Genomic studies of gastric cancer have identified highly recurrent genomic alterations impacting RHO signalling, especially in the diffuse gastric cancer (DGC) histological subtype. Among these alterations are interchromosomal translations leading to the fusion of the adhesion protein CLDN18 and RHO regulator ARHGAP26. It remains unclear how these fusion constructs impact the activity of the RHO pathway and what is their broader impact on gastric cancer development. Herein, we developed a model to allow us to study the function of this fusion protein in the pathogenesis of DGC and to identify potential therapeutic targets for DGC tumours with these alterations.
We built a transgenic mouse model with LSL-CLDN18-ARHGAP26 fusion engineered into the Col1A1 locus where its expression can be induced by Cre recombinase. Using organoids generated from this model, we evaluated its oncogenic activity and the biochemical effects of the fusion protein on the RHOA pathway and its downstream cell biological effects in the pathogenesis of DGC.
We demonstrated that induction of CLDN18-ARHGAP26 expression in gastric organoids induced the formation of signet ring cells, characteristic features of DGC and was able to cooperatively transform gastric cells when combined with the loss of the tumour suppressor geneTrp53. CLDN18-ARHGAP26 promotes the activation of RHOA and downstream effector signalling. Molecularly, the fusion promotes activation of the focal adhesion kinase (FAK) and induction of the YAP pathway. A combination of FAK and YAP/TEAD inhibition can significantly block tumour growth.
These results indicate that the CLDN18-ARHGAP26 fusion is a gain-of-function DGC oncogene that leads to activation of RHOA and activation of FAK and YAP signalling. These results argue for further evaluation of emerging FAK and YAP-TEAD inhibitors for these deadly cancers.

Whether and how the PI3K-AKT pathway, a central node of metabolic homeostasis, is responsible for high-fat-induced non-alcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC) remain a mystery. Characterisation of AKT regulation in this setting will provide new strategies to combat HCC.
Metabolite library screening disclosed that palmitic acid (PA) could activate AKT. In vivo and in vitro palmitoylation assay were employed to detect AKT palmitoylation. Diverse cell and mouse models, including generation of AKT1C77S and AKT1C224S knock-in cells, Zdhhc17 and Zdhhc24 knockout mice and Akt1C224S knock-in mice were employed. Human liver tissues from patients with NASH and HCC, hydrodynamic transfection mouse model, high-fat/high-cholesterol diet (HFHCD)-induced NASH/HCC mouse model and high-fat and methionine/choline-deficient diet (HFMCD)-induced NASH mouse model were also further explored for our mechanism studies.
By screening a metabolite library, PA has been defined to activate AKT by promoting its palmitoyl modification, an essential step for growth factor-induced AKT activation. Biologically, a high-fat diet could promote AKT kinase activity, thereby promoting NASH and liver cancer. Mechanistically, palmitoyl binding anchors AKT to the cell membrane in a PIP3-independent manner, in part by preventing AKT from assembling into an inactive polymer. The palmitoyltransferases ZDHHC17/24 were characterised to palmitoylate AKT to exert oncogenic effects. Interestingly, the anti-obesity drug orlistat or specific penetrating peptides can effectively attenuate AKT palmitoylation and activation by restricting PA synthesis or repressing AKT modification, respectively, thereby antagonising liver tumorigenesis.
Our findings elucidate a novel fine-tuned regulation of AKT by PA-ZDHHC17/24-mediated palmitoylation, and highlight tumour therapeutic strategies by taking PA-restricted diets, limiting PA synthesis, or directly targeting AKT palmitoylation.

Cholangiocarcinoma (CCA) is a heterogeneous malignancy with high mortality and dismal prognosis, and an urgent clinical need for new therapies. Knowledge of the CCA epigenome is largely limited to aberrant DNA methylation. Dysregulation of enhancer activities has been identified to affect carcinogenesis and leveraged for new therapies but is uninvestigated in CCA. Our aim is to identify potential therapeutic targets in different subtypes of CCA through enhancer profiling.
Integrative multiomics enhancer activity profiling of diverse CCA was performed. A panel of diverse CCA cell lines, patient-derived and cell line-derived xenografts were used to study identified enriched pathways and vulnerabilities. NanoString, multiplex immunohistochemistry staining and single-cell spatial transcriptomics were used to explore the immunogenicity of diverse CCA.
We identified three distinct groups, associated with different etiologies and unique pathways. Drug inhibitors of identified pathways reduced tumour growth in in vitro and in vivo models. The first group (ESTRO), with mostly fluke-positive CCAs, displayed activation in estrogen signalling and were sensitive to MTOR inhibitors. Another group (OXPHO), with mostly BAP1 and IDH-mutant CCAs, displayed activated oxidative phosphorylation pathways, and were sensitive to oxidative phosphorylation inhibitors. Immune-related pathways were activated in the final group (IMMUN), made up of an immunogenic CCA subtype and CCA with aristolochic acid (AA) mutational signatures. Intratumour differences in AA mutation load were correlated to intratumour variation of different immune cell populations.
Our study elucidates the mechanisms underlying enhancer dysregulation and deepens understanding of different tumourigenesis processes in distinct CCA subtypes, with potential significant therapeutics and clinical benefits.

Growing evidence indicates that tumour cells exhibit characteristics similar to their lineage progenitor cells. We found that S100 calcium binding protein A10 (S100A10) exhibited an expression pattern similar to that of liver progenitor genes. However, the role of S100A10 in hepatocellular carcinoma (HCC) progression is unclear. Furthermore, extracellular vesicles (EVs) are critical mediators of tumourigenesis and metastasis, but the extracellular functions of S100A10, particularly those related to EVs (EV-S100A10), are unknown.
The functions and mechanisms of S100A10 and EV-S100A10 in HCC progression were investigated in vitro and in vivo. Neutralising antibody (NA) to S100A10 was used to evaluate the significance of EV-S100A10.
Functionally, S100A10 promoted HCC initiation, self-renewal, chemoresistance and metastasis in vitro and in vivo. Of significance, we found that S100A10 was secreted by HCC cells into EVs both in vitro and in the plasma of patients with HCC. S100A10-enriched EVs enhanced the stemness and metastatic ability of HCC cells, upregulated epidermal growth factor receptor (EGFR), AKT and ERK signalling, and promoted epithelial-mesenchymal transition. EV-S100A10 also functioned as a chemoattractant in HCC cell motility. Of significance, S100A10 governed the protein cargos in EVs and mediated the binding of MMP2, fibronectin and EGF to EV membranes through physical binding with integrin αⅤ. Importantly, blockage of EV-S100A10 with S100A10-NA significantly abrogated these enhancing effects.
Altogether, our results uncovered that S100A10 promotes HCC progression significantly via its transfer in EVs and regulating the protein cargoes of EVs. EV-S100A10 may be a potential therapeutic target and biomarker for HCC progression.

OBJECTIVE: N6-methyladenosine (m6A) is tightly associated with the progression of pancreatic ductal adenocarcinoma (PDAC). Another prominent feature of PDAC is metabolic reprogramming, which provides sufficient nutrients to support rapid cell growth via the tumor microenvironment. However, the underlying influences of m6A-associated metabolic genes on the PDAC microenvironment remain poorly understood. Therefore, we sought to construct a survival prediction model using m6A-related genes to clarify the molecular characteristics of PDAC.
METHODS: In the present study, m6A-related metabolic genes were obtained from The Cancer Genome Atlas (TCGA) pancreatic adenocarcinoma dataset and subjected to coexpression analysis. Consensus clustering recognized two distinct subgroups with different immune cell infiltration patterns according to the expression of m6A-associated metabolic genes. Multivariate Cox regression analyses and least absolute shrinkage and selection operator (LASSO) analysis were adopted to create an m6A-related metabolism model. A nomogram including clinical features and the risk score based on the expression of m6A-related metabolism regulators was constructed.
RESULTS: A four-gene signature comprising ATP8B2, GMPS, LDHA and SDR39U1 was built to predict the overall survival (OS) of PDAC patients. This signature also robustly predicted survival in two independent validation cohorts from the International Cancer Genome Consortium (ICGC) and ArrayExpress (E-MTAB-6134). The four-gene signature divided patients into high- and low-risk groups with distinct OS values as verified by the log-rank test. Among the four genes, LDHA was upregulated in both PDAC tissues and cell lines.
CONCLUSIONS: Collectively, we analyzed the immune microenvironment, predicted drug sensitivity and assessed the implications of the mutation landscape based on the crosstalk between m6A regulators and metabolic rewiring, and we also constructed a novel signature based on m6A-associated metabolic genes to predict PDAC prognosis.

CircRNAs have been the focus of research in recent years. They are differentially expressed in various human tumors and can regulate oncogenes and tumor suppressor genes expression through various mechanisms. The diversity, stability, evolutionary conservatism and cell- or tissue-specific expression patterns of circRNAs also endow them with important regulatory roles in promoting or inhibiting tumor cells malignant biological behaviors progression. More interestingly, emerging studies also found that circRNAs can regulate not only other genes expression, but also their parental gene expression and thus influence tumors development. Apart from some conventional features, circRNAs have a certain specificity in the regulation of parental gene expression, with a higher proportion affecting parental gene transcription and easier translation into protein to regulate parental gene expression. CircRNAs are generally thought to be unable to produce proteins and therefore the protein-coding ability exhibited by circRNAs in regulating parental gene expression is unique and indicates that the regulatory effects of parental gene expression by circRNAs are not only a competitive binding relationship, but also a more complex molecular relationship between circRNAs and parental gene, which deserves further study. This review summarizes the molecular mechanisms of circRNAs regulating parental gene expression and their biological roles in tumorigenesis and development, aiming to provide new ideas for the clinical application of circRNAs in tumor-targeted therapy.

Many cancers engage embryonic genes for rapid growth and evading the immune system. SOX9 has been upregulated in many tumours, yet the role of SOX9 in mediating immunosuppressive tumour microenvironment is unclear. Here, we aim to dissect the role of SOX9-mediated cancer stemness attributes and immunosuppressive microenvironment in advanced gastric adenocarcinoma (GAC) for novel therapeutic discoveries.
Bulk RNAseq/scRNA-seq, patient-derived cells/models and extensive functional studies were used to identify the expression and functions of SOX9 and its target genes in vitro and in vivo. Immune responses were studied in PBMCs or CD45+ immune cells cocultured with tumour cells with SOX9high or knockout and the KP-Luc2 syngeneic models were used for efficacy of combinations.
SOX9 is one of the most upregulated SOX genes in GAC and highly expressed in primary and metastatic tissues and associated with poor prognosis. Depletion of SOX9 in patient-derived GAC cells significantly decreased cancer stemness attributes, tumour formation and metastases and consistently increased CD8+ T cell responses when cocultured with PBMCs/CD45+ cells from GAC patients. RNA sequencing identified the leukaemia inhibitory factor (LIF) as the top secreted molecule regulated by SOX9 in tumour cells and was enriched in malignant ascites and mediated SOX9-induced M2 macrophage repolarisation and inhibited T cell function.
Epithelial SOX9 is critical in suppressing CD8+ T cell responses and modified macrophage function in GAC through the paracrine LIF factor. Cotargeting LIF/LIFR and CSF1R has great potential in targeting SOX9-mediated cancer stemness, T cell immunosuppression and metastases suggesting the novel combination therapy against advanced GAC.

The lysyl oxidase-like protein 2 (LOXL2) contributes to tumour progression and metastasis in different tumour entities, but its role in pancreatic ductal adenocarcinoma (PDAC) has not been evaluated in immunocompetent in vivo PDAC models.
Towards this end, we used PDAC patient data sets, patient-derived xenograft in vivo and in vitro models, and four conditional genetically-engineered mouse models (GEMMS) to dissect the role of LOXL2 in PDAC. For GEMM-based studies, K-Ras +/LSL-G12D;Trp53 LSL-R172H;Pdx1-Cre mice (KPC) and the K-Ras +/LSL-G12D;Pdx1-Cre mice (KC) were crossed with Loxl2 allele floxed mice (Loxl2Exon2 fl/fl) or conditional Loxl2 overexpressing mice (R26Loxl2 KI/KI) to generate KPCL2KO or KCL2KO and KPCL2KI or KCL2KI mice, which were used to study overall survival; tumour incidence, burden and differentiation; metastases; epithelial to mesenchymal transition (EMT); stemness and extracellular collagen matrix (ECM) organisation.
Using these PDAC mouse models, we show that while Loxl2 ablation had little effect on primary tumour development and growth, its loss significantly decreased metastasis and increased overall survival. We attribute this effect to non-cell autonomous factors, primarily ECM remodelling. Loxl2 overexpression, on the other hand, promoted primary and metastatic tumour growth and decreased overall survival, which could be linked to increased EMT and stemness. We also identified tumour-associated macrophage-secreted oncostatin M (OSM) as an inducer of LOXL2 expression, and show that targeting macrophages in vivo affects Osm and Loxl2 expression and collagen fibre alignment.
Taken together, our findings establish novel pathophysiological roles and functions for LOXL2 in PDAC, which could be potentially exploited to treat metastatic disease.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) shows a remarkable predilection for liver metastasis. Pro-oncogenic secretome delivery and trafficking via exosomes are crucial for pre-metastatic microenvironment formation and metastasis. This study aimed to explore the underlying mechanisms of how PDAC-derived exosomes (Pex) modulate the liver microenvironment and promote metastasis.
METHODS: C57BL/6 mice were \'educated\' by tail vein Pex injection. The intrasplenic injection liver metastasis and PDAC orthotopic transplantation models were used to evaluate liver metastasis. Stable cell lines CD44v6 (CD44 variant isoform 6) or C1QBP (complement C1q binding protein) knockdown or overexpression was established using lentivirus transfection or gateway systems. A total of 142 patients with PDAC in Huashan Hospital were retrospectively enrolled. Prognosis and liver metastasis were predicted using Kaplan-Meier survival curves and logistic regression models.
RESULTS: Pex tail vein injection induced the deposition of liver fibrotic extracellular matrix, which promoted PDAC liver metastasis. Specifically, the exosomal CD44v6/C1QBP complex was delivered to the plasma membrane of hepatic satellite cells (HSCs), leading to phosphorylation of insulin-like growth factor 1 signalling molecules, which resulted in HSC activation and liver fibrosis. Expression of Pex CD44v6 and C1QBP in PDAC patients with liver metastasis was significantly higher than in PDAC patients without liver metastasis, and simultaneous high expression of exosomal CD44v6 and C1QBP correlated with a worse prognosis and a higher risk of postoperative PDAC liver metastasis.
CONCLUSIONS: The Pex-derived CD44v6/C1QBP complex is essential for the formation of a fibrotic liver microenvironment and PDAC liver metastasis. Highly expressed exosomal CD44v6 and C1QBP are promising biomarkers for predicting prognosis and liver metastasis in patients with PDAC.

BACKGROUND: High tumor mutation burden (TMB) has gradually become a sensitive biomarker for predicting the response to immunotherapy in many cancers, including lung, bladder and head and neck cancers. However, whether high TMB predicts the response to immunotherapy and prognosis in pancreatic ductal adenocarcinoma (PDAC) remained obscure. Hence, it is significant to investigate the role of genes related to TMB (TRGs) in PDAC.
METHODS: The transcriptome and mutation data of PDAC was downloaded from The Cancer Genome Atlas-Pancreatic Adenocarcinoma (TCGA). Five independent external datasets of PDAC were chosen to validate parts of our results. qRT-PCR and immunohistochemical staining were also performed to promote the reliability of this study.
RESULTS: The median overall survival (OS) was significantly increased in TMB_low group compared with the counterpart with higher TMB score after tumor purity adjusted (P = 0.03). 718 differentially expressed TRGs were identified and functionally enriched in some oncogenic pathways. 67 TRGs were associated with OS in PDAC. A prognostic model for the OS was constructed and showed a high predictive accuracy (AUC = 0.849). We also found TMB score was associated with multiple immune components and signatures in tumor microenvironment. In addition, we identified a PDAC subgroup featured with TMBlowMicrosatellite instabilityhigh (MSIhigh) was associated with prolonged OS and a key molecule, ANKRD55, potentially mediating the survival benefits.
CONCLUSIONS: This study analyzed the biological function, prognosis value, implications for mutation landscape and potential influence on immune microenvironment of TRGs in PDAC, which contributed to get aware of the role of TMB in PDAC. Future studies are expected to investigate how these TRGs regulate the initiation, development or repression of PDAC.