CCA Cell Research Articles

Targeting PDGF-CC as a promising therapeutic strategy to inhibit cholangiocarcinoma progression.

Cholangiocarcinoma (CCA) is an aggressive malignancy with limited treatment options and poor prognosis. Platelet-Derived Growth Factor CC (PDGF-CC) has been implicated in the progression of various tumors, but its specific role in CCA is not well understood. This study aims to investigate the expression and function of PDGF-CC in CCA and evaluate its potential as a therapeutic target. We conducted gene expression analysis using the GEPIA database to compare PDGF-CC mRNA levels in CCA tissues and normal tissues. Serum samples from CCA patients were analyzed for PDGF-CC protein levels, and immunohistochemistry was used to assess PDGF-CC expression in tissue samples. The impact of PDGF-CC on CCA cell behavior was examined by knocking out PDGF-CC in HuCCT1 and QBC939 cell lines, followed by assessments of cell proliferation, migration, invasion, and colony formation in vitro. Additionally, the effects of PDGF-CC knockout were evaluated in xenograft models. The therapeutic potential of PDGF-CC inhibition was further explored using pharmacological inhibitors and antibodies. PDGF-CC mRNA and protein levels were significantly elevated in CCA tissues and patient sera compared to normal controls. Immunohistochemical analysis confirmed increased PDGF-CC expression in CCA tissues. High PDGF-CC expression correlated with poor overall survival in CCA patients, as shown by Kaplan-Meier analysis. Functional assays revealed that PDGF-CC knockout significantly reduced proliferation, migration, invasion, and colony formation in HuCCT1 and QBC939 cells, the lines with the highest PDGF-CC levels. In vivo, PDGF-CC knockout markedly decreased tumor growth in xenograft models. Pharmacological inhibition of PDGF-CC mirrored the effects of genetic knockout, suggesting it as a viable therapeutic strategy. This study underscores the critical role of PDGF-CC in CCA progression and supports the potential of PDGF-CC inhibitors as a therapeutic approach. Given the association between high PDGF-CC expression and poor prognosis, targeting PDGF-CC may improve outcomes for CCA patients. Further clinical investigations are warranted to develop PDGF-CC-targeted therapies for CCA.

Lactiplantibacillus plantarum inhibited the growth of primary liver cancer by inducing early apoptosis and senescence, in vitro

Primary liver cancer (PLC), comprising hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), is a severe form of cancer associated with a high mortality and morbidity rate and increasing incidence worldwide. Current treatment options are limited and chemotherapeutics demonstrate strong side effects. New therapies are highly required. Lactobacilli represent the most diverse lactic acid-producing bacteria group and a prominent example of probiotics. Several studies have highlighted the anticancer efficacy of probiotics, especially of Lactiplantibacillus plantarum. However, there are limited studies on its activity on two PLC types, hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). This study evaluated the inhibitory mechanism and properties of L. plantarum ONU 12 (Lp 12) and L. plantarum ONU 355 (Lp 355), isolated from grapes in Ukraine and France, in murine PLC cell lines, in vitro. Strain Lacticaseibacillus casei ATCC 393 (Lc 393) has been taken for a direct comparison, as the most studied probiotic strain. The three Lactobacillus species were used in three forms: as live and heat-killed suspensions, and as sonicated extracts, and tested either as a monotherapy or in combination with standard chemotherapeutics (sorafenib for HCC and gemcitabine for CCA). Cell proliferation and viability were assessed via crystal violet staining assay and cell counting kit-8 assay. The induction of senescence was investigated by senescence-associated β-galactosidase assay. Fluorescence-activated cell sorting analysis was used to determine the apoptotic mechanism behind the inhibitory property of lactobacilli. The results showed that the live suspensions and sonicated extracts of Lp 12, Lp 355, and Lc 393 demonstrated inhibitory properties in CCA and HCC cells after 48 h of incubation. In combinations with standard chemotherapeutics, lactobacilli treatments have shown strong synergistic effects. The combination therapy allowed to reduce the chemotherapeutic doses of gemcitabine from 50 μM to 0.1 and 0.05 μM and sorafenib from 13.8 μM to 6.9 and 3.45 μM. Successful treatment regimes induced early apoptosis and cellular senescence in PLC, as the mechanism of inhibition. Heat-killed suspensions showed no inhibitory effect in none of the cell lines. Both strains, Lp 12 and Lp 355, showed successful results and need further testing in vivo, using autochthonous HCC and CCA models.

Lenvatinib inhibits cholangiocarcinoma progression by targeting the FGF19/PI3K/AKT signaling pathway.

Cholangiocarcinoma (CCA) is known for its high aggressiveness and dismal prognosis, whose effectiveness of systemic therapy remains limited. As a multi-target drug, lenvatinib has exhibited promising effects in many solid tumors. However, the therapeutic role of lenvatinib in CCA is rarely investigated. Here, the in vitro assays including EdU, colony formation, transwell, wound healing, and apoptosis analyses demonstrated that lenvatinib significantly inhibited the proliferation, migration, and invasion, while simultaneously inducing apoptosis of CCA cells. Mechanistically, lenvatinib downregulated the expression of FGF19 and inactivated the PI3K/AKT signaling pathway. Depletion of FGF19 enhanced the anti-tumor effects of lenvatinib, which was attributed to the inhibition of p-PI3K and p-AKT expression in CCA cells. In contrast, overexpression of FGF19 activated the PI3K/AKT signaling pathway, thereby impairing the inhibitory effects of lenvatinib against CCA. In addition, the AKT inhibitor, MK-2206, reinforced the lenvatinib-induced CCA inhibition. Notably, the in vivo experiment confirmed that the subcutaneous tumorigenicity of CCA cells in nude mice was weakened by lenvatinib. Lenvatinib markedly downregulated the expression of FGF19, p-AKT, Ki-67, vimentin, and VEGF in the xenograft tumor tissues. Collectively, these findings demonstrated that lenvatinib inhibits CCA progression by targeting the FGF19/PI3K/AKT signaling pathway. The present study provides novel experimental evidence for the potential clinical application of lenvatinib in CCA, which also highlights the promising role of targeting FGF19 in combined therapeutic approaches for CCA.

Extracellular vesicles of Clonorchis sinensis promote the malignant phenotypes of cholangiocarcinoma via NF-κB/EMT axis.

Clonorchis sinensis infection is an important risk factor for cholangiocarcinoma (CCA). It has been reported that extracellular vesicles (EVs) are involved in the parasite-host interaction, and EVs of C. sinensis (CsEVs) can contribute to biliary injuries and inflammation. However, uncertainty surrounds the function of CsEVs in the progression of CCA. In this study, differential ultracentrifugation was used to separate CsEVs from the culture supernatant of C. sinensis adult worms, and they were then identified by transmission electron microscopy, nanoparticle tracking analysis and proteome assays. CCK8, EdU-488 and colony formation assays were used to explore the effect of CsEVs on the proliferation of CCA cells in vitro. Wound healing assays, transwell assays and in vivo lung metastasis model were conducted to evaluate the migration and invasion abilities. Moreover, the involvement of EMT process, as well as NF-κB and ERK signaling pathway was assessed. Results showed that CsEVs were successfully isolated and could be taken up by CCA cells, which promoted proliferation by accelerating cell cycle progression. In addition, CsEVs could facilitate cell metastasis by triggering the epithelial-mesenchymal transition (EMT). Mechanistically, activation of NF-κB signaling pathway was involved in the CsEVs-mediated EMT, which could be reversed partly by BAY 11-7082 (an inhibitor of NF-κB). In conclusion, these findings suggested that CsEVs could induce the aberrant proliferation and metastasis of CCA cells by stimulating the NF-κB/EMT axis, providing a novel theoretical explanation for liver fluke-associated CCA.

Feedback loop LINC00511-YTHDF2-SOX2 regulatory network drives cholangiocarcinoma progression and stemness.

Cholangiocarcinoma (CCA) was identified as a malignant tumor with rising incidence and mortality rates, and the roles of long noncoding RNA (lncRNA) in CCA remained not entirely clear. In this study, LINC00511 had high expression in CCA, which was closely related to poor prognosis. Knockdown of LINC00511 significantly inhibited cell malignant biological behaviors. It also affected the stemness of CCA, evidenced by decreased SOX2 protein expression. Moreover, the study revealed the interaction of LINC00511, YTHDF2, and SOX2 in CCA. Specifically, LINC00511 facilitated the formation of a complex with YTHDF2 on SOX2 mRNA, which uniquely enhances the mRNA's stability through m6A methylation sites. This stabilization appears crucial for maintaining malignant behaviors in CCA cells. Additionally, LINC00511 modulated SOX2 expression via the PI3K/AKT signaling pathway. Meanwhile, SOX2 can also promote LINC00511 expression as an upstream transcription factor, thereby confirming a positive feedback loop formed by LINC00511, YTHDF2, and SOX2, which plays a significant role in the occurrence and development of CCA. Finally, the study successfully constructed two patient-derived xenograft models, revealing the vital role of LINC00511 in CCA development. In summary, this research provides a comprehensive understanding of the role of LINC00511 in the pathogenesis of CCA.

USP28 promotes tumor progression and glycolysis by stabilizing PKM2/Hif1-α in cholangiocarcinoma.

Ubiquitination is one of the important modification of proteins which can be reversed by deubiquitinating enzymes (DUBs). Ubiquitin specific protease 28 (USP28) belongs to the deubiquitinase family, which plays a cancer-promoting function in many types of cancers such as pancreatic cancer and breast cancer. So far, the molecular function and significance of USP 28 in cholangiocarcinoma remain unclear. In this study, we evaluated the expression of USP28 using tissue microarray (TMA), reverse transcription polymerase chain reaction (qRT-PCR), and online databases. We investigated the effect of USP28 on the progression of CCA through in vitro and in vivo functional experiments. In addition, we explored downstream molecular pathways using Western blotting (WB), immunofluorescence (IF), and mass spectrometry techniques. Here, we found that cholangiocarcinoma tissue had higher USP 28 expression than normal bile duct tissue, and that high USP 28 levels were significantly associated with a malignant phenotype and poorer prognosis in cholangiocarcinoma patients. Both in vitro and in vivo, USP28 could mediate the deubiquitination of PKM2, thereby activating the downstream Hif1-α signaling pathway, promoting glycolysis and energy supply, and finally promoting tumor progression. In summary, USP28 activated downstream Hif1-α by reducing the ubiquitination level of PKM2, furthermore, promoting the level of glycolysis in CCA cells for tumor progression.

CARD9 promotes cholangiocarcinoma by regulating the IL-17A/Hedgehog and the THEM4/AKT/mTOR signaling pathways

Cholangiocarcinoma (CCA) is a highly lethal malignant tumor originating from the bile duct, and its underlying mechanisms are not fully understood. In order to identify key genes in CCA, we downloaded gene expression data from public GSE76297, GSE26566 and TCGA-CHOL datasets. CARD9 was selected as a CCA-related gene from the datasets. Its expression was identified in the CCA tissues via PCR, western blot and immunohistochemistry assays. The loss-and-gain function assay of CARD9 was conducted in CCA cell lines (QBC939 and RBE) and nude mice. This study found a significant upregulation of CARD9 in CCA tissues, which is associated with poor prognosis in patients. Overexpression of CARD9 promotes proliferation and invasion of QBC939 and RBE cells, enhances the growth and development in CCA mouse models, and reduces sensitivity to chemotherapy drugs for CCA. Mechanistically, CARD9 activates the NF-κB and NLRP3 inflammatory signaling pathways, induces excessive release of various inflammatory factors, and triggers a cascade reaction of interleukin-17 (IL-17A). IL-17A promotes the stability of IHH mRNA by regulating HuR, enhances IHH transcription, and activates the Hedgehog signaling pathway to accelerate the progression of CCA. In addition, CARD9 promotes proliferation and invasion of CCA cells by interacting with THEM4, thereby facilitating AKT and mTOR phosphorylation, and accelerating the progression of CCA. Overall, these data suggest that CARD9 is involved in the progression of CCA by regulating the IL-17A/Hedgehog and the THEM4/AKT/mTOR signaling pathways. Therefore, CARD9 may serve as a novel therapeutic target for CCA treatment.

Emerging insights into ferroptosis in cholangiocarcinoma (Review).

Cholangiocarcinoma (CCA) is a malignant tumor that arises within the biliary system, which exhibits a progressively increasing incidence and a poor patient prognosis. A thorough understanding of the molecular pathogenesis that drives the progression of CCA is essential for the development of effective molecular target therapeutic approaches. Ferroptosis is driven by excessive iron accumulation and catalysis, lipid peroxidation and the failure of antioxidant defense systems. Key molecular targets of iron metabolism, lipid metabolism and antioxidant defense systems involve molecules such as transferrin receptor, ACSL4 and GPX4, respectively. Inhibitors of ferroptosis include ferrostatin-1, liproxstatin-1, vitamin E and coenzyme Q10. By contrast, compounds such as erastin, RSL3 and FIN56 have been identified as inducers of ferroptosis. Ferroptosis serves a notable role in the onset and progression of CCA. CCA cells exhibit high sensitivity to ferroptosis and aberrant iron metabolism in these cells increases oxidative stress and iron accumulation. The induction of ferroptosis markedly reduces the ability of CCA cells to proliferate and migrate. Certain ferroptosis agonists, such as RSL3 and erastin, cause lipid peroxide build up and GPX4 inhibition to induce ferroptosis in CCA cells. Current serological markers, such as CA-199, have low specificity and cause difficulties in the diagnosis of CCA. However, novel techniques, such as non-invasive liquid biopsy and assays for oxidative stress markers and double-cortin-like kinase 1, could improve diagnostic accuracy. CCA is primarily treated with surgery and chemotherapy. A close association between the progression of CCA with ferroptosis mechanisms and related regulatory pathways has been demonstrated. Therefore, it could be suggested that multi-targeted therapeutic approaches, such as ferroptosis inducers, iron chelating agents and novel modulators such as YL-939, may improve treatment efficacy. Iron death-related genes, such as GPX4, that are highly expressed in CCA and are associated with a poor prognosis for patients may represent potential prognostic markers for CCA. The present review focused on molecular targets such as p53 and ACSL4, the process of targeted medications in combination with PDT in CCA and the pathways of lipid peroxidation, the Xc-system and GSH-GPX4 in ferroptosis. The present review thus offered novel perspectives to improve the current understanding of CCA.

Notch-Driven Cholangiocarcinogenesis Involves the Hippo Pathway Effector TAZ via METTL3-m6A-YTHDF1

Background and AimsNotch and TAZ are implicated in cholangiocarcinogenesis, but whether and how these oncogenic molecules interact remain unknown. MethodsThe development of CCA was induced by hydrodynamic tail vein (HDTV) injection of oncogenes (NICD/AKT) to the FVB/NJ mice. CCA xenograft was developed by inoculation of human CCA cells into the livers of SCID mice. Tissues and cells were analyzed using qRT-PCR, Western blotting analyses, Immunohistochemistry, ChIP-qPCR and WST-1 cell proliferation Assay. ResultsOur experimental findings show that TAZ is indispensable in NICD-driven cholangiocarcinogenesis. Notch activation induces the expression of METTL3 (Methyltransferase like-3) which catalyzes N6-methyladenosine (m6A) modification of TAZ mRNA and that this mechanism plays a central role in the crosstalk between Notch and TAZ in CCA cells. Mechanistically, Notch regulates the expression of METTL3 through the binding of NICD to its downstream transcription factor CSL in the promoter region of METTL3. METTL3 in turn mediates m6A modification of TAZ mRNA which is recognized by the m6A reader YTHDF1 to enhance TAZ protein translation. We observed that inhibition of Notch signaling decreased the protein levels of both MELLT3 and TAZ. Depletion of METTL3 by shRNAs or by the next generation GapmeR antisense oligonucleotides (ASOs) decreased the level of TAZ protein and inhibited the growth of human CCA cells in vitro and in mice. ConclusionThis study describes a novel Notch-METTL3-TAZ signaling cascade which is important in CCA development and progression. Our experimental results provide new insight into how the Notch pathway cooperates with TAZ signaling in CCA, and the findings may have important therapeutic implications.

MBD2 regulates the progression and chemoresistance of cholangiocarcinoma through interaction with WDR5

BackgroundCholangiocarcinoma (CCA) is a highly malignant, rapidly progressing tumor of the bile duct. Owing to its chemoresistance, it always has an extremely poor prognosis. Therefore, detailed elucidation of the mechanisms of chemoresistance and identification of therapeutic targets are still needed.MethodsWe analyzed the expression of MBD2 (Methyl-CpG-binding domain 2) in CCA and normal bile duct tissues using the public database and immunohistochemistry (IHC). The roles of MBD2 in CCA cell proliferation, migration, and chemoresistance ability were validated through CCK-8, plate cloning assay, wound healing assays and xenograft mouse model. In addition, we constructed a primary CCA mouse model to further confirm the effect of MBD2. RNA-seq and co-IP-MS were used to identify the mechanisms by how MBD2 leads to chemoresistance.ResultsMBD2 was upregulated in CCA. It promoted the proliferation, migration and chemoresistance of CCA cells. Mechanistically, MBD2 directly interacted with WDR5, bound to the promoter of ABCB1, promoted the trimethylation of H3K4 in this region through KMT2A, and activated the expression of ABCB1. Knocking down WDR5 or KMT2A blocked the transcriptional activation of ABCB1 by MBD2. The molecular inhibitor MM-102 targeted the interaction of WDR5 with KMT2A. MM-102 inhibited the expression of ABCB1 in CCA cells and decreased the chemoresistance of CCA to cisplatin.ConclusionMBD2 promotes the progression and chemoresistance of CCA through interactions with WDR5. MM-102 can effectively block this process and increase the sensitivity of CCA to cisplatin.

SRPK Inhibitors Reduce the Phosphorylation and Translocation of SR Protein Splicing Factors, thereby Correcting BIN1, MCL-1 and BCL2 Splicing Errors and Enabling Apoptosis of Cholangiocarcinoma Cells.

Cholangiocarcinoma (CCA) is a malignancy of the bile duct epithelium that is commonly found in the Thai population. CCA has poor prognosis and a low survival rate due to the lack of early diagnosis methods and the limited effectiveness of current treatments. A number of oncogenic spliced-transcripts resulting from mRNA splicing errors have been reported in CCA, and aberrant mRNA splicing is suspected to be a key driver of this cancer type. The hyperphosphorylation of serine/arginine rich-splicing factors (SRSFs) by serine/arginine protein kinases (SRPKs) causes them to translocate to the nucleus where they facilitate gene splicing errors that generate cancer-related mRNA/protein isoforms. The correlation between SRPK expression and the survival of CCA patients was analyzed using data from The Cancer Genome Atlas (TCGA) dataset. The effect of SRPK inhibitors (SRPIN340 and SPHINX31) on two CCA cell lines (KKU-213A and TFK-1) was also investigated. The induction of cell death was studied by Calcein-AM/PI staining, AnnexinV/7AAD staining, immunofluorescence (IF), and Western blotting (WB). The phosphorylation and nuclear translocation of SRSFs was tracked by WB and IF, and the repair of splicing errors was examined by Reverse Transcription-Polymerase Chain Reaction (RT-PCR). High levels of SRPK1 and SRPK2 transcripts, and in particular SRPK1, correlated with shorter survival in CCA patients. SRPIN340 and SPHINX31 increased the number of dead and apoptotic cells in a dose-dependent manner. CCA also showed diffuse expression of cytoplasmic cytochrome C and upregulation of cleaved caspase-3. Moreover, SRSFs showed low levels of phosphorylation, resulting in the accumulation of cytoplasmic SRSF1. To link these phenotypes with aberrant gene splicing, the apoptosis-associated genes Bridging Integrator 1 (BIN1), Myeloid cell leukemia factor 1 (MCL-1) and B-cell lymphoma 2 (BCL2) were selected for further investigation. Treatment with SRPIN340 and SPHINX31 decreased anti-apoptotic BIN1+12A and increased pro-apoptotic MCL-1S and BCL-xS. The SRPK inhibitors SRPIN340 and SPHINX31 can suppress the phosphorylation of SRSFs and their nuclear translocation, thereby producing BIN1, MCL-1 and BCL2 isoforms that favor apoptosis and facilitate CCA cell death.

Augmented Global Protein Acetylation Diminishes Cell Growth and Migration of Cholangiocarcinoma Cells.

We have previously shown that the overexpression of acetyl-CoA carboxylase 1 (ACC1) was associated with the poor prognosis of cholangiocarcinoma (CCA) patients, and suppression of its expression in CCA cell lines deteriorated cell growth. The present study explored the mechanism by which ACC1 inhibition affects global protein acetylation, using genetic knockdown and pharmacological inhibition with an ACC1 inhibitor ND-646 as models. Both ACC1 knockdown and ACC1-inhibitor-treated cells displayed the hyperacetylation of proteins, accompanied by impaired growth and migration. The immunoprecipitation of hyperacetylated proteins using the anti-acetylated lysine antibody, followed by tandem mass spectrometry, identified three potential verification candidates, namely POTE ankyrin domain family member E, peroxisomal biogenesis factor 1, and heat shock protein 90 beta (HSP90B). HSP90 acetylation was the candidate selected for the verification of protein acetylation. To establish the effects of protein hyperacetylation, treatment with suberoylanilide hydroxamic acid (SAHA), a lysine deacetylase inhibitor, was conducted, and this served as an independent model. Decreased tumor growth but increased acetylated protein levels were observed in ACC1-KD xenograft tumors. Hyperacetylated-alleviated cell growth and migration were consistently observed in the SAHA-treated models. The molecular linkage between protein hyperacetylation and the AKT/GSK3β/Snail pathway was demonstrated. This study highlighted the importance of protein acetylation in CCA progression, suggesting that ACC1 and KDAC are potential targets for CCA treatment.

YAP-TEAD inhibition is associated with upregulation of an androgen receptor mediated transcription program providing therapeutic escape.

Cholangiocarcinoma (CCA) is a highly aggressive form of liver cancer and is an increasing cause of cancer-related death worldwide. Despite its increasing incidence globally and alarming mortality, treatment options for CCA have largely remained unchanged, stressing the importance of developing new effective therapies. YAP activation is common in CCA, and its major transcriptional signaling partners are the TEAD proteins. CA3 is a small-molecule YAP-TEAD disrupter discovered utilizing a TEAD reporter assay. Utilizing CCA, gastric cancer cell lines, and patient-derived xenograft models (PDX), we demonstrate that CA3 is effective in inducing cell death and delaying tumor growth in both FGFR2 fusion and wild-type models. CA3 was associated with on-target decreases in YAP-TEAD target gene expression, TEAD reporter activity, and overall TEAD levels. Hippo pathway signaling was not altered as there was no change in YAP phosphorylation status in the cells exposed to CA3. RNA sequencing of gastric cancer and CCA models demonstrated upregulation of an androgen receptor-mediated transcriptional program following exposure to CA3 in five unique models tested. Consistent with this upstream regulator analysis, CA3 exposure in CCA cells was associated with increased AR protein levels, and combinatorial therapy with CA3 and androgen receptor blockade was associated with increased cancer cell death. CA3 behaves functionally as a YAP-TEAD disrupter in the models tested and demonstrated therapeutic efficacy. Exposure to CA3 was associated with compensatory androgen receptor signaling and dual inhibition improved the therapeutic effect.

Prognostic value of PEA3 subfamily gene expression in cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a lethal malignancy with limited treatment options and poor prognosis. The PEA3 subfamily of E26 transformation specific genes: ETV1, ETV4, and ETV5 are known to play significant roles in various cancers by influencing cell proliferation, invasion, and metastasis. To analyze PEA3 subfamily gene expression levels in CCA and their correlation with clinical parameters to determine their prognostic value for CCA. The expression levels of PEA3 subfamily genes in pan-cancer and CCA data in the cancer genome atlas and genotype-tissue expression project databases were analyzed with R language software. Survival curve and receiver operating characteristic analyses were performed using the SurvMiner, Survival, and Procr language packages. The gene expression profiling interactive analysis 2.0 database was used to analyze the expression levels of PEA3 subfamily genes in different subtypes and stages of CCA. Web Gestalt was used to perform the gene ontology/ Kyoto encyclopedia of genes and genomes (GO/KEGG) analysis, and STRING database analysis was used to determine the genes and proteins related to PEA3 subfamily genes. ETV1, ETV4, and ETV5 expression levels were significantly increased in CCA. There were significant differences in ETV1, ETV4, and ETV5 expression levels among the different subtypes of CCA, and predictive analysis revealed that only high ETV1 and ETV4 expression levels were significantly associated with shorter overall survival in patients with CCA. GO/KEGG analysis revealed that PEA3 subfamily genes were closely related to transcriptional misregulation in cancer. In vitro and in vivo experiments revealed that PEA3 silencing inhibited the invasion and metastasis of CCA cells. The expression level of ETV4 may be a predictive biomarker of survival in patients with CCA.

STUB1-mediated K63-linked ubiquitination of UHRF1 promotes the progression of cholangiocarcinoma by maintaining DNA hypermethylation of PLA2G2A

BackgroundCholangiocarcinoma (CCA) is a highly malignant tumor characterized by a lack of effective targeted therapeutic strategies. The protein UHRF1 plays a pivotal role in the preservation of DNA methylation and works synergistically with DNMT1. Posttranscriptional modifications (PTMs), such as ubiquitination, play indispensable roles in facilitating this process. Nevertheless, the specific PTMs that regulate UHRF1 in CCA remain unidentified.MethodsWe confirmed the interaction between STUB1 and UHRF1 through mass spectrometry analysis. Furthermore, we investigated the underlying mechanisms of the STUB1-UHRF1/DNMT1 axis via co-IP experiments, denaturing IP ubiquitination experiments, nuclear‒cytoplasmic separation and immunofluorescence experiments. The downstream PLA2G2A gene, regulated by the STUB1-UHRF1/DNMT1 axis, was identified via RNA-seq. The negative regulatory mechanism of PLA2G2A was explored via bisulfite sequencing PCR (BSP) experiments to assess changes in promoter methylation. The roles of PLA2G2A and STUB1 in the proliferation, invasion, and migration of CCA cells were assessed using the CCK-8 assay, colony formation assay, Transwell assay, wound healing assay and xenograft mouse model. We evaluated the effects of STUB1/UHRF1 on cholangiocarcinoma by utilizing a primary CCA mouse model.ResultsThis study revealed that STUB1 interacts with UHRF1, resulting in an increase in the K63-linked ubiquitination of UHRF1. Consequently, this facilitates the nuclear translocation of UHRF1 and enhances its binding affinity with DNMT1. The STUB1-UHRF1/DNMT1 axis led to increased DNA methylation of the PLA2G2A promoter, subsequently repressing its expression. Increased STUB1 expression in CCA was inversely correlated with tumor progression and overall survival. Conversely, PLA2G2A functions as a tumor suppressor in CCA by inhibiting cell proliferation, invasion and migration.ConclusionsThese findings suggest that the STUB1-mediated ubiquitination of UHRF1 plays a pivotal role in tumor progression by epigenetically silencing PLA2G2A, underscoring the potential of STUB1 as both a prognostic biomarker and therapeutic target for CCA.Graphical

Identification of PRMT5 as a therapeutic target in cholangiocarcinoma

BackgroundCholangiocarcinoma (CCA) is a very difficult-to-treat cancer. Chemotherapies are little effective and response to immune checkpoint inhibitors is limited. Therefore, new therapeutic strategies need to be identified.ObjectiveWe characterised the enzyme...

A novel biomarker GATM suppresses proliferation and malignancy of cholangiocarcinoma cells by modulating the JNK/c-Jun signalling pathways

BackgroundCholangiocarcinoma (CCA) is the second most common primary malignancy of the liver and is associated with poor prognosis. Despite the emerging role of glycine amidinotransferase (GATM) in cancer development, its function in CCA remains elusive. This study investigated the biological significance and molecular mechanisms of GATM in CCA. MethodGATM expression was measured using immunohistochemistry and western blotting. Cell proliferation, migration, and invasion were assessed through CCK-8, EdU, clone formation, wound healing, and Transwell assays. Rescue experiments were performed to determine whether the JNK/c-Jun pathway is involved in GATM-mediated CCA development. Immunoprecipitation and mass spectrometry were performed to screen for proteins that interact with GATM. The role of GATM in vivo was investigated according to the xenograft experiment. ResultGATM expression was downregulated in CCA tissues and cells (p < 0.05) and had a significant suppressive effect on CCA cell proliferation, migration, and invasion in vitro as well as on tumour growth in vivo (p < 0.05); conversely, GATM knockdown promoted these phenotypes (p < 0.05). Notably, GATM inhibited the JNK/c-Jun pathway, and JNK activation abrogated GATM's antitumor effects (p < 0.05). Isocitrate dehydrogenase 1 (IDH1) interacts with GATM, and IDH1 knockdown significantly attenuated GATM protein degradation. Overexpression of IDH1 restored the biological function of CCA by reversing the inhibition of JNK/c-Jun pathway phosphorylation by GATM (p < 0.05). ConclusionGATM acts as a tumour suppressor in CCA by regulating the phosphorylation of the JNK/c-Jun pathway. IDH1 interacted with GATM to regulate CCA progression.

RBM39 Enhances Cholangiocarcinoma Growth Through EZH2-mediated WNT7B/β-catenin Pathway

Background and aimsThe RNA-binding motif protein 39 (RBM39) functions as both an RNA-binding protein and a splicing factor in a variety of cancer types. However, the function of RBM39 in cholangiocarcinoma (CCA) remains undefined. In this study, we aimed to investigate the role of RBM39 in CCA and explore its potential as a therapeutic target. MethodsThe expression of RBM39 in CCA was investigated by analyzing human CCA tumor specimens. CRISPR/Cas9 or shRNA-mediated depletion of RBM39 was performed in vitro and in vivo to document the oncogenic role of RBM39 in CCA. The anti-tumor effect of the RBM39 inhibitor Indisulam in combination with the EZH2 degrader MS177 was assessed in vitro and in vivo. ResultsRBM39 is significantly increased in human CCA tissues and associated with a poor prognosis in CCA patients. Depletion of RBM39 by CRISPR/Cas9 or shRNA inhibited CCA cell proliferation in vitro and prevented CCA development and tumor growth in mice. Mechanistically, our results showed that depletion of RBM39 suppressed EZH2 expression via disrupting its mRNA splicing. RBM39-regulated EZH2 controls WNT7B/β-catenin activity. Pharmacological co-targeting of RBM39 (with Indisulam) and EZH2 (with MS177) resulted in a synergistic antitumor effect, both in vitro and in vivo. ConclusionThis study discloses a novel RBM39-EZH2-β-catenin signaling axis that is crucial for CCA growth. Our findings suggest that simultaneous inhibition of RBM39 and EZH2 presents a promising therapeutic strategy for CCA treatment.

Cannabidiol exhibits potent anti-cancer activity against gemcitabine-resistant cholangiocarcinoma via ER-stress induction in vitro and in vivo

BackgroundFailure of treatment with gemcitabine in most cholangiocarcinoma (CCA) patients is due to drug resistance. The therapeutic potential of natural plant secondary compounds with minimal toxicity, such as cannabidiol (CBD), is a promising line of investigation in gemcitabine-resistant CCA. We aim to investigate the effects of CBD on gemcitabine-resistant CCA (KKU-213BGemR) cells in vitro and in vivo.MaterialsIn vitro, cell proliferation, colony formation, apoptosis and cell cycle arrest were assessed using MTT assay, clonogenicity assay and flow cytometry. The effect of CBD on ROS production was evaluated using the DCFH-DA fluorescent probe. The mechanism exerted by CBD on ER stress-associated apoptosis was investigated by western blot analysis. A gemcitabine-resistant CCA xenograft model was also used and the expression of PCNA and CHOP were evaluated by immunohistochemical analysis.ResultsThe IC50 values of CBD for KKU-213BGemR cells ranged from 19.66 to 21.05 µM. For a non-cancerous immortalized fibroblast cell line, relevant values were 18.29 to 19.21 µM. CBD suppressed colony formation by KKU-213BGemR cells in a dose-dependent manner in the range of 10 to 30 µM. CBD at 30 µM significantly increased apoptosis at early (16.37%) (P = 0.0024) and late (1.8%) stages (P < 0.0001), for a total of 18.17% apoptosis (P = 0.0017), in part by increasing ROS production (P < 0.0001). Multiphase cell cycle arrest significantly increased at G0/G1 with CBD 10 and 20 µM (P = 0.004 and P = 0.017), and at G2/M with CBD 30 µM (P = 0.005). CBD treatment resulted in increased expression of ER stress-associated apoptosis proteins, including p-PERK, BiP, ATF4, CHOP, BAX, and cytochrome c. In xenografted mouse, CBD significantly suppressed tumors at 10 and 40 mg/kg·Bw (P = 0.0007 and P = 0.0278, respectively), which was supported by an increase in CHOP, but a decrease in PCNA expression in tumor tissues (P < 0.0001).ConclusionThe results suggest that CBD exhibits potent anti-cancer activity against gemcitabine-resistant CCA in vitro and in vivo, in part via ER stress-mediated mechanisms. These results indicate that clinical explorative use of CBD on gemcitabine-resistant CCA patients is warranted.

Hypoxia Stimulates PYGB Enzymatic Activity to Promote Glycogen Metabolism and Cholangiocarcinoma Progression.

Cholangiocarcinoma (CCA) displays enhanced glycolysis, pivotal for fulfilling the heightened energy demands intrinsic to its malignant progression. Recent research has indicated that endogenous glycogen rather than exogenous glucose acts as the major carbon source for glycolysis, highlighting the need to better understand the regulation of glycogen homeostasis in CCA. Here, through comprehensive integrative analysis, we identified that glycogen phosphorylase brain form (PYGB), the main enzyme involved in glycogen homeostasis, was markedly upregulated in CCA tissues, serving as an independent prognostic indicator for human CCA patients. Moreover, elevated PYGB expression potentiated cholangiocarcinogenesis and augmented CCA cell proliferation in both organoid and xenograft models. Hypoxia stimulated PYGB activity in a phosphoglycerate kinase 1 (PGK1)-dependent manner, leading to glycogenolysis and the subsequent release of glucose-6-phosphate (G6P) and thereby facilitating aerobic glycolysis. Notably, a virtual screening pinpointed the beta-blocker carvedilol as a potent pharmacological inhibitor of PYGB that could attenuate CCA progression. Collectively, these findings position PYGB as a promising prognostic biomarker and therapeutic target for CCA.