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Purpose: This study investigates the role and effectiveness of the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) in oral cancer, focusing on the clinical relevance of EGFR and myeloid cell leukemia-1 (Mcl-1) in head and neck cancers (HNCs). It aims to explore the molecular mechanism of afatinib, a TKI, in treating human oral cancer., Methods: We conducted an in silico analysis using databases like The Cancer Genome Atlas, Gene Expression Omnibus, and Clinical Proteomic Tumor Analysis Consortium, along with immunohistochemistry staining, to study EGFR and Mcl-1 expression in HNCs. For investigating afatinib's anticancer properties, we performed various in vitro and in vivo analyses, including trypan blue exclusion assay, Western blotting, 4'-6-diamidino-2-phenylindole staining, flow cytometry, quantitative real-time PCR, Mitochondrial membrane potential assay, overexpression vector construction, transient transfection, and a tumor xenograft model., Results: Higher expression levels of EGFR and Mcl-1 were observed in HNC patient tissues compared to normal tissues, with their co-expression significantly linked to poor prognosis. There was a strong correlation between EGFR and Mcl-1 expressions in oral cancer patients. Afatinib treatment induced apoptosis and suppressed Mcl-1 in oral cancer cell lines without the EGFR T790M mutation. The mechanism of afatinib-induced apoptosis involved the EGFR/mTOR/Mcl-1 axis, as shown by the effects of mTOR activator MHY1485 and inhibitor rapamycin. Afatinib also increased Bim expression, mitochondrial membrane permeabilization, and cytochrome c release. It significantly lowered tumor volume without affecting body, liver, and kidney weights., Conclusion: Afatinib, targeting the EGFR/mTOR/Mcl-1 axis, shows promise as a therapeutic strategy for oral cancer, especially in patients with high EGFR and Mcl-1 expressions., Competing Interests: Declarations. Ethics approval and consent to participate: All procedures for clinical samples were performed after obtaining approval from the Institutional Review Board of the Seoul National University Dental Hospital (IRB No. ERI20021). Consent to publish: All authors provided their consent for publication. Competing interests: The authors have no competing interests to declare that are relevant to the content of this article., (© 2024. The Author(s).)

This study analyzed oral squamous cell carcinoma (OSCC) genomes and transcriptomes in relation to perineural invasion (PNI) and prognosis using Cancer Genome Atlas data and validated these results with GSE41613 data. Gene set enrichment analysis (GSEA), gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes were conducted. We identified 22 DNA mutations associated with both overall survival (OS) and PNI. Among them, TGFBR1 and RPS6KA4 mRNAs were overexpressed, while TYRO3 and GPR137 mRNAs were underexpressed in PNI patients. Among the 141 mRNA genes associated with both OS and PNI, we found overlap with PNI-related DNA mutations, including ZNF43, TEX10, TPSD1, and PSD3. In GSE41613 data, TGFBR1, RPS6KA4, TYRO3, GPR137, TEX10 and TPSD1 mRNAs were expressed differently according to the prognosis. The 22 DNA-mutated genes clustered into nervous system development, regulation of DNA-templated transcription, and transforming growth factor beta binding. GSEA analysis of mRNAs revealed upregulation of hallmarks epithelial mesenchymal transition (EMT), TNFα signaling via NF-κB, and IL2 STAT5 signaling. EMT upregulation aligned with the TGFBR1 DNA mutation, supporting its significance in PNI. These findings suggest a potential role of PNI genes in the prognosis of OSCC, providing insights for diagnosis and treatment of OSCC., (© 2024. The Author(s).)

Background: To identify the prognostic markers of oral squamous cell carcinoma (OSCC), the genetic heterogeneity of the pathological stages was investigated., Methods: The data of 295 patients with primary OSCC obtained from the Cancer Genome Atlas were studied. The genetic prognostic landscape of the pathological stages was systematically analyzed by Cox regressions, Fisher's exact tests, and Gene Ontology (GO) enrichment., Results: Stage 4 patients had a poor prognosis in univariate and multivariate Cox models. Transforming growth factor-beta (TGF-β) pathway alterations were found more frequently in stage 4, whereas alterations in cell cycle pathways were significant in stages 1, 2, and 3. The differentially mutated genes were divided into three groups: risk genes of high stage, hazardless genes, and risk genes of low stage. The risk genes of low stage (RNF112, AKR7L, ZSCAN5C, and ZPBP) were independent prognostic factors with stage 4 and treatment modality in multivariate Cox regressions. Additionally, in genetic interaction analysis, NOMO1 and ZNF333 had a high co-occurrence in high stage, and WIZ had high co-occurrence in low stage. In GO enrichment, the prognostic genes were clustered at the functional term of RNA polymerase II transcription, and ZNF333 had an association with RNA transcription., Conclusion: The genetic mutation type and ratio of tumor heterogeneity are different for each stage of OSCC, and stratification of OSCC patients with differential therapeutic efficacy is needed. Risk genes of both high and low stages must be identified in patients diagnosed with low-stage OSCC. Mutations in NOMO1, ZNF333, and WIZ should be considered as potential prognostic markers., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Poly Adenylate Binding Protein Interacting protein 1 (PAIP1) plays a critical role in translation initiation and is associated with the several cancer types. However, its function and clinical significance have not yet been described in oral squamous cell carcinoma (OSCC) and its associated features like lymph node metastasis (LNM). Here, we used the data available from Gene Expression Omnibus (GEO), The Cancer Genome Atlas (TCGA), and Clinical Proteomic Tumor Analysis Consortium (CPTAC) to analyze PAIP1 expression in oral cancer. The publicly available data suggests that PAIP1 mRNA and protein levels were increased in OSCC. The high PAIP1 expression was more evident in samples with advanced stage, LNM, and worse pattern of invasion. Moreover, the in vitro experiments revealed that PAIP1 knockdown attenuated colony forming, the aggressiveness of OSCC cell lines, decreasing MMP9 activity and SRC phosphorylation. Importantly, we found a correlation between PAIP1 and pSRC through the analysis of the IHC scores and CPTAC data in patient samples. Our findings suggest that PAIP1 could be an independent prognostic factor in OSCC with LNM and a suitable therapeutic target to improve OSCC patient outcomes., (© 2022. The Author(s).)

Background: Sedum species are reported to possess diverse pharmacological activities in various solid tumors. However, the anticancer functions of Sedum orizyfolium and its constituents have never been determined in human cancers., Purpose: The present study focused on addressing the inhibition efficacy of the methanol extract of S. orizyfolium (MESO) and its constituents and the molecular mechanism underlying invasion and epithelial-to-mesenchymal transition (EMT) in oral squamous cell carcinoma (OSCC) cell lines., Study Design/methods: After MESO treatment, a wound-healing assay, an invasion assay, and immunocytochemistry were performed in OSCC cell lines, coupled with in silico analysis and immunohistochemistry in OSCC patient samples, to investigate the role of the EMT transcription factor Slug. Trehalose, an active component of MESO, was identified through gas chromatography-mass spectrometry., Results: Among the methanol extracts of 18 various wild plants from South Korea, MESO exhibited the highest anticancer functionality in OSCC cells by downregulating Slug expression. In silico analysis and immunohistochemistry indicated that elevated Slug levels are remarkably associated with tumor progression and invasion in patients with OSCC, suggesting that changes in Slug expression alter EMT progression and invasion in OSCC. Notably, treatment with trehalose, a sugar component of MESO, inhibited invasiveness and Slug expression in OSCC cells., Conclusion: Cumulatively, this study highlighted the beneficial role of MESO and trehalose in the inhibition of invasiveness of OSCC cells via suppression of Slug expression and suggested a new design for potential chemotherapeutic drugs against OSCC., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Background: Vasculogenic mimicry (VM) is the formation of an alternative circulatory system by aggressive tumor cells. The characteristics of VM and its underlying mechanism in oral squamous cell carcinoma (OSCC) remain unclear. This study aims to determine the relationship between VM in OSCC tissues and clinical outcomes and to investigate the biological role of SOX7 in VM in OSCC cells., Methods: CD31/PAS staining was performed to evaluate VM in OSCC tissue. The relationships between VM and clinicopathological variables, and VM and SOX7 levels were analyzed. The correlation between SOX7 levels and cancer cohorts was investigated using in silico analysis. VM formation assay was performed to observe VM in vitro. To investigate the role of SOX7 in VM formation, SOX7 was transiently over-expressed in SCC-9 cells. VM-modulating genes were identified by Western blotting., Results: We found a positive correlation between VM and lymph node metastasis and patient survival in OSCC (p = 0.003). In silico analysis from The Cancer Genome Atlas and Gene Expression Omnibus database showed that down-regulation of SOX7 expression was significantly correlated with OSCC patients (p = 0.0187) and lymph node metastasis (p = 0.0017). We also found that the presence of VM in OSCC tissue was inversely associated with SOX7 expression (p = 0.020). We observed that overexpression of SOX7 impaired VM formation by reducing the expression of VE-cadherin, thereby inhibiting cell migration and invasion., Conclusion: These results suggest that SOX7 plays an important role in the regulation of VM formation and may inhibit OSCC metastasis., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

In this study, we aimed to identify whether tumor budding is associated with the progression and prognosis of oral squamous cell carcinoma (OSCC) and investigate the correlation between tumor budding and regulators of epithelial-mesenchymal transition (EMT). Fifty-six cases of OSCC were selected and their tumor budding status was reviewed using archived hematoxylin and eosin-stained slides. In addition, the expression of EMT regulators was evaluated by immunohistochemistry using antibodies against Snail and Twist. Tumor budding was observed in 19 (33.9%) of the 56 cases of OSCC. Tumor budding was strongly associated with lymph node metastasis (P = .001) and shorter overall survival (P = .002). The expression of Snail and Twist was correlated with lymph node metastasis (P < .001 and .002, respectively) and poorer overall survival (P = .024 and .024, respectively). Tumor budding was significantly associated with the expression of Snail (P = .003) and showed a tendency toward higher expression of Twist (P = .08). Therefore, our results suggest that tumor budding is significantly associated with poor prognosis in patients with OSCC and histologically represents an EMT process in OSCC., (Copyright © 2018. Published by Elsevier Inc.)

Objective: Caffeic acid phenethyl ester (CAPE), a natural honeybee product exhibits a spectrum of biological activities including antimicrobial, anti-inflammatory, antioxidant and antitumor actions. The purpose of this research was to investigate the anticancer potential of CAPE and its molecular mechanism in human oral cancer cell lines (YD15, HSC-4 and HN22 cells)., Design: To determine the apoptotic activity of CAPE and identify its molecular targets, trypan blue exclusion assay, soft agar assay, Western blot analysis, DAPI staining, and live/dead assay were performed., Results: CAPE significantly suppressed transformation of neoplastic cells induced by epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol 13-acetate (TPA) without inhibiting growth. CAPE treatment inhibited cell growth, increased the cleavages of caspase-3 and poly (ADP-ribose) polymerase (PARP), and augmented the number of fragmented nuclei in human oral cancer cell lines. CAPE activated Bax protein causing it to undergo a conformational change, translocate to the mitochondrial outer membrane, and oligomere. CAPE also significantly increased Puma expression and interestingly Puma and Bax were co-localized., Conclusion: Overall, these results suggest that CAPE is a potent apoptosis-inducing agent in human oral cancer cell lines. Its action is accompanied by up-regulation of Bax and Puma proteins., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: SOX7, a member of the SOX family of transcription factors, acts as a tumor suppressor in multiple cancers. Downregulation of SOX7 has been reported in advanced tumors and correlates with poor prognosis. The aims of this study were to investigate the effects of SOX7 on cell proliferation, invasion, and colony formation in oral squamous cell carcinoma (OSCC) cells and to evaluate the effectiveness of SOX7 protein as a prognostic indicator for OSCC patients., Methods: oral squamous cell carcinoma (OSCC) cell lines were treated with SOX7 small interfering RNA or SOX7 peptide, and their effects on cell proliferation, invasiveness, and colony formation were investigated by proliferation, in vitro invasion, and clonogenic assays. SOX7 protein expression in OSCC and normal oral mucosal tissues was examined by immunohistochemistry. Associations between SOX7 protein expression and clinicopathological parameters of OSCC patients were statistically analyzed., Results: SOX7 silencing-induced cell proliferation and invasion in SCC-4 cells. SOX7 peptide treatment inhibited cell proliferation, colony formation, and invasion in SCC-9 and SCC-25 cells. Expression of SOX7 protein was decreased in OSCC tissues compared with normal oral mucosal tissues (P<.001). Negative SOX7 expression in patients with OSCC was significantly associated with positive lymph node metastasis (P=.041), advanced TNM stage (P=.024), and poor prognosis (P=.017)., Conclusions: These results suggest that SOX7 inhibits cell proliferation, colony formation, and invasion in OSCC as a tumor suppressor and that negative SOX7 expression could be a poor prognostic indicator for patients with OSCC., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Several studies have examined the correlation between nestin expression and the degree of tumor invasion in cutaneous melanoma. However, no information has been reported on nestin in primary mucosal melanoma of the head and neck. The present study examined the expression and prognostic significance of nestin in patients with primary mucosal melanoma of the oral cavity. Nestin expression was examined immunohistochemically in 39 patients (six oral melanoma in-situ cases and 33 invasive oral melanoma cases) and analyzed for association with disease progression. Age, sex, anatomic site, stage, level of invasion, regional lymph node metastasis, surgical margin involvement, and treatment modality were also analyzed. In the 33 invasive melanoma cases, invasion depth correlated significantly with prognosis in univariate and multivariate analyses. High-intensity nestin staining was observed in 14 of the 33 cases and a high proportion of nestin-positive cells was observed in 16 cases. In stage III oral melanoma cases, nestin expression was not significantly associated with disease progression. However, in stage IV cases, both the intensity and the proportion of nestin expression were significantly associated with disease progression (P=0.022 and 0.005, respectively). In all 33 invasive cases, multivariate analyses showed that both the intensity and the proportion of nestin were significantly associated with a poor prognosis (P=0.014 and 0.009; hazard ratio, 3.59 and 4.05; 95% confidence interval, 1.29-9.98 and 1.42-11.56, respectively). In conclusion, nestin can be a valuable prognostic indicator in the advanced-stage (stage IV) cases of oral mucosal melanoma.

Objectives: The staging significance of bone invasion is controversial in oral squamous cell carcinoma (OSCC) cases with tumors measuring 4cm or less according to the American Joint Committee on Cancer (AJCC). Our aim was to retrospectively examine a large group of patients with OSCC to determine the staging significance of bone invasion., Materials and Methods: Three hundred and twenty-three patients with primary OSCC were classified based on tumor size. Bone invasion was categorized as absent, one side bone, and both buccal and lingual bones, and analyzed for association with disease progression. Regional lymph node metastasis (N), perineural invasion, vascular invasion, surgical margin involvement, and adjuvant treatment were also analyzed., Results: In all OSCC cases, bone invasion (p=0.007) with stage N, perineural invasion, and surgical margin involvement were significant independent prognostic factors of disease progression. However, in OSCC cases with tumors measuring 4cm or less, bone invasion was not significantly associated with disease progression. Nevertheless, invasion of both buccal and lingual bones was significantly associated with disease progression (p=0.03). In multivariate analysis, both buccal and lingual bone invasion (p=0.04; hazard ratio=3.4; 95% confidence interval, 1.0-11.0), stage N2, and perineural invasion were also independent prognostic factors., Conclusion: Although OSCC bone invasion was an independent prognostic factor, bone invasion in small OSCC was not. However, small OSCC with both buccal and lingual bone invasion had a significantly worse prognosis. The AJCC T system is of limited prognostic value for small OSCC with bone invasion. But other elements should be examined before a modification can be accepted., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Downregulated expression of KiSS-1 has been correlated with tumor progression, metastasis, and patient prognosis in various human malignancies. However, there is no information regarding the expression of KiSS-1 in oral squamous cell carcinoma (OSCC). Our aims were to examine KiSS-1 expression in OSCC tissue samples and cell lines and to determine its prognostic significance. KiSS-1 expression was significantly lower in lymph node (LN) metastases than in primary tumor tissues. Five of six OSCC cell lines showed absence or relatively low expression of KiSS-1. Correlations between KiSS-1 expression and clinicopathological parameters were statistically assessed. There were significant correlations between KiSS-1 expression and LN metastasis (p = 0.007), TNM stage (p = 0.024), and local recurrence (p = 0.012). In the Kaplan-Meier survival analysis, negative KiSS-1 expression significantly correlated with poorer overall survival (OS) and disease-free survival (DFS) (p = 0.000 and 0.000, respectively). Multivariate analysis using Cox regression modeling revealed that KiSS-1 expression was an independent prognostic factor for both OS and DFS (p = 0.001 and 0.000, respectively). Our findings suggested that KiSS-1 downregulation may play a role in tumor progression and metastasis of OSCC and may be a reliable biomarker for predicting clinical outcome in OSCC., (© 2016 APMIS. Published by John Wiley & Sons Ltd.)

The epithelial-to-mesenchymal transition (EMT) plays a vital role in carcinogenesis, invasion, and metastasis of many epithelial tumors including oral squamous cell carcinoma (OSCC), a common malignancy of the head and neck. However, the functional role of the actin-sequestering protein thymosin β4 (Tβ4) in the EMT in OSCCs remains unclear. Thus, we investigated whether overexpression of Tβ4 could induce in vitro tumorigenesis such as cell proliferation and anchorage independency and an EMT-like phenotype in OSCCs. Also, we examined whether it affects invasiveness and cell motility-associated signaling molecules. Tβ4-overexpressing OSCCs, SCC-15&#95;Tβ4 and SCC-25&#95;Tβ4, enhanced cell proliferation and colony formation. In addition, we observed that Tβ4 overexpression induced an EMT-like phenotype, accompanied by a decrease in expression of the epithelial cell marker E-cadherin and an increase in expression of mesenchymal cell markers vimentin and N-cadherin. Also, the expression level of Twist1, an EMT-inducing transcription factor, was significantly enhanced in SCC-15&#95;Tβ4 and SCC-25&#95;Tβ4 cells. Tβ4 overexpression augmented in vitro invasion and MMP-2 activity and enhanced the phosphorylation of paxillin and cortactin and expression of LIMK1. Taken together, these results suggest that Tβ4 overexpression could be one of the causes of tumorigenesis and progression in OSCCs. Further investigation on the Tβ4 molecule would encourage the development of specific targets for cancer treatment.

Background: EP300 gene encoding p300 is a candidate tumor suppressor gene. This study investigated p300 expression and gene alteration in oral squamous cell carcinoma (OSCC) specimens to assess its role in OSCC development., Methods: Genomic DNA extracted from 13 human OSCC cell lines and 40 OSCC patient specimens was subjected to methylation-specific PCR and exon sequencing. Immunohistochemical staining with primary antibodies against p300 and p53 was performed in 48 patients with OSCC. We analyzed the association between the data and clinicopathological factors of OSCC patients., Results: Methylation-specific PCR revealed that the EP300 promoter region was not hypermethylated in OSCC. Only one cell line demonstrated a point mutation at exon 31. On immunohistochemical examination, patients with metastatic lymph nodes (P = 0.009) and advanced clinical stage (P = 0.046) tended to show increased expression of p300. There was no statistically significant relationship between p300 expression and p53 accumulation in OSCC tissue samples. Patient survival was not correlated with p300 expression., Conclusions: EP300 is not a tumor suppressor gene because there was neither epigenetic inactivation of the gene nor a mutation resulting in functional impairment. Based on p300 overexpression and its association with clinical factors in patients with OSCC, it is likely that p300 itself or one of its target genes plays a key role in the aggressive phenotypes of OSCC., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Tumor-infiltrating lymphocytes (TILs) are considered to represent immune reactions of the host to a malignant tumor. Programmed death receptor ligand-1 (PD-L1) is a surface protein that blocks the function of T lymphocytes and is expressed on cancer cells. Tumor-associated fibroblasts (TAFs), which influence tumor growth have also been reported to express PD-L1 and thus inhibit TILs. In the present study, we investigated the densities of CD4(+)/CD8(+) TILs, PD-L1 expression of tumor cells and TAFs in oral squamous cell carcinoma (OSCC). Forty-five cases of OSCC were selected. We evaluated PD-L1 expression and the infiltration degree of each lymphocyte by immunohistochemical examination. These data were analyzed in connection with clinicopathological factors. Peritumoral CD8(+) TILs were observed in every patient with OSCC, and their densities were correlated with lymph node metastasis (P<0.001), tumor size (P=0.003), and clinical stage (P<0.001). PD-L1 expression on OSCC cells was observed in 39 cases and was associated with the lower density of intratumoral CD8(+) TILs (P=0.047). PD-L1 expression of tumors <4cm in size was correlated with the histological grade of the tumor (P=0.022). TAFs were positive for PD-L1 in 18 cases. Peritumoral TILs were significantly associated with tumor size, lymph node metastasis and clinical stage. Though PD-L1 expressed by OSCC cells did not affect patients' survival, its correlation with decreased number of intratumoral TILs suggests that the development of a strategy to block the interactions of PD-L1 with TIL would be a useful tool for inhibiting tumor growth., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Oral squamous cell carcinoma (OSCC), the most common malignancy of the oral cavity, remains a lethal disease in over 50% of cases diagnosed annually, due mostly to late detection of this cancer in its advanced stages despite the easy accessibility of the oral cavity for regular examinations. Cripto-1 is a member of the epidermal growth factor (EGF)-CFC protein family and is involved in the activation of several different signaling pathways during embryonic development and cellular transformation. Although the Cripto-1 protein is overexpressed in several human cancers including breast, colon, cervix, gastric, and pancreatic cancer, no prior study has evaluated Cripto-1 expression in OSCC. Therefore, our aims in this study were to examine Cripto-1 expression in clinical samples of OSCC patients using immunohistochemistry, to analyze the correlation between Cripto-1 expression and clinicopathologic parameters, and to identify the oncogenic roles of Cripto-1 in OSCC cell lines. Both epithelial dysplasia (73.3%) and OSCC (55.5%) tissue samples showed significantly higher expression of Cripto-1 than normal mucosa (20%) (p=0.031). In the OSCC samples, there was a significant correlation between Cripto-1 expression and the histological differentiation of OSCC (p=0.015) and a high PCNA index (p=0.011). The in vitro cell proliferation assays demonstrated that recombinant human Cripto-1 (rhCripto-1) induced both SCC-4 and SCC-25 cells to proliferate as compared with control cells (p<0.05 and p<0.01, respectively). In in vitro migration assays, treatment of SCC-4 and SCC-25 cells with rhCripto-1 protein induced a 2.4-fold and 1.7-fold-increase in cell migration, respectively (p=0.000 and p=0.008, respectively). Taken together, our data suggest that Cripto-1 plays a role in the malignant transformation of the oral mucosa and is involved in the tumorigenesis and progression of OSCC by promoting the growth and migration of malignant cells., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Although >60% of oral cancer cases are not related to human papillomavirus (HPV) infection, most studies of oral carcinogenesis in human cells in vitro are carried out with human oral keratinocytes immortalized by HPV DNA. To explore whether human oral keratinocytes can spontaneously transform without HPV infection, we attempted to establish spontaneously immortalized and tumorigenic-transformed human oral keratinocytes by serial subculture to the post-mitotic stage. Here we report two spontaneously transformed human oral keratinocyte lines from adult human gingival samples. These lines were obviously immortal (>140 passages) and transformed phenotypes in vitro. One of the lines, Spi-HOK1, remained non-tumorigenic in nude mice, whereas the other line, Spt-HOK80, showed tumorigenicity. These lines showed epithelial origi-nality, but did not contain high-risk types of HPV DNAs. On karyotyping, Spi-HOK1 was aneuploid with a unique stable marker chromosome. Both cell lines revealed a mutation in the p53 gene, loss of p21WAF1/Cip1 and overexpression of p-Rb-Ser807/811. These cell lines were resistant to cisplatin-induced apoptosis by suppressing induction of apoptotic proteins. These results clearly demonstrate that spontaneous immortalization and spontaneous tumorigenic transformation of primary human oral keratinocytes can occur in vitro without HPV infection and are associated with chromosomal alterations, p53 mutation and impaired apoptosis. To our knowledge, this is the first report demonstrating that the Spi-HOK1 and Spt-HOK80 lines are novel cell lines that are spontaneously transformed from primary human oral keratinocytes.

Objective: We investigated the prognostic significance of CXC chemokine receptor CXCR-4 expression in patients with oral squamous cell carcinoma (OSCC) and its relationship with matrix metalloproteinase 2 (MMP-2), MMP-9, and Ki-67 expression., Study Design: The CXCR-4, MMP-2, MMP-9, and Ki-67 expression was assessed immunohistochemically in 74 OSCC patients. The results were analyzed in connection with clinicopathologic factors., Results: The CXCR-4 expression was positive in 45 cases and significantly correlated with lymph node metastasis (P = .037), MMP-9 expression (P = .025), and Ki-67 expression (P = .001). Univariate analysis showed that CXCR-4 expression, MMP-9 expression, Ki-67 expression, tumor size, lymph node metastasis, clinical stage, and recurrence positively correlated with prognosis. Multivariate analysis indicated that CXCR-4 expression was an independent prognostic factor for poor survival in patients with OSCC., Conclusion: Expression of CXCR-4 is a significant prognostic indicator for poor survival in patients with OSCC and correlates with expression of MMP-9 and Ki-67. The inhibition of CXCR-4 represents a possible molecular approach to the treatment of OSCC.

Background: The Akt/PKB family of kinases is frequently activated in human cancers, including oral squamous cell carcinoma (OSCC). Akt-induced epithelial-to-mesenchymal transition (EMT) involves downregulation of E-cadherin, which appears to result from upregulation of the transcription repressor Snail. Recently, it was proposed that carcinoma cells, especially in metastatic sites, could acquire the mesenchymal-to-epithelial reverting transition (MErT) in order to adapt the microenvironments and re-expression of E-cadherin be a critical indicator of MErT. However, the precise mechanism and biologic or clinical importance of the MErT in cancers have been little known. This study aimed to investigate whether Akt inhibition would restore the expression of E-cadherin and beta-catenin, reduce that of Vimentin, and induce the MErT in OSCC cells with low or negative expression of E-cadherin. We also investigate whether inhibition of Akt activity would affect the E-cadherin repressors and signaling molecules like NF-kappaB, ERK, and p38., Methods: We screened several OSCC cell lines in order to select suitable cell line models for inducing MErT, using immunoblotting and methylation specific-PCR. We examined whether Akt inhibitor phosphatidylinositol ether lipid analogues (PIA) treatment would restore the expression of E-cadherin and beta-catenin, reduce that of Vimentin, and induce the MErT in KB and KOSCC-25B cells using RT-PCR, immunoblotting, immunofluorescence analysis, and in vitro migration assay. We also investigated whether inhibition of Akt activity would affect the E-cadherin repressors, including Snail, Twist, and SIP-1/ZEB-2 and signaling molecules like NF-kappaB, ERK, JNK, and p38 using RT-PCR, immunoblotting, and immunofluorescence analysis., Results: Of the 7 OSCC cell lines, KB and KOSCC-25B showed constitutively activated phosphorylated Akt and low or negative expression of E-cadherin. Inhibition of Akt activity by PIA decreased NF-kappaB signaling, but did not affect phosphorylation of ERK, JNK, and p38 in KB and KOSCC-25B cells. Akt inhibition led to downregulation of Snail and Twist expression. In contrast, inhibition of Akt activity by PIA did not induce any changes in SIP-1/ZEB-2 expression. PIA treatment induced the expression of E-cadherin and beta-catenin, reduce that of Vimentin, restored their epithelial morphology of a polygonal shape, and reduced tumor cell migration in KB and KOSCC-25B cells, which was the corresponding feature of MErT., Conclusion: All of these findings suggest that Akt inhibition could induce the MErT through decreased NF-kappaB signaling and downregulation of Snail and Twist in OSCC cells. A strategy involving Akt inhibition might be a useful therapeutic tool in controlling cancer dissemination and metastasis in oral cancer patients.

Background: Oral squamous cell carcinomas (OSCCs) are characterized by a high degree of local invasion and a high rate of metastases to cervical lymph nodes. Downregulation of CXCR-4 by siRNA inhibits invasion and growth of breast and colon cancer cells. However, there have been no reports on the downregulation of CXCR-4 by small interfering RNA (siRNA) in oral cancer cells., Methods: We generated two stable CXCR-4-knockdown clones (KBsi and KOSCC-25Bsi) from the KB and KOSCC-25B OSCC cell lines by lentiviral delivery. In vitro invasion and cell proliferation assays were used to investigate the effect of CXCR-4 downregulation on cell proliferation and invasiveness in KBsi and KOSCC-25Bsi. Immunohistochemistry was performed to evaluate the correlation between CXCR-4 expression and proliferation in 26 OSCC tissue samples., Results: CXCR4-knockdown OSCC cells showed reduced invasiveness. The invasiveness of KBsi decreased to 29.5% of the vector-infected controls, and KOSCC-25Bsi decreased to 38.1% of the control vector-infected cells (P < 0.05). The CXCR4-knockdown OSCC cells grew significantly slower than the vector-infected control cells. KBsi and KOSCC-25Bsi cells proliferated at 69.5% and 71.7%, respectively, of the rate of control vector-infected cells (P < 0.05). CXCR-4-positive group had significantly higher PCNA labeling index than CXCR-4-negative group in OSCC tissue samples., Conclusion: These results suggest that the downregulation of CXCR-4 induces anti-proliferative and anti-invasive effects in OSCC and that CXCR-4 might be a useful target molecule for the treatment of OSCC.

A local invasion and lymph node metastasis (LNM) of an oral squamous cell carcinoma (OSCC) has a poor prognosis, and involves the degradation of the extracellular matrix mediated by multiple proteolytic enzymes including membrane type I-matrix metalloproteinase (MT1-MMP). This study aimed to determine the role of MT1-MMP in OSCC, to evaluate the immunohistochemical expression of MT1-MMP with regard to the invasiveness and LNM of the OSCC, and to evaluate the major source of MT1-MMP mRNA and its protein using immunohistochemistry and in situ hybridization. MT1-MMP expression was examined in 46 OSCCs via immunohistochemistry and non-radioisotope in situ hybridization. The relationship between MT1-MMP expression and LNM, as well as the histological invasiveness, was statistically analyzed. The results showed that whereas 12 out of the 18 OSCCs (66.7%) with LNM showed moderate to strong MT1-MMP expression, only nine of the 28 OSCCs (32.1%) without LNM expressed MT1-MMP strongly. MT1-MMP expression was significantly higher with regard to LNM (P=0.022). As the invasion grade became stronger (from grade a to grade d), MT1-MMP was significantly more strongly expressed (P=0.033). These results suggest that MT1-MMP is primarily secreted in the OSCC cells and is involved in the invasiveness of the OSCC and LNM. Moreover, MT1-MMP combined with other markers may be used to predict the metastatic potential of an OSCC.

Human oral squamous cell carcinoma cell lines (KOSCC-11, -25A, -25B, -25C, -25D, -25E, -33A, and -33B) were established by explantation culture from these oral squamous cell carcinomas. The histopathology of the primary tumors, in vitro growth characteristics, epithelial origin, in vitro anchorage-independency, in vivo tumorigenicity, the frequency of human papillomavirus (HPV) infections, and the status of proto-oncogenes, tumor suppressor genes, DNA mismatch repair genes, and microsatellite instability were investigated in the cell lines. KOSCC-11 is a well-differentiated oral squamous cell carcinoma (OSCC) derived from mandibular gingiva. KOSCC-25A, -25B, -25C, -25D, and -25E cell lines were derived from the same OSCC. KOSCC-33A and -33B were established from the same tumor that originated from the maxillary sinus. All tumor lines studied grew as monolayers and showed: i) epithelial origin by the presence of desmosome and keratin; ii) in vitro anchorage-independent growth ability; and iii) tumorigenic potential in nude mice. The cancer cell lines did not contain HPV DNA and did not express viral genes. Northern blot analysis revealed: i) overexpression of EGFR in four cell lines, ii) overexpression of c-H-ras in four cell lines, iii) overexpression of c-myc in three cell lines, iv) decreased expression of TGF-alpha in seven cell lines, and v) decreased expression of c-jun in five cancer cell lines compared with normal human oral keratinocytes. In all KOSCC cell lines and their corresponding tumor tissues, mutations were identified in highly-conserved functional regions of the p53 gene. The KOSCC-11 cell line contained a frameshift mutation and the other cell lines harbored an identical p53 mutation at codon 175 from CGC (Arg) to CTC (Leu). In five cell lines, a significant reduction of p21WAF1/Cip1 protein was evident. Cancer cell lines expressed higher level of Rb protein than normal human oral keratinocytes. DCC, a tumor suppressor gene, was not detected in KOSCC-25C. The KOSCC-33A cell line displayed microsatellite instability and showed a loss of hMSH2 expression. These well-characterized human OSCC cell lines should serve as useful tools for understanding the biological characteristics of oral cancer.