Mouse Oral Squamous Cell Carcinoma Research Articles

Nimotuzumab shows an additive effect to inhibit cell growth of ALA-PDT treated oral cancer cells

Oral squamous cell carcinoma (OSCC) can be characterized by severe functional impairment and a poor prognosis. The epidermal growth factor receptor (EGFR) is highly expressed in OSCC and is a promising target for cancer therapy. In addition, aminolevulinic acid-induced photodynamic therapy (ALA-PDT) has produced robust clinical effects in oral cancer. Here, an EGFR inhibitor, nimotuzumab, was administered to 2 OSCC cell lines, CAL-27 and SCC-25, treated with ALA-PDT. Cell growth, apoptosis, and reactive oxygen species (ROS) generation were used to measure the antitumor activity of the combination therapy. The in vivo effect of nimotuzumab plus ALA-PDT was done using a mouse OSCC xenograft model (SCC-25). EGFR expression was further compared by Western blotting in different groups. We observed that nimotuzumab combined with ALA-PDT could enhance inhibition of OSCC cell growth in vitro and in vivo. We also observed an enhanced effect after combination on cell apoptosis in CAL-27 and SCC-25 cells. Furthermore, combined therapy significantly reduced the protein expression levels of EGFR in vitro. However, we observed that nimotuzumab plus ALA-PDT did not increase ROS generation substantially in OSCC cells compared to the ALA-PDT group alone. These observations indicate that nimotuzumab combined with ALA-PDT has valuable applications for OSCC treatment.

IFIT2-depleted metastatic oral squamous cell carcinoma cells induce muscle atrophy and cancer cachexia in mice.

BackgroundInterferon‐induced protein with tetratricopeptide repeat 2 (IFIT2) is a reported metastasis suppressor in oral squamous cell carcinoma (OSCC). Metastases and cachexia may coexist. The effect of cancer metastasis on cancer cachexia is largely unknown. We aimed to address this gap in knowledge by characterizing the cachectic phenotype of an IFIT2‐depleted metastatic OSCC mouse model.MethodsGenetically engineered and xenograft tumour models were used to explore the effect of IFIT2‐depleted metastatic OSCC on cancer cachexia. Muscle and organ weight changes, tumour burden, inflammatory cytokine profiles, body composition, food intake, serum albumin and C‐reactive protein (CRP) levels, and survival were assessed. The activation of the IL6/p38 pathway in atrophied muscle was measured.ResultsIFIT2‐depleted metastatic tumours caused marked body weight loss (−18.2% vs. initial body weight, P < 0.001) and a poor survival rate (P < 0.01). Skeletal muscles were markedly smaller in IFIT2‐depleted metastatic tumour‐bearing mice (quadriceps: −28.7%, gastrocnemius: −29.4%, and tibialis: −24.3%, all P < 0.001). Tumour‐derived circulating granulocyte‐macrophage colony‐stimulating factor (+772.2‐fold, P < 0.05), GROα (+1283.7‐fold, P < 0.05), IL6 (+245.8‐fold, P < 0.001), IL8 (+616.9‐fold, P < 0.001), IL18 (+24‐fold, P < 0.05), IP10 (+18.8‐fold, P < 0.001), CCL2 (+439.2‐fold, P < 0.001), CCL22 (+9.1‐fold, P < 0.01) and tumour necrosis factor α (+196.8‐fold, P < 0.05) were elevated in IFIT2‐depleted metastatic tumour‐bearing mice. Murine granulocyte colony‐stimulating factor (+61.4‐fold, P < 0.001) and IL6 (+110.9‐fold, P < 0.01) levels were significantly increased in IFIT2‐depleted metastatic tumour‐bearing mice. Serum CRP level (+82.1%, P < 0.05) was significantly increased in cachectic shIFIT2 mice. Serum albumin level (−26.7%, P < 0.01) was significantly decreased in cachectic shIFIT2 mice. An assessment of body composition revealed decreased fat (−81%, P < 0.001) and lean tissue (−21.7%, P < 0.01), which was consistent with the reduced food intake (−19.3%, P < 0.05). Muscle loss was accompanied by a smaller muscle cross‐sectional area (−23.3%, P < 0.05). Muscle atrophy of cachectic IFIT2‐depleted metastatic tumour‐bearing mice (i.v.‐shIFIT2 group) was associated with elevated IL6 (+2.7‐fold, P < 0.05), phospho‐p38 (+2.8‐fold, P < 0.05), and atrogin‐1 levels (+2.3‐fold, P < 0.05) in the skeletal muscle. Neutralization of IL6 rescued shIFIT2 conditioned medium‐induced myotube atrophy (+24.6%, P < 0.01).ConclusionsOur results suggest that the development of shIFIT2 metastatic OSCC lesions promotes IL6 production and is accompanied by the loss of fat and lean tissue, anorexia, and muscle atrophy. This model is appropriate for the study of OSCC cachexia, especially in linking metastasis with cachexia.

DDX3 modulates the tumor microenvironment via its role in endoplasmic reticulum-associated translation

SummaryUsing antibody arrays, we found that the RNA helicase DDX3 modulates the expression of secreted signaling factors in oral squamous cell carcinoma (OSCC) cells. Ribo-seq analysis confirmed amphiregulin (AREG) as a translational target of DDX3. AREG exerts important biological functions in cancer, including promoting cell migration and paracrine effects of OSCC cells and reprogramming the tumor microenvironment (TME) of OSCC in mice. DDX3-mediated translational control of AREG involves its 3′-untranslated region. Proteomics identified the signal recognition particle (SRP) as an unprecedented interacting partner of DDX3. DDX3 and SRP54 were located near the endoplasmic reticulum, regulated the expression of a common set of secreted factors, and were essential for targeting AREG mRNA to membrane-bound polyribosomes. Finally, OSCC-associated mutant DDX3 increased the expression of AREG, emphasizing the role of DDX3 in tumor progression via SRP-dependent, endoplasmic reticulum-associated translation. Therefore, pharmacological targeting of DDX3 may inhibit the tumor-promoting functions of the TME.

Abstract 1910: Augmented secretion of IL-1α from mouse oral squamous cell carcinoma (OSCC) cells caused by the serum deprivation and hypoxia promotes immune-suppressive activity of mesenchymal stromal cells

Abstract Objectives; We have already reported that mouse oral squamous carcinoma (OSCC), Sq-1979-1, cells produce IL-1α which specifically enhances the immunosuppressive activity of co-cultured mesenchymal stromal, 10T1/2, cells (Morimoto-Ito, Open Dent J. 2019). In this report, we evaluated conditions promoting the expression of IL-1α of Sq-1979-1 cells to further enhance immunosuppressive function of the 10T1/2 cells, and also examined the expression in OSCC tissues. Methods; Expression of IL-1α in Sq-1979-1 cells was evaluated by RT-PCR, western blots and enzyme-linked immune sorbent assay (ELISA). Conditioned mediums (CMs) from Sq-1979-1 cells were co-cultured with 10T1/2 cells and anti-CD3 antibody-stimulated mouse spleen cells. The production of interferon (IFN)- γ and IL-1α was evaluated using ELISA. The function of IL-1α in CM was examined by anti- IL-1α antibody. Immunohistochemical analysis of OSCC tissues was performed. Results; The production of IL-1α from Sq-1979-1 cells was specifically enhanced in lower serum (0.5% or 1.0%FBS), or under hypoxia (1.0% oxygen) compared to that in the standard growth condition supplemented with 10% FBS under normoxia. Compared to standard CM, the CMs prepared in 0.5% and 1.0% FBS, or under hypoxia significantly reduced IFN-γ production from stimulated spleen cells co-cultured with 10T1/2 cells. These functions of the CMs completely abolished by anti- IL-1α antibody. Expression of IL-1α in OSCC tissues was prominent in the midst of carcinomatous cellular lesion, or nearby necrotic lesion, where the supply deficiency could be occurred. Conclusions; The promotion of immunosuppressive activity of mesenchymal cells by Sq-1979-1 cells was specifically augmented in lower serum and hypoxic conditions, mediated by increased IL-1α production. Citation Format: Nobuo Kondoh, Akihiro Matsunami, Masako Mizuno-Kamiya, Naoki Umemura, Eiji Takayama, Harumi Kawaki, Kyouhei Ueno, Hiroe Morimoto-Ito, Yasunori Muramatsu, Shinichiro Sumitomo. Augmented secretion of IL-1α from mouse oral squamous cell carcinoma (OSCC) cells caused by the serum deprivation and hypoxia promotes immune-suppressive activity of mesenchymal stromal cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1910.

MKP-1 is required to limit myeloid-cell mediated oral squamous cell carcinoma progression and regional extension

Mitogen-activated protein kinases (MAPKs) require MAPK phosphatases (MKPs) for deactivation of MAPK intracellular signaling. MKP-1 (encoded by Dusp1) is a key negative regulator of MAPKs and prior reports have indicated that MKP-1 regulates oral cancer-associated inflammation and leukocyte infiltration. ObjectiveTo determine the significance of myeloid-based expression of MKP-1 in oral cancer. MethodsThe Cancer Genome Atlas (TCGA) was used to address DUSP1 expression in oral squamous cell carcinoma (OSCC). Syngeneic and carcinogen-induced mouse models using global and myeloid-specific Dusp-1 deficient mice with immunophenotypic, histologic, and transcriptomic analyses and in vitro migration assays. ResultsData from TCGA indicates the DUSP1 expression is inversely related to oral cancer burden and nodal involvement. Using murine models of OSCC, the role of MKP-1 signaling in tumor associated macrophages (TAMs) was assessed. Dusp1-deficient mice had increased tumor burden and TAM infiltrate with increased M2 macrophage polarization. Transcriptomic signatures of TAMs from Dusp1-deficent mice indicated a pro-metastatic phenotype as well as concomitant differences in myeloid-associated genes, cytokine/chemokine signaling, and Notch signaling consistent with tumor progression. In vitro and in vivo assays revealed mouse OSCC cells had a higher migration rate using TAM cell-free supernatant from Dusp1 deficiency mice compared to controls with enhanced regional cervical lymph node metastasis, respectively. To validate TAM studies using implantable mouse models, an OSCC progression model with conditional myeloid-specific Dusp-1 deficient mice demonstrated enhanced OSCC disease progression, characterized by advanced onset, histological stage, and tumor burden. ConclusionMyeloid-based Dusp1-deficiency increases OSCC burden and metastasis through alteration in TAM recruitment, gene profile, and polarity suggesting that MKP-1 could be a viable target to reprogram TAM to limit local/regional OSCC extension.

Augmented secretion of IL-1α from mouse oral squamous cell carcinoma (OSCC) vcells caused by serum deprivation and hypoxia promotes immune suppressive activity of mesenchymal stromal cells

ObjectivesWe have previously reported that mouse oral squamous carcinoma (OSCC), Sq-1979-1, produces interleukin (IL)-1α, which specifically enhances the immunosuppressive activity of co-cultured mesenchymal stromal 10T1/2 cells. This study assessed the conditions promoting the production of IL-1α in Sq-1979-1 cells, which could further enhance the immunosuppressive function of 10T1/2 cells, and evaluated its expression in OSCC tissues. MethodsThe expression of IL-1α was examined by RT-PCR, western blotting, and enzyme-linked immune sorbent assay (ELISA). The interferon (IFN)- γ-producing capability of anti-CD3 antibody-stimulated mouse spleen cells co-cultured with 10T1/2 cells and conditioned medium (CM) from Sq-1979-1 cells was examined by ELISA. The function of IL-1α was examined using an anti-IL1α antibody. Immunohistochemical analysis of the OSCC tissues was performed. ResultsThe production of IL-1α from Sq-1979-1 cells was synergistically enhanced in lower serum (0.5% or 1.0% FBS) at the transcriptional level, and under hypoxia (1.0% oxygen) at the release level compared to that in the control medium supplemented with 10% FBS under normoxia. The IFN-γ-producing capability of stimulated spleen cells co-cultured with 10T1/2 cells was significantly reduced in the CMs prepared with the lower serum or under hypoxia. These functions of CMs were completely abolished by the anti-IL-1α antibody. The expression of IL-1α in OSCC tissues was prominent in the midst of a carcinomatous cellular lesion or a nearby necrotic lesion, where a supply deficiency could occur. Conclusions: IL-1α production by Sq-1979-1 cells was synergistically augmented under low serum and hypoxic conditions, which could promote the immunosuppressive activity of mesenchymal cells.

NOVEL NEUROPILIN 2-TARGETING BIOLOGIC FOR THE TREATMENT OF ORAL SQUAMOUS CELL CARCINOMA

<h3>Background</h3> Oral squamous cell carcinoma (OSCC) is the sixth most common cancer and a leading cause of cancer mortality in some countries. Recently, our laboratory identified the neuropilin-2 (NRP2) transmembrane glycoprotein as a novel target in OSCC and its associated vasculature in patient biopsies and mouse carcinogenesis models. NRP2 is a co-receptor for the angiogenic factor vascular endothelial growth factor (VEGF)-A with VEGFR2 and the lymphangiogenic factors VEGF-C/D with VEGFR3 and is abundantly expressed in growing endothelium. NRP2 also has an alternative ligand called semaphorin-3F (SEMA3F) that competes with VEGF proteins and inhibits angiogenesis, lymphangiogenesis, and cell motility. <h3>Objective</h3> To examine the effects of exogenous SEMA3F protein on tumorigenicity, neovascularization, and metastasis in OSCC models. <h3>Methods and Results</h3> We engineered and purified a biologically active, mutant (noncleavable) SEMA3F protein. In vitro, SEMA3F inhibited the migration and invasion of Nrp2-expressing mouse OSCC cells (WT3) originally isolated from 4NQO-induced tumors. In vivo, WT3 luciferase-labeled cells were injected into the tongues of C57BI/6 mice or human OSCC (HSC3) cells were injected into the tongues of immunodeficient (athymic) mice. Slow-release osmotic pumps containing SEMA3F or vehicle were implanted into mice with established mouse or human OSCC tumors. Systemic SEMA3F protein induced complete regression, as confirmed by bioluminescence imaging and histologic examination, of OSCC in the majority of immunocompetent mice in just 14 days. In immunodeficient mice, systemic SEMA3F treatment inhibited human OSCC tumorigenicity and reduced tumor-associated lymphangiogenesis compared to controls. <h3>Conclusions</h3> SEMA3F is a promising antitumor therapy specifically targeting the NRP2 axis in carcinoma cells and stromal cells.

The aryl hydrocarbon receptor suppresses immunity to oral squamous cell carcinoma through immune checkpoint regulation

Immune checkpoint inhibitors represent some of the most important cancer treatments developed in the last 20 y. However, existing immunotherapy approaches benefit only a minority of patients. Here, we provide evidence that the aryl hydrocarbon receptor (AhR) is a central player in the regulation of multiple immune checkpoints in oral squamous cell carcinoma (OSCC). Orthotopic transplant of mouse OSCC cells from which the AhR has been deleted (MOC1AhR-KO) results, within 1 wk, in the growth of small tumors that are then completely rejected within 2 wk, concomitant with an increase in activated T cells in tumor-draining lymph nodes (tdLNs) and T cell signaling within the tumor. By 2 wk, AhR+ control cells (MOC1Cas9), but not MOC1AhR-KO cells up-regulate exhaustion pathways in the tumor-infiltrating T cells and expression of checkpoint molecules on CD4+ T cells (PD-1, CTLA4, Lag3, and CD39) and macrophages, dendritic cells, and Ly6G+ myeloid cells (PD-L1 and CD39) in tdLNs. Notably, MOC1AhR-KO cell transplant renders mice 100% immune to later challenge with wild-type tumors. Analysis of altered signaling pathways within MOC1AhR-KO cells shows that the AhR controls baseline and IFNγ-induced Ido and PD-L1 expression, the latter of which occurs through direct transcriptional control. These observations 1) confirm the importance of malignant cell AhR in suppression of tumor immunity, 2) demonstrate the involvement of the AhR in IFNγ control of PD-L1 and IDO expression in the cancer context, and 3) suggest that the AhR is a viable target for modulation of multiple immune checkpoints.

Epithelial Mutant p53 Promotes Resistance to Anti-PD-1-Mediated Oral Cancer Immunoprevention in Carcinogen-Induced Mouse Models.

Simple SummaryImmune checkpoint blockade with anti-PD-1 antibodies blocks the development of oral squamous cell carcinomas (OSCCs) in preclinical models. Understanding whether the genetic alterations that accumulate during oral cancer development affect the response to PD-1 inhibitors is critical to identify patients who may benefit from immunoprevention interventions. Using genetically engineered mouse models that develop carcinogen-induced oral tumors that differ on the mutational status of the p53 gene, we demonstrated that expression of gain-of-function mutant p53 in the epithelial cells of the oral lesions promotes resistance to the immunopreventive effects of anti-PD-1. These novel findings could guide patient-specific strategies for oral cancer immunoprevention based on p53 profiling.Oral squamous cell carcinoma (OSCC) develops through the multistep malignant progression of squamous epithelium. This process can be prevented by PD-1 blockade in a mouse model for oral carcinogenesis. OSCCs exhibit a high incidence of p53 mutations that confer oncogenic gain-of-function (GOF) activities that promote resistance to standard therapies and poor clinical outcomes. To determine whether epithelial p53 mutations modulate anti-PD-1-mediated oral cancer immunoprevention, we generated mouse models for oral carcinogenesis by exposing mice carrying epithelial-specific p53 mutations to the carcinogen 4NQO. Consistent with the oncogenic functions of mutant p53, mice with OSCCs expressing the p53R172H GOF mutation developed higher metastasis rates than mice with loss-of-function (LOF) p53 deletion or with wild-type p53. Throughout oral cancer progression, pre-invasive and invasive lesions showed a gradual increase in T-cell infiltration, recruitment of immunosuppressive regulatory T-cells (Tregs), and induction of PD-1/PD-L1 immune checkpoint proteins. Notably, while PD-1 blockade prevented the development of OSCCs in mice with wild-type p53 or p53 deletion, GOF p53R172H abrogated the immunopreventive effects of anti-PD-1, associated with upregulation of IL17 signaling and depletion of exhausted CD8 cells in the microenvironment of the p53R172H tumors. These findings sustain a potential role for p53 profiling in personalized oral cancer immunoprevention.

Downregulation of ceramide synthase 1 promotes oral cancer through endoplasmic reticulum stress

C18 ceramide plays an important role in the occurrence and development of oral squamous cell carcinoma. However, the function of ceramide synthase 1, a key enzyme in C18 ceramide synthesis, in oral squamous cell carcinoma is still unclear. The aim of our study was to investigate the relationship between ceramide synthase 1 and oral cancer. In this study, we found that the expression of ceramide synthase 1 was downregulated in oral cancer tissues and cell lines. In a mouse oral squamous cell carcinoma model induced by 4-nitroquinolin-1-oxide, ceramide synthase 1 knockout was associated with the severity of oral malignant transformation. Immunohistochemical studies showed significant upregulation of PCNA, MMP2, MMP9, and BCL2 expression and downregulation of BAX expression in the pathological hyperplastic area. In addition, ceramide synthase 1 knockdown promoted cell proliferation, migration, and invasion in vitro. Overexpression of CERS1 obtained the opposite effect. Ceramide synthase 1 knockdown caused endoplasmic reticulum stress and induced the VEGFA upregulation. Activating transcription factor 4 is responsible for ceramide synthase 1 knockdown caused VEGFA transcriptional upregulation. In addition, mild endoplasmic reticulum stress caused by ceramide synthase 1 knockdown could induce cisplatin resistance. Taken together, our study suggests that ceramide synthase 1 is downregulated in oral cancer and promotes the aggressiveness of oral squamous cell carcinoma and chemotherapeutic drug resistance.

The anti-tumor effects of evodiamine on oral squamous cell carcinoma (OSCC) through regulating advanced glycation end products (AGE) / receptor for advanced glycation end products (RAGE) pathway

ABSTRACT Evodiamine (EVO) is emerging as a novel anti-tumor drug, which is involved in the inhibition of cell proliferation and apoptosis. High-Mobility Group Box 1 (HMGB1)/RAGE is involved in invasive behavior of OSCC cells and angiogenesis. In this study, we evaluated the potential of EVO in OSCC in vitro and in vivo. We found that RAGE silencing suppressed HSC-4 cell proliferation and invasion, and tube formation of HUVEC. EVO showed marked inhibitory effects on the malignant behaviors of HSC-4 cells in a dose-dependent manner. Further experiments revealed that the RAGE overexpression was able to markedly block the effects of EVO on cell proliferation and invasion, and tube formation. By analyzing the expression of High-Mobility Group Box 1 (HMGB1) and RAGE in HSC-4 cells, the result showed that EVO slightly reduced HMBG1 levels and dramatically decreased RAGE levels, while RAGE overexpression did have no marked influences on HMBG1 levels. The anti-tumor effects of EVO were further confirmed in mouse oral squamous cell carcinoma xenograft models. Remarkable anti-tumor effects of EVO were also demonstrated, as presented by reduced tumor size and levels of HMBG1 and RAGE in tumor tissue of mouse oral squamous cell carcinoma xenograft models. The results demonstrated that EVO has a direct binding effect on HMGB1, but it may be involved in degrading the protein. More importantly, it can reduce the activity of RAGE pathway by affecting the binding between HMBG1 and RAGE. To conclude, EVO inhibited proliferation, invasion and angiogenesis of OSCC through affecting the downstream signal transduction system of AGE/RAGE by targeting RAGE.

Genomic landscape and clonal architecture of mouse oral squamous cell carcinomas dictate tumour ecology

To establish whether 4-nitroquinoline N-oxide-induced carcinogenesis mirrors the heterogeneity of human oral squamous cell carcinoma (OSCC), we have performed genomic analysis of mouse tongue lesions. The mutational signatures of human and mouse OSCC overlap extensively. Mutational burden is higher in moderate dysplasias and invasive SCCs than in hyperplasias and mild dysplasias, although mutations in p53, Notch1 and Fat1 occur in early lesions. Laminin-α3 mutations are associated with tumour invasiveness and Notch1 mutant tumours have an increased immune infiltrate. Computational modelling of clonal dynamics indicates that high genetic heterogeneity may be a feature of those mild dysplasias that are likely to progress to more aggressive tumours. These studies provide a foundation for exploring OSCC evolution, heterogeneity and progression.

Long non-coding RNA ELDR enhances oral cancer growth by promoting ILF3-cyclin E1 signaling.

Oral squamous cell carcinoma (OSCC) is the sixth most common cancer with a 5-year overall survival rate of 50%. Thus, there is a critical need to understand the disease process, and to identify improved therapeutic strategies. Previously, we found the long non-coding RNA (lncRNA) EGFR long non-coding downstream RNA (ELDR) induced in a mouse tongue cancer model; however, its functional role in human oral cancer remained unknown. Here, we show that ELDR is highly expressed in OSCC patient samples and in cell lines. Overexpression of ELDR in normal non-tumorigenic oral keratinocytes induces cell proliferation, colony formation, and PCNA expression. We also show that ELDR depletion reduces OSCC cell proliferation and PCNA expression. Proteomics data identifies the RNA binding protein ILF3 as an interacting partner of ELDR. We further show that the ELDR-ILF3 axis regulates Cyclin E1 expression and phosphorylation of the retinoblastoma (RB) protein. Intratumoral injection of ELDR-specific siRNA reduces OSCC and PDX tumor growth in mice. These findings provide molecular insight into the role of ELDR in oral cancer and demonstrate that targeting ELDR has promising therapeutic potential.

Preclinical Assessment of the Effectiveness of Magnetic Resonance Molecular Imaging of Extradomain-B Fibronectin for Detection and Characterization of Oral Cancer.

Oral squamous cell carcinoma (OSCC) has not seen a substantial improvement in patient survival despite therapeutic advances, making accurate detection and characterization of the disease a clinical priority. Here, we aim to demonstrate the effectiveness of magnetic resonance imaging (MRI) with the targeted MRI contrast agent MT218 specific to extradomain-B fibronectin (EDB-FN) in the tumor microenvironment for detection and characterization of aggressive OSCC tumors. EDB-FN expression was evaluated in human normal tongue and OSCC specimens with immunohistochemistry. Invasiveness of human CAL27, HSC3, and SCC4 OSCC cells was analyzed with spheroid formation and transwell assays. EDB-FN expression in the cells was analyzed with semiquantitative real-time PCR, western blotting, and a peptide binding study with confocal microscopy. Contrast-enhanced MRI with MT218 was performed on subcutaneous OSCC mouse models at a dose of 0.04mmol/kg, using gadoteridol (0.1mmol/kg) as a control. Strong EDB-FN expression was observed in human untreated primary and metastatic OSCC, reduced expression in treated OSCC, and little expression in normal tongue tissue. SCC4 and HSC3 cell lines demonstrated high invasive potential with high and moderate-EDB-FN expression, respectively, while CAL27 showed little invasive potential and low-EDB-FN expression. In T1-weighted MRI, MT218 produced differential contrast enhancement in the subcutaneous tumor models in correlation with EDB-FN expression in the cancer cells. Enhancement in the high-EDB-FN tumors was greater with MT218 at 0.04mmol/kg than gadoteridol at 0.1mmol/kg. The results suggest EDB-FN has strong potential as an imageable biomarker for aggressive OSCC. MRMI results demonstrate the effectiveness of MT218 and the potential for differential diagnostic imaging of oral cancer for improving the management of the disease.

P120-catenin suppresses proliferation and tumor growth of oral squamous cell carcinoma via inhibiting nuclear phospholipase C-γ1 signaling.

p120-catenin (p120) serves as a stabilizer of the calcium-dependent cadherin-catenin complex and loss of p120 expression has been observed in several types of human cancers. The p120-dependent E-cadherin-β-catenin complex has been shown to mediate calcium-induced keratinocyte differentiation via inducing activation of plasma membrane phospholipase C-γ1 (PLC-γ1). On the other hand, PLC-γ1 has been shown to interact with phosphatidylinositol 3-kinase enhancerin the nucleus and plays a critical role in epidermal growth factor-induced proliferation of oral squamous cell carcinoma (OSCC) cells. To determine whether p120 suppresses OSCC proliferation and tumor growth via inhibiting PLC-γ1, we examined effects of p120 knockdown or p120 and PLC-γ1 double knockdown on proliferation of cultured OSCC cells and tumor growth in xenograft OSCC in mice. The results showed that knockdown of p120 reduced levels of PLC-γ1 in the plasma membrane and increased levels of PLC-γ1 and its signaling in the nucleus in OSCC cells and OSCC cell proliferation as well as xenograft OSCC tumor growth. However, double knockdown of p120 and PLC-γ1 or knockdown of PLC-γ1 alone did not have any effect. Immunohistochemical analysis of OSCC tissue from patients showed a lower expression level of p120 and a higher expression level of PLC-γ1 compared withthat of adjacent noncancerous tissue. These data indicate that p120 suppresses OSCC cell proliferation and tumor growth by inhibiting signaling mediated by nuclear PLC-γ1.

Semaphorin 3F: A Novel Biologic Treatment for Oral Squamous Cell Carcinoma

Oral squamous cell carcinoma (OSCC) accounts for the majority of all oral malignancies. The overall survival rate for patients diagnosed with advanced‐stage OSCC (with metastasis) is only 39%, and the prognosis for these patients has not improved dramatically despite advances in chemotherapeutic, radiotherapeutic, and surgical protocols. As such, there remains an urgent need to develop new therapies for OSCC to improve outcome. Recently, our laboratory identified the Neuropilin‐2 (NRP2) transmembrane receptor as a novel target in OSCC and its associated vasculature in human patient biopsies, human OSCC xenografts, and mouse carcinogenesis models of OSCC. NRP2 is an ideal drug target as it is not expressed in the oral epithelium nor the normal vasculature after birth, hence systemic toxicities are not expected. However, NRP2 is dramatically upregulated on the cell surface in OSCC and its associated tumor endothelium. In embryonic endothelium, NRP2 acts as a co‐receptor with VEGFR2 for the angiogenic factor VEGF‐A and as a co‐receptor with VEGFR3 for the lymphangiogenic factors VEGF‐C/D to regulate the developmental processes of angiogenesis and lymphangiogenesis, respectively. Alternately, NRP2 can bind Semaphorin‐3F (SEMA3F), a secreted chemorepulsive and inhibitory protein, which mediates cellular guidance and vascular patterning. SEMA3F is an inhibitor of the VEGFA/C/D signaling axes as it competes with binding to NRP2 as well as directly signals through the NRP2/PlexinA1 complex to collapse the cellular cytoskeleton and inhibit motility. Our hypothesis is that SEMA3F may potently inhibit OSCC progression by targeting NRP2 in tumor cells and neovessels in the tumor microenvironment. Our in vitro results demonstrate that SEMA3F exogenous protein inhibits the migration, invasion, and proliferation of Nrp2‐expressing mouse OSCC cells isolated from 4NQO‐induced tumors. In vivo, systemic SEMA3F protein delivered continuously via slow‐release mini‐osmotic pumps induces regression, as confirmed by bioluminescence imaging and histologic examination, in established syngeneic luciferase‐labeled OSCC implanted in the tongue of C57BL/6 mice. In human OSCC xenografts implanted orthotopically in immunodeficient mice, systemic SEMA3F treatment inhibits tumorigenicity and reduces tumor‐associated lymphangiogenesis compared to control (vehicle) treatment. Taken together, these preclinical data suggest that SEMA3F may be a powerful anti‐angiogenic and anti‐tumor therapy specifically targeting the NRP2 axis in carcinoma cells and stromal cells in the tumor microenvironment.Support or Funding InformationNone

Neuropilin 2 Drives Tumor Lymphangiogenesis in Oral Squamous Cell Carcinoma

Oral squamous cell carcinoma (OSCC) is the sixth most common cancer worldwide. The majority (67%) of patients with this aggressive cancer already exhibit local or distant metastases at the time of initial diagnosis. Understanding the molecular mechanisms that regulate OSCC progression and metastasis is paramount to developing novel therapeutic strategies for this disease. Enhanced tumor‐associated lymphangiogenesis correlates with increased lymph node metastasis and adversely affects prognosis in OSCC. Neuropilin 2 (NRP2) is an endothelial cell membrane receptor that binds the lymphangiogenic factors, VEGF‐C and VEGF‐D, in complex with VEGFR3 to stimulate the sprouting of lymphatic vessels during developmental lymphangiogenesis. However, once vessels are properly guided and patterned, NRP2 expression is downregulated and not upregulated until times of need such as during inflammation‐, ischemia‐, or cancer‐related neovascularization. Our hypothesis is that NRP2 plays a role in OSCC progression and metastasis. Results from immunohistochemical staining demonstrate that normal human and mouse oral mucosa are devoid of NRP2 expression. Conversely, NRP2 is upregulated in the epithelium in late stages of oral dysplasia and in OSCC and its associated endothelium. Several human OSCC cell lines express NRP2 in vitro and in vivo when implanted orthotopically in the tongue of immunodeficient mice. In fact, NRP2 expression levels in the cancer cells correlate with the degree of tumor‐associated lymphatic vessel density within the tumors. OSCC mouse intralingual isografts show reduced tumor angiogenesis and lymphangiogenesis in adult syngeneic Nrp2‐deficient mice compared to Nrp2‐intact littermates. Taken together, our data suggest that NRP2 may be useful as a late‐stage marker of oral epithelial dysplasia and highlight the importance of the NRP2 axis in effective tumor neovascularization in OSCC. Our ongoing studies using the 4NQO carcinogenesis model in mutant mice lacking Nrp2 specifically in epithelial cells will further elucidate the role of NRP2 in OSCC tumorigenesis.

NDRG1 deficiency is associated with regional metastasis in oral cancer by inducing epithelial-mesenchymal transition.

Regional metastasis is the single most important prognostic factor in oral squamous cell carcinoma (OSCC). Abnormal expression of N-myc downstream-regulated genes (NDRGs) has been identified to occur in several tumor types and to predict poor prognosis. In OSCC, the clinical significance of deregulated NDRG expression has not been fully established. In this study, NDRG1 relevance was assessed at gene and protein levels in 100 OSCC patients followed up by at least 10 years. Survival outcome was analyzed using a multivariable analysis. Tumor progression and metastasis was investigated in preclinical model using oral cancer cell lines (HSC3 and SCC25) treated with epidermal growth factor (EGF) and orthotopic mouse model of metastatic murine OSCC (AT84). We identified NDRG1 expression levels to be significantly lower in patients with metastatic tumors compared with patients with local disease only (P = 0.001). NDRG1 expression was associated with MMP-2, -9, -10 (P = 0.022, P = 0.002, P = 0.042, respectively) and BCL2 (P = 0.035). NDRG1 lower expression was able to predict recurrence and metastasis (log-rank test, P = 0.001). In multivariable analysis, the expression of NDRG1 was an independent prognostic factor (Cox regression, P = 0.013). In invasive OSCC cells, NDRG1 expression is diminished in response to EGF and this was associated with a potent induction of epithelial-mesenchymal transition phenotype. This result was further confirmed in an orthotopic OSCC mouse model. Together, this data support that NDRG1 downregulation is a potential predictor of metastasis and approaches aimed at NDRG1 signaling rescue can serve as potential therapeutic strategy to prevent oral cancer progression to metastasis.

Mouse Tumor-Bearing Models as Preclinical Study Platforms for Oral Squamous Cell Carcinoma.

Preclinical animal models of oral squamous cell carcinoma (OSCC) have been extensively studied in recent years. Investigating the pathogenesis and potential therapeutic strategies of OSCC is required to further progress in this field, and a suitable research animal model that reflects the intricacies of cancer biology is crucial. Of the animal models established for the study of cancers, mouse tumor-bearing models are among the most popular and widely deployed for their high fertility, low cost, and molecular and physiological similarity to humans, as well as the ease of rearing experimental mice. Currently, the different methods of establishing OSCC mouse models can be divided into three categories: chemical carcinogen-induced, transplanted and genetically engineered mouse models. Each of these methods has unique advantages and limitations, and the appropriate application of these techniques in OSCC research deserves our attention. Therefore, this review comprehensively investigates and summarizes the tumorigenesis mechanisms, characteristics, establishment methods, and current applications of OSCC mouse models in published papers. The objective of this review is to provide foundations and considerations for choosing suitable model establishment methods to study the relevant pathogenesis, early diagnosis, and clinical treatment of OSCC.

ERK Activation Modulates Cancer Stemness and Motility of a Novel Mouse Oral Squamous Cell Carcinoma Cell Line.

We established the NHRI-HN1 cell line from a mouse tongue tumor induced by 4-nitroquinoline 1-oxide (4-NQO)/arecoline, with further selection for cell stemness via in vitro sphere culture, to evaluate potential immunotherapies for oral squamous cell carcinoma (OSCC) in East and Southeast Asia. In vivo and in vitro phenotypic characterization, including tumor growth, immune modulator administration, gene expression, morphology, migration, invasion, and sphere formation assays, were conducted. NHRI-HN1 cells are capable of generating orthotopic tumors in syngeneic mice. Interestingly, immune stimulation via CpG oligodeoxynucleotide (CpG-ODN) dramatically reduced the tumor growth in NHRI-HN1 cell-injected syngeneic mice. The pathways enriched in genes that were differentially expressed in NHRI-HN1 cells when compared to non-tumorigenic cells were similar to those that were identified when comparing human OSCC and non-tumorous tissues. NHRI-HN1 cells have characteristics of epithelial–mesenchymal transition (EMT), including enhanced migration and invasion. NHRI-HN1 cells showed aggressive cell growth and sphere formation. The blockage of extracellular signal-regulated kinase (ERK) activation suppressed cell migration and reduced stemness characteristics in NHRI-HN1 cells, similar to human OSCC cell lines. Our data suggest that NHRI-HN1 cells, showing tumorigenic characteristics of EMT, cancer stemness, and ERK activation, are sufficient in modeling human OSCC and also competent for use in investigating oral cancer immunotherapies.