Gene expression analysis of head and neck cancer development
Microarray analysis was performed on 32 head and neck keratinocytes cultures using Affymetrix U133A/B genechips. The panel of cultures included normal cells, mortal and immortal cultures of dysplastic keratinocytes and mortal and immortal cultures from carcinomas, all grown to a standard protocol. The overall GEP revealed that many of the well-established HNSCC molecular markers associated with motility and invasion were up-regulated in the mortal cells, particularly in the mortal carcinomas. Immortal NHSCC cells showed elevated expression of cell-cycle markers and loss of differentiation markers. In addition, a small number of common changes in gene expression in all the carcinomas, regardless of replicative fate, were identified. This included several transcription factors. A series of 49 novel gene expression changes consistently associated with immortality in dysplastic keratinocytes and SCCs were identified. The list included genes involves in cell cycle control, signalling, cellular metabolism and maintenance of cellular structure. Validation of the expression of these genes by western blot demonstrated that, in general, the protein expression of genes agreed with the RNA expression level from the microarray data. However, some heterogeneity was evident. The mortal and immortal gene expression signatures were validated by IHC in the tumours from which the cultures were derived. The tumours that gave rise to immortal cell cultures demonstrated a relatively uniform pattern of staining in relation to the novel markers of immortality. However, those tumours which gave rise to mortal cultures exhibited significant heterogeneity of gene expression pattern, with areas characteristic of both the mortal and immortal phenotype present. These novel markers give us further insight into the mechanisms and importance of keratinocytes immortalization. Surrogate markers of immortality could therefore be valuable for assessment of prognosis and therapy if confirmed in larger in vivo studies.