Oral Cancer Mouse Research Articles

Abstract 3996: MDSC depletion combined with CTLA-4 blockade cause tumor regression in a syngeneic model of oral cavity cancer

Abstract Carcinogen-associated head and neck cancers are a heterogeneous group of aggressive cancers with a high recurrence rate and poor 5-year survival. Many of these cancers are T-cell inflamed but only a subset of patients respond to checkpoint blocking immunotherapy. Local immunosuppression mediated by both the tumor cells and other infiltrating immune cells are likely a major mechanism of resistance to immunotherapy. The mouse oral cancer (MOC) model includes genetically defined cell lines derived from carcinogen-induced tumors that generate tumors with variable T-cell inflamed phenotypes when transplanted into wild-type C57BL/6 mice. Mice bearing MOC1 tumors display a T-cell inflamed phenotype but do not significantly respond to PD-L1 checkpoint inhibition alone, which models a significant subset of human cancers from the oral cavity. Conversely, MOC2 tumors are non-T-cell inflamed. Using these MOC models, we have demonstrated an inverse relationship between accumulation of granulocytic MDSCs (gMDSCs) and effector immune cells (CD8/4+ T-cells, and NK cells) in the tumor microenvironment. Functionally, these gMDSCs suppressed CD8+ T-cell proliferation, IFNγ production, and antigen-specific killing. Depletion of these gMDSCs with a systemic anti-Ly6G antibody (1A8) did not change CD8/4+ T-cell or NK cell accumulation but restored tumor T-cell and NK cell activation and draining lymph node antigen-specific T-lymphocyte activation that was lost with tumor progression. Depletion of gMDSCs also significantly sensitized established MOC1 tumors to CTLA4-based checkpoint inhibition. CTLA-4 blockade alone induced tumor regression in a subset of mice and a marked delay in others. However, gMDSC depletion plus CTLA-4 blockade resulted in complete tumor rejection and development of immunologic memory in all treated mice. Peripheral gMDSCs were found to express the chemokine receptor CXCR2 but no other myeloid chemokine receptors, and expression of CXCR2 ligands CXCL1 and CXCL2 increased within the tumor microenvironment during tumor progression. Macrophage chemotaxis components CSF1R and CSF1 were not significantly expressed in these models. TCGA data analysis demonstrated an MDSC-rich gene expression profile in >60% of head and neck cancers, along with significant CTLA4, CXCR2, CXCL1, and CXCL8 (IL-8) expression within both HPV-positive and negative head and neck cancers. Functional inhibition or elimination of MDSCs from the tumor microenvironment may significantly reverse local immunosuppression and enhance responses to checkpoint inhibition in patients with head and neck cancer. Citation Format: Paul Esteban Clavijo, Ellen Moore, Jianhong Chen, Ruth Davis, Carter Van Waes, Zhong Chen, Clint Allen. MDSC depletion combined with CTLA-4 blockade cause tumor regression in a syngeneic model of oral cavity cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3996. doi:10.1158/1538-7445.AM2017-3996

Abstract 2016: Novel dual cIAP1/XIAP antagonist ASTX660 activity in preclinical models of human papillomavirus(+) and (-) head and neck squamous cell carcinoma

Abstract Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer with a five-year survival of ~50%. HNSCC are linked to human papillomavirus (HPV+) or tobacco carcinogenesis (HPV-). The Cancer Genome Atlas recently analyzed 279 HNSCC, revealing that 30% overexpress FADD (Fas-Associated Death Domain), with or without BIRC2/3 genes encoding cellular Inhibitor of Apoptosis Proteins 1/2 (cIAP1/2), which are critical components of the Tumor Necrosis Factor (TNF) Receptor signaling pathways that determine cell death or survival. The frequency of such mutations provides a window for potential therapeutics, as IAP antagonists have been shown to switch cancer cell TNFα signaling from being pro-survival to pro-apoptotic. We recently showed that birinapant, a second mitochondrial activator of caspases (SMAC) mimetic that promotes IAP degradation, sensitizes HNSCC to cell death by TNFα and eradicates tumors in combination with radiation in HPV- HNSCC models overexpressing FADD+/-BIRC2. ASTX660 (developed by Astex Pharmaceuticals) is a novel dual cIAP1/XIAP antagonist currently in clinical trials for treating advanced solid tumors and lymphomas. The objective of the present study is to determine the therapeutic effects of ASTX660 in HPV+/- HNSCC preclinical models. ASTX660 at nanomolar concentrations was found to potently inhibit cell proliferation (measured using XTT assays) and induce apoptosis (Annexin/7-AAD flow cytometry), and displayed combinatorial activity with TNFα, TRAIL, or cisplatin in multiple human HPV+/- HNSCC cell lines. Western blotting showed near complete degradation of cIAP1 expression at nanomolar concentrations in human HNSCC cell lines. Flow cytometry revealed that ASTX660 in combination with either TNFα or cisplatin reduced MHC-I expression and increased PD-L1 expression, suggesting that potential synergistic efficacy could be observed in combination with immune checkpoint inhibitors. Our results demonstrate that ASTX660 is a potential therapeutic agent for both HPV+/- HNSCC. Experiments evaluating the anti-tumor effects of ASTX660 as a monotherapy and in combination with radiation, cisplatin, and anti-PD-1 therapies are ongoing, using both human HNSCC xenograft and syngeneic mouse oral cancer (MOC) models. Supported by NIDCD intramural projects (ZIA-DC-000016, 73 and 74). Citation Format: Roy Xiao, Yi An, Adeeb Derakhshan, Zhong Chen, Nicole C. Schmitt, Carter Van Waes. Novel dual cIAP1/XIAP antagonist ASTX660 activity in preclinical models of human papillomavirus(+) and (-) head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2016. doi:10.1158/1538-7445.AM2017-2016

Abstract 5648: Combined immune checkpoint targeting with anti-PD-1 plus anti-CD40 antibodies as the most effective approach to eradicate head and neck squamous cell carcinomas (HNSCCs) in mouse models

Abstract Introduction: Immunotherapy with single agent anti-PD-1 antibody confers modest overall survival benefits in patients with recurrent/metastatic HNSCCs. Combined immune checkpoint targeting has shown promising results for melanomas and other solid tumors. Thus, evaluation of combination immunotherapy in animal models specific to HNSCCs could inform prioritization and design of human clinical trials for this disease. We conducted this pre-clinical study to test the hypothesis that combining anti-PD-1 with either anti-CTLA-4 or co-stimulatory immune checkpoint agonist antibodies would be more effective than anti-PD-1 monotherapy in a mouse model of oral carcinoma. We also aimed at identifying the combination regimen with highest anti-tumor activity. Methods: C57BL/6 mice were subcutaneously grafted with 2x106 mouse oral cancer 1 (MOC1) cells derived from a carcinogen-induced tumor molecularly similar to human HNSCC. After 25 days, mice were randomized into one of 12 treatment groups (IgG, or antibodies targeting PD-1, CTLA-4, CD40, OX-40, GITR, 4-1BB, PD-1+CTLA-4, PD1+CD40, PD-1+OX-40, PD-1+GITR, or PD-1+4-1BB), N=8-9 mice/group. Antibodies were administered every 4 days for 3 doses. Tumors were measured twice per week. The primary endpoint was overall survival. Mice were sacrificed when tumor diameter was >2 cm or tumor ulceration was >5 mm. Survival curves were computed using the Kaplan-Meier method and compared using the log-rank (Mantel-Cox) test. Results: In analogy to human clinical trials, anti-PD-1 therapy led to a modest improvement in survival compared to IgG [HR=0.41; 95%CI 0.06-0.53; P=0.015], indicating that this model recapitulates, in part, the effects of immunotherapy in HNSCC patients; none of the animals survived long term. Monotherapy with anti-CTLA-4, anti-CD40, anti-OX-40, or anti-4-1BB also improved survival compared to IgG (P=0.0001, 0.003, 0.02, and 0.003, respectively), but were not more effective than anti-PD-1 alone (P=0.09, 0.12, 0.72, and 0.77, respectively). Combination therapy did not prolong survival compared to anti-PD-1 alone, with the exception of anti-PD-1+ anti-CTLA4 [HR=0.36; 95% CI 0.07-0.6; P=0.016], and anti-PD-1+ anti-CD40 [HR=0.125, 95% CI 0.02-0.24; P=0.0003]. Notably, 50% of mice receiving anti-PD-1+ anti-CD40 antibodies had complete tumor eradication and are alive and disease-free 120 days after tumor grafting. Conclusions: Single agent immunotherapy targeting PD-1, CTLA-4, CD40, OX-40 and 4-1BB modestly improved survival in a mouse model of oral cancer. Combination with anti-PD-1+ anti-CTLA4 and anti-PD-1+ anti-CD40 antibodies outperformed anti-PD-1 monotherapy. Complete responses were exclusively seen with PD-1+CD40 dual targeting. This regimen should be prioritized for evaluation in HNSCC clinical trials. Citation Format: Jose Augusto Monteiro de Oliveira Novaes, Alissa R. Poteete, Marlese A. Pisegna, Uma Giri, Fahao Zhang, Patrick Hwu, John V. Heymach, William N. William. Combined immune checkpoint targeting with anti-PD-1 plus anti-CD40 antibodies as the most effective approach to eradicate head and neck squamous cell carcinomas (HNSCCs) in mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5648. doi:10.1158/1538-7445.AM2017-5648

Abstract 2637: Anti-tumor activity of cisplatin is enhanced by PD-1 blockade in preclinical models of head and neck squamous cell carcinoma

Abstract Rationale: Cisplatin, which remains the most commonly used systemic drug for head and neck squamous cell carcinoma (HNSCC), reduces numbers of circulating immune cells. However, recent studies suggest that cisplatin may enhance some aspects of anti-tumor immunity in the tumor microenvironment. We previously found that cisplatin increases expression of major histocompatibility (MHC) class I and programmed death ligand 1 (PD-L1) in HNSCC cell lines in vitro. Methods: In the current study, we investigated other components of the antigen processing machinery (APM) by flow cytometry following treatment with cisplatin. Using the syngeneic mouse oral cancer 1 (MOC1) model of HNSCC, we then investigated the effects of cisplatin and anti-PD-1 antibody, alone or in combination, on tumor growth and survival. Results: Using three different HNSCC cell lines, we found that levels of the APM components LMP2, ERp57 and calreticulin increased in all cell lines treated with cisplatin, whereas TAP1/2 increased dramatically only in one cisplatin-treated cell line with wildtype TP53 (UMSCC-74A). In MOC1 tumor-bearing mice, tumor growth was partially delayed by treatment with cisplatin and anti-PD-1 antibody alone and substantially delayed when these agents were given in combination, with resultant increased survival. Comparison of different treatment schedules suggested that concurrent administration is more effective than giving cisplatin one day prior to anti-PD-1 or giving anti-PD-1 one day prior to cisplatin. Conclusions: Despite upregulating PD-L1 on tumor cells and decreasing levels of circulating immune cells, cisplatin may enhance some aspects of anti-tumor immunity in the tumor microenvironment. While no mice were cured with concurrent cisplatin and anti-PD-1 antibody, tumor growth was substantially delayed. These results suggest a rationale for combining cisplatin and anti-PD-1 monoclonal antibodies, perhaps with radiation or other therapies, in additional preclinical studies and clinical trials of HNSCC. Supported by NIDCD intramural project ZIA-DC-DC000090. Citation Format: Linda Tran, Clint T. Allen, So-Jin Park, Roy Xiao, Carter Van Waes, Nicole C. Schmitt. Anti-tumor activity of cisplatin is enhanced by PD-1 blockade in preclinical models of head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2637. doi:10.1158/1538-7445.AM2017-2637

PPARα modulates gene expression profiles of mitochondrial energy metabolism in oral tumorigenesis.

Metabolic reprogramming plays a crucial role in the development of cancer. The aim of this study was to explore the effect of fenofibrate, an agonist of peroxisome proliferator-activated receptor alpha (PPARα), on gene expression profiles of mitochondrial energy metabolism. Our results showed that PPARα expression was negatively correlated with tumor progression in an oral cancer mouse model. Activation of PPARα through fenofibrate suppressed migration of oral cancer cells. Differential protein profiling demonstrated that expressions of genes related to mitochondrial energy metabolism were either up-regulated (Atp5g3, Cyc1, Ndufa5, Ndufa10, and Sdhd) or down-regulated (Cox5b, Ndufa1, Ndufb7, and Uqcrh) through PPARα activation and response. Our results indicate that PPARα exhibits a great potential for anti-oral cancer therapies by modulating cancer cell mitochondrial energy metabolism.

Fenofibrate Suppresses Oral Tumorigenesis via Reprogramming Metabolic Processes: Potential Drug Repurposing for Oral Cancer.

One anticancer strategy suggests targeting mitochondrial metabolism to trigger cell death through slowing down energy production from the Warburg effect. Fenofibrate is a clinical lipid-lowering agent and an effective anticancer drug. In the present study, we demonstrate that fenofibrate provided novel mechanisms for delaying oral tumor development via the reprogramming of metabolic processes. Fenofibrate induced cytotoxicity by decreasing oxygen consumption rate (OCR) that was accompanied with increasing extracellular acidification rate (ECAR) and reducing ATP content. Moreover, fenofibrate caused changes in the protein expressions of hexokinase II (HK II), pyruvate kinase, pyruvate dehydrogenase, and voltage-dependent anion channel (VDAC), which are associated with the Warburg effect. In addition, fenofibrate reprogrammed the metabolic pathway by interrupting the binding of HK II to VDAC. In an oral cancer mouse model, fenofibrate exhibited both preventive and therapeutic efficacy on oral tumorigenesis. Fenofibrate administration suppressed the incidence rate of tongue lesions, reduced the tumor sizes, decreased the tumor multiplicity, and decreased the immunoreactivities of VDAC and mTOR. The molecular mechanisms involved in fenofibrate's ability to delay tumor development included the down-regulation of mTOR activity via TSC1/2-dependent signaling through activation of AMPK and inactivation of Akt, or via a TSC1/2-independent pathway through direct suppression of raptor. Our findings provide a molecular rationale whereby fenofibrate exerts anticancer and additional beneficial effects for the treatment of oral cancer patients.

Fusogenic-oligoarginine peptide-mediated silencing of the CIP2A oncogene suppresses oral cancer tumor growth in vivo

Intracellular delivery and endosomal escape of functional small interfering RNAs (siRNAs) remain major barriers limiting the clinical translation of RNA interference (RNAi)-based therapeutics. Recently, we demonstrated that a cell-penetrating endosome-disruptive peptide we synthesized, termed 599, enhanced the intracellular delivery and bioavailability of siRNAs designed to target the CIP2A oncoprotein (siCIP2A) into oral cancer cells and consequently inhibited oral cancer cell invasiveness and anchorage-independent growth in vitro. Thus, to further assess the therapeutic potential of the 599 peptide in mediating RNAi-based therapeutics for oral cancer and its prospective applicability in clinical settings, the objective of the current study was to determine whether intratumoral dosing of the 599 peptide–siCIP2A complex could induce silencing of CIP2A and consequently impair tumor growth using a xenograft oral cancer mouse model. Our results demonstrate that the 599 peptide is able to protect siRNAs from degradation by serum and ribonucleases in vitro and upon intratumoral injection in vivo, confirming the stability of the 599 peptide–siRNA complex and its potential for therapeutic utility. Moreover, 599 peptide-mediated delivery of siCIP2A to tumor tissue induces CIP2A silencing without any associated toxicity, consequently resulting in reduction of the mitotic index and significant inhibition of tumor growth. Together, these data suggest that the 599 peptide carrier is a clinically effective mediator of RNAi-based cancer therapeutics.

Abstract LB-106: Fusogenic-oligoarginine peptide-mediated silencing of the CIP2A oncogene suppresses oral cancer tumor growth in vivo

Abstract Intracellular delivery and endosomal escape of functional small interfering RNAs (siRNAs) remain major barriers limiting the clinical translation of RNA interference (RNAi)-based therapeutics. Recently, we demonstrated that a endosome-disruptive peptide we synthesized termed, 599, could enhance the intracellular delivery and bioavailability of siRNAs designed to target the CIP2A oncoprotein (siCIP2A) into oral cancer cells and consequently inhibit oral cancer cell invasiveness and anchorage-independent growth in vitro. Thus, to further assess the therapeutic potential of the 599 peptide in mediating RNAi-based therapeutics for oral cancer and its prospective applicability in clinical settings, the objective of the current study was to determine whether intratumoral dosing of the 599+siCIP2A complex could induce silencing of CIP2A and consequently impair tumor growth using a xenograft oral cancer mouse model. Our results demonstrated that the 599 peptide was able to protect siRNAs from degradation by serum and ribonucleases in vitro, confirming the stability of the 599+siRNA complex and its potential for in vivo utility. Moreover, 599 peptide-mediated delivery of siCIP2A to tumor tissue induced CIP2A silencing without any associated toxicity, consequently resulting in reduction of the mitotic index and significant inhibition of tumor growth. Together, these data suggest that the 599 peptide carrier could be a clinically effective mediator of RNAi-based cancer therapeutics. Citation Format: Angela Alexander-Bryant, Anca Dumitriu, Christopher Attaway, Hong Yu, Andrew Jakymiw. Fusogenic-oligoarginine peptide-mediated silencing of the CIP2A oncogene suppresses oral cancer tumor growth in vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-106. doi:10.1158/1538-7445.AM2015-LB-106

Orthotopic non-metastatic and metastatic oral cancer mouse models

Oral cancer is characterized by high morbidity and mortality with a predisposition to metastasize to different tissues, including lung, liver, and bone. Despite progress in the understanding of mutational profiles and deregulated pathways in oral cancer, patient survival has not significantly improved over the past decades. Therefore, there is a need to establish in vivo models that recapitulate human oral cancer metastasis to evaluate therapeutic potential of novel drugs. Here we report orthotopic tongue cancer nude mouse models to study oral cancer growth and metastasis using human metastatic (UMSCC2) and non-metastatic (CAL27) cell lines, respectively. Transduction of these cell lines with lentivirus expressing red fluorescent protein (DsRed) followed by injection into tongues of immunodeficient mice generated orthotopic tongue tumors that could be monitored for growth and metastasis by fluorescence measurement with an in vivo Imaging System (IVIS 200). The growth rates of CAL27-DsRed induced tumors were higher than UMSCC2-DsRed tumors after day 15, while UMSCC2-DsRed tumors revealed metastasis beginning on day 21. Importantly, UMSCC2 tumors metastasized to a number of tissues including the submandibular gland, lung, kidney, liver, and bone. Further, immunohistochemical analyses of tongue tumors induced by CAL27 and UMSCC2 cells revealed elevated expression of components of protumorigenic pathways deregulated in human cancers, including Cyclin D1, PCNA, Ki-67, LSD1, LOXL2, MT-MMP1, DPAGT1, E-cadherin, OCT4A, and H3K4me1/2. These orthotopic mouse models are likely to be useful tools for gaining insights into the activity and mechanisms of novel oral cancer drug candidates.

Genomic analysis to define molecular basis of aggressiveness in a mouse model of oral cancer.

To investigate the molecular basis underlying aggressive behavior in oral squamous cell carcinoma (OSCC), our laboratory developed a carcinogen-induced mouse oral cancer (MOC) cell line model that encompasses the growth and metastasis spectrum of its human counterpart. We performed next-generation sequencing (NGS) and gene expression microarray profiles to explore the genomic and transcriptional backgrounds of the differential MOC line phenotypes, as well as, the cross-species relevance of the model. Here we describe the comparative analysis of NGS (www.ncbi.nlm.nih.gov/biosample?LinkName=bioproject_biosample_all&from_uid=247825) and expression microarray (www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE50041) data from the MOC lines and corresponding human data, as described in our recent publication [1].

Demethylating Drugs as Novel Analgesics for Cancer Pain

In this study, we evaluated the analgesic potential of demethylating drugs on oral cancer pain. Although demethylating drugs could affect expression of many genes, we focused on the mu-opioid receptor (OPRM1) gene pathway, because of its role in pain processing. We determined the antinociceptive effect of OPRM1 re-expression in a mouse oral cancer model. Using a mouse oral cancer model, we determined whether demethylating drugs produced antinociception through re-expression of OPRM1. We then re-expressed OPRM1 with adenoviral transduction and determined if, and by what mechanism, OPRM1 re-expression produced antinociception. To determine the clinical significance of OPRM1 on cancer pain, we quantified OPRM1 methylation in painful cancer tissues and nonpainful contralateral normal tissues of patients with oral cancer, and nonpainful dysplastic tissues of patients with oral dysplasia. We demonstrated that OPRM1 was methylated in cancer tissue, but not normal tissue, of patients with oral cancer, and not in dysplastic tissues from patients with oral dysplasia. Treatment with demethylating drugs resulted in mechanical and thermal antinociception in the mouse cancer model. This behavioral change correlated with OPRM1 re-expression in the cancer and associated neurons. Similarly, adenoviral-mediated OPRM1 re-expression on cancer cells resulted in naloxone-reversible antinociception. OPRM1 re-expression on oral cancer cells in vitro increased β-endorphin secretion from the cancer, and decreased activation of neurons that were treated with cancer supernatant. Our study establishes the regulatory role of methylation in cancer pain. OPRM1 re-expression in cancer cells produces antinociception through cancer-mediated endogenous opioid secretion. Demethylating drugs have an analgesic effect that involves OPRM1.

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.

A surprising cross-species conservation in the genomic landscape of mouse and human oral cancer identifies a transcriptional signature predicting metastatic disease.

Improved understanding of the molecular basis underlying oral squamous cell carcinoma (OSCC) aggressive growth has significant clinical implications. Herein, cross-species genomic comparison of carcinogen-induced murine and human OSCCs with indolent or metastatic growth yielded results with surprising translational relevance. Murine OSCC cell lines were subjected to next-generation sequencing (NGS) to define their mutational landscape, to define novel candidate cancer genes, and to assess for parallels with known drivers in human OSCC. Expression arrays identified a mouse metastasis signature, and we assessed its representation in four independent human datasets comprising 324 patients using weighted voting and gene set enrichment analysis. Kaplan-Meier analysis and multivariate Cox proportional hazards modeling were used to stratify outcomes. A quantitative real-time PCR assay based on the mouse signature coupled to a machine-learning algorithm was developed and used to stratify an independent set of 31 patients with respect to metastatic lymphadenopathy. NGS revealed conservation of human driver pathway mutations in mouse OSCC, including in Trp53, mitogen-activated protein kinase, phosphoinositide 3-kinase, NOTCH, JAK/STAT, and Fat1-4. Moreover, comparative analysis between The Cancer Genome Atlas and mouse samples defined AKAP9, MED12L, and MYH6 as novel putative cancer genes. Expression analysis identified a transcriptional signature predicting aggressiveness and clinical outcomes, which were validated in four independent human OSCC datasets. Finally, we harnessed the translational potential of this signature by creating a clinically feasible assay that stratified patients with OSCC with a 93.5% accuracy. These data demonstrate surprising cross-species genomic conservation that has translational relevance for human oral squamous cell cancer. Clin Cancer Res; 20(11); 2873-84. ©2014 AACR.

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.

ADAM17‐mediated CD44 cleavage promotes orasphere formation or stemness and tumorigenesis in HNSCC

CD44, an extracellular matrix (ECM) receptor, has been described as a cancer stem cell marker in multiple cancers, including head and neck squamous cell carcinoma (HNSCC). HNSCC orasphere formation or stemness was characterized by cleavage of CD44, and thus we hypothesized that this proteolytic processing may be critical to stemness and tumorigenesis. We tested this hypothesis by examining the mechanisms that regulate this process in vitro and in vivo, and by exploring its clinical relevance in human specimens. Sphere assays have been used to evaluate stemness in vitro. Spheres comprised of HNSCC cells or oraspheres and an oral cancer mouse model were used to examine the significance of CD44 cleavage using stable suppression and inhibition approaches. These mechanisms were also examined in HNSCC specimens. Oraspheres exhibited increased levels of CD44 cleavage compared to their adherent counterparts. Given that disintegrin and metalloproteinase domain-containing protein 17 (ADAM17) is a major matrix metalloproteinase known to cleave CD44, we chemically inhibited and stably suppressed ADAM17 expression in HNSCC cells and found that these treatments blocked CD44 cleavage and abrogated orasphere formation. Furthermore, stable suppression of ADAM17 in HNSCC cells also diminished tumorigenesis in an oral cancer mouse model. Consistently, stable suppression of CD44 in HNSCC cells abrogated orasphere formation and inhibited tumorigenesis in vivo. The clinical relevance of these findings was confirmed in matched primary and metastatic human HNSCC specimens, which exhibited increased levels of ADAM17 expression and concomitant CD44 cleavage compared to controls. CD44 cleavage by ADAM17 is critical to orasphere formation or stemness and HNSCC tumorigenesis.

An Indirubin Derivative, Indirubin-3′-Monoxime Suppresses Oral Cancer Tumorigenesis through the Downregulation of Survivin

Oral cancer is the fourth most common cause of death from cancer in Taiwanese men. Indirubin-3′-monoxime (I3M), a potent cyclin-dependent kinase inhibitor, has therapeutic effects in other cancer cells. In this study, we carried out in vitro assays to test cell viability, cell cycle progression, apoptosis, cell migration and invasion in this cancer type. In addition, using an oral tumorigenic animal model, we examined target gene and protein expression using real time qPCR, immunoblotting and immunohistochemical staining. Our results demonstrate that I3M has an anti-proliferative effect in both Cal-27 and HSC-3 oral cancer cell lines and that treatment of Cal-27 and HSC-3 cells with I3M results in apoptosis through the activation of cytochrome c. In addition, I3M interrupts the cell cycle in Cal-27 cells in a dose-dependent manner by arresting cells in the G2/M phase. We also found that I3M suppresses migration and invasion in Cal-27 cells by inhibiting the expression of focal adhesion kinase, urokinase-type plasminogen inhibitor, and matrix metalloproteinase 9. Moreover, we identified survivin as a target protein in I3M-treated oral cancer cells. Using an oral cancer mouse model, we demonstrate that topical application of an adhesive gel composed of I3M and poly(vinyl alcohol) (I3M/PVA) has dose-dependent anti-tumorigenic effects. Following treatment, the expression of survivin protein and mRNA was downregulated in cancerous tissues. Furthermore, plasma survivin levels were also reduced in the I3M-treated mice. These results suggest that topical application of I3M, a drug synthesized from indirubin, which is found in Qing-Dai – has therapeutic potential for treating oral cancer.

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.

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

Analgesia Targeting IB4-Positive Neurons in Cancer-Induced Mechanical Hypersensitivity

Cancer patients often suffer from pain and most will be prescribed μ-opioids. μ-opioids are not satisfactory in treating cancer pain and are associated with multiple debilitating side effects. Recent studies show that μ and δ opioid receptors are separately expressed on IB4 (−) and IB4 (+) neurons, which control thermal and mechanical pain, respectively. In this study we investigated IB4 (+) and IB4 (−) neurons in mechanical and thermal hypersensitivity in an orthotopic mouse oral cancer model. We used a δ opioid receptor agonist and a P2X3 antagonist to target IB4 (+) neurons and to demonstrate that this subset plays a key role in cancer-induced mechanical allodynia, but not in thermal hyperalgesia. Moreover, selective removal of IB4 (+) neurons using IB4-saporin impacts cancer-induced mechanical but not thermal hypersensitivity. Our results demonstrate that peripherally administered pharmacological agents targeting IB4 (+) neurons, such as a selective δ-opioid receptor agonist or P2X3 antagonist, might be useful in treating oral cancer pain. PerspectiveTo clarify the mechanisms of oral cancer pain, we examined the differential role of IB4 (+) and IB4 (−) neurons. Characterization of these 2 subsets of putative nociceptors is important for further development of effective clinical cancer pain relief.

Comparative analysis of tumor-infiltrating lymphocytes in a syngeneic mouse model of oral cancer.

To perform a comparative analysis of infiltrating immune cells in a newly developed C57BL/6 background syngeneic transplantable mouse oral cancer (MOC) model. Scientific study in an academic medical center. Use of carcinogen-induced tumorigenesis, tissue culture, cell line transplantation, and flow cytometric analysis techniques. Previously, the authors established a series of cell line models that displayed dichotomous growth phenotypes when transplanted into immunocompetent mice. They now show that the indolent growth pattern of the MOC1-generated tumors is associated with increased baseline and inducible major histocompatibility complex class I expression and increased CD8(+) T-cell infiltration into the tumor microenvironment. Conversely, the aggressive and metastatic pattern of MOC2-generated tumors has decreased basal and inducible class I expression and is associated with FOXP3(+)CD4(+) regulatory T-cell infiltration. Delayed primary tumor growth after targeted monoclonal antibody therapy of these FOXP3(+) regulatory cells further suggests that these immune cells contribute to the aggressive phenotype of MOC2. 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.