Accumulating evidence has proved that deregulation of ΔNp63 expression plays an oncogenic role in head and neck squamous cell carcinomas (HNSCCs). Besides p63, the type 1-insulin-like growth factor (IGF) signalling pathway has been implicated in HNSCC development and progression. Most insulin/IGF1 signalling converges intracellularly onto the protein adaptor insulin receptor substrate-1 (IRS-1) that transmits signals from the receptor to downstream effectors, including the PI3K/AKT and the MAPK kinase pathways, which, ultimately, promote proliferation, invasion, and cell survival. Here we report that p63 directly controls IRS1 transcription and cellular abundance and fosters the PI3K/AKT and MAPK downstream signalling pathways. Inactivation of ΔNp63 expression indeed reduces tumour cell responsiveness to IGF1 stimulation, and inhibits the growth potential of HNSCC cells. In addition, a positive correlation was observed between p63 and IRS1 expression in human HNSCC tissue arrays and in publicly available gene expression data. Our findings indicate that aberrant expression of ΔNp63 in HNSSC may act as an oncogenic stimulus by altering the IGF signalling pathway.

Nuclear integrity and mechanical stability of the nuclear envelope (NE) are conferred by the nuclear lamina, a meshwork of intermediate filaments composed of A- and B-type lamins, supporting the inner nuclear membrane and playing a pivotal role in chromatin organization and epigenetic regulation. During cell senescence, nuclear alterations also involving NE architecture are widely described. In the present study, we utilized induced pluripotent stem cells (iPSCs) upon prolonged in vitro culture as a model to study aging and investigated the organization and expression pattern of NE major constituents. Confocal and four-dimensional imaging combined with molecular analyses, showed that aged iPSCs are characterized by nuclear dysmorphisms, nucleoskeletal components (lamin A/C-prelamin isoforms, lamin B1, emerin, and nesprin-2) imbalance, leading to impaired nucleo-cytoplasmic MKL1 shuttling, actin polymerization defects, mitochondrial dysfunctions, SIRT7 downregulation and NF-kBp65 hyperactivation. The observed age-related NE features of iPSCs closely resemble those reported for premature aging syndromes (e.g., Hutchinson-Gilford progeria syndrome) and for somatic cell senescence. These findings validate the use of aged iPSCs as a suitable cellular model to study senescence and for investigating therapeutic strategies aimed to treat premature aging.

Several transcription factors, including the master regulator of the epidermis, p63, are involved in controlling human keratinocyte proliferation and differentiation. Here, we report that in normal keratinocytes, the expression of FOXM1, a member of the Forkhead superfamily of transcription factors, is controlled by p63. We observe that, together with p63, FOXM1 strongly contributes to the maintenance of high proliferative potential in keratinocytes, whereas its expression decreases during differentiation, as well as during replicative-induced senescence. Depletion of FOXM1 is sufficient to induce keratinocyte senescence, paralleled by an increased ROS production and an inhibition of ROS-scavenger genes (SOD2, CAT, GPX2, PRDX). Interestingly, FOXM1 expression is strongly reduced in keratinocytes isolated from old human subjects compared with young subjects. FOXM1 depletion sensitizes both normal keratinocytes and squamous carcinoma cells to apoptosis and ROS-induced apoptosis. Together, these data identify FOXM1 as a key regulator of ROS in normal dividing epithelial cells and suggest that squamous carcinoma cells may also use FOXM1 to control oxidative stress to escape premature senescence and apoptosis.

Reprogramming of human fibroblasts into induced pluripotent stem cells (iPSCs) leads to mitochondrial rejuvenation, making iPSCs a candidate model to study the mitochondrial biology during stemness and differentiation. At present, it is generally accepted that iPSCs can be maintained and propagated indefinitely in culture, but no specific studies have addressed this issue. In our study, we investigated features related to the 'biological age' of iPSCs, culturing and analyzing iPSCs kept for prolonged periods in vitro. We have demonstrated that aged iPSCs present an increased number of mitochondria per cell with an altered mitochondrial membrane potential and fail to properly undergo in vitro neurogenesis. In aged iPSCs we have also found an altered expression of genes relevant to mitochondria biogenesis. Overall, our results shed light on the mitochondrial biology of young and aged iPSCs and explore how an altered mitochondrial status may influence neuronal differentiation. Our work suggests to deepen the understanding of the iPSCs biology before considering their use in clinical applications.

Mandibular hypoplasia, Deafness and Progeroid features with concomitant Lipodystrophy is a rare, genetic, premature aging disease named MDPL Syndrome, due to almost always a de novo variant in POLD1 gene, encoding the DNA polymerase δ. In previous in vitro studies, we have already described several hallmarks of aging, including genetic damage, telomere shortening, cell senescence and proliferation defects. Since a clear connection has been reported between telomere shortening and mitochondria malfunction to initiate the aging process, we explored the role that mitochondrial metabolism and activity play in pathogenesis of MDPL Syndrome, an aspect that has not been addressed yet. We thus evaluated mtDNA copy number, assessing a significant decrease in mutated cells. The expression level of genes related to mitochondrial biogenesis and activity also revealed a significant reduction, highlighting a mitochondrial dysfunction in MDPL cells. Even the expression levels of mitochondrial marker SOD2, as assessed by immunofluorescence, were reduced. The decrease in this antioxidant enzyme correlated with increased production of mitochondrial ROS in MDPL cells, compared to WT. Consistent with these data, Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) analysis revealed in MDPL cells fewer mitochondria, which also displayed morphological abnormalities. Accordingly, we detected autophagic vacuoles containing partially digested mitochondria. Overall, our results demonstrate a dramatic impairment of mitochondrial biogenesis and activity in MDPL Syndrome. Administration of Metformin, though unable to restore mitochondrial impairment, proved efficient in rescuing nuclear abnormalities, suggesting its use to specifically ameliorate the premature aging phenotype.

The transcription factor p53 is a key player in the tumour suppressive DNA damage response and a growing number of target genes involved in these pathways has been identified. p53 has been shown to be implicated in controlling cell motility and its mutant form enhances metastasis by loss of cell directionality, but the p53 role in this context has not yet being investigated. Here, we report that ZNF185, an actin cytoskeleton-associated protein from LIM-family of Zn-finger proteins, is induced following DNA-damage. ChIP-seq analysis, chromatin crosslinking immune-precipitation experiments and luciferase assays demonstrate that ZNF185 is a bona fide p53 target gene. Upon genotoxic stress, caused by DNA-damaging drug etoposide and UVB irradiation, ZNF185 expression is up-regulated and in etoposide-treated cells, ZNF185 depletion does not affect cell proliferation and apoptosis, but interferes with actin cytoskeleton remodelling and cell polarization. Bioinformatic analysis of different types of epithelial cancers from both TCGA and GTEx databases showed a significant decrease in ZNF185 mRNA level compared to normal tissues. These findings are confirmed by tissue micro-array IHC staining. Our data highlight the involvement of ZNF185 and cytoskeleton changes in p53-mediated cellular response to genotoxic stress and indicate ZNF185 as potential biomarker for epithelial cancer diagnosis.

Induced pluripotent stem cells (iPSCs) hold great promise for developing personalized regenerative medicine, however characterization of their biological features is still incomplete. Moreover, changes occurring in long-term cultured iPSCs have been reported, suggesting these as a model of cellular aging. For this reason, we addressed the ultrastructural characterization of iPSCs, with a focus on possible time-dependent changes, involving specific cell compartments. To this aim, we comparatively analysed cultures at different timepoints, by an innovative electron microscopic technology (FIB/SEM). We observed progressive loss of cell-to-cell contacts, associated with increased occurrence of exosomes. Mitochondria gradually increased, while acquiring an elongated shape, with well-developed cristae. Such mitochondrial maturation was accompanied by their turnover, as assessed by the presence of autophagomes (undetectable in young iPSCs), some containing recognizable mitochondria. This finding was especially frequent in middle-aged iPSCs, while being occasional in aged cells, suggesting early autophagic activation followed by a decreased efficiency of the process with culturing time. Accordingly, confocal microscopy showed age-dependent alterations to the expression and distribution of autophagic markers. Interestingly, responsivity to rapamycin, highest in young iPSCs, was almost lost in aged cells. Overall, our results strongly support long-term cultured iPSCs as a model for studying relevant aspects of cellular senescence, involving intercellular communication, energy metabolism, and autophagy.

p53, with its family members p63 and p73, have been shown to promote myoblast differentiation by regulation of the function of the retinoblastoma protein and by direct activation of p21Cip/Waf1 and p57Kip2, promoting cell cycle exit. In previous studies, we have demonstrated that the TAp63γ isoform is the only member of the p53 family that accumulates during in vitro myoblasts differentiation, and that its silencing led to delay in myotube fusion. To better dissect the role of TAp63γ in myoblast physiology, we have generated both sh-p63 and Tet-On inducible TAp63γ clones. Gene array analysis of sh-p63 C2C7 clones showed a significant modulation of genes involved in proliferation and cellular metabolism. Indeed, we found that sh-p63 C2C7 myoblasts present a higher proliferation rate and that, conversely, TAp63γ ectopic expression decreases myoblasts proliferation, indicating that TAp63γ specifically contributes to myoblasts proliferation, independently of p53 and p73. In addition, sh-p63 cells have a defect in mitochondria respiration highlighted by a reduction in spare respiratory capacity and a decrease in complex I, IV protein levels. These results demonstrated that, beside contributing to cell cycle exit, TAp63γ participates to myoblasts metabolism control.

Ageing of human skin is associated with phenotypic changes in the cutaneous cells; the major functional markers of ageing occur as consequences of dermal and epidermal cell senescence and of structural and compositional remodeling of normally long-lived dermal extracellular matrix proteins. Understanding the contribution of the dermal cells in skin ageing is a key question, since this tissue is particularly important for skin integrity and its properties can affect the epidermis. Several microRNAs have been shown to be involved in the regulation of pathways involved in cellular senescence and exerted important effects on tissues ageing. In this study, we demonstrate that the expression of miR-152 and miR-181a increased during the human dermal fibroblasts senescence and that their overexpression, is sufficient to induce cellular senescence in early-passage cells. The increase of these miRNAs during cells senescence was accompanied by a decrease in integrin α5 and collagen XVI expression at mRNA and/or protein levels resulting in reduced cellular adhesion and suggesting extracellular matrix remodeling. These findings indicate that changes in miRNAs expression, by modulating the levels of adhesion proteins and extra-cellular matrix components, such as integrin α5 and collagen XVI, could contribute to the compositional remodelling of the dermis and epidermis occurring during skin aging.