Expression of Folate Transport Proteins in a Mouse Model of 4NQO‐Induced Oral Cancer

Abstract

IntroductionHead and neck cancer is the sixth most common cancer worldwide. Risk factors include tobacco exposure, alcohol use, male sex, and a poor diet. Administration of 4‐nitroquinoline‐1‐oxide (4NQO) in drinking water causes oral cancer in mice, and mimics the effects of tobacco use. Folate is essential for nucleotide biosynthesis as well as epigenetic regulation. Folate is imported by folate receptors (FR), proton‐coupled folate transporters (PCFT), and the reduced folate carrier (RFC). This study aimed to investigate the effects of a high saturated fat (HF) or low fat (LF) diet, sex, and 4NQO treatment on expression of folate transport proteins in mice tongues. We hypothesized that there would be a decreased expression of these proteins in the HNSCC tongue samples compared to the control tongue samples.Materials and MethodsFive week old mice (C57BI/6, 36 per sex) were provided either a low saturated fat diet (10 kcal% fat; LF) or a high fat diet (60 kcal% fat, HF). After one week, mice were then randomly assigned to 17 weeks on one of three water treatment groups: water alone (control); propylene glycol in water (1.25%, PG‐water); or 4NQO in PG‐water (50 mg/ml; 4NQO). After 17 weeks, the mice were provided water alone for six more weeks but maintained on their diet. Tongues from euthanized animals were fixed in formalin and processed for histological examination. Immunohistochemistry was performed to detect the expression of FR, PCFT, and RFC in these samples. Slides were visually graded, and the total score for the expression of each protein was acquired by adding the intensity grade (0–3) and distribution grade (0–3). Statistical analysis was performed using Graphpad Prism 7.0. All animal work was in compliance with the Institutional Animal Care and Use Committee at Midwestern University, Downers Grove, IL.ResultsCompared to the control tongues, there was decreased expression of all three folate transport proteins in tumors (p<0.001). The total score (intensity + distribution) in oral cancer versus control tissue for FR, RFC, and PCFT, respectively were: 3.21 ± 0.10 vs 4.48 + 0.14; 4.38 ± 0.11 vs 5.13 ± 0.10; and 4.22 ± 0.11 vs 5.65 ± 0.69. All slides displayed a 100% distribution rate of FR, PCFT, and RFC in the epithelium. There were no significant differences in folate transport protein expression in males versus females. In the 4NQO tongues, a statistically significant decrease in FR expression was observed between the HF diet group and the LF group (3.97 ± 0.20 vs 3.05 ± 0.10, p <0.0002). No differences were observed in the expression of RFC and PCFT proteins with regards to diet or sex.ConclusionWe measured decreased expression in all three folate transport proteins in oral cancers when compared to normal tongue epithelium. We speculate that mutations leading to decreased folate import can promote carcinogenesis through epigenetic mechanisms.Support or Funding InformationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.