Background and aim: RuvBL1 belongs to the highly conserved AAA+ ATPases.It is deregulated in various human cancers and its expression correlates with a worse prognosis in HCC patients.We previously found that RuvBL1 haploinsufficiency impairs the PI3 K/ Akt/mTOR pathway in liver.Given the relevance of mTOR pathway hyperactivation in HCC, we hypothesized that RuvBL1 genetic targeting could reduce mTOR-driven hepatocarcinogenesis. Material and methods: Ptenhep-/-and Ruvbl1hep+/-mice were crossed to generate Ptenhep-/-Ruvbl1hep+/-mice.The impact of RuvBL1 haploinsufficiency on NASH development was assessed by histology at 12 weeks of age.Metabolic and inflammatory markers were evaluated by qPCR and IHC.mTOR pathway was analysed by WB of liver lysates.PPARalpha transcriptional activity was evaluated by luciferase reporter assay.The identification of RuvBL1-protein interactions was achieved by MS proteomics analysis of RuvBL1 immunoprecipitation.AML-12 PTEN KO cells were generated by CRISPR-Cas9 genome editing.The impact of RuvBL1 haploinsufficiency on HCC development was assessed by multiplicity evaluation of macroscopic tumours and by histological classification by Edmondson-Steiner grading system at 15 months of age.Results: Ptenhep-/-Ruvbl1hep+/-developed significantly less steatosis, fibrosis, and inflammation compared to Ptenhep-/mice.The mTOR-driven lipogenic targets were similarly expressed in the two mice models.However, Ppara and its target CPT1 was increased in Ptenhep-/-Ruvbl1hep+/-.The spontaneous and insulin-induced accumulation of lipid droplets in PTEN KO AML-12 cells was completely abrogated by RuvBL1 inhibition with CB-6644.Inhibition of RuvBL1 activity by CB-6644 increased PPARalpha transcriptional activity in AML-12 WT and PTEN KO.Analysis of RuvBL1-IP in AML-12 revealed that RuvBL1 interacts with members of the lysosomal AMPK complex.Furthermore, p-AMPK and p-RAPTOR were increased in Ptenhep-/-Ruvbl1hep+/-compared to Ptenhep-/-mice.Finally, Ptenhep-/-Ruvbl1hep+/-mice aged to 15 months showed better survival than Ptenhep-/-which developed significantly more HCC and of higher grade.qPCR analysis showed a significant upregulation of key lipolytic genes, such as Cpt1a, Acadl, Acadvl and Ppara, in Ptenhep-/-Ruvbl1hep+/-at 15 months of age.Conclusions: RuvBL1 targeting reduces mTOR hyperactivation hampering NASH-HCC progression in Ptenhep-/-mice, likely promoting the switch from mTOR-driven lipogenesis to AMPKinduced fatty acid catabolism.

Abstract Background Neutrophil extracellular traps (NETs) consist of DNA filaments and cytoplasmic protein granules, extruded by neutrophils after PAD4-dependent activation. NETs release occurs early during inflammation in various immune-related diseases, further aggravating tissue injury and contributing to fibrosis. Our aim was to investigate the potential profibrotic effect of NETs in Crohn’s disease (CD). Methods NETs and activated fibroblasts were labelled by multiplex immunofluorescence staining on unaffected, inflamed and fibrotic ileum derived from patients with stricturing CD undergoing ileocaecal resection. Intestinal fibroblasts isolated from unaffected CD ileum were co-cultured with NETs for 24 hours to analyse their transcriptome and quantify collagen released with SIRCOL®. Fibroblast migratory activity was investigated with a scratch test. Immunofluorescence intensity of collagen and fibroblast activation protein (FAP) was quantified using the Operetta® Analysis System. Transfection of fibroblasts with NF-kB-luciferase reporter plasmid was performed to evaluate the TLR2/NF-kB pathway, and a specific TLR2 inhibitor (TL2-C29) tested. Finally, mice with selective deletion of PAD4 in neutrophils (PAD4fl/flMRP8Cre+) were subjected to chronic Dextran Sulphate Sodium (DSS) colitis to assess the role of PAD4-mediated NETosis in intestinal fibrosis development. Results We found spatial tissue distribution of clusters of NETs adjacent to FAP+ fibroblasts in ileal ulcerations in patients with active CD. Transcriptomics demonstrated upregulation of various profibrotic processes in fibroblasts stimulated with NETs, including TGFβ-receptor signalling pathway, positive regulation of collagen metabolic process, and TLR-signalling pathways. This correlated with increased proliferation rate (p<0.0001), slower wound healing capability (p<0.0001) and higher collagen release in the medium (p<0.01) in the NET-treated group, as well as higher expression of collagens and FAP. Among TLR genes, TLR2 was found upregulated after stimulation with NETs (p=0.02). Transfection data showed significant upregulation (p<0.05) of NF-kB in the NETs-treated group, whereas its expression and soluble collagen release decreased with the addition of the TLR2 antagonist TL2-C29. In line, a significantly lower amount of collagen deposition was observed in the colon of PAD4fl/flMRP8Cre+ mice subjected to chronic DSS colitis, as well as modulation of MMP2/TIMP2 balance and reduced fibroblast activation. Conclusion NETs may represent an early trigger of intestinal fibroblast activation via the TLR2/NF-kB axis. As NETs are early players during inflammation, blocking PAD4 may reduce inflammation and thus fibrogenesis in CD.

RuvBL1 is an AAA+ ATPase involved in multiple cellular activities including proliferation, chromatin remodeling, gene expression and translation. High RuvBL1 expression correlates with a worse prognosis in HCC and other human tumors. Emerging data suggests that RuvBL1 might exert co-chaperone functions.Aim of this study is to investigate the relations between RuvBL1 and molecular chaperones in HCC.Gene expression analysis of the human HCC samples in the TCGA_LIHC cohort shows that RuvBL1 significantly correlates with the expression of dozens of HSPs family members, with all the TCP-1 ring complex (TRiC) genes and with the transcription factor HSF1. Combining gene expression data from the LIHC_TCGA with publicly available CHIP-seq dataset (CHIP-Atlas and HSF1base) we identified a subset of potential common targets of RuvBL1 and HSF1, which includes several HSPs and all the TRiC genes. Reactome analysis revealed that regulation of cytosolic and mitochondrial translation were among the top enriched pathway regulated by shared RUVBL1 and HSF1 targets. The expression of selected shared targets was evaluated by qPCR in AML-12 and Huh7 cells treated with the RUVBL1/2 ATPase inhibitor CB6644 under basal and stressed conditions. The Heat Shock (HS)- or mitochondrial UPR (mtUPR)-induced expression of HSP90AA1(Hsp90alpha), HSPE1(Hsp10), HSPH1(Hsp110) was abrogated by treatment with CB6644. HSPA8 (Hsp70a8) was induced by mtUPR but not by HS, HSPA4(Hsp70a4) and all the TRiC genes were not induced by either stresses but were nonetheless downregulated by CB6644. HSF1 transcriptional activity, measured in AML-12 clones stably expressing a HSE-Nanoluc reporter, was readily induced by HS or mtUPR and completely abrogated by CB6644. Finally, proximity ligation assay revealed a close interaction of RuvBL1 and Hsf1 proteins in the nucleus of Huh7 cells.In conclusion, RUVBL1 and HSF1 appears to coordinate the expression of several chaperone genes involved in cytosolic and mitochondrial translation. Targeting RuvBL1/2 activity impairs HSF1-mediated stress-response.

IntroductionRuvBL1 belongs to the highly conserved AAA+ ATPases. It is deregulated in various human cancers and its expression correlates with a worse prognosis in HCC patients. We previously found that RuvBL1 haploinsufficiency impairs the PI3K/Akt/mTOR pathway in liver.AimGiven the relevance of mTOR pathway hyperactivation in HCC, we hypothesized that RuvBL1 genetic targeting could reduce mTOR-driven hepatocarcinogenesis.Material and Methods ResultsPtenhep-/- and Ruvbl1hep+/- mice were crossed to generate Ptenhep-/−Ruvbl1hep+/- mice. NASH was assessed by histology at 12 weeks of age. Metabolic and inflammatory markers were evaluated by qPCR and IHC. mTOR pathway was analysed by WB of liver lysates. PPARalpha activity was evaluated by luciferase reporter assay. RuvBL1 interactome was evaluated by MS proteomics of RuvBL1-IP. HCC development was assessed by macroscopic tumour count and by histology. AML-12 PTEN KO cells were generated by CRISPR-Cas9 genome editing.Ptenhep-/−Ruvbl1hep+/- developed significantly less steatosis, fibrosis, and inflammation compared to Ptenhep-/- mice. The mTOR-driven lipogenic targets were similarly expressed in the two mice models. However, Ppara and its target CPT1 was increased in Ptenhep-/−Ruvbl1hep+/−. The spontaneous and insulin-induced accumulation of lipid droplets in PTEN KO AML-12 cells was completely abrogated by RuvBL1 inhibition with CB-6644. Inhibition of RuvBL1 activity by CB-6644 increased PPARalpha transcriptional activity in AML-12 WT and PTEN KO. Analysis of RuvBL1-IP in AML-12 revealed that RuvBL1 interacts with members of the lysosomal AMPK complex. Furthermore, p-AMPK and p-RAPTOR were increased in Ptenhep-/−Ruvbl1hep+/− compared to Ptenhep-/−mice. Finally, Ptenhep-/−Ruvbl1hep+/−mice aged to 15 months showed better survival than Ptenhep-/− which developed significantly more HCC and of higher grade. qPCR analysis showed a significant upregulation of key lipolytic genes, such as Cpt1a, Acadl, Acadvl and Ppara, in Ptenhep-/−Ruvbl1hep+/- at 15 months of age.ConclusionRuvBL1 targeting reduces mTOR hyperactivation hampering NASH-HCC progression in Ptenhep-/- mice, likely promoting the switch from mTOR-driven lipogenesis to AMPK-induced fatty acid catabolism

s of the 29 th National Congress of Digestive Diseases / Digestive and Liver Disease 55S2 (2023) S77-S209 with an increase in body mass index and waist-to-hip ratio (p<0.0001 for all).Low/high-density lipoprotein, glucose, insulin, homeostatic model assessment for insulin resistance, and transaminases worsened at T2 (insulin: p=0.0003, glucose: p=0.0007; p<0.0001 for the others).The non-invasive tools assessments revealed a NAFLD fibrosis score, liver stiffness measurement, and controlled attenuation parameter impairment at T2 (p<0.0001all).The BIA evidenced an increase in Fat Mass (FM) and a reduction of Free Fat Mass (FFM), Body Cell Mass (BCM), and Extracellular Mass (ECM) at T2 in comparison to the other study time points.During the confinement, the overall HCC and Milan-out staged HCC occurrence revealed HR:2.521, p=0.01, and HR:13.78,p=0.0009 respectively.Contrariwise to the modifications of the biochemical parameters, a significant association between body composition parameters modification during the study evaluations with HCC overall and HCC Milan-out criteria occurrence was highlighted.

RuvBL1 is a AAA+ ATPase involved in multiple cellular activities, such as cell proliferation, chromatin remodeling, DNA repair, transcription, translation and mTOR pathway activity. High RuvBL1 expression in HCC correlates with worse prognosis. We previously demonstrated that RuvBL1 is a key regulator of liver metabolism and glucose homeostasis, suggesting that this ATPase may also participate in the metabolic rewiring of HCC cells. We therefore aimed at dissecting RuvBL1 role in HCC cell metabolism. Metabolomics performed by GC/MS in RuvBL1-silenced Huh7 cells highlighted altered intermediates of glucose, TCA and aminoacid metabolism. Pathway enrichment analysis of modulated metabolites showed a significant association with processes related to cancer metabolic reprogramming and centered in mitochondria. RuvBL1 targeting by RNAi or by the selective inhibitor CB-6644 significantly impaired OXPHOS and ATP production in a dose- and time-dependent manner in AML-12, Hepa1-6, Huh7, Hep3B and HepG2 cell lines. Mitochondrial morphology assessed by Mitotracker and TEM was severely affected by RuvBL1 inhibition, that caused a reduction in matrix electrondensity, disruption of the cristae, swelling and fragmentation. Using superresolution STED microscopy and immunoglold/TEM, we detected RuvBL1 within mitochondria. Thus, we performed MS analysis of RuvBL1 co-immunoprecipitated complexes from purified mitochondria. Gene ontology analysis of mitochondrial RuvBL1-interactome revealed that this ATPase impact on TCA, aminoacid, purines, and lipid metabolism, mito-ribosome assembly, mitochondrial transmembrane transport, and membrane organization. Intriguingly, several members of the MIB complex (SAMM50, Mic19, Mic60), which plays a crucial role in shaping mitochondrial cristae, were identified in the RuvBL1-interactome. In the TCGA-LIHC dataset, RuvBL1 expression positively correlates with 9 out of 14 principal members of the MIB complex and genes differentially expressed between HCC with high- vs low-RuvBL1 were significantly enriched in mitochondria-related GO terms. Our data uncover a novel localization and function of RuvBL1 in mitochondria, and suggest that RuvBL1 overexpression support mitochondria-related processes in HCC. RuvBL1 is a AAA+ ATPase involved in multiple cellular activities, such as cell proliferation, chromatin remodeling, DNA repair, transcription, translation and mTOR pathway activity. High RuvBL1 expression in HCC correlates with worse prognosis. We previously demonstrated that RuvBL1 is a key regulator of liver metabolism and glucose homeostasis, suggesting that this ATPase may also participate in the metabolic rewiring of HCC cells. We therefore aimed at dissecting RuvBL1 role in HCC cell metabolism. Metabolomics performed by GC/MS in RuvBL1-silenced Huh7 cells highlighted altered intermediates of glucose, TCA and aminoacid metabolism. Pathway enrichment analysis of modulated metabolites showed a significant association with processes related to cancer metabolic reprogramming and centered in mitochondria. RuvBL1 targeting by RNAi or by the selective inhibitor CB-6644 significantly impaired OXPHOS and ATP production in a dose- and time-dependent manner in AML-12, Hepa1-6, Huh7, Hep3B and HepG2 cell lines. Mitochondrial morphology assessed by Mitotracker and TEM was severely affected by RuvBL1 inhibition, that caused a reduction in matrix electrondensity, disruption of the cristae, swelling and fragmentation. Using superresolution STED microscopy and immunoglold/TEM, we detected RuvBL1 within mitochondria. Thus, we performed MS analysis of RuvBL1 co-immunoprecipitated complexes from purified mitochondria. Gene ontology analysis of mitochondrial RuvBL1-interactome revealed that this ATPase impact on TCA, aminoacid, purines, and lipid metabolism, mito-ribosome assembly, mitochondrial transmembrane transport, and membrane organization. Intriguingly, several members of the MIB complex (SAMM50, Mic19, Mic60), which plays a crucial role in shaping mitochondrial cristae, were identified in the RuvBL1-interactome. In the TCGA-LIHC dataset, RuvBL1 expression positively correlates with 9 out of 14 principal members of the MIB complex and genes differentially expressed between HCC with high- vs low-RuvBL1 were significantly enriched in mitochondria-related GO terms. Our data uncover a novel localization and function of RuvBL1 in mitochondria, and suggest that RuvBL1 overexpression support mitochondria-related processes in HCC.

Background and Aims RuvBL1 is a highly conserved AAA+ ATPases. It is deregulated in various human cancers and its expression correlates with a worse prognosis in HCC patients. We previously found that liver haploinsufficiency of RuvBL1 impairs the PI3K/Akt/mTOR pathway. We thus hypothesized that genetic targeting of RuvBL1 could reduce mTOR driven hepatocarcinogenesis. Method Ptenhep-/- and Ruvbl1hep+/- mice were crossed to generate Ptenhep-/−Ruvbl1hep+/- mice. NASH was assessed by histology at 12 weeks of age. Metabolic and inflammatory markers were evaluated by qPCR and IHC. mTOR pathway was analysed by WB of liver lysates. PPARalpha activity was evaluated by luciferase reporter assay. RuvBL1 interactome was evaluated by MS proteomics of RuvBL1-IP. HCC development was assessed by macroscopic tumour count and by histology. AML-12 PTEN KO cells were generated by CRISPR-Cas9 genome editing. Results Ptenhep-/−Ruvbl1hep+/- developed significantly less steatosis, fibrosis, and inflammation compared to Ptenhep-/- mice. The mTOR-driven lipogenic targets were similarly expressed in the two mice models. However, Ppara and its target CPT1 was increased in Ptenhep-/−Ruvbl1hep+/−. Inhibition of RuvBL1 activity by CB-6644 increased PPARalpha transcriptional activity in AML-12 hepatocytic cell line. Analysis of RuvBL1-IP in AML-12 and Hepa1-6 cells revealed that RuvBL1 interacts with members of the lysosomal AMPK complex. Furthermore, p-AMPK and p-RAPTOR were increased in Ptenhep-/−Ruvbl1hep+/− compared to Ptenhep-/−mice. The spontaneous and insulin-induced accumulation of lipid droplets in PTEN KO AML-12 cells was completely abrogated by RuvBL1 inhibition with CB-6644. Finally, Ptenhep-/−Ruvbl1hep+/−mice aged to 15 months showed better survival than Ptenhep-/− which developed significantly more HCC and of higher grade. qPCR analysis showed a significant upregulation of key lipolytic genes, such as Cpt1a, Acadl, Acadvl and Ppara, in Ptenhep-/−Ruvbl1hep+/- at 15 months of age. Conclusion RuvBL1 targeting reduces mTOR hyperactivation hampering NASH-HCC progression in Ptenhep-/- mice, likely promoting the switch from mTOR-driven lipogenesis to AMPK-induced fatty acid catabolism. RuvBL1 is a highly conserved AAA+ ATPases. It is deregulated in various human cancers and its expression correlates with a worse prognosis in HCC patients. We previously found that liver haploinsufficiency of RuvBL1 impairs the PI3K/Akt/mTOR pathway. We thus hypothesized that genetic targeting of RuvBL1 could reduce mTOR driven hepatocarcinogenesis. Ptenhep-/- and Ruvbl1hep+/- mice were crossed to generate Ptenhep-/−Ruvbl1hep+/- mice. NASH was assessed by histology at 12 weeks of age. Metabolic and inflammatory markers were evaluated by qPCR and IHC. mTOR pathway was analysed by WB of liver lysates. PPARalpha activity was evaluated by luciferase reporter assay. RuvBL1 interactome was evaluated by MS proteomics of RuvBL1-IP. HCC development was assessed by macroscopic tumour count and by histology. AML-12 PTEN KO cells were generated by CRISPR-Cas9 genome editing. Ptenhep-/−Ruvbl1hep+/- developed significantly less steatosis, fibrosis, and inflammation compared to Ptenhep-/- mice. The mTOR-driven lipogenic targets were similarly expressed in the two mice models. However, Ppara and its target CPT1 was increased in Ptenhep-/−Ruvbl1hep+/−. Inhibition of RuvBL1 activity by CB-6644 increased PPARalpha transcriptional activity in AML-12 hepatocytic cell line. Analysis of RuvBL1-IP in AML-12 and Hepa1-6 cells revealed that RuvBL1 interacts with members of the lysosomal AMPK complex. Furthermore, p-AMPK and p-RAPTOR were increased in Ptenhep-/−Ruvbl1hep+/− compared to Ptenhep-/−mice. The spontaneous and insulin-induced accumulation of lipid droplets in PTEN KO AML-12 cells was completely abrogated by RuvBL1 inhibition with CB-6644. Finally, Ptenhep-/−Ruvbl1hep+/−mice aged to 15 months showed better survival than Ptenhep-/− which developed significantly more HCC and of higher grade. qPCR analysis showed a significant upregulation of key lipolytic genes, such as Cpt1a, Acadl, Acadvl and Ppara, in Ptenhep-/−Ruvbl1hep+/- at 15 months of age. RuvBL1 targeting reduces mTOR hyperactivation hampering NASH-HCC progression in Ptenhep-/- mice, likely promoting the switch from mTOR-driven lipogenesis to AMPK-induced fatty acid catabolism.

Hepatocellular carcinoma (HCC) is currently a major challenge in medicine for its poor prognosis and lack of effective therapeutic options. RuvBL1 is a AAA+ ATPase whose expression correlates with a worse prognosis in HCC patients. It associates with several multiprotein complexes regulating key cellular processes such as cell proliferation, chromatin remodeling, DNA repair and mTOR pathway activity. We have previously demonstrated that RuvBL1 haploinsufficiency impairs liver metabolism and glucose homeostasis suggesting that RuvBL1 could be involved in the metabolic rewiring of HCC.

Introduction: HCC is the second cause of cancer-related death worldwide. In the last years, the role of nuclear receptors in hepatocarcinogenesis has received great attention. COUP-TF2 regulates important biological processes and. studies indicate that is a pro-oncogenic factor but its role in HCC is still controversial.

Hepatocellular carcinoma (HCC) is currently a major challenge in medicine for its poor prognosis and lack of effective therapeutic options. The AAA+ ATPase RuvBL1 associates with Hsp90 in several multiprotein complexes regulating key cellular pathways such as cell proliferation, gene expression, chromatin remodeling and telomere maintenance. In several human cancer, including HCC, RuvBL1 overexpression correlates with a poor prognosis. Despite the several functions potentially regulated by RuvBL1, its role in the onset and progression of HCC is still unknown. We had previously generated a RuvBL1 hepatocyte-specific knock-out mouse model and observed by TEM major structural alterations in mitochondria of hepatocytes. Moreover, by a 2D-proteomic screening, we found that several key mitochondrial proteins and Hsp90β were significantly reduced in RuvBL1-silenced hepatoma cells in vitro.