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Previous findings have indicated a marked upregulation of SNORA71A in gallbladder cancer (GBC) tissues compared to normal samples. However, the precise role and molecular mechanisms of SNORA71A in GBC remain largely unknown. Moreover, gemcitabine (GEM) drug resistance has been found to lead to unfavorable outcomes and recurrence in GBC patients. Therefore, this study aims to investigate the impact of SNORA71A on GBC and explore its potential effects on the sensitivity of GBC cells to GEM. RT-qPCR was conducted to assess SNORA71A level in matched normal and GBC tissues. Cell proliferation was examined through CCK-8 and 5-ethynyl-2'-deoxyuridine (EdU) assays. Additionally, the expression of proteins in GBC cells was analyzed using western blot assay. The level of SNORA71A was notably higher in GBC tissues relative to normal tissues. SNORA71A overexpression led to increased GBC cell proliferation and invasion. Conversely, SNORA71A deficiency strongly suppressed GBC cell proliferation and invasion and triggered cell apoptosis and ferroptosis. Additionally, downregulation of SNORA71A obviously enhanced the antiproliferative and anti-invasive effects of GEM on GBC cells, whereas these changes were reversed by inhibiting ferroptosis. Furthermore, deficiency of SNORA71A further augmented the GEM-induced downregulation of p-Akt, Nrf2, and GPX4 in NOZ cells; however, these effects were reversed by ferroptosis inhibition. Collectively, these findings suggested that downregulation of SNORA71A may increase the sensitivity of GBC cells to GEM by triggering ferroptosis through inhibiting the AKT/NRF2/GPX4 signaling pathway.

Gallbladder cancer (GBC) is highly aggressive and has poor prognosis, with most patients only diagnosed at an advanced stage. Furthermore, treatment options are limited, and their effect is unsatisfactory. Bromodomain-containing protein (BRD) is an epigenetic regulator that plays a carcinogenic role in several tumors, including squamous cell lung cancer, acute myeloid leukemia, synovial sarcoma, and malignant rhabdomyosarcoma. However, the expression, biological function, and molecular mechanisms of action of BRD9 in GBC are still unknown. Kaplan-Meier analysis, qRT-PCR, and analysis of clinical features were used to assess the clinical significance of BRD9 in GBC. Cell Counting Kit-8 and colony formation assays were performed to determine the effects of BRD9 on cell growth. The functional role of BRD9 in GBC was explored using qRT-PCR, western blotting, siRNA, and CHIP-qPCR. mRNA sequencing was performed to explore the underlying mechanisms of BRD9, and a nude mouse model of GBC was established to explore the anti-tumor effects of the BRD9 inhibitor I-BRD9 in vivo. BRD9 expression was elevated in GBC tissues compared with adjacent non-tumor tissues, and high BRD9 expression was associated with poor prognosis in patients with GBC. BRD9 knockdown by siRNA significantly decreased cell growth. Targeting BRD9 with I-BRD9 inhibited the proliferation of GBC cells without significant toxic effects. Additionally, I-BRD9 treatment suppressed CST1 expression in GBC cell lines, thereby inhibiting the PI3K-AKT pathway. The transcription factor FOXP1 was found to interact with BRD9 to regulate CST1 expression. Collectively, these results suggest that BRD9 may be a promising biomarker and therapeutic target for GBC., Competing Interests: Competing interests The authors declare no competing interests. Ethics approval and consent to participate Animal experiments involving nude mice met the requirements and were approved by Xinhua Hospital of Shanghai Jiao Tong University School of Medicine (Animal experiment approval number: XHEC–NSFC–2021-093, approval date: February 24, 2021). The study was approved by the Ethics Committee of Xinhua Hospital, Shanghai Jiaotong University School of Medicine. Informed consent was obtained from the patients for this study. Consent for publication All authors have seen and approved the manuscript and consent publication., (© 2024. The Author(s).)

Gallbladder cancer (GBC) presents as an aggressive malignancy with poor patient outcome. Like other epithelial cancers, the mechanisms of GBC cancer progression remain vague and efforts in finding targeted therapies fall below expectations. This study combined proteomic analysis of formalin-fixed paraffin-embedded (FFPE) GBC samples, functional and molecular characterization of potential oncogenes and identification of potential therapeutic strategies for GBC. We identified Carcinoembryonic Antigen-related Cell Adhesion Molecule 6 (CEACAM6) as one of the significantly most upregulated proteins in GBC. CEACAM6 overexpression has been observed in other cancer entities but the molecular function remains unclear. Our functional analyses in vitro and in vivo mouse models revealed that CEACAM6 supported the initial steps of cancer progression and metastasis by decreasing cell adhesion and promoting migration and invasion of GBC cells. Conversely, CEACAM6 knockdown abolished GBC aggressiveness by increasing cell adhesion while reducing cell migration, cell proliferation, and colony formation. BirA-BioID followed by mass-spectrometry revealed Integrin Beta-1 (ITGB1) and Protein Kinase C Delta (PRKCD) as direct molecular and functional partners of CEACAM6 supporting GBC cell migration. ERK and AKT signaling and their downstream target genes were regulated by CEACAM6 and thus the treatment with AKT inhibitor capivasertib or ERK inhibitor ulixertinib mitigated the CEACAM6-induced migration. These findings demonstrate that CEACAM6 is crucially involved in gallbladder cancer progression by promoting migration and inhibiting cell adhesion through ERK and AKT signaling providing specific options for treatment of CEACAM6-positive cancers., (© 2024. The Author(s).)

Circular RNAs (circRNAs) have been shown to play important roles in tumour development and tumour immunology. However, genome-wide characterisation of circRNAs and their roles in the immunology and immunotherapy of gallbladder carcinoma (GBC) has been lacking. We present a comprehensive characterisation of the circRNA landscape in GBC, revealing GBC-specific circRNAs. Our analysis found that circRNAs are significantly enriched in cell proliferation and are involved in cancer-related hallmarks. In particular, circAATF was upregulated in GBC, which was positively correlated with AATF mRNA expression, and promoted GBC cell growth. Through integrating computational and experimental approaches, we revealed that circAATF is positively associated with the CD4 + T cell abundance and PD-L1 level, and enhances the clinical benefits of anti-PD-L1 immunotherapy for GBC. We further demonstrate that circAATF elevates the PD-L1 level by activating phosphorylated AKT and acting as a sponge for miR-142-5p. CircAATF is positively associated with CD4 + T cells and PD-L1 levels and shows potential to aid anti-PD-L1 immunotherapy for GBC. Our study provides insights into roles of circAATF in the tumour development and immunology of GBC and accelerates the development of therapeutic strategies for GBC immunotherapy. HIGHLIGHTS: We present a comprehensive characterisation of circRNA landscape in gallbladder carcinoma (GBC). CircAATF is positively associated with CD4 + T cell abundance and PD-L1 expression and is shown to promote PD-L1 treatment in mouse model. CircAATF can elevate PD-L1 level through phosphorylated AKT and linear AATF, which upregulates PD-L1 by acting as a sponge of miR-142-5p., (© 2024 The Author(s). Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

Gallbladder cancer (GBC) is a common malignant cancer in the biliary system, which poses a serious threat to human health. It is urgent to explore ideal drugs for the treatment of GBC. Matrine is the main active ingredient of Sophora flavescentis, with a wide range of biological activities encompassing anti-inflammatory, antiviral, immunomodulatory, and anti-tumor. However, the underlying mechanism by which Matrine treats GBC is still unclear. The purpose of this study is to investigate the anti-tumor effects of Matrine on GBC in vivo and in vitro and to clarify the potential regulatory mechanisms. Here, we found that Matrine had a significant killing effect on GBC through CCK8 and flow cytometry, including arrest of cell cycle, inhibition of GBC cell, and induction of apoptosis. Further in vivo studies confirmed the inhibitory effect of Matrine on tumor growth in NOZ xenografted nude mouse. At the same time, Matrine also significantly suppressed the migration and invasion of GBC cells through scratch and Transwell experiments. In addition, by detecting the mRNA and protein levels of epithelial-mesenchymal transition (EMT) and matrix metalloproteinases, Matrine furtherly substantiated the inhibitory role on invasion and migration of GBC. From a mechanistic perspective, network pharmacology analysis suggests that the potential targets of Matrine in the treatment of GBC are enriched in the PI3K/AKT signaling pathway. Subsequently, Matrine effectively decreased the abundance of p-PI3K and p-AKT protein in vivo and in vitro. More importantly, PI3K activator (740 Y-P) antagonized the anti-tumor effect of Matrine, while PI3K inhibitor (LY294002) increased the sensitivity of Matrine for GBC. Based on the above findings, we conclude that Matrine inhibits the invasion and migration of GBC by regulating PI3K/AKT signaling pathway. Our results indicate the crucial role and regulatory mechanism of Matrine in suppressing the growth of GBC, which provides a theoretical basis for Matrine to be a candidate drug for the treatment and research of GBC., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

In this study, we successfully established a novel gallbladder cancer cell line, designated as GBC-X1, derived from a primary tumor of a gallbladder cancer patient. By comprehensively analyzing the cell line's phenotype, molecular characteristics, biomarkers, and histological characteristics, we confirmed that GBC-X1 serves as a valuable model for investigating the pathogenesis of gallbladder cancer and developing therapeutic agents. GBC-X1 has been continuously cultured for one year, with over 60 stable passages. Morphologically, GBC-X1 exhibits typical features of epithelial tumors. The population doubling time of GBC-X1 is 32 h. STR analysis validated a high consistency between GBC-X1 and the patient's primary tumor. Karyotype analysis revealed an abnormal hypertetraploid karyotype for GBC-X1, characterized by representative karyotypes of 98, XXXX del (4) p (12) del (5) p (21) der (10). Under suspension culture conditions, GBC-X1 efficiently forms tumor balls, while subcutaneous inoculation of GBC-X1 cells into NXG mice leads to xenograft formation with a rate of 80%. Drug sensitivity testing demonstrated that GBC-X1 is resistant to oxaliplatin and sensitive to 5-FU, gemcitabine, and paclitaxel. Immunohistochemistry revealed positive expression of CK7, CK19, E-cadherin, MMP-2, CD44, SOX2, and TP53 in GBC-X1 cells, weak positive expression of Vimentin, and a Ki67 positive rate of 35%. Our research highlights GBC-X1 as a novel gallbladder cancer cell line and emphasizes its potential as an effective experimental model for investigating the pathogenesis of gallbladder cancer and drug development., (© 2024. The Author(s).)

Background: Gallbladder cancer (GBC) is a highly aggressive malignant tumor with a poor prognosis. Despite being first described two centuries ago, there are no targeted therapies available beyond conventional cytotoxic therapy. Epidemiological studies have shown that the incidence of gallbladder cancer is higher in females than males. This suggests that the gallbladder may be a female sex hormone-responsive organ, and these hormones might be involved in the pathogenesis of gallbladder cancer. Therefore, we aimed to analyze the expression of ERα and PR in GBC and correlate their expression with clinicopathological variables and overall survival., Patients and Methods: A total of 235 histopathologically diagnosed GBC cases were included in this hospital-based cross-sectional study. Clinicopathological data were collected, and the expression of ERα and PR was evaluated by immunohistochemistry., Results: The mean age of this study population was 55.47 ± 8.45 with range 28-87 years. Females were predominated over male with a male-to-female ratio of 1:3.5. Positive nuclear expression of the ERα and PR was found in 13 (5.5%) and eight (3.4%) cases, respectively. Apart from nuclear staining, cytoplasmic expression of ERα and PR was found in three (1.2%) and 31 (13.2%) cases, respectively. Higher percentage of positive nuclear expression of ER was found in < 50 years age (p value = 0.04), parity > 4 (p value = 0.02), advanced pT stage (T3) (p value = 0.01), lymphovascular invasion (p value = 0.02), and liver invasion (p value = 0.04) which were statistically significant. Higher percentage of PR expression was also observed in < 50 years age (p value = 0.01), and tumor associated with gallstone (p value = 0.04). There was no significant correlation between cytoplasmic expression of ER, PR, and clinicopathological variables. In multivariate analysis, there was no significant correlation between ER or PR positive expression and overall survival., Conclusion: Although nuclear expression of ERα was significantly associated with progressive disease factors but the positive expression was found in very small percentage of GBC cases. So anti-hormone therapy might be an option in patient with ER α positive gallbladder carcinoma., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Gallbladder cancer (GBC) is an aggressive and lethal malignancy with a poor prognosis. Long noncoding RNAs (lncRNAs) and natural products have emerged as key orchestrators of cancer pathogenesis through widespread dysregulation across GBC transcriptomes. Functional studies have revealed that lncRNAs interact with oncoproteins and tumor suppressors to control proliferation, invasion, metastasis, angiogenesis, stemness, and drug resistance. Curcumin, baicalein, oleanolic acid, shikonin, oxymatrine, arctigenin, liensinine, fangchinoline, and dioscin are a few examples of natural compounds that have demonstrated promising anticancer activities against GBC through the regulation of important signaling pathways. The lncRNAs, i.e., SNHG6, Linc00261, GALM, OIP5-AS1, FOXD2-AS1, MINCR, DGCR5, MEG3, GATA6-AS, TUG1, and DILC, are key players in regulating the aforementioned processes. For example, the lncRNAs FOXD2-AS1, DILC, and HOTAIR activate oncogenes such as DNMT1, Wnt/β-catenin, BMI1, and c-Myc, whereas MEG3 and GATA6-AS suppress the tumor proteins NF-κB, EZH2, and miR-421. Clinically, specific lncRNAs can serve as diagnostic or prognostic biomarkers based on overexpression correlating with advanced TNM stage, metastasis, chemoresistance, and poor survival. Therapeutically, targeting aberrant lncRNAs with siRNA or antisense oligos disrupts their oncogenic signaling and inhibits GBC progression. Overall, dysfunctional lncRNA regulatory circuits offer multiple avenues for precision medicine approaches to improve early GBC detection and overcome this deadly cancer. They have the potential to serve as novel biomarkers as they are detectable in bodily fluids and tissues. These findings enhance gallbladder treatments, mitigating resistance to chemo- and radiotherapy., Competing Interests: Declarations. Ethical approval: Not applicable. Consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Aberrant expression of G protein-coupled receptor class C group 5 member A (GPRC5A) has been reported in multiple cancers and is closely related to patient prognosis. However, the mechanistic role of GPRC5A in gallbladder cancer (GBC) remains unclear. Here, we determined tumor expression levels of GPRC5A and the molecular mechanisms by which GPRC5A regulates gallbladder cancer metastasis. We found that GPRC5A was significantly upregulated in GBC, correlating with poorer patient survival. Knocking down GPRC5A inhibited GBC cell metastasis both in vitro and in vivo. GRPRC5A knockdown resulted in downregulation of TNS4 expression through the JAK2-STAT3 axis. Clinically, GPRC5A expression positively correlated with TNS4. Finally, STAT3 bound to TNS4's promoter region, inducing its expression. Overall, GPRC5A showed high expression in GBC tissues, associated with poor patient prognosis. Our findings first demonstrate that the GPRC5A-JAK2-STAT3-TNS4 pathway promotes GBC cell metastasis, suggesting potential therapy targets., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Gallbladder carcinoma (GBC) is a malignant hepatobiliary cancer characterized by an intricate tumor microenvironments (TME) and heterogeneity. The traditional GBC 2D culture models cannot faithfully recapitulate the characteristics of the TME. Three-dimensional (3D) bioprinting enables the establishment of high-throughput and high-fidelity multicellular GBC models. In this study, we designed a concentric cylindrical tetra-culture model to reconstitute the spatial distribution of cells in tumor tissue, with the inner portion containing GBC cells, and the outer ring containing a mixture of endothelial cells, fibroblasts, and macrophages. We confirmed the survival, proliferation, biomarker expression and gene expression profiles of GBC 3D tetra-culture models. Hematoxylin-eosin (HE) and immunofluorescence staining verified the morphology and robust expression of GBC/endothelial/fibroblast/macrophage biomarkers in GBC 3D tetra-culture models. Single-cell RNA sequencing revealed two distinct subtypes of GBC cells within the model, glandular epithelial and squamous epithelial cells, suggesting the mimicry of intratumoral heterogeneity. Comparative transcriptome profile analysis among various in vitro models revealed that cellular interactions and the TME in 3D tetra-culture models reshaped the biological processes of tumor cells to a more aggressive phenotype. GBC 3D tetra-culture models restored the characteristics of the TME as well as intratumoral heterogeneity. Therefore, this model is expected to have future applications in tumor biology research and antitumor drug development., (Creative Commons Attribution license.)

Aims: Advanced gallbladder carcinoma (AGBC) carries a poor prognosis with dismal survival. There are no data regarding HER2/ERBB2 expression in AGBC. This study evaluated the overexpression of HER2/ERBB2 in cytological aspirates from AGBCs to identify potential patients for whom anti-HER2 targeted therapies can benefit., Methods: This prospective, case-control study was performed on 50 primary AGBC cases. A detailed cytomorphological assessment, followed by immunocytochemistry (ICC) for HER2/ERBB2, was performed on AGBC cell blocks. A similar number of age-matched and gender-matched resected chronic cholecystitis specimens were included as controls. Fluorescence in situ hybridisation (FISH) was performed in equivocal cases., Results: A total of 10 (20%) cases showed positive (3+), 19 (38%) equivocal (2+) expression and 21 (42%) were negative on HER2/ERBB2 ICC. None of the equivocal cases demonstrated HER2 amplification by FISH. Among the controls, none showed positive (3+) immunoexpression, 23 (46%) demonstrated equivocal expression and 27 (54%) were negative. On statistical analysis, HER2/ERBB2 overexpression was significantly associated with AGBC compared with the controls. Of all the clinical, radiological and cytomorphological parameters, the predominant papillary or acinar arrangements of the tumour cells were significantly associated with HER2/ERBB2 overexpression., Conclusions: This is the first study to evaluate the expression of HER2/ERBB2 on cytological aspirates in AGBC using ICC and FISH. HER2/ERBB2 overexpression(20%) was significantly associated with AGBC. Furthermore, predominant papillary or acinar arrangements of tumour cells in the cytological smears were significantly associated with HER2/ERBB2 overexpression. They can serve as potential predictors of HER2/ERBB2 overexpression to select AGBC patients for anti-HER2 targeted therapies., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Objective: Elucidating complex ecosystems and molecular features of gallbladder cancer (GBC) and benign gallbladder diseases is pivotal to proactive cancer prevention and optimal therapeutic intervention., Design: We performed single-cell transcriptome analysis on 230 737 cells from 15 GBCs, 4 cholecystitis samples, 3 gallbladder polyps, 5 gallbladder adenomas and 16 adjacent normal tissues. Findings were validated through large-scale histological assays, digital spatial profiler multiplexed immunofluorescence (GeoMx), etc. Further molecular mechanism was demonstrated with in vitro and in vivo studies., Results: The cell atlas unveiled an altered immune landscape across different pathological states of gallbladder diseases. GBC featured a more suppressive immune microenvironment with distinct T-cell proliferation patterns and macrophage attributions in different GBC subtypes. Notably, mutual exclusivity between stromal and immune cells was identified and remarkable stromal ecosystem (SC) heterogeneity during GBC progression was unveiled. Specifically, SC1 demonstrated active interaction between Fibro-iCAF and Endo-Tip cells, correlating with poor prognosis. Moreover, epithelium genetic variations within adenocarcinoma (AC) indicated an evolutionary similarity between adenoma and AC. Importantly, our study identified elevated olfactomedin 4 (OLFM4) in epithelial cells as a central player in GBC progression. OLFM4 was related to T-cell malfunction and tumour-associated macrophage infiltration, leading to a worse prognosis in GBC. Further investigations revealed that OLFM4 upregulated programmed death-ligand 1 (PD-L1) expression through the MAPK-AP1 axis, facilitating tumour cell immune evasion., Conclusion: These findings offer a valuable resource for understanding the pathogenesis of gallbladder diseases and indicate OLFM4 as a potential biomarker and therapeutic target for GBC., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Gallbladder cancer (GBC) is an aggressive cancer with poor prognosis. PARP inhibitors (PARPi) target PARP enzymes and have shown efficacy in patients with breast cancer gene (BRCA) mutations. Immunotherapy, especially immune checkpoint inhibitors (ICIs), has transformed cancer treatment. However, the combined impact of PARPi and ICIs in GBC remains unclear. We present a groundbreaking case of a GBC patient with BRCA2 mutations who received combination therapy with PARPi and ICIs after failing multiple lines of treatment. Next-generation sequencing (NGS-Seq) identified BRCA gene mutations. To further investigate potential mechanisms, we developed a PARP1-BRCA1-BRCA2 pathway-related risk score (PBscore) system to evaluate the impact of PARPi on the tumor immune microenvironment via RNA-Seq data. Gene expression and functional analysis identified potential mechanisms associated with the PBscore. Experimental validation assessed the impact of the combination therapy on the tumor microenvironment using multiplexed immunofluorescence imaging and immunohistochemistry in patients with BRCA gene wild type or mutations. RNA-Seq analysis revealed correlations between PBscore, immune checkpoint levels, tumor-infiltrating immune cells (TIICs), and the cancer-immunity cycle. Multiplexed immunofluorescence imaging validated that low PBscore patients might have an active tumor microenvironment. Furthermore, upon drug resistance, we observed an upregulation of negative immune checkpoints such as CEACAM1, indicating that the tumor immune microenvironment becomes suppressed after resistance. Our study revealed that PBscore could serve as a biomarker to predict immunotherapy efficacy, offering a promising alternative for BRCA2-mutated GBC patients., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Liver metastasis is common in patients with gallbladder cancer (GBC), imposing a significant challenge in clinical management and serving as a poor prognostic indicator. However, the mechanisms underlying liver metastasis remain largely unknown. Here, we report a crucial role of tyrosine aminotransferase (TAT) in liver metastasis of GBC. TAT is frequently up-regulated in GBC tissues. Increased TAT expression is associated with frequent liver metastasis and poor prognosis of GBC patients. Overexpression of TAT promotes GBC cell migration and invasion in vitro, as well as liver metastasis in vivo. TAT knockdown has the opposite effects. Intriguingly, TAT promotes liver metastasis of GBC by potentiating cardiolipin-dependent mitophagy. Mechanistically, TAT directly binds to cardiolipin and leads to cardiolipin externalization and subsequent mitophagy. Moreover, TRIM21 (Tripartite Motif Containing 21), an E3 ubiquitin ligase, interacts with TAT. The histine residues 336 and 338 at TRIM21 are essential for this binding. TRIM21 preferentially adds the lysine 63 (K63)-linked ubiquitin chains on TAT principally at K136. TRIM21-mediated TAT ubiquitination impairs its dimerization and mitochondrial location, subsequently inhibiting tumor invasion and migration of GBC cells. Therefore, our study identifies TAT as a novel driver of GBC liver metastasis, emphasizing its potential as a therapeutic target., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Gallbladder cancer (GBC) is a rare but very aggressive most common digestive tract cancer with a high mortality rate due to delayed diagnosis at the advanced stage. Moreover, GBC progression shows asymptomatic characteristics making it impossible to detect at an early stage. In these circumstances, conventional therapy like surgery, chemotherapy, and radiotherapy becomes refractive. However, few studies reported some molecular markers like KRAS (Kirsten Rat Sarcoma) mutation, upregulation of HER2/neu, EGFR (Epidermal Growth Factor Receptor), and microRNAs in GBC. However, the absence of some specific early diagnostic and prognostic markers is the biggest hurdle for the therapy of GBC to date. The present study has been designed to identify some specific molecular markers for precise diagnosis, and prognosis, for successful treatment of the GBC. By In Silico a network-centric analysis of two microarray datasets; (GSE202479) and (GSE13222) from the Gene Expression Omnibus (GEO) database, shows 50 differentially expressed genes (DEGs) associated with GBC. Further network analysis revealed that 12 genes are highly interconnected based on the highest MCODE (Molecular Complex Detection) value, among all three genes; TRIP13 (Thyroid Receptor Interacting Protein), NEK2 (Never in Mitosis gene-A related Kinase 2), and TPX2 (Targeting Protein for Xklp2) having highest network interaction with transcription factors and miRNA suggesting critically associated with GBC. Further survival analysis data corroborate the association of these genes; TRIP13, NEK2, and TPX2 with GBC. Thus, TRIP13, NEK2, and TPX2 genes are significantly correlated with a greater risk of mortality, transforming them from mere biomarkers of the GBC for early detections and may emerge as prognostic markers for treatment., (© 2024. The Author(s).)

Long non-coding RNAs (lncRNAs) are nucleotide sequences that participate in different biological processes and are associated with different pathologies, including cancer. Long intergenic non-protein-coding RNA 662 (LINC00662) has been reported to be involved in different cancers, including colorectal, prostate, and breast cancer. However, its role in gallbladder cancer has not yet been described. In this article, we hypothesize that LINC00662 has an important role in the acquisition of aggressiveness traits such as a stem-like phenotype, invasion, and chemoresistance in gallbladder cancer. Here, we show that LINC00662 is associated with larger tumor size and lymph node metastasis in patients with gallbladder cancer. Furthermore, we show that the overexpression of LINC00662 promotes an increase in CD133 + /CD44 + cell populations and the expression of stemness-associated genes. LINC00662 promotes greater invasive capacity and the expression of genes associated with epithelial-mesenchymal transition. In addition, the expression of LINC00662 promotes resistance to cisplatin and 5-fluorouracil, associated with increased expression of chemoresistance-related ATP-binding cassette (ABC) transporters in gallbladder cancer (GBC) cell lines. Finally, we show that the mechanism by which LINC00662 exerts its function is through a decrease in microRNA 335-5p (miR-335-5p) and an increase in octamer-binding transcription factor 4 (OCT4) in GBC cells. Thus, our data allow us to propose LINC00662 as a biomarker of poor prognosis and a potential therapeutic target for patients with GBC.

TGF-β1 plays a pivotal role in the metastatic cascade of malignant neoplasms. N6-methyladenosine (m 6 A) stands as one of the most abundant modifications on the mRNA transcriptome. However, in the metastasis of gallbladder carcinoma (GBC), the effect of TGF-β1 with mRNA m 6 A modification, especially the effect of mRNA translation efficiency associated with m 6 A modification, remains poorly elucidated. Here we demonstrated a negative correlation between FOXA1 and TGF-β1 expression in GBC. Overexpression of FOXA1 inhibited TGF-β1-induced migration and epithelial-mesenchymal transition (EMT) in GBC cells. Mechanistically, we confirmed that TGF-β1 suppressed the translation efficiency of FOXA1 mRNA through polysome profiling analysis. Importantly, both in vivo and in vitro experiments showed that TGF-β1 promoted m 6 A modification on the coding sequence (CDS) region of FOXA1 mRNA, which was responsible for the inhibition of FOXA1 mRNA translation by TGF-β1. We demonstrated through MeRIP and RIP assays, dual-luciferase reporter assays and site-directed mutagenesis that ALKBH5 promoted FOXA1 protein expression by inhibiting m 6 A modification on the CDS region of FOXA1 mRNA. Moreover, TGF-β1 inhibited the binding capacity of ALKBH5 to the FOXA1 CDS region. Lastly, our study confirmed that overexpression of FOXA1 suppressed lung metastasis and EMT in a nude mice lung metastasis model. In summary, our research findings underscore the role of TGF-β1 in regulating TGF-β1/FOXA1-induced GBC EMT and metastasis by inhibiting FOXA1 translation efficiency through m 6 A modification., (© 2024. The Author(s).)

The aim of this investigation was to evaluate the differential expression of the sterol O-acyltransferase 1 (SOAT1) protein in gallbladder cancer tissues and cells, investigate the impact of Avastin on the proliferation, migration, invasion capabilities of gallbladder cancer cells, and its potential to induce cell apoptosis. Immunohistochemical analysis of samples from 145 gallbladder cancer patients was conducted, along with analysis of SOAT1 protein, mRNA expression levels, and cholesterol content in gallbladder cancer cell lines SGC-996, NOZ, and gallbladder cancer (GBC)-SD using Western blot and q-PCR techniques. Furthermore, the effects of Avastin on the proliferation, migration, and invasion capabilities of these gallbladder cancer cell lines were studied, and its ability to induce cell apoptosis was evaluated using flow cytometry, Western blot, and immunohistochemical methods. Additionally, gene expression and pathway analysis were performed, and the synergistic therapeutic effects of Avastin combined with gemcitabine were tested in a gallbladder cancer xenograft model. The study found that SOAT1 expression was significantly upregulated in GBC tissues and positively correlated with lymph node metastasis and TNM staging. In vitro experiments demonstrated that Avastin significantly inhibited the proliferation, migration, and invasion capabilities of SGC-996 and GBC-SD cell lines and induced apoptosis. RNA sequencing analysis revealed multiple differentially expressed genes in cells treated with Avastin, primarily enriched in biological pathways such as signaling transduction, malignant tumors, and the immune system. In vivo, experiments confirmed that Avastin could effectively suppress tumor growth in a gallbladder cancer xenograft model and enhanced the treatment efficacy when used in combination with gemcitabine. Overall, these findings provide new insights and strategies for targeted therapy in gallbladder cancer., (© 2024 Wiley Periodicals LLC.)

Gallbladder cancer is a rare but fatal malignancy. However, the mechanisms underlying gallbladder carcinogenesis and its progression are poorly understood. The function of m6A modification and its regulators was still unclear for gallbladder cancer. The current study seeks to investigate the function of YTH m6A RNA-binding protein 1 (YTHDF1) in gallbladder cancer. Transcriptomic analysis and immunochemical staining of YTHDF1 in gallbladder cancer tissues revealed its upregulation compared to paracancerous tissues. Moreover, YTHDF1 promotes the proliferation assays, Transwell migration assays, and Transwell invasion assays of gallbladder cancer cells in vitro. And it also increased tumour growth in xenograft mouse model and metastases in tail vein injection model in vivo. In vitro, UHRF1 knockdown partly reversed the effects of YTHDF1 overexpression. Mechanistically, dual-luciferase assays proved that YTHDF1 promotes UHRF1 expression via direct binding to the mRNA 3'-UTR in a m6A-dependent manner. Overexpression of YTHDF1 enhanced UHRF1 mRNA stability, as demonstrated by mRNA stability assays, and Co-IP studies confirmed a direct interaction between YTHDF1 and PABPC1. Collectively, these findings provide new insights into the progression of gallbladder cancer as well as a novel post-transcriptional mechanism of YTHDF1 via stabilizing target mRNA., (© 2024 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Background: Human umbilical cord mesenchymal stem cell (hucMSC)-derived exosomes have been reported to be effective in the treatment of cancer. The miR-214-3p is a suppressor miRNA that has been extensively studied and has been proposed as a diagnostic and prognostic biomarker in some cancers., Objectives: The aim of this study was to investigate whether the regulatory mechanism of hucMSC-derived exosomal miR-214-3p with GLUT1 and ACLY affects the proliferation and apoptosis of gallbladder cancer (GBC) cells., Material and Methods: We found that the target genes of miR-214-3p on the TargetScan website contain GLUT1 and ACLY, and the targeting relationship was verified using luciferases. The GBC-SD cells overexpressing GLUT1 and ACLY were constructed to determine proliferation, apoptosis, migration, and other cellular activities., Results: We identified hucMSCs and exosomes, and found that the exosomes contained miR-214-3p. Furthermore, TargetScan predicted that miR-214-3p had base interactions with ACLY. Dual luciferase assays showed that miR-214-3p could inhibit ACLY (p < 0.05). The results of quantitative reverse transcription polymerase chain reaction (RT-qPCR) and western blot showed that exosomal miR-214-3p could inhibit the expression of ACLY and GLUT1 (p < 0.05). Exosomal miR-214-3p can inhibit the proliferation, cloning and migration of GBC-SD cells (p < 0.05). The apoptosis of GBC-SD cells was increased (p < 0.05). The GBC-SD cells overexpressing ACLY and GLUT1 could reverse the efficacy of miR-214-3p., Conclusions: Exosomal miR-214-3p can inhibit the downstream expression of ACLY and GLUT1. The ACLY and GLUT1 could affect the proliferation and apoptosis of GBC-SD cells.

Gallbladder cancer (GBC) is among the most common malignancies of biliary tract system due to its limited treatments. The immunotherapeutic targets for T cells are appealing, however, heterogeneity of T cells hinds its further development. We systematically construct T cell atlas by single-cell RNA sequencing; and utilized the identified gene signatures of high&#95;CNV&#95;T cells to predict molecular subtyping towards personalized therapeutic treatments for GBC. We identified 12 T cell subtypes, where exhausted CD8 + T cells, activated/exhausted CD8 + T cells, and regulatory T cells were predominant in tumors. There appeared to be an inverse relationship between Th17 and Treg populations with Th17 levels significantly reduced, whereas Tregs were concomitantly increased. Furthermore, we first established subtyping criterion to identify three subtypes of GBC based on their pro-tumorigenic microenvironments, e.g., the type 1 group shows more M2 macrophages infiltration, while the type 2 group is infiltrated by highly exhausted CD8 + T cells, B cells and Tregs with suppressive activities. Our study provides valuable insights into T cell heterogeneity and suggests that molecular subtyping based on T cells might provide a potential immunotherapeutic strategy to improve GBC treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Gallbladder cancer (GBC) is an extremely lethal malignancy with aggressive behaviors, including liver or distant metastasis; however, the underlying mechanisms driving the metastasis of GBC remain poorly understood. In this study, it is found that DNA methyltransferase DNMT3A is highly expressed in GBC tumor tissues compared to matched adjacent normal tissues. Clinicopathological analysis shows that DNMT3A is positively correlated with liver metastasis and poor overall survival outcomes in patients with GBC. Functional analysis confirms that DNMT3A promotes the metastasis of GBC cells in a manner dependent on its DNA methyltransferase activity. Mechanistically, DNMT3A interacts with and is recruited by YAP/TAZ to recognize and access the CpG island within the CDH1 promoter and generates hypermethylation of the CDH1 promoter, which leads to transcriptional silencing of CDH1 and accelerated epithelial-to-mesenchymal transition. Using tissue microarrays, the association between the expression of DNMT3A, YAP/TAZ, and CDH1 is confirmed, which affects the metastatic ability of GBC. These results reveal a novel mechanism through which DNMT3A recruitment by YAP/TAZ guides DNA methylation to drive GBC metastasis and provide insights into the treatment of GBC metastasis by targeting the functional connection between DNMT3A and YAP/TAZ., (© 2024 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

This study uncovered the potential clinical value and molecular driving mechanisms of circular RNAs (circRNAs) in gallbladder cancer (GBC). Differentially expressed circRNAs in GBC cells were screened by high-throughput sequencing. CircRNA&#95;CDKN1A (circBase ID: hsa&#95;circ&#95;0076194) was knocked out in BGC-SD cells through transfection with sh-circRNA&#95;CDKN1A. Then, proliferation was investigated via CCK8 and EdU assays, apoptosis via flow cytometry, migration via wound healing assays, and invasion via Transwell assays. Bioinformatics analysis of circRNA&#95;CDKN1A-related signaling pathways was performed using MetScape and g:Profiler. Results showed that the knockdown of circRNA&#95;CDKN1A enhanced the proliferation, migration, and invasion of GBC cells and inhibited apoptosis. In addition, knocking out circRNA&#95;CDKN1A promoted GBC cell proliferation and enhanced the dry indices of the OCT4 protein and CD34 expression levels. The knockdown of circRNA&#95;CDKN1A activated the epithelial-mesenchymal transition pathway. Bioinformatics analysis revealed that the biological role of circRNA&#95;CDKN1A in GBC cells involved the NF-κB pathway. LY2409881, which is an NF-κB inhibitor, reversed the effects induced by the knockdown of circRNA&#95;CDKN1A in GBC-SD cells. In summary, the knockdown of circRNA&#95;CDKN1A promoted the progression of GBC by activating the NF-κB signaling pathway. For the first time, this study revealed the mechanism of circRNA&#95;CDKN1A-mediated regulatory action in GBC and identified the newly discovered circRNA&#95;CDKN1A-NF-κB signaling axis as a potentially important candidate for clinical therapy and prognostic diagnosis of GBC., (© 2024 John Wiley & Sons Ltd.)

Recent findings in cancer research have pointed towards the bidirectional interaction between circadian and hypoxia pathways. However, little is known about their crosstalk mechanism. In this work, we aimed to investigate this crosstalk at a network level utilizing the omics information of gallbladder cancer. Differential gene expression and pathway enrichment analysis were used for selecting the crucial genes from both the pathways, followed by the construction of a logical crosstalk model using GINsim. Functional circuit identification and node perturbations were then performed. Significant node combinations were used to investigate the temporal behavior of the network through MaBoSS. Lastly, the model was validated using published in vitro experimentations. Four new positive circuits and a new axis viz. BMAL1/ HIF1αβ/ NANOG, responsible for stemness were identified. Through triple node perturbations viz.a. BMAL:CLOCK (KO or E1) + P53 (E1) + HIF1α (KO); b. P53 (E1) + HIF1α (KO) + MYC (E1); and c. HIF1α (KO) + MYC (E1) + EGFR (KO), the model was able to inhibit cancer growth and maintain a homeostatic condition. This work provides an architecture for drug simulation analysis to entrainment circadian rhythm and in vitro experiments for chronotherapy-related studies. Insight Box. Circadian rhythm and hypoxia are the key dysregulated processes which fuels-up the cancer growth. In the present work we have developed a gallbladder cancer (GBC) specific Boolean model, utilizing the RNASeq data from GBC dataset and tissue specific interactions. This work adequately models the bidirectional nature of interactions previously illustrated in experimental papers showing the effect of hypoxia on dysregulation of circadian rhythm and the influence of this disruption on progression towards metastasis. Through the dynamical study of the model and its response to different perturbations, we report novel triple node combinations that can be targeted to efficiently reduce GBC growth. This network can be used as a generalized framework to investigate different crosstalk pathways linked with cancer progression., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

A simple near-infrared (NIR) spectroscopic scheme enabling direct measurement of organic phase extracted from human bile with no spectral interference from the extraction solvent was demonstrated for identification of gallbladder (GB) cancer. This scheme is used to recognize the different lipid contents in bile samples from GB cancer patients using NIR spectroscopy for disease identification. To this end, the extraction solvent should provide an absorption-free NIR region to observe peaks of related metabolite. For this purpose, deuterated chloroform (CDCl 3 ) is uniquely suited as an extraction medium because it has few absorption peaks in the 4380-4100 cm -1 range, where intense peaks for lipids and cholesterol are located. This exploratory study used 37 bile samples (obtained from five normal subjects and nine GB polyp, 11 gallstone, six hepatocellular carcinoma (HCC), and six GB cancer patients). The transmission NIR spectra of the organic phases extracted using CDCl 3 in a commercial glass vial were directly measured. The peak intensities of the GB cancer samples were lower than those of the other samples, and the differences were statistically significant, with a confidence interval greater than 99.0%. The lower lipid and cholesterol contents in the organic phases of the GB cancer samples were effectively identified in the corresponding NIR spectra. Therefore, the proposed NIR scheme is simpler and faster than the previous infrared (IR) measurement approach that requires solvent drying to highlight the buried metabolite peaks under a solvent absorption band., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Context: Gallbladder cancer (GBC) is the most common malignant tumour of the biliary tract. Isoalantolactone (IAL), an active sesquiterpene lactone compound isolated from the roots of Inula helenium L. (Asteraceae), has antitumour effects., Objective: This study investigates the effects of IAL on GBC., Materials and Methods: In vitro , NOZ and GBC-SD cells were treated with IAL (0, 10, 20 and 40 μM) for 24 h. The DMSO-treated cells were selected as a control. Cell proliferation, migration, invasion and apoptosis were measured by the CCK-8 assay, transwell assay, flow cytometry and western blot. In vivo , subcutaneous tumour xenografts were constructed by injecting nude mice (BALB/C) with 5 × 10 6 NOZ cells. Mice were divided into the control group (equal amount of DMSO), the IAL group (10 mg/kg/day) and the IAL + Ro 67-7476 group (IAL, 10 mg/kg/day; Ro 67-7476, 4 mg/kg/day). The study duration was 30 days., Results: Compared with the DMSO group, cell proliferation of NOZ (IC 50 15.98 μM) and GBC-SD (IC 50 20.22 μM) was inhibited by about 70% in the IAL 40 μM group. Migration and invasion were suppressed by about 80%. Cell apoptosis rate was increased about three-fold. The phosphorylation level of ERK was decreased to 30-35%. Tumour volume and weight (about 80% reduction) were suppressed by IAL in vivo . Moreover, the effects of IAL were abolished by Ro 67-7476 in vitro and in vivo ., Discussion and Conclusions: Our findings indicate that IAL could inhibit GBC progression in vitro and in vivo by inhibiting the ERK signalling pathway.

Background: Oct4 has critical role in maintaining pluripotency, proliferative potential, and self-renewal capacity in embryonic stem and germ cells. Although Oct4 has been shown to be upregulated in many cancers, its clinical significance in gallbladder carcinoma is poorly understood., Methods: We studied the expression profile of Oct4 in 61 GBC and 30 chronic cholecystitis (as control) using real time RT-PCR, western blotting, and immunohistochemistry. The expression data was correlated with clinico-pathological parameters. The diagnostic utility was assessed through ROC curve, and prognostic value was analyzed by Kaplan-Meier method., Results: Oct4 was significantly upregulated at mRNA as well as protein levels. The higher mRNA expression shows significant association with late stage, late T stage, and higher grade of tumor. A significant positive correlation was also observed with stage, T stage, and tumor grade. Sum score analysis of protein expression shows positive correlation with stage and the presence or absence of gallstone in tumor samples. The ROC curve analysis revealed the moderate diagnostic potential of Oct4. Kaplan-Meier analysis showed that patients having higher expression of Oct4 were having low mean survival compared with the patients with lower Oct4 expression., Conclusion: In conclusion, our data suggests that higher expression of Oct4 may serve as potential biological indicator for tumor aggressiveness and poor prognosis of GBC., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

N6-methyladenosine (m6A) is the most abundant internal modification in eukaryotic RNA and involved in the carcinogenesis of various malignancies. However, the functions and mechanisms of m6A in gallbladder cancer (GBC) remain unclear. In this study, we investigated the role and underlying mechanism of the RNA-binding protein YT521-B homology domain-containing family protein 2 (YTHDF2), an m6A reader, in GBC. Herein, we detected that YTHDF2 was remarkably upregulated in GBC tissues compared to normal gallbladder tissues. Functionally, YTHDF2 overexpression promoted the proliferation, tumor growth, migration, and invasion of GBC cells while inhibiting the apoptosis in vitro and in vivo. Conversely, YTHDF2 knockdown induced opposite results. Mechanistically, we further investigated the underlying mechanism by integrating RNA immunoprecipitation sequencing (RIP-seq), m6A-modified RIP-seq, and RNA sequencing, which revealed that death-associated protein kinase 3 (DAPK3) is a direct target of YTHDF2. YTHDF2 binds to the 3'-UTR of DAPK3 mRNA and facilitates its degradation in an m6A-dependent manner. DAPK3 inhibition restores the tumor-suppressive phenotype induced by YTHDF2 deficiency. Moreover, the YTHDF2/DAPK3 axis induces the resistance of GBC cells to gemcitabine. In conclusion, we reveal the oncogenic role of YTHDF2 in GBC, demonstrating that YTHDF2 increases the mRNA degradation of the tumor suppressor DAPK3 in an m6A-dependent way, which promotes GBC progression and desensitizes GBC cells to gemcitabine. Our findings provide novel insights into potential therapeutic strategies for GBC., (© 2023 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Background: Although disc large associated protein family (DLGAP5) has been reported to be involved in a variety of tumor pathologic processes, its expression and mechanism in gallbladder cancer (GBC) are still uncertain. Macrophages were divided into M1 and M2 macrophages. TAM is more closely defined as M2 polarized macrophages, which plays a key role in cancer progression., Objective: To clarify the role of disc large associated protein family (DLGAP5) in gallbladder cancer (GBC) progression and investigate the mechanism., Methods: Differential genes in 10 normal paracancer tissues and 10 GBC tissues in GSE139682 from NCBI-GEO were analyzed by R language. Bioinformation analysis and clinical sample analysis were performed to detect DLGAP5 expression in GBC and its correlation with prognosis. CCK-8, EDU, transwell, wound closure, and Immunoblot were performed to detect its effects on the function of GBC cells. GST-pulldown showed the direct interact between DLGAP5 and cAMP. Macrophage polarization assay was further conducted to detect the effects of DLGAP5 on macrophage M2 polarization. The tumor growth assays were further conducted to confirm its role in mice., Results: Biological analysis and clinical samples confirmed that DLGAP5 was increased in GBC and strongly related to poor prognosis in patients with GBC. After overexpression of DLGAP5 in GBC cell lines, such as GBC-SD and NOZ cells, cell proliferation and migration were enhanced, and macrophages were polarized to M2. However, after DLGAP5 is knocked down, there is opposite effect. Mechanistically, DLGAP5 promotes the growth and migration of GBC-SD and NOZ cells and the M2 polarization of THP-1-derived macrophages by activating cyclic adenosine monophosphate (cAMP) pathway. In vivo, GBC-SD with DLGAP5 knockdown was subcutaneously injected into nude mice. It was found that after DLGAP5 knockdown, both tumor volume and tumor were reduced, and indicators related to proliferation and M2 polarization decreased., Conclusion: Our study shows that DLGAP5 is significantly elevated in GBC and is strongly related to poor prognosis in patients with GBC. DLGAP5 promotes GBC proliferation, migration, and M2 polarization of macrophages through cAMP pathway, which provides a theoretical basis for the treatment of GBC and may become a promising therapeutic target., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The granulocyte-colony-stimulating factor (G-CSF) glycoprotein stimulates precursor cell proliferation and differentiation in the bone marrow. Various G-CSF-producing tumors have been reported; they showed early progression and an extremely poor prognosis. Here, we report a case of G-CSF-producing gallbladder cancer with lymph node metastasis. In addition, we reviewed 30 previous case reports of G-CSF-producing gallbladder cancers to elucidate the characteristics and most appropriate treatment. During a routine visit to her local doctor for monitoring of diabetes and hypertension, a 68-year-old female was found to have an elevated white-blood-cell (WBC) count and C-reactive protein (CRP) level, and a gallbladder mass. Laboratory tests revealed a high serum G-CSF level, and imaging revealed a tumor of the gallbladder with regional lymphadenopathy. We diagnosed a G-CSF-producing gallbladder cancer and performed liver resection of segment IVa/V: regional lymph node dissection with extrahepatic bile duct resection. Pathologically, the tumor was a poorly differentiated squamous cell carcinoma. G-CSF immunostaining for tumor cells was positive. She is alive without recurrence at 16 months after surgery. If a patient exhibits a gallbladder tumor, with an elevated WBC count and CRP level but no symptoms of infection, a G-CSF-producing gallbladder cancer should be suspected; radical resection should be performed immediately after diagnosis., (© 2023. Japanese Society of Gastroenterology.)

The prognosis of gallbladder cancer (GBC) remains poor, and a better understanding of GBC molecular mechanisms is important. Genome sequencing of human GBC has demonstrated that loss-of-function mutations of E74-like ETS transcription factor 3 (ELF3) are frequently observed, with ELF3 considered to be a tumour suppressor in GBC. To clarify the underlying molecular mechanisms by which ELF3 suppresses GBC development, we performed in vivo analysis using a combination of autochthonous and allograft mouse models. We first evaluated the clinical significance of ELF3 expression in human GBC tissues and found that low ELF3 expression was associated with advanced clinical stage and deep tumour invasion. For in vivo analysis, we generated Pdx1-Cre; Kras G12D ; Trp53 R172H ; Elf3 f/f (KPCE) mice and Pdx1-Cre; Kras G12D ; Trp53 R172H ; Elf3 wt/wt (KPC) mice as a control and analysed their gallbladders histologically. KPCE mice developed larger papillary lesions in the gallbladder than those developed by KPC mice. Organoids established from the gallbladders of KPCE and KPC mice were analysed in vitro. RNA sequencing showed upregulated expression of epiregulin (Ereg) in KPCE organoids, and western blotting revealed that EGFR/mechanical targets of rapamycin complex 1 (mTORC1) were upregulated in KPCE organoids. In addition, ChIP assays on Elf3-overexpressing KPCE organoids showed that ELF3 directly regulated Ereg. Ereg deletion in KPCE organoids (using CRISPR/Cas9) induced EGFR/mTORC1 downregulation, indicating that ELF3 controlled EGFR/mTORC1 activity through regulation of Ereg expression. We also generated allograft mouse models using KPCE and KPC organoids and found that KPCE organoid allograft tumours exhibited poorly differentiated structures with mTORC1 upregulation and mesenchymal phenotype, which were suppressed by Ereg deletion. Furthermore, EGFR/mTORC1 inhibition suppressed cell proliferation and epithelial-mesenchymal transition in KPCE organoids. Our results suggest that ELF3 suppresses GBC development via downregulation of EREG/EGFR/mTORC1 signalling. EGFR/mTORC1 inhibition is a potential therapeutic option for GBC with ELF3 mutation. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland., (© 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.)

Gallbladder cancer (GBC) is a type of rare but highly aggressive cancer with a dismal prognosis. Runt-related transcription factor 3 (RUNX3), a member of the runt-domain family, and its promoter methylation have been widely observed in a variety of human malignancies. However, the biological function and underlying mechanism of RUNX3 in GBC remain elusive. In this study, bisulfate sequencing PCR (BSP), Western blot, and qPCR were applied to identify the expression level and DNA methylation level of RUNX3 in GBC tissues and cells. The transcriptional relationship between RUNX3 and Inhibitor of growth 1 (ING1) was validated by dual-luciferase reporter assay and ChIP assay. A series of gain-of-function and loss-of-function assays were performed to detect the function and the regulatory relationship of RUNX3 in vitro and in vivo. RUNX3 was aberrantly downregulated in GBC cells and tissues caused by DNA Methyltransferase 1 (DNMT1)-mediated methylation, and downregulation of RUNX3 is associated with poor prognosis of GBC patients. Functional experiments reveal that RUNX3 can induce ferroptosis of GBC cells in vitro and in vivo. Mechanistically, RUNX3 induces ferroptosis by activating ING1 transcription, thereby repressing SLC7A11 in a p53-dependent manner. In conclusion, the downregulation of RUNX3 is mediated by DNA methylation, which promotes the pathogenesis of gallbladder cancer through attenuating SLC7A11-mediated ferroptosis. This study gives novel insights into the role of RUNX3 in the ferroptosis of GBC cells, which may contribute to developing potential treatment targets for GBC., Competing Interests: Declaration of Competing Interest The authors assert that no competing interests exist in relation to this work., (Copyright © 2023. Published by Elsevier Inc.)

The pathogenesis of gallbladder cancer is complex, involving environmental and genetic risk factors. The matrix metallopeptidase 14 (MMP14) alters the tumor microenvironment and promotes tumorigenesis. In this study, we have characterized the role of the MMP14 promoter variants rs1004030 and rs1003049 in gallbladder cancer pathogenesis. Previously, we have shown the association of rs1004030 and rs1003049 with GBC and allele-specific differential expression of MMP14 in GBC patients. These variants reside within the cis-regulatory element (CRE) with high DNase and H3K4me3 signals, suggesting an active regulatory role in MMP14 expression. The luciferase-based reporter assay showed the role of promoter variants on expression levels in two GBC cell lines. Deleting the 119 bp promoter region surrounding the variants rs1004030 and rs1003049 by CRISPR-Cas9 genome editing resulted in reduced MMP14 expression in G415 cells. Electrophoretic mobility shift assay shows the presence of risk allele 'C'/'G' at rs1004030 and rs1003049 and create binding sites for transcription factors SOX10 and MYB, respectively. Further, stable knockdown of these transcription factors in G415 and TGBC1TKB cells showed reduced expression of MMP14. However, in both GBC cells, ectopic expression of these transcription factors increased the expression of MMP14. Rescue of MYB and SOX10 expression levels showed a significant increase in luciferase activity only in risk allele-carrying constructs. In conclusion, our study unveils a mechanistic role of the MMP14 promoter variants rs1004030 and rs1003049 in gallbladder cancer., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Gallbladder cancer (GBC) is the most aggressively malignant tumor in the bile duct system. The prognosis for patients with GBC is extremely poor. Ponicidin is a diterpenoid compound extracted and purified from the traditional Chinese herb Rabdosia rubescens, and showed promising anti-cancer effects in a variety of tumors. However, Ponicidin has not been investigated in GBC., Methods: CCK-8, colony formation assay and EdU-488 DNA synthesis assay were performed to investigate the effect of Ponicidin on GBC cells proliferation. Cell invasion and migration assays and wound-healing assay were used to explore the effect of Ponicidin on invasion and migration ability of GBC cells. mRNA-seq was adopted to explore the underlying mechanisms. Western blot and immunohistochemical staining were conducted to detect the protein level. CHIP assay and dual-luciferase assay were used to validate binding motif. Nude mouse model of GBC was used to assess the anti-tumor effect and safety of Ponicidin., Results: Ponicidin inhibited the proliferation and cell invasion and migration of GBC cells in vitro. Moreover, Ponicidin exerted anti-tumor effects by down-regulating the expression of MAGEB2. Mechanically, Ponicidin upregulated the FOXO4 expression and promoted it to accumulate in nucleus to inhibit the transcript of MAGEB2. Furthermore, Ponicidin suppressed tumor growth in the nude mouse model of GBC with excellent safety., Conclusion: Ponicidin may be a promising agent for the treatment of GBC effectively and safely., Competing Interests: Declaration of Competing Interest There are no conflicts of interest to declare., (Copyright © 2023 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Despite of the high lethality of gallbladder cancer (GBC), little is known regarding molecular regulation of the tumor immunosuppressive microenvironment. Here, we determined tumor expression levels of YKL-40 and the molecular mechanisms by which YKL-40 regulates escape of anti-tumor immune surveillance. We found that elevated expression levels of YKL-40 in plasma and tissue were correlated with tumor size, stage IV and lymph node metastasis. Single cell transcriptome analysis revealed that YKL-40 was predominantly derived from M2-like subtype of infiltrating macrophages. Blockade of M2-like macrophage differentiation of THP-1 cells with YKL-40 shRNA resulted in reprogramming to M1-like macrophages and restricting tumor development. YKL-40 induced tumor cell expression and secretion of growth differentiation factor 15 (GDF15), thus coordinating to promote PD-L1 expression mediated by PI3K, AKT and/or Erk activation. Interestingly, extracellular GDF15 inhibited intracellular expression of GDF15 that suppressed PD-L1 expression. Thus, YKL-40 disrupted the balance of pro- and anti-PD-L1 regulation to enhance expression of PD-L1 and inhibition of T cell cytotoxicity, leading to tumor immune evasion. The data suggest that YKL-40 and GDF15 could serve as diagnostic biomarkers and immunotherapeutic targets for GBC., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Gallbladder cancer (GBC) performs strongly invasive and poor prognosis, and adenocarcinoma is the most common histological type in it. Statistically, the 5-year survival rate of patients with advanced GBC is less than 5%. Such dismal outcome might be caused by chemotherapy resistance and native biology of tumor cells, regardless of emerging therapeutic strategies. Early diagnosis, depending on biomarkers, receptors and secretive proteins, is more important than clinical therapy, guiding the pathologic stage of cancer and the choice of medication. Therefore, it is in urgent need to understand the specific pathogenesis of GBC and strive to find promising novel biomarkers for early screening in GBC. Non-coding RNAs (ncRNAs), especially microRNAs (miRNAs, miRs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs), are confirmed to participate in and regulate the occurrence and development of GBC. Exceptionally, lncRNAs and circRNAs could act as competing endogenous RNAs (ceRNAs) containing binding sites for miRNAs and crosstalk with miRNAs to target regulatory downstream protein-coding messenger RNAs (mRNAs), thus affecting the expression levels of specific proteins to participate in and regulate the development and progression of GBC. It follows that ncRNAs may become promising biomarkers and potential therapeutic targets for GBC. In this review, we mainly summarize the recent research progress of miRNAs and lncRNAs in regulating the development and progression of GBC, chemoresistance, and predicting the prognosis of patients, and highlight the potential applications of the lncRNA/circRNA-miRNA-mRNA cross-regulatory networks in early diagnosis, chemoresistance, and prognostic evaluation, aiming to better understand the pathogenesis of GBC and develop new diagnostic and therapeutic strategies., (© 2022. The Author(s), under exclusive licence to Federación de Sociedades Españolas de Oncología (FESEO).)

Orthodenticle homeobox (OTX1) is reported to be involved in numerous cancers, but the expression level and molecular function of OTX1 in gallbladder cancer (GBC) remain unknown. Here, we found the elevated level of OTX1 associated with poor prognosis in human gallbladder cancer. In vitro and in vivo studies of human gallbladder cancer cell lines demonstrated that overexpression of OTX1 promoted cell proliferation, whereas the downregulation inhibited it. Additionally, we found a tight correlation between the serum level of taurodeoxycholic acid (TDCA) and OTX1 expression. TDCA-induced activation of YAP1 by phosphorylation inhibition contributed to the transcriptional activation of OTX1. Mechanistically, we identified that OTX1 activated AKT signaling pathway by transactivating the expression of IFITM3 and thus promoted the proliferation of GBC cells. Taken together, our results showed that TDCA-YAP1-dependent expression of OTX1 regulated IFITM3 and affected GBC proliferation via the AKT signaling pathway. Our experiments also suggested that OTX1 is a novel therapeutic target for GBC., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Treatment options for advanced gallbladder cancer (GBC) are scarce and usually rely on cytotoxic chemotherapy, but the effectiveness of any regimen is limited and recurrence rates are high. Here, we investigated the molecular mechanisms of acquired resistance in GBC through the development and characterization of two gemcitabine-resistant GBC cell sublines (NOZ GemR and TGBC1 GemR). Morphological changes, cross-resistance, and migratory/invasive capabilities were evaluated. Then, microarray-based transcriptome profiling and quantitative SILAC-based phosphotyrosine proteomic analyses were performed to identify biological processes and signaling pathways dysregulated in gemcitabine-resistant GBC cells. The transcriptome profiling of parental and gemcitabine-resistant cells revealed the dysregulation of protein-coding genes that promote the enrichment of biological processes such as epithelial-to-mesenchymal transition and drug metabolism. On the other hand, the phosphoproteomics analysis of NOZ GemR identified aberrantly dysregulated signaling pathways in resistant cells as well as active kinases, such as ABL1, PDGFRA, and LYN, which could be novel therapeutic targets in GBC. Accordingly, NOZ GemR showed increased sensitivity toward the multikinase inhibitor dasatinib compared to parental cells. Our study describes transcriptome changes and altered signaling pathways occurring in gemcitabine-resistant GBC cells, which greatly expands our understanding of the underlying mechanisms of acquired drug resistance in GBC.

Background: Gallbladder carcinoma is the most common cancer of the biliary tract, and the immune checkpoint blockade showed promising efficacy in the treatment of advanced gallbladder carcinoma. However, the underlying mechanisms remain unknown., Methods: Single-cell RNA sequencing was used to reveal immune cell dynamics in an anti-PD-1 responder with gallbladder carcinoma liver metastases. Gene set variation analysis, pseudotime analysis, single-cell regulatory network inference and clustering analysis, and CellChat analysis were used to identify the functions of each cell cluster. Immunohistochemistry and multicolored immunohistochemistry analysis were applied to confirm the intratumoral cell types, and the prognostic value of CXCL13+CD8+T cells in patients with gallbladder carcinoma liver metastases with immunotherapy was evaluated. Four biliary tract carcinoma and 3 immunotherapy bulk RNA-seq datasets were analyzed to investigate the prognostic value of CXCL13+CD8+T cells and SPP1+TAMs., Result: A total of 19,648 high-quality single-cell transcriptome data were obtained from liver metastasis before and after aPD-1 therapy. We discovered improved cytotoxic activity in CD8+T cells and enhanced proinflammatory phenotypes in myeloid cells. The identified SPP1+TAMs were related to poor prognosis. The increased effector/memory T cells represented characteristics similar to exhausted T cells in transitory status after aPD-1therapy, which may play a crucial role in the antitumor immune response. We further revealed that CXCL13+T cells in a high subtype of biliary tract carcinoma were characterized as a 'hot tumor' profile with high immune scores, correlated to the immunostimulatory context with favorable survival, and can predict effective responses to immunotherapy., Conclusions: Our study provided an overview of immune cell dynamics in gallbladder carcinoma liver metastases after aPD-1 treatment and highlighted the importance of CXCL13+T cells in biliary tract carcinoma and effective responses to immunotherapy, which would advance the understanding and treatment of the disease., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Association for the Study of Liver Diseases.)

Cell adhesion molecule 4 (CADM4) is involved in intercellular interactions and is a tumor-suppressor candidate. The role of CADM4 in gallbladder cancer (GBC) has not been reported. Therefore, the clinicopathological significance and prognostic value of CADM4 expression in GBC were evaluated in the present study. Immunohistochemistry (IHC) was performed on 100 GBC tissues to assess CADM4 expression at the protein level. The association between CADM4 expression and the clinicopathological characteristics of GBC was analyzed, and the prognostic significance of CADM4 expression was evaluated. Low CADM4 expression was significantly associated with advanced T category ( p = 0.010) and high AJCC stage ( p = 0.019). In a survival analysis, low CADM4 expression was associated with shorter overall survival (OS; p = 0.001) and recurrence-free survival (RFS; p = 0.018). In univariate analyses, low CADM4 expression was associated with shorter OS ( p = 0.002) and RFS ( p = 0.023). In multivariate analyses, low CADM4 expression was an independent prognostic factor of OS ( p = 0.013). Low CADM4 expression was associated with tumor invasiveness and poor clinical outcomes in patients with GBC. CADM4 may play an important role in cancer progression and patient survival and can be used as a potential prognostic marker of GBC.

To demonstrate the infrared (IR)-based bile analysis as a reliable screening tool for gall bladder (GB) cancer, we analyzed a sample set of 37 diverse bile samples (five normal, 18 GB polyp, six hepatocellular carcinoma (HCC), and eight GB cancer subjects). Bile samples of normal subjects (control) and HCC patients were newly included to examine if IR-based bile analysis could be expanded to identify HCC. Concentrations of three bile acids and eight bile salts in the aqueous phase samples were determined in parallel and lipidomic analysis of nine lipid classes in the organic phase samples was performed using liquid chromatography-mass spectrometry. Concentrations of bile salts were lower and relative abundances of bile salts were dissimilar between GB cancer samples and remained group samples. Also, the levels of lipids such as phosphatidylcholines and phosphatidylethanolamines were again lower and their relative abundances in the organic phase of GB cancer samples were different from those of other samples. IR spectral features of the aqueous, organic, and amphiphilic aggregate phases were individually characteristic, while not descriptive enough for the thorough identification of GB cancer. Nonetheless, since they were mutually complementary to represent different metabolites in bile, the use of three phase-merged spectra was synergetic to yield the superior discrimination of GB cancer., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The microRNAs (miRNAs) in circulation could serve as biomarkers for cancer detection. Gallbladder carcinoma (GBC) is mostly asymptomatic; therefore, using microRNAs (miRNAs) as an early diagnostic biomarker could be a valuable tool. We aimed to identify the tumor-associated miR-1, miR130, miR-146, miR-182, and miR-21expression in serum as a biomarker for early detection of GBC and identify their possible diagnostic role. The study group comprised of paired serum and tissue samples from 34 GBC, 19 cholecystitis (CC), 21 normal controls (uninflamed gall bladder), and additional 29 serum-only samples of GBC. Total RNA was isolated using a commercially available RNA isolation kit (Applied Biosystem, USA) and reverse transcribed using Advanced Taqman MicroRNA reverse transcription kit. The relative expression of miRNAs was analyzed using Quantitative real-time polymerase chain reaction. The diagnostic potential of these miRNAs was assessed by ROC analysis. In paired samples, the trend towards up and down regulation for miR-182, miR-21, miR-1, miR-130, and miR-146 was similar in both tissue and sera of GBC. The expression pattern of serum miR-1, miR130, and miR-146 gradually decreased from normal control (NC) to CC to GBC, while miR-21 and miR-182 gradually increased from NC to CC to GBC. The miR-1, miR-121, miR-182, and miR-146 significantly differed between CC vs. early stage and early stage vs. NC. Among these miRNAs, the sensitivity of miR-1 (85.71 %) was the highest, and the specificity of miR-21 was the highest (92.73 %). The combined sensitivity for miRNAs ranged from 73.13 % (CI: 60.90-83.24 %) to 98.63 % (CI: 89.0-99.61 %); however, the specificity was lower. In stage I&II vs. III&IV discrimination, the diagnostic sensitivity of miR-1 was highest (89.36 %, CI: 76.90-96.45). The two miRNAs, in combination, increase the diagnostic sensitivity. Circulating serum miRNAs may provide a new approach for clinical application. Panels of specific circulating miRNA, which require further validation, could be potential non-invasive diagnostic biomarkers for GBC in combination with abnormal radio diagnostic scans., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier GmbH. All rights reserved.)

Gallbladder carcinoma (GBC), an aggressive malignant tumor of the biliary system, is characterized by high cellular heterogeneity and poor prognosis. Fewer data have been reported in GBC than other common cancer types. Multi-omics data will contribute to the understanding of the molecular mechanisms of cancer, cancer diagnosis and prognosis. Herein, to provide better understanding of the molecular events in GBC pathogenesis, we developed GBCdb ( http://tmliang.cn/gbc/ ), a user-friendly interface for the query and browsing of GBC-associated genes and RNA interaction networks using published multi-omics data, which also included experimentally supported data from different molecular levels. GBCdb will help to elucidate the potential biological roles of different RNAs and allow for the exploration of RNA interactions in GBC. These resources will provide an opportunity for unraveling the potential molecular features of Gallbladder carcinoma., (© 2023. The Author(s).)

Gallbladder cancer (GBC) ranks seventh among the gastrointestinal cancers. Messenger RNAs (mRNAs) could regulate the progression of GBC. For the purpose of exploring the targets for GBC treatment, RNA sequencing was used to identify the differential expressed mRNAs between GBC and adjacent tissues. Next, CCK8 assay was used to assess the cell viability, and cell proliferation was investigated by colony formation assay. Flow cytometry was performed to evaluate the cell apoptosis. Protein and mRNA expression were analyzed by western blot and RT-qPCR, respectively. Transwell was performed to evaluate the cell metastasis. GBC-derived exosomes were isolated with ultracentrifugation. To evaluate the function of exosomes in GBC, in vivo model of GBC was constructed. The data revealed IGF2BP3 was identified to be upregulated in GBC, and IGF2BP3 silencing was able to decrease GBC cell proliferation by promoting the apoptosis. The migration and invasion of GBC cells were reduced by IGF2BP3 knockdown. Silencing of IGF2BP3 obviously suppressed the level of p-STAT3 in GBC cells. Meanwhile, GBC cell-derived exosomes notably promoted macrophage M2 polarization via carrying IGF2BP3, and then the polarized macrophages promoted the malignant behavior of GBC cells. Furthermore, exosomes markedly promoted the tumor growth of GBC via promoting macrophage M2 polarization. In summary, knockdown of IGF2BP3 suppressed the malignant behavior of GBC cells. Additionally, knockdown of IGF2BP3 modified tumor microenvironment during the progression of GBC. Thus, these findings might provide a new theoretical basis for exploring a strategies against GBC., (© 2022 Wiley Periodicals LLC.)

Ubiquitin-specific protease 3 (USP3), a kind of cysteine protease, is a crucial family member of deubiquitinating enzymes. USP3 is aberrantly expressed in several tumors, which may contribute to cancer progression. However, the role of USP3 in gallbladder cancer (GBC) is still unknown. In the current study, we detected the expression of USP3 in GBC tissues, measured its contribution to the cell proliferation in GBC progression, and further studied the underlying mechanism of USP3 in GBC through pyruvate kinase L/R (PKLR; a kind of glycolytic enzyme). We found that the expression of USP3 in GBC tissues were higher than that of adjacent tissues, and the protein levels of USP3 and PKLR were positively correlated. Additionally, overexpressed USP3 significantly promoted cell proliferation in vitro and tumor growth in vivo, while the silencing of USP3 inhibited proliferation and tumor growth. Glycolysis in GBC cells ws promoted by the USP3 overexpression and inhibited bye USP3 downregulation. Moreover, the loss of USP3 promoted the ubiquitination and weakened the stability of PKLR. Results of the rescue assay confirmed that PKLR knockdown suppressed USP3-induced oncogenic activity in USP3 overexpressed GBC cells. These findings imply that USP3 is an essential positive regulator in GBC progression, and USP3-PKLR plays a vital role in the progression and metabolism of GBC., (© 2022. The Author(s), under exclusive licence to United States and Canadian Academy of Pathology.)

Background: tRNA-derived fragments (tRFs) are newly discovered noncoding RNAs and regulate tumor progression via diverse molecular mechanisms. However, the expression and biofunction of tRFs in gallbladder cancer (GBC) have not been reported yet., Methods: The expression of tRFs in GBC was detected by tRF and tiRNA sequencing in GBC tissues and adjacent tissues. The biological function of tRFs was investigated by cell proliferation assay, clonal formation assay, cell cycle assay, and xenotransplantation model in GBC cell lines. The molecular mechanism was discovered and verified by transcriptome sequencing, fluorescence in situ hybridization (FISH), target gene site prediction, and RNA binding protein immunoprecipitation (RIP)., Results: tRF-3013b was significantly downregulated in GBC compared with para-cancer tissues. Decreased expression of tRF-3013b in GBC patients was correlated with poor overall survival. Dicer regulated the production of tRF-3013b, and its expression was positively correlated with tRF-3013b in GBC tissues. Functional experiments demonstrated that tRF-3013b inhibited GBC cell proliferation and induced cell-cycle arrest. Mechanically, tRF-3013b exerted RNA silencing effect on TPRG1L by binding to AGO3, and then inhibited NF-κB. TPRG1L overexpression could rescue the effects of tRF-3013b on GBC cell proliferation., Conclusions: This study indicated that Dicer-induced tRF-3013b inhibited GBC proliferation by targeting TPRG1L and repressed NF-κB, pointing to tRF-3013b as a novel potential therapeutic target of GBC., (© 2022. The Author(s).)

Lithocholic acid (LCA), one of the most common metabolic products of bile acids (BAs), is originally synthesized in the liver, stored in the gallbladder, and released to the intestine, where it assists absorption of lipid-soluble nutrients. LCA has recently emerged as a powerful reagent to inhibit tumorigenesis; however, the anti-tumor activity and molecular mechanisms of LCA in gallbladder cancer (GBC) remain poorly acknowledged. Here, we analyzed serum levels of LCA in human GBC and found that LCA was significantly downregulated in these patients, and reduced LCA levels were associated with poor clinical outcomes. Treatment of xenografts with LCA impeded tumor growth. Furthermore, LCA treatment in GBC cell lines decreased glutaminase (GLS) expression, glutamine (Gln) consumption, and GSH/GSSG and NADPH/NADP + ratios, leading to cellular ferroptosis. In contrast, GLS overexpression in tumor cells fully restored GBC proliferation and decreased ROS imbalance, thus suppressing ferroptosis. Our findings reveal that LCA functions as a tumor-suppressive factor in GBC by downregulating GLS-mediated glutamine metabolism and subsequently inducing ferroptosis. This study may offer a new therapeutic strategy tailored to improve the treatment of GBC., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Inc.)

Background: Dual specificity phosphatase 4 (DUSP4), which regulates the mitogen activated protein kinases, has emerged as a tumor suppressor gene in several human malignancies., Aims and Objectives: In this study, we investigated the clinicopathologic significance and the prognostic role of DUSP4 in gallbladder adenocarcinoma. Materials and methods: DUSP4 expression was evaluated immunohistochemically in tissue microarray from 110 gallbladder adenocarcinoma samples and scored by H score system. The cut off (H score <170) was determined by ROC curve analysis. Results: Low expression of DUSP4 expression was observed in 57 (51.8%) out of 110 gallbladder adenocarcinoma samples. Low expression of DUSP4 expression was significantly associated with high histologic grade (P = 0.017), high pT stage (P = 0.002) and high AJCC stage (P = 0.007). Kaplan Meier survival curves revealed that patients with low expression of DUSP4 expression had significantly worse cancer specific survival (P = 0.024, log rank test). However, there was no significant association between DUSP4 expression and recurrence free survival., Conclusions: In conclusion, gallbladder adenocarcinoma with low expression of DUSP4 expression was associated with adverse clinicopathologic characteristics and poor patient outcome.patient outcome., Competing Interests: None

Background: Gallbladder cancer (GBC) is a mortal malignancy with limited therapeutic strategies. We aimed to develop novel immune scoring systems focusing on B7-H3, B7-H4, and HHLA2. We further investigated their potential clinical effects in predicting survival and immunotherapeutic efficacy for GBC., Methods: This was a retrospective cohort study in a single center that explored the expression characteristics of B7-H3, B7-H4, and HHLA2. The immune scoring nomograms for prognostic were developed via logistic regression analyses. Their performance was evaluated using the Harrell concordance index (C-index) and decision curves analysis (DCA), and validated with calibration curves., Results: B7-H3, B7-H4, and HHLA2 manifested with a relatively high rate of co-expression patterns in GBC tissues. They were associated with worse clinicopathological stage, suppression of immune microenvironment, and unfavorable prognosis in postoperative survival. B7 stratification established based on B7-H3, B7-H4, and HHLA2 was an independent prognostic predictor (p<0.05 in both groups). Moreover, immune stratification was also successfully constructed based on B7 stratification and the density of CD8 + TILs (all p<0.001). The prediction models were developed based on B7-/or immune stratification combined with the TNM/or Nevin staging system. These novel models have excellent discrimination ability in predicting survival and immunotherapeutic efficacy for GBC patients by DCA and clinical impact plots. Finally, dynamic nomograms were developed for the most promising clinical prediction models (B7-TNM model and Immune-TNM model) to facilitate prediction., Conclusions: Immune scoring systems focusing on B7-H3, B7-H4, and HHLA2 may effectively stratify the prognosis of GBC. Prognostic nomograms based on novel immune scoring systems may potentially predict survival and immunotherapeutic efficacy in GBC. Further valid verification is necessary., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lv, Han, Wu, Liang, Li, Zhang, Lang, Zhong, Fu, Yu, Xu and Tian.)

Purpose: This study aimed to investigate the efficiency of our chemically synthesized TT-00420, a novel spectrum-selective multiple protein kinase inhibitor, in cultured cells and animal models of gallbladder cancer (GBC) and explore its potential mechanism., Methods: Multiple GBC models were established to assess the anti-tumor efficiency, toxicity, and pharmacokinetics of TT-00420. Integrated transcriptomic, proteomic and phosphoproteomic analysis was conducted to identify potential downstream effectors of TT-00420. Western blotting, qRT-PCR, nuclear-cytoplasm separation, and immunofluorescence were performed to confirm the multi-omic results and explore the molecular mechanism of TT-00420. Immunohistochemistry was used to detect FGFR1 and p-FGFR1 expression levels in GBC samples. Autodock software was utilized to investigate the potential binding mode between the TT-00420 and the human FGFR1., Results: We found that TT-00420 exerted potent growth inhibition of GBC cell lines and multiple xenograft models. Treatment of mice with 15 mg/kg TT-00420 via gavage displayed a half-life of 1.8 h in the blood and rapid distribution to the liver, kidneys, lungs, spleen, and tumors at 0.25 h, but no toxicity to these organs over 2 weeks. Multi-omic analysis revealed c-Jun as a potential downstream effector after TT-00420 treatment. Mechanistically, TT-00420 showed rigorous ability to block FGFR1 and its downstream JNK-JUN (S63/S73) signaling pathway, and induce c-Jun S243-dependent MEK/ERK reactivation, leading to FASLG-dependent tumor cell death. Finally, we found that FGFR1 and p-FGFR1 expression was elevated in GBC patients and these levels correlated with decreased patient survival., Conclusions: TT-00420 shows potent antitumor efficacy and may serve as a novel agent to improve GBC prognosis., (© 2022. Springer Nature Switzerland AG.)