CBFB cooperates with p53 to maintain TAp73 expression and suppress breast cancer.

The CBFB gene is frequently mutated in several types of solid tumors. Emerging evidence suggests that CBFB is a tumor suppressor in breast cancer. However, our understanding of the tumor suppressive function of CBFB remains incomplete. Here, we analyze genetic interactions between mutations of CBFB and other highly mutated genes in human breast cancer datasets and find that CBFB and TP53 mutations are mutually exclusive, suggesting a functional association between CBFB and p53. Integrated genomic studies reveal that TAp73 is a common transcriptional target of CBFB and p53. CBFB cooperates with p53 to maintain TAp73 expression, as either CBFB or p53 loss leads to TAp73 depletion. TAp73 re-expression abrogates the tumorigenic effect of CBFB deletion. Although TAp73 loss alone is insufficient for tumorigenesis, it enhances the tumorigenic effect of NOTCH3 overexpression, a downstream event of CBFB loss. Immunohistochemistry shows that p73 loss is coupled with higher proliferation in xenografts. Moreover, TAp73 loss-of-expression is a frequent event in human breast cancer tumors and cell lines. Together, our results significantly advance our understanding of the tumor suppressive functions of CBFB and reveal a mechanism underlying the communication between the two tumor suppressors CBFB and p53.

FDXR regulates TP73 tumor suppressor via IRP2 to modulate aging and tumor suppression.

Ferredoxin reductase (FDXR) is a mitochondrial flavoprotein that initiates electron transport from NADPH to several cytochromes P450 via two electron carriers, ferredoxin 1 (FDX1) and FDX2. FDXR is the sole ferredoxin reductase in humans and plays a critical role in steroidogenesis and biosynthesis of heme and iron-sulfur clusters. However, much less is known about the role of FDXR in cancer. Here, we show that FDXR plays a role in tumorigenesis by modulating expression of the tumor suppressor p73. By using genetically modified mouse models, we recently showed that mice deficient in either Fdxr or Trp73 had a shorter lifespan and were prone to spontaneous tumors as compared with wild-type (WT) mice. Interestingly, compound Trp73 +/- ;Fdxr +/- mice lived longer and developed fewer tumors when compared with Fdxr +/- or Trp73 +/- mice. Moreover, we found that cellular senescence was increased in Trp73 +/- and Fdxr +/- mouse embryonic fibroblasts (MEFs), which was further increased in Trp73 +/- ;Fdxr +/- MEFs, as compared with that in WT MEFs. As FDXR is regulated by p73, we examined whether there was a feedback regulation between p73 and FDXR. Indeed, we found that Trp73 expression was decreased by loss of Fdxr in MEFs and that FDXR is required for p73 expression in multiple human cancer cell lines independent of p53. Mechanistically, we found that loss of FDXR, via FDX2, increased expression of iron-binding protein 2 (IRP2), which subsequently repressed TP73 mRNA stability. We also showed that TP73 transcript contained an iron response element in its 3'UTR, which was required for IRP2 to destabilize TP73 mRNA. Together, these data reveal a novel regulation of p73 by FDXR via IRP2 and that the FDXR-p73 axis plays a critical role in aging and tumor suppression. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Transcription factor p73 regulates Th1 differentiation.

Inter-individual differences in T helper (Th) cell responses affect susceptibility to infectious, allergic and autoimmune diseases. To identify factors contributing to these response differences, here we analyze in vitro differentiated Th1 cells from 16 inbred mouse strains. Haplotype-based computational genetic analysis indicates that the p53 family protein, p73, affects Th1 differentiation. In cells differentiated under Th1 conditions in vitro, p73 negatively regulates IFNγ production. p73 binds within, or upstream of, and modulates the expression of Th1 differentiation-related genes such as Ifng and Il12rb2. Furthermore, in mouse experimental autoimmune encephalitis, p73-deficient mice have increased IFNγ production and less disease severity, whereas in an adoptive transfer model of inflammatory bowel disease, transfer of p73-deficient naïve CD4+ T cells increases Th1 responses and augments disease severity. Our results thus identify p73 as a negative regulator of the Th1 immune response, suggesting that p73 dysregulation may contribute to susceptibility to autoimmune disease.

p73 regulates ependymal planar cell polarity by modulating actin and microtubule cytoskeleton.

Planar cell polarity (PCP) and intercellular junctional complexes establish tissue structure and coordinated behaviors across epithelial sheets. In multiciliated ependymal cells, rotational and translational PCP coordinate cilia beating and direct cerebrospinal fluid circulation. Thus, PCP disruption results in ciliopathies and hydrocephalus. PCP establishment depends on the polarization of cytoskeleton and requires the asymmetric localization of core and global regulatory modules, including membrane proteins like Vangl1/2 or Frizzled. We analyzed the subcellular localization of select proteins that make up these modules in ependymal cells and the effect of Trp73 loss on their localization. We identify a novel function of the Trp73 tumor suppressor gene, the TAp73 isoform in particular, as an essential regulator of PCP through the modulation of actin and microtubule cytoskeleton dynamics, demonstrating that Trp73 is a key player in the organization of ependymal ciliated epithelia. Mechanistically, we show that p73 regulates translational PCP and actin dynamics through TAp73-dependent modulation of non-musclemyosin-II activity. In addition, TAp73 is required for the asymmetric localization of PCP-core and global signaling modules and regulates polarized microtubule dynamics, which in turn set up the rotational PCP. Therefore, TAp73 modulates, directly and/or indirectly, transcriptional programs regulating actin and microtubules dynamics and Golgi organization signaling pathways. These results shed light into the mechanism of ependymal cell planar polarization and reveal p73 as an epithelial architect during development regulating the cellular cytoskeleton.

Sustained protein synthesis and reduced eEF2K levels in TAp73-\- mice brain: a possible compensatory mechanism.

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.

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.

p73 is required for appropriate BMP-induced mesenchymal-to-epithelial transition during somatic cell reprogramming.

The generation of induced pluripotent stem cells (iPSCs) by somatic cell reprogramming holds great potential for modeling human diseases. However, the reprogramming process remains very inefficient and a better understanding of its basic biology is required. The mesenchymal-to-epithelial transition (MET) has been recognized as a crucial step for the successful reprogramming of fibroblasts into iPSCs. It has been reported that the p53 tumor suppressor gene acts as a barrier of this process, while its homolog p63 acts as an enabling factor. In this regard, the information concerning the role of the third homolog, p73, during cell reprogramming is limited. Here, we derive total Trp73 knockout mouse embryonic fibroblasts, with or without Trp53, and examine their reprogramming capacity. We show that p73 is required for effective reprogramming by the Yamanaka factors, even in the absence of p53. Lack of p73 affects the early stages of reprogramming, impairing the MET and resulting in altered maturation and stabilization phases. Accordingly, the obtained p73-deficient iPSCs have a defective epithelial phenotype and alterations in the expression of pluripotency markers. We demonstrate that p73 deficiency impairs the MET, at least in part, by hindering BMP pathway activation. We report that p73 is a positive modulator of the BMP circuit, enhancing its activation by DNp73 repression of the Smad6 promoter. Collectively, these findings provide mechanistic insight into the MET process, proposing p73 as an enhancer of MET during cellular reprogramming.

Recombinant adeno-associated virus expressing a p53-derived apoptotic peptide (37AA) inhibits HCC cells growth in vitro and in vivo.

Recent studies have confirmed that a p53-derived apoptotic peptide (37AA) could act as a tumor suppressor inducing apoptosis in multiple tumor cells through derepressing p73. However, the tumor suppressive effects of recombinant adeno-associated virus (rAAV) expressing 37AA on HCC cells are still unknown. In this study, we successfully constructed a recombinant rAAV expressing 37AA. In vitro and in vivo assays showed that transfection of NT4-37AA/rAAV in HCC cells strongly suppressed cell proliferation, induced apoptosis, and up-regulated the cellular expression of p73. NT4-37AA/rAAV transfection markedly slowed Huh-7 xenografted tumor growth in murine. Pretreatment of HCC cells with p73 siRNA abrogated these effects of NT4-37AA/rAAV. Furthermore, we found that expression of p73 was upregulated and the formation of P73/iASSP complex was prevented when 37AA was introduced into HCC cells. Taken together, these results indicate that introduction of 37AA into HCC cells with a rAAV vector may lead to the development of broadly applicable agents for the treatment of HCC, and the mechanism may, at least in part, be associated with the upregulation of p73 expression and reduced level of P73/iASSP complex.

The p53 family members have distinct roles during mammalian embryonic development.

The p53 tumor suppressor is a member of a multi-protein family, including the p63 and p73 transcription factors. These proteins can bind to the same consensus sites in DNA and activate the same target genes, suggesting that there could be functional redundancy between them. Indeed, double mutant mice heterozygous for any two family member-encoding genes display enhanced cancer phenotypes relative to single heterozygous mutants. However, whether the family members play redundant roles during embryonic development has remained largely unexplored. Although p53-/-; p73-/- mice are born and manifest phenotypes characteristic of each of the single mutants, the consequences of combined deficiency of p63 and either p53 or p73 have not been elucidated. To examine the functional overlap of p53 family members during development, we bred and analyzed compound mutant embryo phenotypes. We discovered that double knockout embryos and five allele knockout embryos only displayed obvious defects accounted for by loss of single p53 family members. Surprisingly, at mid-gestation (E11), we identified a single viable triple knockout embryo that appeared grossly normal. Together, these results suggest that the p53 family is not absolutely required for early embryogenesis and that p53 family members are largely non-redundant during early development.

TAp73 upregulates IL-1ß in cancer cells: Potential biomarker in lung and breast cancer?

p73 is a transcription factor belonging to the p53 tumour suppressor family. p73-/- mice exhibit a range of phenotypes including neurological, reproductive and inflammatory defects. Although the role of p73 in the control of genomic stability explains part of these phenotypes, a clear mechanism of how p73 participates in the inflammatory response is still elusive. Interleukin-1ß (IL-1ß) has a crucial role in mediating the inflammatory response. Because of its high potency to induce inflammation, the activation and secretion of IL-1ß is tightly regulated by large protein complexes, named inflammasomes. Inflammasomes regulate activation of proinflammatory caspase-1, which in turn proteolytically processes its substrates, including pro-IL-1ß. Caspase-1 gene transcription is strongly activated by p53 protein family members including p73. Here, we have addressed whether p73 might be directly involved in IL-1ß regulation and therefore in the control of the inflammatory response. Our results show that TAp73ß upregulates pro-IL-1ß mRNA and processed IL-1ß protein. In addition, analysis of breast and lung cancer patient cohorts demonstrated that interaction between p73 and IL-1ß predicts a negative survival outcome in these human cancers.

p73 and FoxJ1: Programming Multiciliated Epithelia.

The mysteriously diverse phenotypes in mice lacking the p53 homolog p73 are recently unified by new analysis showing p73 is required for formation of multiciliated epithelia. p73 directly activates FoxJ1, the central transcriptional driver for multiciliation, and induces a host of genes critical for ciliogenesis.

p73 Is Required for Multiciliogenesis and Regulates the Foxj1-Associated Gene Network.

We report that p73 is expressed in multiciliated cells (MCCs), is required for MCC differentiation, and directly regulates transcriptional modulators of multiciliogenesis. Loss of ciliary biogenesis provides a unifying mechanism for many phenotypes observed in p73 knockout mice including hydrocephalus; hippocampal dysgenesis; sterility; and chronic inflammation/infection of lung, middle ear, and sinus. Through p73 and p63 ChIP-seq using murine tracheal cells, we identified over 100 putative p73 target genes that regulate MCC differentiation and homeostasis. We validated Foxj1, a transcriptional regulator of multiciliogenesis, and many other cilia-associated genes as direct target genes of p73 and p63. We show p73 and p63 are co-expressed in a subset of basal cells and suggest that p73 marks these cells for MCC differentiation. In summary, p73 is essential for MCC differentiation, functions as a critical regulator of a transcriptome required for MCC differentiation, and, like p63, has an essential role in development of tissues.

p73 is required for ependymal cell maturation and neurogenic SVZ cytoarchitecture.

The adult subventricular zone (SVZ) is a highly organized microenvironment established during the first postnatal days when radial glia cells begin to transform into type B-cells and ependymal cells, all of which will form regenerative units, pinwheels, along the lateral wall of the lateral ventricle. Here, we identify p73, a p53 homologue, as a critical factor controlling both cell-type specification and structural organization of the developing mouse SVZ. We describe that p73 deficiency halts the transition of the radial glia into ependymal cells, leading to the emergence of immature cells with abnormal identities in the ventricle and resulting in loss of the ventricular integrity. p73-deficient ependymal cells have noticeably impaired ciliogenesis and they fail to organize into pinwheels, disrupting SVZ niche structure and function. Therefore, p73 is essential for appropriate ependymal cell maturation and the establishment of the neurogenic niche architecture. Accordingly, lack of p73 results in impaired neurogenesis. Moreover, p73 is required for translational planar cell polarity establishment, since p73 deficiency results in profound defects in cilia organization in individual cells and in intercellular patch orientation. Thus, our data reveal a completely new function of p73, independent of p53, in the neurogenic architecture of the SVZ of rodent brain and in the establishment of ependymal planar cell polarity with important implications in neurogenesis. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 730-747, 2016.