The human bronchial epithelial immortalized cell line Beas-2B was purchased from the American Type Culture Collection and cultured in Bronchial Epithelial Basal Medium (BEBM; Lonza) with 10% fetal bovine serum (FBS; Corning, Inc.) and 1% penicillin/streptomycin (Gibco; Thermo Fisher Scientific, Inc.). The lung cancer cell lines NCI-H1299, NCI-H358, A-549 and NCI-H23 were purchased from Zhong Qiao Xin Zhou Biotechnology Co., Ltd. NCI-H1299, NCI-H358 and NCI-H23 were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS and 1% penicillin/streptomycin; A-549 was grown in F-12K medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS and 1% penicillin/streptomycin. All cells were maintained in a humidified incubator with 5% CO₂ at 37°C.

To detect the protein level of 14-3-3ơ in LUAD tissue, a total of four LUAD tissue samples and non-tumor adjacent tissues samples were derived from biopsy samples and pathologically confirmed as LUAD from December 2020 to August 2021. Specific information on the patients is provided in Table I. The present study was performed according to the Declaration of Helsinki and approved by Ethics Committee of the People's Hospital of Beilun District [Ningbo, China; no. 2021-23(YS)]. Each patient provided written informed consent prior to surgery.

To detect the relationship between 14-3-3ơ and prognosis of patients with LUAD, a total of 106 paraffin-embedded LUAD specimens from the archives of the Department of Pathology at People's Hospital of Beilun (Ningbo, China) were included in the present study. All patients had undergone surgery from February 2002 to October 2003. All patients were pathologically confirmed as LUAD. Furthermore, they had no chemotherapy, radiation therapy or history of surgery. The patients, 66 males (62.3%) and 40 females (37.7%), ranged in age from 39 to 77 years (mean, 59 years). All patients received radical surgery for primary tumor and lymph nodes, and postoperative cisplatin-based adjuvant chemotherapy. The patients did not receive any radiotherapy or chemotherapy prior to surgery. The histological type and stage were determined according to the classification for NSCLC by the World Health Organization and the International Union against Cancer Tumor-Nodes-Metastasis staging system (32). All patients provided written informed consent for tissue use prior to surgery and this study was approved by the Institute Research Ethics Committee of People's Hospital of Beilun District (Ningbo, China). All patients had follow-up records for over 6 years. After the completion of therapy, patients were observed every 3 months for the first 3 years and every 6 months thereafter. Overall survival (OS) was defined as the time from diagnosis to the date of death, or at the latest date if patients were still alive. Progression-free survival (PFS) was determined from the first day of treatment to the earliest signs of disease progression as identified by CT or MRI, or death from any cause.

Plasmids of HA-14-3-3σ were obtained from Professor Jie Xu from The Second Affiliated Hospital of the Third Military Medical University (Army Medical University, Chongqing, China). The targeting plasmids were delivered into NCI-H1299 LUAD cells using the Lipofectamine^® 2000 transfection reagent (Cat No: 11668019; Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. siRNA designed to target 14-3-3σ (si-14-3-3σ) was purchased from Sangon Biotech Co., Ltd., including si-14-3-3σ-1# (5′-GGAUCCCACUCUUCUUGCA-3′) and si-14-3-3σ-2# (5′-GACCAUGUUUCCUCUCAAU-3′). The sequence for the scrambled control siRNA was 5′-AUUGUAUGCGAUCGCAGACUU-3′. For transient transfection, control and si-14-3-3σ siRNA (#1 and #2) were mixed with Lipofectamine^® 2000 and then added to the cell culture medium of A-549 LUAD cells according to the manufacturer's protocol. Finally, western blot analysis was used to assess the transfection efficiency.

Cells were seeded into 96-well plates in triplicate at an initial density of 0.2×10⁴ cells/well. Cells were processed using the ATP-lite assay (cat. no. 6016739; Perkin-Elmer, Inc.) to assess cell proliferation at various time-points (24, 48, 72, 96 and 120 h) according to the manufacturer's protocol (33). Luminescence was read using a LD 400 plate reader (Beckman Coulter, Inc.). Each experiment was performed in triplicate.

After transduction with targeting plasmids or small interfering (si)RNA, the stably transfected A-549 or NCI-H1299 cells were seeded into 6-cm dishes in triplicate at a density of 0.5×10³ cells/well and incubated at 37°C for 14 days. The colonies were fixed with 4% paraformaldehyde, stained with crystal violet and then counted (33).

A standard soft agar assay was done as detailed previously (34). In brief, 5×10³ of parental cells or their transfectants (14-3-3σ-overexpressing or 14-3-3σ silenced, along with the vector controls), were suspended in 0.33% agar (cat. no. A5431; MilliporeSigma) containing 10% FCS in 6-cm dishes. After culture at 37°C for 14 days, the cells were stained with p-iodonitrotetrazolium (cat. no. V900870; 1 mg/ml; MilliporeSigma) overnight at 37°C and the colonies with cell numbers of more than eight were counted in five randomly selected areas from each dish.

Total protein was extracted from cells using RIPA lysis buffer (50 mM Tris-pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate and 0.1% SDS; cat. no. P0013B; Beyotime Institute of Biotechnology) supplemented with phosphatase (cat. no. B15001; Bimake) and protease inhibitors (cat. no. B14001; Bimake). BCA assay kit (cat. no. P0011; Beyotime Institute of Biotechnology) was used to determine the protein concentration and 40 µg protein was loaded onto the gel per lane. The equal amounts of total protein were separated by SDS-PAGE (8–15%) and then transferred onto nitrocellulose (NC) membranes (EMD Millipore). The NC membranes were blocked with 5% nonfat milk (cat. no. P0216; Beyotime Institute of Biotechnology) for 1 h at room temperature, followed by incubation with primary antibodies overnight at 4°C. The following primary antibodies were used: 14-3-3σ (cat. no. sc-100638; 1:1,000 dilution; Santa Cruz Biotechnology, Inc.), poly (ADP-ribose) polymerase (PARP; cat. no. 13371-1-AP; 1:1,000 dilution; ProteinTech Group, Inc.), Caspase-3 (cat. no. 66470-2-Ig; 1:1,000 dilution; ProteinTech Group, Inc.), GAPDH (cat. no. 60004-1-Ig; 1:1,000 dilution; ProteinTech Group, Inc.) and hemagglutinin (HA)-tag (cat. no. 51064-2-AP; 1:1,000 dilution; ProteinTech Group, Inc.). The NC membranes were then incubated with the following corresponding secondary antibodies: Goat anti-rabbit IgG (cat. no. SA00001-2; 1:2,000 dilution; ProteinTech Group, Inc.); goat anti-mouse IgG (cat. no. SA00001-1; 1:2,000 dilution; ProteinTech Group, Inc.); goat anti-rat IgG (cat. no. SA00001-15; 1:2,000 dilution; ProteinTech Group, Inc.) for 1 h at room temperature. ECL reagent (cat. no. 32106; Thermo Fisher Scientific, Inc.) was used to detect the signals.

IHC analysis was used to study 14-3-3σ protein expression in human LUAD tissues and normal adjacent tissues as controls. The slides were dewaxed in xylene, rehydrated with an alcohol gradient, immersed in 3% hydrogen peroxide for 10 min to block endogenous peroxidase activity and subjected to antigen retrieval by pressure cooking in Tris/EDTA (pH=8.0) for 10 min. The slides were then incubated with 14-3-3σ primary antibody overnight at 4°C. After incubation with the secondary antibody for 30 min at 37°C, the specimens were stained with a DAB staining kit (cat. no. ab64238; Abcam). Finally, the sections were counterstained with hematoxylin, dehydrated and fixed. The brown granules in the cytoplasm were considered as positive staining for 14-3-3σ. For the evaluation of nuclear and cytoplasmic staining, the staining intensity was scored as follows (35): Negative (score 0); weak (score 1); moderate (score 2) and strong (score 3). The degree of staining was divided into four categories according to the percentage of stained cells in the field: Negative (score 0%), 0-25% (score 1), 26-75% (score 2), 56-100% (score 3) and 76-100% (score 4). The results reported as the expression score were the product of the above two scores. Protein expression was then evaluated by jointly assessing the intensity and extent of staining. IHC staining was assessed and scored by two independent researchers (JL and CDZ), who were blinded to the clinicopathological data.

A receiver operating characteristic (ROC) curve analysis was performed for the selection of the cutoff value of the 14-3-3σ IHC score for OS and PFS, as described previously (36). In brief, the sensitivity and specificity of the outcome being studied for each score were plotted to generate an ROC curve. The score localized closest to the point of maximum sensitivity and specificity, the point on the curve (0.0, 1.0), was selected as the cutoff score leading to the highest number of tumors correctly classified as with or without the respective outcome. To facilitate the ROC curve analysis, the patients' outcome characteristics were dichotomized by survival [death vs. other outcome (censored, alive or death from other causes)].

All experimental procedures using mice were performed in accordance with protocols approved by the Laboratory Animal Welfare and Ethics Committee of The Third People's Hospital of Yunnan Province (Kunming, China). A total of eight BALB/C nude mice (nu/nu; male; aged, 4–6 weeks; body weight, 18–20 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and each experimental group consisted of four mice. In brief, 5×10⁵ NCI-H1299 cells stably transfected with Vector or HA-14-3-3σ were mixed 1:1 with Matrigel^® in a total volume of 0.2 ml and injected subcutaneously into the right flank of each mouse (37). Tumor size and mouse body weight were measured twice a week with a vernier caliper and precise analytical balance, respectively. On day 28 after tumor cell injection, mice were euthanized by carbon dioxide asphyxiation (30% vol/min) when certain tumors reached the size limits set by the guidelines ‘Tumor Induction in Mice and Rats-UCSF Animal Care and Use’ (38). The largest sizes of tumor in the study were no bigger than 16 mm. At 10 min after the animals were euthanized by carbon dioxide asphyxiation, the absence of vital signs was checked to confirm that the animals had died. In this study, the nude mice did not suddenly lose 20-25% weight (cachexia); therefore, no other humane endpoint was used in this experiment except for tumor size limitation. Tumors were excised, weighed and frozen or fixed in 4% paraformaldehyde and paraffin-embedded for IHC analysis. The mean tumor volume (TV) was calculated according to the following equation: TV=(L × W²)/2, where L is the length and W is the width of the tumor.

IHC staining of mouse tumors was performed as described above. In brief, after deparaffinization, rehydration, antigen retrieval and blocking, the tissue slides were incubated overnight at 4°C with the indicated antibodies. The following primary antibodies were used: Ki-67 (1:1,000 dilution; cat. no. 9449; Cell Signaling Technology, Inc.), p21 (1:200 dilution; cat. no. 10355-1-AP; ProteinTech Group, Inc.), p27 (1:200 dilution; cat. no. 25614-1-AP; ProteinTech Group, Inc.) and cleaved-Caspase-3 (Asp175) (1:200 dilution; cat. no. 9664; Cell Signaling Technology, Inc.).

All data were expressed as the mean ± standard deviation and statistical charts were prepared using GraphPad Prism 7.0 software (GraphPad Software, Inc.) and SPSS 20.0 (IBM Corporation). Transcripts per million (TPM) values were used and survival analysis was applied for assessing differential expression of 14-3-3σ mRNA levels in UALCAN. For survival analysis, the χ² test or Fisher's exact test was used to assess the association between 14-3-3σ protein expression and clinicopathological variables. The multivariate Cox proportional hazards model was used to estimate the hazard ratios and 95% confidence intervals for patient outcomes. The associations between 14-3-3σ protein expression and OS or PFS were determined by Kaplan-Meier analysis. The log-rank test was performed to assess differences in survival probability between patient subsets. Differences between two groups were analyzed using an unpaired Student's t-test, while the expression of 14-3-3σ in tumor tissues and matched normal tissues was compared using a paired Student's t-test. Comparisons of multiple groups were performed using one-way ANOVA followed by Dunnett's or Tukey's test. P<0.05 was considered to indicate a statistically significant difference.

It was previously reported that 14-3-3σ expression is present in LUAD (27), but the biofunction role of 14-3-3ơ and its clinicopathological/prognostic significance in LUAD have remained elusive. In the present study, 14-3-3σ mRNA expression was analyzed in LUAD using the data from UALCAN (http://ualcan.path.uab.edu.). Significant differences were identified between LUAD tissues and their normal counterparts (Fig. 1A). For the dataset from UALCAN, Kaplan-Meier survival analysis also indicated that patients with higher 14-3-3σ mRNA expression had poor overall survival (Fig. 1B). Next, the protein expression of 14-3-3σ was examined in LUAD and adjacent normal tissues by western blot analysis (Fig. 1C), and it was observed that 14-3-3σ was upregulated in LUAD tissues, whereas its expression was low in the normal adjacent tissue. Consistent with this result, the expression of 14-3-3σ in four established LUAD cell lines (A-549, NCI-H1299, NCI-H358 and NCI-H23) and a human bronchial epithelial cell line (Beas-2B) was analyzed by western blot analysis, indicating that 14-3-3σ was highly expressed in LUAD cell lines (Fig. 1D). Taken together, 14-3-3σ may function as an oncogene for LUAD.

Consistent with the western blot results, IHC analysis revealed that 14-3-3σ was present in the cytoplasm and highly expressed in LUAD tissues (Fig. 2Aa₁ and a₂), while it was weakly expressed in normal paired lung tissues (Fig. 2Ac₁ and c₂). Next, ROC curve analysis was employed to determine a cutoff score for 14-3-3σ expression to predict survival (Fig. 2B). The cutoff score for 14-3-3σ to predict OS and PFS in patients with LUAD (n=106) was 3.8 (P<0.001) and 4.1 (P=0.017), respectively. Therefore, a 14-3-3σ expression score of 4.0 (>4.0 vs. ≤4.0) was selected as the unified cutoff point for survival analysis in patients with LUAD. The ROC-derived 14-3-3σ cutoff score for patients with LUAD was 4 and the cohort (n=106) was divided into a high (52/106, 49.1%) and a low (54/106, 50.9%) expression group. Kaplan-Meier analysis further indicated that high expression of 14-3-3σ predicted an inferior OS and PFS in patients with LUAD (P<0.001, Fig. 2C).

The association between 14-3-3σ expression and the clinical characteristics of patients with LUAD was then examined. As presented in Table II, high expression of 14-3-3σ in LUAD tissues was significantly positively associated with the tumor stage (P=0.001). In addition, high expression of 14-3-3σ was associated with tumor size (P=0.036) and recurrence (P<0.001, Table II). However, no association between 14-3-3σ and other patient characteristics was observed, including patient age, sex or node stage.

To avoid the influence of various factors in the univariate analysis, the expression of 14-3-3σ as well as other parameters were examined in a multivariate Cox regression analysis (Table III). Among the patients with LUAD, 14-3-3σ was indeed determined to be a significant independent prognostic factor for OS (hazard ratio, 4.878; 95% CI, 2.895-8.219; P<0.001; Table III) and PFS (hazard ratio, 3.041; 95% CI, 1.878-4.923; P<0.001; Table III). Furthermore, the node stage was also identified as an independent prognostic parameter for OS (hazard ratio, 2.396; 95% CI, 1.115-5.148; P=0.025; Table III) and PFS (hazard ratio, 2.471; 95% CI, 1.168-5.228; P=0.018; Table III) in the patients with LUAD. However, other important prognostic factors, including age, sex, smoking, tumor stage and metastasis, were no significant independent prognostic factors for LUAD according to this analysis, implying that a larger cohort may be required in future studies.

The above results suggested that high expression of 14-3-3σ was associated with progression and poor prognosis of LUAD. Thus, the biological function of 14-3-3σ in LUAD was further investigated in vitro. First, 14-3-3σ plasmid was transfected into NCI-H1299 LUAD cells (Fig. 3A), as the native cell line expressed 14-3-3σ at a low level (Fig. 1D), and it was indicated that overexpression of 14-3-3σ promoted cell proliferation and survival, as measured by ATP-lite (Fig. 3B) and colony formation assays (Fig. 3C), respectively. Furthermore, the anchorage-independent growth of NCI-H1299 cells, measured by the soft agar assay, was increased upon 14-3-3σ overexpression (Fig. 3D). Thus, 14-3-3σ appeared to have growth-promoting activity in LUAD cells.

To further validate 14-3-3σ as an oncogene for LUAD, siRNA-based knockdown experiments were performed in A-549 LUAD cells, as the native cell line has high expression of 14-3-3σ (Fig. 1D). The results indicated that 14-3-3σ depletion suppressed cell proliferation and the colony-forming ability of the cells (Fig. 4A-C). The nature of survival inhibition upon 14-3-3σ depletion was demonstrated to be induction of apoptosis, as evidenced by an increase in PARP cleavage and Caspase-3 cleavage (Fig. 4D). Furthermore, the anchorage-independent growth of A-549 LUAD cells was inhibited by up to 60% on 14-3-3σ depletion (Fig. 4E). Thus, growth suppression upon 14-3-3σ depletion may be mediated via apoptosis induction.

Next, the above in vitro findings were validated in a xenograft model. NCI-H1299 cells with stable overexpression of 14-3-3σ were inoculated into the right flank of nude mice and, as expected, overexpression of 14-3-3σ profoundly promoted tumor growth in vivo, increasing the tumor size and weight compared to the vector control (Fig. 5A-C). Likewise, IHC staining of tumor tissues also revealed that overexpression of 14-3-3σ markedly promoted tumor growth (increase of Ki-67 and decrease of p21 and p27) and inhibited apoptosis (decrease of cleaved-Caspase-3) (Fig. 5D). Collectively, the results of the in vitro cell experiments and in vivo xenograft models consistently demonstrated that overexpression of 14-3-3σ significantly promoted the growth of LUAD. At the same time, clinical data analysis also confirmed that 14-3-3σ high expression was positively correlated with poor prognosis of patients with LUAD.