De Marino, Elena (2025) Arf at the crossroad between cancer and differentiation. [Tesi di dottorato]

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Abstract

The p14/p19ARF (Alternative Reading Frame), encoded by the Cdkn2A locus, is recognized as one of the most critical tumor suppressors due to its well-established role in cancer suppression. Recent findings, however, reveal an emerging role for ARF in development, differentiation, and in the regulation of stem cell selfrenewal, positioning it at the crossroads between cancer biology and cellular differentiation. Progenitor cell homeostasis is crucial not only for physiological processes, such as organ development, but also for pathological events, including cancer. Progenitor cells (PCs), which arise from the asymmetric division of stem cells within cellular niches, are particularly essential for regenerating organs and tissues with high cellular turnover, such as the pancreas. Dysregulation of PC homeostasis is central to both healthy tissue function and disease, as disruptions can contribute to tumorigenesis. Notably, the multi-step molecular progression to pancreatic cancer frequently involves the inactivation of key tumor suppressor genes, including p14/p19ARF. This PhD project focuses on studying ARF's role in balancing progenitor cell proliferation and quiescence during in vitro pancreatic differentiation. Previous findings from our laboratory indicated that p19ARF levels increase at both transcriptional and protein levels during in vitro pancreatic differentiation, suggesting its potential involvement in this process. Therefore, to explore ARF’s role in progenitor cell homeostasis, by using the CRISPR-Cas9 technology, I introduced mutations in exon 1 of p19Arf in mESCs. I obtained several clones with biallelic and mono allelic mutations confirmed by sequencing. Remarkably, p19Arf mutated clones showed impairment in the differentiation process toward PPCs with a more prolonged expression of stemness factors, thus suggesting an important role of p19Arf in the regulation of the stemness phenotype and PPCs homeostasis. These observations could help to clarify p19Arf role in differentiation processes and cancer susceptibility.

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