Oral Squamous Cell Carcinoma Tumor Research Articles

Prognostic significance of fascin and Wilm’s tumor 1 (WT1) in oral squamous cell carcinomas – case control study

Aims: To elucidate the role of fascin and WT1 expression in oral squamous cell carcinoma (OSCC) by correlation with clinicopathological parameters.Subjects and methods: Paraffin sections of 27 OSCC tissue were immunohistochemically stained with fascin and WT1 using the avidin-biotin-peroxidase staining method. Correlations between fascin and WT1 and various clinicopathological features, and prognosis were studied.Results: Immunohistochemical study revealed significant increase of fascin and WT1 in OSCC in relation to control group (p< 0.000). Receiver operating characteristic (ROC) curve for fascin and WT1 were conducted for detection of recurrence. Fascin showed fair area under curve (AUC) (AUC=0.66), with sensitivity of 63.6 % and specificity of 50.0 % at cutoff value of 42.5 %. WT1 also showed fair AUC (AUC=0.63), with sensitivity of 63.6 % and specificity 68.8 % at cutoff value of 40.0 %. ROC curve for WT1 and fascin were conducted for detection of lymph node infiltration. While fascin showed excellent AUC (AUC=0.865), cutoff value of 52.5 %, with sensitivity of 70.0 % and specificity of 94.1%. WT1 also showed excellent AUC (AUC=0.791), cutoff value of 42.2 %, with sensitivity of 70.0 % and specificity 70.6%. Significant associations were detected between median fascin cutoff value at 45% and well differentiated tumor (P=0.04), T3&T4 tumor size (P= 0.01), LN infiltration (P=0.05), and tumor-node metastasis 3 and 4 (TNM3 &TNM4) staging (P=0.02). Significant associations were found between the median cutoff value of WT1 and moderately differentiated tumor (P=0.02), and LN infiltration (P=0.01). The 2-year survival rate of patients with fascin of ≥ 45% and WT1 ≥ 40% were nonsignificantly higher than that of patients with fascin of < 45% and WT1 <40% (P=0.09, P=0.55). Univariate analysis demonstrated that Fascin, WT1 were significant risk factors of LN (p=0.005, 0.02) but not considered as risk factors of tumor recurrence. Conclusion: Fascin and WT1 have oncogenic effects playing an important role in progression of OSCC. Overexpression of them contributes to a more aggressive clinical course. Understanding their role on OSCC and other tumors will facilitate the development of new treatment strategies.

TARGETING THE METABOLIC SIGNALING NETWORK FOR IMMUNE CELL LONGEVITY AND FUNCTION IN ORAL CANCER THERAPY

Oral squamous cell carcinoma (OSCC), which includes cancer of the oral cavity and oropharynx, is a significant health issue. Despite aggressive multimodality therapies, OSCC has a poor five year survival rate (60%), which results in ~300,000 deaths each year worldwide1. OSCC is associated with human papillomavirus (HPV) infection, and abusive intake of tobacco and alcohol 2–3. OSCC evades the immune recognition and anti‐tumor immune response, by exploiting the immune system's regulatory immune suppressive mechanisms 2–3. A key mechanism of tumor immune suppression has been depicted as T‐cell exhaustion 4–7. In this case, T‐cell's effector function is restrained due to the activation of checkpoints, such as PD‐1 by its ligand PD‐L1, expressed on cancers cells, T‐cells, natural killer cells and macrophages 4–7. Immune checkpoint inhibitors have demonstrated revolutionary restoration of T‐cell mediated anti‐tumor immunity in OSCC 4–7. Despite this success, the overall response rate of these immune therapies in OSCC is only ~20 percent 4–7. New therapeutic options to prevent and treat OSCC are urgently needed. The unrestrained growth of cancer cells and the effector function of immune cells are often supported by the same metabolic pathway, namely aerobic glycolysis8. We believe that by taking advantage of the unique metabolic requirement of tumor cells and the cancer‐fighting immune cells, we can discover new insights into how such a relationship regulates anti‐tumor immunity. Our aim is to elucidate the role of immune and cancer cell metabolic properties in regulating the efficacy of the anti‐tumor response. Targeting both the metabolic signaling pathways of the OSCC tumor cells and tumor infiltrating immune cells has the potential to achieve cancer remission by both halting OSCC progression and enhancing the longevity of the anti‐tumor immune response.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.

Gene microarray analysis revealed a potential crucial gene RACK1 in oral squamous cell carcinoma (OSCC)

ABSTRACTOral squamous cell carcinoma (OSCC) is the sixth most common cancer worldwide, which appears as a consequence of multiple molecular genetic events in various chromosomes and genes. In order to unveil the possible mechanisms underlying OSCC tumorigenesis, the OSCC-related gene expression variance and the gene interaction network should be further investigated. Herein, we conducted the NimbleGen Human Gene Expression Microarray to analyze expression heterogeneity between OSCC primary tumor tissue and its adjacent normal tissue from two patients. A total number of 7872 out of 32,448 detected genes are differentially expressed in OSCC. Gene ontology (GO) analysis demonstrated that these differentially expressed transcripts were critical in a series of metabolic processes, cancer-related signal pathways, and biological regulations. KEGG signaling pathway enrichment suggested a number of pathways (metabolic process and immune response) which are frequently enrolled during cancer progression. 15 most differential regulated genes between OSCC tumor and non-tumor were confirmed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Furthermore, the interaction network analysis of these confirmed genes by STRING database showed the two subunits of RACK1 had direct interaction with 14 differential proteins. This bioinformatics research lends support about the critical role of RACK1 which functions as a key node protein driving OSCC development.

NIPA-like domain containing 1 is a novel tumor-promoting factor in oral squamous cell carcinoma.

In our previous global gene expression analysis, we identified NIPA-like domain containing 1 (NIPAL1), which encodes a magnesium transporter, as one of the most overexpressed genes in recurrent oral squamous cell carcinoma (OSCC). Although has been NIPAL1 linked with gout pathogenesis, little is known about its expression and function in human malignancies. In this study, we examined NIPAL1 expression in 192 cases of OSCC by immunohistochemistry and performed a functional analysis of human OSCC cells. NIPAL1 immunostaining was observed in 39 of 192 OSCC patients (20.3%). NIPAL1 expression correlated significantly with cancer cell intravsation (P = 0.0062), as well as with poorer disease-free survival in a Kaplan-Meier analysis (P < 0.0001). Moreover, a multivariate Cox proportional hazards model analysis revealed that NIPAL1 expression was an independent predictor of disease-free survival in OSCC (P < 0.0001). In a functional analysis, NIPAL1 regulated the growth and adhesion of OSCC tumor cells and endothelial cells. Our findings suggest that NIPAL1 might be a novel factor promoting OSCC tumorigenesis, as well as a useful molecular marker of OSCC.

In vitro and in silico validation of CA3 and FHL1 downregulation in oral cancer

BackgroundAberrant methylation is a frequent event in oral cancer.MethodsIn order to better characterize these alterations, a search for genes downregulated by aberrant methylation in oral squamous cell carcinoma (OSCC) was conducted through the mining of ORESTES dataset. Findings were further validated in OSCC cell lines and patients’ samples and confirmed using TCGA data. Differentially expressed genes were identified in ORESTES libraries and validated in vitro using RT-PCR in HNSCC cell-lines and OSCC tumor samples. Further confirmation of these results was performed using mRNA expression and methylation data from The Cancer Genome Atlas (TCGA) data.ResultsFrom the set of genes selected for validation, CA3 and FHL1 were downregulated in 60% (12/20) and 75% (15/20) of OSCC samples, respectively, and in HNSCC cell lines. The treatment of cell lines JHU-13 and FaDu with the demethylating agent 5'-aza-dC was efficient in restoring CA3 and FHL1 expression. TCGA expression and methylation data on OSCC confirms the downregulation of these genes in OSCC samples and also suggests that expression of CA3 and FHL1 is probably regulated by methylation. The downregulation of CA3 and FHL1 observed in silico was validated in HNSCC cell lines and OSCC samples, showing the feasibility of integrating different datasets to select differentially expressed genes in silico.ConclusionsThese results showed that the downregulation of CA3 and FHL1 data observed in the ORESTES libraries was validated in HNSCC cell lines and OSCC samples and in a large cohort of samples from the TCGA database. Moreover, it suggests that expression of CA3 and FHL1 could probably be regulated by methylation having an important role the oral carcinogenesis.

A Real-Time Near-Infrared Fluorescence Imaging Method for the Detection of Oral Cancers in Mice Using an Indocyanine Green-Labeled Podoplanin Antibody.

Podoplanin is distinctively overexpressed in oral squamous cell carcinoma than oral benign neoplasms and plays a crucial role in the pathogenesis and metastasis of oral squamous cell carcinoma but its diagnostic application is quite limited. Here, we report a new near-infrared fluorescence imaging method using an indocyanine green (ICG)–labeled anti-podoplanin antibody and a desktop/a handheld ICG detection device for the visualization of oral squamous cell carcinoma–xenografted tumors in nude mice. Both near-infrared imaging methods using a desktop (in vivo imaging system: IVIS) and a handheld device (photodynamic eye: PDE) successfully detected oral squamous cell carcinoma tumors in nude mice in a podoplanin expression–dependent manner with comparable sensitivity. Of these 2 devices, only near-infrared imaging methods using a handheld device visualized oral squamous cell carcinoma xenografts in mice in real time. Furthermore, near-infrared imaging methods using the handheld device (PDE) could detect smaller podoplanin-positive oral squamous cell carcinoma tumors than a non-near-infrared, autofluorescence-based imaging method. Based on these results, a near-infrared imaging method using an ICG-labeled anti-podoplanin antibody and a handheld detection device (PDE) allows the sensitive, semiquantitative, and real-time imaging of oral squamous cell carcinoma tumors and therefore represents a useful tool for the detection and subsequent monitoring of malignant oral neoplasms in both preclinical and some clinical settings.

Genomic and epigenetic signatures associated with survival rate in oral squamous cell carcinoma patients.

Purpose: Although oral squamous cell carcinoma (OSCC) presents great mortality and morbidity worldwide, the mechanisms behind its clinical behavior remain unclear. Biomarkers are needed to forecast patients' survival and, among those patients undergoing curative therapy, which are more likely to develop tumor recurrence/metastasis. Demonstrating clinical relevance of these biomarkers could be crucial both for surveillance and in helping to establish adjuvant therapy strategies. We aimed to identify genomic and epigenetic biomarkers of OSCC prognosis as well as to explore a noninvasive strategy to perform its detection.Methods: OSCC tumor and non-tumor tissue samples and cells scrapped from the tumor surface were genomic and epigenetically evaluated by Methylation-Specific Multiplex Ligation-dependent Probe Amplification technique.Results: Copy number alterations in ATM, CASR, TP73, CADM1, RARB, CDH13, PAX5, RB1 genes and GATA5, PAX6, CADM1 and CHFR promoter methylation were shown to be associated with worse OSCC patients' survival. Copy number alterations in BRCA1, CDKN2A, CHFR, GATA5, PYCARD, STK11, TP53, VHL genes and GATA5, CADM1, KLLN, MSH6, PAX5, WT1 promoter methylation were shown to be associated with development of metastasis/relapses during or after OSCC patients' treatment. We also found a good agreement in the status of CDKN2A promoter methylation evaluated noninvasively or in the tumor tissue.Conclusions: Genomic and epigenetic signatures were validated in a larger and geographically separate cohort, from TCGA database, which reinforce their clinical applicability. Noninvasive methodologies for detection of these signatures require further studies before translation in to clinical practice.

Cyclin D1 overexpression associated with activation of STAT3 in oral carcinoma patients from South India.

Our previous study showed that overexpression of cyclin D1 protein is associated with poor prognosis in oral squamous cell carcinoma (OSCC) patients. Regarding the alteration in the transactivating pathway regulating cyclin D1 expression is still unclear in OSCC from our population. The major objective of this study is to understand the alteration associated with the transactivation pathway regulating the cyclin D1 overexpression in OSCC patients from our population. Alteration in the transactivation pathway regulating cyclin D1 expression was evaluated in tumor sample from OSCC patients. The findings were further validated using in vitro knockdown model in OSCC cell line. Results from the patients' samples showed that the Phospho-STAT3 has a significant association with cyclin D1 overexpression in OSCC tumor samples. Further knockdown in vitro studies using SCC66 showed a significant correlation between STAT3 and cyclin D1 in OSCC. The results from this study showed that in our population the cyclin D1 overexpression is associated with hyperactivation of STAT3 pathway. Our previous result has shown that the cyclin D1 protein overexpression is associated with poor prognosis in OSCC patients. Hence, STAT3 pathway will be better target for the patients with increased cyclin D1 in OSCC patients from our population.

Long non-coding RNA MALAT1 promotes oral squamous cell carcinoma development via microRNA-125b/STAT3 axis.

Oral squamous cell carcinoma (OSCC), as the most common type of oral cancer, is responsible for almost 3% of all malignant tumors worldwide. Non-coding RNAs such as lncRNAs and microRNAs have been involved in many cancers including OSCC. Recently, lncRNA metastasis-associated lung adenocarcinoma transcript-1 (MALAT1) has been reported to play an oncogenic role in OSCC metastasis. However, the underlying mechanism of MALAT1 in regulating OSCC progression remains unclear. The aim of this study was to investigate the specific role of MALAT1 in OSCC development. It was observed that MALAT1 was upregulated in OSCC cell lines. Inhibition of MALAT1 can prevent OSCC proliferation while overexpressing MALAT1 promoted OSCC progression. In addition, bioinformatics search was used to identify that miR-125b was a direct target of MALAT1, which indicated a negative correlation between MALAT1 and miR-125b. Besides these, STAT3 was predicted as a binding target of miR-125b in OSCC. Overexpression of MALAT1 was able to suppress the tumor inhibitory effect of miR-125b mimics via upregulating STAT3. Moreover, the function of MALAT1 in OSCC development was further investigated by using in vivo assays. The established nude mice models revealed that downregulated MALAT1 greatly inhibited OSCC tumor growth and reversely upregualated MALAT1 promoted OSCC development via miR-125b/STAT3 axis, respectively. In conclusion, MALAT1 can function as a competing endogenous RNA (ceRNA) to modulate STAT3 expression by absorbing miR-125b in OSCC and could be used as a novel therapeutic target in OSCC diagnosis and treatment.

Inhibition of BMP signaling overcomes acquired resistance to cetuximab in oral squamous cell carcinomas

Despite expressing high levels of the epidermal growth factor receptor (EGFR), a majority of oral squamous cell carcinoma (OSCC) patients show limited response to cetuximab and ultimately develop drug resistance. However, mechanism underlying cetuximab resistance in OSCC is not clearly understood. Here, using a mouse orthotopic xenograft model of OSCC, we show that bone morphogenic protein-7-phosphorylated Smad-1, -5, -8 (BMP7-p-Smad1/5/8) signaling contributes to cetuximab resistance. Tumor cells isolated from the recurrent cetuximab-resistant xenograft models exhibited low EGFR expression but extremely high levels of p-Smad1/5/8. Treatment with the bone morphogenic protein receptor type 1 (BMPRI) inhibitor, DMH1 significantly reduced cetuximab-resistant OSCC tumor growth, and combined treatment of DMH1 and cetuximab remarkably reduced relapsed tumor growth in vivo. Importantly, p-Smad1/5/8 level was elevated in cetuximab-resistant patients and this correlated with poor prognosis. Collectively, our results indicate that the BMP7-p-Smad1/5/8 signaling is a key pathway to acquired cetuximab resistance, and demonstrate that combination therapy of cetuximab and a BMP signaling inhibitor as potentially a new therapeutic strategy for overcoming acquired resistance to cetuximab in OSCC.

Astrocyte elevated gene-1 promotes tumour growth and invasion by inducing EMT in oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is a common human malignancy with a high incidence rate and poor prognosis. Although astrocyte elevated gene 1 (AEG-1) expression is up-regulated in various human cancers and plays an important role in carcinogenesis and tumour progression, the impact of AEG-1 on the development and progression of OSCC remains unclear. Accordingly, this study aims to clarify the biological significance of AEG-1 in OSCC. We found AEG-1 to be overexpressed in OSCC tissues compared to normal oral mucosa. Knockdown or overexpression of AEG-1 in OSCC cell lines showed that AEG-1 is important for tumour growth, apoptosis, drug tolerance, and maintaining epithelial-mesenchymal transition (EMT)-mediated cell migration and invasion in vitro. Moreover, in a xenograft-mouse model generated by AEG-1-overexpressing SCC15 cells, we found that higher expression of AEG-1 promoted tumour growth, angiogenesis, and EMT in vivo. These findings provide mechanistic insight into the role of AEG-1 in regulating OSCC tumour growth, apoptosis, drug tolerance, and invasion, as well as AEG-1-induced activation of p38 and NF-κB signalling, suggesting that AEG-1 is an important prognostic factor and therapeutic target for OSCC.

EGFR and Cortactin: Markers for potential double target therapy in oral squamous cell carcinoma.

Survival periods of patients following surgical therapy of oral squamous cell carcinoma (OSCC) have previously been demonstrated to decrease over recent decades. Epidermal growth factor receptor (EGFR) and Cortactin are molecular markers that are important in tumour progression and development, and interact within the EGF pathway. Although EGFR antibody therapy exists, sufficient efforts for increased survival are still lacking due to the present limited response rates. The aim of the present study was to examine the association between EGFR and Cortactin expression on survival rates of OSCC patients and to determine whether EGFR and Cortactin expression levels are associated with advanced tumor sizes and lymphnode-metastases. In total, 222 OSCC patients were included in the study. EGFR and Cortactin expression in tumor tissue was evaluated by immunohistochemistry. Cox regression was used for survival analysis. Categories were tested for associations by using cross tabs (Chi-square test). Groups were compared by the non-parametric Mann Whitney U-test. Probabilities of less than 0.05 were considered significant and significant expression of Cortactin was observed in Advanced Union Internationale Contre le Cancer stage (P=0.032), including advanced tumour stage (P=0.021) and lymph node metastasis (P=0.049). High Cortactin expression was significantly associated with poorer survival rates (P=0.037). Further Cortactin expression was not associated with extracapsular spread, however EGFR exhibited a significant association (P=0.034). Neither EGFR nor Cortactin expression was correlated to grading. EGFR and Cortactin co-expression was demonstrated to be significantly associated with poorer survival rates in OSCC patients, suggesting that identification of predictive biomarkers for adjuvant therapies are of primary concern in OSCC. In particular, efficient dual-target therapy may act as an appropriate therapy to improve survival time for patients at advanced OSCC tumor stages.

A 3D printed microfluidic perfusion device for multicellular spheroid cultures

The advent of 3D printing technologies promises to make microfluidic organ-on-chip technologies more accessible for the biological research community. To date, hydrogel-encapsulated cells have been successfully incorporated into 3D printed microfluidic devices. However, there is currently no 3D printed microfluidic device that can support multicellular spheroid culture, which facilitates extensive cell–cell contacts important for recapitulating many multicellular functional biological structures. Here, we report a first instance of fabricating a 3D printed microfluidic cell culture device capable of directly immobilizing and maintaining the viability and functionality of 3D multicellular spheroids. We evaluated the feasibility of two common 3D printing technologies i.e. stereolithography (SLA) and PolyJet printing, and found that SLA could prototype a device comprising of cell immobilizing micro-structures that were housed within a microfluidic network with higher fidelity. We have also implemented a pump-free perfusion system, relying on gravity-driven flow to perform medium perfusion in order to reduce the complexity and footprint of the device setup, thereby improving its adaptability into a standard biological laboratory. Finally, we demonstrated the biological performance of the 3D printed device by performing pump-free perfusion cultures of patient-derived parental and metastatic oral squamous cell carcinoma tumor and liver cell (HepG2) spheroids with good cell viability and functionality. This paper presents a proof-of-concept in simplifying and integrating the prototyping and operation of a microfluidic spheroid culture device, which will facilitate its applications in various drug efficacy, metabolism and toxicity studies.

Fatty acid synthase as a potential therapeutic target in feline oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is an aggressive and treatment-resistant malignancy in both feline and human patients. Recent work has demonstrated aberrant expression of fatty acid synthase (FASN) and an increased capacity for lipogenesis in human OSCC and other cancers. In human OSCC, inhibition of FASN decreased cell viability and growth in vitro, and diminished tumour growth and metastasis in murine preclinical models. This study aimed to characterize FASN as a therapeutic target in feline OSCC. Immunohistochemistry revealed high FASN expression in primary feline OSCC tumours, and FASN expression was detected in OSCC cell lines (3 feline and 3 human) by immunoblotting and quantitative real-time-polymerase chain reaction (qRT-PCR). Orlistat, a FASN inhibitor, substantially reduced cell viability in both feline and human OSCC lines, although feline cell lines consistently displayed higher sensitivity to the drug. FASN mRNA expression among cell lines mirrored sensitivity to orlistat, with feline cell lines expressing higher levels of FASN. Consistent with this observation, diminished sensitivity to orlistat treatment and decreased FASN mRNA expression were observed in feline OSCC cells following incubation under hypoxic conditions. Treatment with orlistat did not potentiate sensitivity to carboplatin in the cell lines investigated; instead, combinations of the 2 drugs resulted in additive to antagonistic effects. Our results suggest that FASN inhibition is a viable therapeutic target for feline OSCC. Furthermore, cats may serve as a spontaneous large animal model for human oral cancer, although differences in the regulation of lipogenesis between these 2 species require further investigation.

Activated spleen tyrosine kinase promotes malignant progression of oral squamous cell carcinoma via mTOR/S6 signaling pathway in an ERK1/2-independent manner.

Spleen tyrosine kinase (SYK), a non-receptor cytoplasmic tyrosine enzyme, is well known for its ability in certain pathways through immune receptors. Recently, SYK role in cancer has been widely studied. SYK plays a dual role as a tumor suppressor and tumor promoter. Nevertheless, its role in oral squamous cell carcinoma (OSCC) has not been fully investigated. In the current study, samples from OSCC tumors and adjacent normal counterparts were collected and SYK expression was evaluated by real-time qPCR. SYK mRNA expression in tumors was higher than the normal tissues. And high SYK expression was confirmed by immunohistochemistry analysis and closely related to worse overall survival. The expression of SYK mRNA and protein was detected in 2 of 4 OSCC cell lines. SYK pharmacological suppression and RNAi-mediated knockdown inhibited proliferation, migration, and invasion of SYK-positive cells by reducing phosphorylated ERK1/2 and mTOR levels. One inhibitor of MEK, PD98059, also suppressed the same cancer-associated phenotypes of SYK-positive cells by decreasing phosphorylated ERK1/2 but increasing phosphorylated mTOR. Piceatannol, one pharmacological inhibitor of SYK, attenuated tumor growth in vivo. Overall, our results revealed a novel mechanism triggered by SYK to increase OSCC tumoriogenesis and tumor progression.

Apolipoprotein E Promotes Invasion in Oral Squamous Cell Carcinoma

Oral squamous cell carcinoma (OSCC) patients generally have a poor prognosis, because of the invasive nature of these tumors. In comparing transcription profiles between OSCC tumors with a more invasive (worst pattern of tumor invasion 5) versus a less invasive (worst pattern of tumor invasion 3) pattern of invasion, we identified a total of 97 genes that were overexpressed at least 1.5-fold in the more invasive tumor subtype. The most functionally relevant genes were assessed using invitro invasion assays with an OSCC cell line (UM-SCC-1). Individual siRNA knockdown of 15 of these 45 genes resulted in significant reductions in tumor cell invasion compared to a nontargeting siRNA control. One gene whose knockdown had a strong effect on invasion corresponded to apolipoprotein E (APOE). Both matrix degradation and the number of mature invadopodia were significantly decreased with APOE knockdown. APOE knockdown also resulted in increased cellular cholesterol, consistent with APOE's role in regulating cholesterol efflux. APOE knockdown resulted in decreased levels of phospho-extracellular signal-regulated kinase 1/2, phospho-c-Jun N-terminal kinase, and phospho-cJun, as well as decreased activator protein 1 (AP-1) activity. Expression of matrix metalloproteinase 7 (MMP7), an AP-1 target, was also significantly decreased. Our findings suggest that APOE protein plays a significant role in OSCC tumor invasion because of its effects on cellular cholesterol and subsequent effects on cell signaling and AP-1 activity, leading to changes in the expression of invasion-related proteins, including MMP7.

Histochemical analysis of collagen reorganization at the invasive front of oral squamous cell carcinoma tumors.

The aim of the present study was to: (a) analyze the nature of collagen with respect to cohesive and discohesive invasive tumor front (ITF) of oral squamous cell carcinoma (OSCC); (b) analyze the clinicopathological correlation with the nature of collagen at the ITF of OSCC; and (c) correlate the nature of collagen with Broder's and Bryne's histological grading system of OSCC. Tissue sections of 29 OSCC with ITF were stained with hematoxylin-eosin and picrosirius red staining for evaluation under a polarized microscope. Tumors with a cohesive front had a thick collagen fiber, are predominantly organized, red-yellow in color, well packed, and show strong birefringence (P<.005). A gradual change in the nature of the collagen fiber was observed in the discohesive tumor front, where the collagen fibers were thin, disorganized, yellow-orange to green-yellow in color, loosely packed, and with weak birefringence (P<.005). A cohesive tumor front with organized collagen fibers prevents tumor invasion and metastasis. As it is inhibits an increase in tumor size, it is associated with the initial stage of tumor (I & II), whereas in a discohesive tumor front, the fibers might enhance the movement of tumor cells, resulting in invasion and metastasis.

Identification of differentially expressed genes through a meta-analysis approach for oral cancer classification.

We propose a meta-analysis scheme for identifying differentially expressed genes in Oral Squamous Cell Carcinoma (OSCC) from different microarray studies. We detect a subset of relevant features and further classify samples under two experimental conditions (i.e healthy and cancer samples) for better patient stratification. A well-established meta-analysis method is adopted and gene expression data sets are derived from a public functional genomics data repository. Our primary aim is the accurate identification of up- and down-regulated genes in order to extract valuable biological information concerning the changes in expression between healthy and cancer samples. According to our results and the extracted informative gene list, a high classification accuracy of healthy and OSCC tumors is achieved with as few genes as possible. Furthermore, the proposed scheme implies that the combination of datasets from different origins may reduce the estimated percentage of false predictions, while the power of gene identification and disease classification is increased.

Abstract 3933: Characterization of oncogenic activity of interferon-induced protein with tetratricopeptide repeats 1 and 3 in human oral squamous cell carcinoma progression

Abstract Interferon-induced protein with tetratricopeptide repeats (IFITs) family has multiple tetratricopeptide repeats with helix-turn-helix structural motifs that mediate a variety of protein-protein interactions. There are four IFIT genes that have been identified namely, ISG56/IFIT1, ISG54/IFIT2, ISG60/IFIT3, and ISG58/IFIT5. IFIT proteins have been documented to involve in a variety of cellular functions, such as cell proliferation, migration, virus-induced translation initiation, replication and double-stranded RNA signaling. Our previous study has demonstrated that IFIT2 inhibits oral squamous cell carcinoma (OSCC) metastasis. However, little is known about IFIT1 and IFIT3 in the progression of OSCC. In the present study, we are investigating the functional role of IFIT1 and IFIT3 genes in OSCC tumor progression. Immunohistochemical analysis of 147 patients derived tumor specimens with OSCC revealed a strong correlation between IFIT1 and IFIT3 proteins (p&amp;lt;0.001). Clinicoplathological characteristics correlation of IFIT1 and IFIT3 expression levels were positively associated with regional lymph node invasion and nerve invasion. IFIT1 was also associated with venous invasion. Ectopic expression of IFIT1 and IFIT3 proteins in Ca9-22 and SAS cells inducing PKC, EGFR, and AKT activation, resulting in OSCC cells resistance to cisplatin but highly susceptible to tyrosine kinase inhibitor gefitinib, whereas IFIT1 and IFIT3 knockdown SCC25 cells have shown opposite effect. Interestingly, IFIT1 and IFIT3 expression is associated with enhanced cell invasion activity and altered expression of epithelial-mesenchymal transition markers. These data suggested that IFIT1 and IFIT3 may serve as potential biomarkers for the OSCC treatment. The study on how IFIT1 and IFIT3 cause PKC, EGFR, and AKT activation and resistance to DNA damaging agents is ongoing. Our further investigations may explain the molecular signaling mechanism of IFIT1 and IFIT3 in OSCC tumor progression. Citation Format: Vijaya Kumar Pidugu, Ai-Hsin Yen, Ying-Chen Chen, Mei-Maan Wu, Chung-Ji Liu, Te-Chang Lee. Characterization of oncogenic activity of interferon-induced protein with tetratricopeptide repeats 1 and 3 in human oral squamous cell carcinoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3933. doi:10.1158/1538-7445.AM2017-3933