Oral Squamous Cell Carcinoma Tumor Progression Research Articles

Upregulation of miRNA-196a and miRNA-196b correlates with Bryne’s prognostic score in oral squamous cell carcinoma

BackgroundmicroRNA(miRNA)-196a and miRNA-196b expression has been found to be dysregulated and involved in tumorigenesis and tumor progression in array of cancers through different targets. The role of these miRNAs together in clinical application is not always consistent and, its prognostic value in oral squamous cell carcinoma (OSCC) is still elusive. This study was performed to investigate the correlation of these miRNAs expression with histological grades of OSCC according to Bryne's histological grading system, to predict prognosis and to evaluate their relationship with clinico-pathological data. MethodsReal-time quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) was done to evaluate the expression levels of miRNA-196a and miRNA-196b in 75 pairs of OSCC tissue matched with adjacent normal mucosa, used as a control. ResultsmiRNA-196a and 196b expression in OSCC was significantly higher than that in corresponding adjacent normal tissues (p > 0.001). Also, a significant differential correlation was found in between the expression levels of these two miRNAs (Pearson correlation test r = 0.676, p-value<0.0001). The increased expression of these miRNAs was more frequently observed in OSCC tissues with advanced clinical and pathological TNM stages (IVa and IVb, pIVb respectively, p-value<0.0001). Significant correlation was found between miRNA-196a upregulation and moderate prognostic score (p-value<0.0001) in comparison with good and poor prognostic score of histological grades of OSCC. Sensitivity and specificity for miRNA-196a were 95 % and 85 %, respectively (AUC = 1, 95 % CI = 0.617–0.850; p 0.001), while for miRNA-196b were 94 % and 86 %, respectively (AUC = 0.808, 95 % CI = 0.701–0.916; p0.0001). ConclusionsThese findings suggest that the increased expression of miRNA-196a and 196b may play an important role in tumor progression in OSCC. miRNA-196a might be a useful marker for predicting the clinical outcome of OSCC, especially for advanced stages. In conclusion, our data demonstrate for the first time that these miRNAs may serve as a potent prognostic marker for tumor progression. We further highlight miRNA-196a and miRNA-196b as a promising predictor of prognostic assessment in OSCC.

Role of Stromal Fibroblast–Induced WNT7A Associated with Cancer Cell Migration Through the AKT/CLDN1 Signaling Axis in Oral Squamous Cell Carcinoma

Wnt signaling plays a crucial role in the progression of various cancers, including oral squamous cell carcinoma (OSCC). However, the tumor microenvironment (TME) regulating Wnt signaling has not yet been fully elucidated. In this study, we investigated whether cancer-associated fibroblasts (CAFs), the primary components of the TME, activate Wnt signaling and promote tumor progression in OSCC. We conducted a Transwell coculture assay using human OSCC cell lines and normal human dermal fibroblasts (NHDFs). NHDFs stimulated WNT7A expression in several OSCC cell lines, especially HO-1-N-1 and HSC-5. An immunohistochemical study using 122 human OSCC samples indicated that high WNT7A expression in tumor cells was significantly associated with invasion depth and poor prognosis. Moreover, WNT7A expression in OSCC cells was positively correlated with α–smooth muscle actin expression in CAFs. WNT7A knockdown in OSCC cells demonstrated that OSCC cells cocultured with NHDFs significantly promoted tumor cell migration and invasion, which was dependent on WNT7A expression in OSCC cells. We also isolated HSC-5 cells from the coculture and conducted microarray analysis to investigate the factors that promote tumor progression induced by WNT7A. Among the various differentially expressed genes, we identified a downregulated gene encoding CLDN1 and confirmed that WNT7A negatively regulated CLDN1 expression in OSCC cells and CLDN1 knockdown in OSCC cells promoted their migration. Phosphokinase array analysis showed that WNT7A activates protein kinase B (AKT) phosphorylation. Activating AKT signaling using the SC79 agonist induced CLDN1 downregulation in OSCC cells. In the coculture assay, the AKT inhibitor MK2206 significantly recovered CLDN1 expression downregulated by WNT7A, resulting in OSCC cell migration suppression. These results suggest that CAFs stimulate OSCC cells to produce WNT7A, following CLDN1 expression downregulation by activating AKT signaling, promoting cancer cell migration. These findings highlight the importance of molecular therapies targeting the TME in OSCC.

Analysis of the protumoral mechanisms of F.nucleatumon growth, epithelial-mesenchymal transition and the expression of immunosuppressive markers in cell line of OSCC

Abstract Background: Oral squamous cell carcinoma (OSCC) is the most common manifestation of oral cancer. It has been proposed that periodontal pathogens contribute to OSCC progression, and preliminary data from our laboratory has identified Fusobacterium nucleatum (F.n) and Epithelial Mesenchymal Transition (EMT) markers in the secretome of biopsies from patients with OSCC in comparison with healthy biopsies from the oral cavity. However, the mechanisms modulated by the tumoral bacteriome in OSCC remains not fully understood. Methods: Oral cancer cells (HSC3) were infected with F.n using a MOI of 100 to evaluate the effect of the bacteria on tumor growth, expression of EMT markers and immunomodulatory properties. Results: After infection, we confirmed the presence of bacteria inside and surrounding the cancer cells by confocal microscopy. Then, a significant increment in the size of F.n-infected tumor spheroids was found at 3-, 6- and 10-days post-infection. Related to EMT markers, MMP-9 transcripts were significantly elevated in infected cells, whereas E-cadherin transcripts were significantly downregulated post infection. In addition, the Human XL Oncology array kit revealed that several cancer proteins such as MMP-3, GM-CSF, FOXO1/FKHR, Cathepsina D-S, Amphiregulin, SerpinB5/Maspin and EGFR were upregulated and others like ICAM-1, DLL-1 and ERB2 were downregulated after F.n-infection. Finally, the expression of Galectin-9 measured by flow cytometry was significantly increased in infected cells than control cancer cells. Conclusions: The periodontal bacterium F.n promotes tumor progression of OSCC through increased tumor growth, acquisition of ETM-associated markers and Galectin-9 upregulation. Fondecyt Regular Project 1211480

Characterization of DJ-1, PTEN, and p-Akt as Prognostic Biomarkers in the Progression of Oral Squamous Cell Carcinoma.

Objective Oral cancer has a five-year survival rate of 68%, and the methods used to assess it still relyheavily on morphology. Protein biomarkers can potentially increase the predictive power of histopathological evaluation. This study aims to examine the expression of three closely linked proteins implicated in the pathogenesis of oral squamous cell carcinoma (OSCC); protein deglycase (DJ-1), an oncogene, phosphatase, and tensin homolog (PTEN), a tumor suppressor gene, and phosphorylated protein kinase B (p-Akt), the activated form of a vital serine/threonine kinase, which is involved in the oncogenesis of several human malignancies, throughout the tumor progression steps to establish their potential as prognostic biomarkers. Study design Western blot analysis was carried out using four different cell lines representing the successive steps of OSCC progression, including normal oral keratinocytes, dysplastic oral keratinocytes, locally invasive OSCC, and metastatic OSCC. Results DJ-1 expression was found to be upregulated gradually throughout the successive steps of OSCC progressionfrom normal to dysplastic to locally invasive to metastatic OSCC. PTEN expression showed an overall opposite trend. Interestingly, a significant downregulation of p-Akt was seen in the locally invasive OSCC cells, although it was followed by a significant increase in p-Akt expression in the metastatic OSCC cell line, which is consistent with the role of p-Akt in the motility and migration of cancer cells. Conclusion This study documented trends in expression patterns of three important signaling molecules, DJ-1, PTEN, and p-Akt, in normal, premalignant, and malignant oral keratinocytes. The oncogenic DJ-1 and tumor suppressor PTEN were expressed in a manner consistent with their respective roles in tumorigenesis, while p-Akt only showed a significant upregulation in the metastatic OSCC cells. Overall, all three proteins exhibited unique trends throughout the progressive stages of OSCC tumor progression, thereby adding to their potential utility as prognostic biomarkers for oral cancer patients.

Elastin remodeling: Does it play a role in priming the malignant phenotype of oral mucosa?

The extracellular matrix (ECM) is a dynamic tissue that provides nutrition and support to overlying epithelium. During tumorigenesis, the tumor microenvironment (TME) dysregulates the ECM. This is reflected by morphological changes seen in collagen and elastic fibers and is thought to facilitate metastasis. To study the degradation of elastic fibers in different grades of oral squamous cell carcinoma (OSCC) and in oral epithelial dysplasia (OED) using histochemistry and to correlate it to the TNM stage of OSCC. Tumor cores from 38 cases of OSCC (well-differentiated[15], moderately differentiated[14], and poorly differentiated[9]) and 15 incisional biopsies of OED were analyzed. Hematoxylin-eosin and Verhoeff's-Van Gieson (VVG) stains were used. The stained sections were assessed for morphological changes in elastic fibers. Data were analyzed using Statistical Package for Social Sciences (SPSS) version 22 software. Fisher's exact, Kruskal-Wallis, one-way ANOVA, and Turkey post hoc tests were used to establish significance (P ≤ 0.05). Spearman's correlation test was used to correlate elastin fiber degradation with TNM stage of OSCC. All grades of OSCC showed absence of elastic fibers around the tumor islands. Elastic fiber degradation (fragmented and clumped type fibers) increased proportionately with the grade and TNM stage of OSCC. In OED, A significant reduction in the amount of elastic fibers with increasing grade was noted. A positive correlation was noted between elastin degradation and grade and stage of OSCC. Therefore, it may be implicated in tumor progression of OSCC.

Metabolomic Analysis Reveals that SPHK1 Promotes Oral Squamous Cell Carcinoma Progression through NF-κB Activation.

Metabolic disorders are significant in the occurrence and development of malignant tumors. Changes of specific metabolites and metabolic pathways are molecular therapeutic targets. This study aims to determine the metabolic differences between oral squamous cell carcinoma (OSCC) tissues and paired adjacent noncancerous tissues (ANT) through liquid chromatography-mass spectrometry (LC-MS). SPHK1 is a key enzyme in sphingolipid metabolism. This study also investigates the potential role of SPHK1 in OSCC. This study used LC-MS to analyze metabolic differences between OSCC tissues and paired ANT. Principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) were applied to explain the significance of phospholipid metabolism pathways in the occurrence and development of OSCC. Through further experiments, we confirmed the oncogenic phenotypes of SPHK1 in vitro and in vivo, including proliferation, migration, and invasion. The sphingolipid metabolic pathway was significantly activated in OSCC, and the key enzyme SPHK1 was significantly upregulated in oral cancer tissues, predicting poor OSCC prognosis. In this study, SPHK1 overexpression was associated with high-grade malignancy and poor OSCC prognosis. SPHK1 targeted NF-κB by facilitating p65 expression to regulate OSCC tumor progression and promote metastasis. This study identified metabolic differences between OSCC and paired ANT, explored the carcinogenic role of overexpressed SPHK1, and revealed the association of SPHK1 with poor OSCC prognosis. SPHK1 targets NF-κB signaling by facilitating p65 expression to regulate tumor progression and promote tumor metastasis, providing potential therapeutic targets for diagnosing and treating oral tumors.

Assessment of Serum Urea, Creatinine and Uric Acid in Oral Cancer.

Background: Oral squamous cell carcinoma (OSCC) is a common malignancy worldwide, leading to significant disease-associated social and financial burdens. The investigation of underlying mechanisms involved in carcinogenesis and tumor progression in OSCC might provide new therapeutic perspectives with an impact on disease control and patient survival. Our study aims to investigate the interrelation between metabolic processes, expressed through final catabolism products and clinicopathological characteristics in OSCC. Materials and methods: This is a single cancer comparative retrospective study investigating metabolic byproducts, namely serum urea, creatinine and uric acid, detected at the moment of diagnosis in patients with OSCC, in comparison to healthy controls. Clinical and paraclinical data regarding exposure to risk factors, disease staging and pathological characteristics were collected for all patients. Subjects with co-existing systemic or metabolic diseases, or with a history of malignancy, were excluded from the study. Subsequently, the metabolic byproducts revealing significant changes in OSCC patients were considered for a correlation analysis with the disease clinico-pathological characteristics. Results: Blood levels for urea, creatinine and uric acid were determined in a total of 225 subjects: 145 patients diagnosed with OSCC and 80 healthy control subjects admitted to our hospital between 2016 and 2021. The comparative analysis between groups revealed that the serum urea level was significantly lower in OSCC patients (p = 0.0344). Serum creatinine and uric acid did not reveal significant differences between groups. Furthermore, in advanced stages of the disease (stages III and IV), the blood level of urea was significantly lower compared to incipient OSCC (stages I and II) (p = 0.003). We found a negative correlation of serum urea levels with smoking (p = 0.0004) and cervical lymph node metastasis (p = 0.0070), and a positive correlation with aging (p = 0.0000). We found no significant correlation of serum urea with primary tumor size (p = 0.5061) and patient survival (p = 0.2932). Conclusions: Decreased serum urea levels are detected in patients with advanced OSCC, in correlation with lymph node metastasis. The invasive features of tumor cells in OSCC might be promoted in association with dysregulation of protein catabolism processes, facilitating aggressive behavior in OSCC.

Circular RNA circFOXO3 regulates KDM2A by targeting miR-214 to promote tumor growth and metastasis in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is a pathological type of oral cancer, which accounts for over 90% of oral cancers. It has been widely shown that circRNA is involved in the regulation of multiple malignant oral diseases including OSCC. However, the mechanism underlying how circRNA regulates OSCC is still not clearly elucidated. In this article, we report circFOXO3 promotes tumor growth and invasion of OSCC by targeting miR‐214 which specifically degrades the lysine demethylase 2A (KDM2A). CircRNA sequencing was conducted in OSCC tumor and tumor‐side tissues, and the expression of circFOXO3 is found to be markedly increased in tumor tissues. CircFOXO3 is also highly expressed in several OSCC cell lines compared with human oral keratinocytes. Transwell assay and colony formation showed that knockdown of circFOXO3 prevents the invasion and proliferation of oral cancer cells. Via bioinformatic research, miR‐214 was found to be the target of circFOXO3 and correlate well with circFOXO3 both in vitro and in vivo. KDM2A was then validated by database analysis and luciferase assay to be the direct target of miR‐214. KDM2A helps to promote tumor invasiveness and proliferation of OSCC. Collectively, our results proved that circFOXO3 sponges miR‐214 to up‐regulate the expression of KDM2A, thus promotes tumor progression in OSCC.

Expression of “periostin” in leukoplakia and oral submucous fibrosis: An immunohistochemical study

Aim: Periostin is an extracellular matrix protein secreted in the tumor microenvironment. It is frequently overexpressed in various human cancers, including oral squamous cell carcinoma (OSCC). The role of periostin in carcinogenesis of OSCC is yet to be established. The present study was aimed at determining “Periostin” expression in leukoplakia and the oral submucous fibrosis (OSMF) to relate with its role in malignant transformation of these disorders to OSCC. Materials and Methods: A total of 63 cases were categorized into three groups, namely Group I: OSCC (n = 21), Group II: Leukoplakia (n = 21), and Group III: Oral submucous fibrosis (n = 21); these groups were subjected to immunohistochemical analysis by using the periostin antibody. Kruskal–Wallis test and Mann–Whitney U tests were used to assess the significant differences in the periostin expression between groups (SPSS software version 23.0). Results: Results indicated that there was significant difference in the periostin levels between the groups (P < 0.005), indicating a fairly strong relationship between periostin expression among groups. A significant difference between the different grades of leukoplakia was observed, whereas there were no significant differences between subgroups of the OSCC and OSMF. Conclusion: The present study justifies periostin as a key stromal element in tumor progression of OSCC. The potential role of periostin in malignant transformation of oral potentially malignant disorders (OPMD) has been suggested.

MiR-155-5p promotes oral cancer progression by targeting chromatin remodeling gene ARID2

BackgroundDysregulation of miRNAs is associated with aberrant migration and invasion by suppressing relevant target genes in multiple cancers, including oral squamous cell carcinoma (OSCC). Accumulating evidence suggests that microRNA-155-5p is involved in carcinogenesis and tumor progression. However, the exact function and molecular mechanism of miR-155-5p in OSCC remain unclear. This study aimed to investigate the function of miR-155-5p and the molecular mechanisms underlying the influencing progression of OSCC. MethodsThe miR-155-5p expression level in the OSCC tissues and oral cancer cell lines were determined by the qRT-PCR. Gain-of-function and knockdown approach were used to examine the effect of miR-155-5p on cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of OSCC. The luciferase reporter assay was applied to confirm the AT-rich interactive domain 2 (ARID2) as a potential target of miR-155-5p, and the rescue experiment was employed to verify the roles of the miRNA-155-5p-ARID2 axis in OSCC progression. Immunohistochemical staining was used to detect ARID2 expression in another cohort sample tissues from OSCC patients. ResultsMiR-155-5p was significantly upregulated in OSCC tissues and cell lines. The miR-155-5p expression level was positively correlated with tumor size, TNM stage, histological grade and lymph node metastasis of OSCC patients. Functional assays demonstrated that miR-155-5p enhanced OSCC cell proliferation, migration and invasion. Mechanistically, ARID2 was identified as a direct target and functional effector of miR-155-5p in OSCC. Furthermore, ARID2 overexpression could rescue the aberrant biological function by overexpressed miR-155-5p in OSCC cells. Notably, we showed that ARID2 could be used as an independent prognosis factor in OSCC. ConclusionsOur results suggest that miR-155-5p facilitates tumor progression of OSCC by targeting ARID2, and miR-155-5p-ARID2 axis may be a potential therapeutic target of OSCC.

Ginkgolic Acid, a SUMO-1 Inhibitor, Inhibits the Progression of Oral Squamous Cell Carcinoma by Alleviating SUMOylation of SMAD4

Small ubiquitin-related modifiers (SUMO) represent a class of ubiquitin-like proteins that are conjugated, like ubiquitin, by a set of enzymes to form cellular regulatory proteins, and play key roles in the control of cell proliferation, differentiation, and apoptosis. We found that ginkgolic acid (GA) can significantly reduce cell vitality in a dose- and time-dependent manner and can also accelerate cyto-apoptosis in both Tca8113 and Cal-27 cells. Migration and wound-healing assays were executed to determine the anti-migration effect of GA in oral squamous cell carcinoma (OSCC) cell lines. GA represses transforming growth factor-β1 (TGF-β1)-induced epithelial-mesenchymal transition (EMT) markers in OSCC cell lines. This investigation is the first evidence that GA suppresses TGF-β1-induced SUMOylation of SMAD4. We show that GA affects the phosphorylation of SMAD2/3 protein and the release of SMAD4. In the xenograft mouse model, the OSCC progression was reduced by GA, effectively suppressing the growth of tumors. In addition, siSMAD4 improved cell migration and viability, which was inhibited by GA in Tca8113 cells. GA suppresses tumorigenicity and tumor progression of OSCC through inhibition of TGF-β1-induced enhancement of SUMOylation of SMAD4. Thus, GA could be a promising therapeutic for OSCC.

Abstract C123: Loss of tumor suppressor NDRG2 promotes cell proliferation and migration/invasion of oral squamous cell carcinoma cells

Abstract More than 90% of oral cancers are diagnosed as oral squamous cell carcinoma (OSCC). Although there has been much understanding of the molecular pathogenesis of OSCC, the majority of patients still die from local recurrence and metastasis of tumors. Recently, N-myc downstream-regulated gene 2 (NDRG2) has been known as a tumor suppressor gene in various types of malignancies, but its function and molecular mechanism in OSCC are very little known. To verify the functional roles of NDRG2 in OSCC, we screened six OSCC cell lines first using western blotting. Among the 6 cell lines, HN22 cell line showing high expression of NDRG2 was selected and treated with anti-NDRG2 small interfering RNA (siRNA). The effect of NDRG2 on cell proliferation was evaluated at 24h, 48h, and 72h after treatment of 50nM of anti-NDRG2 siRNA. In addition, in vitro migration and invasion assays were performed after same treatment. Western blot analysis showed variable levels of NDRG2 in 6 OSCC cell lines. Normal human oral keratinocyte as a positive control expressed very high level of NDRG2. Among 6 OSCC cell lines, three cell lines had very low level or absence of NDRG2, but remaining three cell lines including HN22 cell had moderate to high level of NDRG2. After 24h treatment of anti-NDRG2 siRNA on the HN22 cells, NDRG2 expression was significantly downregulated. In cell proliferation assay after treatment of 50nM of anti-NDRG2 siRNA, there was a significant increase in cell growth of HN22 cells when compared with the control (p=.005). In wound healing assay, NDRG2-downregulated HN22 cell line showed much more closure of wound area when compared with the control (p=.007). From the invasion assay after treatment, there was a significant increase in invasion ability of HN22 cells (p&amp;lt;.001). These date indicate that loss of function of NDRG2 as a tumor suppressor can enhance the cell growth, migration, and invasion of OSCC cells. Our results suggest that loss of NDRG2 expression plays a role in tumor progression of OSCC by enhancing tumor cell growth, motility, and invasiveness. Therefore, recovering loss of function of NDRG2 may be a new therapeutic strategy for OSCC treatment. Citation Format: Hye-Jung Yoon, Chi-Hyun Ahn, Ji-Hoon Kim. Loss of tumor suppressor NDRG2 promotes cell proliferation and migration/invasion of oral squamous cell carcinoma cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C123. doi:10.1158/1535-7163.TARG-19-C123

RANKL triggers resistance to TRAIL-induced cell death in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) occurs as a malignancy of the oral cavity. RANK ligand (RANKL) is essential for osteoclast formation/bone resorption. Recently, we showed autoregulation of receptor activator of nuclear factor-κB ligand (RANKL) stimulates OSCC cell proliferation. OSCC cells show resistance to tumor necrosis factor related apoptosis inducing ligand (TRAIL) treatment. Therefore, we hypothesize that RANKL promotes resistance for TRAIL induction of OSCC apoptotic cell death. In this study, SCC14A and SCC74A cells cultured with TRAIL revealed high-level expression of RANKL which increased resistance to TRAIL inhibition of tumor cell proliferation. RANKL stimulation inhibited terminal deoxynucleotidyl transferase dUTP nick end labeling positive staining in TRAIL-treated cells. CRISPR/Cas-9 knockout of RANKL (RANKL-KO) increased caspase-9, caspase-3 activity and cytochrome c release in OSCC cells. RANKL inhibited proapoptotic proteins BAD and BAX expression. TRAIL treatment suppressed the SQSTM1/p62 and RANKL restored the expression. Interestingly, RANKL alone significantly increased proteasome activity. RANKL-KO in OSCC cells inhibited autophagic activity as evidenced by decreased light chain 3B-II and beclin-1 expression. Thus, RANKL stimulation of OSCC tumor cells triggered resistance for TRAIL-induced OSCC cell death. Taken together, blockade of RANKL may inhibit OSCC tumor progression and enhance the potential of TRAIL induced OSCC tumor cell apoptosis.

Abstract 2098: IFIT1 and IFIT3 modulate the drug response in human oral squamous cell carcinoma through interaction and activation of Hsp90

Abstract Interferon-induced protein with tetratricopeptide repeats (IFITs) family, well-known interferon-stimulated genes, have multiple tetratricopeptide repeats with helix-turn-helix structural motifs that mediate a variety of protein-protein interactions. Four IFIT genes have been identified in humans: IFIT1, IFIT2, IFIT3, and IFIT5. While the importance of IFIT1 and IFIT3 in the prognosis of cancer has been reported, the molecular basis of IFIT1 and IFIT3 in cancer progression remains unexplored. We have previously shown that high IFIT1 and IFIT3 expression associated with advanced T-stage, lymph node metastasis, perineural invasion, lymphovascular invasion, extranodal extension, and poor overall survival rate in oral squamous cell carcinoma (OSCC) patients. Also, we have demonstrated that high IFIT1 or IFIT3 expression promoted OSCC invasion and metastasis by EGFR signaling via enhancing EGFR endocytic recycling. Intriguingly, our recent studies have revealed the involvement of IFIT1 and IFIT3 in the resistance to chemotherapeutic agents in OSCC cells. The failure of chemotherapy or targeted therapy was often associated with the intrinsic or acquired drug resistance in cancer cells. In the present study, we intended to explore the biological role of IFIT1 and IFIT3 on drug resistance in OSCC cells. Our results showed that ectopic expression of IFIT1 or IFIT3 proteins in OSCC cells significantly increased the resistance to various therapeutic drugs such as cisplatin, carboplatin, 5-FU, and doxorubicin. Whereas, silencing of IFIT1 and IFIT3 by shRNA enhanced the sensitivity to these drugs. We further demonstrated that enhanced expression of IFIT1 and IFIT3 significantly increased the levels of C-terminal phosphorylation of Hsp90 and activation of its client proteins, such as EGFR, AKT, p38, and SAPK/JNK. Using mass spectrophotometric, immunoprecipitation and immunofluorescence analyses, we identified the interactions of IFIT1 and IFIT3 with Hsp90. Suppression of Hsp90 by a specific inhibitor resulted in decreased activation of its downstream targets in IFIT1- or IFIT3-overexpressing cells. These results suggested a novel mechanism of IFIT1 and IFIT3 in activating Hsp90 and several downstream signaling regulators which are crucial for OSCC tumor progression and drug resistance. Thus, IFIT1 and IFIT3 may act as co-chaperones and serve as potentially important prognostic biomarkers for OSCC progression. Citation Format: Vijaya Kumar Pidugu, Meei-Maan wu, Hima Bindu Pidugu, Te-Chang Lee. IFIT1 and IFIT3 modulate the drug response in human oral squamous cell carcinoma through interaction and activation of Hsp90 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2098.

Amplification of the EGFR and CCND1 Are Coordinated and Play Important Roles in the Progression of Oral Squamous Cell Carcinomas.

Oral squamous cell carcinoma (OSCC) is a common cancer in Taiwan and worldwide. To provide some clues for clinical management of OSCC, 72 advanced-stage OSCCs were analyzed using two microarray platforms (26 cases with Affymetrix 500 K and 46 cases with Affymetrix SNP 6.0). Genomic identification of significant targets in cancer analyses were used to identify significant copy number alterations (CNAs) using a q-value cutoff of 0.25. Among the several significant regions, 12 CNAs were common between these two platforms. Two gain regions contained the well-known oncogenes EGFR (7p11.2) and CCND1 (11q13.3) and several known cancer suppressor genes, such as FHIT (3p14.2–p12.1), FAT1 (4q35.1), CDKN2A (9p21.3), and ATM (11q22.3–q24.3), reside within the 10 deletion regions. Copy number gains of EGFR and CCND1 were further confirmed by fluorescence in situ hybridization and TaqMan CN assay, respectively, in 257 OSCC cases. Our results indicate that EGFR and CCND1 CNAs are significantly associated with clinical stage, tumor differentiation, and lymph node metastasis. Furthermore, EGFR and CCND1 CNAs have an additive effect on OSCC tumor progression. Thus, current genome-wide CNA analysis provides clues for future characterization of important oncogenes and tumor suppressor genes associated with the behaviors of the disease.

MicroRNA‑199a‑5p suppresses migration and invasion in oral squamous cell carcinoma through inhibiting the EMT‑related transcription factor SOX4.

MicroRNAs (miRs) are small, non-coding RNAs that can act as oncogenes or tumor suppressor genes in human cancer. Recent studies have revealed that miR-199a-5p is abnormally expressed in various types of human cancer; however, the potential role of miR-199a-5p in oral squamous cell carcinoma (OSCC) remains elusive. The present study investigated the role of miR-199a-5p in OSCC cells and explored the potential molecular mechanism. Reverse transcription-quantitative polymerase chain reaction was used to measure miR-199a-5p expression in OSCC tissues and adjacent normal oral epithelial tissues. Cell invasion and migration were evaluated using Transwell invasion and wound-healing assays in OSCC cells post-transfection with miR-199a-5p mimics or negative control mimics. In addition, a luciferase reporter assay was conducted to identify the target gene of miR-199a-5p in OSCC cells. The results demonstrated that miR-199a-5p expression was significantly downregulated in OSCC tissues and cell lines, and was associated with tumor progression in OSCC. Furthermore, overexpression of miR-199a-5p inhibited cell invasion and migration, and blocked the epithelial-mesenchymal transition (EMT) cascade. Notably, the results revealed that the EMT-related transcription factor SRY-box 4 (SOX4) was a direct target gene of miR-199a-5p, as determined by the direct binding of miR-199a-5p with the 3′-untranslated region of SOX4. In addition, knockdown of SOX4 by small interfering RNA-SOX4 suppressed proliferation, migration and invasion of OSCC cells. Conversely, overexpression of SOX4 rescued the suppressive effects of miR-199a-5p on cell migration and invasion. Collectively, these data indicated that miR-199a-5p may inhibit the migration and invasion of OSCC cells via targeting the EMT-related transcription factor SOX4, thus suggesting that miR-199a-5p may serve as a prognostic biomarker and therapeutic target in the treatment of OSCC.

Aberrant Collagen Cross-linking in Human Oral Squamous Cell Carcinoma

Tumor progression is a complex process involving extracellular matrix (ECM) remodeling and stiffening. However, the mechanisms that govern these processes and their roles in tumor progression are still poorly understood. In this study, we performed bioinformatics, immunohistochemical, and biochemical analyses to examine if collagen cross-linking is associated with tumor stage and regional lymph node metastasis (RLNM) in oral squamous cell carcinoma (OSCC). We found that the genes encoding key enzymes for cross-linking are frequently overexpressed in oral, head, and neck cancers. Specifically, the enzymes lysyl hydroxylase 2 (LH2) or lysyl oxidase (LOX) and LOX-like 2 (LOXL2) were significantly upregulated in late-stage tumors and associated with poor patient prognosis. The protein levels of these enzymes in the primary human OSCC were also significantly increased in late-stage tumors and markedly elevated in the RLNM-positive tumors. Notably, while overall LOX/LOXL2-catalyzed collagen cross-links were enriched in late-stage and RLNM-positive tumors, LH2-mediated stable cross-links were significantly increased. To our knowledge, this is the first study to investigate the association of collagen cross-linking and expression of key enzymes regulating this process with OSCC stage. The data indicate a critical role for collagen cross-linking in OSCC tumor progression and metastasis, which may provide insights into development of novel therapeutic strategies to prevent OSCC progression.

The Multifarious Functions of Pyruvate Kinase M2 in Oral Cancer Cells.

Head and neck cancers, including oral squamous cell carcinoma (OSCC), are the sixth most common malignancies worldwide. OSCC frequently leads to oral dysfunction, which worsens a patient’s quality of life. Moreover, its prognosis remains poor. Unlike normal cells, tumor cells preferentially metabolize glucose by aerobic glycolysis. Pyruvate kinase (PK) catalyzes the final step in glycolysis, and the transition from PKM1 to PKM2 is observed in many cancer cells. However, little is known about PKM expression and function in OSCC. In this study, we investigated the expression of PKM in OSCC specimens and performed a functional analysis of human OSCC cells. We found that the PKM2/PKM1 ratio was higher in OSCC cells than in adjacent normal mucosal cells and in samples obtained from dysplasia patients. Furthermore, PKM2 expression was strongly correlated with OSCC tumor progression on immunohistochemistry. PKM2 expression was higher during cell growth, invasion, and apoptosis in HSC3 cells, which show a high energy flow and whose metabolism depends on aerobic glycolysis and oxidative phosphorylation. PKM2 expression was also associated with the production of reactive oxygen species (ROS) and integration of glutamine into lactate. Our results suggested that PKM2 has a variety of tumor progressive functions in OSCC cells.

Regulation of proliferation and cell cycle by protein regulator of cytokinesis 1 in oral squamous cell carcinoma

Protein regulator of cytokinesis 1 (PRC1), a microtubule-associated protein, has emerged as a critical regulator of proliferation and apoptosis, acting predominantly in numerous tumors. However, its function in oral squamous cell carcinoma (OSCC) is still unknown. To establish the roles of PRC1 in OSCC, 95 oral clinical samples (54 OSCC, 24 oral leukoplakia [OLK], and 17 normal oral mucosa) and seven oral cell lines (6 OSCC and 1 normal oral cell lines) were analyzed using a series of molecular and genomic assays both in vivo and in vitro were conducted in this study. Herein, we provide evidence demonstrating that expression of PRC1 closely correlates with the degree of epithelial dysplasia in OLK (n = 24) (p < 0.001), and the poor differentiation, large tumor volume, lymph node metastasis, and high-clinical stage in OSCC (n = 54) (p < 0.05), illustrating that PRC1 has a promotive influence on tumor progression in OSCC. Simultaneously, we observed that PRC1 knockdown in OSCC cell lines caused G2/M phase arrest (p < 0.05), inhibited cell proliferation in vitro (p < 0.05) and tumor growth in vivo (p < 0.001). Furthermore, the effects of PRC1 on the regulation of proliferation and cell cycle transition in OSCC samples were mediated by p53. The p53/PRC1/EGFR signaling pathway was found to be implicated in the tumor progression of OSCC. Based on our data, we demonstrate that PRC1 is a key factor in regulating proliferation and the cell cycle, pointing to the potential benefits of PRC1-targeted therapies for OSCC.

Autoregulation of RANK ligand in oral squamous cell carcinoma tumor cells.

Oral squamous cell carcinoma (OSCC) is the most common malignancy among oral cancers and shows potent activity for local bone invasion. Receptor activator of nuclear factor κB (RANK) ligand (RANKL) is critical for bone-resorbing osteoclast formation. We previously demonstrated that OSCC tumor cells express high levels of RANKL. In this study, confocal microscopy demonstrated RANKL specific receptor, RANK expression in OSCC tumor cell lines (SCC1, SCC12, and SCC14a). We also confirmed the expression of RANK and RANKL in primary human OSCC tumor specimens. However, regulatory mechanisms of RANKL expression and a functional role in OSCC tumor progression are unclear. Interestingly, we identified that RANKL expression is autoregulated in OSCC tumor cells. The RANKL specific inhibitor osteoprotegerin (OPG) treatment to OSCC cells inhibits autoregulation of RANKL expression. Further, we showed conditioned media from RANKL CRISPR-Cas9 knockout OSCC cells significantly decreased osteoclast formation and bone resorption activity. In addition, RANKL increases OSCC tumor cell proliferation. RANKL treatment to OSCC cells demonstrated a dose-dependent increase in RANK intracellular adaptor protein, TRAF6 expression, and activation of IKK and IκB signaling molecules. We further identified that transcription factor NFATc2 mediates autoregulation of RANKL expression in OSCC cells. Thus, our results implicate RANKL autoregulation as a novel mechanism that facilitates OSCC tumor cell growth and osteoclast differentiation/bone destruction.