The primary objective of this study is to understand the regulatory factor(s) responsible for the observed over-expression of CYP1B1 in head and neck squamous cell carcinoma (HNSCC) and associated pre-malignant tissue (both hyperplastic and dysplastic) compared to normal tissue. CYP1B1 is regulated by several key transcription factors such as the aryl hydrocarbon receptor (AhR) and the AhR nuclear translocator (ARNT) complex (AhR/ARNT) which binds to dioxin responsive elements in the promoter/enhancer region. DNA hypermethylation of CpG islands in the promoters of genes is a well studied epigenetic alteration of the genome and is known to play a significant role in cancer, including HNSCC. However, the authors have demonstrated that the over-expression of CYP1B1 in prostate cancer is associated with hypomethlyation of the CpG sites contained in key promoter elements in the CYP1B1 gene. This study seeks to establish whether a correlation between CYP1B1 expression and gene hypomethylation also extends to HNSCC against a background of epigenetic alteration usually resulting in gene hypermethylation. In order to study the CYP1B1 phenotype in vitro and in vivo a panel of 8 HNSCC cell lines (UTSCC5, 8, 9, 10, 14, 16a, 16b, and 24a) were selected from primary tumours from patients with different clinical characteristics: sex M/F 6/2, age 25 to 81 (median 58); location 5 SCC linguae; 2 larnyx, 1 linguae; site tongue, larynx, neck; grade G1 to G3; lesion 7 primary and 1 metastatic. To evaluate the constitutive and inducible expression of CYP1B1 mRNA all HNSCC cell lines were treated with the aryl hydrocarbon agonist TCDD (typically 10 nM for 24 h then harvested on day 7) and/or the demethylating agent 5-Aza-dC (typically 0 to 5 μM for 7 days). Parallel experiments were performed in vivo with the corresponding HNSCC human tumour xenografts. Mice were treated with 5-Aza-dC (5 mg/kg, i.p. for 4 days) alone or with TCDD (5 to 50 μg/kg i.p. for 24 h). For both in vitro and in vivo experiments total RNA/DNA was extracted for RT-PCR and bisulfite modified DNA sequencing. All 8 HNSCC cell lines exhibited constitutive expression of CYP1B1 mRNA in vitro and in vivo which was inducible by TCDD. Furthermore, 5-Aza-dC did not change the constitutive nor TCDD-inducible level of CYP1B1 mRNA thus providing indirect evidence for hypomethylation of the CYP1B1 gene. DNA bisulphite modified sequencing confirmed that the CpG islands in the enhancer/promoter region of the CYP1B1 gene in all HNSCC cells were indeed hypomethylated. Despite the fact that the primary HNSCC cells exhibit completely different clinical characteristics the CYP1B1 gene is 'globally' hypomethylated against a background of gene alteration usually characterised by DNA hypermethylation. This work is supported by the NIH and Cancer Research UK.

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