A luminal intermediate cell state maintains long-term prostate homeostasis and contributes to tumorigenesis.

Cellular heterogeneity poses tremendous challenges for developing cell-targeted therapies and biomarkers of clinically significant prostate cancer. The origins of this heterogeneity within normal adult and aging tissue remain unknown, leaving cellular states and transcriptional programs that allow expansions of malignant clones unidentified. To define cell states that contribute to early cancer development, we performed clonal analyses and single cell transcriptomics of normal prostate from genetically-engineered mouse models. We uncovered a luminal transcriptional state with a unique "basal-like" Wnt/p63 signaling ( luminal intermediate , LumI) which contributes to the maintenance of long-term prostate homeostasis. Moreover, LumI cells greatly expand during early stages of tumorigenesis in several mouse models of prostate cancer. Genetic ablation of p63 in vivo in luminal cells reduced the formation of aggressive clones in mouse prostate tumor models. Finally, the LumI cells and Wnt signaling appear to significantly increase in human aging prostate and prostate cancer samples, highlighting the importance of this hybrid cell state for human pathologies with potential translational impact.

Sustained glymphatic transport and impaired drainage to the nasal cavity observed in multiciliated cell ciliopathies with hydrocephalus.

BACKGROUND: Hydrocephalus (increased ventricular size due to CSF accumulation) is a common finding in human ciliopathies and in mouse models with genetic depletion of the multiciliated cell (MCC) cilia machinery. However, the contribution of MCC to CSF dynamics and, the mechanism by which impaired MCC function leads to hydrocephalus remains poorly understood. The aim of our study was to examine if defects in MCC ciliogenesis and cilia-generated CSF flow impact central nervous system (CNS) fluid homeostasis including glymphatic transport and solute waste drainage. METHODS: We used two distinct mouse models of MCC ciliopathy: MCC-specific CEP164 conditional knockout mice (FOXJ1-Cre;CEP164fl/fl (N = 10), 3-month-old) and p73 knock-out (p73-/- (N = 8), 5-month-old) mice. Age-matched, wild-type littermates for each of the mutants served as controls. Glymphatic transport and solute drainage was quantified using in vivo T1 mapping by magnetic resonance imaging (MRI) after CSF infusion of gadoteric acid. Brain morphometry and aquaporin 4 expression (AQP4) was also assessed. Intracranial pressure (ICP) was measured in separate cohorts. RESULTS: In both of the two models of MCC ciliopathy we found the ventriculomegaly to be associated with normal ICP. We showed that FOXJ1-Cre;CEP164fl/fl mice with hydrocephalus still demonstrated sustained glymphatic transport and normal AQP4 expression along capillaries. In p73-/- mice glymphatic transport was even increased, and this was paralleled by an increase in AQP4 polarization around capillaries. Further, solute drainage via the cribriform plate to the nasal cavity was severely impaired in both ciliopathy models and associated with chronic rhinitis and olfactory bulb hypoplasia. CONCLUSIONS: The combination of sustained glymphatic transport, impaired solute drainage via the cribriform plate to the nasal cavity and hydrocephalus has not previously been reported in models of MCC ciliopathy. Our data enhance our understanding of how different types of ciliopathies contribute to disruption of CNS fluid homeostasis, manifested in pathologies such as hydrocephalus.

Statin as anti-cancer therapy in autochthonous T-lymphomas expressing stabilized gain-of-function mutant p53 proteins.

An important component of missense mutant p53 gain-of-function (mutp53 GOF) activities is the ability of stabilized mutp53 proteins to upregulate the mevalonate pathway, providing a rationale for exploring the statin family of HMG-CoA reductase inhibitors as anticancer agents in mutp53 tumors. In this small exploratory study we report on the effects of statin treatment in autochthonous mouse models of clinically advanced T-cell lymphoma expressing two different GOF mutp53 alleles. We find that Rosuvastatin monotherapy shows a modest, p53 allele-selective and transient anti-tumor effect in autochthonous T-lymphomas expressing the p53 R248Q DNA contact mutant, but not in tumors expressing the p53 R172H conformational mutant. p53 null mice also do not benefit. In vitro statin sensitivity is not a strong predictor for in vivo sensitivity, while subcutaneous allografts are. Future explorations of statins in combination therapies are justified to improve its anti-tumor effects and to better define the most statin-sensitive alleles and tumor types among mutp53-stabilized cancers.

Tissue-specific roles of p73 in development and homeostasis.

p73 (TP73) belongs to the p53 family of transcription factors. Its gene locus encodes two opposing types of isoforms, the transcriptionally active TAp73 class and the dominant-negative DNp73 class, which both play critical roles in development and homeostasis in an astonishingly diverse array of biological systems within specific tissues. While p73 has functions in cancer, this Review focuses on the non-oncogenic activities of p73. In the central and peripheral nervous system, both isoforms cooperate in complex ways to regulate neural stem cell survival, self-renewal and terminal differentiation. In airways, oviduct and to a lesser extent in brain ependyma, TAp73 is the master transcriptional regulator of multiciliogenesis, enabling fluid and germ cell transport across tissue surfaces. In male and female reproduction, TAp73 regulates gene networks that control cell-cell adhesion programs within germinal epithelium to enable germ cell maturation. Finally, p73 participates in the control of angiogenesis in development and cancer. While many open questions remain, we discuss here key findings that provide insight into the complex functions of this gene at the organismal, cellular and molecular level.

Cajal-Retzius neurons are required for the development of the human hippocampal fissure.

Cajal-Retzius neurons (CRN) are the main source of Reelin in the marginal zone of the developing neocortex and hippocampus (HC). They also express the transcription factor p73 and are complemented by later-appearing GABAergic Reelin+ interneurons. The human dorsal HC forms at gestational week 10 (GW10), when it develops a rudimentary Ammonic plate and incipient dentate migration, although the dorsal hippocampal fissure (HF) remains shallow and contains few CRN. The dorsal HC transforms into the indusium griseum (IG), concurrently with the rostro-caudal appearance of the corpus callosum, by GW14-17. Dorsal and ventral HC merge at the site of the former caudal hem, which is located at the level of the future atrium of the lateral ventricle and closely connected with the choroid plexus. The ventral HC forms at GW11 in the temporal lobe. The ventral HF is wide open at GW14-16 and densely populated by large numbers of CRNs. These are in intimate contact with the meninges and meningeal blood vessels, suggesting signalling through diverse pathways. At GW17, the fissure deepens and begins to fuse, although it is still marked by p73/Reelin+ CRNs. The p73KO mouse illustrates the importance of p73 in CRN for HF formation. In the mutant, Tbr1/Reelin+ CRNs are born in the hem but do not leave it and subsequently disappear, so that the mutant cortex and HC lack CRN from the onset of corticogenesis. The HF is absent, which leads to profound architectonic alterations of the HC. To determine which p73 isoform is important for HF formation, isoform-specific TAp73- and DeltaNp73-deficient embryonic and early postnatal mice were examined. In both mutants, the number of CRNs was reduced, but each of their phenotypes was much milder than in the global p73KO mutant missing both isoforms. In the TAp73KO mice, the HF of the dorsal HC failed to form, but was present in the ventral HC. In the DeltaNp73KO mice, the HC had a mild patterning defect along with a shorter HF. Complex interactions between both isoforms in CRNs may contribute to their crucial activity in the developing brain.

Non-oncogenic roles of TAp73: from multiciliogenesis to metabolism.

The p53 family of transcription factors (p53, p63 and p73) covers a wide range of functions critical for development, homeostasis and health of mammals across their lifespan. Beside the well-established tumor suppressor role, recent evidence has highlighted novel non-oncogenic functions exerted by p73. In particular, p73 is required for multiciliated cell (MCC) differentiation; MCCs have critical roles in brain and airways to move fluids across epithelial surfaces and to transport germ cells in the reproductive tract. This novel function of p73 provides a unifying cellular mechanism for the disparate inflammatory and immunological phenotypes of p73-deficient mice. Indeed, mice with Trp73 deficiency suffer from hydrocephalus, sterility and chronic respiratory tract infections due to profound defects in ciliogenesis and complete loss of mucociliary clearance since MCCs are essential for cleaning airways from inhaled pollutants, pathogens and allergens. Cross-species genomic analyses and functional rescue experiments identify TAp73 as the master transcriptional integrator of ciliogenesis, upstream of previously known central nodes. In addition, TAp73 shows a significant ability to regulate cellular metabolism and energy production through direct transcriptional regulation of several metabolic enzymes, such as glutaminase-2 and glucose-6 phosphate dehydrogenase. This recently uncovered role of TAp73 in the regulation of cellular metabolism strongly affects oxidative balance, thus potentially influencing all the biological aspects associated with p73 function, including development, homeostasis and cancer. Although through different mechanisms, p63 isoforms also contribute to regulation of cellular metabolism, thus indicating a common route used by all family members to control cell fate. At the structural level, the complexity of p73's function is further enhanced by its ability to form heterotetramers with some p63 isoforms, thus indicating the existence of an intrafamily crosstalk that determines the global outcome of p53 family function. In this review, we have tried to summarize all the recent evidence that have emerged on the novel non-oncogenic roles of p73, in an attempt to provide a unified view of the complex function of this gene within its family.

A Novel In Vitro CypD-Mediated p53 Aggregation Assay Suggests a Model for Mitochondrial Permeability Transition by Chaperone Systems.

Tissue necrosis as a consequence of ischemia-reperfusion injury and oxidative damage is a leading cause of permanent disability and death worldwide. The complete mechanism by which cells undergo necrosis upon oxidative stress is not understood. In response to an oxidative insult, wild-type p53 has been implicated as a central regulatory component of the mitochondrial permeability transition (mPT), triggering necrosis. This process is associated with cellular stabilization and translocation of p53 into the mitochondrial matrix. Here, we probe the mechanism by which p53 activates the key mPT regulator cyclophilin D (CypD). We explore the involvement of Trap1, an Hsp90-related mitochondrial matrix protein and a member of the mitochondrial unfolded protein response, and its ability to suppress mPT in a p53-dependent manner. Our study finds that catalytically active CypD causes strong aggregation of wild-type p53 protein (both full-length and isolated DNA-binding domain) into amyloid-type fibrils in vitro. The responsible CypD residues for this activity were mapped by NMR to the active site amino acids R55, F60, F113, and W121. The data also present a new proline isomerization assay for CypD by monitoring the aggregation of p53 as an indicator of CypD activity. Moreover, we find that the inhibition of Trap1 by the mitochondria-specific HSP90 ATPase antagonist Gamitrinib strongly sensitizes primary mouse embryonic fibroblasts to mPT and permeability transition pore opening in a p53- and CypD-dependent manner. We propose a mechanism by which the influx of unfolded p53 into the mitochondrial matrix in response to oxidative stress indirectly activates the normally inhibited CypD by displacing it from Trap1 complexes. This activates CypD's isomerase activity. Liberated CypD then isomerizes multiple proteins including p53 (causing p53 aggregation) and the structural components of the mPTP pore, inducing pore opening. This working model can now be tested in the future.

TAp73 is a central transcriptional regulator of airway multiciliogenesis.

Motile multiciliated cells (MCCs) have critical roles in respiratory health and disease and are essential for cleaning inhaled pollutants and pathogens from airways. Despite their significance for human disease, the transcriptional control that governs multiciliogenesis remains poorly understood. Here we identify TP73, a p53 homolog, as governing the program for airway multiciliogenesis. Mice with TP73 deficiency suffer from chronic respiratory tract infections due to profound defects in ciliogenesis and complete loss of mucociliary clearance. Organotypic airway cultures pinpoint TAp73 as necessary and sufficient for basal body docking, axonemal extension, and motility during the differentiation of MCC progenitors. Mechanistically, cross-species genomic analyses and complete ciliary rescue of knockout MCCs identify TAp73 as the conserved central transcriptional integrator of multiciliogenesis. TAp73 directly activates the key regulators FoxJ1, Rfx2, Rfx3, and miR34bc plus nearly 50 structural and functional ciliary genes, some of which are associated with human ciliopathies. Our results position TAp73 as a novel central regulator of MCC differentiation.

MDM2 Associates with Polycomb Repressor Complex 2 and Enhances Stemness-Promoting Chromatin Modifications Independent of p53.

The MDM2 oncoprotein ubiquitinates and antagonizes p53 but may also carry out p53-independent functions. Here we report that MDM2 is required for the efficient generation of induced pluripotent stem cells (iPSCs) from murine embryonic fibroblasts, in the absence of p53. Similarly, MDM2 depletion in the context of p53 deficiency also promoted the differentiation of human mesenchymal stem cells and diminished clonogenic survival of cancer cells. Most of the MDM2-controlled genes also responded to the inactivation of the Polycomb Repressor Complex 2 (PRC2) and its catalytic component EZH2. MDM2 physically associated with EZH2 on chromatin, enhancing the trimethylation of histone 3 at lysine 27 and the ubiquitination of histone 2A at lysine 119 (H2AK119) at its target genes. Removing MDM2 simultaneously with the H2AK119 E3 ligase Ring1B/RNF2 further induced these genes and synthetically arrested cell proliferation. In conclusion, MDM2 supports the Polycomb-mediated repression of lineage-specific genes, independent of p53.

Mutant p53 Amplifies Epidermal Growth Factor Receptor Family Signaling to Promote Mammary Tumorigenesis.

UNLABELLED: The EGFR family (ErbB2/Her2 and EGFR/ErbB1/Her1) often modulates the transcriptional program involved in promoting mammary tumorigenesis. In humans, the majority of ErbB2-positive sporadic breast cancers harbor p53 mutations, which correlate with poor prognosis. Also, the extremely high incidence of ErbB2-positive breast cancer in women with p53 germline mutations (Li-Fraumeni syndrome) suggests a key role of mutant p53 specifically in ErbB2-mediated mammary tumorigenesis. To examine the role of mutant p53 during ErbB2-mediated mammary tumorigenesis, a mutant p53 allele (R172H) was introduced into the (MMTV)-ErbB2/Neu mouse model system. Interestingly, we show in heterozygous p53 mice that mutant p53 R172H is a more potent activator of ErbB2-mediated mammary tumorigenesis than simple loss of p53. The more aggressive disease in mutant p53 animals was reflected by earlier tumor onset, increased mammary tumor multiplicity, and shorter survival. These in vivo and in vitro data provide mechanistic evidence that mutant p53 amplifies ErbB2 and EGFR signaling to promote the expansion of mammary stem cells and induce cell proliferation. IMPLICATIONS: This study identifies mutant p53 as an essential player in ErbB2 and EGFR-mediated mammary tumorigenesis and indicates the potential translational importance of targeting mutant p53 in this subset of patients with breast cancer.

p53Ψ is a transcriptionally inactive p53 isoform able to reprogram cells toward a metastatic-like state.

Although much is known about the underlying mechanisms of p53 activity and regulation, the factors that influence the diversity and duration of p53 responses are not well understood. Here we describe a unique mode of p53 regulation involving alternative splicing of the TP53 gene. We found that the use of an alternative 3' splice site in intron 6 generates a unique p53 isoform, dubbed p53Ψ. At the molecular level, p53Ψ is unable to bind to DNA and does not transactivate canonical p53 target genes. However, like certain p53 gain-of-function mutants, p53Ψ attenuates the expression of E-cadherin, induces expression of markers of the epithelial-mesenchymal transition, and enhances the motility and invasive capacity of cells through a unique mechanism involving the regulation of cyclophilin D activity, a component of the mitochondrial inner pore permeability. Hence, we propose that p53Ψ encodes a separation-of-function isoform that, although lacking canonical p53 tumor suppressor/transcriptional activities, is able to induce a prometastatic program in a transcriptionally independent manner.

TAp73 is essential for germ cell adhesion and maturation in testis.

A core evolutionary function of the p53 family is to protect the genomic integrity of gametes. However, the role of p73 in the male germ line is unknown. Here, we reveal that TAp73 unexpectedly functions as an adhesion and maturation factor of the seminiferous epithelium orchestrating spermiogenesis. TAp73 knockout (TAp73KO) and p73KO mice, but not ΔNp73KO mice, display a "near-empty seminiferous tubule" phenotype due to massive premature loss of immature germ cells. The cellular basis of this phenotype is defective cell-cell adhesions of developing germ cells to Sertoli nurse cells, with likely secondary degeneration of Sertoli cells, including the blood-testis barrier, which leads to disruption of the adhesive integrity and maturation of the germ epithelium. At the molecular level, TAp73, which is produced in germ cells, controls a coordinated transcriptional program of adhesion- and migration-related proteins including peptidase inhibitors, proteases, receptors, and integrins required for germ-Sertoli cell adhesion and dynamic junctional restructuring. Thus, we propose the testis as a unique organ with strict division of labor among all family members: p63 and p53 safeguard germ line fidelity, whereas TAp73 ensures fertility by enabling sperm maturation.

Direct reprogramming by oncogenic Ras and Myc.

Genetically or epigenetically defined reprogramming is a hallmark of cancer cells. However, a causal association between genome reprogramming and cancer has not yet been conclusively established. In particular, little is known about the mechanisms that underlie metastasis of cancer, and even less is known about the identity of metastasizing cancer cells. In this study, we used a model of conditional expression of oncogenic KrasG12D allele in primary mouse cells to show that reprogramming and dedifferentiation is a fundamental early step in malignant transformation and cancer initiation. Our data indicate that stable expression of activated KrasG12D confers on cells a large degree of phenotypic plasticity that predisposes them to neoplastic transformation and acquisition of stem cell characteristics. We have developed a genetically tractable model system to investigate the origins and evolution of metastatic pancreatic cancer cells. We show that metastatic conversion of KrasG12D-expressing cells that exhibit different degrees of differentiation and malignancy can be reconstructed in cell culture, and that the proto-oncogene c-Myc controls the generation of self-renewing metastatic cancer cells. Collectively, our results support a model wherein non-stem cancer cells have the potential to dedifferentiate and acquire stem cell properties as a direct consequence of oncogene-induced plasticity. Moreover, the disturbance in the normally existing dynamic equilibrium between cancer stem cells and non-stem cancer cells allows the formation of cancer stem cells with high metastatic capacity at any time during cancer progression.

Inhibition of topoisomerase IIα: novel function of wedelolactone.

The naturally occurring coumestan wedelolactone has been previously shown to reduce growth of various cancer cells. So far, the growth-suppressing effect of wedelolactone has been attributed to the inhibition of the NFκB transcription factor and/or androgen receptors. We found that wedelolactone suppressed growth and induced apoptosis of androgen receptor-negative MDA-MB-231 breast cancer cells at concentrations that did not inhibit the NFκB activity. The cells responded to wedelolactone by the S and G2/M phase cell cycle arrest and induction of the DNA damage signaling. Wedelolactone interacted with dsDNA and inhibited the activity of DNA topoisomerase IIα. We conclude that wedelolactone can act as growth suppressor independently of NFκB and androgen receptors.

Loss of p73 promotes dissemination of Myc-induced B cell lymphomas in mice.

Mice engineered to express c-Myc in B cells (Emu-myc mice) develop lethal lymphomas in which the gene encoding the p53 tumor suppressor is frequently mutated. Whether the p53 homolog p73 also functions as a tumor suppressor in vivo remains controversial. Here we have shown that p73 loss does not substantially affect disease onset and mortality in Emu-myc mice. However, it does alter the phenotype of the disease. Specifically, p73 loss decreased nodal disease and increased widespread extranodal dissemination. We further found that p53 acted as the dominant tumor suppressor during the onset of Emu-myc-driven B cell lymphomagenesis, while p73 modulated tumor dissemination and extranodal growth. Immunophenotyping and expression profiling suggested that p73 loss allowed increased maturation of malignant B cells and deregulated genes involved in lymphocyte homing and dissemination of human lymphomas. Consistent with this, p73 expression was frequently downregulated in a large cohort of human mature aggressive B cell lymphomas, and both the incidence and degree of p73 downregulation in these tumors correlated with their extranodal dissemination status. These data indicate that p73 is a modifier of Myc-driven lymphomas in mice, favoring tumor dissemination, and suggest that p73 could be a biomarker for human B cell lymphoma dissemination, a notion that can now be tested in clinicopathologic correlation studies.

Targeted deletion of p73 in mice reveals its role in T cell development and lymphomagenesis.

Transcriptional silencing of the p73 gene through methylation has been demonstrated in human leukemias and lymphomas. However, the role of p73 in the malignant process remains to be explored. We show here that p73 acts as a T cell-specific tumor suppressor in a genetically defined mouse model, and that concomitant ablation of p53 and p73 predisposes mice to an increased incidence of thymic lymphomas compared to the loss of p53 alone. Our results demonstrate a causal role for loss of p73 in progression of T cell lymphomas to the stage of aggressive, disseminated disease. We provide evidence that tumorigenesis in mice lacking p53 and p73 proceeds through mechanisms involving altered patterns of gene expression, defects in early T cell development, impaired apoptosis, and the ensuing accumulation of chromosomal aberrations. Collectively, our data imply that tumor suppressive properties of p73 are highly dependent on cellular context, wherein p73 plays a major role in T cell development and neoplasia.

A tautomerase-null macrophage migration-inhibitory factor (MIF) gene knock-in mouse model reveals that protein interactions and not enzymatic activity mediate MIF-dependent growth regulation.

Macrophage migration-inhibitory factor (MIF) is an upstream regulator of innate immunity and a potential molecular link between inflammation and cancer. The unusual structural homology between MIF and certain tautomerases, which includes both a conserved substrate-binding pocket and a catalytic N-terminal proline (Pro1), has fueled speculation that an enzymatic reaction underlies MIF's biologic function. To address the functional role of the MIF tautomerase activity in vivo, we created a knock-in mouse in which the endogenous mif gene was replaced by one encoding a tautomerase-null, Pro1-->Gly1 MIF protein (P1G-MIF). While P1G-MIF is completely inactive catalytically, it maintains significant, albeit reduced, binding to its cell surface receptor (CD74) and to the intracellular binding protein JAB1/CSN5. P1G-MIF knock-in mice (mif(P1G/P1G)) and cells derived from these mice show a phenotype in assays of growth control and tumor induction that is intermediate between those of the wild type (mif(+/+)) and complete MIF deficiency (mif(-)(/)(-)). These data provide genetic evidence that MIF's intrinsic tautomerase activity is dispensable for this cytokine's growth-regulatory properties and support a role for the N-terminal region in protein-protein interactions.

E2F1 plays a direct role in Rb stabilization and p53-independent tumor suppression.

To better understand the role of E2F1 in tumor formation, we analyzed spontaneous tumorigenesis in p53(-/-)E2F1(+/+) and p53(-/-)E2F1(-/-) mice. We show that the combined loss of p53 and E2F1 leads to an increased incidence of sarcomas and carcinomas compared to the loss of p53 alone. E2F1-deficient tumors show wide chromosomal variation, indicative of genomic instability. Consistent with this, p53(-/-)E2F1(-/-) primary fibroblasts have a reduced capacity to maintain genomic stability when exposed to S-phase inhibitors or genotoxic drugs. A major mechanism of E2F1's contribution to genomic integrity lies in mediating stabilization and engagement of the Rb protein.

p73 suppresses polyploidy and aneuploidy in the absence of functional p53.

Previous studies showed that p53 plays a central role in G1 and DNA damage checkpoints, thus contributing to genomic stability. We show here that p73 also plays a role in genomic integrity but this mechanism is manifest only when p53 is lost. Isolated p73 loss in primary cells does not induce genomic instability. Instead, it results in impaired proliferation and premature senescence due to compensatory activation of p53. Combined loss of p73 and p53 rescues these defects, but at the expense of exacerbated genomic instability. This leads to rapid increase in polyploidy and aneuploidy, markedly exceeding that of p53 loss alone. Constitutive deregulation of cyclin-Cdk activities and excess failure of the G2/M DNA damage checkpoint appear to fuel increased ploidy abnormalities upon p53/p73 loss, while primary mitotic defects do not play a causal role. These data indicate that p73 is essential for suppressing polyploidy and aneuploidy when p53 is inactivated.