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ISSN : 1225-1577(Print)
ISSN : 2384-0900(Online)
The Korean Journal of Oral and Maxillofacial Pathology Vol.47 No.5 pp.93-103
DOI : https://doi.org/10.17779/KAOMP.2023.47.5.001

The Expression of PTTG1 as a Biomarker and Impact on Invasion and Growth of Oral Squamous Cell Carcinoma

Yeonjun Lee1), Gyeongwon Park1), Shihyun Kim2), Suyeon Park2), Jongho Choi2)*
1)Research Institute of Oral Science, College of Dentistry, Gangneung-Wonju National University, Gangneung-si, Gangwon-do, 25457, Republic of Korea
2)Department of Oral Pathology, College of Dentistry, Gangneung-Wonju National University, Gangneung-si, Gangwon-do, 25457, Republic of Korea
#

These authors contributed equally to this work.


* Correspondence: Jongho Choi Ph.D., Department of Oral Pathology, College of Dentistry Gangneung-Wonju National University, 7Jukheon-gil, Gangneung-si, Gangwon-do, Korea Tel: +82-33-640-2231 Email: jhchoi@gwnu.ac.kr
September 11, 2023 October 4, 2023 October 13, 2023

Abstract


Oral squamous cell carcinoma (OSCC) is the most common type of oral cancer, and it has been steadily increasing in worldwide. Pituitary tumor transforming gene 1 (PTTG1) has been known as oncogene in a verity of cancers. Nevertheless, the expression and role of PTTG1 in OSCC progression remains largely unexplored. In this study, clinical datasets were analyzed to assess the genetic impact of PTTG1 on OSCC progression and to identify its functional roles in OSCC cell lines. We analyzed the expression of PTTG1 in head and neck squamous cell carcinoma (HNSC) and OSCC using databases form the Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO). To investigate the effect of PTTG1 on proliferation and migration abilities in OSCC cell lines, following the knockdown of PTTG1 in HSC-2 and SCC-9 cell lines, we analyzed the proliferation and metastatic abilities of OSCC cells using EdU and Boyden chamber assays. Our database analysis revealed that PTTG1 was significantly overexpressed in tumor tissues compared to normal tissues. Moreover, its expression correlated with clinical parameters of OSCC. In vitro experiments demonstrated that depletion of PTTG1 suppressed the ability of cell proliferation and migration in both HSC-2 and SCC-9 cell lines. In conclusion, our study suggests that PTTG1 may act as an oncogene in OSCC. These findings provide new insights into the mechanisms and clinical implications of PTTG1 expression in OSCC patients.


Oral squamous cell carcinoma , Pituitary tumor transforming gene 1 , Metastasis , Proliferation

바이오마커로의 PTTG1의 발현과 구강 편평세포 암종의

이연준1), 박경원1), 김시현2), 박수연2), 최종호2)*
1)강릉원주대학교 치의학과
2)강릉원주대학교 치과대학 병리학교실

초록

    Ⅰ. INTRODUCTION

    HNSC is the most prevalent malignant cancer in the head and neck region.1) It ranks as the sixth most common cancer globally, with 890,000 new diagnoses and 450,000 deaths recorded in 2018.2) Notably, HNSC survivors experience profound psychological distress and reduced quality of life. They hold the second-highest rate of suicide (63.4 cases per 100,000 individuals) among cancer survivors, surpassed only by pancreatic cancer patients (86.4 cases per 100,000 individuals) in contrast with survivors from other cancer types (23.6 cases per 100,000 individuals).2) OSCC specifically refers to HNSC found in the oral region. OSCC accounts for over 90% of oral cancer cases, with its incidence notably on the rise in recent times.3,4,5) Early-stage OSCC patients have a relatively favorable 5-year survival rate between 80% to 90%. However, for those diagnosed in advanced stages, this rate drops to between 40% and 50%.6) Tragically, late-stage diagnoses correlate with heightened mortality.7) Despite advances in medical technology enhancing cancer management, overall survival for OSCC has seen only a modest 5% improvement, underscoring the need for better approaches.8) One challenge is that OSCC often progresses from a premalignancy to an invasive state without distinct symptoms, making early detection challenging.9) While considerable efforts have been directed at early OSCC diagnosis, the absence of specific markers often results in diagnostic delays, leading to late-stage diagnoses and poorer prognoses.10) Genomic markers hold promise for cancer detection and outcome prediction. However, current genomic data associated with OSCC growth remains imprecise.11) To address the high mortality resulting from delayed diagnosis, it's pivotal to identify genomic prognostic markers related to cell proliferation and growth in OSCC. Such discoveries could spearhead the development of early diagnostic tools and targeted therapies.12)

    The pituitary tumor transforming gene (PTTG) family was first isolated and identified from the pituitary gland cancer cells of rats as a gene inducing cellular transformation in mice.13) Subsequent studies have categorized the PTTG family into three distinct genes: PTTG1, PTTG2, and PTTG3P.14) Of all the members in the PTTG family implicated in carcinogenesis, PTTG1 demonstrates a notably higher predisposition and is prominently overexpressed in various cancers.14,15) In clear cell renal cell carcinoma, PTTG1 exhibits elevated expression levels on tumor cells and is associated with high-grade tumor cells and an unfavorable patient prognosis.16) In breast cancer, tissue samples from patients reveal significantly higher PTTG1 expression compared to normal tissues.17) In colorectal cancer, Qinggui Ren et al. observed PTTG1 overexpression in cancer cells, and they found that its expression levels positively correlate with clinical stage.18) In the head and neck region, specifically where the oral cavity is situated, the expression of PTTG1, PTTG2, and the PTTG3P pseudogene varies based on mutations in the TP53 gene, a well-known oncogene. Notably, PTTG1 presents with high expression levels and is linked to poor prognosis in these cases.19) However, comprehensive studies examining both the mechanistic effects of PTTG1 on proliferation, migration, and invasion in OSCC cells, and the clinical significance of its expression, remain limited.

    In present study, therefore, we undertook a comprehensive analysis of PTTG1 expression using clinical datasets for both HNSCC and OSCC. Additionally, we conducted in vitro studies to examine the functional effects of PTTG1 expression on the growth and metastasis of OSCC, based on its presence or absence. Furthermore, we explored its potential as a therapeutic biomarker.

    Ⅱ. MATERIALS AND METHODS

    1. Data sources

    The 992 gene expression profiles and their clinical-pathological data from 500 HNSC patient samples and 44 noncancerous samples, were retrieved from The Cancer Genome Atlas (TCGA) database (https://portal.gdc.cancer.gov/repository). In case of OSCC patients, gene expression profiles and clinical- pathological data from 329 samples and 32 noncancerous samples were categorized and retrieved form TCGA data (https://portal.gdc.cancer.gov/repository). To visualize the data as a form of volcano plots, UCSC Xena (https://xenabrowser.net/) was used, and for heat maps, dot plot and paired plots, MBatch of MD Anderson Cancer Center (https://bioinforma tics.mdanderson.org/public-software/mbatch/) was used.

    2. Detection of differentially expressed genes (DEG) analysis

    In this study, DEseq2(http://www.bioconductor.org) was applied to uncover DEG in HNSC and OSCC samples comparing noncancerous samples.20,21) The adjusted p-value was used in DESeq2 to minimize false positives, following the Benjamini and Hochberg method.21) Differentially expressed genes (DEGs) were filtered according to the criteria of adj.P < 0.001 and |log2(fold-change) | > 1 threshold.22,23,24)

    3. Validation of risk score model

    Based on clinical data from TCGA-HNSC and -OSCC, univariate Cox analysis of overall survival (OS) was analyzed using R survival analysis packages (https://cran.r-project. org/) with prognostic values and by time-dependent receiver- operating characteristic (ROC) curve.25,26) Hazard ratios (HR) were obtained through Cox proportional hazards regression analysis, and p-values were obtained using the Log-rank test to compare the overall survival (OS) between the PTTG1 high- and low-expression groups. To present the results visually, the Kaplan-Meier method was employed for plotting, and median survival values were included for a more intuitive comparison.

    4. Cell culture

    The cell lines used in this study were obtained from the Japan Research Biological Resources Cell Bank (JRBR, Ibaraki, Osaka, Japan) for HSC-2 and the American Type Culture Collection (ATCC, Manassas, VA, USA) for SCC-9, respectively. The HSC-2 cell line was cultured with Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Carlsbad, CA, USA) contained with 10% fetal bovine serum (FBS, Gibco, Waltham, MA, USA) and 1% penicillin/ streptomycin(P/S) in 5% CO2 at 37 ℃ incubator. The small interfering RNA (siRNA) used to knock down PTTG1 in HSC-2 and SCC-9 cell lines was small interfering RNA (siRNA)-PTTG1 (siPTTG1, Bioneer, Seoul, Korea). For the PTTG1 transfection experiment, the two cell lines were initially cultured in serum-free OPTI-MEM (Gibco) culture medium for 24 h. Subsequently, they were treated with 50 nM of siPTTG1 (Bioneer) for 6 h. After incubation, the culture medium was replaced, and the cells were further incubated 5% CO2 at 37 ℃ for 24 h.

    5. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis

    Total RNA was extracted from OSCC cell lines treated with siPTTG1 using the TRIzol reagent (Invitrogen). For cDNA synthesis, 500 μg of Superscript III Reverse Transcriptase (Invitrogen) was used, following the manufacturer’s instructions. Next, qRT-PCR was performed using a CFX-96 Touch Real-Time PCR system (Bio-Rad Laboratories, Hercules, CA, USA) to confirm the expression of PTTG1 after transfection. For the PCR amplification, the conditions were as follows: 95 ℃ for 3 min (Initial denaturation step), followed by 40 cycles of 95 ℃ for 15 s (denaturation step) and 60 ℃ for 30 s (annealing/extension step), 95 ℃ for 15 s, 65 ℃ for 5 s and 95 ℃ for 30 s (final step). The expression of PTTG1 was normalized to that of GAPDH. The following primer sequences were used: PTTG1 forward, 5'-TGA CTC AGG CTG GAA GAT TTG-3'; PTTG1 reverse, 5'-GGT GGG AGA AGC AAA GGT ATA G-3'; GAPDH forward, 5'-CAA AGT TGT CAT GGA TGA CC-3'; GAPDH reverse, 5'-CCA TGG AGA AGG CTG GGG-3'. All experiments were performed at least three times.

    6. 5-Ethynyl-2′-deoxyuridine (EdU) assay

    To evaluate the proliferative ability of transfected cell lines, the Click-IT™ 5-ethynyl-2′-deoxyuridine (EdU) cell proliferation kit was used (Abcam, Waltham, MA, USA) as the manufacturer's guidelines. Cells from siPTTG1-transfected and control groups were seeded on coverslips in 6-well culture plates with 10 μM EdU and incubated for 2 h under previously specified conditions. Post-incubation, cells were rinsed with phosphate-buffered saline (PBS) and fixed using 4% paraformaldehyde (PFA) in PBS. After discarding PFA, they were washed with 0.01% Tween-20 in PBS (PBS-T). For permeabilization, 0.5% Triton® X-100 in PBS was used for 15 min, followed by another wash with PBS-T. The Click-iT® reaction cocktail, formulated with the provided 1X Click-iT® EdU reaction buffer and additional components, was added to the cells and incubated for 30 min in a light-shielded environment. Following this, cells were washed again with PBS-T, stained using 4′,6-diamidino-2-phenylindole (DAPI) for 10 min, and imaged using an Olympus U-TV microscope (Olympus, Tokyo, Japan). ImageJ software (National Institutes of Health, Bethesda, MD, USA) was utilized for EdU positive cell counting.

    7. Migration and invasion assay using Transwell assays

    For the Transwell assay, 24-well cell culture plates from Corning, Inc. (Corning, NY, USA) equipped with 8.0 μm-pore Transwell inserts were used. Distinct from the migration assay, the invasion assay incorporated Matrigel-coated membranes from BD Biosciences (San Jose, CA, USA) to mimic the biological basement membrane. Procedures were conducted following the manufacturer's instructions. Cells from HSC-2 and SCC-9 were divided into siPTTG1- transfected and control groups and were cultured in Opti-MEM (Gibco) within the inserts, maintaining previously described culture conditions. The upper chamber of the insert contained Opti-MEM, while the lower chamber held medium supplemented with 10% FBS. Following incubation, cells were fixed and stained using Mayer's hematoxylin histological reagent from Dako (Santa Clara, CA, USA) and then rinsed with phosphate-buffered saline (PBS). Resulting cell distributions were imaged under an Olympus U-TV microscope (Olympus, Tokyo, Japan), and quantification was analyzed using Cell Counting Plugin of ImageJ software (National Institutes of Health, Bethesda, MD, USA).

    8. Statistical analysis

    Statistical analyses in present study were performed with GraphPad Prism version 9.0 (GraphPad Software Inc., La Jolla, CA, USA). To compare the gene expression level of PTTG1 between noncancerous and cancer samples, the log2 (TPM+1) values were subjected to Welch’s t-test in the HNSC and OSCC cohort. To compare noncancerous and staged samples, outlier elimination using the Interquartile Range (IQR) method.27) was conducted. Furthermore, to assess the significant differences in HNSC and OSCC depend on stages, Welch’s t-test was used to compare between noncancerous and cancer samples, and the Kruskal Wallis test was used to compare all groups. All in vitro assays were repeated at least three times, and the data are presented as mean ± standard error of the mean. The significant differences in control and siPTTG1 treat groups were analyzed using on-way analysis of variance or Student t-test.

    Ⅲ. RESULTS

    1. PTTG1 expression is elevated in tumor patients and is associated with cell cycle mechanisms.

    For the initial investigation into the potential mechanism of PTTG1 in HNSC and OSCC, the heatmaps of mRNA expression were explored (Figure 1A). 111 (HNSC) and 106 (OSCC) genes consistently exhibited high expression. Among them, the top 107 (HNSC) and 104 (OSCC) DEGs are highlighted on the heatmap, showing correlation with the cell cycle log2 FC > 1 and adj.p < 0.001. Since PTTG1 has been reported to contribute tmor development and progression in association with the cell cycle in various cancer studies.28,29,30) In HNSC tissues, the expression levels of CTCFL, PTTG1, PAK2, ABCB1, CETN1, HACE1, NUDC, PSMD6, and THBS1 genes, which have involved with the cell cycle, were elevated compared to normal tissues. Similarly, in OSCC tissues, HMGA2, PTTG1, NUP37, HSP90AA1, CDKL1, GAK, NBN, PRKACB and SSNA1 genes exhibited increased expression relative to normal tissues (Figure 1a). Furthermore, the results of the volcano plot, used for comparative analysis in tumor tissues, indicated that 107 (HNSC) and 104 (OSCC) genes were overexpressed whereas 73 (HNSC) and 79 (OSCC) genes exhibited reduced expression (Figure 1b). Comparative analysis of paired samples demonstrated elevated expression of PTTG1 in tumor tissues compared to their corresponding normal tissues in both HNSC (n=86) and OSCC (n=64) tissues (Figure 1c).

    2. PTTG1 overexpression affects overall survival (OS) in OSCC, but not in HNSC.

    Next, we investigated how the expression of PTTG1 changes in different tumor stages, aiming to confirm the observed differences. Violin plot indicates that PTTG1 expression was significantly upregulated in stage I, II, III and IV in HNSC tissues compared to normal tissues, but no significant differences were observed depend on HNSC stages (Figure 2a). In OSCC tissues, PTTG1 expression was statisticallyher than that of normal tissues, similar to HNSC. However, in each stage, PTTG1 expression was significantly higher both stage II and III compared to stage I (p < 0.05). Furthermore, PTTG1 expression was observed to be higher in stage IV than in stage II (p < 0.05) (Figure 2b). To examine the impact of PTTG1 on overall survival (OS), patients from the TCGA database were divided into two group: one with higher expression of PTTG1 and the other with lower expression of PTTG1 based on the calculated cut-off value from the point maximizing the function in ROC : f(c) = sensitivity(c) + specificity(c) – 1. Patients with high expression of PTTG1 were weakly associated with poor OS in HNSC (Figure 2c, p = 0.14). In contrast, patients with high expression of PTTG1 were significantly linked to OS in OSCC (Figure 2d, p < 0.05). Our findings indicate that PTTG1 is upregulated in both HNSC and OSCC; however, its role in tumor development differs between the two types of tumor.

    3. The knockdown of PTTG1 reduces the proliferation metastasis ability of OSCC cell lines.

    Before initiating the experiment, PTTG1 mRNA expression in OSCC cell lines was verified using qRT-PCR after treatment with siRNA-PTTG1. Our findings revealed a statistically significant decrease in PTTG1 mRNA expression in the HSC-2 cell line treated with siPTTG1 compared to the mock control (Figure 3a). The EdU assay showed that the number of EdU-positive HSC-2 cells was remarkably decreased in cells treated with siPTTG1 compared to that of mock control (Figure 3b). Quantitative analysis further showed that a statistically significant reduction in HSC-2 cells treated with siPTTG1 compared to the mock control cells (Figure 3c). Similarly, to the results in HSC-2 cells, the number of EdU-positive cells was notably diminished in SCC-9 cells with PTTG1 knockdown, compared to the mock control cells (Figure 3d, 3e and 3f).

    To investigate the impact of PTTG1 on metastatic potential, we performed the migration and invasion assay. Our results showed that the numbers of HSC-2 and SCC-9 cells migrated from upper to the lower insert were markedly reduced after siPTTG1 treatment compared to those of mock control (Figure 4a). Following siPTTG1 treatment, a significant reduction in the number of cells invading from the upper insert to the lower insert was observed in both cell lines, mirroring the findings of the migration experiment, compared to the mock control cells (Figure 4b). Our results suggest that silencing PTTG1 inhibits the proliferative and metastatic potential of OSCC cell lines.

    Ⅳ. DISCUSSION

    The landscape of bioinformatics methodologies has seen a pronounced evolution concomitant with recent technological advancements.31) Bioinformatics techniques have continually progressed within the field of biological investigation, assuming a vital role, particularly in oncology.31) The implementation of bioinformatics, characterized by the employment of computational methodologies and the sophisticated interpretation of extensive biological datasets, has become invaluable in the curation and management of clinical and pathological data32) A salient exemplar in this domain is The Cancer Genome Atlas (TCGA), a pioneering initiative that furnishes research data, encompassing gene expression delineations extracted from a myriad of patient tissues spanning various geopolitical regions. This archive has been instrumental in facilitating the derivation of statistical predictions pertinent to oncogenic factors, realized through meticulous data scrutiny.31,33) However, in research focused on OSCC, the depth of analysis related to progression and classification is limited by the lack of accessible data.34) Therefore, in these present studies, we investigated HNSC to explain the OSCC, since HNSC has massive databases for deeper analysis.

    To validate the potential of the PTTG1 gene as a biomarker in OSCC, a comparative analysis of the TCGA datasets was used for both HNSC and OSCC. Our bioinformatics results showed that the expression of PTTG1 was elevated in patient tissues with HNSC and OSCC, indicating the potential of PTTG1 as prognostic marker for HNSC and OSCC. However, a difference was observed between HNSC and OSCC, as the expression of PTTG1 exhibiting an increase depend on progression of OSCC. Moreover, the expression of the overexpressed PTTG1 also had an impact on the survival ratio of OSCC patients. Therefore, we analyzed the proliferation ability depend on PTTG1 expression in two OSCC cell lines to investigate whether these findings were consistently applicable in vitro experiment, and showed that silence of PTTG1 reduced the proliferation ability of OSCC cell lines. Furthermore, in vitro experiments using two OSCC cell lines demonstrated that a significant reduction in the metastatic potential of the cells was observed following PTTG1 silencing. These findings suggest that the expression of PTTG1 not only plays a crucial role in the progression of OSCC but also regulates the metastatic ability of OSCC.

    In this study, we emphasize the overexpression of PTTG1 in OSCC patients, underscoring its close association with OSCC development, metastatic potential, and its influence on the survival rate of OSCC patients. However, this study still has some limitations: Firstly, similar to many studies focusing on OSCC patents,35) the correlation between PTTG1 and metastatic cases remains inconclusive because of a limited number of patient datasets. Secondly, in vitro studies were solely conducted on OSCC cell lines, we are unable to impact conclusion involved with HNSC. These limitations should be addressed and clarified in subsequent studies.

    In summary, our study strongly suggests that PTTG1 functions as an oncogene. Furthermore, PTTG1 may be a novel marker for predicting the progression of OSCC. These findings provide a new paradigm in the field of early diagnosis for OSCC.

    Figure

    KAOMP-47-5-93_F1.gif

    Identification of differentially expressed RNAs in tumor and normal tissues. (a) The heat map indicates the expression patterns of DEG that effectively distinguish between normal and tumor tissues (left: HNSC tissues, right: OSCC tissues). (b) The volcano plot illustrates the DEGs, with red indicating upregulation and blue indicating downregulation in tumor versus normal tissues (left: HNSC tissues, right: OSCC tissues). (c) The expression of PTTG1 in normal and paired tumor tissues of HNSC (left, TCGA) and OSCC (right, TCGA). * indicates the significant differences vs. normal tissues (p < 0.05).

    KAOMP-47-5-93_F2.gif

    The correlation between PTTG1 expression and survival prognosis of different stage on TCGA database. Correlation between PTTG1 expression and (a) HNSC or (b) OSCC tumor stage in the violin plot. Correlation between PTTG1 expression and OS of (c) HNSC or (d) OSCC. Tumor cases were divided into two groups based on individual expression levels. The survival maps and Kaplan-Meier curves of two different tumors were showed. *, # and + indicates the significant differences (p < 0.05).

    KAOMP-47-5-93_F3.gif

    The effect of PTTG1 in proliferation ability of OSCC cell lines. (a) The mRNA expression of PTTG1 in HSC-2 cells treated with siPTTG1 compared to mock control cells. (b) Representative images comparing the cell proliferation of HSC-2 cells treated with mock control and siPTTG1 using EdU assay (Scale bar: 200 μm) (c) Graph indicating number of proliferating cells (d) The mRNA expression of PTTG1 in SCC-9 cells treated with siPTTG1 compared to mock control cells. (e) Representative images comparing the cell proliferation of SCC-9 cells treated with mock control and siPTTG1 using EdU assay (Scale bar: 200 μm) (f) Graph indicating number of proliferating cells. * represents a significant difference between control cells and siPTTG1 (p < 0.05). Mock: mock control; siPTTG1: siRNA-PTTG1. EdU: 5-ethynyl-2’-deoxyuridien; DAPI: 4’, 6-diamidino-2-phenylindole.

    KAOMP-47-5-93_F4.gif

    The effect of PTTG1 in metastatic potential of OSCC cell lines. (a) The number of migrated HSC-2 (upper channel) and SCC-9 (lower channel) cells treated with siPTTG1 compared to mock control using migration assay. (b) The number of invaded HSC-2 (upper channel) and SCC-9 (lower channel) cells treated with siPTTG1 compared to mock control using invasion assay. Eight objectives were randomly selected to count cells using Image J software. * represents a significant difference between control cells and siPTTG1 (p < 0.05). Mock: mock control; siPTTG1: siRNA-PTTG1.

    Table

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