p53 regulates expression of nuclear envelope components in cancer cells.

Nuclear organisation and architecture are essential for the maintenance of genomic integrity as well as for the epigenetic regulations and gene expression. Disruption of lamin B1, major structural and functional member of the nuclear lamina, is observed in human laminopathies and in sporadic cancers, and leads to chromosomal rearrangements and alterations of gene expression. The tumour suppressor p53 has been shown to direct specific transcriptional programmes by regulating lamin A/C, however its relationship with lamin B1 has remained elusive. Here, we show that loss of p53 correlates with increased expression of members belonging to the nuclear pore complex and nuclear lamina and directly regulates transcription of lamin B1. We show that the genomic loci of a fraction of p53-dependent genes physically interact with lamin B1 and Nup210. This observation provides a possible mechanistic explanation for the p53-depedent changes of chromatin accessibility, with the consequent influence of expression and rearrangement of these genomic sites in pancreatic cancer. Overall, these data suggest a potential functional and biochemical regulatory network connecting p53 and nuclear architecture.

Extracellular serine empowers epidermal proliferation and psoriasis-like symptoms.

The contribution of nutrient availability to control epidermal cell proliferation, inflammation, and hyperproliferative diseases remains unknown. Here, we studied extracellular serine and serine/glycine metabolism using human keratinocytes, human skin biopsies, and a mouse model of psoriasis-like disease. We focused on a metabolic enzyme, serine hydroxymethyltransferase (SHMT), that converts serine into glycine and tetrahydrofolate-bound one­carbon units to support cell growth. We found that keratinocytes are both serine and glycine auxotrophs. Metabolomic profiling and hypoxanthine supplementation indicated that SHMT silencing/inhibition reduced cell growth through purine depletion, leading to nucleotide loss. In addition, topical application of an SHMT inhibitor suppressed both keratinocyte proliferation and inflammation in the imiquimod model and resulted in a decrease in psoriasis-associated gene expression. In conclusion, our study highlights SHMT2 activity and serine/glycine availability as an important metabolic hub controlling both keratinocyte proliferation and inflammatory cell expansion in psoriasis and holds promise for additional approaches to treat skin diseases.

Metabolic regulation by p53 prevents R-loop-associated genomic instability.

Gene-environment interactions can perturb the epigenome, triggering network alterations that participate in cancer pathogenesis. Integrating epigenomics, transcriptomics, and metabolic analyses with functional perturbation, we show that the tumor suppressor p53 preserves genomic integrity by empowering adequate levels of the universal methyl donor S-adenosylmethionine (SAM). In p53-deficient cells, perturbation of DNA methylation promotes derepression of heterochromatin, massive loss of histone H3-lysine 9 methylation, and consequent upregulation of satellite RNAs that triggers R-loop-associated replication stress and chromosomal aberrations. In p53-deficient cells, the inadequate SAM level underlies the inability to respond to perturbation because exogenous reintroduction of SAM represses satellite elements and restores the ability to cope with stress. Mechanistically, p53 transcriptionally controls genes involved in one-carbon metabolism, including Slc43a2, the methionine uptake transporter that is critical for SAM synthesis. Supported by clinical data, our findings shed light on the role of p53-mediated metabolism in preventing unscheduled R-loop-associated genomic instability.

p53-driven lipidome influences non-cell-autonomous lysophospholipids in pancreatic cancer.

Adaptation of the lipid metabolism participates  in cancer pathogenesis, facilitating energy storage and influencing cell fate and control of molecular signalling. The tumour suppressor protein p53 is a molecular hub of cell metabolism, supporting antioxidant capabilities and counteracting oncogene-induced metabolic switch. Despite extensive work has described the p53-dependent metabolic pathways, a global profiling of p53 lipidome is still missing. By high-throughput untargeted lipidomic analysis of pancreatic ductal adenocarcinoma (PDAC) cells, we profile the p53-dependent lipidome, revealing intracellular and secreted lysophospholipids as one of the most affected class. Lysophospholipids are hydrolysed forms of phospholipids that results from phospholipase activity, which can function as signalling molecules, exerting non-cell-autonomous effects and instructing cancer microenvironment and immunity. Here, we reveal that p53 depletion reduces abundance of intracellular lysophosphatidyl-choline, -ethanolamine and -serine and their secretion in the extracellular environment. By integrating this with genomic and transcriptomic studies from in vitro models and human PDAC patients, we identified potential clinically relevant candidate p53-dependent phospholipases. In particular PLD3, PLCB4 and PLCD4 expression is regulated by p53 and chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) indicates a direct transcriptional control on their chromatin accessible genomic loci. Consistently, PLD3, PLCB4 and PLCD4 expression correlates with p53 mutational status in PDAC patients, and these genes display prognostic significance. Overall, our data provide insights into lipidome rewiring driven by p53 loss and identify alterations of lysophospholipids as a potential molecular mechanism for p53-mediated non-cell-autonomous molecular signalling that instructs cancer microenvironment and immunity during PDAC pathogenesis.

p53 mutations define the chromatin landscape to confer drug tolerance in pancreatic cancer.

Somatic inactivation of p53 (TP53) mainly occurs as missense mutations that lead to the acquisition of neomorphic mutant protein forms. p53 mutants have been postulated to exert gain-of-function (GOF) effects, including promotion of metastasis and drug tolerance, which generally contribute to the acquisition of the lethal phenotype. Here, by integrating a p53R270H -dependent transcriptomic analysis with chromatin accessibility (ATAC-seq) profiling, we shed light on the molecular basis of a p53 mutant-dependent drug-tolerant phenotype in pancreatic cancer. p53R270H finely tunes chromatin accessibility in specific genomic loci, orchestrating a transcriptional programme that participates in phenotypic evolution of the cancer. We specifically focused on the p53R270H -dependent regulation of the tyrosine kinase receptor macrophage-stimulating protein receptor (MST1r). MST1r deregulation substantially impinged on drug response in the experimental model, recapitulating the p53R270H -dependent phenotype, and strongly correlated with p53 mutant and aggressive phenotype in pancreatic cancer patients. As cellular plasticity in the final stages of the evolution of pancreatic cancer seems to predominantly originate from epigenetic mechanisms, we propose that mutant p53 participates in the acquisition of a lethal phenotype by fine-tuning the chromatin landscape.

NUAK2 and RCan2 participate in the p53 mutant pro-tumorigenic network.

Most inactivating mutations in TP53 gene generates neomorphic forms of p53 proteins that experimental evidence and clinical observations suggest to exert gain-of-function effects. While massive effort has been deployed in the dissection of wild type p53 transcriptional programme, p53 mutant pro-tumorigenic gene network is still largely elusive. To help dissecting the molecular basis of p53 mutant GOF, we performed an analysis of a fully annotated genomic and transcriptomic human pancreatic adenocarcinoma to select candidate players of p53 mutant network on the basis their differential expression between p53 mutant and p53 wild-type cohorts and their prognostic value. We identified NUAK2 and RCan2 whose p53 mutant GOF-dependent regulation was further validated in pancreatic cancer cellular model. Our data demonstrated that p53R270H can physically bind RCan2 gene locus in regulatory regions corresponding to the chromatin permissive areas where known binding partners of p53 mutant, such as p63 and Srebp, bind. Overall, starting from clinically relevant data and progressing into experimental validation, our work suggests NUAK2 and RCan2 as novel candidate players of the p53 mutant pro-tumorigenic network whose prognostic and therapeutic interest might attract future studies.

Understanding p53 tumour suppressor network.

The mutation of TP53 gene affects half of all human cancers, resulting in impairment of the regulation of several cellular functions, including cell cycle progression and cell death in response to genotoxic stress. In the recent years additional p53-mediated tumour suppression mechanisms have been described, questioning the contribution of its canonical pathway for tumour suppression. These include regulation of alternative cell death modalities (i.e. ferroptosis), cell metabolism and the emerging role in RNA stability. Here we briefly summarize our knowledge on p53 "canonical DNA damage response" and discuss the most relevant recent findings describing potential mechanistic explanation of p53-mediated tumour suppression.

The C terminus of p73 is essential for hippocampal development.

The p53 family member p73 has a complex gene structure, including alternative promoters and alternative splicing of the 3' UTR. This results in a complex range of isoforms whose biological relevance largely remains to be determined. By deleting exon 13 (which encodes a sterile α motif) from the Trp73 gene, we selectively engineered mice to replace the most abundantly expressed C-terminal isoform, p73α, with a shorter product of alternative splicing, p73ß. These mice (Trp73Δ13/Δ13 ) display severe neurodevelopmental defects with significant functional and morphological abnormalities. Replacement of p73α with p73ß results in the depletion of Cajal-Retzius (CR) cells in embryonic stages, thus depriving the developing hippocampus of the pool of neurons necessary for correct hippocampal architecture. Consequently, Trp73Δ13/Δ13 mice display severe hippocampal dysgenesis, reduced synaptic functionality and impaired learning and memory capabilities. Our data shed light on the relevance of p73 alternative splicing and show that the full-length C terminus of p73 is essential for hippocampal development.

P73 C-terminus is dispensable for multiciliogenesis.

The p53 family transcriptional factor p73 plays a pivotal role in development. Ablation of p73 results in severe neurodevelopmental defects, chronic infections, inflammation and infertility. In addition to this, Trp73-\- mice display severe alteration in the ciliated epithelial lining and the full-length N-terminal isoform TAp73 has been implicated in the control of multiciliogenesis transcriptional program. With our recently generated Trp73Δ13/Δ13 mouse model, we interrogate the physiological role of p73 C-terminal isoforms in vivo. Trp73Δ13/Δ13 mice lack exon 13 in Trp73 gene, producing an ectopic switch from the C-terminal isoforms p73α to p73ß. Trp73Δ13/Δ13 mice show a pattern of expression of TAp73 comparable to the wild-type littermates, indicating that the α to ß switch does not significantly alter the expression of the gene in this cell type. Moreover, Trp73Δ13/Δ13 do not display any significant alteration in the airway ciliated epithelium, suggesting that in this context p73ß can fully substitute the function of the longer isoform p73α. Similarly, Trp73Δ13/Δ13 ciliated epithelium of the brain ependyma also does appear defective. In this district however expression of TAp73 is not detectable, indicating that expression of the gene might be compensated by alternative mechanisms. Overall our work indicates that C-terminus p73 is dispensable for the multiciliogenesis program and suggests a possible tissue-specific effect of p73 alternative splicing.

Long non-coding RNA uc.291 controls epithelial differentiation by interfering with the ACTL6A/BAF complex.

The mechanisms that regulate the switch between epidermal progenitor state and differentiation are not fully understood. Recent findings indicate that the chromatin remodelling BAF complex (Brg1-associated factor complex or SWI/SNF complex) and the transcription factor p63 mutually recruit one another to open chromatin during epidermal differentiation. Here, we identify a long non-coding transcript that includes an ultraconserved element, uc.291, which physically interacts with ACTL6A and modulates chromatin remodelling to allow differentiation. Loss of uc.291 expression, both in primary keratinocytes and in three-dimensional skin equivalents, inhibits differentiation as indicated by epidermal differentiation complex genes down-regulation. ChIP experiments reveal that upon uc.291 depletion, ACTL6A is bound to the differentiation gene promoters and inhibits BAF complex targeting to induce terminal differentiation genes. In the presence of uc.291, the ACTL6A inhibitory effect is released, allowing chromatin changes to promote the expression of differentiation genes. Thus, uc.291 interacts with ACTL6A to modulate chromatin remodelling activity, allowing the transcription of late differentiation genes.

ZNF185 is a p63 target gene critical for epidermal differentiation and squamous cell carcinoma development.

Development and maintenance of healthy stratified epithelia require the coordination of complex transcriptional programmes. The transcription factor p63, a member of the p53 family, plays a crucial role in epithelial development and homeostasis. Analysis of the p63-dependent transcriptome indicated that one important aspect of p63 functions in epithelial development is the regulation of cell-cell and cell-matrix adhesion programmes. However, limited knowledge exists on the relevant cell-cell adhesion molecules involved in physiological epithelial formation. Similarly, limited data are available to understand if deregulation of the cell-cell adhesion programme is important in tumour formation. Here, using the epidermis as an experimental model with the RNA sequencing approach, we identify a novel p63-regulated gene induced during differentiation, ZNF185. ZNF185 is an actin-cytoskeleton-associated Lin-l 1, Isl-1 and Mec-3 (LIM) domain-containing protein, whose function is poorly known. We found that p63 binds to a specific enhancer region, promoting its expression to sustain epithelial differentiation. ZNF185 silencing strongly impaired keratinocyte differentiation according to gene array analysis. ZNF185 is detected at the cell-cell periphery where it physically interacts with E-cadherin, indicating that it is important to maintain epithelial integrity beyond its pro-differentiation role. Interestingly, poorly differentiated, including head and neck, cervical and oesophageal, squamous cell carcinomas display loss of ZNF185 expression. Together, these studies reinforce that p63 is a crucial gene for maintaining epithelial tissue integrity and support the deregulation of the cell-cell adhesion programme,which plays a critical role in carcinoma development.

TAp73 regulates ATP7A: possible implications for ageing-related diseases.

The p53 family member p73 controls a wide range of cellular function. Deletion of p73 in mice results in increased tumorigenesis, infertility, neurological defects and altered immune system. Despite the extensive effort directed to define the molecular underlying mechanism of p73 function a clear definition of its transcriptional signature and the extent of overlap with the other p53 family members is still missing. Here we describe a novel TAp73 target, ATP7A a member of a large family of P-type ATPases implicated in human neurogenerative conditions and cancer chemoresistance. Modulation of TAp73 expression influences basal expression level of ATP7A in different cellular models and chromatin immunoprecipitation confirmed a physical direct binding of TAp73 on ATP7A genomic regions. Bioinformatic analysis of expression profile datasets of human lung cancer patients suggests a possible implication of TAp73/ATP7A axis in human cancer. These data provide a novel TAp73-dependent target which might have implications in ageing-related diseases such as cancer and neurodegeneration.

Kruppel-like factor 4 regulates keratinocyte senescence.

Keratinocyte replicative senescence has an important role in time-related changes of epidermis. Previous studies demonstrated that miRNAs play key roles in inhibiting proliferation and in the acquisition of the keratinocyte senescent phenotype as well as in individual ageing. Kruppel-like factor 4 is a transcription factor with dual functions in keratinocytes, being a stemness factor and a pro-differentiation factor. Interestingly, in skin squamous cell carcinomas KLF4 expression is strongly down-regulated or absent. While KLF4 involvement in senescence and ageing has not been investigated yet. Here, we show that Klf4 protein decreases during keratinocyte replicative senescence and during physiological skin aging, while its mRNA level does not change. We demonstrated that the senescence-associated miR-34a regulates post-transcriptionally Klf4 expression. KLF4 silencing is sufficient to induce a senescent phenotype in primary keratinocytes and ectopic miR-34a over-expression phenocopies this result. Our findings identify a novel regulatory loop between miR-34a and KLF4 during keratinocytes replicative senescence. This regulatory loop, beside aging, may play a role in age-related pathologies.

Metabolic pathways regulated by p63.

The transcription factor p63 belongs to the p53-family and is a master regulator of proliferative potential, lineage specification, and differentiation in epithelia during development and tissue homeostasis. In cancer, p63 contribution is isoform-specific, with both oncogenic and tumour suppressive roles attributed, for ΔNp63 and TAp63, respectively. Recently, p53 and TAp73, in line with other tumour suppressor genes, have emerged as important regulators of energy metabolism and metabolic reprogramming in cancer. To date, p63 contributions in controlling energy metabolism have been partially investigated; given the extensive interaction of the p53 family members, these studies have potential implications in tumour cells for metabolic reprogramming. Here, we review the role of p63 isoforms, TAp63 and ΔNp63, in controlling cell metabolism, focusing on their specific metabolic target genes and their physiological/functional context of action.

Ultraconserved long non-coding RNA uc.63 in breast cancer.

Transcribed-ultraconserved regions (T-UCRs) are long non-coding RNAs (lncRNA) encoded by a subset of long ultraconserved stretches in the human genome. Recent studies revealed that the expression of several T-UCRs is altered in cancer and growing evidences underline the importance of T-UCRs in oncogenesis, offering also potential new strategies for diagnosis and prognosis. We found that overexpression of one specific T-UCRs named uc.63 is associated with bad outcome in luminal A subtype of breast cancer patients. uc.63 is localized in the third intron of exportin-1 gene (XPO1) and is transcribed in the same orientation of its host gene. Interestingly, silencing of uc.63 induces apoptosis in vitro. However, silencing of host gene XPO1 does not cause the same effect suggesting that the transcription of uc.63 is independent of XPO1. Our results reveal an important role of uc.63 in promoting breast cancer cells survival and offer the prospect to identify a signature associated with poor prognosis.

FOXM1 regulates proliferation, senescence and oxidative stress in keratinocytes and cancer cells.

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.

Vascular ageing and endothelial cell senescence: Molecular mechanisms of physiology and diseases.

Ageing leads to a progressive deterioration of structure and function of all organs over the time. During this process endothelial cells undergo senescence and manifest significant changes in their properties, resulting in impairment of the vascular functionality and neo-angiogenic capability. This ageing-dependent impairment of endothelial functions is considered a key factor contributing to vascular dysfunctions, which is responsible of several age-related diseases of the vascular system and other organs. Several mechanisms have been described to control ageing-related endothelial cell senescence including microRNAs, mitochondrial dysfunction and micro environmental stressors, such as hypoxia. In this review, we attempt to summarize the recent literature in the field, discussing the major mechanisms involved in endothelial cell senescence. We also underline key molecular aspects of ageing-associated vascular dysfunction in the attempt to highlight potential innovative therapeutic targets to delay the onset of age-related diseases.