Modeling epithelial homeostasis and perturbation in three-dimensional human esophageal organoids.

Background: Esophageal organoids from a variety of pathologies including cancer are grown in Advanced Dulbecco's Modified Eagle Medium-Nutrient Mixture F12 (hereafter ADF). However, the currently available ADF-based formulations are suboptimal for normal human esophageal organoids, limiting the ability to compare normal esophageal organoids with those representing a given disease state. Methods: We have utilized immortalized normal human esophageal epithelial cell (keratinocyte) lines EPC1 and EPC2 and endoscopic normal esophageal biopsies to generate three-dimensional (3D) organoids. To optimize ADF-based medium, we evaluated the requirement of exogenous epidermal growth factor (EGF) and inhibition of transforming growth factor-(TGF)-ß receptor-mediated signaling, both key regulators of proliferation of human esophageal keratinocytes. We have modeled human esophageal epithelial pathology by stimulating esophageal 3D organoids with interleukin (IL)-13, an inflammatory cytokine, or UAB30, a novel pharmacological activator of retinoic acid signaling. Results: The formation of normal human esophageal 3D organoids was limited by excessive EGF and intrinsic TGFß receptor-mediated signaling. In optimized HOME0, normal human esophageal organoid formation was improved, whereas IL-13 and UAB30 induced epithelial changes reminiscent of basal cell hyperplasia, a common histopathologic feature in broad esophageal disease conditions including eosinophilic esophagitis. Conclusions: HOME0 allows modeling of the homeostatic differentiation gradient and perturbation of the human esophageal epithelium while permitting a comparison of organoids from mice and other organs grown in ADF-based media.

Effects of methylene blue photodynamic therapy on oral carcinoma and leukoplakia cells.

Objective: Methylene blue (MB) is a readily available and affordable substrate that can be used as a photosensitizer for photodynamic therapy (PDT). The objective of this study was to determine if PDT with MB can downregulate matrix metalloproteinases (MMPs) related to oral carcinoma. Methods: Cell cultures of oral squamous cell carcinoma (CA-9-22), oral leukoplakia (MSK-Leuk1), and immortalized keratinocytes (Rhek-1A) were photosensitized with MB and treated with PDT. MMP-9 gene expression was interrogated via qRT-PCR. The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to confirm the efficacy of MB PDT. Results: MMP-9 gene expression was found to be significantly decreased in oral carcinoma, leukoplakia, and immortalized keratinocytes with use of MB PDT. Conclusion: This work demonstrates that MB-mediated PDT can downregulate MMPs which are critical to the invasion and metastasis of oral cancer. These results suggest that MB PDT could be a clinically significant and cost-effective treatment for oral leukoplakia and carcinoma. Level of Evidence: NA.

Preclinical evidence for pioglitazone and bexarotene combination in oral cancer chemoprevention.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) requires new treatments and targeted approaches to improve survival. The peroxisome proliferator-activated receptor γ (PPARγ) and retinoic X receptor alpha (RXRα) nuclear receptor pathways may be targetable with repurposed Food and Drug Administration (FDA)-approved agents for prevention and treatment. METHODS: Oral cancer and leukoplakia cell lines were treated with the PPARγ agonist (pioglitazone) and RXRα activator (bexarotene). PPARγ activation, cellular proliferation, apoptosis activity and phenotype, including the pharmacodynamic marker, involucrin (IVL), were subsequently analyzed using a reporter gene assay, genomic data, MTT assay and western blot. RESULTS: Microarray analysis of HNSCC tumor versus normal tissue shows IVL expression is significantly increased in normal tissue compared to HNSCC tumors (p < 0.0001). In MSK Leuk1 and CA 9-22 cell lines, pioglitazone increases PPARγ DNA binding activity and IVL promoter activity in a dose dependent manner (p < 0.01 and p < 0.0001). Combination treatment with pioglitazone and bexarotene increases PPARγ DNA binding activity and IVL promoter activity (p < 0.01 and p < 0.0001). MTT analysis shows decreases in cell proliferation when cells are treated with pioglitazone and bexarotene. Decreases in cell proliferation are significant to at least p < 0.05 for all combination versus single agent treatments. Western blot on whole-cell lysate from cells treated with pioglitazone and bexarotene alone or in combination for IVL showed increased protein levels with combination treatment. CONCLUSIONS: Targeting the PPARγ/RXRα heterodimer with pioglitazone and bexarotene was effective in this preclinical project. This was functional in both preneoplastic and oral cancer cell lines. A better understanding of the molecular mechanism on downstream effects on cellular proliferation could potentially have implications clinically, both in oral preneoplasia and possibly head and neck cancer; however, more research needs to be done to explore the potential these medications have in chemoprevention.

Inhibition of mTOR signaling and clinical activity of metformin in oral premalignant lesions.

BACKGROUNDThe aberrant activation of the PI3K/mTOR signaling circuitry is one of the most frequently dysregulated signaling events in head and neck squamous cell carcinoma (HNSCC). Here, we conducted a single-arm, open-label phase IIa clinical trial in individuals with oral premalignant lesions (OPLs) to explore the potential of metformin to target PI3K/mTOR signaling for HNSCC prevention.METHODSIndividuals with OPLs, but who were otherwise healthy and without diabetes, underwent pretreatment and posttreatment clinical exam and biopsy. Participants received metformin for 12 weeks (week 1, 500 mg; week 2, 1000 mg; weeks 3-12, 2000 mg daily). Pretreatment and posttreatment biopsies, saliva, and blood were obtained for biomarker analysis, including IHC assessment of mTOR signaling and exome sequencing.RESULTSTwenty-three participants were evaluable for response. The clinical response rate (defined as a ≥50% reduction in lesion size) was 17%. Although lower than the proposed threshold for favorable clinical response, the histological response rate (improvement in histological grade) was 60%, including 17% complete responses and 43% partial responses. Logistic regression analysis revealed that when compared with never smokers, current and former smokers had statistically significantly increased histological responses (P = 0.016). Remarkably, a significant correlation existed between decreased mTOR activity (pS6 IHC staining) in the basal epithelial layers of OPLs and the histological (P = 0.04) and clinical (P = 0.01) responses.CONCLUSIONTo our knowledge this is the first phase II trial of metformin in individuals with OPLs, providing evidence that metformin administration results in encouraging histological responses and mTOR pathway modulation, thus supporting its further investigation as a chemopreventive agent.TRIAL REGISTRATIONNCT02581137FUNDINGNIH contract HHSN261201200031I, grants R01DE026644 and R01DE026870.

Chemoprevention of Lung Carcinogenesis by Dietary Nicotinamide and Inhaled Budesonide.

Nicotinamide, the amide form of vitamin B3, and budesonide, a synthetic glucocorticoid used in the treatment of asthma, were evaluated to determine their individual and combinational chemopreventive efficacy on benzo(a)pyrene-induced lung tumors in female A/J mice. Nicotinamide fed at a dietary concentration of 0.75% significantly inhibited tumor multiplicity. Nicotinamide by aerosol inhalation at doses up to 15 mg/kg/day did not result in a statistically significant reduction in tumor multiplicity. Finally, dietary nicotinamide was administered with aerosol budesonide and tumor multiplicity reduced by 90% at 1 week and 49% at 8 weeks post last carcinogen dose. We conclude nicotinamide is an effective and safe agent for lung cancer dietary prevention at both early- and late-stage carcinogenesis and that efficacy is increased with aerosol budesonide. Combination chemoprevention with these agents is a well-tolerated and effective strategy which could be clinically advanced to human studies.

PPARγ-Mediated p21 Induction in Aerodigestive Preneoplastic Cell Lines.

INTRODUCTION: Oral leukoplakia is defined as a mucous membrane disorder characterized by white patches that cannot be scraped off. Leukoplakia is the most frequent, potentially premalignant oral mucosa disorder and a good candidate for chemopreventive therapies. Pioglitazone activates peroxisome proliferator-activated receptor gamma (PPARγ), which forms a complex with nuclear cofactors and regulates gene expression of a variety of cell-cycle proteins and is currently being tested preclinically and clinically in aerodigestive cancer prevention. METHODS: In the present study, we hypothesized that pioglitazone would decrease proliferation of human leukoplakia cells (MSK Leuk1) and transformed bronchial epithelial cells (BEAS-2B) through regulatory changes of G1 checkpoint protein regulators, p21 and cyclin-D1. MSK Leuk1 and BEAS-2B cells were treated with pioglitazone and assayed for cell proliferation and p21 transcriptional activity. RESULTS: We discovered pioglitazone significantly inhibited cell proliferation in a dose-dependent fashion. We also observed p21 protein induction after treatment with pioglitazone, which was preceded by measurable increases in p21 mRNA induction. CONCLUSIONS: We conclude the PPARγ activator, pioglitazone, can activate p21, which is associated with decreased proliferation in 2 aerodigestive preneoplastic cell lines. In addition, the p21 gene may be a potential hypothesis-driven biomarker in translational studies of pioglitazone as a chemoprevention agent for aerodigestive cancer.

Effects of talactoferrin alpha on lung adenoma prevention in A/J mice June 2, 2016.

Talactoferrin alpha is a promising non-toxic solid tumor cancer agent that met with success in the treatment of early-stage lung cancer clinically in humans. It is well-tolerated, anddendritic cell-stimulation is a target. We tested the efficacy of this agent in a chemoprevention setting in A/J mice. All groups received benzo[a]pyrene (B[a]P) by oral gavage in three doses of 3 mg/kg body weight over the course of one week. Animals were then randomized into 5 groups of 24 mice per group based on weight. Experimental diets oftalactoferrin alpha (Agennix Inc., Indianapolis, IN), at 1.40% and 0.42% of the diet, were started one week or eight weeks after the last dose of B[a]P. Animals were continued on the feeding schedule, weighed weekly, and monitored for toxicity. The study was concluded 16 weeks after administration of B[a]P. The agent was well-tolerated for the duration of the experiment and there was no observable toxicity or weight change. The average number of adenomas per animal was 14.04 ± 0.93 (N=24) in the control group, 18.14 ± 1.45 (N=22) in the early low-dose group, 16.70 ± 1.30 (N=23) in the late low-dose group, 15.09 ± 1.41 (N=23) in the early high-dose group and 14.46 ± 1.21 (N=24) in the late high-dose group. We conclude talactoferrinalpha is well-tolerated. However, it did not inhibit carcinogenesis at a dose of 1.4% or 0.42% of the diet, which equates to human doses of 1.12 g/kg/day or 0.336 g/kg/day.

Thiazolidinediones abrogate cervical cancer growth.

Peroxisome proliferator-activated receptor gamma (PPAR γ) is activated by thiazolidinedione drugs (TZDs) and can promote anti-cancer properties. We used three TZDs (pioglitazone, rosiglitazone, and ciglitazone) to target cervical cancer cell lines and a nude mouse animal model. Each agent increased activation of PPAR γ, as judged by a luciferase reporter gene assay in three HPV-associated cell lines (CaSki, SiHa, and HeLa cells) while decreasing cellular proliferation in a dose-dependent manner. They also promoted Oil Red O accumulation in treated cell lines and upregulated the lipid differentiation marker adipsin. Interestingly, xenograft HeLa tumors in nude mice treated with 100mg/kg/day pioglitazone exhibited decreased growth compared to control mice or mice treated with standard cervical chemotherapy. In conclusion, TZDs slow tumor cell growth in vitro and in vivo with decreases in cell proliferation and increases in PPAR γ and adipsin. These agents may be interesting treatments or treatment adjuncts for HPV-associated cancers or perhaps even precancerous conditions.

Fixed-Dose Combinations of Pioglitazone and Metformin for Lung Cancer Prevention.

Combination treatment with pioglitazone and metformin is utilized clinically in the treatment of type II diabetes. Treatment with this drug combination reduced the development of aerodigestive cancers in this patient population. Our goal is to expand this treatment into clinical lung cancer chemoprevention. We hypothesized that dietary delivery of metformin/pioglitazone would prevent lung adenoma formation in A/J mice in a benzo[a]pyrene (B[a]P)-induced carcinogenesis model while modulating chemoprevention and anti-inflammatory biomarkers in residual adenomas. We found that metformin (500 and 850 mg/kg/d) and pioglitazone (15 mg/kg/d) produced statistically significant decreases in lung adenoma formation both as single-agent treatments and in combination, compared with untreated controls, after 15 weeks. Treatment with metformin alone and in combination with pioglitazone resulted in statistically significant decreases in lung adenoma formation at both early- and late-stage interventions. Pioglitazone alone resulted in significant decreases in adenoma formation only at early treatment intervention. We conclude that oral metformin is a viable chemopreventive treatment at doses ranging from 500 to 1,000 mg/kg/d. Pioglitazone at 15 mg/kg/d is a viable chemopreventive agent at early-stage interventions. Combination metformin and pioglitazone performed equal to metformin alone and better than pioglitazone at 15 mg/kg/d. Because the drugs are already FDA-approved, rapid movement to human clinical studies is possible. Cancer Prev Res; 10(2); 116-23. ©2017 AACR.

Functional activation of PPARγ in human upper aerodigestive cancer cell lines.

Upper aerodigestive cancer is an aggressive malignancy with relatively stagnant long-term survival rates over 20 yr. Recent studies have demonstrated that exploitation of PPARγ pathways may be a novel therapy for cancer and its prevention. We tested whether PPARγ is expressed and inducible in aerodigestive carcinoma cells and whether it is present in human upper aerodigestive tumors. Human oral cancer CA-9-22 and NA cell lines were treated with the PPAR activators eicosatetraynoic acid (ETYA), 15-deoxy-δ- 12,14-prostaglandin J2 (PG-J2), and the thiazolidinedione, ciglitazone, and evaluated for their ability to functionally activate PPARγ luciferase reporter gene constructs. Cellular proliferation and clonogenic potential after PPARγ ligand treatment were also evaluated. Aerodigestive cancer specimens and normal tissues were evaluated for PPARγ expression on gene expression profiling and immunoblotting. Functional activation of PPARγ reporter gene constructs and increases in PPARγ protein were confirmed in the nuclear compartment after PPARγ ligand treatment. Significant decreases in cell proliferation and clonogenic potential resulted from treatment. Lipid accumulation was induced by PPARγ activator treatment. 75% of tumor specimens and 100% of normal control tissues expressed PPARγ RNA, and PPARγ protein was confirmed in 66% of tumor specimens analyzed by immunoblotting. We conclude PPARγ can be functionally activated in upper aerodigestive cancer and that its activation downregulates several features of the neoplastic phenotype. PPARγ expression in human upper aerodigestive tract tumors and normal cells potentially legitimizes it as a novel intervention target in this disease. © 2016 Wiley Periodicals, Inc.

Safety and Preclinical Efficacy of Aerosol Pioglitazone on Lung Adenoma Prevention in A/J Mice.

Pioglitazone is a PPARγ agonist commonly prescribed for the clinical treatment of diabetes. We sought to expand its use to lung cancer prevention in a benzo[a]pyrene (B[a]P) mouse model with direct lung delivery via inhalation. Initially, we conducted inhalational toxicity experiments with 0, 15, 50, 150, and 450 µg/kg body weight/day pioglitazone in 40 A/J mice. We examined the animals for any physical toxicity and bronchoalveolar lavage fluids for inflammatory and cytotoxicity markers. Doses up to and including 450 µg/kg bw/d failed to demonstrate toxicity with aerosol pioglitazone. For chemoprevention experiments, A/J mice were randomized to treatment groups of inhaled doses of 0, 50, 150, or 450 µg/kg bw/d pioglitazone 1 or 8 weeks after the last dose of B[a]P. For the early treatment group, we found up to 32% decrease in lung adenoma formation with 450 µg/kg bw/d pioglitazone. We repeated the treatments in a second late-stage experiment and found up to 44% decreases in lung adenoma formation in doses of pioglitazone of 150 and 450 µg/kg bw/day. Both the early- and the late-stage experiments demonstrated biologically relevant and statistically significant decreases in adenoma formation. We conclude that aerosol pioglitazone is well-tolerated in the A/J mouse model and a promising chemoprevention agent for the lower respiratory tract. Cancer Prev Res; 10(2); 124-32. ©2016 AACR.

N-methylnitrosourea­induced carcinoma as a model for laryngeal carcinogenesis.

BACKGROUND: Preclinical animal models to study laryngeal cancer are nonexistent. The purpose of this study was to describe a novel mice laryngeal cancer model. METHODS: A total of 18 six-week-old A/J mice were used. Animals underwent microdirect laryngoscopy, superficial larynx scratching, and instillation of N-methyl-N-nitrosourea (MNU) at 2 different concentrations (15 µL and 30 µL) or dimethyl sulfoxide (DMSO) to the control group directly to the larynx. Mice received a total of 5 instillations of MNU or DMSO at 1-week intervals. Mice were euthanized at 20 and 30 weeks after the last intervention and laryngeal histology was analyzed. RESULTS: Laryngeal instillation of MNU caused a 60% cancer conversion in the study group. CONCLUSION: Our findings demonstrate the feasibility of developing a murine laryngeal carcinogenesis model using direct topical instillation of MNU. This is the first murine model of laryngeal cancer and has great potential for evaluating new agents for chemoprevention and treatment for laryngeal carcinoma.

Quantitative proteomic analysis of oral brush biopsies identifies secretory leukocyte protease inhibitor as a promising, mechanism-based oral cancer biomarker.

A decrease in the almost fifty percent mortality rate from oral cancer is needed urgently. Improvements in early diagnosis and more effective preventive treatments could affect such a decrease. Towards this end, we undertook for the first time an in-depth mass spectrometry-based quantitative shotgun proteomics study of non-invasively collected oral brush biopsies. Proteins isolated from brush biopsies from healthy normal tissue, oral premalignant lesion tissue (OPMLs), oral squamous cell carcinoma (OSCC) and matched control tissue were compared. In replicated proteomic datasets, the secretory leukocyte protease inhibitor (SLPI) protein stood out based on its decrease in abundance in both OPML and OSCC lesion tissues compared to healthy normal tissue. Western blotting in additional brushed biopsy samples confirmed a trend of gradual decreasing SLPI abundance between healthy normal and OPML tissue, with a larger decrease in OSCC lesion tissue. A similar SLPI decrease was observed in-vitro comparing model OPML and OSCC cell lines. In addition, exfoliated oral cells in patients' whole saliva showed a loss of SLPI correlated with oral cancer progression. These results, combined with proteomics data indicating a decrease in SLPI in matched healthy control tissue from OSCC patients compared to tissue from healthy normal tissue, suggested a systemic decrease of SLPI in oral cells correlated with oral cancer development. Finally, in-vitro experiments showed that treatment with SLPI significantly decreased NF-kB activity in an OPML cell line. The findings indicate anti-inflammatory activity in OPML, supporting a mechanistic role of SLPI in OSCC progression and suggesting its potential for preventative treatment of at-risk oral lesions. Collectively, our results show for the first time the potential for SLPI as a mechanism-based, non-invasive biomarker of oral cancer progression with potential in preventive treatment.

Enforced expression of nuclear factor kappa B in p53 deficient keratinocytes induces cell cycle, angiogenic potential and tumorigenesis.

Multiple genetic mutations with subsequent molecular events are required for progression of normal epithelial cells to cancer, with p53 mutations being a very common event in squamous carcinogenesis. Upregulation of nuclear factor kappa B (NF-κB) is an associated feature of malignancy, however studies have not examined purposeful overexpression of the NF-κB p65 subunit in in vitro models of oral carcinogenesis. Our objective is to demonstrate that NF-κB p65 transfection into p53 deficient Rhek keratinocytes produces carcinogenic progression. We constitutively over-expressed NF-κB p65 in Rhek keratinocytes, previously immortalized by SV 40 thus inactivating p53, and studied NF-κB dependent events. NF-κB p65 overexpression provided functional upregulation of NF-κB and produced cyclin D1-mediated proliferation and interleukin 8 transcription and secretion. Consequently, we demonstrated tumorigenesis in athymic mice with NF-κB p65 overexpressing cells. We conclude NF-κB p65 overexpression in p53 inactivated immortalized keratinocytes produces tumorigenesis, and that this single alteration in NF-κB expression on a p53 inactivated background is sufficient for squamous carcinogenesis features, thus providing evidence that p65 may act as a gain of function oncogene in this setting.

The effect of indomethacin on paclitaxel sensitivity and apoptosis in oral squamous carcinoma cells: the role of nuclear factor-κB inhibition.

OBJECTIVE: To investigate new strategies to intensify chemosensitivity in head and neck squamous cell carcinoma. DESIGN: Oral squamous carcinoma cells were examined for nuclear factor-κB (NF-κB) activation and binding activity by paclitaxel, an agent currently used in head and neck cancer chemotherapy. Electromobility shift assays were used to assess the effect of indomethacin on NF-κB binding activity. Cell proliferation assays were used to study cell sensitivity to paclitaxel. To examine whether cytotoxicity could be increased by specifically inhibiting NF-κB, a dominant negative cell line, inhibitor kappa B-alpha (IκBα), was stably expressed in CA-9-22 cells. RESULTS: Paclitaxel possessed the capacity to functionally activate NF-κB, as demonstrated by luciferase reporter gene assays and electromobility shift assay. Indomethacin was able to inhibit paclitaxel-mediated NF-κB activation and promote apoptosis of paclitaxel-treated cells at 24 hours. Indomethacin augmented the paclitaxel cell-killing effect. The dominant negative IκBα cell line exhibited increased chemosensitization to paclitaxel by 2- to 10-fold. CONCLUSIONS: Paclitaxel has the capacity to activate NF-κB in oral squamous carcinoma cells. Indomethacin can reverse this activation to decrease cell proliferation and increase apoptosis. Treatment strategies that combine paclitaxel with indomethacin may have therapeutic benefits attributable to paclitaxel chemosensitization through NF-κB inhibition.

Tobacco carcinogen mediated up-regulation of AP-1 dependent pro-angiogenic cytokines in head and neck carcinogenesis.

Tobacco is notably genotoxic and associated with head and neck carcinogenesis. Cigarette carcinogens have the capacity to alter early response gene expression in tobacco-related malignancies via genes such as nuclear factor kappa B (NFκB). A number of early response gene activation events are also facilitated by fos/jun activator protein 1 (AP-1) associated pathways. In the present study, we hypothesize that tobacco products may induce microenvironment alterations, promoting angiogenesis and providing a permissive environment for head and neck cancer progression. In an in vitro analysis, we employed immortalized oral keratinocyte (HOK-16B) and laryngeal squamous carcinoma (UM-SCC-11A) cells to investigate interleukin (IL)-8 and vascular endothelial growth factor (VEGF) induction by cigarette smoke condensate (CSC). IL-8 and VEGF expression is based on interactions between NFκB, AP-1, and NF-IL6. We identified at least 1.5-fold dose-dependent induction of AP-1, VEGF, and IL-8 promoter/reporter gene activity after 24 h exposure to CSC. Next, we stably transfected UM-SCC-11A cells with A-Fos, a dominant negative AP-1 protein. Treatment with CSC of the A-Fos cell lines compared to empty vector controls significantly down-regulated AP-1, VEGF, and IL-8 promoter/reporter gene expression. We also performed ELISAs and discovered significant up-regulation of IL-8 and VEGF secretion by UMSCC 11A after treatment with phorbol 12-myristate 13-acetate, tumor necrosis factor alpha, and CSC, which was down-regulated by the A-Fos dominant negative protein. We conclude tobacco carcinogens up-regulate AP-1 activity and AP-1 dependent IL-8 and VEGF gene expression in head and neck cancer. This up-regulation may promote an angiogenic phenotype favoring invasion in both premalignant and squamous cancer cells of the head and neck.

Cigarette smoke condensate induces nuclear factor kappa-b activity and proangiogenic growth factors in aerodigestive cells.

OBJECTIVES/HYPOTHESIS: Aerodigestive cancer risk of both lung and head and neck cancers has been linked to the genotoxic effects of tobacco use. These effects include upregulation of nuclear factor kappa-B (NFkappaB) and its downstream products associated with both lung and head and neck cancer malignant progression. STUDY DESIGN: Bench Research. METHODS: In the present study we examined the effects of cigarette smoke condensate on functional activation of NFkappaB in human papillomavirus (HPV)-transformed oral cavity cells (HOK 16B cells) and transformed bronchial epithelium (Beas2B cells) using the head and neck squamous cancer cell line, UMSCC 38, as a comparison. Luciferase reporter gene assays with two types of transiently transfected NFkappaB reporter genes were employed and downstream NFkappaB-dependent products, interleukin-6, interleukin-8, and vascular endothelial growth factor, were assayed by enzyme-linked immunosorbent assay. RESULTS: All cell lines were able to dose dependently activate NFkappaB reporter genes after exposure to cigarette smoke condensate (P < .05). However, the HPV premalignant, transformed cell line had a much more robust NFkappaB response (3.45-fold) versus the squamous cancer cell line (1.62-fold) and SV40 transformed Beas2B (1.83). Both NFkappaB reporter genes had similar response curves. CONCLUSIONS: This study demonstrates cigarette smoke products might be more potent promoters of an NFkappaB-dependent progression from HPV+ premalignancy to cancer rather than after tumors are established. Future studies should focus on abrogating NFkappaB increases during malignant progression and premalignancy. This might be even more relevant in the HPV+ patient with premalignancy.

TNF-alpha drives matrix metalloproteinase-9 in squamous oral carcinogenesis.

OBJECTIVES/HYPOTHESIS: It is well known that invasion is a seminal event in the progression of oral and other head and neck carcinoma sites. We have previously demonstrated tumor necrosis factor (TNF)-alpha and its dependent cytokines are upregulated in saliva during oral carcinogenesis. TNF-dependent events stimulate nuclear factor (NF)-kappaB and many NF-kappaB-dependent genes are associated with cancer progression. MATERIALS AND METHODS: In the present study, we examined NF-kappaB stimulation of matrix metalloproteinase (MMP)-9 in a precancerous keratinocyte cell line that models leukoplakia (Rhek cells). We stimulated Rhek cells with both TNF-alpha and phorbol myristate acetate, known stimulants of NF-kappaB. We then assayed MMP-9 transcription and secretion by luciferase reporter genes, quantitative real-time polymerase chain reaction, and fluorometric enzyme-linked immunosorbent serologic assay. RESULTS: We discovered that the MMP-9 promoter was significantly stimulated by phorbol myristate acetate and TNF-alpha on luciferase reporter gene assays. Further, we uncovered that functional MMP-9 promoter activation was accompanied by significant increases in MMP-9 gene expression, as judged by quantitative real-time polymerase chain reaction. Functional activation of the MMP-9 protein was stimulated by TNF-alpha and PMA on a fluorescent enzyme-linked immunosorbent serologic assay. Finally, we searched our salivary proteomic database for increases in MMP-9 and discovered it was the third most significant protein in salivas of oral cavity cancer patients over normal controls. CONCLUSIONS: We conclude the milieu cytokine, TNF-alpha, has the capacity to provide stimulation of events related to early invasion of oral cavity cancer, as judged by its ability to stimulate MMP-9.

The potential role of neutrophils in promoting the metastatic phenotype of tumors releasing interleukin-8.

In the last decade, several groups have shown a direct correlation between the inappropriate or ectopic release of interleukin (IL)-8 by tumor cells in vitro and their growth and metastatic potential using in vivo models of tumor growth. IL-8 is a potent neutrophil chemoattractant. Neutrophils, as "early responders" to wounds and infections, release enzymes to remodel the extracellular matrix of the tissues through which they migrate to reach the site of the wound or infection. It is proposed that the host's cellular response to IL-8 released by tumor cells enhances angiogenesis and contributes to tumor growth and progression. The activities released by the responding neutrophils could serve as enablers of tumor cell migration through the extracellular matrix, helping them enter the vasculature and journey to new, metastatic sites. The reactive oxygen species produced by neutrophilic oxidases to kill invading organisms have the potential to interact with tumor cells to attenuate their apoptotic cascade and increase their mutational rate. It is proposed that the increase in metastatic potential of tumors ectopically releasing IL-8 is, in part, attributable to their ability to attract neutrophils. Discussed here are possible mechanisms by which the neutrophils responding to ectopic IL-8 contribute to the in vivo growth, progression, and metastatic potential of tumor cells. Possible targets are also presented for the development of therapies to attenuate the effects of the ectopic IL-8 release by tumor cells.

Atypical methylation of the interleukin-8 gene correlates strongly with the metastatic potential of breast carcinoma cells.

Previously, we have shown that a strong correlation exists between the metastatic potential of breast carcinoma cell lines and their ectopic expression of IL-8. The undifferentiated, highly metastatic cell lines with high metastatic potential produce much more IL-8 than their differentiated lower metastatic counterparts. After eliminating the possibility that transcription factor activity was responsible for differences in IL-8 release, we examined the IL-8 gene for possible epigenetic modifications. Here, we report an aberrant methylation pattern that may be responsible for the differences in IL-8 release between the high and low metastatic cell lines. We determined that none of the deoxycytidylate-phosphate-deoxyguanylate (CpG) sites in the reported IL-8 promoter were methylated in either cell type. Much further upstream in the IL-8 gene, two CpG sites were identified that are differentially methylated. These two sites were fully methylated in the high metastatic cell lines, which produce large quantities of IL-8 and remain unmethylated in the low metastatic cell lines where the IL-8 gene is relatively silent. The DNA methylation results presented here differ from the common epigenetic paradigm in which methylation of promoter CpG islands silences gene expression, suggesting that there are additional epigenetic control mechanisms that as yet have not been fully appreciated or explored.