Abstract 2464: Dysregulated DeltaNp63alpha modulates p16 expression, blocks senescence, and promotes malignant conversion of keratinocytes
Authors: Ha Linan , Ponnamperuma Roshini M. , Jay Steven , Ricci Stacey , Conde Betty , Weinberg Wendy C. 
Published: February 2011

Abstract
P63 is critical for squamous epithelial development, and elevated levels of the p63 isoform ΔNp63 were reported in squamous cell cancers of the head, neck, skin, lung and cervix. Our lab has previously shown that transient overexpression of ΔNp63α via adenoviral transduction in primary mouse epidermal keratinocytes is associated with enhanced cell proliferation and inhibition of differentiation. However, long-term biological effects of sustained ΔNp63α dysregulation and its contribution to squamous cancer pathogenesis remain unknown. Here, lentiviruses were developed to drive long-term overexpression of ΔNp63α in an in vivo grafting model of experimental skin cancer. Lenti-GFP- and lenti-ΔNp63α-expressing keratinocytes consistently formed normal skin following grafting to nude mice (10 grafted mice/group). Lenti-GFP /v-rasHa keratinocytes developed benign tumors (well-differentiated papillomas) in 4/15 grafted mice. In contrast, lenti- ΔNp63α/v-ras-Ha keratinocytes formed undifferentiated carcinomas in 20/20 mice. The average volume of lenti-ΔNp63α/v-rasHa tumors was 831.9mm3, compared to 25mm3 in the lenti-GFP/v-rasHa group, consistent with increased BrdU incorporation detected by immunohistochemistry. To understand the mechanism of ΔNp63α-induced malignant transformation, we further studied in vitro cell proliferation and senescence in the presence of ΔNp63α dysregulation. Proliferation status was assessed by BrdU uptake. The S-phase population was consistently higher in lenti-ΔNp63α cultures compared to lenti-GFP control cultures (18.18%, 11.12% and 5.12% at days 5, 10, and 14, respectively, versus 17.58%, 4.12% and 1.59%). Moreover, keratinocytes expressing lenti-GFP, but not lenti-ΔNp63α, underwent spontaneous replicative senescence by day 14, as evidenced by the expression of SA-β-gal or the presence of nuclear foci of heterochromatin protein 1γ (HP1γ). Lenti-ΔNp63α also blocked oncogene-induced senescence typically seen within 14 days following introduction of v-rasHa. Both replicative and oncogene-induced senescence were accompanied by an upregulation of p16, which corresponded with a decreased nuclear level of E2F1, starting at day 7 post-infection in lenti-GFP keratinocytes. The upregulation of p16 is much delayed and attenuated in lenti-ΔNp63α cells. The relationship between ΔNp63α and p16 was further confirmed following transient adenoviral-mediated expression of ΔNp63α, which delayed p16 induction until ΔNp63α decreased to endogenous levels. The modulation of p16 by ΔNp63α was shown by RT-PCR to be regulated at the transcriptional level. Taken together, our findings suggest that long-term overexpression of ΔNp63α, as seen in human squamous cell cancers, promotes keratinocyte proliferation and inhibits senescence, thereby facilitating keratinocyte transformation.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2464.