Mutant TRP53 exerts a target gene-selective dominant-negative effect to drive tumor development.

Mutations in Trp53, prevalent in human cancer, are reported to drive tumorigenesis through dominant-negative effects (DNEs) over wild-type TRP53 function as well as neomorphic gain-of-function (GOF) activity. We show that five TRP53 mutants do not accelerate lymphomagenesis on a TRP53-deficient background but strongly synergize with c-MYC overexpression in a manner that distinguishes the hot spot Trp53 mutations. RNA sequencing revealed that the mutant TRP53 DNE does not globally repress wild-type TRP53 function but disproportionately impacts a subset of wild-type TRP53 target genes. Accordingly, TRP53 mutant proteins impair pathways for DNA repair, proliferation, and metabolism in premalignant cells. This reveals that, in our studies of lymphomagenesis, mutant TRP53 drives tumorigenesis primarily through the DNE, which modulates wild-type TRP53 function in a manner advantageous for neoplastic transformation.

The Neurodevelopmental Disorders Associated Gene Rnf113a Regulates Survival and Differentiation Properties of Neural Stem Cells.

RNF113A (Ring Finger Protein 113A) is genetically associated with autism spectrum disorders and X-linked trichothiodystrophy (TTD) syndrome. Loss-of-function mutations in human RNF113A are causally linked to TTD, which is characterized by abnormal development of the central nervous system (CNS) and mental retardation. How the loss of RNF113A activity affects brain development is not known. Here we identify Rnf113a1 as a critical regulator of cell death and neurogenesis during mouse brain development. Rnf113a1 gene exhibits widespread expression in the embryonic CNS. Knockdown studies in embryonic cortical neural stem/progenitor cells (NSCs) and the mouse cortex suggest that Rnf113a1 controls the survival, proliferation, and differentiation properties of progenitor cells. Importantly, Rnf113a1 deficiency triggers cell apoptosis via a combined action on essential regulators of cell survival, including p53, Nupr1, and Rad51. Collectively, these observations establish Rnf113a1 as a regulatory factor in CNS development and provide insights into its role in neurodevelopmental defects associated with TTD and autism.

Supplementing culture medium with the weak acid, 5,5-dimethyl-2,4-oxazolidinedione (DMO) limits the development of aneuploid mouse embryos through a Trp53-dependent mechanism.

PURPOSE: This study was designed to determine if DMO limits in vitro development of aneuploid-enriched mouse embryos by activating a Trp53-dependent mechanism. METHODS: Mouse cleavage-stage embryos were treated with reversine to induce aneuploidy or vehicle to generate controls, and then cultured in media supplemented with DMO to reduce the pH of the culture media. Embryo morphology was assessed by phase microscopy. Cell number, mitotic figures, and apoptotic bodies were revealed by staining fixed embryos with DAPI. mRNA levels of Trp53, Oct-4, and Cdx2 were monitored by quantitative polymerase chain reactions (qPCRs). The effect of Trp53 on the expression of Oct-4 and Cdx2 was assessed by depleting Trp53 using Trp53 siRNA. RESULTS: Aneuploid-enriched late-stage blastocysts were morphologically indistinguishable from control blastocysts but had fewer cells and reduced mRNA levels of Oct-4 and Cdx2. Adding 1 mM DMO to the culture media during the 8-cell to blastocyst transition reduced the formation of aneuploid-enriched late-stage blastocysts but not control blastocysts and further suppressed the levels of Oct-4 and Cdx2 mRNA. Trp53 RNA levels in aneuploid-enriched embryos that were exposed to DMO were > twofold higher than controls, and Trp53 siRNA levels reduced the levels of Trp53 and increased levels of Oct-4 and Cdx2 mRNA by > twofold. CONCLUSION: These studies suggest that the development of morphologically normal aneuploid-enriched mouse blastocysts can be inhibited by adding low amounts of DMO to the culture media, which results in elevated levels of Trp53 mRNA that suppresses Oct-4 and Cdx2 expression.

Loss of Brca1 and Trp53 in adult mouse mammary ductal epithelium results in development of hormone receptor-positive or hormone receptor-negative tumors, depending on inactivation of Rb family proteins.

BACKGROUND: Breast cancer is a heterogenous disease with several histological and molecular subtypes. Models that represent these subtypes are essential for translational research aimed at improving clinical strategy for targeted therapeutics. METHODS: Different combinations of genetic aberrations (Brca1 and Trp53 loss, and inhibition of proteins of the Rb family) were induced in the mammary gland by injection of adenovirus expressing Cre recombinase into the mammary ducts of adult genetically engineered mice. Mammary tumors with different genetic aberrations were classified into molecular subtypes based on expression of molecular markers and RNAseq analysis. In vitro potency assays and Western blots were used to examine their drug sensitivities. RESULTS: Induction of Brca1 and Trp53 loss in mammary ductal epithelium resulted in development of basal-like hormone receptor (HR)-negative mammary tumors. Inhibition of Rb and Trp53 loss or the combination of Rb, Trp53 and Brca1 aberrations resulted in development of luminal ductal carcinoma positive for ER, PR, and Her2 expression. HR positivity in tumors with Rb, Trp53 and Brca1 aberrations indicated that functionality of the Rb pathway rather than Brca1 status affected HR status in these models. Mammary tumor gene expression profiles recapitulated human basal-like or luminal B breast cancer signatures, but HR-positive luminal cancer models were endocrine resistant and exhibited upregulation of PI3K signaling and sensitivity to this pathway inhibition. Furthermore, both tumor subtypes were resistant to CDK4/6 inhibition. CONCLUSIONS: Examination of molecular expression profiles and drug sensitivities of tumors indicate that these breast cancer models can be utilized as a translational platform for evaluation of targeted combinations to improve chemotherapeutic response in patients that no longer respond to hormone therapy or that are resistant to CDK4/6 inhibition.

E3 ubiquitin ligase MDM2 acts through p53 to control respiratory progenitor cell number and lung size.

The respiratory lineage initiates from the specification of NKX2-1+ progenitor cells that ultimately give rise to a vast gas-exchange surface area. How the size of the progenitor pool is determined and whether this directly impacts final lung size remains poorly understood. Here, we show that epithelium-specific inactivation of Mdm2, which encodes an E3 ubiquitin ligase, led to lethality at birth with a striking reduction of lung size to a single vestigial lobe. Intriguingly, this lobe was patterned and contained all the appropriate epithelial cell types. The reduction of size can be traced to the progenitor stage, when p53, a principal MDM2 protein degradation target, was transiently upregulated. This was followed by a brief increase of apoptosis. Inactivation of the p53 gene in the Mdm2 mutant background effectively reversed the lung size phenotype, allowing survival at birth. Together, these findings demonstrate that p53 protein turnover by MDM2 is essential for the survival of respiratory progenitors. Unlike in the liver, in which genetic reduction of progenitors triggered compensation, in the lung, respiratory progenitor number is a key determinant factor for final lung size.

The histone acetyltransferase inhibitor Nir regulates epidermis development.

In addition to its function as an inhibitor of histone acetyltransferases, Nir (Noc2l) binds to p53 and TAp63 to regulate their activity. Here, we show that epidermis-specific ablation of Nir impairs epidermal stratification and barrier function, resulting in perinatal lethality. Nir-deficient epidermis lacks appendages and remains single layered during embryogenesis. Cell proliferation is inhibited, whereas apoptosis and p53 acetylation are increased, indicating that Nir is controlling cell proliferation by limiting p53 acetylation. Transcriptome analysis revealed that Nir regulates the expression of essential factors in epidermis development, such as keratins, integrins and laminins. Furthermore, Nir binds to and controls the expression of p63 and limits H3K18ac at the p63 promoter. Corroborating the stratification defects, asymmetric cell divisions were virtually absent in Nir-deficient mice, suggesting that Nir is required for correct mitotic spindle orientation. In summary, our data define Nir as a key regulator of skin development.

Postmortem expression of apoptosis-related genes in the liver of mice and their use for estimation of the time of death.

PURPOSE: A major challenge in forensic medicine is to estimate the postmortem interval (PMI). Several approaches had been tried to determine the time of death, including physical and chemical changes. This study aims to explore the postmortem changes in the expression of apoptosis-related genes in the liver of mice and to use these changes for estimation of the PMI. METHODS: Hepatic tissue was collected from sacrificed mice immediately after death (the control group) and at 3, 6, 9, 12, 18, and 24 hours after death. Four apoptosisrelated genes were selected as target genes, which are Caspase 3 (Casp3), B cell leukemia/ lymphoma 2 (Bcl2), BCL2-associated X protein (Bax), and Transformation related protein 53 (Trp53), and their relative expression was measured using quantitative PCR. miR-122 was used as a reference gene for normalization of the Ct (threshold cycle) values of the target genes. RESULTS: The results revealed that the postmortem expression of Casp3 increased in a time-dependent manner; the expression of Bax increased from 3 to 18 hours followed by a decrease at 24 hours after death; the expression of Bcl2 decreased in a time-dependent manner after death; the expression of Trp53 increased from 3 to 6 hours and then started to decrease from 9 to 24 hours after death. CONCLUSION: Based on the observed changes in the expression level of these genes, mathematical models were established to estimate the PMI. Further research is needed to investigate these markers and mathematical models in human tissues.

Inactivation of TRP53, PTEN, RB1, and/or CDH1 in the ovarian surface epithelium induces ovarian cancer transformation and metastasis.

Ovarian cancer (OvCa) remains the most common cause of death from gynecological malignancies. Genetically engineered mouse models have been used to study initiation, origin, progression, and/or mechanisms of OvCa. Based on the clinical features of OvCa, we examined a quadruple combination of pathway perturbations including PTEN, TRP53, RB1, and/or CDH1. To characterize the cancer-promoting events in the ovarian surface epithelium (OSE), Amhr2cre/+ mice were used to ablate floxed alleles of Pten, Trp53, and Cdh1, which were crossed with TgK19GT121 mice to inactivate RB1 in KRT19-expressing cells. Inactivation of PTEN, TRP53, and RB1 with or without CDH1 led to the development of type I low-grade OvCa with enlarged serous papillary carcinomas and some high-grade serous carcinomas (HGSCs) in older mice. Initiation of epithelial hyperplasia and micropapillary carcinoma started earlier at 1 month in the triple mutations of Trp53, Pten, and Rb1 mice as compared to 2 months in quadruple mutations of Trp53, Pten, Rb1, and Cdh1 mice, whereas both genotypes eventually developed enlarged proliferating tumors that invaded into the ovary at 3-4 months. Mice with triple and quadruple mutations developed HGSC and/or metastatic tumors, which disseminated into the peritoneal cavity at 4-6 months. In summary, inactivation of PTEN, TRP53, and RB1 initiates OvCa from the OSE. Additional ablation of CDH1 further increased persistence of tumor dissemination and ascites fluid accumulation enhancing peritoneal metastasis.

Salmonella enterica serovar Typhimurium inhibits the innate immune response and promotes apoptosis in a ribosomal/TRP53-dependent manner in swine neutrophils.

Neutrophils are the first barriers for resisting the invasion, proliferation, and damage caused by Salmonella Typhimurium. However, the mechanisms that control this resistance are not completely understood. In this study, we established an in vitro Salmonella infection model in porcine neutrophils, and analyzed the cellular transcriptome by deep sequencing and flow cytometry. The results showed that ribosomal gene transcription was inhibited, and two of these genes, RPL39 and RPL9, were related to TRP53 activation. Furthermore, several important innate immunity genes were also inhibited. Knock-down of RPL39 and RPL9 by siRNA caused an approximate fourfold up-regulation of TRP53. Knock-down of RPL39 and RPL9 also resulted in a significant down-regulation of IFNG and TNF, indicating an inhibition of the innate immune response. Silencing of RPL39 and RPL9 also resulted in the up-regulation of FAS, RB1, CASP6, and GADD45A, which play roles in cell cycle arrest and apoptosis. Neutrophils were either first treated with RPL39 siRNA, RPL9 siRNA, TRP53 activator, or TRP53 inhibitor, and then infected with Salmonella. Knock-down of RPL39 and RPL9, or treatment with TRP53 activator, can increase the intracellular proliferation of Salmonella in neutrophils. We speculate that much of the Salmonella virulence can be attributed to the enhancement of cell cycle arrest and the inhibition of the innate immune response, which allows the bacteria to successfully proliferate intracellularly.

S100a4 upregulation in Pik3caH1047R;Trp53R270H;MMTV-Cre-driven mammary tumors promotes metastasis.

BACKGROUND: PIK3CA mutations are frequent in human breast cancer. Pik3caH1047R mutant expression in mouse mammary gland promotes tumorigenesis. TP53 mutations co-occur with PIK3CA mutations in human breast cancers. We previously generated a conditionally activatable Pik3caH1047R;MMTV-Cre mouse model and found a few malignant sarcomatoid (spindle cell) carcinomas that had acquired spontaneous dominant-negative Trp53 mutations. METHODS: A Pik3caH1047R;Trp53R270H;MMTV-Cre double mutant mouse breast cancer model was generated. Tumors were characterized by histology, marker analysis, transcriptional profiling, single-cell RNA-seq, and bioinformatics. Cell lines were developed from mutant tumors and used to identify and confirm genes involved in metastasis. RESULTS: We found Pik3caH1047R and Trp53R270H cooperate in driving oncogenesis in mammary glands leading to a shorter latency than either alone. Double mutant mice develop multiple histologically distinct mammary tumors, including adenocarcinoma and sarcomatoid (spindle cell) carcinoma. We found some tumors to be invasive and a few metastasized to the lung and/or the lymph node. Single-cell RNA-seq analysis of the tumors identified epithelial, stromal, myeloid, and T cell groups. Expression analysis of the metastatic tumors identified S100a4 as a top candidate gene associated with metastasis. Metastatic tumors contained a much higher percentage of epithelial-mesenchymal transition (EMT)-signature positive and S100a4-expressing cells. CRISPR/CAS9-mediated knockout of S100a4 in a metastatic tumor-derived cell line disrupted its metastatic potential indicating a role for S100a4 in metastasis. CONCLUSIONS: Pik3caH1047R;Trp53R270H;MMTV-Cre mouse provides a preclinical model to mimic a subtype of human breast cancers that carry both PIK3CA and TP53 mutations. It also allows for understanding the cooperation between the two mutant genes in tumorigenesis. Our model also provides a system to study metastasis and develop therapeutic strategies for PIK3CA/TP53 double-positive cancers. S100a4 found involved in metastasis in this model can be a potential diagnostic and therapeutic target.

Mutations in the murine homologue of TUBB5 cause microcephaly by perturbing cell cycle progression and inducing p53-associated apoptosis.

Microtubules play a crucial role in the generation, migration and differentiation of nascent neurons in the developing vertebrate brain. Mutations in the constituents of microtubules, the tubulins, are known to cause an array of neurological disorders, including lissencephaly, polymicrogyria and microcephaly. In this study we explore the genetic and cellular mechanisms that cause TUBB5-associated microcephaly by exploiting two new mouse models: a conditional E401K knock-in, and a conditional knockout animal. These mice present with profound microcephaly due to a loss of upper-layer neurons that correlates with massive apoptosis and upregulation of p53. This phenotype is associated with a delay in cell cycle progression and ectopic DNA elements in progenitors, which is dependent on the dosage of functional Tubb5. Strikingly, we report ectopic Sox2-positive progenitors and defects in spindle orientation in our knock-in mouse line, which are absent in knockout animals. This work sheds light on the functional repertoire of Tubb5, reveals that the E401K mutation acts by a complex mechanism, and demonstrates that the cellular pathology driving TUBB5-associated microcephaly is cell death.

Rapid generation and selection of Cas9-engineering TRP53 R172P mice that do not have off-target effects.

BACKGROUND: Genetic mutations cause severe human diseases, and suitable animal models to study the regulatory mechanisms involved are required. The CRISPR/Cas9 system is a powerful, highly efficient and easily manipulated tool for genetic modifications. However, utilization of CRISPR/Cas9 to introduce point mutations and the exclusion of off-target effects in mice remain challenging. TP53-R175 is one of the most frequently mutated sites in human cancers, and it plays crucial roles in human diseases, including cancers and diabetes. RESULTS: Here, we generated TRP53-R172P mutant mice (C57BL/6 J, corresponding to TP53-R175P in humans) using a single microinjection of the CRISPR/Cas9 system. The optimal parameters comprised gRNA selection, donor designation (silent mutations within gRNA region), the concentration of CRISPR components and the cellular sites of injection. TRP53-R172P conversion was genetically and functionally confirmed. Combination of TA cloning and Sanger sequencing helped identify the correctly targeted mice as well as the off-target effects in the engineered mice, which provide us a strategy to select the on-target mice without off-target effects quickly and efficiently. CONCLUSIONS: A single injection of the this optimized CRISPR/Cas9 system can be applied to introduce particular mutations in the genome of mice without off-target effects to model various human diseases.

Lactobacillus rescues postnatal neurobehavioral and microglial dysfunction in a model of maternal microbiome dysbiosis.

Increasing reports of pregnancy events leading to maternal microbiome dysbiosis (MMD) show strong correlates with atypical neurodevelopmental outcomes. However, the mechanism(s) driving microbiome-mediated behavioral dysfunction in offspring remain understudied. Here, we demonstrate the presence of a novel gut commensal bacterium strain, Lactobacillus murinus HU-1, was sufficient to rescue behavioral deficits and brain region-specific microglial activationobserved in MMD-reared murine offspring. We furtheridentified a postnatal window of susceptibility that could prevent social impairments with timed maternal administration of the symbiotic bacterium. Moreover, MMD increased expression of microglial senescence genes, Trp53 and Il1ß, and Cx3cr1 protein in the prefrontal cortex, which correlated with dysfunctional modeling of synapses and accompanied dysbiosis-induced microglial activation. MMD male offspring harboring Lactobacillus murinus HU-1 or lacking Cx3cr1 showed amelioration of these effects. The current study describes a new avenue of influence by which maternally transferred Lactobacillus drives proper development of social behavior in the offspring through microglia-specific regulation of Cx3cr1 signaling.

Increased susceptibility to mammary carcinogenesis and an opposite trend in endometrium in Trp53 heterozygous knockout female mice by backcrossing the BALB/c strain onto the background C3H strain.

Patients with dominantly inherited Li-Fraumeni syndrome have a loss-of-function mutation in TP53 and develop diverse mesenchymal and epithelial neoplasms at multiple sites. Trp53 +/- female mice with the BALB/c background provide unique characteristics for the study of breast cancer in Li-Fraumeni syndrome; however, we previously found that female C3H-Trp53+/ - mice did not spontaneously develop mammary tumors. Therefore, we obtained F1 and N2-N4 female mice by backcrossing the BALB/c strain and examined the incidence of mammary and other tumors in lifetime studies. Malignant lymphomas, osteosarcomas, and uterine adenocarcinomas spontaneously developed in approximately 20% or more of Trp53+/ - mice with the C3H background. In contrast, the incidence of uterine adenocarcinomas showed a tendency to decrease, while that of mammary adenocarcinomas gradually increased in mice with the BALB/c strain backcross. Wild-type BALB/c female mice are predisposed to a wide spectrum of neoplasms, including mammary tumors, partly due to genetic factors, whereas uterine tumors are uncommon not only in BALB/c mice but also C3H mice. Thus, genetic factors appear to contribute to a strain-specific predisposition to malignant neoplasms in Trp53+/- mice, and further studies are needed to clarify the detailed mechanisms.

A new genetically engineered mouse model of choroid plexus carcinoma.

Choroid plexus carcinomas (CPCs) are highly malignant brain tumours predominantly found in children and associated to poor prognosis. Improved therapy for these cancers would benefit from the generation of animal models. Here we have created a novel mouse CPC model by expressing a stabilised form of c-Myc (MycT58A) and inactivating Trp53 in the choroid plexus of newborn mice. This induced aberrant proliferation of choroid plexus epithelial cells, leading to aggressive tumour development and death within 150 days. Choroid plexus tumours occurred with a complete penetrance in all brain ventricles, with prevalence in the lateral and fourth ventricles. Histological and cellular analysis indicated that these tumours were CPCs resembling their human counterparts. Comparison of gene expression profiles of CPCs and non-neoplastic tissues revealed profound alterations in cell cycle regulation and DNA damage responses, suggesting that dysregulation of cell division and DNA checkpoint pathways may represent key vulnerabilities. This novel animal model of CPC provides an invaluable tool to elucidate the mechanism of CPC formation and to develop successful therapies against this devastating paediatric cancer.

Differential DNA damage signalling and apoptotic threshold correlate with mouse epiblast-specific hypersensitivity to radiation.

Between implantation and gastrulation, mouse pluripotent epiblast cells expand enormously in number and exhibit a remarkable hypersensitivity to DNA damage. Upon low-dose irradiation, they undergo mitotic arrest followed by p53-dependent apoptosis, whereas the other cell types simply arrest. This protective mechanism, active exclusively after E5.5 and lost during gastrulation, ensures the elimination of every mutated cell before its clonal expansion and is therefore expected to greatly increase fitness. We show that the insurgence of apoptosis relies on the epiblast-specific convergence of both increased DNA damage signalling and stronger pro-apoptotic balance. Although upstream Atm/Atr global activity and specific γH2AX phosphorylation are similar in all cell types of the embryo, 53BP1 recruitment at DNA breaks is immediately amplified only in epiblast cells after ionizing radiation. This correlates with rapid epiblast-specific activation of p53 and its transcriptional properties. Moreover, between E5.5 and E6.5 epiblast cells lower their apoptotic threshold by enhancing the expression of pro-apoptotic Bak and Bim and repressing the anti-apoptotic Bcl-xL. Thus, even after low-dose irradiation, the cytoplasmic priming of epiblast cells allows p53 to rapidly induce apoptosis via a partially transcription-independent mechanism.

Histone deacetylase 1 and 2 regulate Wnt and p53 pathways in the ureteric bud epithelium.

Histone deacetylases (HDACs) regulate a broad range of biological processes through removal of acetyl groups from histones as well as non-histone proteins. Our previous studies showed that Hdac1 and Hdac2 are bound to promoters of key renal developmental regulators and that HDAC activity is required for embryonic kidney gene expression. However, the existence of many HDAC isoforms in embryonic kidneys raises questions concerning the possible specificity or redundancy of their functions. We report here that targeted deletion of both the Hdac1 and Hdac2 genes from the ureteric bud (UB) cell lineage of mice causes bilateral renal hypodysplasia. One copy of either Hdac1 or Hdac2 is sufficient to sustain normal renal development. In addition to defective cell proliferation and survival, genome-wide transcriptional profiling revealed that the canonical Wnt signaling pathway is specifically impaired in UB(Hdac1,2-/-) kidneys. Our results also demonstrate that loss of Hdac1 and Hdac2 in the UB epithelium leads to marked hyperacetylation of the tumor suppressor protein p53 on lysine 370, 379 and 383; these post-translational modifications are known to boost p53 stability and transcriptional activity. Genetic deletion of p53 partially rescues the development of UB(Hdac1,2-/-) kidneys. Together, these data indicate that Hdac1 and Hdac2 are crucial for kidney development. They perform redundant, yet essential, cell lineage-autonomous functions via p53-dependent and -independent pathways.

A systems genetics approach identifies Trp53inp2 as a link between cardiomyocyte glucose utilization and hypertrophic response.

Cardiac failure has been widely associated with an increase in glucose utilization. The aim of our study was to identify factors that mechanistically bridge this link between hyperglycemia and heart failure. Here, we screened the Hybrid Mouse Diversity Panel (HMDP) for substrate-specific cardiomyocyte candidates based on heart transcriptional profile and circulating nutrients. Next, we utilized an in vitro model of rat cardiomyocytes to demonstrate that the gene expression changes were in direct response to substrate abundance. After overlaying candidates of interest with a separate HMDP study evaluating isoproterenol-induced heart failure, we chose to focus on the gene Trp53inp2 as a cardiomyocyte glucose utilization-specific factor. Trp53inp2 gene knockdown in rat cardiomyocytes reduced expression and protein abundance of key glycolytic enzymes. This resulted in reduction of both glucose uptake and glycogen content in cardiomyocytes stimulated with isoproterenol. Furthermore, this reduction effectively blunted the capacity of glucose and isoprotereonol to synergistically induce hypertrophic gene expression and cell size expansion. We conclude that Trp53inp2 serves as regulator of cardiomyocyte glycolytic activity and can consequently regulate hypertrophic response in the context of elevated glucose content.NEW & NOTEWORTHY Here, we apply a novel method for screening transcripts based on a substrate-specific expression pattern to identify Trp53inp2 as an induced cardiomyocyte glucose utilization factor. We further show that reducing expression of the gene could effectively blunt hypertrophic response in the context of elevated glucose content.

Combined deletion of Vhl, Trp53 and Kif3a causes cystic and neoplastic renal lesions.

The von Hippel-Lindau (VHL) tumour suppressor gene is bi-allelically inactivated in the majority of cases of clear cell renal cell carcinoma (ccRCC); however, Vhl knockout mouse models do not recapitulate human ccRCC, implying that additional mutations are required for tumour formation. Mutational inactivation of VHL sensitises renal epithelial cells to lose the primary cilium in response to other mutations or extracellular stimuli. Loss of cilia is believed to represent a second hit in VHL mutant cells that causes the development of cystic lesions that, in some cases, can progress to ccRCC. Supporting this idea, genetic ablation of the primary cilium by deletion of the kinesin family member 3A (Kif3a) gene cooperates with loss of Vhl to accelerate cyst formation in mouse kidneys. Additionally, aged Vhl/Trp53 double-mutant mice develop renal cysts and tumours at a relatively low incidence, suggesting that there is a genetic cooperation between VHL and TP53 mutation in the development of ccRCC. Here we generated renal epithelium-specific Kif3a/Trp53 and Vhl/Kif3a/Trp53 mutant mice to investigate whether primary cilium deletion would accelerate the development of cystic precursor lesions or cause their progression to ccRCC. Longitudinal microcomputed tomography (µCT) imaging and histopathological analyses revealed an increased rate of cyst formation, increased proportion of cysts with proliferating cells, higher frequency of atypical cysts as well as the development of neoplasms in Vhl/Kif3a/Trp53 mutant kidneys compared to Kif3a/Trp53 or Vhl/Kif3a mutant kidneys. These findings demonstrate that primary cilium loss, in addition to Vhl and Trp53 losses, promotes the transition towards malignancy and provide further evidence that the primary cilium functions as a tumour suppressor organelle in the kidney. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Transcriptomic analysis reveals sex-specific differences in the expression of Dcl1 and Fis1 genes in the radio-adaptive response of thymocytes to TRP53-mediated apoptosis.

BACKGROUND: Radio-Adaptive Response (RAR) is a biological defense mechanism whereby exposure to low dose ionizing radiation (IR) mitigates the detrimental effects of high dose irradiation. RAR has been widely observed in vivo using as endpoint less induction of apoptosis. However, sex differences associated with RAR and variations between males and females on global gene expression influenced by RAR have not been still investigated. In addition, the response to radiation-induced apoptosis is associated with phosphorylation of TRP53 at both the serine 15 (ser-18 in the mouse) and serine 392 (ser-389 in mice) residues, but the role of these two phosphorylated forms in male and female RAR remains to be elucidated. RESULTS: We analyzed the effect of administering priming low dose radiation (0.075 Gy of X-rays) prior to high dose radiation (1.75 Gy of γ-rays) on the level of caspase-3-mediated apoptosis and on global transcriptional expression in thymocytes of male and female mice. Here, we provide the first evidence of a differential sex effect of RAR on the reduction of thymocyte apoptosis with males showing lesser levels of caspase-3-mediated apoptosis than females. Analysis of transcriptomic profiles of 1944 genes involved in apoptosis signaling in radio-adapted thymocytes identified 17 transcripts exhibiting differential expression between both sexes. Among them, Dlc1 and Fis1 are closely related to the apoptosis mediated by the TRP53 protein. Our data demonstrate that overexpression of Dlc1 and Fis1 occur concomitantly with a highest accumulation of phosphoserine-18-TRP53 and caspase-3 in radio-adapted thymocytes of female mice. In an opposite way, both down-modulation of Fis1 and phosphoserine-389-TRP53 accumulation appear to be associated with protection from thymocyte apoptosis mediated by caspase-3 in males. CONCLUSIONS: Transcriptomic analysis performed in this work reveals for the first time sex-specific differences in gene expression influenced by RAR. Our results also suggest a sex-dependent dual role for phosphoserine-18-TRP53 and phosphoserine-389-TRP53 in the regulation of the radio-adaptive response in mouse thymocytes.