Tumor and adjacent healthy tissue specimens from 32 patients (24 males and 8 females) with OSCC were obtained from Ganenung-Wonju National University Dental Hospital (Gangneung), Keimyung University Dongsan Hospital (Daegu), Pusan National University (Pusan), Inje Paik University (Pusan), and Korea Biobank Network members (Yongin). All tissue samples were obtained with written informed consent of patients aged 50-75 years (mean age: 62) before surgery, and OSCC was histologically diagnosed by pathologists using tissue fragments from the oral region. The tissues were divided into two sections. One section was fixed in 10% formalin overnight at 25°C for immunohistochemical (IHC) staining. The other section was frozen immediately in liquid nitrogen, stored at −80°C and was used for real time-PCR and Western blotting. In total, 32 paired individual samples were randomly pooled into four samples, each containing an equal mass of tissues, for analysis. The present study was approved (approval no. GWNUIRB-2020-26-1) by the Committee for Ethical Review of Research of Gangneung-Wonju National University (Gangneung-si, Korea) and conducted from December 2020 to February 2023.

The p53 mutant human OSCC cell lines HSC-2, SCC-9 and YD-10B were obtained from the Japanese Collection of Research Bioresources Cell Bank, the American Type Culture Collection and the Korean Cell Line Bank, respectively. All OSCC cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen; Thermo Fisher Scientific, Inc.) supplemented with 10% (w/v) fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin (P/S; Gibco; Thermo Fisher Scientific, Inc.). The cell lines were incubated at 37°C with 5% CO₂.

For the transient knockdown of PTTG1, small interfering (si)RNA (Invitrogen; Thermo Fisher Scientific, Inc) was used to silence PTTG1 expression. A negative vehicle siRNA plasmid was used as a control (Table SI). Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) was used for transfection following the manufacturer's protocol. Briefly, OSCC cell lines were transfected with 25 nM PTTG1 siRNA in serum-free media until the cells reached 30–40% confluence. After 6 h of incubation at 37°C in a 5% CO₂ atmosphere, the medium was replaced with a culture medium, and the cells were incubated overnight before harvesting.

Total RNA was extracted from OSCC cells transfected with the negative control vehicle or with the PTTG1 siRNA using the TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The isolated RNA was used for cDNA synthesis using the Accupower^® RocketScript™️ Cycle RT PreMix kit (Bioneer, Inc.) according to the manufacturer's protocol. The thermocycling conditions for cDNA amplification were as follows: 50°C for 60 min, 95°C for 5 min, and hold at 4°C. The RT-qPCR was conducted using the CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, Inc.) and the following thermocycling conditions: 95°C for 3 min, followed by 40 cycles of 95°C for 15 sec, 60°C for 30 sec, 95°C for 15 sec, 65°C for 5 sec, and 95°C for 30 sec. The RT-qPCR was performed using primers specific for PTTG1 (forward, 5′-TGACTCAGGCTGGAAGATTTG-3′ and reverse, 5′-GGTGGGAGAAGCAAAGGTATAG-3′), p21 (forward, 5′-AGGTGGACCTGGAGACTCTCAG-3′ and reverse, 5′-TCCTCTTGGAGAAGATCAGCCG-3′) and GAPDH (forward, 5′-CAAAGTTGTCATGGATGACC-3′ and reverse, 5′-CCATGGAGAAGGCTGGGG-3′). The 2^−ΔΔCq method was used for the relative quantification of gene expression (29). The mRNA levels of the target genes were normalized to that of GAPDH. Independent experiments were conducted in triplicates.

For protein isolation, OSCC samples (vehicle control and siRNA) were lysed in Laemmli buffer supplemented with a protease inhibitor cocktail (Roche Diagnostics). The protein concentration was measured using a BCA kit (Thermo Fisher Scientific, Inc.). An equal amount of proteins (50 µg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using an 8–15% acrylamide gel at 100 V for ~2 h and 30 min. The separated proteins were transferred to 0.45-µm nitrocellulose membranes (Bio-Rad Laboratories, Inc.) at 20 V overnight. The membranes were blocked in 5% (w/v) bovine serum albumin (BSA; Sigma-Aldrich; Merck KGaA) dissolved in phosphate-buffered saline (PBS) containing 0.01% Tween-20 (PBS-T) for 1 h at room temperature (RT, 15–25°C) and then washed with 0.05% PBST. Next, the membranes were incubated with the following primary antibodies overnight at 4°C: Rabbit anti-PTTG1 (cat. no. GTX111938; GeneTex, Inc.), anti-p21 (cat. no. 2947; Cell Signaling Technology, Inc.), anti-p53 (cat. no. 2527; Cell Signaling Technology, Inc.), anti-retinoblastoma (Rb; cat. no. 9390; Cell Signaling Technology, Inc.), anti-phosphorylated Rb (cat. no. 8180; Cell Signaling Technology, Inc.), anti-proliferating cell nuclear antigen (PCNA; cat. no. 2586; Cell Signaling Technology, Inc.), mouse anti-Caspase-7 (Cas-7; cat no. sc-56063; Santa Cruz Biotechnology, Inc.), anti-cleaved (c-) Cas-7 (cat. no. 8438; Cell Signaling Technology, Inc.), anti-c-poly (ADP-ribose) polymerase (PARP; cat. no. 5625; Cell Signaling Technology, Inc.), anti-PARP (cat. no. 9542; Cell Signaling Technology, Inc.), mouse anti-cyclin D1 (cat. no. sc-450; Santa Cruz Biotechnology, Inc.), mouse anti-cyclin E (cat. no. sc-247; Santa Cruz Biotechnology, Inc.), mouse anti-cyclin B1 (cat. no. sc-245; Santa Cruz Biotechnology, Inc.), anti-phosphorylated histone H2AX (γH2AX; cat. no. 80312; Cell Signaling Technology, Inc.), anti-phosphorylated ATR (cat. no. 2853; Cell Signaling Technology, Inc.) anti-ATR (cat. no. 2790; Cell Signaling Technology, Inc.), anti-phosphorylated ataxia telangiectasia mutant (p-ATM; cat. no. 13050; Cell Signaling Technology, Inc.) and anti-ATM (cat. no. 2873; Cell Signaling Technology, Inc.); all primary antibodies were diluted at 1:1,000 with BSA. GAPDH (cat. no. LF-PA0018; AbFrontier Co., Ltd.; 1:3,000 dilutions in BSA) was used as a loading control. After washing three times with PBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies against rabbit (cat. no. 7074P2; Cell Signaling Technology, Inc.) or mouse IgG (cat. no. 7076P2; Cell Signaling Technology, Inc.) at a dilution of 1:5,000 in BSA for 1 h at RT. Protein bands were visualized using a FUSION Solo S imaging system (Vilber China) with an enhanced chemiluminescence reagent (cat. no. WBLUF0100; Merck KGaA). Protein expression was quantified in triplicate using ImageJ software (version 1.53a; National Institutes of Health), and the intensity of the bands was calculated as fold change. The expression levels of phosphorylated proteins were normalized to the total form of each protein. This experiment was repeated thrice.

Paraffin-embedded OSCC tissues were sectioned into 4-µm sections and placed onto coated slides and fixed in 4% paraformaldehyde overnight at RT. Next, the sections were dewaxed in xylene for 20 min and dehydrated in a graded ethanol series for 1 min each. After incubating in 3% H₂O₂ for 30 min, the samples were washed with PBS and incubated in sodium sulfate buffer (pH 6.0) at RT for antigen retrieval. The samples were blocked with 5% (w/v) BSA for 30 min at RT and washed three times with PBS. Subsequently, the samples were incubated with rabbit anti-PTTG1 antibody (cat. no. GTX111938; GeneTex, Inc.; 1:500 dilutions in BSA) at 4°C overnight. Tissues were washed three times with PBS and incubated with biotinylated goat anti-rabbit (1:1,00; cat. no. 7074P2; Cell Signaling Technology, Inc.) or anti-mouse IgG (1:1,000; cat. no. 7076P2; Cell Signaling Technology, Inc.) for 1 h at RT. The samples were washed thrice with PBS and incubated with diaminobenzidine (DAB; Abcam). Subsequently, the samples were counterstained with hematoxylin (Dako; Agilent Technologies, Inc.) and dehydrated using a graded ethanol and xylene series before mounting in Canada balsam mixture (cat. no. 2525-4405; Sigma-Aldrich; Merck KGaA). All observations were performed using an upright microscope (BX53; Olympus Corporation) equipped with an objective lens (×10).

To explore chromosomal damage in OSCC cells, 3×10⁴ cells were cultured in Opti-MEM media (Gibco; Thermo Fisher Scientific, Inc.) on coverslips and transfected with 25 nM vehicle control or siRNA-PTTG1 at a density of 30–40%. The following day, the cells were transferred to culture media, cultured overnight, fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 (Sigma-Aldrich; Merck KGaA) for 15 min at RT, and then washed twice with PBS for 3 min. After incubation with the primary antibody against γH2AX (1:500; cat. no. 80312; Cell Signaling Technology, Inc.) at 4°C overnight, the coverslips were washed with PBS and incubated with a secondary goat anti-mouse antibody conjugated with Alexa Fluorescence 488 (1:1,000; cat. no. A32723; Invitrogen; Thermo Fisher Scientific, Inc.). Images were acquired using a confocal microscope (STELLARIS 5; Leica Microsystems, Inc.) equipped with an oil objective lens (×63). Z-stacks were used in three confocal images and merged using the ImageJ software. The experiment was performed in triplicate.

To detect cell proliferation in OSCC, an EdU staining Proliferation kit (cat. no. C10337; Fluor 488, Invitrogen; Thermo Fisher Scientific, Inc.) was used following the manufacturer's protocol. A total of 5×10⁴ cells were plated on 12-mm coverslips in serum-free media and incubated overnight. The cells were transfected with vehicle control or siRNA-PTTG1 at a density of 30–40% in Opti-MEM media (Gibco; Thermo Fisher Scientific, Inc.) for 6 h and the culture medium was changed. After 24 h, 2 µg/ml EdU was added, and the cells were cultured in 5% CO₂ at 37°C for 2 h. The cells were fixed, permeabilized with 4% paraformaldehyde and 0.5% Triton X-100 (Sigma-Aldrich; Merck KGaA) for 15 min at RT, stained with 500 µl Click-iT reaction buffer for 1 h at RT, and mounted with a Fluorshield Mounting Medium containing DAPI (cat. no. ab104139; Abcam) for 15 min at RT. For quantification, at least seven images were randomly obtained using a fluorescence microscope (BX53; Olympus Corporation) with a ×40 objective lens. The experiments were performed in triplicate.

To investigate the cellular senescence of the OSCC cells, a SA-β-gal staining kit (cat. no. 9860; Cell Signaling Technology, Inc.) was used following the manufacturer's protocol. The cells were seeded in 6-well plates at 5×10⁴ cells per well, allowed to attach overnight, treated with either vehicle control or siRNA-PTTG1 at 25 µM in Opti-MEM media (Gibco; Thermo Fisher Scientific, Inc.) for 6 h, and cultured overnight. The cells were then fixed in the fixation solution for 15 min, washed twice with PBS, and incubated with SA-β-gal staining solution for 12 h at 37°C. For quantification, at least seven images were randomly acquired using an inverted light microscope (BX53; Olympus Corporation) at ×40 magnification. Independent experiments were conducted in triplicate.

A total of 2×10⁶ HSC-2 and SCC-9 cells treated with vehicle control or siRNA-PTTG1 were randomly divided and injected into independent groups of 5 BALB/c-nu/nu male nude mice (total n=20; mean weight, 16±1 g; ORIENT BIO, Inc.) aged 5-6 weeks, respectively. The mice were housed in a pathogen-free animal facility with a 12 h light/dark cycle at 20–24°C with 45–55% humidity and allowed free access to food and water. All animals were examined for tumor formation and growth every 3 days. To produce OSCC xenograft models, 100 µl of the tumor resuspended in Dulbecco's Phosphate Buffered Saline (D-PBS) was injected subcutaneously into the flank. After ~3 weeks, when the tumor size approached 10² mm³, and the tumor volume was calculated by V=(L × W²)/2 where V=volume (mm³); L=the largest dimension (mm); W=perpendicular diameter (mm), the mice were anesthetized via inhalation of isoflurane (IFRAN LIQ., Hana Pharm Co., Ltd.) with an induction concentration of 4–5% and a maintenance concentration of 2–3%, and the tumors xenografts form each mouse were removed. The humane endpoint of the experiment was reached when any of the following criteria applies: The tumor's largest diameter reaches <20 mm, the tumor weight exceeds 10% of the animals' body weight, the body weight losses >20% of animals' body weight or the animal stops eating and is deemed to be in poor health. After the experiment was completed, all mice were sedated with a CO₂/O₂ mixture (70/30%). Once the mice lost consciousness, they were euthanized with 100% CO₂. Euthanasia was performed in a chamber for 5 min, and death was confirmed by monitoring breathing, heart beating and pupil dilation for an additional 5 min. Nude mouse tumors were explanted for protein and RNA extraction, embedded in paraffin blocks, and frozen. The present study was approved (approval no. GWNUIRB-2020-36-1) by the Committee for Ethical Review of Research of Gangneung-Wonju National University (Gangneung-si, Korea) and conducted from December 2020 to January 2023.

To observe cell death in vivo, a TUNEL assay was performed using the in situ Cell Death Detection kit (cat. no. 12156792910; Roche Diagnostics) following the manufacturer's protocol. Briefly, frozen tissues were cryo-sectioned at a thickness of 7 µm using a cryostat microtome (Leica Microsystems, Inc.). The sections were fixed on slides with 4% paraformaldehyde for 20 min at RT and then washed thrice with PBS. The slides were then permeabilized with 0.1% sodium citrate for 2 min on ice and washed thrice with PBS. The samples were added to the TUNEL reaction mixture, incubated at 37°C, without a CO₂ incubator, for 1 h, and washed thrice with PBS. For quantification, at least seven images were obtained, and the samples were analyzed using a fluorescence microscope (BX53; Olympus Corporation) at ×40 magnification. The experiments were performed in triplicate.

The MTT assay was performed to analyze the effect of doxorubicin (DOX) on DNA damage mediated by the expression of PTTG1 and p21 in OSCC cell lines. Briefly, OSCC cells were seeded in a 96-well plate and cultured at 37°C in a 5% CO₂ humidified incubator. After 24 h, the cells were treated with 0.05, 0.5, 5, 50 and 100 µM of DOX (cat. no. D4193; Tokyo Chemical Industry Co., Ltd.) for 24 h and 200 µl DMSO (cat. no. 276855; Sigma-Aldrich; Merck KGaA) for 30 min. Absorbance at 570 nm was then measured using and enzyme-linked immunosorbent assay (ELISA) microplate reader (Molecular Devices, LLC). All experiments were repeated at least three times.

All assays were performed in triplicate, and the data are presented as the mean ± standard error (SE) of the mean. Differences in test variables between the control and experimental groups were analyzed using an unpaired Student's t-test or one-way ANOVA, followed by Dunnett's post hoc test. *P<0.05 was considered to indicate a statistically significant difference. Statistical analyses were performed using SPSS (version 20.0; IBM Corp.) and GraphPad Prism 9 (GraphPad Software; Dotmatics).

To investigate the expression and potential role of PTTG1 in OSCC, IHC was performed on 32 pairs of OSCC and adjacent healthy tissue samples. PTTG1 expression was significantly higher in OSCC tissues than in healthy tissues (Fig. 1A). Samples from patients with OSCC were further examined using RT-qPCR and western blot analysis which confirmed that PTTG1 was significantly overexpressed in tumors compared with that in healthy tissues (Fig. 1B and C; protein fold change: 2.10, 2.14, 2.10 and 2.00, respectively, compared with non-tumor tissues). In addition, PTTG1 was expressed in HSC-2 and SCC-9, two OSCC cell lines. Interestingly, the expression of the anti-oncogene p21, which is associated with several key characteristics of cellular senescence (i.e., a modified transcriptome, DNA damage, and SASP) (30,31) exhibited an opposite pattern in HSC-2 and SCC-9 cell lines in contrast to that of PTTG1 (Fig. 1D and E; protein fold change: 1.44 and 0.56, respectively, between HSC-2 and SCC-9 cells). The results of the independent analyses indicated that PTTG1 is an oncogene highly expressed in both OSCC tissues and cells.

To evaluate the effect of PTTG1 on OSCC cell proliferation, the expression of PTTG1 was inhibited using siRNA. The mRNA and protein expression of PTTG1 was significantly downregulated in HSC-2, SCC-9 and YD-10B cells transfected with siRNA-PTTG1 compared with levels in those transfected with the vehicle control (Figs. 2A and S1A). Furthermore, the expression of PCNA, a DNA replication marker (32), was significantly reduced in both OSCC cell lines transfected with siRNA-PTTG1 compared with levels in those transfected with the vehicle control (Figs. 2B and S1B). The percentage of DNA synthesis was significantly suppressed in HSC-2 and SCC-9 cells transfected with siRNA-PTTG1, leading to a decrease in cell viability, compared with that in cells transfected with the vehicle control (Fig. 2C and D). Additionally, cyclins D1, B1 and E1, which are responsible for cell proliferation, cell division and DNA replication, respectively, were significantly downregulated in both OSCC cell lines transfected with siRNA-PTTG1 compared with levels in those transfected with the vehicle control (Figs. 2E and S1C). Aberrant changes in the cell cycle in cancer cells lead to an imbalance between cell survival and apoptosis (33). In the present study, the expression ratio of apoptotic markers (including c-Cas-7 and c-PARP) was significantly increased in both OSCC cell lines transfected with siRNA-PTTG1 compared with levels in those transfected with the vehicle control (Figs. 2F and S1D). These findings suggested that PTTG1 depletion in OSCC cells restricts cell proliferation and promotes cell death through cell cycle arrest.

p21 is a cyclin-dependent kinase inhibitor and a senescence regulator in senescent cells, where its activation maintains stable cell cycle arrest (25). To determine whether cell cycle arrest caused by PTTG1 results in cellular senescence, alterations in senescent phenotypes were evaluated following PTTG1 knockdown. The mRNA and protein expression of p21 was significantly increased in both cell lines transfected with siRNA-PTTG1 compared with levels in those transfected with the vehicle control (Fig. 3A and B). Moreover, intracellular SA-β-gal significantly accumulated in both OSCC cell lines transfected with siRNA-PTTG1 compared with that in those transfected with vehicle control (Fig. 3C). To understand the mechanism underlying apoptosis induction in senescent cells following PTTG1 knockdown and to test whether an increased DNA damage response (DDR) causes this effect after knockdown, the phosphorylation levels of ATM and ATR, which are involved in the DDR, were evaluated. It was found that PTTG1 knockdown in the two OSCC cell lines led to a substantial increase in the phosphorylation of these proteins (Fig. 3D). Furthermore, there was a significant increase in the number of γH2AX foci in the nucleus of PTTG1 knockdown cells compared with that in those transfected with the vehicle control, where only a few γH2AX foci were observed (Fig. 3E and F). In the YD-10B cell line, it was observed that γH2AX expression and SA-β-gal accumulation increased compared with the vehicle control after PTTG1 knockdown (Fig. S2). Furthermore, in the HSC-2 and SCC-9 cell lines, PTTG1 knockdown resulted in decreased p53 expression and increased p-Rb expression compared to the vehicle control (Fig. S3). These findings suggested that loss of PTTG1 promotes DNA damage in a p21-dependent manner, followed by cellular senescence regardless of cell types.

To verify the effect of PTTG1 on tumor progression, 6-week-old nude mice were subcutaneously injected with 2×10⁶ vehicle control-treated or PTTG1-knockdown HSC-2 or SCC-9 cells. Tumor growth, weight, DNA damage and apoptosis were analyzed to determine the effect of PTTG1 on tumor progression (Fig. 4A). Compared with that in the vehicle control-treated group, a steady reduction in tumor size was observed in the groups injected with PTTG1-knockdown HSC-2 and SCC-9 cell lines starting from 15 days after injection (Fig. 4B). The largest tumor observed in the SCC-9 VC group had a maximum diameter of 14.06 mm and a volume of 12.6 mm (V=1116 mm³). Moreover, the groups injected with the PTTG1-knockdown cell lines showed a statistically significant decrease in tumor weight 3 weeks after cell injection compared with that in the vehicle control group (Fig. 4C). Immunoblotting results revealed that PTTG1 knockdown significantly reduced the expression of PCNA, a cell proliferation marker, and elevated the expression of p21 (senescence), c-Cas-7 (apoptosis), markers of γH2AX p-ATR and p-ATM (DNA damage), compared with that in the vehicle control-treated group (Fig. 4D). To examine whether the reduction in tumor growth by PTTG1 knockdown was associated with DNA damage-dependent cancer cell apoptosis, a TUNEL assay was performed to measure DNA breaks in cells undergoing apoptosis (34). In the vehicle control group, the nuclei of cells present in the tumor tissues exhibited a negligible number of DNA breaks. Conversely, tumor tissues injected with PTTG1-knockdown HSC-2 and SCC-9 cells displayed a significant increase in DNA breaks, similar to the effects observed with DNA-damaging chemicals in vitro (Figs. 4E, F and S4). Thus, these findings suggested that PTTG1 expression plays a crucial role in regulating tumor growth and apoptosis and that these effects are mediated by DNA damage.