p63, a homologue of the transcription factor p53, is essential for maintaining the proliferative capacity of epithelial stem cells. The p63 gene yields two major isoforms transcribed from dual promoters at its N-terminus and at least three different splice variants at its C-terminus. The TAp63 isoform functions as a p53-like tumor suppressor and transactivates p53 gene targets. In contrast, ΔNp63α predominates in epithelial stem cells and regulates their proliferative potential, whilst also acting as an oncogene by suppressing the function of both p53 and TAp63 in a dominant-negative manner. ΔNp63α is frequently upregulated in human epithelial cancers and has been theorized to play a role in tumorigenesis. Our prior studies found that an aberrantly spliced ΔNp63α isoform heterozygous for a lack of exon 4-coded sequences (hereafter referred to as Δ4) appeared to be specifically expressed in squamous cell carcinomas, suggesting a potential link between this new isoform and a specific type of cancer. Our study aimed to elucidate the involvement of this aberrantly spliced p63 isoform in the initiation and/or development of epithelial cancer. We established a p63+/Δ4 mouse model to obtain a baseline for the effect of one aberrant allele on both embryonic and adult mouse development. We found that p63+/Δ4 mouse embryos developed normally and that over a period of 74 weeks, the p63+/Δ4 mice developed normally and lived lifespans equivalent to their wildtype counterparts, though a small proportion of the p63+/Δ4 colony showed minor issues with inflammation. After our initial observations, we established an experimental mouse model to test the effect of oncogenic stimulation on p63+/Δ4 mice. In the first model, we administered the oral carcinogen 4-nitroquinoline 1-oxide to mirror the histological and molecular changes seen in human oral carcinogenesis. We observed significant dysplasia in the tongue epithelia of p63+/Δ4 mice compared to that of wildtype. With this data, we developed the hypothesis that expression of Δ4 accelerates tumorigenesis in the presence of oncogenic stimulation. To further test this possibility, we generated a second mouse model: a triple transgenic Cre-inducible mouse model under the control of epithelial cell-specific promotor gene, keratin 14 (K14). This cross introduced a mutated Kras gene to the p63+/Δ4 lineage, as well as to control wildtype mice bred with the same Cre elements. Upon administration of tamoxifen, genetic recombination caused expression of both mutant Kras G12D and Δ4 in K14-positive tissues and subsequent malignant transformation. Among these K14-positive tissues were the oral epithelia, which presented with dysplasia more severe than that of control mice expressing either Kras or Δ4 alone and which matched the phenotype-genotype associations observed in our 4NQO experiment. Lastly, we attempted to create an antibody specific to Δ4 that could eliminate the need for our current two-antibody identification strategy. Though we did not succeed, we found value in elucidating the genetic sequence requirements to create the artificial peptide that we used for mouse immunization. We attempted to optimize established hybridoma protocols and developed a Δ4-specific screening process to determine viable candidates for expansion. In conclusion, this study is the first characterization of a hitherto unknown mutant splice variant of a gene vital to stem cell maintenance and epithelial development. It is the first demonstration of this gene mutation’s responsibility for the acceleration of squamous cell carcinoma, which carries implications for potential therapeutic treatments. / 2026-02-12T00:00:00Z