Overexpression of Lipocalins and Pro-Inflammatory Chemokines and Altered Methylation of PTGS2 and APC2 in Oral Squamous Cell Carcinomas Induced in Rats by 4-Nitroquinoline-1-Oxide

January Overexpression of Lipocalins and Pro- Inflammatory Chemokines and Altered Methylation of PTGS2 and APC2 in Oral Squamous Cell Carcinomas Induced in Rats by 4-Nitroquinoline-1-Oxide Xinjian Peng 0 1 2 3 Wenping Li 0 1 2 3 William D. Johnson 0 1 2 3 Karen E. O. Torres 0 1 2 3 David L. McCormick 0 1 2 3 0 Funding: This work was supported by internal re- search and development funds from IIT Research In- stitute. The funders had no role in study design, data collection and analysis, decision to publish, or prepa- ration of the manuscript 1 Data Availability Statement: All relevant data are within the paper and its Supporting Information files and microarray data are available in the National Cen- ter for Biotechnology Information Gene Expression Omnibus (GEO) with accession GSE51125 2 Academic Editor: Muy-Teck Teh, Queen Mary Uni- versity of London, UNITED KINGDOM 3 1 Life Sciences Group, IIT Research Institute , Chicago, Illinois, 60616 , United States of America , 2 GenUs Biosystems, Northbrook, Illinois, 60062 , United States of America Oral squamous cell carcinomas (OSCC) induced in F344 rats by 4-nitroquinoline-1-oxide (4-NQO) demonstrate considerable phenotypic similarity to human oral cancers. Gene expression studies (microarray and PCR) were coupled with methylation analysis of selected genes to identify molecular markers of carcinogenesis in this model and potential biochemical and molecular targets for oral cancer chemoprevention. Microarray analysis of 11 pairs of OSCC and site-matched phenotypically normal oral tissues from 4-NQO-treated rats identified more than 3500 differentially expressed genes; 1735 genes were up-regulated in rat OSCC versus non-malignant tissues, while 1803 genes were down-regulated. In addition to several genes involved in normal digestion, genes demonstrating the largest fold increases in expression in 4-NQO-induced OSCC include three lipocalins (VEGP1, VEGP2, LCN2) and three chemokines (CCL, CXCL2, CXCL3); both classes are potentially druggable targets for oral cancer chemoprevention and/or therapy. Down-regulated genes in 4-NQO-induced OSCC include numerous keratins and keratin-associated proteins, suggesting that alterations in keratin expression profiles may provide a useful biomarker of oral cancer in F344 rats treated with 4-NQO. Confirming and extending our previous results, PTGS2 (cyclooxygenase-2) and several cyclooxygenase-related genes were significantly up-regulated in 4-NQO-induced oral cancers; up-regulation of PTGS2 was associated with promoter hypomethylation. Rat OSCC also demonstrated increased methylation of the first exon of APC2; the increased methylation was correlated with down-regulation of this tumor suppressor gene. Overexpression of pro-inflammatory chemokines, hypomethylation of PTGS2, and hypermethylation of APC2 may be causally linked to the etiology of oral cancer in this model. - Competing Interests: Co-author Karen E. O. Torres is an employee of a commercial company GenUs Biosystems, whose major contribution to our work was to perform data analysis, and co-author David McCormick is a PLOS ONE Editorial Board Member (Section Editor). This does not alter the authors adherence to PLOS ONE Editorial policies and criteria. More than 250,000 new cases of oral squamous cell carcinoma (OSCC) are diagnosed each year around the world, and more than 125,000 people die of the disease [1, 2]. In the United States, it is estimated that approximately 28,000 new cases of cancer of the tongue, gum, lip, or mouth will be diagnosed in 2014, and that approximately 8,000 people will die from these malignancies [3]. The most important risk factors for human oral cancer are use of tobacco (including smokeless tobacco) and alcohol [46]. It is estimated that consumption of tobacco and alcohol is responsible for approximately 75% of oral cancer cases in the United States, and that tobacco and alcohol may act synergistically to induce oral neoplasia [4, 5]. Exposure to human papillomavirus (HPV) is an emerging factor in oral cancer etiology, and has been identified as a major risk factor in younger individuals as well as in non-smokers and non-drinkers [7, 8]. In spite of continuing efforts to improve surgical and pharmacologic approaches to treat oral cancer, the 5-year survival rate for oral cancer patients has remained between 50% and 60% since the mid-1970s [9]. On this basis, primary prevention efforts aimed at decreasing exposure to major risk factors for oral cancer, and secondary prevention efforts involving oral cancer chemoprevention are necessary to reduce mortality and the substantial morbidity that is associated with this neoplasm. The oral cavity provides an attractive site for clinical efforts in cancer prevention, as site accessibility and the existence of grossly identifiable preneoplastic lesions (such as leukoplakia and erythroplakia) facilitate the evaluation of disease progression and chemopreventive drug efficacy. Oral preneoplastic lesions demonstrate a variety of genetic alterations [10, 11], some of which may be critical determinants of lesion progression from preneoplasia to invasive oral cancer. High quality in vivo carcinogenesis models that demonstrate biological congruity with human oral cancer are essential elements of studies to identify molecular targets for oral cancer prevention and to evaluate the efficacy and safety of novel agents and regimens designed to inhibit or retard oral carcinogenesis. An experimental model in which invasive OSCC are induced in the tongue of F344 rats by 4-nitroquinoline 1-oxide (4-NQO) has been used widely in studies of cancer chemoprevention [1214]. Studies performed in our laboratory [14] demonstrate that invasive oral cancers induced by administration of 4-NQO (20 ppm in the drinking water for ten weeks) develop in four to six months after the first exposure to chemical carcinogen, and demonstrate highly reproducible incidence and latency patterns. Importantly, this model generates invasive malignancies in an anatomic site in which cancers are commonly seen in humans [12, 14]; 4-NQO-induced oral cancers in rats also demonstrate considerable phenotypic similarity to human oral cancers [12]. In the present studies, microarray and PCR approaches were used to identify molecular alterations that are associated with 4-NQO-induced oral carcinogenesis in the F344 rat. The goals of these studies were to identify molecular pathways that could serve as useful targets for oral cancer chemoprevention, and to identify potential biomarkers for carcinogenesis in this site. Additional studies were performed to identify the molecular mechanisms responsible for the differential expression of selected genes in rat oral cancers induced by 4-NQO. We report that oral cancers induced in the rat tongue by 4-NQO demonstrate differential expression of numerous genes, some of which appear to provide suitable targets for pharmacologic interventions directed at oral cancer che (...truncated)