Carcinogen 4-nitroquinoline 1-oxide Research Articles

Autophagy Contributes to Homeostasis in Esophageal Epithelium Where High Autophagic Vesicle Level Marks Basal Cells With Limited Proliferation and Enhanced Self-Renewal Potential

Background & AimsAutophagy plays roles in esophageal pathologies both benign and malignant. Here, we aim to define the role of autophagy in esophageal epithelial homeostasis. MethodsWe generated tamoxifen-inducible, squamous epithelial-specific Atg7 (autophagy related 7) conditional knockout mice to evaluate effects on esophageal homeostasis and response to the carcinogen 4-nitroquinoline 1-oxide (4NQO) using histological and biochemical analyses. We FACS sorted esophageal basal cells based upon fluorescence of the autophagic vesicle (AV)-identifying dye Cyto-ID, then subjected these cells to transmission electron microscopy, image flow cytometry, 3D organoid assays, RNA-Sequencing (RNA-Seq), and cell cycle analysis. 3D organoids were subjected to passaging, single cell (sc) RNA-Seq, cell cycle analysis, and immunostaining. ResultsGenetic autophagy inhibition in squamous epithelium resulted in increased proliferation of esophageal basal cells under homeostatic conditions and also was associated with significant weight loss in mice treated with 4NQO that further displayed perturbed epithelial tissue architecture. Esophageal basal cells with high AV level (Cyto-IDHigh) displayed limited organoid formation capability upon initial plating but passaged more efficiently than their counterparts with low AV level (Cyto-IDLow). RNA-Seq suggested increased autophagy in Cyto-IDHigh esophageal basal cells along with decreased cell cycle progression, the latter of which was confirmed by cell cycle analysis. scRNA-Seq of 3D organoids generated by Cyto-IDLow and Cyto-IDHigh cells identified expansion of 3 cell populations, and enrichment of G2/M-associated genes in the Cyto-IDHigh group. Ki67 expression was also increased in organoids generated by Cyto-IDHigh cells, including in basal cells localized beyond the outermost cell layer. ConclusionsAutophagy contributes to maintenance of the esophageal proliferation-differentiation gradient. Esophageal basal cells with high AV level exhibit limited proliferation and generate 3D organoids with enhanced self-renewal capacity.

Spermidine Suppresses Oral Carcinogenesis through Autophagy Induction, DNA Damage Repair, and Oxidative Stress Reduction

Autophagy has been proposed to play a dual role in cancer-as a tumor suppressor in early stages and oncogenicin late stages of tumorigenesis. This study investigated the role of autophagy in oral carcinogenesis using the model of oral squamous cell carcinoma (OSCC) induced by carcinogen 4-nitroquinoline 1-oxide (4NQO), mimicking molecular and histopathologic aspects of human OSCC. The induction of autophagy by spermidine (SPD) treatment reduced the severity of lesions and the incidence of OSCC in mice exposed to 4NQO. On the other hand, autophagy inhibition by chloroquine treatment had no protection. The comet assay indicated that SPD reduced 4NQO-induced DNA damage, likely related to the activation of DNA repair and the decrease of reactive oxygen species. As sphingolipid alterations have been reported in OSCC, sphingolipids in the tongue and plasma of animals were analyzed and plasma C16 ceramide levels were shown to increase proportionally to lesion severity, indicating its potential as a biomarker. Mice exposed to 4NQO plus SPD had lower levels of C16 ceramide than the 4NQO group, which indicated SPD's ability to prevent the 4NQO-induced carcinogenesis. Together, these data indicate that activation of autophagy has a tumor suppressor role during the early stages of oral carcinogenesis. Because of its ability to induce autophagy accompanied by reduced oxidative stress and DNA damage, SPD may have a protective action against chemically induced oral cancer.

A method for scoring 4-nitroquinoline 1-oxide-induced murine esophageal squamous neoplasia.

A mouse model for esophageal squamous cell carcinoma (ESCC) is induced by oral administration of the carcinogen 4-nitroquinoline 1-oxide (4-NQO). There is not an objective method for determining histopathologic severity of disease in this model. We aim to create a clearly defined and easily applied scoring system that can quantify the severity of 4-NQO-induced murine ESCC. Fifteen wild-type C57BL/6J mice were treated with 4-NQO for 8 (n = 8) or 16 (n = 7) weeks, while the rest (n = 9) were treated with vehicle, as 8 weeks of 4-NQO typically results in dysplasia and 16 weeks in carcinoma. We identified histologic abnormalities of the esophagus in this model and developed metrics to grade severity of dysplasia, papillomas, and invasion. Scores were then calculated using quantitative digitized image analysis for measuring depth and extent of each feature within the entire sample. Each feature was also assigned a weight based on its relation to cancer severity. Histology scores were significantly different in the three groups, suggesting that this method can discriminate dysplasia from carcinoma. This model can be applied to any mouse treated with 4-NQO.

Exposure to Eosinophilic Esophagitis Limits Esophageal Carcinogenesis in a Murine Model

IntroductionEpidemiological data suggest that eosinophilic esophagitis (EoE) patients do not develop esophageal cancer. Here, we paired murine models of EoE and esophageal squamous cell carcinoma (ESCC) to perform the first functional interrogation of the relationship between EoE and esophageal cancer.MethodsC57B6 mice were treated with MC903/Ovalbumin (OVA) to induce EoE and the esophageal carcinogen 4‐nitroquinoline 1‐oxide (4NQO) to induce ESCC according to the experimental scheme shown in Fig 1A. Esophagi of mice treated with vehicle controls (n=6), MC903/OVA alone (n=4), 4NQO alone (n=10), and MC903 in combination with 4NQO (n=5) were dissected. Tumor frequency was recorded and tumor load (tumor size x tumor number) was determined under a dissecting microscope. H&E stained tissues were evaluated for esophageal lesions. CD45‐positive immune cells were isolated from the spleens of mice with EoE or naïve controls then co‐cultured with the murine‐derived esophageal cell line AKR. After 24‐72 hours of co‐culture, live/dead dye was used to assess AKR viability.Results10/10 mice treated with 4NQO alone developed ESCC as expected in this robust tumor model. In mice co‐treated with MC903/OVA and 4NQO, ESCC was detected in 4/5 mice with a significant decrease in tumor load as compared to mice treated with 4NQO alone (Fig 1B, C). 6/6 mice treated with MC903/OVA alone and 4/4 mice treated with propylene glycol (vehicle for 4NQO) failed to display tumors (Fig 1C). We further evaluated esophageal epithelium of 4NQO‐treated animals to characterize distribution of ESCC lesions. While squamous dysplasia (pre‐malignant), early ESCC and late ESCC were present in all tumor‐bearing animals; there was a decrease in early ESCC and a lack of late ESCC lesions in 4NQO‐treated mice that were exposed to MC903/OVA‐induced EoE (Fig 1D). Moreover, the total percentage of esophageal epithelium occupied by neoplastic lesions was significantly diminished in mice co‐treated with 4NQO and MC903/OVA (Fig 1E). To investigate how EoE limits esophageal carcinogenesis, we exposed AKR esophageal tumor cells to CD45‐positive cells isolated from spleens of strain‐matched donor mice with or without EoE (Fig 2A). We found a robust induction of cell death in tumor cells exposed to immune cells from EoE donor mice compared to those exposed to immune cells from vehicle‐treated controls at 48 hours (n=3, p<0.05) (Fig 2B, C).ConclusionsThese data indicate that exposure to EoE activates an anti‐tumoral immune response that limits esophageal carcinogenesis. These studies have the potential for significant implications related to the clinical care of patients affected by EoE and ESCC.

Oral cancer induced TRPV1 sensitization is mediated by PAR2 signaling in primary afferent neurons innervating the cancer microenvironment

Oral cancer patients report sensitivity to spicy foods and liquids. The mechanism responsible for chemosensitivity induced by oral cancer is not known. We simulate oral cancer-induced chemosensitivity in a xenograft oral cancer mouse model using two-bottle choice drinking and conditioned place aversion assays. An anatomic basis of chemosensitivity is shown in increased expression of TRPV1 in anatomically relevant trigeminal ganglion (TG) neurons in both the xenograft and a carcinogen (4-nitroquinoline 1-oxide)-induced oral cancer mouse models. The percent of retrograde labeled TG neurons that respond to TRPV1 agonist, capsaicin, is increased along with the magnitude of response as measured by calcium influx, in neurons from the cancer models. To address the possible mechanism of TRPV1 sensitivity in tongue afferents, we study the role of PAR2, which can sensitize the TRPV1 channel. We show co-expression of TRPV1 and PAR2 on tongue afferents and using a conditioned place aversion assay, demonstrate that PAR2 mediates oral cancer-induced, TRPV1-evoked sensitivity in an oral cancer mouse model. The findings provide insight into oral cancer-mediated chemosensitivity.

Generation and Application of Inducible Chimeric RNA ASTN2-PAPPAas Knockin Mouse Model.

Chimeric RNAs (chiRNAs) play many previously unrecognized roles in different diseases including cancer. They can not only be used as biomarkers for diagnosis and prognosis of various diseases but also serve as potential therapeutic targets. In order to better understand the roles of chiRNAs in pathogenesis, we inserted human sequences into mouse genome and established a knockin mouse model of the tamoxifen-inducible expression of ASTN2-PAPPA antisense chimeric RNA (A-PaschiRNA). Mice carrying the A-PaschiRNA knockin gene do not display any apparent abnormalities in growth, fertility, histological, hematopoietic, and biochemical indices. Using this model, we dissected the role of A-PaschiRNA in chemical carcinogen 4-nitroquinoline 1-oxide (4NQO)-induced carcinogenesis of esophageal squamous cell carcinoma (ESCC). To our knowledge, we are the first to generate a chiRNA knockin mouse model using the Cre-loxP system. The model could be used to explore the roles of chiRNA in pathogenesis and potential targeted therapies.

Abstract 1752: Exploring the relation of eosinophilic esophagitis (EoE) with esophageal cancer

Abstract A negative correlation between allergic inflammation and cancer risk has been identified in a variety of organs via population-based studies; however, functional investigations are necessary to define the relationship between allergy and cancer and determine the feasibility of therapeutic approaches leveraging allergic inflammation for cancer prevention and therapy. Esophageal cancer is amongst the most aggressive forms of human cancer. Reflux esophagitis is a primary risk factor for esophageal adenocarcinoma. Eosinophilic esophagitis (EoE) represents a distinct, food allergen-mediated type of esophagitis characterized by esophageal eosinophilia. In contrast to patients with reflux esophagitis, epidemiological data indicates that EoE patients fail to develop esophageal cancer despite the presence of chronic esophageal inflammation. To investigate the functional relationship between EoE and esophageal cancer, we have paired the food allergen-driven MC903/Ovalbumin (OVA) model of EoE with esophageal squamous cell carcinoma induced by the carcinogen 4-nitroquinoline 1-oxide (4NQO). 10/10 mice treated with 4NQOalone developed ESCC as expected in this robust carcinogen-driven tumor model. In mice co-treated with MC903/OVA and 4NQO, esophageal tumors were detected in 4/5 mice with a significant decrease in tumor load as compared to mice treated with 4NQO alone. Notably, 6/6 mice treated withMC903/OVA alone and 4/4 mice treated with propylene glycol (vehicle for 4NQO) failed to display tumors. To examine the possibility that EoE may induce an anti-tumor inflammatory milieu, we exposed primary esophageal tumor cells to CD45positive immune cells isolated from the peripheral blood of strain- matched donor mice with or without food allergen-mediated EoE. These experiments revealed a robust induction of apoptotic cell death in tumor cells exposed to immune cells from EoE donor mice as compared to those exposed to immune cells from naïve controls. Finally, in order to investigate how EoE and cancer may differentially impact esophageal biology, single cell RNA-sequencing was performed and further identified differences in the esophageal epithelial and immune cell compartments in mice with EoE as compared to those with cancer. The innovative approaches employed here, including combining mouse models of EoE and esophageal cancer capability, establish a robust experimental platform to define the relationship between EoE and esophageal cancer which will be further used for future functional investigations to define the precise cellular and molecular mechanisms through which EoE inflammation limits carcinogenesis. As allergic disorders promote systemic inflammation, findings from the current study may be applicable to cancers occurring in organs other than the esophagus. Citation Format: Mohammad Faujul Kabir, Adam Karami, Anbin Mu, Kelly A. Whelan. Exploring the relation of eosinophilic esophagitis (EoE) with esophageal cancer [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 1752.

Simultaneous Expression of Th1- and Treg-Associated Chemokine Genes and CD4+, CD8+, and Foxp3+ Cells in the Premalignant Lesions of 4NQO-Induced Mouse Tongue Tumorigenesis.

Simple SummaryOral squamous cell carcinoma (OSCC), the most common oral malignancy, severely impacts patient quality of life because of oro-facial destruction. OSCC is preceded by oral premalignant lesions (OPLs). Moreover, lower T cell infiltration in OPLs is associated with OSCC, suggesting that T cell-mediated adaptive immunity protects against malignant transformation. In this study, we used the carcinogen 4NQO, which mimics tobacco-related carcinogenesis, in a mouse model to examine the gene expression kinetics of chemokines/cytokines during OPL and OSCC development. Our results demonstrate that both Th1- and Treg-associated chemokines were simultaneously expressed in 4NQO-induced OPL, with their expression correlating with the infiltration of CD8+ and Foxp3+ cells, respectively. These results indicate that antitumor immune responses and immunosuppression are simultaneously initiated during OLP development.Chemokines and cytokines in the tumor microenvironment influence immune cell infiltration and activation. To elucidate their role in immune cell recruitment during oral cancer development, we generated a mouse tongue cancer model using the carcinogen 4-nitroquinoline 1-oxide (4NQO) and investigated the carcinogenetic process and chemokine/cytokine gene expression kinetics in the mouse tongue. C57/BL6 mice were administered 4NQO in drinking water, after which tongues were dissected at 16 and 28 weeks and subjected to analysis using the RT2 Profiler PCR Array, qRT-PCR, and pathologic and immunohistochemical analyses. We found that Th1-associated chemokine/cytokine (Cxcl9, Cxcl10, Ccl5, and Ifng) and Treg-associated chemokine/cytokine (Ccl17, Ccl22, and Il10) mRNA levels were simultaneously increased in premalignant lesions of 4NQO-treated mice at 16 weeks. Additionally, although levels of Gata3, a Th2 marker, were not upregulated, those of Cxcr3, Ccr4, and Foxp3 were upregulated in the tongue tissue. Furthermore, immunohistochemical analysis confirmed the infiltration of CD4+, CD8+, and Foxp3+ cells in the tongue tissue of 4NQO-treated mice, as well as significant correlations between Th1- or Treg-associated chemokine/cytokine mRNA expression and T cell infiltration. These results indicate that CD4+, CD8+, and Foxp3+ cells were simultaneously recruited through the expression of Th1- and Treg-associated chemokines in premalignant lesions of 4NQO-induced mouse tongue tissue.

Abstract PO048: CD40/anti-PD-1 sequential immunotherapy outperforms multiple immunotherapy combinations in an oral cancer prevention mouse model

Abstract PD-1 checkpoint inhibitors pembrolizumab and nivolumab are approved for the treatment of recurrent or metastatic oral squamous cell carcinoma (OSCC). Previously we have demonstrated that PD-1/PD-L1 pathway blockade can reduce the progression of oral premalignant lesions (OPL) to carcinoma in the 4-NQO carcinogen-induced murine model of OSCC. Furthermore, pembrolizumab is currently being evaluated for prevention of OSCC in patients with high risk OPLs in the Immune Prevention of Oral Cancer clinical trial (NCT02882282). Building on these earlier studies, here we have conducted pre-clinical evaluation of other possible targets including CD40, OX40, and 4-1BB for the immunoprevention of OSCC, and determined the impact of combined or sequential treatment with PD-1/PD-L1 pathway blockade. We treated C57BL/6 mice with the carcinogen 4-nitroquinoline 1-oxide for 8 weeks in drinking water. Eight weeks after discontinuing 4-NQO, mice were treated with either IgG (n: 20), used as control group, anti-PD-1 monotherapy, concomitant anti-PD-1+OX4, anti-PD-1+CD40, anti-PD-1+4-1BB, or OX40, CD40, and 4-1BB followed by anti-PD-1 therapy (n:10 treatment groups). Mice were sacrificed 8 weeks after treatment. We assessed serial H&E-stained cross sections of tongues harvested at the same time point for total OSCC area. Tumor draining lymph nodes (TDLN) lymphocytes were analyzed by flow cytometry. Anova and Mann-Whitney test were used for statistical comparison. Treatment of mice harboring OPLs with CD40 followed by anti-PD-1 immunotherapy resulted in a decrease in the median total OSCC area when compared with our IgG control group (p= 0.034). Treatment with anti-PD-1 monotherapy showed a trend towards reducing progression of OPL to OSCC. Concomitant therapy with CD40 and anti-PD-1 along with the other combination immunotherapy regimens did not demonstrate any significant reduction in the progression of OPL to OSCC. Treatment with sequential CD40+anti-PD-1 increased both the CD8+PD-1+ lymphocyte population and the MFI values for PD-1 in the CD4+Foxp3- population in TDLN. When the TDLN of both concomitant and sequential 4-1BB+anti-PD-1 treatment groups were analyzed, a decrease in the CD4+PD-L1+ population, and an increase in both CD8+PD-1+ population and CD8/CD4+PD-L1+ ratio was found, although this change in the immune milieu was not associated to a reduction of OPL progression to OSCC. Targeting CD40 and PD-1 pathways sequentially, but not concomitantly, during the OPL stage led to a reduction in the progression of OPL to OSCC. The TDLN analysis of this treatment group revealed an increase in both CD8+PD-1+ lymphocytes and MFI values for PD-1 in CD4+Foxp3- lymphocytes. No other treatment group was able to significantly reduce progression of OPL to OSCC or showed a similar modulation of the lymphocyte population. These results support further investigation on how sequential CD40+anti-PD-1 immunotherapy hinder tumor immune evasion, making it a potential regimen for prevention of OSCC. Citation Format: Jose A. Monteiro de Oliveira Novaes, Alissa Poteete, Marlese Pisegna, William N. William, John V. Heymach. CD40/anti-PD-1 sequential immunotherapy outperforms multiple immunotherapy combinations in an oral cancer prevention mouse model [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO048.

Following the adverse outcome pathway from micronucleus to cancer using H2B-eGFP transgenic healthy stem cells

In vitro assessment of genotoxicity as an early warning tool for carcinogenicity mainly relies on recording cytogenetic damages (micronuclei, nucleoplasmic bridges) in tumour-derived mammalian cell lines like V79 or CHO. The forecasting power of the corresponding standardised test is based on epidemiological evidence between micronuclei frequencies and cancer incidence. As an alternative to destructive staining of nuclear structures a fish stem cell line transgenic for a fusion protein of histone 2B (H2B) and enhanced green fluorescent protein (eGFP) was established. The cells are derived from koi carp brain (KCB) and distinguish from mammalian culturable cells by non-tumour-driven self-renewal. This technology enables the analysis of genotoxic- and malign downstream effects in situ in a combined approach. In proof-of concept-experiments, we used known carcinogens (4-Nitroquinoline 1-oxide, colchicine, diethylstilbestrol, ethyl methanesulfonate) and observed a significant increase in micronuclei (MNi) frequencies in a dose-dependent manner. The concentration ranges for MNi induction were comparable to human/mammalian cells (i.e. VH-16, CHL and HepG2). Cannabidiol caused the same specific cytogenetic damage pattern as observed in human cells, in particular nucleoplasmic bridges. Metabolic activation of aflatoxin B1 and cyclophosphamide could be demonstrated by pre-incubation of the test compounds using either conventional rat derived S9 mix as well as an in vitro generated biotechnological alternative product ewoS9R. The presented high throughput live H2B-eGFP imaging technology using non-transformed stem cells opens new perspectives in the field of in vitro toxicology. The technology offers experimental access to investigate the effects of carcinogens on cell cycle control, gene expression pattern and motility in the course of malign transformation. The new technology enables the definition of Adverse Outcome Pathways leading to malign cell transformation and contributes to the replacement of animal testing. Summary: Complementation of genotoxicity testing by addressing initiating events leading to malign transformation is suggested. A vertebrate cell model showing "healthy" stemness is recommended, in contrast to malign transformed cells used in toxicology/oncocology.

Cytoprotective effects of (E)-N-(2-(3, 5-dimethoxystyryl) phenyl) furan-2-carboxamide (BK3C231) against 4-nitroquinoline 1-oxide-induced damage in CCD-18Co human colon fibroblast cells.

Stilbenes are a group of chemicals characterized with the presence of 1,2-diphenylethylene. Previously, our group has demonstrated that synthesized (E)-N-(2-(3, 5-dimethoxystyryl) phenyl) furan-2-carboxamide (BK3C231) possesses potential chemopreventive activity specifically inducing NAD(P)H:quinone oxidoreductase 1 (NQO1) protein expression and activity. In this study, the cytoprotective effects of BK3C231 on cellular DNA and mitochondria were investigated in normal human colon fibroblast, CCD-18Co cells. The cells were pretreated with BK3C231 prior to exposure to the carcinogen 4-nitroquinoline 1-oxide (4NQO). BK3C231 was able to inhibit 4NQO-induced cytotoxicity. Cells treated with 4NQO alone caused high level of DNA and mitochondrial damages. However, pretreatment with BK3C231 protected against these damages by reducing DNA strand breaks and micronucleus formation as well as decreasing losses of mitochondrial membrane potential (ΔΨm) and cardiolipin. Interestingly, our study has demonstrated that nitrosative stress instead of oxidative stress was involved in 4NQO-induced DNA and mitochondrial damages. Inhibition of 4NQO-induced nitrosative stress by BK3C231 was observed through a decrease in nitric oxide (NO) level and an increase in glutathione (GSH) level. These new findings elucidate the cytoprotective potential of BK3C231 in human colon fibroblast CCD-18Co cell model which warrants further investigation into its chemopreventive role.

Specific Deletion of p16INK4a with Retention of p19ARF Enhances the Development of Invasive Oral Squamous Cell Carcinoma

The cyclin-dependent kinase inhibitor 2A (CDKN2A)/alternate reading frame (ARF) locus consists of two overlapping tumor suppressor genes, p16INK4a and p14ARF (p19ARF in mice), encoding two unrelated proteins in alternative reading frames. Previous reports suggest that p16INK4a and p14ARF alterations independently exhibit differential roles, and p16INK4a is more closely associated with a poor prognosis in oral cancer. However, the role of p16INK4a-specific loss in oral squamous cell carcinogenesis remains unclear. The authors assessed chemical carcinogen 4-nitroquinoline 1-oxide (4NQO)-induced multistep oral squamous cell carcinogenesis in mice carrying p16INK4a-specific loss with retention of the p19ARF gene (p16INK4a-/-). 4NQO-treated p16-/- mice exhibited a higher incidence and multiplicity of oral squamous cell carcinoma (OSCC) development relative to 4NQO-treated wild-type mice. 4NQO-treated p16INK4a-/- OSCC cells exhibited higher proliferation and up-regulation of Arf, transcription factor E2f1, tumor protein p63 (tp63), and oncogenic ΔNp63, an isoform p63, compared with observations in 4NQO-treated wild-type OSCC cells. Furthermore, the overexpression of oncogenic ΔNp63 was associated with human OSCC. In conclusion, these results in mice indicate the biological significance of p16INK4a-specific loss with retention of p19ARF in oral squamous cell carcinogenesis, and ΔNp63 may be a potential target for OSCC.

MTA1 promotes tumorigenesis and development of esophageal squamous cell carcinoma via activating the MEK/ERK/p90RSK signaling pathway.

Metastasis-associated protein 1 (MTA1) is upregulated in multiple malignancies and promotes cancer proliferation and metastasis, but whether and how MTA1 promotes esophageal squamous cell carcinoma (ESCC) tumorigenesis remain unanswered. Here, we established an ESCC model in MTA1 transgenic mice induced by the chemical carcinogen 4-nitroquinoline 1-oxide (4-NQO) and found that MTA1 promotes ESCC tumorigenesis in mice. MTA1 overexpression was observed in ESCC cells and clinical ESCC samples. Overexpressed MTA1 increased colony formation and the invasiveness and migration of ESCC cells, whereas knock down of MTA1 in ESCC cells significantly decreased colony formation, invasion and migration in vitro and inhibited the growth of xenograft tumors in vivo. RNA sequencing (RNA-seq) analysis combined with western blot assays revealed that MTA1 promotes carcinogenesis by enhancing MEK/ERK/p90RSK signaling. The phosphorylation of MEK, ERK and their downstream target p90RSK was significantly decreased after MTA1 knockdown in ESCC cells and was increased in MTA1-overexpressing cells. Moreover, colony formation, invasion and migration potential were dramatically suppressed when cells overexpressing MTA1 were treated with MEK (PD0325901) or ERK (SCH772948) inhibitors. In conclusion, MTA1 plays a pivotal oncogenic role in ESCC tumorigenesis and development through activating the MEK/ERK/p90RSK pathway.

Abstract 4665: Lineage-tracing technology to understand the molecular events in a mouse model of tongue squamous cell carcinoma (SCC) carcinogenesis

Abstract Oral cavity squamous cell carcinomas (OCSCCs) account for over 450,000 cases per year worldwide, present a high metastatic potential, and have a survival rate ≤ 50%. There is a high incidence of OCSCC, but the mechanisms of carcinogenesis remain unclear. Therefore, the aim of this research was to delineate the mutational landscape in developing tumors and OCSCCs by combining a lineage tracing approach and a mouse model of tongue carcinogenesis, previously established by our group. We used K14-CreERTAM; Rosa26LacZ mice, which allowed the long term basal stem cells of the epithelium to be permanently marked at the time of a two-day tamoxifen treatment. We then induced carcinogenesis by treating mice for 10 weeks with the carcinogen 4-nitroquinoline 1-oxide (4-NQO), which recapitulates key phenotypic and molecular features observed in human OCSCC. We performed Laser Capture Microdissection (LCM) to isolate and retrieve high-quality genomic DNA exclusively from each clone of permanently labeled, long-lived LacZ+ stem cells; each clone of cells arises from one LacZ+ stem cell. We then performed exome sequencing of 3 to 5 mice per group, sacrificed immediately after the 10-week treatment with 4-NQO (pre-neoplastic), and mice sacrificed >19 weeks after the end of 4-NQO treatment (SCC). As a reference genome for alignment and germline mutation exclusion, we included 3 mice of the same genetic background that did not receive 4-NQO. After 10 weeks of 4-NQO treatment the tongues were hyperplastic, with occasional necrosis and mild atypia, whereas all mice evaluated >19 weeks post 4-NQO treatment showed tongue SCCs. The total numbers of somatic mutations and LOH (loss of heterozygosity) identified using the mutation calling tool VarScan were 75% (4615±2170 vs 1148±76) higher in SCC compared to pre-neoplastic tongue samples. Among the mutations predicted as high and moderate impact, the levels of SNVs (single nucleotide variants) vs. indels were similar between SCCs and pre-neoplastic samples, 98.4% and 96.5%, respectively. By detailed analysis of the mutation types we showed that the pre-neoplastic tissues exhibited a much higher proportion of LOH events than the SCCs, 34.4% and 7.6%, respectively. Some of the genes affected by LOH in pre-neoplastic tissues were identified as tumor suppressors. Our findings suggest that a predominance of LOH in pre-neoplastic tongue tissue may result in loss of tumor suppressor genes, potentially defining the early steps of carcinogenesis in tongue SCCs. In conclusion, following the fate of long term tongue epithelial stem cells has allowed us to identify genomic perturbations occurring in the tongues of 4-NQO-treated mice and to find potential early biomarkers for OCSCC. Grant ID: R01CA205258; Funding Agency: NIH/NCI. Citation Format: Marta Melis, Tuo Zhang, Theresa Scognamiglio, Lorraine J. Gudas. Lineage-tracing technology to understand the molecular events in a mouse model of tongue squamous cell carcinoma (SCC) carcinogenesis [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 4665.

Abstract 1268: Anti-PD-1 monotherapy outperforms multiple immunotherapy combination strategies in an oral cancer prevention mouse model

Abstract Oral premalignant lesion (OPL) expression of PD-L1 is associated with increased cancer risk. These data suggest that immune evasion is already present at the OPL stage and warrant an evaluation of immune modulatory therapies for cancer prevention. We conducted this preclinical study in a carcinogen-induced mouse model of oral cancer to evaluate the efficacy of multiple immunomodulatory strategies, given at a time point when only OPLs are present, to reduce the incidence of oral squamous cell carcinoma (OSCC). We treated C57BL/6 mice with the carcinogen 4-nitroquinoline 1-oxide (4-NQO) for 8 weeks in drinking water. Eight weeks after discontinuing 4-NQO treatment, a group of mice (N=6) was sacrificed for assessment of invasive and non-invasive tongue lesions. A second group of mice was treated with either anti-PD-1 (N=40), anti-CTLA-4 (N=20), anti-OX40 (N=20), anti-PD-1 + anti-CTLA-4 (N=20), anti-PD-1 + anti-OX40 (N=20) antibodies, or IgG (N=40) for a total of 3 doses every 3 days, initiating 8 weeks after the cessation of 4-NQO. Antibodies used in combination were given on the same day . Mice were sacrificed 56 days after the last dose of treatment. We assessed serial H&E-stained cross sections of the tongues harvested at that same time point for development of OPLs and cancer and measured the OSCC area of each tongue using aperio imagescope software. Mann-Whitney and Fisher's exact tests were used for statistical comparisons between groups. Eight weeks after 4NQO cessation, 100% of mice developed tongue hyperplasia or dysplasia and invasive lesions were not identified, indicating this to be an ideal time point to initiate treatment strategies addressing OPLs. Tongues from mice treated with anti-PD-1 antibody displayed a decrease in the mean OSCC area when compared with mice treated with IgG (mean 3.53 mm2 versus 6.62 mm2, respectively; p= 0.018). At this time point, invasive oral cancers had developed in 75% versus 50% of IgG and anti-PD-1 treated mice, respectively (p=0.03). There were also non-significant decreases in the mean OSCC area in tongues from mice treated with anti-CTLA-4 (mean = 5.07 mm2), anti-OX40 (mean = 3.79 mm2) and anti-PD-1 + anti-CTLA-4 (mean = 3.86 mm2) antibodies when compared to mice treated with IgG (mean = 6.62 mm2) control. In the group treated with anti-PD-1 + anti-OX40 combination therapy, there was an increase in the mean OSCC area when compared to anti-PD-1 monotherapy (mean 3.53 mm2 versus 9.03 mm2), respectively (p= 0.01). Short-term anti-PD-1 immune checkpoint inhibitor therapy in the context of OPLs led to a reduction in the incidence of oral cancer. When comparing the mean area of OSCC in each group, anti-PD-1 monotherapy was superior to IgG and all other treatment strategies. Paradoxically, combining anti-OX40 and anti-PD-1 antibodies created an antagonizing effect on the therapy and increased total tumor burden when compared with anti-PD-1 monotherapy. Citation Format: Jose Augusto Monteiro de Oliveira Novaes, Marlese A. Pisegna, Alissa Poteete, Fahao Zhang, John V. Heymach, William N. William. Anti-PD-1 monotherapy outperforms multiple immunotherapy combination strategies in an oral cancer prevention mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1268.

Abstract 5203: Bitter melon extract modulates immune signaling in prevention of tobacco associated carcinogen induced oral carcinogenesis

Abstract Oral squamous cell carcinoma (OSCC) is a complex malignancy and tobacco habit is one of the main etiological factors. Our previous study showed potential chemopreventive effect of bitter melon (Momordica charantia) extract (BME) in different pre-clinical cancer models like head and neck, breast, prostate. The aim of this study is to understand the molecular mechanism of BME in prevention of carcinogen induced in-vivo oral carcinogenesis. For this purpose, we induced oral cancer in immunocompetent mice by using tobacco associated carcinogen 4-nitroquinoline 1-oxide (4-NQO), and treated with BME through their drinking water. We observed that BME feeding significantly reduced the incidence of 4-NQO induced oral cancer. Histological analysis showed moderate dysplasia to invasive squamous cell carcinoma in 4NQO treated mice tongue, whereas no remarkable changes were seen in the BME-fed mice. RNA-seq data showed differential expression of several genes during carcinogenesis and during prevention of the cancer. We previously showed that BME treatment decreases the infiltrating regulatory T (Treg) cells by inhibiting FoxP3+ populations in the tumors and in spleens, suggesting an immunomodulatory role of BME in prevention. Here, we observed elevated expression of pro-inflammatory genes s100a9, IL23a, IL-1β and immune check point gene PDCD1/PD1 of the pathway, during oral cancer development. Interestingly, BME treatment significantly reduced the expression of these molecules. Subsequently, we observed the enhancement of MMP9 during carcinogenesis, which was reduced in BME-fed mouse tongue tissues. Taken together, our observation strongly demonstrated a chemopreventive effect of BME on carcinogen induced oral cancer. Citation Format: Subhayan Sur, Robert Steele, Ratna B. Ray. Bitter melon extract modulates immune signaling in prevention of tobacco associated carcinogen induced oral carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5203.

Dek overexpression in murine epithelia increases overt esophageal squamous cell carcinoma incidence.

Esophageal cancer occurs as either squamous cell carcinoma (ESCC) or adenocarcinoma. ESCCs comprise almost 90% of cases worldwide, and recur with a less than 15% five-year survival rate despite available treatments. The identification of new ESCC drivers and therapeutic targets is critical for improving outcomes. Here we report that expression of the human DEK oncogene is strongly upregulated in esophageal SCC based on data in the cancer genome atlas (TCGA). DEK is a chromatin-associated protein with important roles in several nuclear processes including gene transcription, epigenetics, and DNA repair. Our previous data have utilized a murine knockout model to demonstrate that Dek expression is required for oral and esophageal SCC growth. Also, DEK overexpression in human keratinocytes, the cell of origin for SCC, was sufficient to cause hyperplasia in 3D organotypic raft cultures that mimic human skin, thus linking high DEK expression in keratinocytes to oncogenic phenotypes. However, the role of DEK over-expression in ESCC development remains unknown in human cells or genetic mouse models. To define the consequences of Dek overexpression in vivo, we generated and validated a tetracycline responsive Dek transgenic mouse model referred to as Bi-L-Dek. Dek overexpression was induced in the basal keratinocytes of stratified squamous epithelium by crossing Bi-L-Dek mice to keratin 5 tetracycline transactivator (K5-tTA) mice. Conditional transgene expression was validated in the resulting Bi-L-Dek_K5-tTA mice and was suppressed with doxycycline treatment in the tetracycline-off system. The mice were subjected to an established HNSCC and esophageal carcinogenesis protocol using the chemical carcinogen 4-nitroquinoline 1-oxide (4NQO). Dek overexpression stimulated gross esophageal tumor development, when compared to doxycycline treated control mice. Furthermore, high Dek expression caused a trend toward esophageal hyperplasia in 4NQO treated mice. Taken together, these data demonstrate that Dek overexpression in the cell of origin for SCC is sufficient to promote esophageal SCC development in vivo.

Abstract A077: Sustained immune response to anti-PD-1 therapy for oral premalignant lesions (OPLs) in a carcinogen-induced oral cancer mouse model

Abstract PD-L1 expression occurs in OPLs in humans and is associated with increased cancer risk (William et al., ASCO 2017). These data suggest a contribution of the PD-1/PD-L1 axis to immune evasion of OPLs, and warrant evaluation of anti-PD-1 immunotherapy for cancer prevention. We conducted this preclinical study in a carcinogen-induced mouse model of oral cancer to test the hypothesis that a short course of anti-PD-1 therapy, given at a time point when only OPLs are present (and before invasive cancer development), could lead to enhanced and sustained immune responses. We treated C57BL/6 mice with the carcinogen 4-nitroquinoline 1-oxide (4-NQO) for 8 weeks in drinking water. Eight weeks after 4-NQO discontinuation, a group of mice (N=6) was sacrificed for assessment of invasive and noninvasive tongue lesions. A second group of mice was treated with either anti-PD-1 antibody (N=10) or IgG (N=10) for a total of 3 doses every 3 days initiating 8 weeks after the cessation of 4-NQO. We sacrificed these animals 56 days after the last dose of treatment, harvested cervical lymph nodes, and prepared and stained cell suspensions with fluorochrome-labeled antibodies against CD45, CD3, CD8, Granzyme B (GZB), PD-1, TIM-3, Lag-3, ZombieNirt (live/dead discrimination) for flow cytometric evaluation. We also assessed serial H&E-stained cross-sections of the tongues harvested at that same time point for development of OPLs and cancers. Student’s t-test and Fisher's exact test were used for statistical comparisons between groups. At 8 weeks after 4NQO cessation, 6 out of 6 mice developed tongue hyperplasia or dysplasia. No invasive lesions were identified, indicating this to be an ideal time point to study/initiate treatment strategies addressing OPLs. Anti-PD-1 therapy led to a sustained increase in the proportion of CD8+|GZB+ cytotoxic T lymphocytes (CTLs) when compared to IgG (mean 26.9% ± 4.4% versus 14.4% ± 2.3%, respectively; p= 0.025) in cervical lymph nodes harvested 56 days after treatment discontinuation. We also observed an increase in the proportion of CD8+|PD-1+ CTLs (mean 22.3% ± 2.08% versus 13.3% ± 1.42%, respectively; p= 0.0026) and a trend towards an increase in CD8+|TIM-3+|LAG-3+ CTLs (mean 5.9% ± 1.5% versus 2.5% ± 0.7%, respectively; p= 0.0733). This immune profile is indicative of activated CTLs with limited/no signs of exhaustion. At this late sacrifice time point, frankly invasive oral cancers had developed in 70% vs 40% of IgG and anti-PD1-treated mice, respectively (p=0.36). Short-term anti-PD-1 immune checkpoint inhibitor therapy in the context of OPLs led to a sustained increase in the population of granzyme B+ CTLs in cervical lymph nodes, accompanied by a trend towards a reduction in the incidence of oral cancer. These results suggest that immune checkpoint-targeted therapy can reverse immune suppression already present at the preinvasive stage, and support the ongoing Immune Prevention of Oral Cancer (IPOC) clinical trial evaluating pembrolizumab in high-risk OPLs (NCT02882282). Additional preclinical studies are under way to assess if an immune response to anti-PD1 therapy can occur at earlier time points, and lead to pronounced prevention of oral cancers at a stage when the (irreversible) malignant transformation process induced by the potent 4-NQO carcinogen is not yet far advanced. Citation Format: Jose Augusto Monteiro de Oliveira Novaes, Taghreed Hirz, Marlese A. Pisegna, Patrick Hwu, John V. Heymach, William N. William. Sustained immune response to anti-PD-1 therapy for oral premalignant lesions (OPLs) in a carcinogen-induced oral cancer mouse model [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A077.

Deficiency of IL-18 Aggravates Esophageal Carcinoma Through Inhibiting IFN-γ Production by CD8+T Cells and NK Cells.

To investigate the potential role of interleukin-18 (IL-18) in immunomodulation during tumorigenesis of esophageal carcinoma and elucidate the underlying molecular mechanism, we employed IL-18 knockout mice for this purpose. Carcinogen 4-nitroquinoline 1-oxide (4NQO) was administrated in drinking water to induce occurrence of esophageal squamous cell carcinoma (ESCC). T cell activation as indicated by the surface CD molecules was analyzed with flow cytometry. The serous content of interferon-γ (IFN-γ) along with other cytokines was determined by inflammatory human cytokine cytometric bead array. The cytotoxicity assay was performed by co-culture of tumor cells with immune cells and relative cell viability was determined by lactate dehydrogenase (LDH) assay. Apoptotic cells were stained with Annexin-V/propidium iodide (PI) and analyzed by flow cytometry. Cell proliferation was measured with Cell Counting Kit-8 (CCK-8) assay. Our data demonstrated that deficiency of IL-18 promoted the progression and development of 4NQO-induced ESCC. Loss of IL-18 suppressed the activation of T cells in the esophagus. Deficiency of IL-18 inhibited the IFN-γ production by CD8+ T cells and natural killer (NK) cells. Absence of IL-18 inhibited the cytotoxicity of CD8+ T cells and NK cell in vitro. Moreover, deficiency of IL-18 promoted the apoptosis of CD8+ T cells and inhibited the proliferation of CD8+ T cells in vitro. Our data elucidated the immunomodulatory role of IL-18 during tumorigenesis of ESCC, whose deficiency compromised antitumor immunity and contributed to immune escape of esophageal carcinoma. Our results also indicated the therapeutic potential of exogenous IL-18 against ESCC, which warrants further investigations.

P120-Catenin Is Required for Dietary Calcium Suppression of Oral Carcinogenesis in Mice.

Previous studies have shown that dietary calcium suppresses oral carcinogenesis, but the mechanism is unclear. p120-catenin (p120) is a cytoplasmic protein closely associated with E-cadherin to form the E-cadherin-β-catenin complex and may function as a tumor suppressor in the oral epithelium. To determine whether p120 is involved in the mechanism by which dietary calcium suppresses oral carcinogenesis, The normal, low, or high calcium diet was fed control mice (designated as floxed p120 mice) or mice in which p120 was specifically deleted in the oral squamous epithelium during the adult stage (designated as p120cKO mice). All mice were exposed to a low dose of oral cancer carcinogen 4-nitroquinoline 1-oxide and rates of oral squamous cell carcinoma (OSCC) and proliferation and differentiation in the cancerous and non-cancerous oral epithelium of these mice were examined. The results showed that the low calcium diet increased rates of OSCC and proliferation of the non-cancerous oral epithelium and decreased differentiation of the non-cancerous oral epithelium, but had no effect on cancerous oral epithelium. In contrast, the high calcium diet had opposite effects. However, the effect of the dietary calcium on the rates of OSCC, proliferation, and differentiation of the non-cancerous epithelium were not seen in p120cKO mice. Based on these results, we conclude that p120 is required for dietary calcium suppression of oral carcinogenesis and oral epithelial proliferation and dietary calcium induction of oral epithelial differentiation. J. Cell. Physiol. 232: 1360-1367, 2017. © 2016 Wiley Periodicals, Inc.