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C/CAAT enhancer binding protein alpha (C/EBPα) is a bZIP transcription factor involved in cell cycle control and cellular differentiation, which makes this gene a good tumor suppressor. In fact, C/EBPα has been shown to be silenced in some cancer types. For example, C/EBPα silencing by mutations substantially contributes to the malignant transformation evidenced in acute myeloid leukemia. More recently, epigenetic mechanisms have been found to regulate C/EBPα expression in non-small cell lung cancer. In this study, we investigated the potential for C/EBPα tumor suppressor activity in HNSCC. We found through immunohistochemistry that C/EBPα is downregulated in 33/48 (69%) of the HNSCC tumor epithelium samples studied. Mutation analysis of 39 HNSCC patient samples and 10 HNSCC cell lines indicated no changes in the C/EBPα coding sequence. Deletion analysis revealed loss of heterozygosity (LOH) in 39/60 (65%) HNSCC tumor epithelium samples at D19S245 (a marker 200 Kb from the C/EBPα locus). Using COBRA and bisulfite sequencing, we show for the first time that the decreased C/EBPα expression in HNSCC is in part due to promoter methylation of an upstream regulatory region (-1399bp to -1253bp). Demethylating treatment (i.e. with 5-aza-2’-deoxycytidine) is able to significantly restore C/EBPαmRNA expression in three of five HNSCC cell lines tested. Furthermore, increased involucrin expression (a squamous cell differentiation marker that is transcriptionally activated by C/EBPα) subsequent to 5-aza-2’-deoxycytidine treatment suggests that C/EBPα reexpression induces differentiation. C/EBPα overexpression in a HNSCC cell line revealed its ability to provide tumor suppressor activity in HNSCC in vitro and in vivo. In contrast, silencing C/EBPα revealed an increase in cell proliferation. In conclusion, we have identified C/EBPα as a putative novel tumor suppressor in HNSCC that is downregulated by either genetic deletions and/or promoter methylation.

Keywords: HNSCC, C/EBPα, methylation, and tumor suppressor.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA