Parthenolide inhibits the progression of intrahepatic cholangiocarcinoma by promoting ferroptosis through inhibiting UBD

Background: The incidence of intrahepatic cholangiocarcinoma (ICC) is increasing globally, and its prognosis has not improved substantially in recent years. Understanding the pathogenesis of ICC may provide a theoretical basis for its treatment. In this study, we investigated the effects and underlying mechanisms of fucosyltransferase 5 (FUT5) on the malignant progression of ICC. Methods: FUT5 expression in ICC samples and adjacent nontumor tissues was compared using quantitative real-time polymerase chain reaction and immunohistochemical assays. We performed cell counting kit-8, colony formation, and migration assays to determine whether FUT5 influenced the proliferation and mobility of ICC cells. Finally, mass spectrometry was performed to identify the glycoproteins regulated by FUT5. Results: FUT5 mRNA was significantly upregulated in most ICC samples compared with corresponding adjacent nontumor tissues. The ectopic expression of FUT5 promoted the proliferation and migration of ICC cells, whereas FUT5 knockdown significantly suppressed these cellular properties. Mechanistically, we demonstrated that FUT5 is essential for the synthesis and glycosylation of several proteins, including versican, β3 integrin, and cystatin 7, which may serve key roles in the precancer effects of FUT5. Conclusions: FUT5 is upregulated in ICC and promotes ICC development by promoting glycosylation of several proteins. Therefore, FUT5 may serve as a therapeutic target for the treatment of ICC.

Hepatocellular carcinoma (HCC), a prevalent and aggressive malignancy, is one of the most common malignancies worldwide. Various studies show that ubiquitin D (UBD) is overexpresses in different cancer types and may serve as a potential prognostic factor. Although catenin alpha 3 (CTNNA3) is a tumour suppressor in HCC, the relationship between UBD and CTNNA3 in HCC remains unclear. This study aims to explore the role of UBD in HCC and its relationship with CTNNA3 in HCC cells. In this experimental study, UBD expression in clinical samples was analysed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry. Protein levels of epithelialmesenchymal transition (EMT) markers were evaluated by Western blot. Cellular behaviours that included proliferation, colony formation, migration, and invasion were assessed using the CCK-8, colony formation, EdU, and transwell assays. UBD-mediated ubiquitination of CTNNA3 was examined by in vitro ubiquitination assays. There was a significant elevation in UBD expression in the HCC patients, which correlated with poor prognosis. Knockdown of UBD suppressed the proliferation, colony formation, and EMT of the HCC cells. UBD reduced CTNNA3 expression in HCC cells by promoting the ubiquitination and degradation of CTNNA3. Decreased CTNNA3 expression in HCC patients was associated with poor overall survival. Silencing of CTNNA3 repressed HCC proliferation, migration, invasion, and EMT. CTNNA3 deficiency counteracted the inhibitory effects of UBD knockdown on the malignant behaviour of the HCC cells. UBD plays an oncogenic role in HCC by encouraging proliferation and EMT through promoting CTNNA3 degradation. These findings suggest that targeting the UBD/CTNNA3 axis could be a potential therapeutic strategy for HCC management.

Background To explore Ubiquitin D (UBD) and autophagy in hepatocellular carcinoma (HCC) and the key role of Olaparib targeting UBD in treating HCC. Research design and methods Bioinformatics analysis was conducted to study UBD expression in HCC tissues. qRT-PCR and Western blot measured UBD mRNA/protein levels, autophagy markers, and Gln metabolism proteins in HCC tissues. Cellular thermal shift assay (CETSA) confirmed Olaparib-UBD interaction. A xenograft tumor model was established to observe tumor growth in mice, with qRT-PCR and western blot used to measure UBD expression levels in tumor tissues and Immunohistochemistry (IHC) used to assess expression of Microtubule-associated protein light chain 3 (LC3), sequestosome 1 (P62), solute carrier family 1 member 5 (SLC1A5), and glutaminase (GLS). Results UBD was highly expressed in HCC tissues (p = 7.6e-11). UBD could negatively regulate autophagy levels by activating Gln metabolism. Olaparib could target and downregulate UBD expression, promoting HCC cell autophagy by regulating Gln metabolism pathways. Olaparib treatment in xenograft mice overexpressing UBD significantly reduced tumor growth (p < 0.05), inhibited Gln metabolism pathways, and enhanced HCC cell autophagy. Conclusions Olaparib targeted UBD to promote autophagy in HCC by inhibiting Gln metabolism pathways.

Background: BTC represents the second most common primary cancer of the hepatobiliary system and is associated with a poor prognosis. Activation of PI3K/mTOR and hedgehog pathways has been demonstrated in biliary cancer. Activated mTOR/S6K1 pathway promotes Gli1 transcriptional activity and oncogenesis through S6K1-mediated Gli phosphorylation. Recently, synergistic antitumor efficacy was demonstrated with the combination of hedgehog and mTOR inhibitors, particularly in cases with mTOR/S6K pathway in esophageal cancer (Cancer Cell 2012; 21(3):374-87). Methods: Gene expression profiling was performed on 10 human biliary tract cancer cell lines (MMNK-1, Mz-ChA-1, Sk-ChA-1, KKU-M213, KKU-M214, KKU-M055, KKU-M139, M213L5H, and M213LOH) by real-time qRT-PCR. CellTiter-Glo® luminescent cell viability assay, colony formation, and stem cells morphology spheres were conducted to examine the effects of the combination of Rapamycin and Vismodegib on cell proliferation. Western-blot assay was used to examine the related protein expression. Fluorescence-activated cell sorting (FACS) was used to examine the effects of the combination on BTC stem cells. Results: Based on Gli1 and S6K gene expression, the Mz-ChA-1 and Sk-ChA-1 cell lines were chosen to investigate the effects of the combination of Rapamycin and Vismodegib on BTC (Table 1). Combination of Rapamycin and Vismodegib exhibited the synergistic antiproliferative effect on Mz-ChA-1 cells. The combination of Rapamycin and Vismodegib inhibited the Mz-ChA-1 cell proliferation, colony formation and stem cell sphere formation. This combination therapy decreases the NANOG, OTC3/4, and E-Cadherin gene expression in Mz-ChA-1 stem cells. Furthermore, the drug combination can decrease the phospho-p70S6K and Gli1 protein and increase cleaved caspase-3 expression in Mz-ChA-1 cells. No such effect was observed in Sk-ChA-1 cells.Conclusions: Crosstalk between the mTOR pathway and HH pathway provides a new avenue for combination therapy in BTC with hedgehog and mTOR inhibitors. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C171. Citation Format: Mingxin Zuo, Asif Rashid, Chaitanya Churi, Mien-Chie Hung, Milind Javle. Novel therapeutic strategies targeting the hedgehog signaling pathway and mTOR pathway in biliary tract cancer (BTC). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C171.

Septin 2 accelerates the progression of biliary tract cancer and is negatively regulated by mir-140-5p

Background: Intrahepatic cholangiocarcinoma (ICC) is a prevalent type of cancer originating from epithelial cells of the bile duct within the liver. The molecular mechanisms underlying ICC proliferation, invasion, and metastasis remain unclear. Recently, N6-methyladenosine (m6A) RNA methylation has been associated with tumor progression. Methyltransferase 3 (METTL3) is a crucial methyltransferase for m6A. However, its biological significance and the regulatory mechanisms underlying ICC invasion and metastasis remain poorly understood. Therefore, we aimed to explore the role of METTL3 in ICC progression and its potential mechanisms. Methods: We analyzed the expression of METTL3 in ICC using bioinformatics, quantitative reverse transcription polymerase chain reaction (qRT-PCR), and immunohistochemistry analysis. Furthermore, we evaluated the impact of METTL3 on ICC cell proliferation and metastasis in vivo and in vitro . For mechanistic studies, we used RNA-Seq to screen the crucial downstream targets of METTL3 in ICC cells. Furthermore, we examined the regulatory impact of METTL3 on the phenotype of ICC cells through S100 calcium-binding protein A4 (S100A4) using qRT-PCR, Western blot, and rescue experiments. Finally, we assessed the effect of METTL3 on S100A4 stability by mediating m6A modification using the methylated RNA immunoprecipitation qPCR (MeRIP-qPCR) and messenger RNA (mRNA) degradation experiments. Results: The expression of METTL3 was upregulated in patients with ICC ( p &lt; 0.05). Moreover, METTL3 knockdown inhibited the proliferation, migration, and invasion ability of ICC cells ( p &lt; 0.05). Mechanistically, METTL3 mediated m6A modification of S100 calcium-binding protein A4 ( S100A4 ) mRNA and inhibited S100A4 mRNA decay in an m6A-dependent manner ( p &lt; 0.05), thus promoting the proliferation and metastasis of ICC. Conclusions: METTL3 promotes ICC proliferation and metastasis by mediating S100A4 mRNA degradation, suggesting that METTL3 may be a potential target for treating ICC.

Activating Mutations in PTPN3 Promote Cholangiocarcinoma Cell Proliferation and Migration and Are Associated With Tumor Recurrence in Patients

AimCervical cancer (CC) is the fourth most common cancer among women worldwide. We aimed to explore the role of hsa_circ_000543 played in CC.MethodsThe hsa_circ_000543 expressions in CC tissues and cells were measured by qRT-PCR. The correlation of hsa_circ_000543 expression and the clinical features of CC patients were analyzed by SPSS 20.0. The up- or down-regulated plasmids of hsa_circ_000543 were respectively transfected into CC cells. Cell proliferation, apoptosis and colony formation were detected through CCK-8 assay, flow cytometry and cell colony formation assay, respectively. The cell migration and invasion were evaluated by Transwell assay. The underlying molecular mechanism of hsa_circ_000543 was studied by bioinformatic prediction tools and luciferase reporter assay. Rescue experiments were performed to validate the regulation mechanism of hsa_circ_000543/miR-567/ZNF268 axis in CC.ResultsHsa_circ_000543 was over-expressed in CC tissues and cells. The high expression of hsa_circ_000543 indicated poor prognosis of CC patients. Hsa_circ_000543 promoted cell proliferation, colony formation, migration and invasion, as well as inhibited cell apoptosis in CC cells. Hsa_circ_000543 directly targeted miR-567/ZNF268 in CC cell lines. In CC tumor tissues and cells, the hsa_circ_000543 expression was negatively correlated with miR-567 expression and showed a positive correlation with ZNF268 expression. The rescue experiments revealed that hsa_circ_000543 mediated cell proliferation, apoptosis, colony formation, migration and invasion of CC cells via regulating miR-567/ZNF268 axis.ConclusionHsa_circ_000543 regulated CC cell activities through binding miR-567 and therefore enhancing ZNF268 expression.

Ferroptosis, a regulated form of cell death characterized by iron accumulation and lipid peroxidation, has gained increasing attention as a therapeutic target in cancer. Polyethylene glycol-liposomal doxorubicin (PLD), a nanocarrier formulation with improved pharmacological properties, shows promise in breast cancer therapy, yet the molecular mechanisms underlying its effects on ferroptosis remain unclear. Breast cancer cell lines (MDA-MB-231 and MCF-7) were treated with PLD to evaluate its anti-tumor effects. Cell viability, colony formation, and migration assays were performed, while lipid ROS accumulation was assessed using BODIPY-C11, and oxidative stress markers (MDA, Fe, GSH, and SOD) were quantified. Western blotting and immunofluorescence were used to examine NRF2/xCT/GPX4 signaling, and molecular docking predicted the interactions of PLD with KEAP1 and NRF2. Functional roles were further validated through NRF2 overexpression and KEAP1-R483S mutation. PLD exerts potent anti-breast cancer effects by suppressing cell viability, colony formation, and migration in MDA-MB-231 and MCF-7 cells. Mechanistically, PLD induces ferroptosis, evidenced by increased lipid ROS, elevated MDA and Fe levels, decreased GSH content and SOD activity, and downregulation of xCT and GPX4. NRF2 overexpression attenuates these effects by restoring antioxidant defenses, reducing lipid peroxidation and iron accumulation, and partially rescuing cell proliferation and migration. Molecular docking further revealed stable interactions of PLD with KEAP1 and NRF2, with Arg483 identified as a key residue mediating KEAP1-NRF2 and PLD-KEAP1 binding, suggesting that KEAP1 modulates NRF2 stability and cellular susceptibility to ferroptosis. Collectively, these results indicate that PLD partially inhibits breast cancer growth through KEAP1/NRF2-mediated ferroptosis, highlighting a novel mechanism underlying its anti-tumor activity.

Cholangiocarcinoma (CHOL) is often diagnosed at an advanced stage; therefore, exploring its key regulatory factors is important for earlier diagnosis and treatment. This study aimed to identify the mechanisms of long non-coding RNA (lncRNA) TMPO Antisense RNA 1 (TMPO-AS1), microRNA let-7 g-5p, and high-mobility group A1 (HMGA1) proteins in CHOL. Our results, through quantitative real-time PCR and Western blot detection, showed that TMPO-AS1 and HMGA1 were overexpressed while let-7 g-5p was underexpressed in CHOL. Cell function experiments in CHOL cells revealed that TMPO-AS1 knockdown inhibited cell proliferation, colony formation, and cell migration, but induced apoptosis. TMPO-AS1 knockdown also suppressed tumor growth in vivo. Together with luciferase assay and Western blotting, we found that TMPO-AS1 could sponge let-7 g-5p to promote HMGA1 expression. Moreover, HMGA1 overexpression attenuated the effect of TMPO-AS1 downregulation in CHOL cells. Overall, our findings identified the oncogenic effect of TMPO-AS1 on CHOL cells, which may put forward a novel methodology for CHOL diagnosis and therapy.

BackgroundThe orphan G protein-coupled receptor (GPCR) GPR137 is implicated in the proliferation of various tumor cells. However, its role and underlying mechanisms in medulloblastoma (MB), particularly the sonic hedgehog (SHH) subtype, remain unclear. This study aimed to investigate the function of GPR137 in SHH-MB and its potential regulation of ferroptosis via the Wnt/β-catenin pathway.MethodsA GPR137-knockdown Daoy cell line was constructed using lentiviral shRNA. Cell proliferation, invasion, and colony formation were assessed using Cell Counting Kit-8 (CCK-8), wound healing, and colony formation assays, respectively. Ferroptosis was induced with erastin, and its inhibition was achieved with ferrostatin-1. Key ferroptosis markers, including lipid peroxidation products [malondialdehyde (MDA), 4-hydroxynonenal (4-HNE)], reactive oxygen species (ROS), glutathione (GSH) levels, and labile iron (Fe²+) were measured. The expression of ferroptosis-related proteins (GPX4, xCT) and Wnt/β-catenin pathway components was analyzed by western blot and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Rescue experiments were performed by overexpressing β-catenin in GPR137-knockdown cells.ResultsGPR137 knockdown significantly inhibited Daoy cell proliferation, invasion, and colony formation. It synergized with erastin to aggravate ferroptosis, leading to increased cell death, elevated levels of MDA, 4-HNE, ROS, and Fe²+, alongside decreased GSH and downregulated GPX4 and xCT protein expression. These pro-ferroptotic effects were reversed by ferrostatin-1. Mechanistically, GPR137 depletion downregulated key mRNAs (β-catenin, c-jun, c-myc, cyclin D1, Axin2) and the protein level of β-catenin in the Wnt pathway. Crucially, β-catenin overexpression significantly alleviated the enhanced ferroptosis phenotype caused by GPR137 knockdown.ConclusionsOur findings demonstrate that GPR137 deletion promotes ferroptosis in SHH-MB cells by inhibiting the Wnt/β-catenin signaling pathway. This reveals a novel regulatory axis in MB and suggests that targeting GPR137 could be a promising therapeutic strategy for SHH-MB.

To explore whether miR-573 can suppress pancreatic cancer cell proliferation, migration, and invasion by targeting TSPAN1. The expression of miR-573 and TSPAN1 in pancreatic cancer tissues and cells lines was analyzed using RT-qPCR. The human pancreatic cancer cell line PANC‑1 was transfected with miR-573 mimic, pcDNA3.1-TSPAN1, or genOFFTM st-h-TSPAN1. The effects of miR-573 and TSPAN1 on cell proliferation, colony formation, migration, and invasion were analyzed by CCK‑8, colony formation, transwell migration, and invasion assay, respectively. Target genes of miR-573 were screened using bioinformatics tools and confirmed by dual-luciferase reporter assay and real-time PCR. The effects of miR-573 in vivo were observed using tumor xenografts. We found that miR-573 is downregulated and TSPAN1 is upregulated in pancreatic cancer tissues and cells lines. Function assays demonstrated that overexpression of miR-573 inhibited cell proliferation, colony formation, migration, and invasion of pancreatic cancer cells, as well as suppressing tumor growth in vivo. Target genes of miR-573 were predicted using bioinformatics tools and confirmed by dual-luciferase reporter assay and RT-qPCR or western blotting. Downregulation of TSPAN1 also inhibited cell proliferation, colony formation, migration, and invasion of pancreatic cancer cells. Furthermore, overexpression of TSPAN1 attenuated miR-573-induced inhibition of pancreatic cancer cell proliferation and migration. Our findings indicated that miR-573 suppresses pancreatic cancer cell proliferation, migration, and invasion through targeting TSPAN1. TSPAN1 targeted by miR-573 might be apotential therapeutic target for clinical treatment of pancreatic cancer.

Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver tumor and a major cause of cancer mortality worldwide. Integrin β5 (ITGB5) is considered to be involved in the intercellular signal transduction and regulation of tumorigenesis and development. The present study investigated the association between ITGB5 expression levels and the prognosis of ICC, as well as the effects of ITGB5 on the proliferation and invasion of ICC cells. RNA-sequencing transcriptomic profiling data of ICC samples were retrieved from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) databases. Tissue specimens from patients with ICC treated at Taizhou People's Hospital were collected and the ITGB5 expression levels were evaluated using immunohistochemical staining. The biological function of ITGB5 in ICC was investigated using Gene Ontology (GO), Gene Set Enrichment Analysis (GSEA) and in vitro experiments using HuCCT1 cells. After knocking down ITGB5 expression, cell proliferation was detected using Cell Counting Kit-8 assay, while cell invasion was assessed using Transwell assays. According to TCGA dataset, ITGB5 was highly expressed in ICC; however, there was no significant difference in prognosis between patients with high and low ITGB5 expression levels. High expression of ITGB5 was present in the tissues of patients with ICC from the GEO database, which was associated with poor prognosis. Survival analyses of the clinical data obtained in the present study revealed that high expression levels of ITGB5 in patients with ICC were associated with a reduced overall survival. GO and GSEA indicated that genes associated with ITGB5 were enriched in the extracellular matrix-receptor interaction and focal adhesion signaling pathways. Silencing ITGB5 inhibited the proliferation and invasion of ICC cells. In conclusion, ITGB5 may act as an essential regulator of ICC development and progression by influencing the proliferation and invasion of ICC cells. However, future studies with larger sample sizes are required to validate the role of ITGB5 in the prognosis of patients with ICC.

BackgroundCholangiocarcinoma represents a malignant neoplasm originating from the hepatobiliary tree, with a subset of tumors developing inside the liver. Intrahepatic cholangiocarcinomas (ICC) commonly exhibit an asymptomatic presentation, rendering both diagnosis and treatment challenging. Cuproptosis, an emerging regulated cell death pathway induced by copper ions, has garnered attention recently. As cancer cells show altered copper metabolism and comparatively higher copper needs, cuproptosis may play a role in the development of ICC. However, studies investigating this possibility are currently lacking.MethodsSingle-cell and bulk RNA sequence data were analyzed, and correlations were established between the expression of cuproptosis-related molecules and ICC patient survival. Genes with predicting survival were used to create a CUPT score using Cox and LASSO regression and tumor mutation burden (TMB) analysis. The CIBERSORT software was employed to characterize immune cell infiltration within the tumors. Furthermore, immune infiltration prediction, biological function enrichment, and drug sensitivity analyses were conducted to explore the potential implications of the cuproptosis-related signature. The effects of silencing solute carrier family 39 member 4 gene (SLC39A4) expression using siRNA were investigated using assays measuring cell proliferation, colony formation, and cell migration. Key genes of cuproptosis were detected by western blotting.ResultsThe developed CUPT score divided patients into high and low CUPT score groups. Those with a low score had significantly better prognosis and longer survival. In contrast, high CUPT scores were associated with worse clinical outcomes and significantly higher TMB. Comparisons of the two groups also indicated differences in the immune infiltrate present in the tumors. Finally, we were able to identify 95 drugs potentially affecting the cuproptosis pathway. Some of these might be effective in the treatment of ICC. The in vitro experiments revealed that suppressing the expression of SLC39A4 in ICC cell lines resulted in reduced cell proliferation, colony formation, and cell migration. It also led to an increase in cell death and the upregulation of key genes associated with cuproptosis, namely ferredoxin 1 (FDX1) and dihydrolipoyl transacetylase (DLAT). These findings strongly suggest that this cuproptosis-associated molecule may play a pivotal role in the development and metastasis of ICC.ConclusionsChanges in the expression of a cuproptosis-related gene signature can predict the clinical prognosis of ICC with considerable accuracy. This supports the notion that cuproptosis influences the diversity and complexity of the immune microenvironment, mutational landscape, and biological behavior of ICC. Understanding this pathway better may hold promise for the development of innovative strategies in the management of this disease.

323 Background: Adiponectin is the key adipokine, which plays an important role in health and disease such as obesity, diabetes, and cancer. Adiponectin is reduced in different tumor types, especially in obesity-related cancer, and recent studies showed that Adiponectin had a potential anti-cancer effect. Obesity is a risk factor for various tumor diseases including cholangiocarcinoma (CC), the second most common primary hepatic cancer. The aim of this study is to investigate for the first time the anti-cancer effect of AdipoR agonist in CC cell lines and a CC engineered mouse model. Methods: Human CC cell lines (TFK-1 and SZ-1) and CC engineered mice (Alb-Cre/KRASG12D/p53L/L) were used to investigate the anti-cancer effects of an AdipoR agonist (2-(4-Benzoylphenoxy)-N-[1-(phenylmethyl)-4-piperidinyl]-acetamide). Cell proliferation, migration, invasion, colony formation, apoptosis assay were applied to evaluate the effect of AdipoR agonist in vitro. In addition, important cancer signalling pathways and targets were analysed by Western Blot. Mice (n = 12) were treated with AdipoR or verhicle and tumor burden and survival were monitored. Results: AdipoR agonist suppressed proliferation, migration, invasion, colony formation and apoptosis in CC cells. AdipoR agonist regulated the expression of different proteins such as EMT markers, pAMPK, pSTAT3, and PARP, which have pivotal functions in cholangiocarcinogenesis. AdipoR agonist also prolonged survival time in a CC engineered mouse model. Conclusions: Our data revealed that AdipoR agonist inhibited successfully cell proliferation, migration, invasion, colony formation and apoptosis in vitro, and prolonged mice survival in vivo through regulation of crucial signaling pathways in cholangiocarcinogenesis. These results suggested that AdipoR agonist might become a new potential candidate for CC treatment in the future.