Characterization of Opa interacting protein 5 as a new biomarker and therapeutic target for oral cancer
- Authors:
- Published online on: January 31, 2022 https://doi.org/10.3892/ijo.2022.5317
- Article Number: 27
Abstract
Introduction
Oral cancer is defined as a group of cancers originating from the oral cavity or lip and represents one of the most common malignancies worldwide (1). In 2020, a total of 377,713 new cases were diagnosed globally, and 177,757 patients succumbed to this disease (2). Oral squamous cell carcinoma is the most common pathological type of oral cancer; it represents more than 90% of the cases (3). Some modifiable risk factors have been reported to contribute to the development of oral cancer, including tobacco smoking, alcohol consumption, betel quid, human papillomavirus infection, and poor oral hygiene (4). The overall 5-year survival rate for oral cancer is 75–90% for stage I and 10–22% for stage IV (5). Currently, the standard treatment for oral cancer includes surgical resection, radiotherapy, and chemotherapy with molecular-targeted therapy and immunotherapy using immune checkpoint inhibitors. These treatments are associated with various adverse events, and many patients develop drug resistance during treatment. Thereby, local and regional recurrences are reported in 90% of oral cancers after surgery and radiotherapy (6,7). At present, clinical trials for head and neck squamous cell carcinoma (HNSCC) treated with immune checkpoint inhibitors are ongoing. Anti-PD-1 antibodies, such as nivolumab and pembrolizumab, have been approved by the US Food and Drug Administration for patients with relapsed or metastatic HNSCC, as well as cisplatin-resistant tumors (8). Anti-epidermal growth factor receptor (EGFR) antibodies, such as cetuximab, have been approved for use in combination with radiation therapy in patients with HNSCC (9). Despite these developments, efficacy remains limited, and adverse events have been reported. Therefore, further development of novel therapeutic strategies, such as personalized therapies based on cancer biomarkers and novel molecular-targeted therapies with no or low side effects in patients with oral cancer, are needed.
To identify molecular targets for the diagnosis and treatment of cancer, a genome-wide expression profile analysis and subsequent tissue microarray analysis with solid tumor tissues and normal tissues were performed. Several oncoantigens involved in the development and/or progression of solid cancers were isolated (10–36). This systematic strategy identified Opa interacting protein 5 (OIP5) as a candidate molecular target overexpressed in the majority of oral cancers.
OIP5 encodes a 25-kDa protein and was initially identified in a yeast two-hybrid system (37). OIP5 contains a Yippee domain (38) and a coiled-coil domain (39), suggesting that it has a role in cell proliferation and mitosis-associated processes. OIP5 is a component of the Mis18 complex, which consists of three subcomplexes: Mis18α, Mis18β (OIP5), and Mis18BP1. OIP5 interacts with Holliday junction recognition protein (HJURP) and mediates the loading of newly synthesized centromeric protein A (CENP-A) on the centromere by the Mis18 complex, which is required for accurate chromosome segregation (40,41). Previous findings indicate that OIP5 is overexpressed in various types of human cancers, such as lung and esophageal carcinomas (29), bladder cancer (42), gastric cancer (43), and glioblastoma (44); however, its role in oral carcinogenesis and the clinical significance of OIP5 protein as a tissue biomarker for oral cancer has yet to be clarified.
In the present study, the aim was to determine whether OIP5 plays a significant role in the development of oral cancer and is a putative cancer biomarker and therapeutic target.
Materials and methods
Oral cancer cell lines and clinical tissue samples
The following oral cancer cell lines were used in this study: four tongue squamous cell carcinoma cell lines (SCC9, CAL 27, HSC3, and HSC4), one pharynx squamous cell carcinoma (FaDu), one mouth squamous cell carcinoma (HSC2), and one gingival squamous cell carcinoma (Ca9-22). The human oral mucosa keratinocyte (HOMK) cell line, used as a normal control, was purchased from the Cell Research Corporation (Singapore). The histology of the cell lines and resources are summarized in Table I. All cells were grown in monolayer in an appropriate medium. The oral cancer cell lines were cultured at 37°C in a humidified atmosphere with 5% CO2 in a medium containing 10% fetal bovine serum (FBS) and antibiotics (Thermo Fisher Scientific, Inc.). HOMK cells were grown in a medium supplemented with EpiLife Defined Growth Supplement. In total, 13 oral squamous cell cancer tissue samples (5 females, 8 males; median age, 63 years; age range, 45–74 years) were obtained from ProteoGenex (Inglewood, USA), and a normal tongue tissue was obtained from Clontech (USA). A total of 164 existing formalin-fixed primary oral cancer tissue samples and adjacent normal tissue samples obtained from patients with oral cancer who had undergone surgery between 2004 and 2012 and treated with adjuvant or neoadjuvant chemotherapy at Kumamoto University were used in this study. The clinical stage of the tumor samples was judged according to the Union for International Cancer Control TNM classification (7th Edition of UICC; http://www.inen.sld.pe/portal/documentos/pdf/educacion/13072015_TNM%20Classification.pdf).
The study protocol and the use of existing clinical materials were approved by the Ethics Committees [Kumamoto University; Shiga University of Medical Science (no. G2009-163)] based on the national ethical guidelines for human subjects. It was confirmed that this study was fully ethically compliant, and that informed consent was waived by the ethics committee due to the retrospective nature of the study and the national ethical guidelines.
Real-time PCR
Total RNA was extracted from cultured oral cancer cells and oral cancer tissues using the Maxwell 16 LEV Simply RNA purification kit (Promega Corp.) according to the manufacturer's protocol. The RNA was reverse-transcribed into cDNA using the PrimeScript RT Master Mix (Takara Bio Inc.). Gene expression was measured by real-time PCR using TaqMan Universal Master Mix II and TaqMan assays on a StepOnePlus thermocycler (Applied Biosystems; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The reaction conditions were as follows: initial denaturation for 2 min at 50°C and 10 min at 95°C, followed by 40 cycles of 95°C for 1 sec and 60°C for 20 sec. Each experiment was performed in triplicate. ACTB (Hs01060665_g1) as an internal control and OIP5 (Hs00944000_g1) primers (Applied Biosystems) were used for the PCR reaction. Relative OIP5 mRNA expression was calculated using the 2−ΔΔCt method (45).
Western blot analysis
To prepare whole cell lysates, the cells were lysed on ice in Pierce RIPA buffer (Thermo Fisher Scientific, Inc.) supplemented with 1% protease inhibitor cocktail (Thermo Fisher Scientific, Inc.). After homogenization, the cell lysates were incubated on ice for 30 min and centrifuged at 14,000 × g for 15 min to separate the supernatant from the cellular debris. The concentration of total protein was estimated using the Qubit Protein Assay Kit (Thermo Fisher Scientific, Inc.), and the proteins were mixed with SDS sample buffer, boiled at 100°C for 5 min, and incubated at room temperature for 5 min before loading into 12% Mini-Protean® TGX gels (Bio-Rad Laboratories). After electrophoresis at 200 V for 30 min, the proteins were transferred to Trans-Blot® Turbo 0.2 µm PVDF membranes (Bio-Rad Laboratories). The membranes were blocked with 5% nonfat milk in TBST at room temperature for 1 h. A commercially available rabbit polyclonal antibody to human OIP5 (dilution, 1:1,000; cat. no. 12142-1-AP; ProteinTech Group) was incubated with the membrane at room temperature for 1.5 h, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (GE Healthcare, UK) for 1 h at room temperature. Protein bands were visualized by enhanced chemiluminescence using the Fusion Solo S system (Vilber Lourmat, France).
Immunocytochemistry
The cultured cells were seeded into Lab-Tek II chamber slides (Nalge Nunc Int.). After fixation in 4% formaldehyde solution for 15 min at room temperature, the cells were washed twice with PBS, and the upper part of the well was then removed. To render the cells permeable, PBS containing 0.1% Triton X-100 was added for 2 min at room temperature. The cells were covered with 3% bovine serum albumin (BSA) for 30 min at room temperature to block nonspecific binding. Then, the membrane was incubated with rabbit anti-OIP5 antibody (ca. no. 12142-1-AP; ProteinTech Group) in PBS containing 1% BSA and 0.1% Tween-20 at 4°C overnight in a wet box. After washing with PBS, a goat anti-rabbit secondary antibody conjugated to Alexa 488 (Thermo Fisher Scientific, Inc.) was added for 1 h at room temperature in a wet box in the dark. Nuclei were stained with Vectashield Mounting Medium containing DAPI (Vector Laboratories). OIP5 antibody staining was visualized using fluorescence microscopy (BZ-X710, Keyence, Japan).
Immunohistochemistry and tissue microarray analysis
Tumor tissue microarrays were constructed according to previously published procedures using 164 formalin-fixed primary oral cancer tissues that were surgically resected (63 patients treated with curative surgery and neoadjuvant chemotherapy, and 101 patients treated with curative surgery and adjuvant chemotherapy). The tissue areas selected for sampling were determined by visual alignment with the corresponding hematoxylin and eosin (H&E)-stained sections on the slides. Three, four, or five tissue cores (diameter, 0.6 mm; height, 3–4 mm) taken from donor tumor blocks were placed into recipient paraffin blocks using a tissue microarrayer (Beecher Instruments Inc.). A core of normal oral epithelial tissue was punched in each case, and 5-µm sections of the resulting microarray blocks were used for immunohistochemical analysis.
Tissue microarray slides were deparaffinized in xylene and rehydrated in graded concentrations of ethanol. For antigen retrieval, the slides were boiled in Target Retrieval Solution (pH 6.0) (Dako; Agilent Technologies, Inc.). A rabbit polyclonal anti-OIP5 antibody (cat. no. 12142-1-AP; ProteinTech Group) was added to each slide and incubated overnight after blocking endogenous peroxidase activity in a 0.3% hydrogen peroxide/methanol mixed solution and nonspecific protein binding sites in protein blocking buffer (cat. no. X0909; Dako Cytomation). After primary antibody incubation, sections were incubated with an HRP-labeled anti-rabbit IgG secondary antibody (Dako Cytomation) for 30 min. Chromogen substrate was added to visualize labeled protein, and the specimens were counterstained with H&E. Because the intensity of staining within each tumor tissue core was mostly homogeneous, positivity for OIP5 was assessed semi-quantitatively by three independent investigators without prior knowledge of the clinicopathological data, each of whom recorded staining intensity as positive or negative. Cases were accepted as positive if at least two investigators independently scored them as such.
Cell growth assay
The SCC9 and HSC2 cell lines that did not express endogenous OIP5 were plated at 0.8×106 cells/100-mm dish and transfected with OIP5-expressing plasmid (pCMV6-Entry-Myc-DDK, OriGene, USA) or mock plasmids by FuGENE 6 (Promega Corp.). Cell viability was assessed using MTT assay (Cell Counting Kit-8; Dojindo Laboratories, Japan) 5 days after plasmid transfection.
RNA interference assay
To investigate the biological function of OIP5 in oral cancer cells, small interfering RNAs (siRNAs) (Sigma-Aldrich; Merck KGaA) were transfected into Ca9-22 and CAL 27 cells using Lipofectamine 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The sequences targeting each gene were as follows: si-OIP5-#1, 5′-GGUUCACUCAAAGGCAGUA-3′; si-OIP5-#2, 5′-CUAUUUACCUUUAGGCUGA-3′; control 1: si-LUC, 5′-CGUACGCGGAAUACUUCGA-3′; and control 2: si-EGFP, 5′-GAAGCAGCACGACUUCUUC-3′. Cell numbers and viability were measured by colony formation and MTT assays using Cell Counting Kit-8 solution (Dojindo Laboratories) 6 days after transfection.
Flow cytometry
Cell cycle analysis was performed using a Cycletest Plus DNA reagent kit (BD Biosciences). Ca9-22 and CAL 27 cells, for measuring DNA ploidy, were harvested by trypsinization 72 h after si-OIP5-#2 or si-LUC transfection and suspended at a concentration of 1×106 cells/ml. The sample was filtered through a 50-µm nylon mesh, kept on ice, and protected from light. Cell cycle analysis was conducted within 3 h using a flow cytometer (BD FACSCanto™ II. The DNA content was measured in cells selected from at least 20,000 ungated cells.
Live-cell imaging
The Ca9-22 and CAL 27 cells transfected with si-OIP5-#2 or si-LUC were seeded on 35-mm glass dishes in an appropriate culture medium containing 10% FBS. The Evos FL Auto Cell Imaging System (Life Technologies; Thermo Fisher Scientific, Inc.) time-lapse microscopy was used to monitor changes in cell morphology and death after transfection with OIP5 siRNA. Live-cell imaging was initiated 72 h after si-LUC and si-OIP5 transfection, and images were captured every 15 min for 24 h up to 96 h.
Senescence assay
The cells were plated into 35-mm-diameter culture dishes up to 5 days after siRNA transfection into Ca9-22 and CAL 27 cells and stained with the senescence β-Galactosidase Staining Kit #9860 (Cell Signaling Technology, Inc.). The plates were incubated overnight at 37°C in a non-CO2 containing dry incubator. The percentage of senescence-associated β-galactosidase (SA-β-gal) positivity per 50 cells observed under a light microscope was calculated. Senescence-associated heterochromatic foci (SAHF) were detected by DAPI staining 96 h after siRNA transfection.
Statistical analysis
Statistical analyses were performed using the StatView 5.0 Statistical Program (SAS Institute, Inc.). The significance test analyzing the difference between two groups of cell-based assays was performed using the Student's t-test. One-way ANOVA followed by Tukey's post hoc test was performed to compare the means of each group with those of every other group when performing multiple comparisons. Results are presented as mean ± standard deviation. Each experiment was performed in triplicate. We used contingency tables to analyze the relationship between OIP5 expression and clinicopathological variables (e.g., sex, age, primary tumor region, and pathological TNM stage) in patients with oral cancer. Tumor-specific survival curves were generated on the date of surgery to the time of death from oral cancer or last follow-up observation. Kaplan-Meier curves were calculated for each relevant variable and OIP5 expression in oral tumors. The differences in survival times among the patient subgroups were analyzed using the log-rank test. Univariate and multivariate analyses were performed using the Cox proportional hazard regression model to determine the association between clinicopathological variables and cancer-related mortality. First, we analyzed associations between death and possible prognostic factors, including OIP5 positivity, age, sex, primary region, pT factor, pN factor, and treatment (curative surgery with adjuvant chemotherapy vs. curative surgery with neoadjuvant chemotherapy). Second, a multivariate Cox analysis was performed in a stepwise fashion in which OIP5 positivity was forced into the model along with each significant variable. As significant prognostic factors were continually added to the model, P<0.05 was considered statistically significant and an independent factor.
Database analysis
The gene expression and signal pathways related to OIP5 were screened using the ONCOMINE database (https://www.oncomine.org/resource/login.html), Gene Set Enrichment Analysis (GSEA) database (https://www.gsea-msigdb.org/gsea/msigdb/search.jsp), BioGPS database (http://biogps.org/#goto=welcome), and UALCAN database (http://ualcan.path.uab.edu/). Mutational status of the OIP5 gene was screened using cBioPortal for Cancer Genomics database (http://www.cbioportal.org/).
Results
Expression of OIP5 in oral cancer cells and tissues
We first confirmed that OIP5 mRNA was frequently and significantly overexpressed in many of the oral cancer cell lines, while it was barely detectable in the HOMKs (Fig. 1A). Additionally, OIP5 mRNA was frequently overexpressed in most of the oral cancer tissues, while its expression was hardly detectable in healthy tongue tissues (Fig. 1B). Western blotting revealed that OIP5 protein was expressed in many oral cancer cell lines; however, no expression was detected in the HOMK cells (Fig. 1C). Immunocytochemical analysis revealed that OIP5 protein was localized in the nucleus and cytoplasm of oral cancer cells (Fig. 1D).
Correlation of OIP5 expression with poor clinical outcome for patients with oral cancer
To verify the biological and clinicopathological significance of OIP5 in oral cancer tissues, we examined the expression of OIP5 protein using tissue microarrays containing oral cancer tissues from 164 patients who underwent radical resection. Immunohistochemical staining using anti-OIP5 antibody demonstrated that OIP5 expression was detectable in 120 of 164 (73.2%) oral cancer cases; however, it was barely detectable in the surrounding normal tissues (Fig. 2A). Next, we assessed the association between OIP5 protein expression and clinical parameters. Sex (higher in females; P=0.0202 using Fisher's exact test) and region (higher in the gingiva, buccal mucosa, and others; P=0.0029 using Fisher's exact test) were significantly associated with positive OIP5 expression (Table II). Furthermore, the positivity of OIP5 protein expression was also significantly related to poorer prognosis for patients with oral cancer (P=0.0013 using log-rank test; Fig. 2B). In addition, we also performed univariate analysis to determine the correlation between patient prognosis and other clinicopathological factors, including age (<65 vs. ≥65 years), sex (male vs. female), region (tongue vs. others), pT classification (T1-T2 vs. T3-T4), pN classification (N0 vs. N1-N2), treatment (curative surgery with adjuvant chemotherapy vs. curative surgery with neoadjuvant chemotherapy), and OIP5 expression status (absent vs. positive). Of these parameters, positive OIP5 expression (P=0.0061) and advanced pN stage (P=0.0029) were significantly associated with poor prognosis. Furthermore, multivariate analysis showed that positive OIP5 expression (P=0.0086) and advanced pN factor (P=0.0068) were independent prognostic factors (Table III).
 | Table II.Association between OIP5 protein expression in oral cancer tissues and patient characteristics (N=164). |
| Table III.Cox proportional hazards model analysis of prognostic factors in the patients with oral cancer. |
Enforced OIP5 expression promotes oral cancer cell growth
To evaluate the potential role of OIP5 in tumorigenesis, plasmids expressing OIP5 or mock plasmids were transfected into SCC9 and HSC2 cells that did not express endogenous OIP5. After confirmation of exogenous OIP5 expression by western blot analysis (Fig. 3A and B), cell viability was examined using MTT assay. It was found that the viability of OIP5-overexpressing cells was significantly higher compared than the viability of the cells transfected with the mock vectors (Fig. 3C and D). The colony formation assay also showed that OIP5 overexpression increased the numbers of SCC9 and HSC2 cells (Fig. 3E and F).
Inhibition of oral cancer cell growth by suppression of OIP5 expression
To further assess the role of OIP5 in oral cancer development and progression, siRNAs against OIP5 (si-OIP5-#1 and si-OIP5-#2) along with control siRNAs (si-LUC and si-EGFP) were transfected into oral cancer cell lines (Ca9-22 and CAL 27). After confirming the reduction of OIP5 protein levels using western blot analysis (Fig. 4A and B), we continually performed an MTT assay and colony formation assay to examine the role of OIP5 in tumor proliferation. The MTT assay showed that the suppression of endogenous OIP5 expression by si-OIP5 effectively inhibited oral cancer cell viability (P<0.0001) in the si-OIP5-transfected Ca9-22 and CAL 27 cells compared with si-controls (Fig. 4C and D). In addition, colony formation assays demonstrated that the suppression of OIP5 expression inhibited the growth of Ca9-22 and CAL 27 cells compared with controls (Fig. 4E and F).
Cell cycle arrest by inhibition of OIP5 expression
To examine the effect of OIP5 on cell cycle, flow cytometric analyses of Ca9-22 and CAL 27 cells 72 h after siRNA transfection was performed. Following OIP5 knockdown, we observed that the cell population at the G2/M phase was significantly increased (P<0.0001; Figs. 5A and B and S1). To further clarify the effect of OIP5 suppression on cellular morphology and cell cycle, live-cell imaging of the Ca9-22 and CAL 27 cells was carried out after si-OIP5 or control siRNA transfection. Time-lapse microscopy revealed that the cells were normally divided in control siRNA-transfected cells, whereas the cells failed to divide and subsequently died in si-OIP5-transfected cells (Fig. 5C and D).
Suppression of OIP5 expression induces cellular senescence
To examine whether OIP5 regulates the immortality of oral cancer cells, senescence analysis was performed using acidic β-galactosidase (SA-β-Gal) staining. OIP5 knockdown by si-OIP5 significantly increased the number of cells stained with senescent cell markers of SA-β-gal compared with the controls (P<0.0001) (Fig. 6A-D). In addition, SAHF in the nucleus were also detected in si-OIP5-transfected oral cancer cells (Fig. 6E and F). The results indicate that OIP5 is likely an important molecule for the immortality of oral cancer cells.
Database analysis of OIP5 expression, mutation, and its related pathways
To validate the expression of OIP5 in HNSCC and normal tissues, we investigated the OIP5 data using databases. BioGPS database analysis showed OIP5 mRNA expression in testis is nearly 20 times higher than in other normal tissues. UALCAN database showed among 520 cases of primary HNSCC and 44 cases of normal tissues, OIP5 expression is significantly higher in tumors than in normal tissues, and the methylation levels of OIP5 promotor are likely to be lower in HNSCC tissues compare with that in normal tissues. To examine the mechanism of oncogenic OIP5 activation in oral cancer, we investigated the OIP5 data using the cBioPortal for Cancer Genomics database. Among 523 cases of HNSCC, mutations and deep deletions of OIP5 were detected in only two cases (0.38%) and one case (0.19%), respectively; however, no amplification of OIP5 was observed. To screen the biological role of OIP5, we referred the OIP5 data using pathway analysis. GSEA database revealed that OIP5 expression was associated with cell cycle and chromosome maintenance, as well as the deposition of new CENP-A-containing nucleosomes at the centromere. To examine the importance of the functional association of OIP5 with CENP-A in oral cancer, we confirmed the co-expression of OIP5 protein with CENP-A in HNSCC using the ONCOMINE database (GSE12452 and GSE9844 datasets with co-expression correlation coefficients of 0.818 and 0.663, respectively).
Discussion
Oral cancer is highly malignant and shows resistance to anticancer treatment. Despite progression following oral cancer treatment, only a 5% improvement in overall survival has been achieved over the last 2 decades (46). Targeting specific molecules involved in oral cancer development may be an effective therapeutic approach that exhibits high efficacy and less toxicity if the molecular mechanisms of oral cancer proliferation and survival are well defined. In the present study, a high level of OIP5 expression was detected in most oral cancer cells and tissues, but low or no detectable expression of OIP5 was observed in normal oral cells and tissues. A gene expression database suggested that, except for the testes, OIP5 is expressed at minimal levels in normal tissues and organs, indicating that it is a candidate target for the development of new diagnostics and therapeutics. In addition, OIP5 expression is significantly increased in HNSCC, and the methylation levels of OIP5 promotor are likely to be lower in HNSCC tissues compare with that in normal tissues. Genetic mutation and amplification of OIP5 are not frequent in HNSCC. Therefore, OIP5 overexpression may be caused by an epigenetic mechanism in oral cancer cells. Further analysis of the transcriptional regulation of OIP5 is warranted.
Tissue microarray analysis showed that OIP5 expression was associated with poor clinical outcomes for patients with oral cancer. We found that there was no significant association between the type of oral cancer treatment (neoadjuvant cases and adjuvant cases) and OIP5 protein expression level in oral cancer tissues (positive vs. negative; P=0.8566), thus confirming that neoadjuvant chemotherapy did not affect OIP5 expression. In addition, multivariate analysis showed that positive OIP5 expression was an independent prognostic factor, indicating that OIP5 expression in oral cancer tissues may represent a clinical prognostic indicator that warrants further study in patients receiving chemotherapy for oral cancer.
The exogenous expression of OIP5 in oral cancer cell lines that did not normally express endogenous OIP5 enhanced cell growth. In contrast, OIP5 knockdown led to G2/M cell cycle arrest and subsequent cell death and/or cellular senescence in oral cancer cells, as monitored by live-cell imaging and senescence assays; this suggests that OIP5 may play a role in cell cycle and cellular senescence. Therefore, targeting OIP5 and its pathway with selective small molecule inhibitors and/or gene therapy may be a therapeutic strategy for oral cancer.
Using the database, we found that OIP5 expression was associated with cell cycle and chromosome maintenance as well as the deposition of new CENP-A-containing nucleosomes at the centromere. The old CENP-A nucleosome recruits CENP-C through their interaction. In addition, CENP-C recruits the Mis18 complex containing OIP5 as a component at late anaphase when CDK1 activity is markedly reduced. Thereby, the Mis18 complex recruits the prenucleosomal CENP-A complex through the direct binding of Mis18β and HJURP (41). Previous reports have demonstrated that the centromere recruitment of newly synthesized CENP-A was rapidly abolished by the suppression of OIP5 expression in HeLa cells, followed by defects, such as misaligned chromosomes, anaphase missegregation, and interphase micronuclei (40). Those cell defects could support our results that depletion of OIP5 inhibits oral cancer cell proliferation. To examine the importance of the functional association of OIP5 with CENP-A in oral cancer, we confirmed the co-expression of OIP5 protein with CENP-A using the database. To identify novel pathways related to OIP5 overexpression, further investigation of downstream signaling of OIP5 is important for clarifying the role of OIP5 in oral cancer development.
In summary, OIP5 is likely to function as an oncoprotein in oral cancer and may play an important role in oral cancer proliferation and survival. OIP5 is a putative biomarker that may predict the prognosis of patients with oral cancer. Therefore, targeting OIP5 may be useful for developing novel treatments, including immunotherapies and molecular-targeted therapies, which may exert strong biological effects with minimal adverse effects.
Supplementary Material
Supporting Data
Acknowledgements
Not applicable.
Funding
This research study was supported in part by a Grant-in-Aid for Scientific Research (B), Grant-in-Aid for Challenging Research (Exploratory), and Grant-in-Aid for Scientific Research on Innovative Areas from the Japan Society for the Promotion of Science (JSPS KAKENHI, grant nos. 15H04761, 19H03559, 21K19444 and 16H06277). YD is a member of the Shiga Cancer Treatment Project supported by Shiga Prefecture (Japan) and the International Joint Research Project (FY2016-2021) of the Institute of Medical Sciences (The University of Tokyo).
Availability of data and materials
All data generated or analyzed in this study are included in the published paper.
Authors' contributions
MZ, AT, and YD conceptualized and designed the study. MZ, AT, and YD developed the study methodology. YY, MS, and YD acquired the data (acquiring and managing patients and providing facilities). MZ, AT, BT, and YD analyzed and interpreted the data (statistical analysis, biostatistics, and computational analysis) and confirmed the accuracy. MZ, AT, and YD wrote, reviewed, and/or revised the manuscript. MZ, AT, and YD were involved in administrative, technical, or material support (reporting or organizing data and constructing databases). YD supervised the study. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The present study was approved by the Ethics Committees (Kumamoto University; Shiga University of Medical Science).
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Glossary
Abbreviations
Abbreviations:
|
BSA |
bovine serum albumin |
|
EGFR |
epidermal growth factor receptor |
|
FBS |
fetal bovine serum |
|
H&E |
hematoxylin and eosin |
|
HJURP |
Holliday junction recognition protein |
|
HNSCC |
head and neck squamous cell carcinoma |
|
HOMKs |
human oral mucosa keratinocytes |
|
OIP5 |
opa interacting protein 5 |
|
SAHF |
senescence-associated heterochromatic foci |
|
SaS |
StatView statistical |
References
|
Montero PH and Patel SG: Cancer of the oral cavity. Surg Oncol Clin N Am. 24:491–508. 2015. View Article : Google Scholar : PubMed/NCBI | |
|
Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A and Bray F: Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 71:209–249. 2021. View Article : Google Scholar : PubMed/NCBI | |
|
Dumache R, Rogobete AF, Andreescu N and Puiu M: Genetic and epigenetic biomarkers of molecular alterations in oral carcinogenesis. Clin Lab. 61:1373–1381. 2015. View Article : Google Scholar : PubMed/NCBI | |
|
Oji C and Chukwuneke F: Poor oral hygiene may be the sole cause of oral cancer. J Maxillofac Oral Surg. 11:379–383. 2012. View Article : Google Scholar : PubMed/NCBI | |
|
da Silva SD, Hier M, Mlynarek A, Kowalski LP and Alaoui-Jamali MA: Recurrent oral cancer: Current and emerging therapeutic approaches. Front Pharmacol. 3:1492012. View Article : Google Scholar : PubMed/NCBI | |
|
Kareemaghay S and Tavassoli M: Clinical immunotherapeutic approaches for the treatment of head and neck cancer. Int J Oral Maxillofac Surg. 48:419–436. 2019. View Article : Google Scholar : PubMed/NCBI | |
|
Carvalho ALKL, Kowalski LP, Agra IM, Pontes E, Campos OD and Pellizzon AC: Treatment results on advanced neck metastasis (N3) from head and neck squamous carcinoma. Otolaryngol Head Neck Surg. 132:862–868. 2005. View Article : Google Scholar : PubMed/NCBI | |
|
Ferris RL, Blumenschein G Jr, Fayette J, Guigay J, Colevas AD, Licitra L, Harrington K, Kasper S, Vokes EE, Even C, et al: Nivolumab for recurrent squamous-cell carcinoma of the head and neck. N Engl J Med. 375:1856–1867. 2016. View Article : Google Scholar : PubMed/NCBI | |
|
Cripps C, Winquist E, Devries MC, Stys-Norman D and Gilbert R; Head Neck Cancer Disease Site Group, : Epidermal growth factor receptor targeted therapy in stages III and IV head and neck cancer. Curr Oncol. 17:37–48. 2010. View Article : Google Scholar : PubMed/NCBI | |
|
Daigo Y and Nakamura Y: From cancer genomics to thoracic oncology: Discovery of new biomarkers and therapeutic targets for lung and esophageal carcinoma. Gen Thorac Cardiovasc Surg. 56:43–53. 2008. View Article : Google Scholar : PubMed/NCBI | |
|
Daigo Y, Takano A, Teramoto K, Chung S and Nakamura Y: A systematic approach to the development of novel therapeutics for lung cancer using genomic analyses. Clin Pharmacol Ther. 94:218–223. 2013. View Article : Google Scholar : PubMed/NCBI | |
|
Ishikawa N, Daigo Y, Takano A, Taniwaki M, Kato T, Hayama S, Murakami H, Takeshima Y, Inai K, Nishimura H, et al: Increases of amphiregulin and transforming growth factor-alpha in serum as predictors of poor response to gefitinib among patients with advanced non-small cell lung cancers. Cancer Res. 65:9176–9184. 2005. View Article : Google Scholar : PubMed/NCBI | |
|
Ishikawa N, Daigo Y, Yasui W, Inai K, Nishimura H, Tsuchiya E, Kohno N and Nakamura Y: ADAM8 as a novel serological and histochemical marker for lung cancer. Clin Cancer Res. 10:8363–8370. 2004. View Article : Google Scholar : PubMed/NCBI | |
|
Kakiuchi S, Daigo Y, Ishikawa N, Furukawa C, Tsunoda T, Yano S, Nakagawa K, Tsuruo T, Kohno N, Fukuoka M, et al: Prediction of sensitivity of advanced non-small cell lung cancers to gefitinib (Iressa, ZD1839). Hum Mol Genet. 13:3029–3043. 2004. View Article : Google Scholar : PubMed/NCBI | |
|
Kato T, Daigo Y, Hayama S, Ishikawa N, Yamabuki T, Ito T, Miyamoto M, Kondo S and Nakamura Y: A novel human tRNA-dihydrouridine synthase involved in pulmonary carcinogenesis. Cancer Res. 65:5638–5646. 2005. View Article : Google Scholar : PubMed/NCBI | |
|
Kikuchi T, Daigo Y, Katagiri T, Tsunoda T, Okada K, Kakiuchi S, Zembutsu H, Furukawa Y, Kawamura M, Kobayashi K, et al: Expression profiles of non-small cell lung cancers on cDNA microarrays: Identification of genes for prediction of lymph-node metastasis and sensitivity to anti-cancer drugs. Oncogene. 22:2192–2205. 2003. View Article : Google Scholar : PubMed/NCBI | |
|
Suzuki C, Daigo Y, Ishikawa N, Kato T, Hayama S, Ito T, Tsuchiya E and Nakamura Y: ANLN plays a critical role in human lung carcinogenesis through the activation of RHOA and by involvement in the phosphoinositide 3-kinase/AKT pathway. Cancer Res. 65:11314–11325. 2005. View Article : Google Scholar : PubMed/NCBI | |
|
Kakiuchi S, Daigo Y, Tsunoda T, Yano S, Sone S and Nakamura Y: Genome-wide analysis of organ-preferential metastasis of human small cell lung cancer in mice. Mol Cancer Res. 1:485–499. 2003.PubMed/NCBI | |
|
Taniwaki M, Daigo Y, Ishikawa N, Takano A, Tsunoda T, Yasui W, Inai K, Kohno N and Nakamura Y: Gene expression profiles of small-cell lung cancers: Molecular signatures of lung cancer. Int J Oncol. 29:567–575. 2006.PubMed/NCBI | |
|
Oshita H, Nishino R, Takano A, Fujitomo T, Aragaki M, Kato T, Akiyama H, Tsuchiya E, Kohno N, Nakamura Y and Daigo Y: RASEF is a novel diagnostic biomarker and a therapeutic target for lung cancer. Mol Cancer Res. 11:937–951. 2013. View Article : Google Scholar : PubMed/NCBI | |
|
Hayama S, Daigo Y, Yamabuki T, Hirata D, Kato T, Miyamoto M, Ito T, Tsuchiya E, Kondo S and Nakamura Y: Phosphorylation and activation of cell division cycle associated 8 by aurora kinase B plays a significant role in human lung carcinogenesis. Cancer Res. 67:4113–4122. 2007. View Article : Google Scholar : PubMed/NCBI | |
|
Ishikawa N, Daigo Y, Takano A, Taniwaki M, Kato T, Tanaka S, Yasui W, Takeshima Y, Inai K, Nishimura H, et al: Characterization of SEZ6L2 cell-surface protein as a novel prognostic marker for lung cancer. Cancer Sci. 97:737–745. 2006. View Article : Google Scholar : PubMed/NCBI | |
|
Kato T, Sato N, Hayama S, Yamabuki T, Ito T, Miyamoto M, Kondo S, Nakamura Y and Daigo Y: Activation of Holliday junction recognizing protein involved in the chromosomal stability and immortality of cancer cells. Cancer Res. 67:8544–8553. 2007. View Article : Google Scholar : PubMed/NCBI | |
|
Suzuki C, Takahashi K, Hayama S, Ishikawa N, Kato T, Ito T, Tsuchiya E, Nakamura Y and Daigo Y: Identification of Myc-associated protein with JmjC domain as a novel therapeutic target oncogene for lung cancer. Mol Cancer Ther. 6:542–551. 2007. View Article : Google Scholar : PubMed/NCBI | |
|
Takahashi K, Furukawa C, Takano A, Ishikawa N, Kato T, Hayama S, Suzuki C, Yasui W, Inai K, Sone S, et al: The neuromedin U-growth hormone secretagogue receptor 1b/neurotensin receptor 1 oncogenic signaling pathway as a therapeutic target for lung cancer. Cancer Res. 66:9408–9419. 2006. View Article : Google Scholar : PubMed/NCBI | |
|
Taniwaki M, Takano A, Ishikawa N, Yasui W, Inai K, Nishimura H, Tsuchiya E, Kohno N, Nakamura Y and Daigo Y: Activation of KIF4A as a prognostic biomarker and therapeutic target for lung cancer. Clin Cancer Res. 13:6624–6631. 2007. View Article : Google Scholar : PubMed/NCBI | |
|
Yamabuki T, Takano A, Hayama S, Ishikawa N, Kato T, Miyamoto M, Ito T, Ito H, Miyagi Y, Nakayama H, et al: Dikkopf-1 as a novel serologic and prognostic biomarker for lung and esophageal carcinomas. Cancer Res. 67:2517–2525. 2007. View Article : Google Scholar : PubMed/NCBI | |
|
Fujitomo T, Daigo Y, Matsuda K, Ueda K and Nakamura Y: Identification of a nuclear protein, LRRC42, involved in lung carcinogenesis. Int J Oncol. 45:147–156. 2014. View Article : Google Scholar : PubMed/NCBI | |
|
Koinuma J, Akiyama H, Fujita M, Hosokawa M, Tsuchiya E, Kondo S, Nakamura Y and Daigo Y: Characterization of an Opa interacting protein 5 involved in lung and esophageal carcinogenesis. Cancer Sci. 103:577–586. 2012. View Article : Google Scholar : PubMed/NCBI | |
|
Nguyen MH, Koinuma J, Ueda K, Ito T, Tsuchiya E, Nakamura Y and Daigo Y: Phosphorylation and activation of cell division cycle associated 5 by mitogen-activated protein kinase play a crucial role in human lung carcinogenesis. Cancer Res. 70:5337–5347. 2010. View Article : Google Scholar : PubMed/NCBI | |
|
Hayama S, Daigo Y, Kato T, Ishikawa N, Yamabuki T, Miyamoto M, Ito T, Tsuchiya E, Kondo S and Nakamura Y: Activation of CDCA1-KNTC2, members of centromere protein complex, involved in pulmonary carcinogenesis. Cancer Res. 66:10339–10348. 2006. View Article : Google Scholar : PubMed/NCBI | |
|
Takano A, Ishikawa N, Nishino R, Masuda K, Yasui W, Inai K, Nishimura H, Ito H, Nakayama H, Miyagi Y, et al: Identification of nectin-4 oncoprotein as a diagnostic and therapeutic target for lung cancer. Cancer Res. 69:6694–6703. 2009. View Article : Google Scholar : PubMed/NCBI | |
|
Kobayashi Y, Takano A, Miyagi Y, Tsuchiya E, Sonoda H, Shimizu T, Okabe H, Tani T, Fujiyama Y and Daigo Y: Cell division cycle-associated protein 1 overexpression is essential for the malignant potential of colorectal cancers. Int J Oncol. 44:69–77. 2014. View Article : Google Scholar : PubMed/NCBI | |
|
Thang PM, Takano A, Yoshitake Y, Shinohara M, Murakami Y and Daigo Y: Cell division cycle associated 1 as a novel prognostic biomarker and therapeutic target for oral cancer. Int J Oncol. 49:1385–1393. 2016. View Article : Google Scholar : PubMed/NCBI | |
|
Daigo K, Takano A, Thang PM, Yoshitake Y, Shinohara M, Tohnai I, Murakami Y, Maegawa J and Daigo Y: Characterization of KIF11 as a novel prognostic biomarker and therapeutic target for oral cancer. Int J Oncol. 52:155–165. 2018.PubMed/NCBI | |
|
Nakamura M, Takano A, Thang PM, Tsevegjav B, Zhu M, Yokose T, Yamashita T, Miyagi Y and Daigo Y: Characterization of KIF20A as a prognostic biomarker and therapeutic target for different subtypes of breast cancer. Int J Oncol. 57:277–288. 2020. View Article : Google Scholar : PubMed/NCBI | |
|
Williams JM, Chen GC, Zhu L and Rest RF: Using the yeast two-hybrid system to identify human epithelial cell proteins that bind gonococcal Opa proteins: Intracellular gonococci bind pyruvate kinase via their Opa proteins and require host pyruvate for growth. Mol Microbiol. 27:171–186. 1998. View Article : Google Scholar : PubMed/NCBI | |
|
Stellfox ME, Nardi IK, Knippler CM and Foltz DR: Differential binding partners of the Mis18α/β YIPPEE domains regulate mis18 complex recruitment to centromeres. Cell Rep. 15:2127–2135. 2016. View Article : Google Scholar : PubMed/NCBI | |
|
Nardi IK, Zasadzińska E, Stellfox ME, Knippler CM and Foltz DR: Licensing of centromeric chromatin assembly through the mis18α-mis18β heterotetramer. Mol Cell. 61:774–787. 2016. View Article : Google Scholar : PubMed/NCBI | |
|
Fujita Y, Hayashi T, Kiyomitsu T, Toyoda Y, Kokubu A, Obuse C and Yanagida M: Priming of centromere for CENP-A recruitment by human hMis18alpha, hMis18beta, and M18BP1. Dev Cell. 12:17–30. 2007. View Article : Google Scholar : PubMed/NCBI | |
|
Wang J, Liu X, Dou Z, Chen L, Jiang H, Fu C, Fu G, Liu D, Zhang J, Zhu T, et al: Mitotic regulator Mis18β interacts with and specifies the centromeric assembly of molecular chaperone holliday junction recognition protein (HJURP). J Biol Chem. 289:8326–8336. 2014. View Article : Google Scholar : PubMed/NCBI | |
|
He X, Hou J, Ping J, Wen D and He J: Opa interacting protein 5 acts as an oncogene in bladder cancer. J Cancer Res Clin Oncol. 143:2221–2233. 2017. View Article : Google Scholar : PubMed/NCBI | |
|
Nakamura Y, Tanaka F, Nagahara H, Ieta K, Haraguchi N, Mimori K, Sasaki A, Inoue H, Yanaga K and Mori M: Opa interacting protein 5 (OIP5) is a novel cancer-testis specific gene in gastric cancer. Ann Surg Oncol. 14:885–892. 2007. View Article : Google Scholar : PubMed/NCBI | |
|
He J, Zhao Y, Zhao E, Wang X, Dong Z, Chen Y, Yang L and Cui H: Cancer-testis specific gene OIP5: A downstream gene of E2F1 that promotes tumorigenesis and metastasis in glioblastoma by stabilizing E2F1 signaling. Neuro Oncol. 20:1173–1184. 2018. View Article : Google Scholar : PubMed/NCBI | |
|
Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCq method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI | |
|
Chinn SB and Myers JN: Oral cavity carcinoma: Current management, controversies, and future directions. J Clin Oncol. 33:3269–3276. 2015. View Article : Google Scholar : PubMed/NCBI |
