Dysregulated Delta Np63 alpha Inhibits Expression of Ink4a/arf, Blocks Senescence, and Promotes Malignant Conversion of Keratinocytes

p63 is critical for squamous epithelial development, and elevated levels of the Delta Np63 alpha isoform are seen in squamous cell cancers of various organ sites. However, significant controversy exists regarding the role of p63 isoforms as oncoproteins or tumor suppressors. Here, lentiviruses were developed to drive long-term overexpression of Delta Np63 alpha in primary keratinocytes. Elevated levels of Delta Np63 alpha in vitro promote long-term survival and block both replicative and oncogene-induced senescence in primary keratinocytes, as evidenced by the expression of SA-beta-gal and the presence of nuclear foci of heterochromatin protein 1 gamma. The contribution of Delta Np63 alpha to cancer development was assessed using an in vivo grafting model of experimental skin tumorigenesis that allows distinction between benign and malignant tumors. Grafted lenti-Delta Np63 alpha keratinocytes do not form tumors, whereas lenti-GFP/v-ras(Ha) keratinocytes develop well-differentiated papillomas. Lenti-Delta Np63 alpha/v-ras(Ha) keratinocytes form undifferentiated carcinomas. The average volume of lenti-Delta Np63 alpha/v-ras(Ha) tumors was significantly higher than those in the lenti-GFP/v-ras(Ha) group, consistent with increased BrdU incorporation detected by immunohistochemistry. The block in oncogene-induced senescence corresponds to sustained levels of E2F1 and phosphorylated AKT, and is associated with loss of induction of p16(ink4a)/p19(arf). The relevance of p16(ink4a)/p19(arf) loss was demonstrated in grafting studies of p19(arf)-null keratinocytes, which develop malignant carcinomas in the presence of v-ras(Ha) similar to those arising in wildtype keratinocytes that express lenti-Delta Np63 alpha and v-ras(Ha). Our findings establish that Delta Np63 alpha has oncogenic activity and its overexpression in human squamous cell carcinomas contributes to the malignant phenotype, and implicate its ability to regulate p16(ink4a)/p19(arf) in the process.

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