The development of cancer results from a multistep process in which genetic and phenotypic mutations must overcome many highly regulated pathways to cause malignancy. This study focuses on characterizing the genetic and biological alterations associated with squamous cell carcinoma development.
Biological characterization of normal, pre-neoplastic and neoplastic cells indicated that disruption of the differentiation pathway appeared to be an early event in skin neoplasia. Normal keratinocytes (HEKs), pre-neoplastic IEC-1 cells and the neoplastic SCC-25 and COLO-16 responded differently to stimulators of keratinocyte differentiation. As expected, HEKs were sensitive to mediators of differentiation and treatment with these agents resulted in biological and molecular alterations associated with the differentiated phenotype. In contrast, the SCC-25 and COLO-16 were not sensitive to treatment with differentiation inducers. The IEC-1
cells appeared to be sensitive to the effects of differentiation inducers, however, these cells were not able to undergo an irreversible growth arrest suggesting that the differentiation program of pre-neoplastic cells is altered.
To identify the possible genetic alterations associated with the observed differences between normal, pre-neoplastic and neoplastic cells, HEKs, IEC-1 cells, and two neoplastic cell lines, SCC-25 and COLO-16 were grown as raft cultures and their gene expression profiles screened using cDNA arrays. These data indicated that the expression levels of at least 37 genes were significantly (P ≤ 0.05; 1.9% of genes screened) altered in neoplastic cells compared to normal cells. Of these genes, 10 genes were up-regulated and 27 genes were downregulated in the neoplastic cells. In addition, 51% of the genes altered in the neoplastic cells were already altered in the pre-neoplastic IEC-1 cells.
Immunohistochemical staining of patient tumours were used to verify the cDNA array analysis. This analysis indicated that alterations in genes associated with extracellular matrix production, apoptosis and ROS scavenging are disrupted in pre-neoplastic cells whilst later stages of neoplasia are associated with alterations in gene expression for genes involved in DNA repair or EGF receptor/MAPKK/MAPK/AP-1 signalling. Subsequent functional analysis of the alterations in expression of the EGF receptor/MAPKK/MAPK/AP-1 genes suggested they were unlikely to contribute to the neoplastic phenotype.