P16 upregulation is required to restrict the clonal proliferation of aerodigestive epithelium

Abstract

Problem: Field cancerization in the upper aerodigestive tract involves the loss of p16INK4a function in clonally expanded epithelial cells that have an increased likelihood of progressing to invasive squamous cell carcinoma. The putative upstream signals that regulate p16INK4a’s ability to elicit growth arrest and curtail clonal expansion remain elusive and suggest that p16INK4a’s loss may merely confer a growth advantage relative to the surrounding wild-type cells. Our objective was to determine if clonal expansion is the result of increased growth rates in cells lacking p16INK4a, or a consequence of these cells’ inability to upregulate p16INK4a protein expression. Methods: An in vitro model system based on a head and neck squamous cell carcinoma (HNSCC) cell line that expresses basal levels of p16INK4a was constructed allowing low, steady state or upregulated p16INK4a expression. Acute loss of either basal or high levels of p16INK4a was attained through viral delivery of short hairpin RNA. Subsequent cell cycle and growth effects were determined using immunofluorescence, FACS, and growth curves. Results: Inhibition of basal p16INK4a levels had no effect on cell growth despite a slight increase in S-phase cells (42.0% vs 33.8%). In contrast, inhibition of upregulated p16INK4a levels resulted in a clear growth advantage (23.8-fold increase in population doublings), as well as a dramatic 2.3-fold increase in S-phase cells. Similar results were also seen in human primary cells with high levels of p16INK4a expression due to serial passage, where the p16INK4a RNAi knockdown allowed continued clonal expansion as compared to controls. Conclusion: Clonal expansion of p16INK4a deleted aerodigestive epithelial cells results when these cells fail to respond to p16 upregulation, rather than a competitive growth advantage over cells expressing basal levels of p16INK4a. Significance: Future identification of the signals that regulate p16INK4a expression will enhance our understanding of field cancerization and allow the development of molecular therapeutics against premalignant disease and second primaries. Support: JWR, Daniel Miller Professorship in Otology and Laryngology, Harvard Medical School and Massachusetts Eye and Ear Infirmary; JJS, NIH T32 Training grant program.