Co-Expression of p16 and Laminin 5 2 by Microinvasive and Superficial Squamous Cell Carcinomas in Vivo and by Migrating Wound and Senescent Keratinocytes in Culture

The high frequency of mutation, deletion, and promoter silencing of the gene encoding p16 (p16) in premalignant dysplasias and squamous cell carcinomas (SCC) of epidermis and oral epithelium classifies p16 as a tumor suppressor. However, the point during neoplastic progression at which this protein is expressed and presumably impedes formation of an SCC is unknown. Induction of p16 has been found to be responsible for the senescence arrest of normal human keratinocytes in culture, suggesting the possibility that excessive or spatially abnormal cell growth in vivo triggers p16 expression. We examined 73 skin and oral mucosal biopsy specimens immunohistochemically to test this hypothesis. p16 was not detectable in benign hyperplastic lesions, but instead was expressed heterogeneously in some dysplastic and carcinoma in situ lesions and consistently at areas of microinvasion and at superficial margins of advanced SCCs. p16-positive cells in these regions coexpressed the 2 chain of laminin 5, identified previously as a marker of invasion in some carcinomas. Normal keratinocytes undergoing senescence arrest in culture proved to coordinately express p16 and 2 and this was frequently associated with increased directional motility. Keratinocytes at the edges of wounds made in confluent early passage cultures also coexpressed p16 and 2, accompanying migration to fill the wound. These results have identified the point during neoplastic progression in stratified squamous epithelial at which the tumor suppressor p16 is expressed and suggest that normal epithelia may use the same mechanism to generate non-dividing, motile cells for wound repair. (Am J Pathol 2003, 163:477–491) Squamous cell carcinoma (SCC) is the malignancy of the oral mucosal epithelium, the epidermis, and other stratified squamous epithelia. SCCs arise within areas of abnormal, pre-invasive cell growth (dysplasia), which may take months to many years to progress to invasive cancer. A consistent feature of SCC is loss of the ability to express functional p16 (p16) by mutation, deletion, or promoter hypermethylation. p16 is a specific inhibitor of the cyclin D1-dependent kinases cdk4 and cdk6. Normally, in the absence of p16, cyclin D1/cdk4 and cyclin D1/cdk6 complexes phosphorylate and inactivate the Rb protein, permitting E2F-dependent transcription of genes encoding a set of proteins necessary to initiate chromosome replication and ultimately another round of cell division. Most cervicogenital SCCs and a fraction of head and neck SCCs contain human papilloma viruses (HPV) and viral DNA integration as a feature of their neoplastic progression. These tumors and their premalignant dysplasias typically have unaltered p16 alleles and often express p16 protein, presumably as a response to inhibition of Rb function by the HPV E7 viral oncoprotein. Humans and mice that inherit a heterozygous or homozygous loss of function mutation in the p16 gene or that express a p16-insensitive mutant form of cdk4 are predisposed to a variety of spontaneous and carcinogen-induced cancers, but they undergo normal development and form structurally and functionally normal stratified squamous epithelia. These results confirm the tumor suppressor function of p16 and also are consistent with the finding that p16 protein is not expressed as a feature of normal stratified squamous epithelial renewal or differentiation. Importantly, the point during neoplastic progression toward SCC at which p16 protein becomes expressed and functions as a tumor suppressor has remained unknown.