Oral Cancer Model Research Articles

Use of High Frequency Ultrasound to Monitor Cervical Lymph Node Alterations in Mice

Cervical lymph node evaluation by clinical ultrasound is a non-invasive procedure used in diagnosing nodal status, and when combined with fine-needle aspiration cytology (FNAC), provides an effective method to assess nodal pathologies. Development of high-frequency ultrasound (HF US) allows real-time monitoring of lymph node alterations in animal models. While HF US is frequently used in animal models of tumor biology, use of HF US for studying cervical lymph nodes alterations associated with murine models of head and neck cancer, or any other model of lymphadenopathy, is lacking. Here we utilize HF US to monitor cervical lymph nodes changes in mice following exposure to the oral cancer-inducing carcinogen 4-nitroquinoline-1-oxide (4-NQO) and in mice with systemic autoimmunity. 4-NQO induces tumors within the mouse oral cavity as early as 19 wks that recapitulate HNSCC. Monitoring of cervical (mandibular) lymph nodes by gray scale and power Doppler sonography revealed changes in lymph node size eight weeks after 4-NQO treatment, prior to tumor formation. 4-NQO causes changes in cervical node blood flow resulting from oral tumor progression. Histological evaluation indicated that the early 4-NQO induced changes in lymph node volume were due to specific hyperproliferation of T-cell enriched zones in the paracortex. We also show that HF US can be used to perform image-guided fine needle aspirate (FNA) biopsies on mice with enlarged mandibular lymph nodes due to genetic mutation of Fas ligand (Fasl). Collectively these studies indicate that HF US is an effective technique for the non-invasive study of cervical lymph node alterations in live mouse models of oral cancer and other mouse models containing cervical lymphadenopathy.

RAS/PI3K Crosstalk and Cetuximab Resistance in Head and Neck Squamous Cell Carcinoma

Cetuximab, an antibody directed against the EGF receptor, is an effective clinical therapy for patients with head and neck squamous cell cancer (HNSCC). Despite great clinical promise, intrinsic or acquired cetuximab resistance hinders successful treatment outcomes but little is known about the underlying mechanism. To study the role of oncogenic HRAS in cetuximab resistance in HNSCC, the frequency of oncogenic HRAS mutations was determined in a cohort of 180 genomic DNAs from head and neck cancer specimens. We also used a combination of cetuximab-resistant cell lines and a transgenic mouse model of RAS-driven oral cancer to identify an oncogenic RAS-specific gene expression signature that promotes cetuximab resistance. Here, we show that activation of RAS signaling leads to persistent extracellular signal-regulated kinase 1/2 signaling and consequently to cetuximab resistance. HRAS depletion in cells containing oncogenic HRAS or PIK3CA restored cetuximab sensitivity. In our study, the gene expression signature of c-MYC, BCL-2, BCL-XL, and cyclin D1 upon activation of MAPK signaling was not altered by cetuximab treatment, suggesting that this signature may have a pivotal role in cetuximab resistance of RAS-activated HNSCC. Finally, a subset of patients with head and neck cancer with oncogenic HRAS mutations was found to exhibit de novo resistance to cetuximab-based therapy. Collectively, these findings identify a distinct cetuximab resistance mechanism. Oncogenic HRAS in HNSCC promotes activation of ERK signaling, which in turn mediates cetuximab resistance through a specific gene expression signature. Clin Cancer Res; 20(11); 2933-46. ©2014 AACR.

Preclinical Evidence for the Pharmacological Actions of Naringin: A Review

Naringin, chemically 4',5,7- trihydroxyflavanone-7-rhamnoglucoside, is a major flavanone glycoside obtained from tomatoes, grapefruits, and many other citrus fruits. It has been experimentally documented to possess numerous biological properties such as antioxidant, anti-inflammatory, and antiapoptotic activities. In vitro and in vivo studies have further established the usefulness of naringin in various preclinical models of atherosclerosis, cardiovascular disorders, diabetes mellitus, neurodegenerative disorders, osteoporosis, and rheumatological disorders. Apart from this, naringin has also exerted chemopreventive and anticancer attributes in various models of oral, breast, colon, liver, lung, and ovarian cancer. This wide spectrum of biological expediency has been documented to be a result of either the upregulation of various cell survival proteins or the inhibition of inflammatory processes, or a combination of both. Due to the scarcity of human studies on naringin, this review focuses on the various established activities of naringin in in vitro and in vivo preclinical models, and its potential therapeutic applications using the available knowledge in the literature. Additionally, it also encompasses the pharmacokinetic properties of naringin and its inhibition of CYP isoenzymes, and the subsequent drug interactions. Moreover, further clinical research is evidently needed to provide significant insights into the mechanisms underlying the effects of naringin in humans.

Gemcitabine chemotherapy induces phenotypic alterations of tumor cells that facilitate antitumor T cell responses in a mouse model of oral cancer

Gemcitabine (GEM) is a pyrimidine nucleoside analogue that is a new chemotherapeutic agent used for treating various cancers. Because accumulating evidence indicates that GEM may activate host immune responses, its potential as an immune modulator in cancer chemotherapy has generated considerable interest. In the present study, we investigated the antitumor effects of GEM using a mouse oral cancer model using immunological analyses. We examined apoptotic cell death of tumor cells with GEM treatment both in vitro and in vivo. We also investigated whether in vivo administration of GEM affected the distributions of immune cells, tumor-cell surface expression levels of immune accessory molecules and T cell immune responses in tumor-bearing mice. GEM induced significant oral cancer-cell apoptosis in vitro, and in vivo GEM administration markedly attenuated established mouse tumor growth. In vivo GEM administration decreased the numbers of both myeloid-derived suppressor cells (MDSCs) and B cells in tumor-bearing mice and enhanced dendritic cell maturation. Moreover, GEM treatment upregulated tumor-cell surface expressions of several immune accessory molecules and adhesion molecules, including CD80, CD86, CD40, ICAM-1, VCAM-1, and P-selectin. Remarkably, these tumor cells augmented tumor specific T-cell responses. These results suggest that GEM can induce host antitumor immune responses, which would facilitate antitumor effects in the treatment of oral cancer.

Temporal characterization of lymphatic metastasis in an orthotopic mouse model of oral cancer

The overall mortality rate in cases of head and neck squamous cell carcinoma (HNSCC) has not improved over the past 30 years, mostly because of the high treatment failure rate among patients with regionally metastatic disease. To better understand the pathobiologic processes leading to lymphatic metastasis development, there is an urgent need for relevant animal models. HNSCC cell lines were implanted into the tongues of athymic nude mice. Histology, immunohistochemistry, and ex vivo 2-photon microscopy were used to evaluate tumor progress and spread. Orthotopic xenografts of different HNSCC cell lines produced distinct patterns of survival, tumor histology, disease progression rate, and lymph node metastasis development. Remarkably, all injected cell types reached the lymph nodes within 24 hours after injection, but not all developed metastasis. This orthotopic xenograft model closely mimics several characteristics of human cancer and could be extremely valuable for translational studies focusing on lymphatic metastasis development and pathobiology.

Boron biodistribution for BNCT in the hamster cheek pouch oral cancer model: Combined administration of BSH and BPA

Sodium mercaptoundecahydro-closo-dodecaborate (BSH) is being investigated clinically for BNCT. We examined the biodistribution of BSH and BPA administered jointly in different proportions in the hamster cheek pouch oral cancer model. The 3 assayed protocols were non-toxic, and showed preferential tumor boron uptake versus precancerous and normal tissue and therapeutic tumor boron concentration values (70–85ppm). All 3 protocols warrant assessment in BNCT studies to contribute to the knowledge of (BSH+BPA)-BNCT radiobiology for head and neck cancer and optimize therapeutic efficacy.

Gemcitabine induces phenotypic alterations of tumor cells that facilitate antitumor T cell responses in a mouse model of oral cancer

Gemcitabine induces phenotypic alterations of tumor cells that facilitate antitumor T cell responses in a mouse model of oral cancer

Coordinated Targeting of the EGFR Signaling Axis by MicroRNA-27a*

Epidermal growth factor receptor (EGFR) has been characterized as a critical factor in the development and progression of multiple solid tumors, including head and neck squamous cell carcinoma (HNSCC). However, monotherapy with EGFR-specific agents has not been as dramatic as preclinical studies have suggested. Since complex regulation of the EGFR signaling axis might confound current attempts to inhibit EGFR directly, we searched for microRNAs (miRNAs) that may target the EGFR signaling axis. We identified miR-27a (miR-27a-3p) and its complementary or star (*) strand, miR-27a* (miR-27a-5p), as novel miRNAs targeting EGFR, which were significantly downregulated in multiple HNSCC cell lines. Analysis of human specimens demonstrated that miR-27a* is significantly underexpressed in HNSCC as compared to normal mucosa. Increased expression of miR-27a* in HNSCC produced a profound cytotoxic effect not seen with miR-27a. Analysis for potential targets of miR-27a* led to the identification of AKT1 (protein kinase B) and mTOR (mammalian target of rapamycin) within the EGFR signaling axis. Treatment with miR-27a* led to coordinated downregulation of EGFR, AKT1 and mTOR. Overexpression of EGFR signaling pathway components decreased the overall effect of miR-27a* on HNSCC cell viability. Constitutive and inducible expression of miR-27a* in a murine orthotopic xenograft model of oral cavity cancer led to decreased tumor growth. Direct intratumoral injection of miR-27a* inhibited tumor growth in vivo. These findings identify miR-27a* as a functional star sequence that exhibits novel coordinated regulation of the EGFR pathway in solid tumors and potentially represents a novel therapeutic option.

Lactobacillus Salivarius REN Inhibits Rat Oral Cancer Induced by 4-Nitroquioline 1-Oxide

Despite significant advances in cancer therapy, cancer-related mobility and mortality are still rising. Alternative strategies such as cancer prevention thus become essential. Probiotics represent an emerging option for cancer prevention, but studies are limited to colon cancers. The efficiency of probiotics in the prevention of other cancers and the correlative mechanism remains to be explored. A novel probiotics Lactobacillus salivarius REN (L. salivarius REN) was isolated from centenarians at Bama of China, which showed highly potent antigenotoxicity in an initial assay. 4-nitroquioline 1-oxide (4NQO)-induced oral cancer model was introduced to study the anticancer activity of L. salivarius REN in vivo. The results indicated that oral administration of probiotic L. salivarius REN or its secretions could effectively suppress 4NQO-induced oral carcinogenesis in the initial and postinitial stage, and the inhibition was in a dose-dependent manner. A significant decrease of neoplasm incidence (65%-0%) was detected in rats fed with the high dose of L. salivarius REN [5 × 10(10) CFU/kg body weight (bw)/d]. In vivo evidences indicated that the probiotics inhibited 4NQO-induced oral cancer by protecting DNA against oxidative damage and downregulating COX-2 expression. L. salivarius REN treatment significantly decreased the expression of proliferating cell nuclear antigen (PCNA) and induced apoptosis in a dose-dependent manner. Our findings suggest that probiotics may act as potential agents for oral cancer prevention. This is the first report showing the inhibitory effect of the probiotics on oral carcinogenesis.

Astaxanthin inhibits NF-κB and Wnt/β-catenin signaling pathways via inactivation of Erk/MAPK and PI3K/Akt to induce intrinsic apoptosis in a hamster model of oral cancer

BackgroundThe oncogenic transcription factors NF-κB and β-catenin, constitutively activated by upstream serine/threonine kinases control several cellular processes implicated in malignant transformation including apoptosis evasion. The aim of this study was to investigate the chemopreventive effects of astaxanthin, an antioxidant carotenoid, in the hamster buccal pouch (HBP) carcinogenesis model based on its ability to modulate NF-κB and Wnt signaling pathways and induce apoptosis. MethodsWe determined the effect of dietary supplementation of astaxanthin on the oncogenic signaling pathways — NF-κB and Wnt/β-catenin, their upstream activator kinases — Erk/MAPK and PI-3K/Akt, and the downstream event — apoptosis evasion by real-time quantitative RT-PCR, western blot, and immunohistochemical analyses. ResultsWe found that astaxanthin inhibits NF-κB and Wnt signaling by downregulating the key regulatory enzymes IKKβ and GSK-3β. Analysis of gene expression and docking interactions revealed that inhibition of these pathways may be mediated via inactivation of the upstream signaling kinases Erk/Akt by astaxanthin. Astaxanthin also induced caspase-mediated mitochondrial apoptosis by downregulating the expression of antiapoptotic Bcl-2, p-Bad, and survivin and upregulating proapoptotic Bax and Bad, accompanied by efflux of Smac/Diablo and cytochrome-c into the cytosol, and induced cleavage of poly (ADP-ribose) polymerase (PARP). ConclusionsThe results provide compelling evidence that astaxanthin exerts chemopreventive effects by concurrently inhibiting phosphorylation of transcription factors and signaling kinases and inducing intrinsic apoptosis. General significanceAstaxanthin targets key molecules in oncogenic signaling pathways and induces apoptosis and is a promising candidate agent for cancer prevention and therapy.

PP062: Detection of peroxiredoxin 1 and hypoxia in a xenograft model of human oral cancer

Hypoxia is closely related to malignant progress, angiogenesis, invasion, metastasis and tolerance of treatment in malignant tumor. But the precise molecular mechanism of hypoxia is not clear in oral squamous cell carcinoma (OSCC). In this study, for the first time we investigated interrelationship between hypoxia and Prx1 during tumor growth in human OSCC xenograft model. Eighty tumors were divided into four groups according to a mean tumor diameter of 2, 5, 10, and 15 mm. 2 mm group was set as control. 5, 10, and 15 mm were experimental groups. Pimonidazole staining, immunohistochemistry, QRT-PCR, Western blot, ELISA were used to detect hypoxia, MVD, 8-OHdG, Prx1, HO-1 and NF-B genes. The results showed the extent of tumor hypoxia increased compared with control with tumor size increasing (p

CTLA4-mediated inhibitory effect on osteoclastogenesis by Tregs in oral cancer (P2047)

Abstract Purpose: Despite regulatory T cells (Tregs) have been demonstrated to inhibit inflammatory bone destruction through suppressing osteoclast formation in the bone microenvironment of inflammatory diseases, very little work has been done to characterize Tregs in the microenvironment of cancer bone invasion. In the present study, we investigated the mechanism of interaction between Tregs and osteoclasts in a murine model of oral cancer to elucidate the role of Tregs in the bone invasion of oral cancer. Materials and Methods: SCCVII mouse oral squamous cell carcinoma cells were administered into the right masseter region of a C3H/HeN mouse. After 14 days of tumor inoculation, mice were sacrificed and Tregs were purified from the tumors. Tregs were then cocultured with bone marrow CD11b positive cells in the absence or presence of RANKL and M-CSF for 4 days. Secreted tartrate-resistant acid phosphatase (TRACP) 5b was determined as a marker of osteoclast number. Results: Attenuation of osteoclast-derived TRACP5b was observed in the coculture of tumor derived Tregs and bone marrow CD11b positive cells, and this attenuation of TRACP5b was ameliorated by neutralization of cytotoxic T-lymphocyte-associated antigens-4 (CTLA4). Conclusion: These results indicate that Tregs may play a protective role in bone invasion of oral cancer via suppression of osteoclastogenesis, and CTLA4 may be a key regulator for the suppression of osteoclasts by Tregs in oral cancer.

Biodistribution of sodium borocaptate (BSH) for boron neutron capture therapy (BNCT) in an oral cancer model

Boron neutron capture therapy (BNCT) is based on selective accumulation of ¹⁰B carriers in tumor followed by neutron irradiation. We previously proved the therapeutic success of BNCT mediated by the boron compounds boronophenylalanine and sodium decahydrodecaborate (GB-10) in the hamster cheek pouch oral cancer model. Based on the clinical relevance of the boron carrier sodium borocaptate (BSH) and the knowledge that the most effective way to optimize BNCT is to improve tumor boron targeting, the specific aim of this study was to perform biodistribution studies of BSH in the hamster cheek pouch oral cancer model and evaluate the feasibility of BNCT mediated by BSH at nuclear reactor RA-3. The general aim of these studies is to contribute to the knowledge of BNCT radiobiology and optimize BNCT for head and neck cancer. Sodium borocaptate (50 mg ¹⁰B/kg) was administered to tumor-bearing hamsters. Groups of 3-5 animals were killed humanely at nine time-points, 3-12 h post-administration. Samples of blood, tumor, precancerous pouch tissue, normal pouch tissue and other clinically relevant normal tissues were processed for boron measurement by optic emission spectroscopy. Tumor boron concentration peaked to therapeutically useful boron concentration values of 24-35 ppm. The boron concentration ratio tumor/normal pouch tissue ranged from 1.1 to 1.8. Pharmacokinetic curves showed that the optimum interval between BSH administration and neutron irradiation was 7-11 h. It is concluded that BNCT mediated by BSH at nuclear reactor RA-3 would be feasible.

Rab25 regulates invasion and metastasis in head and neck cancer.

Head and neck squamous cell carcinoma (HNSCC) is one of the 10 most common cancers with a 50% five-year survival rate, which has remained unchanged for the past three decades. One of the major reasons for the aggressiveness of this cancer is that HNSCCs readily metastasize to cervical lymph nodes that are abundant in the head and neck region. Hence, discovering new molecules controlling the metastatic process as well as understanding their regulation at the molecular level are essential for effective therapeutic strategies. Rab25 expression level was analyzed in HNSCC tissue microarray. We used a combination of intravital microscopy in live animals and immunofluorescence in an in vitro invasion assay to study the role of Rab25 in tumor cell migration and invasion. In this study, we identified the small GTPase Rab25 as a key regulator of HNSCC metastasis. We observed that Rab25 is downregulated in HNSCC patients. Next, we determined that reexpression of Rab25 in a metastatic cell line is sufficient to block invasion in a three-dimensional collagen matrix and metastasis to cervical lymph nodes in a mouse model for oral cancer. Specifically, Rab25 affects the organization of F-actin at the cell surface, rather than cell proliferation, apoptosis, or tumor angiogenesis. These findings suggest that Rab25 plays an important role in tumor migration and metastasis, and that understanding its function may lead to the development of new strategies to prevent metastasis in oral cancer patients.

Abstract B110: Chemoprevention of rat oral carcinogenesis by licofelone, a dual cyclooxygenase/lipoxygenase inhibitor

Abstract Cyclooxygenase [COX] inhibitors [piroxicam, celecoxib, naproxen, and NO-naproxen] are potent inhibitors of oral carcinogenesis induced in rats by 4-nitroquinoline-1-oxide [NQO]. By contrast, a model 5-lipoxygenase [LOX] inhibitor [zileuton] has no chemopreventive activity in the rat NQO oral cancer model. Bifunctional agents such as licofelone [6-(4-chlorophenyl)-2,2-dimethyl-7-phenyl-2,3-dihydro-1H-pyrrolizin-5-yl]acetic acid] can inhibit both COX and LOX pathways of eicosanoid biosynthesis. As a result of these activities, dual COX/LOX inhibitors (a) may be less toxic than inhibitors of a single pathway of arachidonic acid metabolism, and (b) may prevent redirection of eicosanoid biosynthesis into parallel pathways that stimulate inflammation and carcinogenesis. A chemoprevention efficacy evaluation of dual COX/LOX inhibition was performed using licofelone as a model compound. Squamous cell carcinomas of the tongue were induced in male F344 rats by administration of NQO [20 ppm] in the drinking water for 10 weeks. Beginning two days after completion of NQO administration, groups of 30 rats received daily oral [gavage] administration of licofelone at 0 mg/kg/day [vehicle control], 37.5 mg/kg/day [low dose], or 75 mg/kg/day [high dose]. A fourth experimental group received oral [gavage] administration of 75 mg licofelone/kg beginning 6 weeks post-NQO [high dose, delayed intervention]. Licofelone or vehicle administration was continued until study termination at week 25. When administered beginning two days after NQO, licofelone conferred dose-related protection against oral carcinogenesis [55% and 43% incidences of oral cancer in low and high dose groups, respectively, versus 75% in controls; p < 0.05 for high dose group]. Delayed administration of the high dose of licofelone demonstrated the greatest chemopreventive activity [34% oral cancer incidence versus 75% incidence in controls; p < 0.01]. Licofelone may inhibit oral carcinogenesis by delaying cancer progression; rats receiving the high dose of licofelone starting either 2 days or 6 weeks post-NQO demonstrated (a) decreases in the incidence of the most invasive [+4] cancers [17% in both groups versus 54% in controls; p < 0.01] and (b) increases in the number of rats without invasive malignancies [normal epithelium or epithelial hyperplasia only; 37% and 41% incidence versus 11% in controls; p < 0.01 for both comparisons]. The chemopreventive activity of the low dose of licofelone was marginally significant [0.05 < p < 0.10 for both comparisons]. Licofelone also prevented the pre-terminal body weight loss commonly seen in animals with advanced oral cancer in this model. These data demonstrate that licofelone is an effective inhibitor of oral carcinogenesis induced in rats by NQO, and suggest that licofelone chemopreventive activity results from inhibition or delayed progression of preneoplastic lesions to invasive oral cancers. The chemopreventive activity of delayed administration of licofelone is particularly notable because clinical trials are most likely to involve individuals with preexisting oral lesions. Dual COX/LOX inhibitors may provide an approach to oral cancer prevention that is not limited by the toxicity induced by specific of inhibitors of either COX or LOX. [Supported by NCI-N01-CN-25113] Citation Format: David L. McCormick, Thomas L. Horn, William D. Johnson, Ronald A. Lubet, Vernon E. Steele. Chemoprevention of rat oral carcinogenesis by licofelone, a dual cyclooxygenase/ lipoxygenase inhibitor. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr B110.

A Comparative Analysis of Immune Infiltrating Lymphocytes in a Murine Model of Oral Cancer

Objective: 1) Understand types of infiltrating immune cells that contribute to the tumor microenvironment. 2) Understand the relative difference in immune infiltrates between aggressive and indolent tumor phenotypes in a new syngeneic model of OSCC. Method: This research is a preclinical scientific study focusing on mouse derived oral cavity squamous cell carcinoma that was conducted over 6 months at Washington University in St Louis. This study analyzed tumor infiltrating immune cells by flow cytometry and identified individual infiltrating populations associated with either aggressive or indolent growth. Results: Indolent growing mouse oral cancer (MOC) lines were associated with increased expression of increased MHC class I expression and increased CD8+ T-cell infiltration into the tumor microenvironment. However, aggressive and metastatic MOC lines were associated with decreased class I expression and increased FOXP3+ CD4+ T-cell infiltration and targeting these regulatory T-cells delayed tumor growth in vivo. Conclusion: These data validate that key infiltrating immune cells identified here parallel findings in human head and neck cancer, making this newly developed syngeneic model a critical platform for the continued dissection of tumor-host interactions in head and neck cancer.

Early induction of cytokines/cytokine receptors and Cox2, and activation of NF-κB in 4-nitroquinoline 1-oxide-induced murine oral cancer model

The purpose of this study was to identify the genes induced early in murine oral carcinogenesis. Murine tongue tumors induced by the carcinogen, 4-nitroquinoline 1-oxide (4-NQO), and paired non-tumor tissues were subjected to microarray analysis. Hierarchical clustering of upregulated genes in the tumor tissues revealed an association of induced genes with inflammation. Cytokines/cytokine receptors induced early were subsequently identified, clearly indicating their involvement in oral carcinogenesis. Hierarchical clustering also showed that cytokine-mediated inflammation was possibly linked with Mapk6. Cox2 exhibited the greatest extent (9–18 fold) of induction in the microarray data, and its early induction was observed in a 2h painting experiment by RT-PCR. MetaCore analysis showed that overexpressed Cox2 may interact with p53 and transcriptionally inhibit expression of several downstream genes. A painting experiment in transgenic mice also demonstrated that NF-κB activates early independently of Cox2 induction. MetaCore analysis revealed the most striking metabolic alterations in tumor tissues, especially in lipid metabolism resulting from the reduction of Pparα and Rxrg. Reduced expression of Mapk12 was noted, and MetaCore analysis established its relationship with decreased efficiency of Pparα phosphorylation. In conclusion, in addition to cytokines/cytokine receptors, the early induction of Cox2 and NF-κB activation is involved in murine oral carcinogenesis.

Abstract 2358: ERK1/2 regulation of CD44 modulates aggressiveness in a new mouse model of oral cancer

Abstract Carcinogen-induced oral cavity squamous cell carcinoma (OSCC) causes significant morbidity and mortality and constitutes a global health challenge. To gain further insight into this disease, six unique C57BL/6 mouse OSCC cell lines were generated from 7,12-dimethylbenz(≥)anthracene (DMBA) induced murine primary OSCCs. The cell lines were designated as mouse oral cancer (MOC1, 2, 7, 10, 22 and 23) and 5/6 (excluding MOC23) formed tumors when transplanted into immunocompetent wild-type (WT) mice. Analyses of tumor growth in mice showed that MOC2, MOC7 and MOC10 grew more rapidly and metastasized to regional lymph nodes, while MOC1 and MOC22 grew much slower and did not metastasize. This aggressive growth was correlated with ERK1/2 activation, which in addition, was found to induce CD44 expression- a molecule whose expression is associated with EMT and putative cancer stem cells. Inhibiting MEK, the upstream activator of ERK1/2, led to decreased CD44 expression and promoter activity and also decreased cellular migration and invasion. Conversely, enforced activation of MEK1 led to enhanced CD44 expression and promoter activity. CD44 was required for the aggressive growth since reduction of CD44 levels significantly attenuated in vitro migration and in vivo tumor formation of MOC2 and MOC10 cell lines. These results in the mouse model were extended to freshly resected human OSCC where a strict relationship between ERK1/2 phosphorylation and CD44 expression was also identified. Thus, using a novel immunocompetent mouse model, we identify CD44 as an essential mediator of the aggressive tumor-promoting effects of ERK1/2, which highlight the potential efficacy of targeting this molecular pathway in head and neck cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2358. doi:1538-7445.AM2012-2358

Abstract 5470: Genotoxic effect and epigenetic alterations induced by the environmental carcinogen dibenzo[a, l]pyrene in oral tissues of mice

Abstract Oral cancer is the major form of head and neck squamous cell carcinoma, which is the fifth most common cancer worldwide. Tobacco smoking is one of the leading causes of oral cancer. Dibenzo[a, l]pyrene (DB[a, l]P) is the most potent carcinogenic polycyclic aromatic hydrocarbon found in tobacco smoke. Recently we developed a novel mouse model of oral cancer induced by DB[a, l]P; we also showed the remarkable carcinogenicity and specificity of the fjord region diol epoxide metabolite, (±)-anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a, l]pyrene (DB[a, l]PDE). Using immunohistochemistry (IHC), we have shown over-expression of p53 protein in mice oral squamous cell carcinoma and dysplastic tissues induced by DB[a, l]P and DB[a, l]PDE. Current understanding of molecular pathogenesis of oral cancer in humans suggests that both genetic and epigenetic alterations are crucial and complement each other. Our hypothesis is that both genetic and epigenetic alterations induced by DB[a, l]P can contribute to the development of oral cancer. P53 protein overexpression detected by IHC may result from p53 gene mutation or exposure to genotoxic stress. To determine whether p53 over-expression is in part due to p53 mutations, Exons 5 to 8 of p53 from 5 tumor tissues were analyzed by polymerase chain reaction single-strand conformation polymorphisms (PCR-SSCP) and direct sequencing. G to T transversion was detected in Exon 5, leading to mutation of codon 155 Arg to Leu; A to T transversion was detected in Exon 7, resulting in mutation of codon 232 Lys to stop codon. To test the epigenetic effect of DB[a, l]P, methylation specific PCR and bisulfate sequencing were used to detect methylation alteration of p16 and RAR-β promoters. Promoter hypermethylation of both p16 and RAR-β were detected in tumors induced by DB[a, l]PDE; also in oral tissues of mice treated with DB[a, l]P (24nmol, 3 times per week, for 5 weeks, sacrificed at 48 h, 1, 2 and 4 weeks after last dose). These results suggested that genotoxic DNA adducts derived from DB[a, l]P can induce mutations in critical genes, such as tumor suppressor gene p53; this environmental carcinogen also can induce p16 and RAR-β promoter hypermethylation at early stage, which can contribute to the promotion and progression of oral carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5470. doi:1538-7445.AM2012-5470

Abstract 5469: Detection of deoxyguanosine adducts in the oral tissues of mice treated with the environmental carcinogen dibenzo[a, l]pyrene by LC-MS/MS

Abstract Oral cancer is the major form of head and neck squamous cell carcinoma, which is the fifth most common cancer worldwide. Tobacco smoking is one of the leading causes of oral cancer. Dibenzo[a, l]pyrene (DB[a, l]P) is the most potent carcinogenic polycyclic aromatic hydrocarbon found in tobacco smoke. Our laboratory had developed a mouse model of oral cancer induced by DB[a, l]P. Our working hypothesis is that the stereochemical course of DB[a, l]P metabolism, the conformations of the DNA adducts formed and their removal by mammalian DNA repair enzymes, and their mutagenic properties if not removed in an error-free manner, all play critical roles during the induction of oral carcinogenesis. As an initial study to test our hypothesis, we have previously developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to detect and quantify DB[a, l]PDE-N6-dA adducts in oral tissues of mice treated with DB[a, l]P. We have shown that (-)-anti-cis- and (-)-anti-trans-DB[a, l]PDE-N6-dA adducts were detected from oral tissues of mice treated with DB[a, l]P. In the present study, to further test our hypothesis, we report on the development of a LC-MS/MS method to detect DB[a, l]PDE-N2-dG adducts in vivo. (±)-anti-[15N5]-DB[a, l]PDE-N2-dG adducts were synthesized as internal standards. The stereochemistry of adducts were characterized. Following the addition of internal standards, DNA isolated from oral tissues of mice treated with DB[a, l]P or DB[a, l]PDE was enzymatically hydrolyzed to 2′-deoxyribonucleosides and partially purified by solid-phase extraction. The LC-MS/MS analysis was carried out by monitoring transitions m/z 620 [M+H]+→ m/z 504 [(M+H)+−2′-deoxyribose] for DB[a, l]PDE-N2-dG adducts and m/z 625α m/z 509 for the internal standards. We have detected two N2-dG adducts from oral tissues of mice treated with DB[a, l]P, and four N2-dG adducts from oral tissues of mice treated with (±)-anti-DB[a, l]PDE. Collectively, the in vivo detection of dA and dG adducts indicated that DB[a, l]P is predominantly metabolized to (-)-anti-DB[a, l]PDE in oral tissues of mice. This sensitive LC-MS/MS method, is capable of simultaneously detecting both dA and dG adducts derived from anti-DB[a, l]PDE. Our results indicated that levels of dA adducts are significantly higher than dG adducts in vivo, which are consistent with those reported in literature in organs other than oral tissues, demonstrating that fjord region diol epoxide of DB[a, l]P predominantly form dA adducts than dG adducts. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5469. doi:1538-7445.AM2012-5469