Metabolic and Metabolomic Effects of Metformin in Murine Model of Pulmonary Adenoma Formation.

Epidemiologic studies of diabetic patients treated with metformin identified significantly lower incidences of cancer. From this, there is growing interest in the use of metformin to treat and prevent cancer. Studies have investigated chemopreventive mechanisms including alterations in calorie intake, cancer metabolism, and cell signaling. Repurposing the drug is challenging due to its metabolic effects and non-uniform effects on different types of cancer. In our previously published studies, we observed that benzo[a]pyrene treated mice receiving metformin significantly reduced lung adenomas; however, mice had reduced weight gain. In this study, we compared chemoprevention diets with and without metformin to evaluate the effects of diet vs. effects of metformin. We also performed tandem mass spectrometry on mouse serum to assess metabolomic alterations associated with metformin treatment. In metformin cohorts, the rate of weight gain was reduced, but weights did not vary between diets. There was no weight difference between diets without metformin. Interestingly, caloric intake was increased in metformin treated mice. Metabolomic analysis revealed metabolite alterations consistent with metformin treatment. Based on these results, we conclude that previous reductions in lung adenomas may have been occurred from anticancer effects of metformin rather than a potentially toxic effect such as calorie restriction.

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.

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.

Inhaled fosamprenavir for laryngopharyngeal reflux: Toxicology and fluid dynamics modeling.

Objectives: Approximately 25% of Americans suffer from laryngopharyngeal reflux (LPR), a disease for which no effective medical therapy exists. Pepsin is a predominant source of damage during LPR and a key therapeutic target. Fosamprenavir (FOS) inhibits pepsin and prevents damage in an LPR mouse model. Inhaled FOS protects at a lower dose than oral; however, the safety of inhaled FOS is unknown and there are no inhalers for laryngopharyngeal delivery. A pre-Good Lab Practice (GLP) study of inhaled FOS was performed to assess safety and computational fluid dynamics (CFD) modeling used to predict the optimal particle size for a laryngopharyngeal dry powder inhaler (DPI). Methods: Aerosolized FOS, amprenavir (APR), or air (control) were provided 5 days/week for 4 weeks (n = 6) in an LPR mouse model. Organs (nasal cavity, larynx, esophagus, trachea, lung, liver, heart, and kidney) were assessed by a pathologist and bronchoalveolar lavage cytokines and plasma cardiotoxicity markers were assessed by Luminex assay. CFD simulations were conducted in a model of a healthy 49-year-old female. Results: No significant increase was observed in histologic lesions, cytokines, or cardiotoxicity markers in FOS or APR groups relative to the control. CFD predicted that laryngopharyngeal deposition was maximized with aerodynamic diameters of 8.1-11.5 µm for inhalation rates of 30-60 L/min. Conclusions: A 4-week pre-GLP study supports the safety of inhaled FOS. A formal GLP assessment is underway to support a phase I clinical trial of an FOS DPI for LPR. Level of Evidence: NA.

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.

Using Bland-Altman Analysis to Identify Appropriate Clonogenic Assay Colony Counting Techniques.

OBJECTIVE: Determine the interchangeability of various methods utilized for counting colonies in clonogenic assays. METHODS: Clonogenic assays of 2 head and neck cancer cell lines were counted through 4 different counting modalities: Manual counting pen, via microscope, 1 publicly available automated algorithm, and a semiautomated algorithm presented by the authors. Each method counted individual wells (N = 24). Pen and microscopic counts were performed by 2 observers. Parameters included both low-growth (<150 colonies/well) and high-growth (>150 colonies/well) cell lines. Correlational and Bland-Altman analyses were performed using SPSS software. RESULTS: Interobserver manual pen count correlation R2 value in both growth conditions was 0.902; controlling for only low-growth conditions decreased R2 to 0.660. Correlation of microscopic versus pen counts R2 values for observers 1 and 2 were 0.955 and 0.775, respectively. Comparing techniques, Bland-Altman revealed potential bias with respect to the magnitude of measurement (P < .001) for both observers. Correlation of microscopic counts for both interobserver (R2 = 0.902) and intraobserver (R2 = 0.916) were analyzed. Bland-Altman revealed no bias (P = .489). Automated versus microscopic counts revealed no bias between methodologies (P = .787) and a lower correlation coefficient (R2 = 0.384). Semiautomated versus microscopic counts revealed no bias with respect to magnitude of measurement for either observer (P = .327, .229); Pearson correlation was 0.985 (R2 = 0.970) and 0.965 (R2 = 0.931) for observer 1 and 2. Semiautomated versus manual pen colony counts revealed a significant bias with respect to magnitude of measurement (P < .001). CONCLUSION: Counting with a manual pen demonstrated significant bias when compared to microscopic and semiautomated colony counts; 2 methods were deemed to be interchangeable. Thus, training algorithms based on manual counts may introduce this bias as well. Algorithms trained to select colonies based on size (pixels2) and shape (circularity) should be prioritized. Solely relying on Bland-Altman or correlational analyses when determining method interchangeability should be avoided; they rather should be used in conjunction.

Potential Roles of Activin in Head and Neck Squamous Cell Carcinoma Progression and Mortality.

BACKGROUND/AIM: Activin, a member of the TGF-ß super family of cytokines, is involved in head and neck squamous cell carcinoma (HNSCC). This study examined the constituents of the activin axis in order to further elucidate the role of activin A in HNSCC progression. MATERIALS AND METHODS: Immunohistochemistry (IHC), reverse transcription polymerase chain reaction (RT-PCR), MTT, and matrigel invasion assays, in addition to analysis of the tumor cancer genome atlas (TCGA), were employed. RESULTS: IHC in HNSCC and oral leukoplakia (OPL) lesions demonstrated increased expression of the inhibin subunit ßA (INHBA) (p<0.0001), as well as activin receptor type IB (ACVR1B) (p<0.0032) compared to normal mucosa. TCGA analysis revealed increased INHBA expression was associated with lymph node positive tumors (p=0.024), decreased overall survival (p=0.0167), and decreased promoter methylation (p<0.0001). Concomitant up-regulated expression of gene pathways strongly correlated with INHBA expression demonstrated further deleterious effects on survival (p<0.0148). CONCLUSION: Activin may be an important component of early carcinogenesis in OPL and HNSCC with unfavorable effects on clinical end-points such as survival.

Oral and Inhaled Fosamprenavir Reverses Pepsin-Induced Damage in a Laryngopharyngeal Reflux Mouse Model.

OBJECTIVE: More than 20% of the US population suffers from laryngopharyngeal reflux. Although dietary/lifestyle modifications and alginates provide benefit to some, there is no gold standard medical therapy. Increasing evidence suggests that pepsin is partly, if not wholly, responsible for damage and inflammation caused by laryngopharyngeal reflux. A treatment specifically targeting pepsin would be amenable to local, inhaled delivery, and could prove effective for endoscopic signs and symptoms associated with nonacid reflux. The aim herein was to identify small molecule inhibitors of pepsin and test their efficacy to prevent pepsin-mediated laryngeal damage in vivo. METHODS: Drug and pepsin binding and inhibition were screened by high-throughput assays and crystallography. A mouse model of laryngopharyngeal reflux (mechanical laryngeal injury once weekly for 2 weeks and pH 7 solvent/pepsin instillation 3 days/week for 4 weeks) was provided inhibitor by gavage or aerosol (fosamprenavir or darunavir; 5 days/week for 4 weeks; n = 3). Larynges were collected for histopathologic analysis. RESULTS: HIV protease inhibitors amprenavir, ritonavir, saquinavir, and darunavir bound and inhibited pepsin with IC50 in the low micromolar range. Gavage and aerosol fosamprenavir prevented pepsin-mediated laryngeal damage (i.e., reactive epithelia, increased intraepithelial inflammatory cells, and cell apoptosis). Darunavir gavage elicited mild reactivity and no discernable protection; aerosol protected against apoptosis. CONCLUSIONS: Fosamprenavir and darunavir, FDA-approved therapies for HIV/AIDS, bind and inhibit pepsin, abrogating pepsin-mediated laryngeal damage in a laryngopharyngeal reflux mouse model. These drugs target a foreign virus, making them ideal to repurpose. Reformulation for local inhaled delivery could further improve outcomes and limit side effects. LEVEL OF EVIDENCE: NA. Laryngoscope, 133:S1-S11, 2023.

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.

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.

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.

Retinoids Augment Thiazolidinedione PPARγ Activation in Oral Cancer Cells.

BACKGROUND/AIM: Head and neck squamous cell carcinoma affects nearly 500,000 people annually. Augmenting PPARγ functional activation is linked with multiple anti-carcinogenic processes in aerodigestive cell lines and animal models. PPARγ/RXRα heterodimers may be key partners in this activation. MATERIALS AND METHODS: CA 9-22 and NA cell lines were treated with the PPARγ agonist ciglitazone and/or the RXRα agonist 9-cis-retinoic acid. PPARγ functional activation, cellular proliferation, apoptosis activity, and phenotype were subsequently analyzed. RESULTS: Ciglitazone and 9-cis-retinoic acid independently activated PPARγ and down-regulated the carcinogenic phenotype in vitro. Combination treatment significantly augmented these effects, further decreasing proliferation (p<0.0001), and increasing PPARγ functional activation (p<0.0001), apoptosis (p<0.05), and adipocyte differentiation markers (p<0.0001). CONCLUSION: The efficacy of the combination of ciglitazone and 9-cis-retinoic acid afforded lowering treatment concentrations while maintaining desired therapeutic outcomes, optimistically supporting the feasibility and practicality of this novel treatment option.

Effects of PPAR-γ agonists on oral cancer cell lines: Potential horizons for chemopreventives and adjunctive therapies.

BACKGROUND: Peroxisome proliferator-activated receptor-gamma (PPAR-γ) activators have anti-cancer effects. Our objective was to determine the effect of PPAR-γ ligands 15-deoxy-D12,14 -Prostaglandin J2 (15-PGJ2 ) and ciglitazone on proliferation, apoptosis, and NF-κB in human oral squamous cell carcinoma cell lines. METHODS: NA and CA9-22 cells were treated in vitro with 15-PGJ2 and ciglitazone. Proliferation was measured by MTT colorimetric assay and cell cycle analysis performed via flow cytometry, apoptosis by caspase-3 colorimetric assay and poly-(ADP-ribose) polymerase cleavage on Western blot, and NF-κB activation by luciferase assays. RESULTS: MTT assays demonstrated dose-dependent decreases after 15-PGJ2 treatment in both cell lines, and S-phase cell cycle arrest was also demonstrated. NF-κB luciferase reporter gene activity decreased seven- and eightfold in NA and CA9-22 cells, respectively. Caspase-3 activity increased two- and eightfold in NA and CA9-22 cells, respectively. CONCLUSIONS: Our results suggest these agents, in addition to activating PPAR-γ, can downregulate NF-κB and potentiate apoptosis in oral cancer cells.

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.

Cigarette smoke activates NF kappa B and induces Muc5b expression in mouse middle ear cells.

OBJECTIVES: Cigarette smoke exposure is a significant risk factor in the development of otitis media (OM). Nuclear factor kappa B (NF-kappa B) is a ubiquitous transcription factor known to mediate cigarette smoke effects on gene regulation in multiple cell types. The MUC5B mucin gene contains several putative NF-kappa B sites in its promoter and is the predominant mucin expressed in human OM. We hypothesized that in vitro stimulation of a recently developed model system, murine middle ear epithelial cells (MEEC), with cigarette smoke condensate (CSC) activates NF-kappa B and subsequently induces Muc5b gene expression. METHODS: Luciferase reporter assays, electromobility shift assays (EMSA), and quantitative microplate transcription factor assays (TFA) were performed to evaluate NF-kappaB activation with CSC in immortalized murine MEEC (mMEEC). Reverse transcriptase polymerase chain reaction (RT-PCR) assays and quantitative real time RT-PCR were performed to determine whether time course CSC stimulation upregulates Muc5b mRNA levels in differentiated mMEEC. Luciferase reporter assays were performed to determine whether CSC activates the Muc5b promoter. RESULTS: Reporter assays, EMSA, and TFA demonstrated three- to five-fold dose-dependent activation of NF-kappa B with CSC in mMEEC. CSC stimulation likewise increased Muc5b mRNA abundance and induced reporter activity 1.8- to 4.8-fold in plasmids containing -556 and -255 base pairs upstream of the Muc5b transcriptional start site in mMEEC. CONCLUSIONS: CSC activates NF-kappaB in immortalized MEEC. Furthermore, this activation correlates with CSC-induced Muc5b promoter activation and gene expression. Taken together, these results hint that much as in lung cells, the activation of mucins by cigarette smoke is mediated in part by NF-kappa B.

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.

PPARγ targeted oral cancer treatment and additional utility of genomics analytic techniques.

OBJECTIVE: Peroxisome proliferator-activated receptor γ (PPARγ) agonists have been shown to have anti-proliferative, anti-angiogenic, and proapoptotic effects, leading to interest in their use as cancer therapeutics. Pioglitazone, a U.S. Food and Drug Administration-approved type II diabetes medication and PPARγ agonist, may have a role in adjuvant head-and-neck squamous cell carcinoma treatment or prevention. Therefore, the purpose of this study was: 1) to treat oral cavity cancer cells with the PPARγ activator, pioglitazone, to analyze gene expression changes; and 2) to compare those changes with our preexisting genomic data for development of hypothesis-driven additional basic and clinical studies. STUDY DESIGN: Prospective in vitro. METHODS: We utilized microarray technology, as well as OCPlus (Bioconductor open source software) and Ingenuity Pathway Analysis (Qiagen, Redwood City, CA), to analyze differential gene expression in tumor and pioglitazone-treated tumor cells on a genome-wide level to demonstrate the feasibility of such an approach and determine appropriate sample size for future investigations. RESULTS: We found that approximately 35 samples are required to adequately power future studies. We next discovered that pioglitazone significantly affects Inducible T-Cell Costimulator (iCOS)-Ligand for the T-cell-specific cell surface receptor ICOS (iCOSL) and type II diabetes mellitus pathways as a putative anti-cancer mechanism. CONCLUSION: Genome-wide analysis is possible for the exploration of differential pathway modulation and rapid hypothesis generation. Both inflammation and type II diabetes pathways were significantly altered and therefore might provide unique hypothesis-driven pharmacodynamic parameters for future in vitro or in vivo studies utilizing thiazolidinediones. These techniques could be applied to microarray or other high throughput data from a variety of hypothesis-generating research scenarios in otolaryngology (e.g., middle ear proteomics, sinus microbiome studies). LEVEL OF EVIDENCE: NA. Laryngoscope, 127:E124-E131, 2017.

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.