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An essential function of the epidermis is to provide a physical barrier that prevents the loss of water. Essential mediators of this barrier function include ceramides, cholesterol, and very long chain fatty acids, and their alteration causes human pathologies, including psoriasis and atopic dermatitis. A frameshift mutation in the human ZNF750 gene, which encodes a zinc finger transcription factor, has been shown to cause a seborrhea-like dermatitis. Here, we show that genetic deletion of the mouse homolog ZFP750 results in loss of epidermal barrier function, which is associated with a substantial reduction of ceramides, nonpolar lipids. The alteration of epidermal lipid homeostasis is directly linked to the transcriptional activity of ZFP750. ZFP750 directly and/or indirectly regulates the expression of crucial enzymes primarily involved in the biosynthesis of ceramides. Overall, our study identifies the transcription factor ZFP750 as a master regulator epidermal homeostasis through lipid biosynthesis and thus contributing to our understanding of the pathogenesis of several human skin diseases.

Prostate cancer is the most frequently diagnosed cancer and the fifth leading cause of cancer death among men in 2020. The clinical decision making for prostate cancer patients is based on the stratification of the patients according to both clinical and pathological parameters such as Gleason score and prostate-specific antigen levels. However, these tools still do not adequately predict patient outcome. The aim of this study was to investigate whether ZNF750 could have a role in better stratifying patients, identifying those with a higher risk of metastasis and with the poorest prognosis. The data reported here revealed that ZNF750 protein levels are reduced in human prostate cancer samples, and this reduction is even higher in metastatic samples. Interestingly, nuclear positivity is significantly reduced in patients with metastatic prostate cancer, regardless of both Gleason score and grade group. More importantly, the bioinformatics analysis indicates that ZNF750 expression is positively correlated with better prognosis. Overall, our findings suggest that nuclear expression of ZNF750 may be a reliable prognostic biomarker for metastatic prostate cancer, which lays the foundation for the development of new biological therapies.

The transcription factor p53 has been shown to control the differentiation process of the mesenchymal stem cells (MSCs). As a matter of fact, in vivo p53 loss leads to an unbalance between bone formation versus bone erosion. Using as experimental system, human bone marrow-derived MSCs and mouse bone marrow-derived TAp63-/- MSCs, we have asked whether the other members of the p53 family, p63 and p73, are involved in controlling MSCs osteogenic differentiation. Our results indicate that both during human and mouse MSC-induced osteogenic differentiation, TAp63 isoforms are mainly upregulated in comparison with p73 isoforms. In addition, MSCs derived from TAp63 knock-out mice show delayed osteogenic differentiation. Interestingly, we found that, in contrast to p53, TAp63 trascriptionally regulates osteoprotegerin (OPG or TNFRSF11B) important for bone remodeling and osteoclastogenesis inhibition throughout the RANKL/RANK/OPG pathway. Analysis of the expression of p63 and OPG in breast cancer, which is one of the most common human cancers that metastatizes to bone, showed that p63/OPG pathway is deregulated and that a higher expression of both p63 and OPG is associated with a better patient survival, suggesting that p63/OPG axis may be further investigated as clinical biomarker for breast cancer metastasis. ABBREVIATIONS: MSC, mesenchymal stem cells; OPG, osteoprotegerin; RUNX2, Run-trelated transcription factor 2

Funder: RCUK | Medical Research Council (MRC); doi: https://doi.org/10.13039/501100000265, Funder: Fondazione Luigi Maria Monti IDI-IRCCS (RC), Regulatory noncoding RNAs (ncRNAs) are a class of RNAs transcribed by regions of the human genome that do not encode for proteins. The three main members of this class, named microRNA, long noncoding RNA, and circular RNA play a key role in the regulation of gene expression, eventually shaping critical cellular processes. Compelling experimental evidence shows that ncRNAs function either as tumor suppressors or oncogenes by participating in the regulation of one or several cancer hallmarks, including evading cell death, and their expression is frequently deregulated during cancer onset, progression, and dissemination. More recently, preclinical and clinical studies indicate that ncRNAs are potential biomarkers for monitoring cancer progression, relapse, and response to cancer therapy. Here, we will discuss the role of noncoding RNAs in regulating cancer cell death, focusing on those ncRNAs with a potential clinical relevance.

Cancer cells exhibit dysregulation of critical genes including those involved in lipid biosynthesis, with subsequent defects in metabolism. Here, we show that ELOngation of Very Long chain fatty acids protein 4 (ELOVL4), a rate-limiting enzyme in the biosynthesis of very-long polyunsaturated fatty acids (n-3, ≥28 C), is expressed and transcriptionally repressed by the oncogene MYCN in neuroblastoma cells. In keeping, ELOVL4 positively regulates neuronal differentiation and lipids droplets accumulation in neuroblastoma cells. At the molecular level we found that MYCN binds to the promoter of ELOVL4 in close proximity to the histone deacetylases HDAC1, HDAC2, and the transcription factor Sp1 that can cooperate in the repression of ELOVL4 expression. Accordingly, in vitro differentiation results in an increase of fatty acid with 34 carbons with 6 double bonds (FA34:6); and when MYCN is silenced, FA34:6 metabolite is increased compared with the scrambled. In addition, analysis of large neuroblastoma datasets revealed that ELOVL4 expression is highly expressed in localized clinical stages 1 and 2, and low in high-risk stages 3 and 4. More importantly, high expression of ELOVL4 stratifies a subsets of neuroblastoma patients with good prognosis. Indeed, ELOVL4 expression is a marker of better overall clinical survival also in MYCN not amplified patients and in those with neuroblastoma-associated mutations. In summary, our findings indicate that MYCN, by repressing the expression of ELOVL4 and lipid metabolism, contributes to the progression of neuroblastoma.

The human zinc finger (C2H2-type) protein ZNF750 is a transcription factor regulated by p63 that plays a critical role in epithelial tissues homoeostasis, as well as being involved in the pathogenesis of cancer. Indeed, missense mutations, truncation and genomic deletion have been found in oesophageal squamous cell carcinoma. In keeping, we showed that ZNF750 negatively regulates cell migration and invasion in breast cancer cells; in particular, ZNF750 binds and recruits KDM1A and HDAC1 on the LAMB3 and CTNNAL1 promoters. This interaction, in turn, represses the transcription of LAMB3 and CTNNAL1 genes, which are involved in cell migration and invasion. Given that ZNF750 is emerging as a crucial transcription factor that acts as tumour suppressor gene, here, we show that ZNF750 represses the expression of the small GTPase, Ras-related C3 botulinum toxin substrate 1 (RAC1) in breast cancer cell lines, by directly binding its promoter region. In keeping with ZNF750 controlling RAC1 expression, we found an inverse correlation between ZNF750 and RAC1 in human breast cancer datasets. More importantly, we found a significant upregulation of RAC1 in human breast cancer datasets and we identified a direct correlation between RAC1 expression and the survival rate of breast cancer patient. Overall, our findings provide a novel molecular mechanism by which ZNF750 acts as tumour suppressor gene. Hence, we report a potential clinical relevance of ZNF750/RAC1 axis in breast cancer.

p53 plays a pivotal role in controlling the differentiation of mesenchymal stem cells (MSCs) by regulating genes involved in cell cycle and early steps of differentiation process. In the context of osteogenic differentiation of MSCs and bone homeostasis, the osteoprotegerin/receptor activator of NF-κB ligand/receptor activator of NF-κB (OPG/RANKL/RANK) axis is a critical signaling pathway. The absence or loss of function of p53 has been implicated in aberrant osteogenic differentiation of MSCs that results in higher bone formation versus erosion, leading to an unbalanced bone remodeling. Here, we show by microCT that mice with p53 deletion systemically or specifically in mesenchymal cells possess significantly higher bone density than their respective littermate controls. There is a negative correlation between p53 and OPG both in vivo by analysis of serum from p53+/+, p53+/−, and p53−/− mice and in vitro by p53 knockdown and ChIP assay in MSCs. Notably, high expression of Opg or its combination with low level of p53 are prominent features in clinical cancer lesion of osteosarcoma and prostate cancer respectively, which correlate with poor survival. Intra-bone marrow injection of prostate cancer cells, together with androgen can suppress p53 expression and enhance local Opg expression, leading to an enhancement of bone density. Our results support the notion that MSCs, as osteoblast progenitor cells and one major component of bone microenvironment, represent a cellular source of OPG, whose amount is regulated by the p53 status. It also highlights a key role for the p53-OPG axis in regulating the cancer associated bone remodeling.

Adult neurogenesis is a multistage process by which neurons are generated and integrated into existing neuronal circuits. In the adult brain, neurogenesis is mainly localized in two specialized niches, the subgranular zone (SGZ) of the dentate gyrus and the subventricular zone (SVZ) adjacent to the lateral ventricles. Neurogenesis plays a fundamental role in postnatal brain, where it is required for neuronal plasticity. Moreover, perturbation of adult neurogenesis contributes to several human diseases, including cognitive impairment and neurodegenerative diseases. The interplay between extrinsic and intrinsic factors is fundamental in regulating neurogenesis. Over the past decades, several studies on intrinsic pathways, including transcription factors, have highlighted their fundamental role in regulating every stage of neurogenesis. However, it is likely that transcriptional regulation is part of a more sophisticated regulatory network, which includes epigenetic modifications, non-coding RNAs and metabolic pathways. Here, we review recent findings that advance our knowledge in epigenetic, transcriptional and metabolic regulation of adult neurogenesis in the SGZ of the hippocampus, with a special attention to the p53-family of transcription factors.

Significance Alteration of splicing is emerging as a relevant cause of human disease. The C-terminal region of p73 is subject to complex alternative splicing that can give rise to seven different isoforms. Here, using a newly generated mouse model, we determine the functional consequence of in vivo ectopic switch from the physiologically expressed and most abundant isoform p73α to the shorter p73β isoform. Expression of p73β leads to neurodevelopmental defects with functional and morphological abnormalities in the mouse hippocampus. The ectopic isoform switch results in depletion of Cajal–Retzius (CR) neurons in embryonic stages, leading to aberrant hippocampal architecture. Our study indicates that deregulation in p73 alternative splicing might underlie neurodevelopmental human conditions., 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, p73β. These mice (Trp73Δ13/Δ13) display severe neurodevelopmental defects with significant functional and morphological abnormalities. Replacement of p73α with p73β 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.

The p53 family of transcription factors, including p53, p63, and p73, plays key roles in both biological and pathological processes, including cancer and neural development. Recent Advances: In recent years, a growing body of evidence has indicated that the entire p53 family is involved in the regulation of the central nervous system (CNS) functions as well as in the pathogenesis of several neurological disorders. Mechanistically, the p53 proteins control neuronal cell fate, terminal differentiation, and survival, via a complex interplay among the family members.In this article, we discuss the involvement of the p53 family in neurobiology and in pathological conditions affecting the CNS, including neuroinflammation.Understanding the molecular mechanism(s) underlying the function of the p53 family could improve our general knowledge of the pathogenesis of brain disorders and potentially pave the road for new therapeutic intervention. Antioxid. Redox Signal. 29, 1-14.

Breast cancer is the second leading cause of cancer-related deaths among women, largely due to the progression of a significant fraction of primary tumours to the metastatic stage. Here, we show that zinc-finger protein 750 (ZNF750) opposes the migration and invasion of breast cancer cells by repressing a prometastatic transcriptional programme, which includes genes involved in focal adhesion and extracellular matrix interactions, such as LAMB3 and CTNNAL1. Mechanistically, ZNF750 recruits the epigenetic modifiers KDM1A and HDAC1 to the promoter regions of LAMB3 and CTNNAL1, influencing histone marks and transactivating these genomic sites. Gene expression analysis in cancer patient datasets indicated that ZNF750 and its targets were negative prognostic factors in breast cancer. Together, our findings shed light on the molecular mechanism by which ZNF750 regulates cell migration and invasion, suggesting a role in breast cancer metastasis.

The transcription factor p73 is a member of the p53 family, of which the transactivation domain containing isoform (TAp73) plays key roles in brain development and neuronal stem cells. TAp73 also facilitates homoeostasis and prevents oxidative damage in vivo by inducing the expression of its target genes. Recently, we found that in addition to its role in regulation of transcription, TAp73 also affects mRNA translation. In cultured cells, acute TAp73 depletion activates eEF2K, which phosphorylates eEF2 reducing mRNA translation elongation. As a consequence, there is a reduction in global proteins synthesis rates and reprogramming of the translatome, leading to a selective decrease in the translation of rRNA processing factors. Given the dramatic effects of Tap73 depletion in vitro it was important to determine whether similar effects were observed in vivo. Here, we report the surprising finding that in brains of TAp73 KO mice there is a reduced level of eEF2K, which allows protein synthesis rates to be maintained suggesting a compensation model. These data provide new insights to the role of TAp73 in translation regulation and the eEF2K pathway in the brain.

Significance High-risk neuroblastomas (NBs) show undifferentiated/poorly differentiated morphology as a distinctive feature. We have identified the transcription factor ZNF281 as a factor that can counteract the neuronal differentiation of primary neurons in culture and NB cells. The expression of ZNF281 is inhibited by TAp73 and promoted by MYCN. In turn, ZNF281 inhibits the expression of GDNF and NRP2, two proteins associated with neuronal differentiation. In patients with NB, the expression of ZNF281 is higher in high-risk patients and is associated with worse prognosis. Understanding the molecular mechanisms that regulate neuronal differentiation is relevant for the identification of defects in this process that underlie the development of tumors such as NB, in which an aberrant differentiation arrest has occurred., Derangement of cellular differentiation because of mutation or inappropriate expression of specific genes is a common feature in tumors. Here, we show that the expression of ZNF281, a zinc finger factor involved in several cellular processes, decreases during terminal differentiation of murine cortical neurons and in retinoic acid-induced differentiation of neuroblastoma (NB) cells. The ectopic expression of ZNF281 inhibits the neuronal differentiation of murine cortical neurons and NB cells, whereas its silencing causes the opposite effect. Furthermore, TAp73 inhibits the expression of ZNF281 through miR34a. Conversely, MYCN promotes the expression of ZNF281 at least in part by inhibiting miR34a. These findings imply a functional network that includes p73, MYCN, and ZNF281 in NB cells, where ZNF281 acts by negatively affecting neuronal differentiation. Array analysis of NB cells silenced for ZNF281 expression identified GDNF and NRP2 as two transcriptional targets inhibited by ZNF281. Binding of ZNF281 to the promoters of these genes suggests a direct mechanism of repression. Bioinformatic analysis of NB datasets indicates that ZNF281 expression is higher in aggressive, undifferentiated stage 4 than in localized stage 1 tumors supporting a central role of ZNF281 in affecting the differentiation of NB. Furthermore, patients with NB with high expression of ZNF281 have a poor clinical outcome compared with low-expressors. These observations suggest that ZNF281 is a controller of neuronal differentiation that should be evaluated as a prognostic marker in NB.

The transcription factor p73 has been demonstrated to play a significant role in survival and differentiation of neuronal stem cells. In this report, by employing comprehensive metabolic profile and mitochondrial bioenergetics analysis, we have explored the metabolic alterations in cortical neurons isolated from p73 N-terminal isoform specific knockout animals. We found that loss of the TAp73 or ΔNp73 triggers selective biochemical changes. In particular, p73 isoforms regulate sphingolipid and phospholipid biochemical pathway signaling. Indeed, sphinganine and sphingosine levels were reduced in p73-depleted cortical neurons, and decreased levels of several membrane phospholipids were also observed. Moreover, in line with the complexity associated with p73 functions, loss of the TAp73 seems to increase glycolysis, whereas on the contrary, loss of ΔNp73 isoform reduces glucose metabolism, indicating an isoform-specific differential effect on glycolysis. These changes in glycolytic flux were not reflected by parallel alterations of mitochondrial respiration, as only a slight increase of mitochondrial maximal respiration was observed in p73-depleted cortical neurons. Overall, our findings reinforce the key role of p73 in regulating cellular metabolism and point out that p73 exerts its functions in neuronal biology at least partially through the regulation of metabolic pathways.

As a member of the p53 gene family, p73 regulates cell cycle arrest, apoptosis, neurogenesis, immunity and inflammation. Recently, p73 has been shown to transcriptionally regulate selective metabolic enzymes, such as cytochrome c oxidase subunit IV isoform 1, glucose 6-phosphate dehydrogenase and glutaminase-2, resulting in significant effects on metabolism, including hepatocellular lipid metabolism, glutathione homeostasis and the pentose phosphate pathway. In order to further investigate the metabolic effect of p73, here, we compared the global metabolic profile of livers from p73 knockout and wild-type mice under both control and starvation conditions. Our results show that the depletion of all p73 isoforms cause altered lysine metabolism and glycolysis, distinct patterns for glutathione synthesis and Krebs cycle, as well as an elevated pentose phosphate pathway and abnormal lipid accumulation. These results indicate that p73 regulates basal and starvation-induced fuel metabolism in the liver, a finding that is likely to be highly relevant for metabolism-associated disorders, such as diabetes and cancer.

Zinc-finger proteins (ZNFs) are one of the most abundant groups of proteins and have a wide range of molecular functions. Given the wide variety of zinc-finger domains, ZNFs are able to interact with DNA, RNA, PAR (poly-ADP-ribose) and other proteins. Thus, ZNFs are involved in the regulation of several cellular processes. In fact, ZNFs are implicated in transcriptional regulation, ubiquitin-mediated protein degradation, signal transduction, actin targeting, DNA repair, cell migration, and numerous other processes. The aim of this review is to provide a comprehensive summary of the current state of knowledge of this class of proteins. Firstly, we describe the actual classification of ZNFs, their structure and functions. Secondly, we focus on the biological role of ZNFs in the development of organisms under normal physiological and pathological conditions.

Poor prognosis in lung cancer has been attributed to the presence of lung cancer stem cells (CSCs) which resist chemotherapy and cause disease recurrence. Hence, the strong need to identify mechanisms of chemoresistance and to develop new combination therapies. We have previously shown that Stearoyl-CoA-desaturase 1 (SCD1), the enzyme responsible for the conversion of saturated to monounsaturated fatty acids is upregulated in 3D lung cancer spheroids and is an upstream activator of key proliferation pathways β-catenin and YAP/TAZ. Here we first show that SCD1 expression, either alone or in combination with a variety of CSCs markers, correlates with poor prognosis in adenocarcinoma (ADC) of the lung. Treatment of lung ADC cell cultures with cisplatin enhances the formation of larger 3D tumor spheroids and upregulates CSCs markers. In contrast, co-treatment with cisplatin and the SCD1 inhibitor MF-438 reverts upregulation of CSCs markers, strongly synergizes in the inhibition of 3D spheroids formation and induces CSCs apoptosis. Mechanistically, SCD1 inhibition activates endoplasmic reticulum stress response and enhances autophagy. These data all together support the use of combination therapy with SCD1 inhibitors to achieve better control of lung cancer.

Serine and glycine are amino acids that provide the essential precursors for the synthesis of proteins, nucleic acids and lipids. Employing 3 subsequent enzymes, phosphoglycerate dehydrogenase (PHGDH), phosphoserine phosphatase (PSPH), phosphoserine aminotransferase 1 (PSAT1), 3-phosphoglycerate from glycolysis can be converted in serine, which in turn can by converted in glycine by serine methyl transferase (SHMT). Besides proving precursors for macromolecules, serine/glycine biosynthesis is also required for the maintenance of cellular redox state. Therefore, this metabolic pathway has a pivotal role in proliferating cells, including cancer cells. In the last few years an emerging literature provides genetic and functional evidences that hyperactivation of serine/glycine biosynthetic pathway drives tumorigenesis. Here, we extend these observations performing a bioinformatics analysis using public cancer datasets. Our analysis highlighted the relevance of PHGDH and SHMT2 expression as prognostic factor for breast cancer, revealing a substantial ability of these enzymes to predict patient survival outcome. However analyzing patient datasets of lung cancer our analysis reveled that some other enzymes of the pathways, rather than PHGDH, might be associated to prognosis. Although these observations require further investigations they might suggest a selective requirement of some enzymes in specific cancer types, recommending more cautions in the development of novel translational opportunities and biomarker identification of human cancers. published

TAp73, a member of the p53 family, has been traditionally considered a tumor suppressor gene, but a recent report has claimed that it can promote cellular proliferation. This assumption is based on biochemical evidence of activation of anabolic metabolism, with enhanced pentose phosphate shunt (PPP) and nucleotide biosynthesis. Here, while we confirm that TAp73 expression enhances anabolism, we also substantiate its role in inhibiting proliferation and promoting cell death. Hence, we would like to propose an alternative interpretation of the accumulating data linking p73 to cellular metabolism: we suggest that TAp73 promotes anabolism to counteract cellular senescence rather than to support proliferation.

The generation of viable sperm proceeds through a series of coordinated steps, including germ cell self-renewal, meiotic recombination, and terminal differentiation into functional spermatozoa. The p53 family of transcription factors, including p53, p63, and p73, are critical for many physiological processes, including female fertility, but little is known about their functions in spermatogenesis. Here, we report that deficiency of the TAp73 isoform, but not p53 or ΔNp73, results in male infertility because of severe impairment of spermatogenesis. Mice lacking TAp73 exhibited increased DNA damage and cell death in spermatogonia, disorganized apical ectoplasmic specialization, malformed spermatids, and marked hyperspermia. We demonstrated that TAp73 regulates the mRNA levels of crucial genes involved in germ stem/progenitor cells (CDKN2B), spermatid maturation/spermiogenesis (metalloproteinase and serine proteinase inhibitors), and steroidogenesis (CYP21A2 and progesterone receptor). These alterations of testicular histology and gene expression patterns were specific to TAp73 null mice and not features of mice lacking p53. Our work provides previously unidentified in vivo evidence that TAp73 has a unique role in spermatogenesis that ensures the maintenance of mitotic cells and normal spermiogenesis. These results may have implications for the diagnosis and management of human male infertility. published

The amino acid Glutamine is converted into Glutamate by a deamidation reaction catalyzed by the enzyme Glutaminase (GLS). Two isoforms of this enzyme have been described, and the GLS2 isoform is regulated by the tumor suppressor gene p53. Here, we show that the p53 family member TAp73 also drives the expression of GLS2. Specifically, we demonstrate that TAp73 regulates GLS2 during retinoic acid-induced terminal neuronal differentiation of neuroblastoma cells, and overexpression or inhibition of GLS2 modulates neuronal differentiation and intracellular levels of ATP. Moreover, inhibition of GLS activity, by removing Glutamine from the growth medium, impairs in vitro differentiation of cortical neurons. Finally, expression of GLS2 increases during mouse cerebellar development. Although, p73 is dispensable for the in vivo expression of GLS2, TAp73 loss affects GABA and Glutamate levels in cortical neurons. Together, these findings suggest a role for GLS2 acting, at least in part, downstream of p73 in neuronal differentiation and highlight a possible role of p73 in regulating neurotransmitter synthesis.

Significance p73 is a tumor suppressor protein that also plays a central role in brain development. But how p73 modulates neurite outgrowth and neurite arborization is not clear. Neurotrophins are small molecules, which send signals between cells. Neurotrophins can induce cells to either apoptosis or survival. We show that TAp73 is a transcriptional activator of the p75 neurotrophin receptor (p75 NTR ). TAp73 knockout mice have reduced levels of p75 NTR and show peripheral nerve defect, including reduced myelin thickness and thermal sensitivity. p75 NTR is expressed most strongly in the peripheral nerve, where it is important for nerve development. Relatively limited information exists on p75 NTR gene regulation; by linking the p53/p73 gene family to p75 NTR transcription, we introduce possibly controversial mechanisms of regulation.

p73, a member of the p53 tumor suppressor family, is involved in neurogenesis, sensory pathways, immunity, inflammation, and tumorigenesis. How p73 is able to participate in such a broad spectrum of different biological processes is still largely unknown. Here, we report a novel role of p73 in regulating lipid metabolism by direct transactivation of the promoter of autophagy-related protein 5 (ATG5), a gene whose product is required for autophagosome formation. Following nutrient deprivation, the livers of p73-deficient mice demonstrate a massive accumulation of lipid droplets, together with a low level of autophagy, suggesting that triglyceride hydrolysis into fatty acids is blocked owing to deficient autophagy (macrolipophagy). Compared with wild-type mice, mice functionally deficient in all the p73 isoforms exhibit decreased ATG5 expression and lower levels of autophagy in multiple organs. We further show that the TAp73α is the critical p73 isoform responsible for inducing ATG5 expression in a p53-independent manner and demonstrate that ATG5 gene transfer can correct autophagy and macrolipophagy defects in p73-deficient hepatocytes. These data strongly suggest that the p73–ATG5 axis represents a novel, key pathway for regulating lipid metabolism through autophagy. The identification of p73 as a major regulator of autophagy suggests that it may have an important role in preventing or delaying disease and aging by maintaining a homeostatic control.

// Massimiliano Agostini 1,2 , Margherita Annicchiarico-Petruzzelli 3 , Gerry Melino 1,2 and Alessandro Rufini 4 1 Medical Research Council, Toxicology Unit, Leicester University, Leicester, UK 2 Department of Experimental Medicine and Surgery, University of Rome “Tor Vergata”, Rome, Italy 3 Biochemistry Laboratory IDI-IRCC, Department of Experimental Medicine and Surgery, University of Rome “Tor Vergata”, Rome, Italy 4 Department of Cancer Studies, CRUK Leicester Cancer Centre, University of Leicester, Leicester, UK Correspondence to: Alessandro Rufini, email: // Keywords : p73, p53 family, oxidative stress, metabolism, ROS Received : February 22, 2016 Accepted : April 16, 2016 Published : April 22, 2016 Abstract Reactive oxygen species are involved in both physiological and pathological processes including neurodegeneration and cancer. Therefore, cells have developed scavenging mechanisms to maintain redox homeostasis under control. Tumor suppressor genes play a critical role in the regulation of antioxidant genes. Here, we investigated whether the tumor suppressor gene TAp73 is involved in the regulation of metabolic adaptations triggered in response to oxidative stress. H 2 O 2 treatment resulted in numerous biochemical changes in both control and TAp73 knockout (TAp73-/-) mouse embryonic fibroblasts, however the extent of these changes was more pronounced in TAp73-/- cells when compared to control cells. In particular, loss of TAp73 led to alterations in glucose, nucleotide and amino acid metabolism. In addition, H 2 O 2 treatment resulted in increased pentose phosphate pathway (PPP) activity in null mouse embryonic fibroblasts. Overall, our results suggest that in the absence of TAp73, H 2 O 2 treatment results in an enhanced oxidative environment, and at the same time in an increased pro-anabolic phenotype. In conclusion, the metabolic profile observed reinforces the role of TAp73 as tumor suppressor and indicates that TAp73 exerts this function, at least partially, by regulation of cellular metabolism.

The aim of this study was to determine the perioperative behavior of C-reactive protein (CRP) in Crohn’s disease (CD) patients undergoing elective ileo-cecal (IC) resection and to identify association between perioperative CRP levels and endoscopic recurrence at 1 year. Study hypothesis was that perioperative CRP changes are disease specific and could detect subset of patients with more aggressive pathopysiology. Seventy-five patients undergoing IC resection for CD were prospectively enrolled. Serial CRP levels were assessed: preoperative, postoperative day 1 (POD1) and day 5 (POD5). CD patients’ values were compared against same interval assessments of control groups undergoing right colectomy and appendicectomy. At POD1, the serum concentration increase was significantly higher in CD patients than in controls. Comparing with control groups, CRP levels remained remarkably high and showed a lower reduction in CD at POD5. Difference between groups was statistically significant. Optimal cutoff levels have been identified: serum CRP concentrations of >39.8 mg/l at POD1 and of >23.2 mg/l at POD5 have shown a significant association to endoscopic recurrence when using bivariate correlation. In this preliminary series, binary logistic regression could not demonstrate statistical relationship between endoscopic recurrence and any of the variables evaluated as prognostic factor. This is the only study so far that investigates and confirms a disease-specific upregulation of CRP response in the perioperative period for CD patients undergoing surgery. The postoperative CRP levels and kinetics seem to be related to the grade of mucosal inflammation and recurrence rate according to our 12 months endoscopic evaluation.

Newly generated neurons pass through a series of well-defined developmental stages, which allow them to integrate into existing neuronal circuits. After exit from the cell cycle, postmitotic neurons undergo neuronal migration, axonal elongation, axon pruning, dendrite morphogenesis and synaptic maturation and plasticity. Lack of a global metabolic analysis during early cortical neuronal development led us to explore the role of cellular metabolism and mitochondrial biology during ex vivo differentiation of primary cortical neurons. Unexpectedly, we observed a huge increase in mitochondrial biogenesis. Changes in mitochondrial mass, morphology and function were correlated with the upregulation of the master regulators of mitochondrial biogenesis, TFAM and PGC-1α. Concomitant with mitochondrial biogenesis, we observed an increase in glucose metabolism during neuronal differentiation, which was linked to an increase in glucose uptake and enhanced GLUT3 mRNA expression and platelet isoform of phosphofructokinase 1 (PFKp) protein expression. In addition, glutamate-glutamine metabolism was also increased during the differentiation of cortical neurons. We identified PI3K-Akt-mTOR signalling as a critical regulator role of energy metabolism in neurons. Selective pharmacological inhibition of these metabolic pathways indicate existence of metabolic checkpoint that need to be satisfied in order to allow neuronal differentiation.

Silymarin, a mixture of bioactive flavonolignans isolated from Silybum marianum, exhibits anti-carcinogenic, anti-inflammatory and cytoprotective effects. In this study, the in vitro immunomodulatory activity of silymarin was investigated using CD4+ splenocytes from C57/Bl6 mice. Proliferation assay revealed that silymarin, at 50 microM concentration, significantly inhibited CD4+ cells proliferation. ELISA analyses indicated that silymarin significantly inhibited IL-2 and IFN-gamma production. Immunofluorescence staining performed on the mouse hybridoma T cell line (3DO) revealed a block of nuclear translocation of transcription factor kappaB (NF-kappaB), which is known to be responsible for IL-2 transcriptional activation. Moreover, silymarin inhibited p65/NF-kappaB phosphorylation in CD4+ T cell. These results suggest that silymarin is able to inhibit T cell activation and proliferation, notably acting on pathways of NF-kappaB activation/translocation.

Tsc22d3 coding for glucocorticoid-induced leucine zipper (GILZ) was initially identified as a dexamethasone-responsive gene involved in the control of T lymphocyte activation and apoptosis. However, the physiological role of this molecule and its function in the biological activity of glucocorticoids (GCs) has not been clarified. Here, we demonstrate that GILZ interacts directly with Ras in vitro and in vivo as shown by GILZ and Ras coimmunoprecipitation and colocalization upon PMA activation in primary mouse spleen T lymphocytes and thymus cells. The analysis of GILZ mutants showed that they bound Ras through the tuberous sclerosis complex box (TSC) and, depending on the Ras activation level, formed a trimeric complex with Ras and Raf, which we previously identified as a GILZ binder. As a consequence of these interactions, GILZ diminished the activation of Ras and Raf downstream targets including ERK1/2, AKT/PKB serine/threonine kinase, and retinoblastoma (Rb) phosphorylation and cyclin D1 expression, leading to inhibition of Ras- and Raf-dependent cell proliferation and Ras-induced NIH-3T3 transformation. GILZ silencing resulted in an increase in concanavalin A–induced T cell proliferation and, most notably, inhibition of dexamethasone antiproliferative effects. Together, these findings indicate that GILZ serves as a negative regulator of Ras- and Raf-induced proliferation and is an important mediator of the antiproliferative effect of GCs.

TAp73 is a tumour suppressor transcriptional factor, belonging to p53 family. Alteration of TAp73 in tumours might lead to reduced DNA damage response, cell cycle arrest and apoptosis. Carcinogen-induced TAp73−/− tumours display also increased angiogenesis, associated to hyperactivition of hypoxia inducible factor signaling. Here, we show that TAp73 suppresses BNIP3 expression, directly binding its gene promoter. BNIP3 is a hypoxia responsive protein, involved in a variety of cellular processes, such as autophagy, mitophagy, apoptosis and necrotic-like cell death. Therefore, through different cellular process altered expression of BNIP3 may differently contribute to cancer development and progression. We found a significant upregulation of BNIP3 in human lung cancer datasets, and we identified a direct association between BNIP3 expression and survival rate of lung cancer patients. Our data therefore provide a novel transcriptional target of TAp73, associated to its antagonistic role on HIF signaling in cancer, which might play a role in tumour suppression.

The p53-family member, p73, plays a key role in the development of the central nervous system (CNS), in senescence, and in tumor formation. The role of p73 in neuronal differentiation is complex and involves several downstream pathways. Indeed, in the last few years, we have learnt that TAp73 directly or indirectly regulates several genes involved in neural biology. In particular, TAp73 is involved in the maintenance of neural stem/progenitor cell self-renewal and differentiation throughout the regulation of SOX-2, Hey-2, TRIM32 and Notch. In addition, TAp73 is also implicated in the regulation of the differentiation and function of postmitotic neurons by regulating the expression of p75NTR and GLS2 (glutamine metabolism). Further still, the regulation of miR-34a by TAp73 indicates that microRNAs can also participate in this multifunctional role of p73 in adult brain physiology. However, contradictory results still exist in the relationship between p73 and brain disorders, and this remains an important area for further investigation.

MicroRNAs (miRs) are a class of small noncoding RNAs that suppress the expression of protein-coding genes by repressing protein translation. Although the roles that miRs and the miR processing machinery have in regulating epithelial stem cell biology are not fully understood, their fundamental contributions to these processes have been demonstrated over the last few years. The p53-family member p63 is an essential transcription factor for epidermal morphogenesis and homeostasis. p63 functions as a determinant for keratinocyte cell fate and helps to regulate the balance between stemness, differentiation and senescence. An important factor that regulates p63 function is the reciprocal interaction between p63 and miRs. Some miRs control p63 expression, and p63 regulates the miR expression profile in the epidermis. p63 controls miR expression at different levels. It directly regulates the transcription of several miRs and indirectly regulates their processing by regulating the expression of the miR processing components Dicer and DGCR8. In this review, we will discuss the recent findings on the miR-p63 interaction in epidermal biology, particularly focusing on the ΔNp63-dependent regulation of DGCR8 recently described in the ΔNp63(-/-) mouse. We provide a unified view of the current knowledge and discuss the apparent discrepancies and perspective therapeutic opportunities. published

Melanoma is a widespread cancer with poor prognosis. Female hormones are known to be capable of influencing melanoma progression but clinical data related to pregnancy, oral contraception and hormone replacement therapy are controversial. A few reports show that in vitro progesterone (PG) affects melanoma growth in nuclear progesterone receptor (nPR)-positive and nPR-negative cells, but the experimental protocols used are quite different and the results are not univocal. Further research on this topic is thus needed especially in view of the widespread use of PG in clinical practice. In this study, we used human melanoma cells (A-375), which were cultured in vitro in the presence or absence of a wide range of PG concentrations (from 0.01 to 1000 M) in single treatment. Daily cell count, cell cycle analysis and apoptosis assay were performed. Our results show that the low PG concentrations (from 0.01 to 1.0 M) promote a significant increase of melanoma cell proliferation but this growth-stimulatory effect is not observed at 10 M PG and the higher PG concentrations (i.e. 100 and 1000 M) induce a cell density reduction which is the result of both cell cycle arrest and apoptosis. These findings confirm and extend previous observations reported in the international literature. A higher caution in the clinical use of progesterone is thus mandatory, since PG concentrations capable of stimulating melanoma cell proliferation are very close to those commonly used in a wide spectrum of physio-pathological conditions.

Short p63 isoform, ΔNp63, is crucial for epidermis formation, and it plays a pivotal role in controlling the turnover of basal keratinocytes by regulating the expression of a subset of genes involved in cell cycle and cell adhesion programs. The glycolytic enzyme hexokinase 2 (HK2) represents the first step of glucose utilization in cells. The family of HKs has four isoforms that differ mainly in their tissue and subcellular distribution. The preferential mitochondrial localization of HK2 at voltage-dependent anion channels provides access to ATP generated by oxidative phosphorylation and generates an ADP/ATP recycling mechanism to maintain high respiration rates and low electron leak. Here, we report that ΔNp63 depletion in human keratinocytes impairs mitochondrial basal respiration and increases mitochondrial membrane polarization and intracellular reactive oxygen species. We show ΔNp63-dependent regulation of HK2 expression, and we use ChIP, validated by p63-Chip sequencing genomewide profiling analysis, and luciferase assays to demonstrate the presence of one p63-specific responsive element within the 15th intronic region of the HK2 gene, providing evidence of a direct interaction. Our data support the notion of ΔNp63 as a master regulator in epithelial cells of a combined subset of molecular mechanisms, including cellular energy metabolism and respiration. The ΔNp63-HK2 axis is also present in epithelial cancer cells, suggesting that ΔNp63 could participate in cancer metabolic reprogramming.

BACKGROUND 2-CADO inhibits the growth of several types of cells and causes apoptosis by a mechanism which involves adenosine receptors or cellular uptake or both. METHODS Androgen-independent (PC3) prostate cancer cells were used in the study and proliferation, cell-cycle progression, and apoptosis analyzed. Deoxy-and ribonucleoside triphosphate pools were determined by HPLC. The molecular mechanism was examined by assessing the involvement of DNA synthesizing enzymes in the cellular response. RESULTS 2-CADO treatment dramatically reduced the number of prostate cancer cells and permanently stopped cell-cycle progression in the S-phase. The role of 2-CADO in prostate cancer cells is uptake-mediated and followed by sequential phosphorylations to 2-Cl-ATP that irreversibly inhibits several key-enzymes for DNA biosynthesis. CONCLUSIONS Arrest of DNA synthesis responsible for toxicity of 2-CADO to PC3 cells is due to the lack of substrates for DNA polymerization caused by irreversible inhibition of purine/pyrimidine ribo-and 2-deoxyribonucleotides salvage enzymes. Prostate 66: 1425–1436, 2006. © 2006 Wiley-Liss, Inc.

Activation of serine biosynthesis supports growth and proliferation of cancer cells. Human cancers often exhibit overexpression of phosphoglycerate dehydrogenase (PHGDH), the metabolic enzyme that catalyses the reaction that diverts serine biosynthesis from the glycolytic pathway. By refueling serine biosynthetic pathways, cancer cells sustain their metabolic requirements, promoting macromolecule synthesis, anaplerotic flux and ATP. Serine biosynthesis intersects glutaminolysis and together with this pathway provides substrates for production of antioxidant GSH. In human lung adenocarcinomas we identified a correlation between serine biosynthetic pathway and p73 expression. Metabolic profiling of human cancer cell line revealed that TAp73 activates serine biosynthesis, resulting in increased intracellular levels of serine and glycine, associated to accumulation of glutamate, tricarboxylic acid (TCA) anaplerotic intermediates and GSH. However, at molecular level p73 does not directly regulate serine metabolic enzymes, but transcriptionally controls a key enzyme of glutaminolysis, glutaminase-2 (GLS-2). p73, through GLS-2, favors conversion of glutamine in glutamate, which in turn drives the serine biosynthetic pathway. Serine and glutamate can be then employed for GSH synthesis, thus the p73-dependent metabolic switch enables potential response against oxidative stress. In knockdown experiment, indeed, TAp73 depletion completely abrogates cancer cell proliferation capacity in serine/glycine-deprivation, supporting the role of p73 to help cancer cells under metabolic stress. These findings implicate p73 in regulation of cancer metabolism and suggest that TAp73 influences glutamine and serine metabolism, affecting GSH synthesis and determining cancer pathogenesis.

In mammals, the p53 family comprises two additional members, p63 and p73 (hereafter referred to as TP53, TP63, and TP73, respectively). The usage of two alternative promoters produces protein variants either with (transactivating [TA] isoforms) or without (ΔN isoforms) the N-terminal transactivation domain (TAD). In general, the TA proteins exert TP53-like tumor-suppressive activities through their ability to activate a common set of target genes. The ΔN proteins can act as dominant-negative inhibitors of the transcriptionally active family members. Additionally, they possess intrinsic-specific biological activities due to the presence of alternative TADs, and as a result of engaging a different set of regulators. This review summarizes the current understanding of upstream regulators and downstream effectors of the TP53 family proteins, with particular emphasis on those that are relevant for their role in tumorigenesis. Furthermore, we highlight the existence of networks and cross-talks among the TP53 family members, their modulators, as well as the transcriptional targets.

Highlights • Serine and glycine are essential metabolites for cancer cells. • Serine and glycine provide precursors for macromolecules and antioxidant defence. • Metabolic enzymes of serine and glycine biosynthesis are upregulated in cancer. • Innovative anticancer therapy is aiming to target serine and glycine biosynthesis., Serine and glycine are biosynthetically linked, and together provide the essential precursors for the synthesis of proteins, nucleic acids, and lipids that are crucial to cancer cell growth. Moreover, serine/glycine biosynthesis also affects cellular antioxidative capacity, thus supporting tumour homeostasis. A crucial contribution of serine/glycine to cellular metabolism is through the glycine cleavage system, which refuels one-carbon metabolism; a complex cyclic metabolic network based on chemical reactions of folate compounds. The importance of serine/glycine metabolism is further highlighted by genetic and functional evidence indicating that hyperactivation of the serine/glycine biosynthetic pathway drives oncogenesis. Recent developments in our understanding of these pathways provide novel translational opportunities for drug development, dietary intervention, and biomarker identification of human cancers.

The mir-34 family was originally cloned and characterized in 2007 as a p53 target gene. Almost immediately it became clear that its major role is as a master regulator of tumor suppression. Indeed, when overexpressed, it directly and indirectly represses several oncogenes, resulting in an increase of cancer cell death (including cancer stem cells), and in an inhibition of metastasis. Moreover, its expression is deregulated in several human cancers. In 2013, a miR-34 mimic has become the first microRNA to reach phase 1 clinical trials. Here we review the miR-34 family and their role in tumor biology, and discuss the potential therapeutic applications of miR-34a mimic.

To investigate the role of major histocompatibility complex class I and bone marrow stromal cells on in vitro differentiation of natural killer cells, a CD44(low/-) CD2- population was isolated from mouse bone marrow. This NK-1.1- CD3- LFA-3+ B220+ population, when stimulated with IL-2 and co-cultured with supportive syngeneic stromal cells, generated populations of NK-1.1+ Ly49A+ Ly49C/I+ CD3- mature natural killer cells. The effect of anti-H-2b monoclonal antibodies (mAbs) on this phenomenon was assayed. Pre-adhesion of anti-H-2b mAbs to the stromal cells did not exert any effect, whereas when the same mAbs were pre-adhered to progenitors, there was a inhibition of natural killer cell generation that was maximum when the mAbs were added directly to cultures. In addition, the anti-H-2b mAbs did not inhibit the IL-2-induced proliferation of mature natural killer cells. Allogeneic but not H-2b-deficient stromal cells decreased the expression of Ly-49C/I but not Ly49A, thus suggesting that stromal cell haplotypes qualitatively influence the expression of Ly49s repertoire.

Long-term bone marrow cultures were used to investigate the effect of IL-2, a cytokine widely used in immunotherapy, on natural killer cell differentiation. Specifically, the role of MHC was evaluated by comparing normal B6 and class I-deficient TAP-1-/- mice. The number of cells generated after a 13-day culture was the same in cell cultures from TAP-1-/- or B6 mice but the relative number of natural killer cells, identified as NK-1.1+CD3 cells by flow cytometry analysis, was increased in TAP-1-/- compared to B6 cultures (74.4% and 63.9%, respectively). Addition of an anti-class I mAb determined a strong inhibition of natural killer cell generation in B6 cultures, and its effect was specific since no effect was seen in TAP-1-/- cell cultures. TAP-1-/- natural killer cells or the few natural killer cells escaping the inhibitory effect of anti-class I mAb, were less cytotoxic than total B6 natural killer cells against target cell lines of different haplotype.

The proteins p73 and p63 are members of the p53 protein family and are involved in important developmental processes. Their high sequence identity with the tumor suppressor p53 has suggested that they act as tumor suppressors as well. While p63 has a crucial role in the maintenance of epithelial stem cells and in the quality control of oocytes without a clear role as a tumor suppressor, p73's tumor suppressor activity is well documented. In a recent study we have shown that the transcriptional activity of TAp63α, the isoform responsible for the quality control in oocytes, is regulated by its oligomeric state. The protein forms an inactive, dimeric and compact conformation in resting oocytes, while the detection of DNA damage leads to the formation of an active, tetrameric and open conformation. p73 shows a high sequence identity to p63, including those domains that are crucial in stabilizing its inactive state, thus suggesting that p73's activity might be regulated by its oligomeric state as well. Here, we have investigated the oligomeric state of TAp73α by size exclusion chromatography and detailed domain interaction mapping, and show that in contrast to p63, TAp73α is a constitutive open tetramer. However, its transactivation potential depends on the cellular background and the promoter context. These results imply that the regulation of p73's transcriptional activity might be more closely related to p53 than to p63.