DLK1/DIO3 locus upregulation by a ß-catenin-dependent enhancer drives cell proliferation and liver tumorigenesis.

The CTNNB1 gene, encoding ß-catenin, is frequently mutated in hepatocellular carcinoma (HCC, ∼30%) and in hepatoblastoma (HB, >80%), in which DLK1/DIO3 locus induction is correlated with CTNNB1 mutations. Here, we aim to decipher how sustained ß-catenin activation regulates DLK1/DIO3 locus expression and the role this locus plays in HB and HCC development in mouse models deleted for Apc (ApcΔhep) or Ctnnb1-exon 3 (ß-cateninΔExon3) and in human CTNNB1-mutated hepatic cancer cells. We identified an enhancer site bound by TCF-4/ß-catenin complexes in an open conformation upon sustained ß-catenin activation (DLK1-Wnt responsive element [WRE]) and increasing DLK1/DIO3 locus transcription in ß-catenin-mutated human HB and mouse models. DLK1-WRE editing by CRISPR-Cas9 approach impaired DLK1/DIO3 locus expression and slowed tumor growth in subcutaneous CTNNB1-mutated tumor cell grafts, ApcΔhep HB and ß-cateninΔExon3 HCC. Tumor growth inhibition resulted either from increased FADD expression and subsequent caspase-3 cleavage in the first case or from decreased expression of cell cycle actors regulated by FoxM1 in the others. Therefore, the DLK1/DIO3 locus is an essential determinant of FoxM1-dependent cell proliferation during ß-catenin-driven liver tumorigenesis. Targeting the DLK1-WRE enhancer to silence the DLK1/DIO3 locus might thus represent an interesting therapeutic strategy to restrict tumor growth in primary liver cancers with CTNNB1 mutations.

Fertilization, but Not Post-Implantation Development, Can Occur in the Absence of Sperm and Oocyte Beta1 Integrin in Mice.

Fertilization is a complex process that requires successive stages and culminates in the adhesion/fusion of gamete membranes. If the question of the involvement of oocyte integrins has been swept away by deletion experiments, that of the involvement of sperm integrins remains to be further characterized. In the present study, we addressed the question of the feasibility of sperm-oocyte adhesion/fusion and early implantation in the absence of sperm ß1 integrin. Males and females with ß1 integrin-depleted sperm and oocytes were mated, and fertilization outcome was monitored by a gestational ultrasound analysis. Results suggest that although the sperm ß1 integrin participates in gamete adhesion/fusion, it is dispensable for fertilization in mice. However, sperm- and/or oocyte-originated integrin ß1 is essential for post-implantation development. Redundancy phenomena could be at the origin of a compensatory expression or alternative dimerization pattern.

Increased NOS coupling by the metabolite tetrahydrobiopterin (BH4) reduces preeclampsia/IUGR consequences.

Preeclampsia (PE) is a high-prevalence pregnancy disease characterized by placental insufficiency, gestational hypertension, and proteinuria. Overexpression of the A isoform of the STOX1 transcription factor (STOX1A) recapitulates PE in mice, and STOX1A overexpressing trophoblasts recapitulate PE patients hallmarks in terms of gene expression and pathophysiology. STOX1 overexpression induces nitroso-redox imbalance and mitochondrial hyper-activation. Here, by a thorough analysis on cell models, we show that STOX1 overexpression in trophoblasts alters inducible nitric oxide synthase (iNOS), nitric oxide (NO) content, the nitroso-redox balance, the antioxidant defense, and mitochondrial function. This is accompanied by specific alterations of the Krebs cycle leading to reduced l-malate content. By increasing NOS coupling using the metabolite tetrahydrobiopterin (BH4) we restore this multi-step pathway in vitro. Moving in vivo on two different rodent models (STOX1 mice and RUPP rats, alike early onset and late onset preeclampsia, respectively), we show by transcriptomics that BH4 directly reverts STOX1-deregulated gene expression including glutathione metabolism, oxidative phosphorylation, cholesterol metabolism, inflammation, lipoprotein metabolism and platelet activation, successfully treating placental hypotrophy, gestational hypertension, proteinuria and heart hypertrophy. In the RUPP rats we show that the major fetal issue of preeclampsia, Intra Uterine Growth Restriction (IUGR), is efficiently corrected. Our work posits on solid bases BH4 as a novel potential therapy for preeclampsia.

BCG-trained innate immunity leads to fetal growth restriction by altering immune cell profile in the mouse developing placenta.

Trained immunity is a new concept illustrating that innate immune cells are able to undergo a long-term metabolic and epigenetic reprogramming after infection or vaccination, thus displaying either a pro- or an anti-inflammatory phenotype during a sequential unrelated challenge. Innate immune cells such as natural killer (NK) cells and macrophages constitute a large part of the decidual leukocyte population at the maternal-fetal interface, playing an important role in placental development and as such in fetal growth and development. In this study, we hypothesized that training the innate immune cells before pregnancy could have an impact on pregnancy. To test this hypothesis, we used CBA/J x DBA/2 mouse model to investigate pregnancy outcomes and leukocyte population at the maternal-fetal interface. Although we were not able to show a beneficial effect of LPS-tolerogenic training on fetal resorption, Bacillus Calmette-Guérin (BCG) training, known to prime innate immune cells to be proinflammatory, led to fetal growth restriction, without aggravating the fetal resorption rate. We also found that BCG training led to less NK cells and macrophages at the maternal-fetal interface at the early stage of placentation (E9.5), associated with a down-regulation of Ccr3 and Lif mRNA expression. This induced altered leucocyte population profile can be an explanation for the subsequent fetal growth restriction. These data suggest that preconceptional infections-induced trained immunity could influence pregnancy outcomes.

Cooperation Between the NRF2 Pathway and Oncogenic ß-catenin During HCC Tumorigenesis.

CTNNB1 (catenin beta 1)-mutated hepatocellular carcinomas (HCCs) account for a large proportion of human HCCs. They display high levels of respiratory chain activity. As metabolism and redox balance are closely linked, tumor cells must maintain their redox status during these metabolic alterations. We investigated the redox balance of these HCCs and the feasibility of targeting this balance as an avenue for targeted therapy. We assessed the expression of the nuclear erythroid 2 p45-related factor 2 (NRF2) detoxification pathway in an annotated human HCC data set and reported an enrichment of the NRF2 program in human HCCs with CTNNB1 mutations, largely independent of NFE2L2 (nuclear factor, erythroid 2 like 2) or KEAP1 (Kelch-like ECH-associated protein 1) mutations. We then used mice with hepatocyte-specific oncogenic ß-catenin activation to evaluate the redox status associated with ß-catenin activation in preneoplastic livers and tumors. We challenged them with various oxidative stressors and observed that the ß-catenin pathway activation increased transcription of Nfe2l2, which protects ß-catenin-activated hepatocytes from oxidative damage and supports tumor development. Moreover, outside of its effects on reactive oxygen species scavenging, we found out that Nrf2 itself contributes to the metabolic activity of ß-catenin-activated cells. We then challenged ß-catenin activated tumors pharmacologically to create a redox imbalance and found that pharmacological inactivation of Nrf2 was sufficient to considerably decrease the progression of ß-catenin-dependent HCC development. Conclusion: These results demonstrate cooperation between oncogenic ß-catenin signaling and the NRF2 pathway in CTNNB1-mediated HCC tumorigenesis, and we provide evidence for the relevance of redox balance targeting as a therapeutic strategy in CTNNB1-mutated HCC.

ARID1A loss in adult hepatocytes activates ß-catenin-mediated erythropoietin transcription.

Erythropoietin (EPO) is a key regulator of erythropoiesis. The embryonic liver is the main site of erythropoietin synthesis, after which the kidney takes over. The adult liver retains the ability to express EPO, and we discovered here new players of this transcription, distinct from the classical hypoxia-inducible factor pathway. In mice, genetically invalidated in hepatocytes for the chromatin remodeler Arid1a, and for Apc, the major silencer of Wnt pathway, chromatin was more accessible and histone marks turned into active ones at the Epo downstream enhancer. Activating ß-catenin signaling increased binding of Tcf4/ß-catenin complex and upregulated its enhancer function. The loss of Arid1a together with ß-catenin signaling, resulted in cell-autonomous EPO transcription in mouse and human hepatocytes. In mice with Apc-Arid1a gene invalidations in single hepatocytes, Epo de novo synthesis led to its secretion, to splenic erythropoiesis and to dramatic erythrocytosis. Thus, we identified new hepatic EPO regulation mechanism stimulating erythropoiesis.

Cytosolic PCNA interacts with p47phox and controls NADPH oxidase NOX2 activation in neutrophils.

Neutrophils produce high levels of reactive oxygen species (ROS) by NADPH oxidase that are crucial for host defense but can lead to tissue injury when produced in excess. We previously described that proliferating cell nuclear antigen (PCNA), a nuclear scaffolding protein pivotal in DNA synthesis, controls neutrophil survival through its cytosolic association with procaspases. We herein showed that PCNA associated with p47phox, a key subunit of NADPH oxidase, and that this association regulated ROS production. Surface plasmon resonance and crystallography techniques demonstrated that the interdomain-connecting loop of PCNA interacted directly with the phox homology (PX) domain of the p47phox. PCNA inhibition by competing peptides or by T2AA, a small-molecule PCNA inhibitor, decreased NADPH oxidase activation in vitro. Furthermore, T2AA provided a therapeutic benefit in mice during trinitro-benzene-sulfonic acid (TNBS)-induced colitis by decreasing oxidative stress, accelerating mucosal repair, and promoting the resolution of inflammation. Our data suggest that targeting PCNA in inflammatory neutrophils holds promise as a multifaceted antiinflammatory strategy.

Long-term cardiovascular disorders in the STOX1 mouse model of preeclampsia.

Adverse long-term cardiovascular (CV) consequences of PE are well established in women. However, the mechanism responsible for that risk remains unknown. Here, we mated wild-type female mice of the FVB/N strain to STOX1A-overexpressing mice to mimic severe PE and investigated the long-term consequences on the maternal cardiovascular system. Ultrasonography parameters were analyzed in mice before pregnancy and at 3 and 6 months post-pregnancy. At 6 months post-pregnancy, cardiac stress test induced by dobutamine injection revealed an abnormal ultrasonography Doppler profile in mice with previous PE. Eight months post-pregnancy, the heart, endothelial cells (ECs) and plasma of females were analyzed and compared to controls. The heart of mice with PE showed left-ventricular hypertrophy associated with altered histology (fibrosis). Transcriptomic analysis revealed the deregulation of 1149 genes in purified ECs and of 165 genes in the hearts, many being involved in heart hypertrophy. In ECs, the upregulated genes were associated with inflammation and cellular stress. Systems biology analysis identified interleukin 6 (IL-6) as a hub gene connecting these pathways. Plasma profiling of 33 cytokines showed that, 8 of them (Cxcl13, Cxcl16, Cxcl11, IL-16, IL-10, IL-2, IL-4 and Ccl1) allowed to discriminate mice with previous PE from controls. Thus, PE triggers female long-term CV consequences on the STOX1 mouse model.

ß-catenin-activated hepatocellular carcinomas are addicted to fatty acids.

OBJECTIVES: CTNNB1-mutated hepatocellular carcinomas (HCCs) constitute a major part of human HCC and are largely inaccessible to target therapy. Yet, little is known about the metabolic reprogramming induced by ß-catenin oncogenic activation in the liver. We aimed to decipher such reprogramming and assess whether it may represent a new avenue for targeted therapy of CTNNB1-mutated HCC. DESIGN: We used mice with hepatocyte-specific oncogenic activation of ß-catenin to evaluate metabolic reprogramming using metabolic fluxes on tumourous explants and primary hepatocytes. We assess the role of Pparα in knock-out mice and analysed the consequences of fatty acid oxidation (FAO) using etomoxir. We explored the expression of the FAO pathway in an annotated human HCC dataset. RESULTS: ß-catenin-activated HCC were not glycolytic but intensively oxidised fatty acids. We found that Pparα is a ß-catenin target involved in FAO metabolic reprograming. Deletion of Pparα was sufficient to block the initiation and progression of ß-catenin-dependent HCC development. FAO was also enriched in human CTNNB1-mutated HCC, under the control of the transcription factor PPARα. CONCLUSIONS: FAO induced by ß-catenin oncogenic activation in the liver is the driving force of the ß-catenin-induced HCC. Inhibiting FAO by genetic and pharmacological approaches blocks HCC development, showing that inhibition of FAO is a suitable therapeutic approach for CTNNB1-mutated HCC.

Preeclampsia induced by STOX1 overexpression in mice induces intrauterine growth restriction, abnormal ultrasonography and BOLD MRI signatures.

BACKGROUND: Preeclampsia is a major hypertensive disease caused by pregnancy, inducing proteinuria and increased blood pressure starting from the second half of pregnancy (early preeclampsia) or near the end of pregnancy (late preeclampsia). Pre-symptomatic diagnosis would allow for therapeutic interventions, such as with low-dose aspirin. Among non-invasive methods to explore organ physiology, Doppler ultrasonography (US) and functional blood oxygenation level-dependent (BOLD) MRI (which do not need radioactive contrast agents such as gadolinium) can be used in pregnant women. METHODS: In this study, we used US and BOLD MRI to finely characterize the phenotype of preeclampsia induced by the foeto-placental overexpression of the transcription factor storkhead box 1A (STOX1A) in female mice. RESULTS: We could observe late fetal growth restriction consistent with the placental dysfunction revealed by US and the known association between preeclampsia and intra-uterine growth restriction. On US, uterine and umbilical artery as well as heart and kidney parameters were modified in preeclamptic mice. On BOLD MRI, mean T2* values revealed considerable differences between control and preeclamptic placentas, which suggests altered dynamics of oxygen release and ratio of oxyhemoglobin to deoxyhemoglobin in the model. CONCLUSION: These preliminary pre-clinical results suggest that BOLD MRI could be evaluated as a prognostic/diagnostic tool for preeclampsia.

Cationic microbubbles and antibiotic-free miniplasmid for sustained ultrasound-mediated transgene expression in liver.

Despite the increasing number of clinical trials in gene therapy, no ideal methods still allow non-viral gene transfer in deep tissues such as the liver. We were interested in ultrasound (US)-mediated gene delivery to provide long term liver expression. For this purpose, new positively charged microbubbles were designed and complexed with pFAR4, a highly efficient small length miniplasmid DNA devoid of antibiotic resistance sequence. Sonoporation parameters, such as insonation time, acoustic pressure and duration of plasmid injection were controlled under ultrasound imaging guidance. The optimization of these various parameters was performed by bioluminescence optical imaging of luciferase reporter gene expression in the liver. Mice were injected with 50µg pFAR4-LUC either alone, or complexed with positively charged microbubbles, or co-injected with neutral MicroMarker™ microbubbles, followed by low ultrasound energy application to the liver. Injection of the pFAR4 encoding luciferase alone led to a transient transgene expression that lasted only for two days. The significant luciferase signal obtained with neutral microbubbles decreased over 2days and reached a plateau with a level around 1 log above the signal obtained with pFAR4 alone. With the newly designed positively charged microbubbles, we obtained a much stronger bioluminescence signal which increased over 2days. The 12-fold difference (p<0.05) between MicroMarker™ and our positively charged microbubbles was maintained over a period of 6months. Noteworthy, the positively charged microbubbles led to an improvement of 180-fold (p<0.001) as regard to free pDNA using unfocused ultrasound performed at clinically tolerated ultrasound amplitude. Transient liver damage was observed when using the cationic microbubble-pFAR4 complexes and the optimized sonoporation parameters. Immunohistochemistry analyses were performed to determine the nature of cells transfected. The pFAR4 miniplasmid complexed with cationic microbubbles allowed to transfect mostly hepatocytes compared to its co-injection with MicroMarker™ which transfected more preferentially endothelial cells.