124 Characterization of the cooperative role of co-amplified PIK3Cα and ΔNp63α in squamous tumor initiation and progression

Abstract

The chromosome region between 3q26 and 3q28, which includes proto-oncogenes PIK3CA and TP63, is frequently amplified in a spectrum of squamous cell carcinomas. Using primary murine epidermal keratinocytes and an orthotopic nude mouse grafting model, our lab has previously established that elevated ΔNp63α cooperates with the oncogenic H-Ras pathway to drive malignant progression of H-Ras-initiated tumors. The PI3K pathway operates downstream of Ras and is often activated in cancers by amplification of the wild-type PIK3Cα gene (wt) or by gain-of-function mutation (H1047R or E545K, mt). We asked whether activation of PI3K pathway by overexpressing wt or mt PIK3Cα is sufficient to initiate tumorigenesis and/or cooperate with ΔNp63α to drive malignant progression. H-Ras and wt or mt PIK3Cα were overexpressed individually and in combination with ΔNp63α or vector control in primary mouse keratinocytes by lentiviral gene transduction. The expression of wt or mt PIK3Cα activated downstream target AKT, indicated by the phosphorylation of Ser473 and Thr308 residues. Transduction of PIK3Cα (wt or mt), did not activate Ras/MEK/ERK pathway, and did not induce the typical senescent phenotype observed with H-Ras expression in primary keratinocytes, suggesting that PI3K pathway is not responsible for oncogene induced senescence in this context. Lentiviral driven PIK3C α expression in primary keratinocytes enhanced BrdU incorporation and prolonged cell survival but was not sufficient to support cell passaging. In preliminary in vivo grafting studies in athymic nude mice, keratinocytes transduced with PIK3Cα (wt or mt) did not form tumors or cooperate with ΔNp63α in tumor progression, as is seen with Ras. This multistep model allows for the assessment of additional genes, both co-amplified (e.g. SOX2 and ACTL6A) and on other chromosomes, for their contribution to squamous tumor initiation and progression.