Gastric cancer ranks among the most prevalent malignancies globally and remains a major contributor to cancerrelated mortality. Its progression is profoundly shaped by the tumor microenvironment (TME), a dynamic milieu comprising extracellular matrix components, soluble mediators, and diverse non-malignant immune populations that collectively influence tumor initiation and evolution. Although immunotherapeutic strategies have recently attracted increasing attention in the management of solid tumors, including gastric carcinoma, the determinants of immune evasion in this context are still not fully delineated. The Farnesoid X receptor (FXR), a key nuclear receptor for bile acids, has recently been implicated in regulating TME composition and immune cell recruitment within gastric tumors. Yet, how bile acid signaling contributes to the bidirectional interactions between malignant and immune cells remains largely undefined. This study aimed to investigate the role of FXR in modulating immune-regulatory networks associated with PD-L1/PD-1 in gastric cancer. RNA-seq analysis of paired gastric mucosa from 39 gastric cancer patients revealed significant molecular differences between intestinal and diffuse tumors. FXR was overexpressed in the intestinal subtype and correlated with poor prognosis. MS/MS analysis demonstrated enrichment of tumor tissues by cholic acid and chenodeoxycholic acid compared to non-neoplastic pairs, supporting the hypothesis of dysregulated bile acid metabolism. In vitro, exposure of gastric cancer cell lines and patient-derived organoids to FXR ligands increased PD-L1 expression through direct binding of FXRresponsive elements in the PD-L1 promoter. Targeting FXR may offer a therapeutic approach to improve the effectiveness of immunotherapies in intestinal gastric cancer.

INTRODUCTION:Gastric cancer (GC) is a major clinical challenge, characterized by limited response rates to immune checkpoint inhibitors (ICIs) and persistent immune evasion. Leukemia Inhibitory Factor (LIF), an IL-6 family cytokine, reshapes the tumor microenvironment, yet its contribution to PD-L1-mediated immune suppression in GC has not been investigated. MATERIAL AND METHODS:LIF and PD-L1 expression were quantified in resected GC specimens and matched mucosa by immunohistochemistry and gene expression (Log2), and their associations with clinicopathological variables and survival were evaluated. GC cell lines were exposed to recombinant LIF and to anovel LIF antagonist, LRI-305. Activation of the JAK1/STAT3 pathway, PD-L1 transcription and protein and epithelial-mesenchymal transition (EMT) markers were analyzed. By t-SNE analysis we profiled LIF⁺/PD-L1⁺ cell subsets across myeloid and non-haematopoietic compartments, and by functional assays we have assessed whether LIF blockade modulates T cell activation. RESULTS:LIF expression was significantly elevated in GC tissues and correlates with poor prognosis and increased PD-L1 levels. LIF promotes immune escape by activating the JAK1/STAT3 pathway, leading to transcriptional upregulation of PD-L1 and enhancement of EMT. The t-SNE analysis revealed that LIF⁺/PD-L1⁺ myeloid and non-hematopoietic cells were enriched in the neoplastic mucosa. Pharmacological blockade of LIF signaling effectively suppressed STAT3 phosphorylation and downregulated PD-L1 expression. LRI-305 treatment partially restored immune activation signatures, supporting its potential as a therapeutic adjuvant to ICIs. DISCUSSION:LIF/STAT3 enhances PD-L1 expression and participate to GC immune evasion. Targeting LIF signaling could be a strategy to overcome resistance to immunotherapy.

Endoplasmic reticulum (ER) stress represents a critical pathophysiological condition that plays a central role in the development of various human diseases, including protein misfolding diseases. While the small molecule Vx-445 (Elexacaftor) exhibits robust cellular bioactivity, its cryptic intracellular targets and off-label mechanisms of action remain poorly defined. This study investigates the cytoprotective efficacy and molecular targets of Vx-445 in Thapsigargin-induced ER stress in a neuronal cell model. Integrating biochemical assays, gene expression proteomics, and label-free functional proteomics, we demonstrate that Vx-445 significantly mitigates oxidative stress by reducing intracellular levels of reactive oxygen species, restores calcium homeostasis to baseline levels, and prevents apoptosis by inhibiting cytochrome c release. These phenotypic modifications correlated with changes in proteomic expression and were validated by Drug Affinity Responsive Target Stability (DARTS) analysis to map restored cellular pathways and identify potential protein interaction partners. Together, these findings uncover alternative molecular targets for Vx-445, providing a mechanistic basis for drug repurposing strategies in endoplasmic reticulum stress-related diseases.

Intestinal fibrosis is a severe complication of Crohn's disease for which therapy remains suboptimal. Probiotics are widely used in the treatment of intestinal inflammation, but all major guidelines do not recommend in favor of their use, with the exception of an 8-strains bacterial formula, which is recommended for the treatment of pouch inflammation in ulcerative colitis. Using this 8-strains formulation as a comparator, we characterized a 9-strains probiotic formulation enriched with Lactobacillus rhamnosus and paracasei in a mouse model of intestinal inflammation and fibrosis. Our findings demonstrated that while both formulations exerted similar protective effects in acute colitis, only the 9-strains probiotic attenuates inflammation and fibrosis in chronic colitis. Mechanistically, we found that the 9-strains formulation remodeled the microbiota composition and the structure of microbiota-derived secondary bile acids, leading to the selective enrichment of those bile acids that act as GPBAR1 agonists, including 3-oxo-DCA, which in vitro directly attenuates activation of intestinal fibroblasts. Confirming the role of this pathway, feeding Gpbar1⁻/⁻ mice with 9-strains probiotic formulation abrogates its beneficial effects on inflammation and fibrosis. These findings highlight the importance of microbial metabolites in shaping probiotic efficacy and support the development of probiotic formulations that target host-microbiota interactions through bile acid signaling.

This review examines the structure and the functional role of the signaling network formed by bile acids as their receptors in mediating chemical communication between the intestinal microbiota and host in entero-hepatic disorders, such as inflammatory bowel disease (IBD) and Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD). In the last decade, advances in analytical technologies and bioinformatic approaches, including metabolomics and reverse metabolomics, have greatly expanded our understanding of bile acid metabolism. These studies have shown that the intestinal microbiota transforms primary bile acids synthesized in the liver from cholesterol into a highly diverse family of bioactive mediators. Hundred of “classical” secondary bile acids and Microbial Derived Bile Acids (MDBA) have been identified and characterized for their role in regulating the activity of widely expressed receptors, including the Farnesoid-x-receptor (FXR), the G protein-coupled bile acid receptor (GPBAR1) and the retinoid-related orphan receptor (RORγ). Simultaneous characterization of microbial species expressing bile acid-related genes is emerging as a valuable tool for disease phenotyping. One example is bile salt hydrolase (BSH), an enzyme that catalyzes bile acid deconjugation. In parallel, profiling bile acid composition in biological compartments such as feces and blood may help identify associations between microbiota dysbiosis and diseases including IBD and metabolic dysfunction-associated steatohepatitis (MASH). The review also examines therapeutic approaches that could be developed to exploit this novel conceptual framework.

Quinazolinones have been recently recognized as valuable scaffolds for developing novel therapeutic opportunities. They indeed exhibit structural versatility and a wide range of biological activities, including antifungal, antitubercular, antihypertensive, anticancer, and antiviral ones. In this work, a focused library of new bioactive 4-(3-H)-quinazolinones has been synthesized, their cytotoxic action against DU-145 prostate cancer cells has been detailed, and compound 4k has been revealed as the most active one. Consequently, its interactome has been characterized by a label-free functional proteomics-based platform coupling drug affinity responsive target stability (DARTS) and targeted limited proteolysis-multiple reaction monitoring-mass spectrometry (t-LiP-MRM-MS). This multifaced strategy has been employed to reveal few subunits of the 26S proteasome machinery as the most reliable compound 4k biological targets. This paved the way for the deepening of the protein-ligand interaction using in vitro and in silico bio-orthogonal techniques. Finally, the analysis of its function in living DU-145 cells prompted compound 4k as a novel quinazolinone-bearing inhibitor of the chymotrypsin-like activity of the proteasomal β-5 subunit, stirring this framework for the development of new anticancer drugs.

Extracellular matrix remodelling that occurs in pancreatic ductal adenocarcinoma (PDAC) is considered a promoting factor of cancer growth, immune evasion and therapeutic resistance. Cancer-associated fibroblasts (CAFs) that constitute the dominant stromal population, arise primarily from activated pancreatic stellate cells and display remarkable functional heterogeneity, encompassing inflammatory iCAFs and contractile myCAFs. Although epithelial-stromal communication is central to PDAC biology, the upstream mechanisms that prime tumour cells toward CAF-Activating cells remain incompletely defined. The leukaemia inhibitory factor (LIF), a pleiotropic cytokine of the IL-6 family, is highly expressed in PDAC and has been implicated in tumour progression. However, the role of LIF and LIF receptor (LIFR):gp130 complex in promoting CAF activation is poorly defined. Here, we combined human PDAC transcriptomics, immunofluorescence and epithelial-stromal co-culture assays to define LIF-driven pro-CAF programs and evaluate their pharmacological reversibility. In PDAC cancer cells, MIAPaCa-2 cells, LIF induced a coordinated transcriptional network encompassing inflammatory mediators, paracrine fibroblast-activating signals and ECM/mechanotransductive modules, while repressing stromal-inhibitory genes. These signatures were recapitulated in PDAC tissues, where LIF expression directly correlated with CAF markers and with stromal remodelling genes. On this background, we have developed a novel steroidal LIFR antagonist, LRI310, and evaluate its effects on LIF:LIFR axis. Exposure of PDCA cell lines to LRI310 suppresses STAT3 activation and counteracts effects of LIF on proliferation and CAF-inducing transcriptional programs. Collectively, these findings identify LIF as an important epithelial driver of CAF-oriented transcriptional programs in PDAC and support the development of LIFR antagonism as a promising strategy to modulate the desmoplastic microenvironment

This review delves into the extremely fascinating topic of protein target identification of bioactive marine natural products, with the aim of addressing proteomic and mass spectrometry–based approaches. Specifically, it examines both strategies in which marine compounds are covalently conjugated to a linker to enable the enrichment and identification of interacting proteins from complex mixtures, as well as label-free approaches in which compound–protein interactions are inferred from ligand-induced conformational changes. In addition, protein targets identified through activity-based protein profiling and photoaffinity labeling strategies are discussed. Finally, the molecular mechanisms of action of selected marine metabolites bearing reactive functional groups, as elucidated by mass spectrometry, are described in detail.

Cancer cachexia is a multifactorial metabolic syndrome characterized by progressive skeletal muscle and adipose tissue loss, systemic inflammation, and poor clinical outcomes, and represents a major unmet clinical need in gastric cancer. Growth Differentiation Factor 15 (GDF15) is a key mediator of cachexia-associated anorexia and tissue wasting; however, the upstream mechanisms regulating its expression in gastric cancer remain poorly defined. Leukemia Inhibitory Factor (LIF), a pleiotropic cytokine implicated in tumor progression and metabolic dysregulation, has emerged as a potential regulator of cachexia-related pathways. Here, we investigated the association between LIF in regulating GDF15 expression and its relationship with metabolic, inflammatory, and body composition alterations in gastric cancer. Transcriptomic profiling of paired neoplastic and non-neoplastic gastric mucosa from 61 gastric cancer patients revealed a significant upregulation of both LIF and GDF15 in tumor tissue, with a strong positive correlation between their expression levels. High GDF15 expression was associated with reduced overall survival, a finding validated in independent TCGA-STAD and ACRG cohorts. Intratumoral bile acid profiling uncovered a marked enrichment of primary bile acids and a depletion of secondary bile acids, resulting in reduced levels of bile acids with endogenous LIF receptor (LIFR) antagonist activity; elevated primary, LIFR non-antagonist bile acids were associated with worse survival outcomes. Clinically, increased LIF and GDF15 expression correlated with weight loss, heightened inflammatory burden, reduced serum protein and albumin levels, and impaired body composition in a sub-cohort of 19 patients. Notably, LIF expression showed a significant inverse association with both lumbar skeletal muscle index (L3SMI) and subcutaneous adipose tissue index (SATI). Mechanistically, experimental models demonstrated that LIF enhances proliferative activity in gastric cancer spheroids and exerts paracrine effects that impair myogenic differentiation and suppress hepatic metabolic gene expression. Collectively, these findings identify the LIF/GDF15 axis as a central driver of cancer-associated cachexia in gastric cancer and highlight LIF signaling as a potential therapeutic target.

Introduction:Psoriasis is a chronic inflammatory skin condition driven by activated epidermal keratinocytes, dermal fibroblasts, and immune cell infiltrates, which causes tissue injury. The ecto-5'-nucleotidase CD73/adenosine pathway plays a critical role in controlling inflammatory/immune responses, yet it is dysregulated in psoriasis patients. However, the expression and function of this pathway in psoriasis remain poorly explored. Methods:In this study, we investigated the regulation of CD73 in keratinocytes and examined the anti-inflammatory effects of adenosine in keratinocytes and dermal fibroblasts under psoriatic-like conditions. HaCaT cells and primary normal human epidermal keratinocytes (NHEK) were stimulated with the M5 cytokine cocktail, comprising interleukin (IL)-1α, IL-17A, IL-22, oncostatin M (OSM), and tumor necrosis factor (TNF)-α, to induce a proinflammatory phenotype. Results:M5-treated keratinocytes release IL-1β, IL-6, and IL-8, as well as the antimicrobial peptide S100A9, and exhibit activation of the signal transducer and activator of transcription (STAT), nuclear factor-kappa B (NF-κB), mitogen-activated protein kinase (MAPK), and phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) pathways. We demonstrated that CD73 is upregulated in inflamed keratinocytes, with OSM identified as a regulator of CD73 expression in a Janus kinase/MAPK-dependent manner. High CD73 expression in inflamed keratinocytes is associated with increased adenosine production. In M5-stimulated keratinocytes, adenosine A2A receptors (A2AR) expression is increased, whereas A2BR expression is decreased. Functional analyses revealed that an A2AR agonist, and to a lesser extent an A2BR agonist, reduced IL-8 levels in inflamed keratinocytes. Similarly, M5-treated dermal fibroblasts released IL-1β, IL-6, and IL-8, and exhibited activation of inflammatory signaling pathways. In inflamed dermal fibroblasts, both A2AR and A2BR were upregulated, and IL-8 release was mitigated by an A2AR agonist. Discussion:In conclusion, these results provide new insights into the mechanisms by which the CD73/adenosine axis can be modulated in psoriatic conditions and may guide the development of effective strategies to mitigate inflammation.

The metabolite Glucose-1,6-bisphosphate (Glc-1,6-P2) plays a vital role in human metabolism, and is a crucial activator and stabilizer for phosphomannomutase-2 (PMM2) - mutations within this protein propagate the most common congenital disorder of glycosylation (PMM2-CDG). In vivo, Glc-1,6-P2 is hydrolysed by phosphomannomutase-1 (PMM1), predominantly in the brain, under the influence of inosine monophosphate (IMP). In the present study, we employed knock-out PMM1 in Arg141His/Phe119LeuPMM2 patient-derived fibroblasts and investigated the phenotypic improvement. Increased Glc-1,6-P2 was associated with glycosylation enhancement, confirmed by glycan profiling. Previously identified PMM2-CDG biomarkers, such as LAMP1, PTX3 and lysosomal enzymes showed empirical imrovement- these findings were corroborated by metabolomic and proteomic analysis. Moreover, our results support the potential of Glc-1,6-P2 modulation for PMM2-CDG, potentiating novel perspectives in drug discovery.

BACKGROUND:Genetic polymorphisms in sphingolipid metabolism, particularly involving the orosomucoid-like 3 (ORMDL3) gene, have been associated with asthma risk. Notably, asthma prevalence and severity exhibit pronounced sex differences, emerging in childhood and persisting into adulthood. However, the molecular mechanisms underlying this sexual dimorphism remain incompletely elucidated. OBJECTIVE:We investigated whether ORMDL3 contributes to sex differences in airway function and asthma-like features. METHODS:ORMDL3 expression was measured in lung tissues from healthy male and female human donors and in human bronchial epithelial cells (BEAS-2B) after exposure to 17β-estradiol (E2), Dermatophagoides pteronyssinus 1 (Der p 1), or both. A murine preparation of asthma was used to evaluate sex-dependent differences in ORMDL3 expression, airway responsiveness, and remodeling. Pharmacologic modulation of estrogen signaling and the ORMDL3-sphingosine-1-phosphate (S1P) axis were used. Methods included quantitative real-time PCR, immunostaining, liquid chromatography-tandem mass spectrometry, and airway function measurements. RESULTS:Female lungs exhibited higher ORMDL3 expression than male lungs, and this correlated with elevated forced expiratory volume to forced vital capacity ratios in the same patients. In BEAS-2B cells, E2 significantly upregulated ORMDL3 and altered sphingolipid metabolism by inducing expression of ceramidase, sphingosine kinases 1/2, and S1P receptors. Der p 1 also increased ORMDL3 and triggered epithelial activation via inflammasome signaling, while E2 enhanced IFN-β signaling and MUC5AC expression. Combination Der p 1/E2 synergistically activated sphingolipid, interferon, and inflammasome pathways. These in vitro findings prompted in vivo investigation using a murine asthma preparation, where female mice displayed elevated ORMDL3, sphingosine, and S1P levels, with increased airway hyperresponsiveness and remodeling. Treatment with tamoxifen or E2 normalized airway hyperresponsiveness and S1P signaling across sexes. Allergen sensitization intensified female-biased ORMDL3 expression and airway inflammation. Inhibition of the ORMDL3-S1P axis attenuated asthma-like features only in female animals. CONCLUSION:This study identifies ORMDL3 as an estrogen-responsive regulator of airway responsiveness that may contribute to sex-related differences in asthma features through modulation of sphingolipid metabolism.

To further extend the structure-activity relationships (SARs) of the previously published ubiquitin-specific protease 7 (USP-7) inhibitor STIRUR-41, a small library of 5-aminopyrazoles 1a-d and 2a-d is designed and synthesized. The chemical identity of the desired structure is confirmed by nuclear magnetic resonance and single crystal X-ray diffraction analyses. All novel derivatives are tested as potential USP-7 inhibitors and compounds 1a-d block enzyme activity in a dose-dependent manner and with lower IC50 values compared to the lead compound STIRUR-41. Notably, 1d, bearing a meta-trifluoromethylphenyl group linked to the carbamate moiety, proved to be the most active candidate. Conversely, compounds belonging to series 2, which possess greater steric hindrance, exhibit no activity. The most effective compounds of series 1 are noncytotoxic across a panel of tumor and normal cell lines at 10 μM concentration. For the most active compound 1d, a parallel artificial membrane permeability assay is also performed, as well as docking and molecular dynamics simulations.

Boswellia serrata, commonly known as Indian olibanum or Indian frankincense, is a medicinal plant recognized for its significant anti-inflammatory, analgesic, and anticancer activities. In our investigation, the activity of its n-hexane extract was evaluated on targets never explored for Boswellia, mainly involved in hepatic fibrosis and cancer development. Since this extract exhibited a significant antagonistic activity on the interaction between leukemia inhibitory factor (LIF) and its receptor (LIFR), it was subjected to an untargeted metabolomic analysis using a high-resolution mass spectrometry-based approach combined with molecular networking. An unambiguous assignment of several Boswellia triterpenoid metabolites was then achieved upon isolation and NMR spectroscopic investigation to accurately identify the bioactive Boswellia components responsible for the n-hexane extract activity on the LIF/LIFR system. Key active metabolites, including boswellic acids and their derivatives and a small library of semisynthetic analogues, demonstrated potential inhibitory activity toward LIF/LIFR interaction. In particular, α-boswellic acid (1) emerged as a LIFR antagonist, able to reduce the expression of col1α1 and α-SMA in LX-2 cells. Furthermore, computational studies highlighted the role of the carboxyl group in engaging a network of electrostatic and hydrogen bond interactions within residues of human LIFR (hLIFR) binding site. This finding suggests the potential use of Boswellia in hepatic fibrosis and sheds light on a relatively novel target for liver fibrosis therapy.

Fibrosis is a pathological process characterized by excessive deposition of the extracellular matrix (ECM) within tissues. Chronic fibrotic disorders involving the lungs, liver, intestine, and kidneys represent a major cause of morbidity and mortality and remain a major unmet therapeutic need. In the liver, the development of pathological ECM depends on the activation of key cell targets, i.e., the hepatic stellate cells (HSC). HSCs express the leukemia inhibitory factor receptor (LIFR), which promotes fibrosis, and a bile acid-activated receptor, GPBAR1, which attenuates HSC activation. Herein, we report the design and synthesis of a new class of 4,9-estradien-3,17-dione derivatives acting as dual LIFR inhibitors and GPBAR1 agonists. In silico and pharmacological characterization of these dual modulators led to the identification of compound 2o as a first-in-class LIFR/GPBAR1 modulator that reverses liver fibrosis in vitro and in vivo. These findings demonstrate the therapeutic potential of LIFR/GPBAR1 hybrid molecules in human fibrotic disorders.

BACKGROUND:Primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC) are immune-mediated cholestatic disorders characterized by progressive biliary inflammation and fibrosis, for which treatment options remain limited, underscoring the need for novel therapeutic targets. The leukemia inhibitory factor (LIF) is an IL-6-related cytokine that dysregulates the communication between epithelial cells and extracellular matrices by binding a heterodimeric complex formed by LIF receptor (LIFR) and gp130. The role of the LIF/LIFR system in PSC and PBC and its potential as a therapeutic target remain unclear. METHODS:We investigated LIF/LIFR system alteration in PSC and PBC and assessed the therapeutic potential of LIFR antagonism in a genetic mouse model of PSC (Abcb4-/- mice). Single-cell transcriptomics analyses were performed to evaluate LIF and LIFR expression in human liver samples. Whole liver RNA-seq and immunostaining were used to assess LIF/LIFR levels and correlation with fibrotic and immune markers. The effects of LIFR antagonism were evaluated in vitro using LRI-310, a steroidal LIFR antagonist, on human cholangiocytes, HSCs, endothelial cells, and macrophages. In vivo, LRI-310 was administered to Abcb4-/- mice, and effects on liver injury, cholestasis, fibrosis, leukocyte infiltration, and gene expression were assessed. RESULTS:LIF expression s enriched in human cholangiocytes, while LIFR is predominantly expressed by HSCs, endothelial cells, and macrophages. Whole liver RNAseq analysis and liver sections immunostaining demonstarted that increased LIF expression correlates with expression of markers of hepatic fibrosis and immune activation in PSC and PBC patients. LRI-310, a steroidal LIFR antagonist, attenuated human cholangiocytes activation and expression of inflammatory mediators, as well as the activation of liver sinusoidal cells and hepatic fibroblasts. In Abcb4-/- mice administration of LRI-310 mitigated liver injury, cholestasis, liver leukocytes infiltration and reduced the expression of biomarkers associated with fibrosis, inflammation, and bile acid dismetabolism. CONCLUSION:Cholangiocyte-derived LIF promotes the formation of a pro-inflammatory and pro-fibrotic niche centred on damaged cholangiocytes. LIFR antagonism reverses fibrosis and immune dysregulation in Abcb4-/- mice, supporting the development of anti-LIFR therapies in human cholangiopathies.