Abstract 1598: Grainyhead-like 2 (GRHL2) regulates the expression of forkhead box transcription factor M1 (FoxM1) in human oral cancer cells and determines tumorigenicity

Abstract GRHL2 is a proto-oncogene that regulates epithelial proliferation and differentiation through transcriptional regulation of large number of target genes. The current study was performed to determine its role in oral carcinogenesis through regulation of forkhead box 1 (FoxM1), which is a known oncogene for many solid tumors. GRHL2 expression was highly elevated in oral squamous cell carcinoma (OSCC) tissues compared with normal oral epithelium (NHOM), and its protein expression is localized in the proliferative cells at the outer border of invasive islands of OSCC. In cultured OSCCs, GRHL2 and FoxM1 levels were both highly upregulated compared with normal human oral keratinocytes (NHOK), supporting their roles in oral carcinogenesis. When GRHL2 was knocked down in SCC4 cells by RNA interference, the cells have lost their proliferative capacity and colony formation when plated at low density. GRHL2 knockdown also resulted in marked loss of tumor spheroid formation, which is hallmark of cancer self-renewal. GRHL2 knockdown also led to loss of tumorigenic ability of cells upon xenograft transplantation in immunocompromised mice. GRHL2 appeared to determine the epithelial phenotype in OSCC cells. GRHL2 knockdown led to loss of E-cadherin and miR-200 family genes, which are epithelial markers. These data indicate that GRHL2 is required to maintain the transformed phenotype in OSCC. We also noted that GRHL2 knockdown led to drastic loss of FoxM1 expression, suggesting that GRHL2 may regulate FoxM1. To test this possibility, we performed in vivo binding assay by chromatin immunoprecipitation (ChIP), which showed GRHL2 binding at the proximal promoter of FoxM1. Luciferase reporter plasmid under FoxM1 promoter showed that GRHL2 directly regulates the gene promoter activity. FoxM1 knockdown resulted in loss of OSCC proliferation and tumor spheroid formation, while its overexpression conferred transformed phenotype in non-tumorigenic human oral keratinocytes (HOKs). Therefore, our data indicate that GRHL2 is required for the maintenance of transformed phenotype and that it plays a role in oral carcinogenesis through regulation of FoxM1. This study was supported in part by the grants from NIDCR/NIH (DE18295 and DE18959) and Jack Weichman Endowed Fund. Note: This abstract was not presented at the meeting. Citation Format: Wei Chen, Jin-Kyu Yi, Ki-Hyuk Shin, Reuben Kim, Shebli Mehrazarin, No-Hee Park, Mo K. Kang. Grainyhead-like 2 (GRHL2) regulates the expression of forkhead box transcription factor M1 (FoxM1) in human oral cancer cells and determines tumorigenicity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1598. doi:10.1158/1538-7445.AM2014-1598