[show abstract][hide abstract] ABSTRACT: 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.
Proceedings of the National Academy of Sciences 01/2014; · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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 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, 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 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.
[show abstract][hide abstract] ABSTRACT: It is well known that protein tyrosine phosphatases (PTPs) that become oxidized due to exposure to reactive oxygen species (ROS) undergo a conformational change and are inactivated. However, whether PTPs can actively regulate ROS levels in order to prevent PTP inhibition has yet to be investigated. Here, we demonstrate that PTP non-receptor type 12 (PTPN12) protects cells against aberrant ROS accumulation and death induced by oxidative stress. Murine embryonic fibroblasts (MEFs) deficient in PTPN12 underwent increased ROS-induced apoptosis under conditions of antioxidant depletion. Cells lacking PTPN12 also showed defective activation of FOXO1/3a, transcription factors required for the upregulation of several antioxidant genes. PTPN12-mediated regulation of ROS appeared to be mediated by phosphoinositide-dependent kinase-1 (PDK1), which was hyperstimulated in the absence of PTPN12. As tight regulation of ROS to sustain survival is a key feature of cancer cells, we examined PTPN12 levels in tumors from a cohort of breast cancer patients. Patients whose tumors showed high levels of PTPN12 transcripts had a significantly poorer prognosis. Analysis of tissues from patients with various breast cancer subtypes revealed that more triple-negative breast cancers, the most aggressive breast cancer subtype, showed high PTPN12 expression than any other subtype. Furthermore, both human breast cancer cells and mouse mammary epithelial tumor cells engineered to lack PTPN12 exhibited reduced tumorigenic and metastatic potential in vivo that correlated with their elevated ROS levels. The involvement of PTPN12 in the antioxidant response of breast cancer cells suggests that PTPN12 may represent a novel therapeutic target for this disease.Oncogene advance online publication, 25 February 2013; doi:10.1038/onc.2013.24.
[show abstract][hide abstract] ABSTRACT: p53 functions as a transcription factor involved in cell-cycle control, DNA repair, apoptosis and cellular stress responses. However, besides inducing cell growth arrest and apoptosis, p53 activation also modulates cellular senescence and organismal aging. Senescence is an irreversible cell-cycle arrest that has a crucial role both in aging and as a robust physiological antitumor response, which counteracts oncogenic insults. Therefore, via the regulation of senescence, p53 contributes to tumor growth suppression, in a manner strictly dependent by its expression and cellular context. In this review, we focus on the recent advances on the contribution of p53 to cellular senescence and its implication for cancer therapy, and we will discuss p53's impact on animal lifespan. Moreover, we describe p53-mediated regulation of several physiological pathways that could mediate its role in both senescence and aging.Oncogene advance online publication, 18 February 2013; doi:10.1038/onc.2012.640.
[show abstract][hide abstract] ABSTRACT: Mitochondria are the dominant source of the cellular energy requirements through oxidative phosphorylation, but they are also central players in apoptosis. Nutrient availability may have been the main evolutionary driving force behind these opposite mitochondrial functions: production of energy to sustain life and release of apoptotic proteins to trigger cell death. Here, we explore the link between nutrients, mitochondria and apoptosis with known and potential implications for age-related decline and metabolic syndromes.
[show abstract][hide abstract] ABSTRACT: 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.
Genes & development 09/2012; 26(18):2009-14. · 12.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: The transcription factor p73 belongs to the p53 family of tumour suppressors and similar to other family members, transcribed as different isoforms with opposing pro- and anti-apoptotic functions. Unlike p53, p73 mutations are extremely rare in cancers. Instead, the pro-apoptotic activities of transcriptionally active p73 isoforms are commonly inhibited by over-expression of the dominant negative p73 isoforms. Therefore the relative ratio of different p73 isoforms is critical for the cellular response to a chemotherapeutic agent. Here, we analysed the expression of N-terminal p73 isoforms in cell lines and mouse tissues. Our data showed that the transcriptionally competent TAp73 isoform is abundantly expressed in cancer cell lines compared to the dominant negative ΔNp73 isoform. Interestingly, we detected higher levels of ΔNp73 in some mouse tissues, suggesting that ΔNp73 may have a physiological role in these tissues.
[show abstract][hide abstract] ABSTRACT: The transcription factor p73 belongs to the p53 family of tumour suppressors and similar to other family members, transcribed as different isoforms with opposing pro- and anti-apoptotic functions. Unlike p53, p73 mutations are extremely rare in cancers. Instead, the pro-apoptotic activities of transcriptionally active p73 isoforms are commonly inhibited by over-expression of the dominant negative p73 isoforms. Therefore the relative ratio of different p73 isoforms is critical for the cellular response to a chemotherapeutic agent. Here, we analysed the expression of N-terminal p73 isoforms in cell lines and mouse tissues. Our data showed that the transcriptionally competent TAp73 isoform is abundantly expressed in cancer cell lines compared to the dominant negative DeltaNp73 isoform. Interestingly, we detected higher levels of DeltaNp73 in some mouse tissues, suggesting that DeltaNp73 may have a physiological role in these tissues.
[show abstract][hide abstract] ABSTRACT: 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.
Biochemical and Biophysical Research Communications 10/2011; 414(3):445-50. · 2.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: Recent findings have shown that the development of teeth involves a complex sequence of molecular events in which the p53 family member p63 is involved. Indeed, mice lacking p63 do not have teeth and humans bearing mutations in p63 suffer developmental syndromes that affect tooth morphology and number. Several isoforms of p63 have been described: the use of two different promoters produces longer TAp63 isoforms, or shorter, 5' truncated isoforms known as ΔNp63. The 3' end of primary transcripts is then subject to alternative splicing resulting in three additional isoforms: alpha (α), beta (β) and gamma (γ). Tooth development relies mainly on the activity of the N-terminally truncated ΔNp63 isoforms. Here we review the experimental evidence for the involvement of ΔNp63 in tooth development through its ability to sustain the molecular signalling that orchestrates epithelial-mesenchymal interaction.
[show abstract][hide abstract] ABSTRACT: Proteins containing a caveolin-binding domain (CBD), such as the Rho-GTPases, can interact with caveolin-1 (Cav1) through its caveolin scaffold domain. Rho-GTPases are important regulators of p130(Cas), which is crucial for both normal cell migration and Src kinase-mediated metastasis of cancer cells. However, although Rho-GTPases (particularly RhoC) and Cav1 have been linked to cancer progression and metastasis, the underlying molecular mechanisms are largely unknown. To investigate the function of Cav1-Rho-GTPase interaction in metastasis, we disrupted Cav1-Rho-GTPase binding in melanoma and mammary epithelial tumor cells by overexpressing CBD, and examined the loss-of-function of RhoC in metastatic cancer cells. Cancer cells overexpressing CBD or lacking RhoC had reduced p130(Cas) phosphorylation and Rac1 activation, resulting in an inhibition of migration and invasion in vitro. The activity of Src and the activation of its downstream targets FAK, Pyk2, Ras and extracellular signal-regulated kinase (Erk)1/2 were also impaired. A reduction in α5-integrin expression, which is required for binding to fibronectin and thus cell migration and survival, was observed in CBD-expressing cells and cells lacking RhoC. As a result of these defects, CBD-expressing melanoma cells had a reduced ability to metastasize in recipient mice, and impaired extravasation and survival in secondary sites in chicken embryos. Our data indicate that interaction between Cav1 and Rho-GTPases (most likely RhoC but not RhoA) promotes metastasis by stimulating α5-integrin expression and regulating the Src-dependent activation of p130(Cas)/Rac1, FAK/Pyk2 and Ras/Erk1/2 signaling cascades.
[show abstract][hide abstract] ABSTRACT: p73 is a tumor suppressor belonging to the p53 family of transcription factors. Distinct isoforms are transcribed from the p73 locus. The use of 2 promoters at the N-terminus allows the expression of an isoform containing (TAp73) or not containing (ΔNp73) a complete N-terminal transactivation domain, with the latter isoform capable of a dominant negative effect over the former. In addition, both N-terminal variants are alternatively spliced at the C-terminus. TAp73 is a bona fide tumor suppressor, being able to induce cell death and cell cycle arrest; conversely, ΔNp73 shows oncogenic properties, inhibiting TAp73 and p53 functions. Here, we discuss the latest findings linking p73 to cancer. The generation of isoform specific null mice has helped in dissecting the contribution of TA versus ΔNp73 isoforms to tumorigenesis. The activity of both isoforms is regulated transcriptionally and by posttranslational modification. p73 dysfunction, particularly of TAp73, has been associated with mitotic abnormalities, which may lead to polyploidy and aneuploidy and thus contribute to tumorigenesis. Although p73 is only rarely mutated in cancer, the tumor suppressor actions of TAp73 are inhibited by mutant p53, a finding that has important implications for cancer therapy. Finally, we discuss the expression and role of p73 isoforms in human cancer, with a particular emphasis on the neuroblastoma cancer model. Broadly, the data support the hypothesis that the ratio between TAp73 and ΔNp73 is crucial for tumor progression and therapeutic response.
[show abstract][hide abstract] ABSTRACT: p73, a transcription factor of the p53 family, plays a key role in many biological processes including neuronal development. Indeed, mice deficient for both TAp73 and ΔNp73 isoforms display neuronal pathologies, including hydrocephalus and hippocampal dysgenesis, with defects in the CA1-CA3 pyramidal cell layers and the dentate gyrus. TAp73 expression increases in parallel with neuronal differentiation and its ectopic expression induces neurite outgrowth and expression of neuronal markers in neuroblastoma cell lines and neural stem cells, suggesting that it has a pro-differentiation role. In contrast, ΔNp73 shows a survival function in mature cortical neurons as selective ΔNp73 null mice have reduced cortical thickness. Recent evidence has also suggested that p73 isoforms are deregulated in neurodegenerative pathologies such as Alzheimer's disease, with abnormal tau phosphorylation. Thus, in addition to its increasingly accepted contribution to tumorigenesis, the p73 subfamily also plays a role in neuronal development and neurodegeneration.
[show abstract][hide abstract] ABSTRACT: 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.
Genes & development 03/2010; 24(6):549-60. · 12.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: The Trp53 gene family member Trp73 encodes two major groups of protein isoforms, TAp73 and DeltaNp73, with opposing pro- and anti-apoptotic functions; consequently, their relative ratio regulates cell fate. However, the precise roles of p73 isoforms in cellular events such as tumor initiation, embryonic development, and cell death remain unclear. To determine which aspects of p73 function are attributable to the TAp73 isoforms, we generated and characterized mice in which exons encoding the TAp73 isoforms were specifically deleted to create a TAp73-deficient (TAp73(-/-)) mouse. Here we show that mice specifically lacking in TAp73 isoforms develop a phenotype intermediate between the phenotypes of Trp73(-/-) and Trp53(-/-) mice with respect to incidence of spontaneous and carcinogen-induced tumors, infertility, and aging, as well as hippocampal dysgenesis. In addition, cells from TAp73(-/-) mice exhibit genomic instability associated with enhanced aneuploidy, which may account for the increased incidence of spontaneous tumors observed in these mutants. Hence, TAp73 isoforms exert tumor-suppressive functions and indicate an emerging role for Trp73 in the maintenance of genomic stability.
Genes & Development 10/2008; 22(19):2677-91. · 12.44 Impact Factor
[show abstract][hide abstract] ABSTRACT: p63, a homologue of the tumor suppressor p53, is pivotal for epithelial development, because its loss causes severe epithelial dysgenesis, although no information is so far available on the role of p63 in the thymus. We identified the expression of all p63 isoforms in the developing thymus. The p63(-/-) thymi show severe abnormalities in size and cellularity, even though the organ expresses normal levels of keratins 5 and 8, indicating a p63-independent differentiation of thymic epithelial cells (TEC). TEC were sufficiently developed to allow a significant degree of education to produce CD4/CD8 single- and double-positive T cells. To study the selective contribution of transactivation-active p63 (TAp63) and amino-deleted p63 (DeltaNp63) isoforms to the function of the TEC, we genetically complemented p63(-/-) mice by crossing p63(+/-) mice with transgenic mice expressing either TAp63alpha or DeltaNp63alpha under the control of the keratin 5 promoter. Thymic morphology and cellularity were partially restored by complementation with DeltaNp63, but not TAp63, one downstream effector being fibroblast growth factor receptor 2-IIIb (FgfR2-IIIb). Indeed, FgfR2-IIIb is regulated directly by p63, via its interaction with apobec-1-binding protein-1, and its knockout shows thymic defects similar to those observed in p63(-/-) thymi. In addition, expression of Jag2, a component of the Notch signaling pathway known to be required for thymic development, was enhanced by p63 in vivo genetic complementation. Like Jag2(-/-) thymi, p63(-/-) thymi also show reduced gammadelta cell formation. Therefore, p63, and particularly the DeltaNp63 isoform, is essential for thymic development via enhanced expression of FgfR2 and Jag2. The action of DeltaNp63 is not due to a direct regulation of TEC differentiation, but it is compatible with maintenance of their "stemness," the thymic abnormalities resulting from epithelial failure due to loss of stem cells.
Proceedings of the National Academy of Sciences 08/2007; 104(29):11999-2004. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: The epidermis is a multilayered stratified epithelium, continuously regenerated by differentiating keratinocytes, that requires the transcription factor p63 for its development and maintenance. The TP63 gene encodes two major protein isoforms, TAp63 and DeltaNp63, which have both transactivating and transcriptional repressing activities and regulate a wide range of target genes. TAp63 shows clear pro-apoptotic activity, mediated both by death receptors (CD95, TNF, TRAIL) and mitochondrial (bax, puma) pathways. Conversely, DeltaNp63 protects from apoptosis by directly competing for TAp63 target promoters or sequestering it, forming inactive tetramers. Accordingly, p63 is expressed in epithelial tumors, contributing to both tumorigenesis and chemoresistance. However, the predominant physiological role of p63 is in epithelial development, as demonstrated by the lack of epidermis and other epithelia in p63-deficient mice. The specific role of TAp63 and isoforms in epithelial development remains mostly unclear. Nevertheless, recent work utilizing in vivo genetic complementation of TAp63 and/or DeltaNp63 into a p63 null background has shed new light into the specific functions of the two isoforms and allowed the in vivo validation of several p63 transcriptional targets, originally identified by microarray analysis in in vitro systems. However, despite concerted efforts to address the role of p63 isoforms, several questions remain unanswered. The main aim of this review is to critically discuss the data available in the literature and thoroughly analyze the models proposed.
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: The epidermis, the outer layer of the skin composed of keratinocytes, develops following the action of the transcription factor p63. The mouse Trp63 gene contains two promoters, driving the production of distinct proteins, one with an N-terminal trans-activation domain (TAp63) and one without (DeltaNp63), although their relative contribution to epidermal development is not clearly established. To identify the relative role of p63 isoforms in relation to IKKalpha, also known to be essential for epithelial development, we performed both molecular and in vivo analyses using genetic complementation in mice. We found that the action of TAp63 is mediated at the molecular level by direct and indirect transactivation of IKKalpha and Ets-1, respectively. We also found that DeltaNp63 upregulates IKKalpha indirectly, through GATA-3. Our data are consistent with a role for p63 directly upstream of IKKalpha in epithelial development.