TAp73 depletion accelerates aging through metabolic dysregulation.

Aging is associated with impaired scavenging of reactive oxygen species (ROS). Here, we show that TAp73, a p53 family member, protects against aging by regulating mitochondrial activity and preventing ROS accumulation. TAp73-null mice show more pronounced aging with increased oxidative damage and senescence. TAp73 deletion reduces cellular ATP levels, oxygen consumption, and mitochondrial complex IV activity, with increased ROS production and oxidative stress sensitivity. We show that the mitochondrial complex IV subunit cytochrome C oxidase subunit 4 (Cox4i1) is a direct TAp73 target and that Cox4i1 knockdown phenocopies the cellular senescence of TAp73-null cells. Results indicate that TAp73 affects mitochondrial respiration and ROS homeostasis, thus regulating aging.

Isoform-specific p73 knockout mice reveal a novel role for delta Np73 in the DNA damage response pathway.

Mice with a complete deficiency of p73 have severe neurological and immunological defects due to the absence of all TAp73 and DeltaNp73 isoforms. As part of our ongoing program to distinguish the biological functions of these isoforms, we generated mice that are selectively deficient for the DeltaNp73 isoform. Mice lacking DeltaNp73 (DeltaNp73(-/-) mice) are viable and fertile but display signs of neurodegeneration. Cells from DeltaNp73(-/-) mice are sensitized to DNA-damaging agents and show an increase in p53-dependent apoptosis. When analyzing the DNA damage response (DDR) in DeltaNp73(-/-) cells, we discovered a completely new role for DeltaNp73 in inhibiting the molecular signal emanating from a DNA break to the DDR pathway. We found that DeltaNp73 localizes directly to the site of DNA damage, can interact with the DNA damage sensor protein 53BP1, and inhibits ATM activation and subsequent p53 phosphorylation. This novel finding may explain why human tumors with high levels of DeltaNp73 expression show enhanced resistance to chemotherapy.

TAp73 is required for spermatogenesis and the maintenance of male fertility.

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.

Current understanding of the interplay between extracellular matrix remodelling and gut permeability in health and disease.

The intestinal wall represents an interactive network regulated by the intestinal epithelium, extracellular matrix (ECM) and mesenchymal compartment. Under healthy physiological conditions, the epithelium undergoes constant renewal and forms an integral and selective barrier. Following damage, the healthy epithelium is restored via a series of signalling pathways that result in remodelling of the scaffolding tissue through finely-regulated proteolysis of the ECM by proteases such as matrix metalloproteinases (MMPs). However, chronic inflammation of the gastrointestinal tract, as occurs in Inflammatory Bowel Disease (IBD), is associated with prolonged disruption of the epithelial barrier and persistent damage to the intestinal mucosa. Increased barrier permeability exhibits distinctive signatures of inflammatory, immunological and ECM components, accompanied by increased ECM proteolytic activity. This narrative review aims to bring together the current knowledge of the interplay between gut barrier, immune and ECM features in health and disease, discussing the role of barrier permeability as a discriminant between homoeostasis and IBD.

Proline metabolism shapes the tumor microenvironment: from collagen deposition to immune evasion.

Proline is a nonessential amino acid, and its metabolism has been implicated in numerous malignancies. Together with a direct role in regulating cancer cells' proliferation and survival, proline metabolism plays active roles in shaping the tumor microenvironment (TME). Cancer-associated fibroblasts (CAFs) display high rates of proline biosynthesis to support the production of collagen for the extracellular matrix (ECM). Indeed, impaired proline metabolism in CAFs results in reduced collagen deposition and compromises the growth and metastatic spread of cancer. Moreover, the rate of proline metabolism regulates intracellular reactive oxygen species (ROS) levels, which influence the production and release of cytokines from cancer cells, contributing toward an immune-permissive TME. Hence, targeting proline metabolism is a promising anticancer strategy that could improve patients' outcome and response to immunotherapy.

BRAFV600E-mutated serrated colorectal neoplasia drives transcriptional activation of cholesterol metabolism.

BRAF mutations occur early in serrated colorectal cancers, but their long-term influence on tissue homeostasis is poorly characterized. We investigated the impact of short-term (3 days) and long-term (6 months) expression of BrafV600E in the intestinal tissue of an inducible mouse model. We show that BrafV600E perturbs the homeostasis of intestinal epithelial cells, with impaired differentiation of enterocytes emerging after prolonged expression of the oncogene. Moreover, BrafV600E leads to a persistent transcriptional reprogramming with enrichment of numerous gene signatures indicative of proliferation and tumorigenesis, and signatures suggestive of metabolic rewiring. We focused on the top-ranking cholesterol biosynthesis signature and confirmed its increased expression in human serrated lesions. Functionally, the cholesterol lowering drug atorvastatin prevents the establishment of intestinal crypt hyperplasia in BrafV600E-mutant mice. Overall, our work unveils the long-term impact of BrafV600E expression in intestinal tissue and suggests that colorectal cancers with mutations in BRAF might be prevented by statins.

Glutathione supports lipid abundance in vivo.

Cells rely on antioxidants to survive. The most abundant antioxidant is glutathione (GSH). The synthesis of GSH is non-redundantly controlled by the glutamate-cysteine ligase catalytic subunit (GCLC). GSH imbalance is implicated in many diseases, but the requirement for GSH in adult tissues is unclear. To interrogate this, we developed a series of in vivo models to induce Gclc deletion in adult animals. We find that GSH is essential to lipid abundance in vivo. GSH levels are reported to be highest in liver tissue, which is also a hub for lipid production. While the loss of GSH did not cause liver failure, it decreased lipogenic enzyme expression, circulating triglyceride levels, and fat stores. Mechanistically, we found that GSH promotes lipid abundance by repressing NRF2, a transcription factor induced by oxidative stress. These studies identify GSH as a fulcrum in the liver's balance of redox buffering and triglyceride production.

TAp73 regulates the spindle assembly checkpoint by modulating BubR1 activity.

The role of various p73 isoforms in tumorigenesis has been controversial. However, as we have recently shown, the generation of TAp73-deficient (TAp73(-/-)) mice reveals that TAp73 isoforms exert tumor-suppressive functions, indicating an emerging role for Trp-73 in the maintenance of genomic stability. Unlike mice lacking all p73 isoforms, TAp73(-/-) mice show a high incidence of spontaneous tumors. Moreover, TAp73(-/-) mice are infertile and produce oocytes exhibiting spindle abnormalities. These data suggest a link between TAp73 activities and the common molecular machinery underlying meiosis and mitosis. Previous studies have indicated that the spindle assembly checkpoint (SAC) complex, whose activation leads to mitotic arrest, also regulates meiosis. In this study, we demonstrate in murine and human cells that TAp73 is able to interact directly with several partners of the SAC complex (Bub1, Bub3, and BubR1). We also show that TAp73 is involved in SAC protein localization and activities. Moreover, we show that decreased TAp73 expression correlates with increases of SAC protein expression in patients with lung cancer. Our results establish TAp73 as a regulator of SAC responses and indicate that TAp73 loss can lead to mitotic arrest defects. Our data suggest that SAC impairment in the absence of functional TAp73 could explain the genomic instability and increased aneuploidy observed in TAp73-deficient cells.

Increased mitochondrial proline metabolism sustains proliferation and survival of colorectal cancer cells.

Research into the metabolism of the non-essential amino acid (NEAA) proline in cancer has gained traction in recent years. The last step in the proline biosynthesis pathway is catalyzed by pyrroline-5-carboxylate reductase (PYCR) enzymes. There are three PYCR enzymes: mitochondrial PYCR1 and 2 and cytosolic PYCR3 encoded by separate genes. The expression of the PYCR1 gene is increased in numerous malignancies and correlates with poor prognosis. PYCR1 expression sustains cancer cells' proliferation and survival and several mechanisms have been implicated to explain its oncogenic role. It has been suggested that the biosynthesis of proline is key to sustain protein synthesis, support mitochondrial function and nucleotide biosynthesis. However, the links between proline metabolism and cancer remain ill-defined and are likely to be tissue specific. Here we use a combination of human dataset, human tissue and mouse models to show that the expression levels of the proline biosynthesis enzymes are significantly increased during colorectal tumorigenesis. Functionally, the expression of mitochondrial PYCRs is necessary for cancer cells' survival and proliferation. However, the phenotypic consequences of PYCRs depletion could not be rescued by external supplementation with either proline or nucleotides. Overall, our data suggest that, despite the mechanisms underlying the role of proline metabolism in colorectal tumorigenesis remain elusive, targeting the proline biosynthesis pathway is a suitable approach for the development of novel anti-cancer therapies.

Cell death pathology: the war against cancer.

Programmed cell death was a fundamental discovery, awarded with the Nobel price in 2002 to Sulston, Brenner and Horvitz. Since then it has been clear that alteration of apoptotic pathways is a common feature of tumors, enabling cancer cells to survive chemotherapeutic interventions. Thus, apoptosis is an attractive target in cancer therapy, with the aim to revert the cancer-related alterations of the cell death machinery. Here, we overview the fundamental apoptotic pathways and summarize the attempts to target apoptosis to restore cell death in cancer cells with a special focus on the p53-family and autophagy.

Mass spectrometric detection of KRAS protein mutations using molecular imprinting.

Cancer is a disease of cellular evolution where single base changes in the genetic code can have significant impact on the translation of proteins and their activity. Thus, in cancer research there is significant interest in methods that can determine mutations and identify the significant binding sites (epitopes) of antibodies to proteins in order to develop novel therapies. Nano molecularly imprinted polymers (nanoMIPs) provide an alternative to antibodies as reagents capable of specifically capturing target molecules depending on their structure. In this study, we used nanoMIPs to capture KRAS, a critical oncogene, to identify mutations which when present are indicative of oncological progress. Herein, coupling nanoMIPs (capture) and liquid chromatography-mass spectrometry (detection), LC-MS has allowed us to investigate mutational assignment and epitope discovery. Specifically, we have shown epitope discovery by generating nanoMIPs to a recombinant KRAS protein and identifying three regions of the protein which have been previously assigned as epitopes using much more time-consuming protocols. The mutation status of the released tryptic peptide was identified by LC-MS following capture of the conserved region of KRAS using nanoMIPS, which were tryptically digested, thus releasing the sequence of a non-conserved (mutated) region. This approach was tested in cell lines where we showed the effective genotyping of a KRAS cell line and in the plasma of cancer patients, thus demonstrating its ability to diagnose precisely the mutational status of a patient. This work provides a clear line-of-sight for the use of nanoMIPs to its translation from research into diagnostic and clinical utility.

The Janus-like role of proline metabolism in cancer.

The metabolism of the non-essential amino acid L-proline is emerging as a key pathway in the metabolic rewiring that sustains cancer cells proliferation, survival and metastatic spread. Pyrroline-5-carboxylate reductase (PYCR) and proline dehydrogenase (PRODH) enzymes, which catalyze the last step in proline biosynthesis and the first step of its catabolism, respectively, have been extensively associated with the progression of several malignancies, and have been exposed as potential targets for anticancer drug development. As investigations into the links between proline metabolism and cancer accumulate, the complexity, and sometimes contradictory nature of this interaction emerge. It is clear that the role of proline metabolism enzymes in cancer depends on tumor type, with different cancers and cancer-related phenotypes displaying different dependencies on these enzymes. Unexpectedly, the outcome of rewiring proline metabolism also differs between conditions of nutrient and oxygen limitation. Here, we provide a comprehensive review of proline metabolism in cancer; we collate the experimental evidence that links proline metabolism with the different aspects of cancer progression and critically discuss the potential mechanisms involved.

p73 Regulates Primary Cortical Neuron Metabolism: a Global Metabolic Profile.

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.

Sensitivity of Colorectal Cancer to Arginine Deprivation Therapy is Shaped by Differential Expression of Urea Cycle Enzymes.

Tumors deficient in the urea cycle enzymes argininosuccinate synthase-1 (ASS1) and ornithine transcarbamylase (OTC) are unable to synthesize arginine and can be targeted using arginine-deprivation therapy. Here, we show that colorectal cancers (CRCs) display negligible expression of OTC and, in subset of cases, ASS1 proteins. CRC cells fail to grow in arginine-free medium and dietary arginine deprivation slows growth of cancer cells implanted into immunocompromised mice. Moreover, we report that clinically-formulated arginine-degrading enzymes are effective anticancer drugs in CRC. Pegylated arginine deiminase (ADI-PEG20), which degrades arginine to citrulline and ammonia, affects growth of ASS1-negative cells, whereas recombinant human arginase-1 (rhArg1peg5000), which degrades arginine into urea and ornithine, is effective against a broad spectrum of OTC-negative CRC cell lines. This reflects the inability of CRC cells to recycle citrulline and ornithine into the urea cycle. Finally, we show that arginase antagonizes chemotherapeutic drugs oxaliplatin and 5-fluorouracil (5-FU), whereas ADI-PEG20 synergizes with oxaliplatin in ASS1-negative cell lines and appears to interact with 5-fluorouracil independently of ASS1 status. Overall, we conclude that CRC is amenable to arginine-deprivation therapy, but we warrant caution when combining arginine deprivation with standard chemotherapy.

Circulating tumor DNA in patients with colorectal adenomas: assessment of detectability and genetic heterogeneity.

Improving early detection of colorectal cancer (CRC) is a key public health priority as adenomas and stage I cancer can be treated with minimally invasive procedures. Population screening strategies based on detection of occult blood in the feces have contributed to enhance detection rates of localized disease, but new approaches based on genetic analyses able to increase specificity and sensitivity could provide additional advantages compared to current screening methodologies. Recently, circulating cell-free DNA (cfDNA) has received much attention as a cancer biomarker for its ability to monitor the progression of advanced disease, predict tumor recurrence and reflect the complex genetic heterogeneity of cancers. Here, we tested whether analysis of cfDNA is a viable tool to enhance detection of colon adenomas. To address this, we assessed a cohort of patients with adenomas and healthy controls using droplet digital PCR (ddPCR) and mutation-specific assays targeted to trunk mutations. Additionally, we performed multiregional, targeted next-generation sequencing (NGS) of adenomas and unmasked extensive heterogeneity, affecting known drivers such as APC, KRAS and mismatch repair (MMR) genes. However, tumor-related mutations were undetectable in patients' plasma. Finally, we employed a preclinical mouse model of Apc-driven intestinal adenomas and confirmed the inability to identify tumor-related alterations via cfDNA, despite the enhanced disease burden displayed by this experimental cancer model. Therefore, we conclude that benign colon lesions display extensive genetic heterogeneity, that they are not prone to release DNA into the circulation and are unlikely to be reliably detected with liquid biopsies, at least with the current technologies.

Role of p63 and p73 isoforms on the cell death in patients with hepatocellular carcinoma submitted to orthotopic liver transplantation.

BACKGROUND & AIMS: Patients with hepatocellular carcinoma (HCC) submitted to orthotopic liver transplantation (OLT) have a variable 5-year survival rate limited mostly by tumor recurrence. The etiology, age, sex, alcohol, Child-Pugh, and the immunesuppressor have been associated with tumour recurrence. The expression of ΔNp73 is related to the reduced survival of patients with HCC. The study evaluated the expression of p63 and p73 isoforms and cell death receptors, and their relation to tumour recurrence and survival. The results were in vitro validated in HCC cell lines. METHODS: HCC sections from patients submitted to OLT were used. The in vitro study was done in differentiated hepatitis B virus (HBV)-expressing Hep3B and control HepG2 cells. The expression of cell death receptors and cFLIPS/L, caspase-8 and -3 activities, and cell proliferation were determined in control and p63 and p73 overexpressing HCC cells. RESULTS: The reduced tumor expression of cell death receptors and TAp63 and TAp73, and increased ΔNp63 and ΔNp73 expression were associated with tumor recurrence and reduced survival. The in vitro study demonstrated that HBV-expressing Hep3B vs HepG2 cells showed reduced expression of p63 and p73, cell death receptors and caspase activation, and increased cFLIPL/cFLIPS ratio. The overexpression of TAp63 and TAp73 exerted a more potent pro-apoptotic and anti-proliferative effects in Hep3B than HepG2-transfected cells which was related to cFLIPL upregulation. CONCLUSIONS: The reduction of TAp63 and TAp73 isoforms, rather than alteration of ΔN isoform expression, exerted a significant functional repercussion on cell death and proliferation in HBV-expressing HepB cells.

p63 and p73, members of the p53 gene family, transactivate PKCdelta.

The p53 family comprises three genes that encode for p53, p63 and p73. These genes have a significant degree of sequence homology, especially in the central sequence-specific DNA-binding domain. The high homology among the three DNA-binding domains indicates that these transcription factors have identical residues interacting with DNA, and thus potentially can recognize the same transcriptional targets. In this study, we demonstrate that PKCdelta is induced by p63 and p73 in Saos2 cells. The putative human PKCdelta promoter harbours three p53-like binding sites (RE I, RE II, RE III). In order to confirm the transactivation of PKCdelta by p53 family members, we performed transcription assays using the entire or selected regions of the promoter upstream of a luciferase reporter gene. The results obtained demonstrated that, at least in vitro, the p53 family members tested (TAp63alpha, TAp73alpha, DeltaNp63alpha, but not DeltaNp73alpha) were able to drive transcription from the PKCdelta promoter.

Response to comment on "Cancer chemoprevention: Evidence of a nonlinear dose response for the protective effects of resveratrol in humans and mice".

Low-dose resveratrol did not have the opposite effect on intestinal adenoma development when given in a standard diet instead of a high-fat diet, although we agree on the need for more information on the interaction of diet-derived compounds such as resveratrol and other lifestyle, metabolic and hormonal factors.

Metabolic pathways regulated by TAp73 in response to oxidative stress.

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. H2O2 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, H2O2 treatment resulted in increased pentose phosphate pathway (PPP) activity in null mouse embryonic fibroblasts. Overall, our results suggest that in the absence of TAp73, H2O2 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.