Epigenetic and metabolism in Pancreatic Adenocarcinoma and Keratinocytes Senenscence

In the last decade the epigenetics field has progressively gained greater importance, it has become fundamental to correlate changes in gene expression to changes at the level of the transcriptome and metabolome. Specifically, the close relationship between epigenetic modifications and metabolic shifts in different somatic tissues is still not fully understood. In this study, we have linked epigenetic changes with changes in metabolism by integrating these factors in two distinct scenarios: pancreatic adenocarcinoma and replicative senescence of keratinocytes. Tumor suppressor protein 53 (TP53), is among the crucial players of tumor suppression and maintenance of genome integrity. Missense mutations in this gene lead to the acquisition of new oncogenic functions, collectively referred to as gain of function (GOF) effects. During the initial phase of my PhD, I delved into investigating the gene regulatory network associated with p53 mutants. Simultaneously, a portion of my work involved calibrating a live cell imaging system for epigenetic analysis. To enhance our understanding of the molecular mechanisms underlying the gain-of-function (GOF) exhibited by the p53 mutant, we undertook an exhaustive examination of a meticulously annotated dataset encompassing both genomic and transcriptomic information from human pancreatic adenocarcinoma. Genes of interest were selected on the basis of their differential expression between p53 mutant and p53 wild-type cohorts and their prognostic value. We identified NUAK2 and RCAN2 as candidate players of p53 mutant network. We validated their regulation in cellular models and analyzed Chip-Seq data to identify hypothetical molecular mechanisms through which p53 mutants could modulate their expression. Interestingly, we found that p53 can physically bind RCAN2 gene locus in the regulatory regions where there is known binding of the other p53 mutant interactors as Srebp1 and p63, indicating the existence of this molecular mechanism. Part of my PhD project was directed to calibration of a system able to perform Live Cell Imaging and detect specific epigenetic modification in living cells with confocal microscope. We characterized the role of p53 on the stability of the pericentromeric regions of MajorStellite DNA in the cellular model of Pancreatic Adenocarcinoma (PDAC). We tested engineered proteins with DNA binding domain able to recognize and mark specific epigenetic targets as DNA methylation and histone 3 lysine 9 trimethylation in mouse Major Satellite, pericentromeric regions of DNA repeats in tandem. This system is composed by two distinct modules: the first one specific to recognize the epigenetic modification of H3K9me3 and the second one, able to mark MajorSatellite repeats. Both modules were combined to two domains of the Venus fluorophores capable of emitting a fluorescent signal that can be microscopically tracked. Probes were employed to study the role of the p53 protein in maintaining the stability of pericentromeric regions and genomic integrity in mouse models of PDAC. In particular, our findings demonstrated the pivotal role of p53 in upholding the integrity of heterochromatin structure, especially when faced with epigenetic disruptions like DNA demethylation. Additionally, we observed that p53 plays a crucial role in maintaining genomic stability by ensuring an adequate supply of S-Adenosylmethionine (SAM), the primary methyl group donor in all cell types. This supply helps prevent the depletion of the epigenetic marker H3K9me3 and the subsequent loss of heterochromatin structure. In the absence of p53, the loss of H3K9me3 occurs, leading to the derepression of satellite DNA regions. This derepression further triggers heightened transcription of unscheduled R-loops, thereby escalating genomic instability. In the second part of my PhD project, I dedicated my time to investigating the process of replicative senescence of Human Epidermal Keratinocytes. Cellular senescence is a permanent proliferation arrest during which cells become unresponsive to growth stimuli while remaining metabolically active and exhibiting a secretory phenotype. Many triggers can drive this phenomen including oncogene activation, DNA damage or telomere shortening known as replicative senescence. It remains unclear how metabolic and epigenetic perturbations can influence replicative senescence. We performed in our laboratory the metabolic profile of human epidermal keratinocytes following replicative senescence. In detail, we compared two biological conditions: proliferant (Passage 1) and senescent (Passage 4) keratinocytes. The outcomes analysis revealing a significant increase in methionine sulfoxide in senescent keratinocytes. This observation led us to speculate about an increased oxidative environment. Furthermore we observed alterations in glycerophospholipid and sphingolipid metabolism, indicative of increased membrane remodeling that occurs during aging. Specifically, senescent cells exhibited increased levels of choline, glycerophosphocholine, glycerol 3-phosphate and sphingosine. Finally, concerning the pentose phosphate pathway and glycolytic pathway, we noticed a significant decrease metabolite 6-phosphogluconate suggesting decreased activity in this pathway while the glycolytic intermediates 3-phosphoglycerate and phosphoenolpyruvate were decreased in senescent cells, we saw increase in pyruvate in senescent keratinocytes. These observations indicated that glycolytic pathway was increased in senescent keratinocytes, as evidenced by modifications in tricarboxylic acid cycle, where we identified increased in intermediates succinate, fumarate, and malate while citrate levels were decreased during senescence. Moreover our analysis revealed distinct changes in the methionine pathway within senescent keratinocytes. Notably, there was an increase in methionine levels in these cells, while the levels of S-adenosylhomocysteine experienced a significant decrease. S-Adenosyl-methionine was not detected, thus indicating a possible decrease of cellular level methylation. Based on these observations, we decided to focus our attention on investigating the interplay between metabolic and epigenetic alteration that underlie the process of replicative senescence. In particular, we hypothesize that fluctuations of SAdenosylmethionine could affect DNA and Histone methylation modulating the expression of genes that drive replicative senescence. To validate our hypothesis, we performed RNA sequencing (RNAseq) and differential genome-wide accessibility profiling through the assay for transposase- accessible chromatin using sequencing (ATAC-seq). This allowed us to make a comprehensive comparison between youthful keratinocytes and aged keratinocytes. Our analysis revealed distinct patterns of RNA expression and chromatin accessibility between these two conditions. Notably, 2798 genes displayed significant RNA expression and chromatin accessibility modulation. The GO ontology analysis of genes differentially regulated for RNA and ATAC-seq revealed general dysregulation of cellular metabolic processes. By integrating our metabolomics analysis with the results of ATAC and RNA sequencing we found important modulations in the tetrahydrofolate pathway. Specifically, we found in senescent cells, an increase of folate while several genes of the mitochondrial tetrahydrofolate metabolic process exhibited low RNA expression and parallel ATAC loss. We validated the reduced expression of SHMT2 and MTHDF2, two enzymes of the mitochondrial tetrahydrofolate process, between proliferating (P1) and senescent (P5, P6) keratinocytes. Although further analysis are still ongoing, the expression of different enzymes belonging to the cytoplasmic tetrahydrofolate process (SHMT1, MTHFD1, MTHDFR and MTHFR) seems to be not affected by senescence. Interestingly, by querying the list of genes found in the integration analysis, we observed that the expression of SLC43A2, a member of the solute carrier family methionine transporter, was significantly increased and showed significant ATAC gain in senescent Keratinocytes (P5, P6) compared to proliferative cells. The ongoing experiments are aimed at understanding if increased methionine uptake, by SLC43A2 upregulation, triggers keratinocytes senescence and potentially influences H3K27 trimethylation of specific genes.