Bile exosomal miR-182/183-5p increases cholangiocarcinoma stemness and progression by targeting HPGD and increasing PGE2 generation.

BACKGROUND AIMS: Cholangiocarcinoma (CCA) is a highly lethal malignancy originating from the biliary ducts. Current CCA diagnostic and prognostic assessments cannot satisfy the clinical requirement. Bile detection is rarely performed, and herein, we aim to estimate the clinical significance of bile liquid biopsy by assessing bile exosomal concentrations and components. APPROACH RESULTS: Exosomes in bile and sera from CCA, pancreatic cancer, and common bile duct stone were identified and quantified by transmission electronmicroscopy, nanoparticle tracking analysis, and nanoFCM. Exosomal components were assessed by liquid chromatography with tandem mass spectrometry and microRNA sequencing (miRNA-seq). Bile exosomal concentration in different diseases had no significant difference, but miR-182-5p and miR-183-5p were ectopically upregulated in CCA bile exosomes. High miR-182/183-5p in both CCA tissues and bile indicates a poor prognosis. Bile exosomal miR-182/183-5p is secreted by CCA cells and can be absorbed by biliary epithelium or CCA cells. With xenografts in humanized mice, we showed that bile exosomal miR-182/183-5p promotes CCA proliferation, invasion, and epithelial-mesenchymal transition (EMT) by targeting hydroxyprostaglandin dehydrogenase in CCA cells and mast cells (MCs), and increasing prostaglandin E2 generation, which stimulates PTGER1 and increases CCA stemness. In single-cell mRNA-seq, hydroxyprostaglandin dehydrogenase is predominantly expressed in MCs. miR-182/183-5p prompts MC to release VEGF-A release from MC by increasing VEGF-A expression, which facilitates angiogenesis. CONCLUSIONS: CCA cells secret exosomal miR-182/183-5p into bile, which targets hydroxyprostaglandin dehydrogenase in CCA cells and MCs and increases prostaglandin E2 and VEGF-A release. Prostaglandin E2 promotes stemness by activating PTGER1. Our results reveal a type of CCA self-driven progression dependent on bile exosomal miR-182/183-5p and MCs, which is a new interplay pattern of CCA and bile.

PTPN9 dephosphorylates FGFR2 pY656/657 through interaction with ACAP1 and ameliorates pemigatinib effect in cholangiocarcinoma.

ABSTRACT AND AIM: Cholangiocarcinoma (CCA) is a highly aggressive and lethal cancer that originates from the biliary epithelium. Systemic treatment options for CCA are currently limited, and the first targeted drug of CCA, pemigatinib, emerged in 2020 for CCA treatment by inhibiting FGFR2 phosphorylation. However, the regulatory mechanism of FGFR2 phosphorylation is not fully elucidated. APPROACH AND RESULTS: Here we screened the FGFR2-interacting proteins and showed that protein tyrosine phosphatase (PTP) N9 interacts with FGFR2 and negatively regulates FGFR2 pY656/657 . Using phosphatase activity assays and modeling the FGFR2-PTPN9 complex structure, we identified FGFR2 pY656/657 as a substrate of PTPN9, and found that sec. 14p domain of PTPN9 interacts with FGFR2 through ACAP1 mediation. Coexpression of PTPN9 and ACAP1 indicates a favorable prognosis for CCA. In addition, we identified key amino acids and motifs involved in the sec. 14p-APCP1-FGFR2 interaction, including the "YRETRRKE" motif of sec. 14p, Y471 of PTPN9, as well as the PH and Arf-GAP domain of ACAP1. Moreover, we discovered that the FGFR2 I654V substitution can decrease PTPN9-FGFR2 interaction and thereby reduce the effectiveness of pemigatinib treatment. Using a series of in vitro and in vivo experiments including patient-derived xenografts (PDX), we showed that PTPN9 synergistically enhances pemigatinib effectiveness and suppresses CCA proliferation, migration, and invasion by inhibiting FGFR2 pY656/657 . CONCLUSIONS: Our study identifies PTPN9 as a negative regulator of FGFR2 phosphorylation and a synergistic factor for pemigatinib treatment. The molecular mechanism, oncogenic function, and clinical significance of the PTPN9-ACAP1-FGFR2 complex are revealed, providing more evidence for CCA precision treatment.

Function of mast cell and bile-cholangiocarcinoma interplay in cholangiocarcinoma microenvironment.

OBJECTIVE: The correlation between cholangiocarcinoma (CCA) progression and bile is rarely studied. Here, we aimed to identify differential metabolites in benign and malignant bile ducts and elucidate the generation, function and degradation of bile metabolites. DESIGN: Differential metabolites in the bile from CCA and benign biliary stenosis were identified by metabonomics. Biliary molecules able to induce mast cell (MC) degranulation were revealed by in vitro and in vivo experiments, including liquid chromatography-mass spectrometry (MS)/MS and bioluminescence resonance energy transfer assays. Histamine (HA) receptor expression in CCA was mapped using a single-cell mRNA sequence. HA receptor functions were elucidated by patient-derived xenografts (PDX) in humanised mice and orthotopic models in MC-deficient mice. Genes involved in HA-induced proliferation were screened by CRISPR/Cas9. RESULTS: Bile HA was elevated in CCA and indicated poorer prognoses. Cancer-associated fibroblasts (CAFs)-derived stem cell factor (SCF) recruited MCs, and bile N,N-dimethyl-1,4-phenylenediamine (DMPD) stimulated MCs to release HA through G protein-coupled receptor subtype 2 (MRGPRX2)-Gαq signalling. Bile-induced MCs released platelet-derived growth factor subunit B (PDGF-B) and angiopoietin 1/2 (ANGPT1/2), which enhanced CCA angiogenesis and lymphangiogenesis. Histamine receptor H1 (HRH1) and HRH2 were predominantly expressed in CCA cells and CAFs, respectively. HA promoted CCA cell proliferation by activating HRH1-Gαq signalling and hastened CAFs to secrete hepatocyte growth factor by stimulating HRH2-Gαs signalling. Solute carrier family 22 member 3 (SLC22A3) inhibited HA-induced CCA proliferation by importing bile HA into cells for degradation, and SLC22A3 deletion resulted in HA accumulation. CONCLUSION: Bile HA is released from MCs through DMPD stimulation and degraded via SLC22A3 import. Different HA receptors exhibit a distinct expression profile in CCA and produce different oncogenic effects. MCs promote CCA progression in a CCA-bile interplay pattern.

BMI1 promotes cholangiocarcinoma progression and correlates with antitumor immunity in an exosome-dependent manner.

BACKGROUND: Cholangiocarcinoma (CCA) is a class of malignant tumors originating from bile duct epithelial cells. Due to difficult early diagnosis and limited treatment, the prognosis of CCA is extremely poor. BMI1 is dysregulated in many human malignancies. However, the prognostic significance and oncogenic role of BMI1 in cholangiocarcinoma (CCA) are not well elucidated. METHODS: In the present study, we investigated its clinical importance and the potential mechanisms in the progression of CCA. We detected BMI1 expression in a large CCA cohort. We demonstrated that BMI1 was substantially upregulated in CCA tissues and was identified as an independent prognostic biomarker of CCA. Moreover, overexpression of BMI1 promoted CCA proliferation, migration, and invasion. And BMI1 knockdown could inhibit proliferation and metastases of CCA in vitro and in vitro/vivo validation. Interestingly, we found that CCA-derived exosomes contain BMI1 proteins, which can transfer BMI1 between CCA cells. The unique BMI1-containing exosomes promote CCA proliferation and metastasis through autocrine/paracrine mechanisms. In addition, we demonstrated that BMI1 inhibits CD8+T cell-recruiting chemokines by promoting repressive H2A ubiquitination in CCA cells. CONCLUSIONS: BMI1 is an unfavorable prognostic biomarker of CCA. Our data depict a novel function of BMI1 in CCA tumorigenesis and metastasis mediated by exosomes. Besides, BMI1 inhibition may augment immune checkpoint blockade to inhibit tumor progression by activating cell-intrinsic immunity of CCA.

WDR5 facilitates EMT and metastasis of CCA by increasing HIF-1α accumulation in Myc-dependent and independent pathways.

Cholangiocarcinoma (CCA) is a highly aggressive malignancy with extremely poor prognoses. The oncogenic role and prognostic value of c-Myc in CCA is not well elucidated. WD repeat domain 5 (WDR5) is a critical regulatory factor directly interacting with c-Myc to regulate c-Myc recruitment at chromosomal locations, but the interaction of WDR5 and c-Myc in CCA was uncovered. In our study, we detected WDR5 and c-Myc expression in all CCA types, including intrahepatic (iCCA), perihilar (pCCA), and distal (dCCA) CCA, and evaluated their prognostic significance. Consequently, we demonstrated that WDR5 was significantly correlated with poor prognosis of CCA and that WDR5 and c-Myc co-expression was a more sensitive prognostic factor. With in vitro and in vivo experiments and bioinformatics, we showed that WDR5 interacted with the Myc box IIIb (MBIIIb) motif of c-Myc and facilitated Myc-induced HIF1A transcription, thereby promoting the epithelial-mesenchymal transition (EMT), invasion, and metastasis of CCA. Moreover, WDR5 enhanced hypoxia-inducible factor 1 subunit α (HIF-1α) accumulation by binding with histone deacetylase 2 (HDAC2) and increasing histone 3 lysine 4 acetylation (H3K4ac) deacetylation of the prolyl hydroxylase domain protein 2 (PHD2) promoter, resulting in the attenuation of chromatin opening and PHD2 expression, and eventually leading to HIF-1α stabilization and accumulation. In conclusion, WDR5 facilitated EMT and metastasis of CCA by increasing HIF-1α accumulation in a Myc-dependent pathway to promote HIF-1α transcription and a Myc-independent pathway to stabilize HIF-1α.

AL355338 acts as an oncogenic lncRNA by interacting with protein ENO1 to regulate EGFR/AKT pathway in NSCLC.

BACKGROUND: Non-small cell lung cancer (NSCLC) is a malignancy with considerable morbidity and mortality. Abnormal metabolism is a hallmark of cancer; however, the mechanism of glycolysis regulation in NSCLC progression is not completely understood. Recent studies suggest that some dysregulated long non-coding RNAs (lncRNAs) play important roles in tumor metabolic reprogramming. METHODS: To identify glycolysis-associated-lncRNAs in NSCLC, we compared RNA-sequencing results between high 18F-fluorodeoxyglucose (FDG)-uptake NSCLC tissues and paired paratumor tissues. The transcript abundance of AL355338 in 80 pairs of clinical samples was evaluated by quantitative real-time PCR assay and fluorescence in situ hybridization. The biological role of AL355338 on NSCLC cells were evaluated by functional experiments in vitro and in vivo. Moreover, RNA pull-down, mass spectrometry and RNA immunoprecipitation (RIP) assays were used to identify the protein interacted with AL355338. Co-immunoprecipitation, in situ proximity ligation assays and western blotting were applied to define the potential downstream pathways of AL355338. RESULTS: AL355338 was an upregulated glycolysis-associated lncRNA in NSCLC. Functional assays revealed that AL355338 was critical for promoting aerobic glycolysis and NSCLC progression. Mechanistic investigations showed that AL355338 directly bound with alpha-enolase (ENO1) and enhanced the protein's stability by modulating its degradation and ubiquitination. A positive correlation was observed between AL355338 and ENO1 in NSCLC, and ENO1 was subsequently confirmed to be responsible for the oncogenic role of AL355338. Furthermore, AL355338 was capable of modulating ENO1/EGFR complex interaction and further activating EGFR-AKT signaling. CONCLUSIONS: This study indicates that AL355338 confers an aggressive phenotype to NSCLC, and targeting it might be an effective therapeutic strategy.

Efficacy and safety of 188Re-HEDP in lung cancer patients with bone metastases: a randomized, multicenter, multiple-dose phase IIa study.

PURPOSE: To investigate the pain-relieving effect and safety of three different doses of 188Re-hydroxyethylidine diphosphonate (HEDP) in patients with lung cancer and bone metastases. METHODS: For this randomised, phase 2 and multicenter trial, we enrolled patients with lung carcinoma and multifocal bone metastases and excluded patients who had received bisphosphonates or external-beam radiotherapy within the previous 4 weeks. Fifty-four patients were randomized to receive a single injection of 188Re-HEDP, at doses of 30, 40 or 50 MBq/kg (interval, 12 weeks). Patients were followed-up by assessment of numerical rating scale (NRS) score, global quality of life (QOL) score and adverse events (AEs). ANOVA analysis, Chi-Squared test and LSD-t test were used in this study. RESULTS: Significantly decreased NRS scores relative to baseline were observed in 40 MBq/kg group (Week 0 vs. Week 12: 6.0 ± 1.4 vs. 4.8 ± 2.5, P = 0.033) and 50 MBq/kg group (Week 0 vs. Week 12: 5.5 ± 1.5 vs. 4.5 ± 2.9, P = 0.046). Significant change of global QOL score from baseline was observed in 40 MBq/kg group at week 8 (global QOL score: P = 0.024, pain score: P = 0.041) and 50 MBq/kg group (pain score: P = 0.021) at week 12. No patients withdrew trial because of AEs in three groups. CONCLUSIONS: 188Re-HEDP at dose of 40 and 50 MBq/kg was generally effective to alleviate pain and improve QOL in lung cancer patients with painful bone metastases. 188Re-HEDP was safe and well-tolerated.

Sprouty2 suppresses progression and correlates to favourable prognosis of intrahepatic cholangiocarcinoma via antagonizing FGFR2 signalling.

Fibroblast growth factor receptor 2 (FGFR2) was demonstrated to correlate to the progression and prognosis of intrahepatic cholangiocarcinoma (ICC) by numerous evidences. However, as a well-recognized suppressor of FGFR2 signalling, the clinical significance of Sprouty (SPRY) family of ICC has not been investigated. In our study, the expressions of SPRY1-4 in 20 pairs of fresh tumour tissues were detected with qPCR, and in 108 cases of paraffin-embedded tissues with immunohistochemistry. The prognostic value of SPRY family in ICC was estimated with univariate analysis and multivariate analysis. As a result, SPRY2 was identified as an independent prognostic biomarker predicting favourable prognosis of ICC. High SPRY2 expression was correlated with good differentiation of ICC. With silencing SPRY2 expression, we demonstrated that SPRY2 could suppress FGFR2-induced ERK phosphorylation, migration, invasion and epithelial-mesenchymal transition (EMT) under FGF1 stimulation. By overexpressing SPRY2-wide type or SPRY2-Y55F, the tyrosine-55 of SPRY2 was demonstrated to be essential in suppressing ERK phosphorylation, tumour invasion and EMT of ICC cells. In conclusion, SPRY2 was correlated with favourable prognosis of ICC via suppressing FGFR2-induced ERK phosphorylation, invasion and EMT. The phosphorylation of SPRY2-Y55 was required in this tumour-suppressing function of SPRY2.

Ultrasound contrast agents and ultrasound molecular imaging.

Ultrasound imaging in combination with microbubble contrast agents has demonstrated its potential for molecular imaging of vascular targets. In this article, we systematically review the technologies related to ultrasound molecular imaging. First, we introduce the basic principles of ultrasound molecular imaging. Then, we discuss ultrasound microbubble contrast agents. This includes selection requirements for targeted contrast agent ligands and receptors, targeted ultrasound contrast agent preparation, targeted ultrasound contrast agent ligands connection methods, and microbubble ultrasound contrast agent safety issues. The use of ultrasound molecular imaging in diagnosis and treatment, as well as some issues relating to high intensity focused ultrasound and ultrasound molecular imaging applications in disease diagnosis are also discussed. Applications include detection of inflammation, thrombus, tumors, and neovascularization. Consideration is also given to the acoustic characteristics of microbubbles and the acoustic principles underlying microbubble ultrasound imaging. Several microbubble scattering models are highlighted (including the Rayleigh-Plesset model, the Rayleigh-Plesset-Noltingk-Neppiras-Poritsky model, the Herring model, the Marmottant model, and the non-spherical bubble dynamics model). The interaction between two microbubbles and the effect of radiation on the microbubbles is also discussed. Finally, from an engineering perspective, we summarize the techniques used to improve the quality of ultrasound molecular images (harmonic imaging, perfusion imaging, and deconvolution technology). It is inevitable that ultrasound molecular imaging will continue to mature and can be expected to play an increasingly larger role in diagnosis and treatment of human diseases in the near future.

Establishment and validation of novel predictive models to predict bone metastasis in newly diagnosed prostate adenocarcinoma based on single-photon emission computed tomography radiomics.

PURPOSE: To establish and validate novel predictive models for predicting bone metastasis (BM) in newly diagnosed prostate adenocarcinoma (PCa) via single-photon emission computed tomography radiomics. METHOD: In a retrospective review of the clinical single-photon emission computed tomography (SPECT) database, 176 patients (training set: n = 140; validation set: n = 36) who underwent SPECT/CT imaging and were histologically confirmed to have newly diagnosed PCa from June 2016 to June 2022 were enrolled. Radiomic features were extracted from the region of interest (ROI) in a targeted lesion in each patient. Clinical features, including age, total prostate-specific antigen (t-PSA), and Gleason grades, were included. Statistical tests were then employed to eliminate irrelevant and redundant features. Finally, four types of optimized models were constructed for the prediction. Furthermore, fivefold cross-validation was applied to obtain sensitivity, specificity, accuracy, and area under the curve (AUC) for performance evaluation. The clinical usefulness of the multivariate models was estimated through decision curve analysis (DCA). RESULTS: A radiomics signature consisting of 27 selected features which were obtained by radiomics' LASSO treatment was significantly correlated with bone status (P < 0.01 for both training and validation sets). Collectively, the models showed good predictive efficiency. The AUC values ranged from 0.87 to 0.98 in four models. The AUC values of the human experts were 0.655 and 0.872 in the training and validation groups, respectively. Most radiomic models showed better diagnostic accuracy than human experts in the training and validation groups. DCA also demonstrated the superiority of the radiomics models compared to human experts. CONCLUSION: Radiomics models are superior to humans in differentiating between benign bone and prostate cancer bone metastases; it can be used to facilitate personalized prediction of BM in newly diagnosed PCa patients.