Franciosi, Marco (2024) PARP1 converges with p63 to sustain cell proliferation in cutaneous squamous cell carcinoma. [Tesi di dottorato]

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Abstract

Cutaneous squamous cell carcinoma (cSCC) is the second most common human cancer. It is caused by chronic long-term sun exposure, which leads to an accumulation of somatic mutations in multiple tumour suppressor and oncogenic genes. Given the high incidence of cSCC in the human population, the identification of uncharacterized pathways underlying its pathogenesis is of critical interest. Preliminary results in our laboratory have shown that the transcription factor p63 and the Poly [ADP-ribose] polymerase 1 (PARP1) are overexpressed in cSCC. In this study, we investigated the functional crosstalk between these proteins in cSCC, with a particular focus on PARP1, whose functions are still poorly characterized in the context of cSCC. Experiments carried out in cSCC cell lines demonstrated a physical interaction between these proteins only in the presence of the DNA suggesting the existence of a chromatin-associated complex. Transcriptomic analysis in cSCC cells depleted for these proteins showed a consistent overlap between the differentially expressed genes (DEGs). Interpolation of our RNA-seq data and ChIP-seq that we and others have generated suggested that p63 and PARP1 may co-regulate a subset of target genes. The most significant biological processes affected in the absence of both p63 and PARP1 are mitotic cell cycle and DNA replication. Further analysis revealed an involvement of the E2F family transcription factors. Functional assays performed in cSCC cells depleted for PARP1, confirmed its importance in sustaining cell proliferation in cSCC. In the absence of PARP1 a strong and early induction of the cyclin-dependent kinase inhibitor p21 (CDKN1A) was observed. Cell cycle analysis in the absence of PARP1 showed a reduction in S phase, but double knockdown of PARP1 and p21 only partially rescued the cell cycle transition. These data suggest that mechanisms other than p21 converge to block the cell cycle in the absence of PARP1, and according to our results, PARP1 favours the S phase transition by somehow stimulating E2Fs mediated transcription. However, further experiments are needed to understand whether p63 is directly involved in these dynamics.

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