nedd4-1 (Bioneer Corporation) were used, and the sequences were as follows: siRNA#1, 5’-UAGAGCCUGGCUGGGUUGUUU-3’ and siRNA#2, 5’-UUCAAUUGCCAUCUGAAGUUUAUCC-3’. A commercial scrambled siRNA was used as a negative control (Bioneer Corporation).

Cells were harvested and lysed with RIPA lysis buffer containing protease inhibitors. Protein concentration was measured using the BCA-kit. The samples were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes. The membranes were incubated in the primary antibody solution overnight at 4˚C and horseradish peroxidase-conjugated secondary antibody solution for 1 hour at room temperature. Primary antibodies include PTEN (no. SC-6817-R), actin (no. SC-1615), and Nedd4-1 (no. SC-25508). Goat anti-mouse IgG-HRP (no. SC-2005) and mouse anti-rabbit IgG-HRP (no. SC-2357) were used as secondary antibodies. All primary and secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. A chemiluminescence reagent (Takara Bio Inc.) was applied and the chemiluminescent signals were captured by Amersham Imager 600 (GE Healthcare Biosciences AB).

MTT assay was used to estimate the number of viable cells following transfection with Nedd4-1 and negative control siRNAs. The cells were plated at a density of 2000 or 7000 cells per well in 96-well plates and incubated for 4 hours in media with 0.5 mg/ml MTT (Sigma-Aldrich) after transfection with siRNA. Optical absorbance at 570 nm was recorded by a microplate reader.

Annexin V-FITC/PI staining method was used to quantify apoptotic cell according to the manufacturer’s instructions (FITC annexin V apoptosis detection kit I, BD Pharming). Briefly, siRNA-transfected cells (5 × 10⁵ cells in 100 mm culture dish) were washed in cold phosphate-buffered saline, suspended in binding buffer, and stained with Annexin V-FITC. The stained cells were analyzed by FACSCalibur (BD Bioscience).

Results were shown as mean ± standard deviations or frequencies (%). Proportions of Nedd4-1 overexpression were compared between normal, CIN III, and cancer, and between histologic subtypes of cervical cancer using chi-square test or Fisher’s exact test. Kaplan-Meier survival curve was performed for analyzing PFS and OS according to Nedd4-1 overexpression in normal epithelium and cancer.

All experiments using cervical cancer cell lines were repeated more than three times. Independent t-tests and Mann-Whitney U tests were used to compare results between cells transfected with Nedd4-1 siRNA and negative control. p < 0.05 was considered to be statistically significant. The statistical analysis was performed with SPSS version 21 (IBM Corp., Armonk, NY, USA).

A total of 57 tissue blocks were identified from patients who received radical hysterectomy for cervical cancer from 2007 to 2008. Of them, 49 blocks met the inclusion criteria: 49 slides had cancer tissue, 35 slides had CIN III, 45 slides had normal epithelium, and 31 blocks had all three of them. The baseline characteristics are shown in Table 2 . The histologic subtypes included squamous cell carcinoma (SCC, n = 40, 81.6%), adenocarcinoma (AC, n = 7, 14.3%), and adenosquamous carcinoma (ASC, n = 2, 4.1%). According to 2009 FIGO staging system, most of patients were stage IB (n = 43, 87.8%), and by 2018 FIGO staging, 26 patients (59.2%) were diagnosed as IB. Lymph node metastasis was found in 10 (20.4%) patients. Involvement of parametrium and resection margin were diagnosed in 8 (16.3%) and 4 (8.2%) patients, respectively. Seventeen patients did not receive any adjuvant therapy, while others received chemoradiation, radiation, or chemotherapy after operation.

^aValues are presented as mean ± standard deviation or number (%). Abbreviations: FIGO: International Federation of Gynecology and Obstetrics; CCRT: Chemoradiation; RT: Radiation; CT: Chemotherapy.

Nedd4-1 overexpression was observed in 15.6% of normal epithelium, 37.1% of CIN III, and 79.6% of cancer. The differences in Nedd4-1 overexpression were statistically significant between normal epithelium and CIN III (p = 0.027), between normal epithelium and cancer (p < 0.001), and between CIN III and cancer (p < 0.001). Additionally, the difference was significant between all three types of tissue (p < 0.001) ( Table 3 , Figure 1 ).

^aValues are presented as number (%). ^bp-value between normal and CIN III = 0.027; CIN III and cancer < 0.001; normal and cancer < 0.001; Overall < 0.001. p-values were calculated using chi-square test. Abbreviations: CIN: Cervical intraepithelial neoplasia.

Nedd4-1 overexpression was similar between AC and ASC, between SCC and ASC (p = 0.444 and 1.000, respectively). However, significant differences were observed between SCC and AC (p = 0.018) and between all three histologic types (p = 0.033) ( Table 4 ).

^aValues are presented as number (%). ^bp-value between SCC and AC = 0.018; AC and ASC = 0.444; SCC and ASC = 1.000; Overall = 0.033. p-values were calculated using Fisher’s exact test. Abbreviations: SCC: Squamous cell carcinoma; AC: Adenocarcinoma; ASC: Adenosquamous carcinoma.

Patients were followed up for median of 92.72 months (range, 0.80 - 183.80 months), and 7 patients were diagnosed with recurrence. No statistically significant differences in PFS were observed between patients with and without Nedd4-1 overexpression in both normal epithelium and cancer tissue (p = 0.369 and 0.788, respectively). Four patients died during follow-up, and OS was also similar between patients according to Nedd4-1 overexpression in both normal epithelium and cancer tissue (p = 0.165 and 0.523, respectively) ( Figure 2 ).

Western blot assay was used to evaluate Nedd4-1 and PTEN expression in control and Nedd4-1 siRNA-transfected cells ( Figure 3(A) ). Silencing Nedd4-1 was not related to PTEN expression. As shown in Figure 3(B) , MTT assay was performed to examine the viability of cervical cancer cell upon silencing Nedd4-1. Downregulation of Nedd4-1 significantly decreased cell viability in both HeLa and CaSki cells, compared to negative control (p < 0.05).

Flow cytometry was performed to assess apoptosis following transfection. Figure 4(A) and Figure 4(B) describe the number of apoptotic cells in negative control and siRNA-transfected cells. The proportion of apoptotic cells was 9.3% and 9.4%, respectively, and no differences were found ( Figure 4(C) ).