Scientific Publications by FDA Staff

Mol Carcinog 2014 Sep;53(9):698-710

Dysregulated DeltaNp63alpha negatively regulates the maspin promoter in keratinocytes via blocking endogenous p73 binding.

King KE, Reddi DM, Ponnamperuma RM, Gerdes M, Weinberg WC

While overexpression of the p63 isoform, DeltaNp63alpha, has been reported in squamous cell cancers, the contribution of p63 to cancer pathogenesis remains unclear. We previously demonstrated that overexpressed DeltaNp63alpha aberrantly maintains proliferation of primary mouse keratinocytes under conditions that normally induce growth arrest and differentiation. To identify genes downstream of dysregulated DeltaNp63alpha that may contribute to squamous cancer development and progression, we performed microarray analyses using primary mouse keratinocytes. Herein we report that elevated DeltaNp63alpha differentially regulates genes involved in a variety of cellular functions. Of note, multiple protease inhibitor mRNAs were downregulated including: maspin (serpinB5); plasminogen activator inhibitor-2 (PAI-2; serpinB2); and tissue inhibitor of metalloproteinase-3 (TIMP-3). Correspondingly, secreted TIMP-3 and PAI-2 protein declined in the presence of dysregulated DeltaNp63alpha, however secreted maspin remained stable. Intracellular maspin protein expression decreased in response to overexpressed DeltaNp63alpha, as did PAI-2. In contrast, TIMP-3 protein was not detected intracellularly, supporting a solely extracellular function. Electrophoretic mobility shift assays (EMSAs) using a maspin promoter p53/p63 consensus sequence revealed endogenous transcription factor(s) binding to this sequence in keratinocytes that was disrupted by overexpressed DeltaNp63alpha. This was confirmed by ChIP assays. This binding was interrupted by the addition of antibodies recognizing p73, but not p53 or p63, and significantly diminished in EMSA reactions from p73(-/-) keratinocytes, confirming p73 as a constituent. Physical association between p73/DeltaNp63alpha was observed in control beta-gal overexpressing keratinocytes and was enhanced in the presence of overexpressed DeltaNp63alpha These findings underscore the importance of properly balanced p53 homologs for tissue homeostasis.