Micro-RNAs (miRNAs) are small non-coding RNAs mediating post-transcriptional gene silencing. We conducted a prospective study to identify circulating miRNAs associated with treatment response in patients with ulcerative colitis (UC) undergoing infliximab therapy. Patients with moderate-to-severe UC starting intravenous infliximab were included and classified after 12 months as “responders” (achieving combined clinical-endoscopic remission, i.e. Mayo endoscopic subscore=0 and absence of symptoms) or “non-responders”. Plasma samples were collected at baseline and post-induction. Clinical and endoscopic assessments were performed at baseline and at 12 months or earlier in case of treatment failure. Circulating miRNAs were analysed using the Nanostring Human v3 miRNA Assay. Differential expression analysis, target genes identification, and receiver operating characteristic (ROC) analyses were performed. Twelve patients were included in this study: 6 responders, 6 non-responders (Table 1). Distinct miRNA expression patterns emerged when comparing the two groups. At baseline (Figure 1A), target genes of upregulated miRNAs in responders were mostly involved in immune regulation pathways, particularly T-cell differentiation and complement activation. In contrast, targets of downregulated miRNAs were associated with TGF-β response, epithelial-mesenchymal transition (EMT), epithelial development, and fibroblast proliferation. After induction (Figure 1B), enrichment analysis of target genes of upregulated miRNAs revealed modulation of pathways related to epithelial and T-cell differentiation. Consistent with baseline findings, targets of downregulated miRNAs in responders were significantly enriched in pathways involved in epithelial development, EMT, TGF-β receptor signaling, and hypoxia-associated pathways. Among the evaluated candidates, the best predictors of combined remission were miRNAs of the miR-200 family. At baseline, miR-200b-3p showed a trend towards significance [area under the ROC curve (AUC)=0.833, p = 0.065], while after induction, miR-200a-3p was the most accurate and significant predictor of 12-month combined remission (AUC=0.933, p = 0.017) (Figure 1C-D). Both miRNAs belong to the TGF-β-related regulatory network, consistent with the recurrent involvement of epithelial development and TGF-β signaling across time points. Our pilot study suggests that specific circulating miRNA signatures may serve as early biomarkers of treatment response in UC. Notably, miR-200a-3p emerged as a promising candidate, demonstrating strong predictive performance for 12-month combined remission when assessed at the end of infliximab induction. These preliminary findings warrant validation in larger, prospective cohorts. Conflict of interest: Dr. Innocenti, Tommaso: Speaker fees from Aurora Biofarma, and Ferring. Travel grants from Abbvie, Alfasigma, Celltrion, Eli Lilly, Ferring, Malesci, Pfizer. Pillozzi, Serena: No conflict of interest Scolari, Federico: No conflict of interest Menicacci, Beatrice: No conflict of interest Iamello, Rocco Gabriele: No conflict of interest Picariello, Lucia: No conflict of interest Curto, Armando: No conflict of interest Ceni, Elisabetta: No conflict of interest Bagnoli, Siro: No conflict of interest Polvani, Simone: No conflict of interest Mello, Tommaso: No conflict of interest Milani, Stefano: No conflict of interest Galli, Andrea: No conflict of interest Dragoni, Gabriele: Grant: ECCO Grant 2020 ECCO/AOCC Travel Grant 2021 ECCO IIS Registry Grant 2023 ECCO/IBUS Research Grant 2023 Personal Fees: - Speaker’s fees from: 2020: Novartis 2022: Janssen 2023: Alfasigma, Janssen, Pfizer, and Takeda 2024: Ferring, Johnson & Johnson, Eli Lilly, Pfizer, and Takeda 2025: Abbvie, Alfasigma, Ferring, Eli Lilly, LionHealth, Pfizer, Takeda - Advisory board fees from: 2023: Celltrion Healthcare and Pfizer 2024: AbbVie 2025: AbbVie, Johnson & Johnson

Background & Aims: The AAA+ ATPase RuvBL1 takes part in several biological processes, including chromatin remodelling and DNA repair, ribosome biogenesis, mTOR signalling, and oncogenic transformation. RUVBL1 overexpression correlates with poor survival in patients with hepatocellular carcinoma (HCC). We previously found that RuvBL1 is a key regulator of liver glucose metabolism in mice. Here, we aimed at disentangling the metabolic function of RuvBL1 in HCC cells. Methods: Non-transformed AML-12, primary mouse hepatocytes, HCC cell lines, and RuvBL1hep-/- mice were used (n = 3). RuvBL1 was targeted by RNAi and by inhibition with CB-6644. Metabolomic profiling and mitochondrial functions were assessed by targeted GC/MS, Seahorse analysis, and ATP synthase activity. Mitochondrial morphology and membrane potential were investigated by fluorescence microscopy, high-content imaging, and transmission electron microscopy. Mitochondrial RuvBL1 was detected by WB, super-resolution microscopy, transmission electron microscopy, and proximity ligation assay. Human HCC and normal liver samples from The Cancer Genome Atlas and GTEx databases were used for in-silico analysis (T = 369, N = 160). Results: Targeting RuvBL1 impairs mitochondria-centred metabolic processes, including amino acid metabolism, TCA cycle, and oxidative phosphorylation. Inhibition of RuvBL1/2 activity induces loss of cristae integrity, mitochondrial hyperpolarisation and fragmentation, a phenotype paralleled by the hepatocytes of RuvBL1hep-/- mice. We detected RuvBL1 in proximity to mitochondrial ATP synthase, a previously unreported localisation for this protein. Mechanistically, CB-6644 reduces ATP synthase-RuvBL1 interaction and impairs complex V activity even under a fuelled TCA cycle. In human HCC, higher RUVBL1 expression correlates with gene signatures associated with mitochondrial oxidative phosphorylation (FDR = 5.64e-03), ATP synthase complex (FDR = 6.03e-03), and poorer outcome (p = 2e-07). Conclusions: Targeting RuvBL1 impairs complex V activity, disrupting mitochondrial metabolic functions and structural integrity. The mitochondrial functions of RuvBL1 may inform novel therapeutic strategies in the fight against hepatocellular carcinoma. Impact and implications: Metabolic reprogramming is a key feature driving HCC onset, progression, and plasticity, contributing to treatment resistance and poor prognosis. RUVBL1 overexpression correlates with reduced survival of patients with HCC and has emerged as a potential metabolic modulator. In this study, we found that targeting RuvBL1 impairs its interaction with mitochondrial ATP synthase, disrupting mitochondrial metabolism and cristae structure. In human HCC samples, RUVBL1 expression correlates with hallmark mitochondrial metabolic processes. These findings may inform the development of targeted therapeutic approaches aimed at impairing the metabolic rewiring and plasticity of HCC.

Colorectal cancer (CRC) is the third most common cancer and the second cause of cancer death in the world. Emerging evidence suggests that the short-chain-fatty-acid butyrate diet-assumed or produced by gut microbiota may interfere with CRC. Novel, more focused and effective anti-cancer natural molecules selectively acting on tumour cells are required to improve patients' compliance compared to more aggressive drug-based schemes. This study explored the in vitro anti-cancer effects of a novel green compound consisting of butyrate-glycerides (BMDG) alone or absorbed on tailor-made Biochar (BMDG-Biochar) or on activated-carbon Norit-B (BMDG-Norit), by using two CRC cell lines, HCT116 and HT29. Tailor-made Biochar characterised by a larger share of meso and macroporosity compared to commercially available activated-carbon Norit-B, with micro-pored ultrastructure, displayed superior performances as a BMDG carrier, with higher absorption/release properties. BMDG, in particular when absorbed on Biochar, interfered significantly with CRC cell proliferation compared to BMDG-Norit that showed no effect. Analysis of cell metabolism revealed a superior sensitivity of HCT116 to the inhibitory effect of BMDG-Biochar. This compound specifically induced a shift from a glycolytic metabolism in particular in HCT116 cells where glycolysis supports the aggressive phenotype, towards the mitochondrial respiration that characterises the more differentiated and less aggressive HT29 cells. Biochar's ability to deliver the butyrate-glyceride bioactive mixture and to exert in vitro combined anti-cancer activity in colorectal cancer, interfering with the Warburg effect that characterises the aggressive CRC forms, opens future translational opportunities to develop new orally assumed green molecules as promising anti-cancer strategies for CRC.

Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer-related deaths due to its late diagnosis, limited treatment options, and early metastasis, resulting in a five-year survival rate of less than 10 %. Central to PDAC development are mutations in the KRAS gene, which drive tumor growth through persistent activation of signaling pathways. The microbiota, comprising bacteria, viruses, and fungi, plays a vital role in human health by aiding digestion, supporting immune function, and maintaining microbial balance. Dysbiosis, an imbalance in microbial communities, is linked to various diseases. Recent research highlights the complex interaction between the pancreas and gut microbiota, with evidence suggesting that microbiota influences pancreatic functions, including enzyme secretion and glucose regulation. This review focuses on the role of microbiome dysbiosis in PDAC, influencing tumorigenesis, immune response, and treatment outcomes through complex interactions with genetic mutations and metabolic pathways, and on the role of the microbiota in influencing KRAS-driven tumorigenesis, where dysbiosis may promote tumor progression. This review also aims to emphasize how the gut microbiome has been implicated in modulating the response to immune checkpoint inhibitors (ICIs) across various cancers, with specific bacterial strains linked to improved outcomes. Recent studies suggest that microbial manipulation, such as bacterial ablation, can enhance anti-tumor immunity in PDAC models by altering the tumor microenvironment (TME). The TME remains a major barrier, and ongoing research is exploring new combinations and targets to overcome resistance and improve the efficacy of ICIs in PDAC. Further research is needed to fully understand this relationship.

Tertiary lymphoid structures (TLSs) have been associated with favorable clinical outcome and improved responses to immune checkpoint inhibitors in soft-tissue sarcomas (STSs). However, due to their rarity and high heterogeneity, information regarding the mechanisms involved in TLS formation and contribution to antitumor immunity in STS are extremely elusive. To address this gap, we integrated immunohistochemistry and transcriptomic analyses from 31 treatment-naïve STS specimens from an independent cohort of patients with primary or locally recurrent disease. Further validation was conducted using external bulk, single-cell and spatial transcriptomics data from 5 publicly available datasets. We found TLSs to be highly heterogeneous in terms of amount, localization, maturation status and cellular composition, and corroborated previous findings showing that their presence, along with B cells in the STS microenvironment, are good indicators of favorable prognosis. Transcriptomic analysis showed that high expression of germinal center (GC) B cell-related genes was associated with TLS presence and with an upregulation of signatures specific for T helper 17 (Th17) cells in STS and other cancer types. Conversely, genes signatures discriminating for immunosuppressive M2-like macrophages were enriched in tumors with low expression of GC B cell-related genes. Immunohistochemistry showed distinct spatial patterns for Th17-like cells and M2-like macrophages within TLS areas, with IL17A+ cells predominantly localized within intratumoral mature TLSs, and CD163+ macrophages mainly observed in immature TLSs. Integrating these findings, we identified tumors with high expression of GC B cell- and Th17 cell-related signatures together with low fractions of M2-like macrophages, to have superior survival outcomes. Herein, our findings point out to two cellular players (Th17-like cells and M2-like macrophages) with potentially opposite roles in STS-associated TLS formation and maturation, thus providing the basis for future research efforts aiming at the development of therapeutic immunological interventions to enhance TLS-mediated antitumor immunity in STS.

Hyponatremia is the most common electrolyte disorder in cancer patients and is associated with a worse outcome. This finding has also been reported in patients with metastatic renal cell carcinoma (mRCC). We have previously demonstrated that low extracellular sodium concentrations ([Na+]) increase cell proliferation and invasion in several human cancer cell lines. The aim of the present study was to evaluate in vitro the effects of mild [Na+] alterations on two mRCC cell lines, ACHN and Caki-1. After growth in reduced extracellular [Na+], we observed that even mild reductions of [Na+] significantly enhanced different key cancer cell features, including proliferation, invasion and migration. Furthermore, we observed a reduction in reactive oxygen species (ROS) levels in low [Na+], with a significant increase of the antioxidant Nrf2/HMOX-1 pathway. Since an excess of ROS causes cell death, this finding clarifies the role of Nrf2/HMOX-1 in maintaining the balance between oxidant and antioxidant species in the tumor environment, promoting cell survival. Although further clinical studies are needed, aiming for instance to determine whether serum [Na+] correction improves the outcome of patients with mRCC, our findings suggest that attention should be deserved to serum [Na+] in this setting.

The incidence and mortality of pancreatic adenocarcinoma (PC) are expected to increase in the coming years, with survival rates remaining poor due to limited treatment options. KRAS mutations, present in over 70% of PC cases, drive aggressive tumor behavior through metabolic reprogramming and immune evasion; however, clinically effective inhibitors for the most common mutations are still lacking. In this study, we analyzed RNA sequencing data from TCGA datasets, comparing tumor versus normal pancreatic tissues and stratifying samples based on KRAS mutation status. Our findings reveal significant dysregulation of the peroxisome proliferator-activated receptor (PPAR) signaling pathway in PC, particularly in the context of KRAS mutations. These findings were validated through RT-qPCR in an independent cohort of primary samples. Key genes, including CD36, FABP4, PLIN1, PLIN4, SCD5, and ACSLs, were consistently downregulated in tumor tissues, with further reductions observed in KRAS-mutated samples. Overall, this study highlights the critical role of PPAR pathway disruption in KRAS-mutated PC, which should be further addressed to improve current treatment strategies.

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.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

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.

Background and Aims During early phases of inflammation, activated neutrophils extrude neutrophil extracellular traps (NETs) in a peptidyl arginine deiminase 4 (PAD4)-dependent manner, aggravating tissue injury and remodeling. In this study, we investigated the potential pro-fibrotic properties and signaling of NETs in Crohn's disease (CD).Methods NETs and activated fibroblasts were labeled on resected ileum from CD patients by multiplex immunofluorescence staining. NETs-treated human primary intestinal fibroblasts were analyzed by bulk RNA sequencing to uncover cell signaling pathways, and by high-throughput imaging to assess collagen production and migratory activity. Consequentially, TLR2/NF-kappa B pathway was evaluated by transfection of CCD-18Co fibroblasts with an NF-kappa B-luciferase reporter plasmid, incorporating C29 to block TLR2 signaling. A chronic dextran sulfate sodium (DSS) mouse model was used to define the specific role of PAD4 deletion in neutrophils (MRP8-Cre, Pad4fl/fl).Results Immunofluorescence showed spatial colocalization of NETs and activated fibroblasts in ileal ulcerations of CD patients. Transcriptomic analysis revealed upregulation of pro-fibrotic genes and activation of Toll-like receptor signaling pathways in NETs-treated fibroblasts. NETs treatment induced fibroblast proliferation, diminished migratory capability, and increased collagen release. Transfection experiments indicated a substantial increase in an NF-kappa B expression with NETs, whereas C29 led to decreased expression and release of collagen. In line, a significant reduction in collagen content was observed in the colon of MRP8-Cre, Pad4fl/fl mice subjected to chronic DSS colitis.Conclusions NETs potentially serve as an initial stimulus for pathological activation of fibroblasts within the intestine via the TLR2/NF-kappa B pathway. Given their early involvement in inflammation, inhibition of PAD4 might offer a strategy to modulate both inflammation and fibrogenesis in CD.

We have previously demonstrated that the vasopressin type 2 receptor (AVPR2) antagonist tolvaptan reduces cell proliferation and invasion and triggers apoptosis in different human cancer cell lines. To study this effect in vivo, a xenograft model of small cell lung cancer was developed in Fox1nu/nu nude mice through the subcutaneous inoculation of H69 cells, which express AVPR2. One group of mice (n = 5) was treated with tolvaptan for 60 days, whereas one group (n = 5) served as the control. A reduced growth was observed in the tolvaptan group in which the mean tumor volume was significantly smaller on day 60 compared to the control group. In the latter group, a significantly lower survival was observed. The analysis of excised tumors revealed that tolvaptan effectively inhibited the cAMP/PKA and PI3K/AKT signaling pathways. The expression of the proliferative marker proliferating cell nuclear antigen (PCNA) was significantly lower in tumors excised from tolvaptan-treated mice, whereas the expression levels of the apoptotic marker caspase-3 were higher than those in control animals. Furthermore, tumor vascularization was significantly lower in the tolvaptan group. Overall, these findings suggest that tolvaptan counteracts tumor progression in vivo and, if confirmed, might indicate a possible role of this molecule as an adjuvant in anticancer strategies.