Dataset Information

Epigenetic deregulation of MLF1 drives intrahepatic cholangiocarcinoma progression through EGFR/AKT and Wnt/β-catenin signaling.

ABSTRACT:

Background

Intrahepatic cholangiocarcinoma (iCCA) is an aggressive malignancy with multiple etiologies and is largely refractory to current treatment strategies. Myeloid leukemia factor 1 (MLF1) is associated with human cancer progression. Nevertheless, the function of MLF1 in iCCA remains unknown.

Methods

We performed expression analyses of MLF1 in human iCCA. In vitro and in vivo experiments were conducted to investigate the role of MLF1 in iCCA progression. The upstream regulatory mechanism of MLF1 upregulation in iCCA was deciphered by luciferase and DNA methylation analyses.

Results

MLF1 was significantly upregulated in clinical iCCA tissue specimens and human iCCA cell lines. MLF1 was positively correlated with KRT19 and MUC1 expression and epithelial-mesenchymal transition (EMT) gene set enrichment score in clinical iCCA. High MLF1 expression was independently associated with worse prognoses in iCCA patients after curative resection. In addition, experimental knockdown of MLF1 attenuated, while overexpression of MLF1 promoted the proliferation, invasiveness, and growth of iCCA cells in vitro and in vivo. Mechanically, MLF1 comodulated EGFR/AKT and Wnt/β-catenin signalings through regulating EGFR, AKT, WNT3, and p-GSK3β expression. Promoter CpG sites' hypermethylation-induced downregulation of miR-29c-3p contributed to MLF1 upregulation in iCCA patients. The upregulation of DNA methyltransferase (DNMT)1, 3A, and 3B downregulated miR-29c-3p by dictating promoter DNA methylation pattern. MiR-29c-3p showed therapeutic potential by targeting MLF1 in iCCA.

Conclusions

Our results demonstrated that hypermethylation-mediated miR-29c-3p downregulation contributes to MLF1 upregulation in iCCA, which resulted in tumor cells' proliferation and metastasis through comodulating EGFR/AKT and Wnt/β-catenin signalings.

SUBMITTER: Tang Z

PROVIDER: S-EPMC10368384 | biostudies-literature | 2023 Aug

REPOSITORIES: biostudies-literature

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Publications

Epigenetic deregulation of MLF1 drives intrahepatic cholangiocarcinoma progression through EGFR/AKT and Wnt/β-catenin signaling.

Tang Zengwei Z Tang Zengwei Z Yang Yuan Y Chen Wen W Liang Tingbo T

Hepatology communications 20230724 8

<h4>Background</h4>Intrahepatic cholangiocarcinoma (iCCA) is an aggressive malignancy with multiple etiologies and is largely refractory to current treatment strategies. Myeloid leukemia factor 1 (MLF1) is associated with human cancer progression. Nevertheless, the function of MLF1 in iCCA remains unknown.<h4>Methods</h4>We performed expression analyses of MLF1 in human iCCA. In vitro and in vivo experiments were conducted to investigate the role of MLF1 in iCCA progression. The upstream regulat ...[more]

PMID: 37486965

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Project description:BackgroundIntrahepatic cholangiocarcinoma (ICC) accounts for about 15% of primary liver cancer, and the incidence rate has been rising in recent years. Surgical resection is the best treatment for ICC, but the 5-year survival rate is less than 30%. ICC signature genes are crucial for the early diagnosis of ICC, so it is especially important to find its signature genes and therapeutic drug. Here, we studied that bufalin targeting CAMKK2 promotes mitochondrial dysfunction and inhibits the occurrence and metastasis of intrahepatic cholangiocarcinoma through Wnt/β-catenin signal pathway.MethodsIC50 of bufalin in ICC cells was determined by CCK8 and invasive and migratory abilities were verified by wound healing, cell cloning, transwell and Western blot. IF and IHC verified the expression of CAMKK2 between ICC patients and normal subjects. BLI and pull-down demonstrated the binding ability of bufalin and CAMKK2. Bioinformatics predicted whether CAMKK2 was related to the Wnt/β-catenin pathway. SKL2001, an activator of β-catenin, verified whether bufalin acted through this pathway. In vitro and in vivo experiments verified whether overexpression of CAMKK2 affects the proliferative and migratory effects of ICC. Transmission electron microscopy verified mitochondrial integrity. Associated Ca2+ levels verified the biological effects of ANXA2 on ICC.ResultsIt was found that bufalin inhibited the proliferation and migration of ICC, and CAMKK2 was highly expressed in ICC, and its high expression was positively correlated with poor prognosis.CAMKK2 is a direct target of bufalin, and is associated with the Wnt/β-catenin signaling pathway, which was dose-dependently decreased after bufalin treatment. In vitro and in vivo experiments verified that CAMKK2 overexpression promoted ICC proliferation and migration, and bufalin reversed this effect. CAMKK2 was associated with Ca2+, and changes in Ca2+ content induced changes in the protein content of ANXA2, which was dose-dependently decreasing in cytoplasmic ANXA2 and dose-dependently increasing in mitochondrial ANXA2 after bufalin treatment. In CAMKK2 overexpressing cells, ANXA2 was knocked down, and we found that reversal of CAMKK2 overexpression-induced enhancement of ICC proliferation and migration after siANXA2.ConclusionsOur results suggest that bufalin targeting CAMKK2 promotes mitochondrial dysfunction and inhibits the proliferation and migration of intrahepatic cholangiocarcinoma through Wnt/β-catenin signal pathway. Thus, bufalin, as a drug, may also be used for cancer therapy in ICC in the future.

Project description:Background & aimsIntrahepatic cholangiocarcinoma (ICC) is a devastating liver cancer with extremely high intra- and inter-tumoral molecular heterogeneity, partly due to its diverse cellular origins. We investigated clinical relevance and the molecular mechanisms underlying hepatocyte (HC)-driven ICC development.MethodsExpression of ICC driver genes in human diseased livers at risk for ICC development were examined. The sleeping beauty and hydrodynamic tail vein injection based Akt-NICD/YAP1 ICC model was used to investigate pathogenetic roles of SRY-box transcription factor 9 (SOX9) and yes-associated protein 1 (YAP1) in HC-driven ICC. We identified DNA methyltransferase 1 (DNMT1) as a YAP1 target, which was validated by loss- and gain-of-function studies, and its mechanism addressed by chromatin immunoprecipitation sequencing.ResultsCo-expression of AKT and Notch intracellular domain (NICD)/YAP1 in HC yielded ICC that represents 13% to 29% of clinical ICC. NICD independently regulates SOX9 and YAP1 and deletion of either, significantly delays ICC development. Yap1 or TEAD inhibition, but not Sox9 deletion, impairs HC-to-biliary epithelial cell (BEC) reprogramming. DNMT1 was discovered as a novel downstream effector of YAP1-TEAD complex that directs HC-to-BEC/ICC fate switch through the repression of HC-specific genes regulated by master regulators for HC differentiation, including hepatocyte nuclear factor 4 alpha, hepatocyte nuclear factor 1 alpha, and CCAAT/enhancer-binding protein alpha/beta. DNMT1 loss prevented NOTCH/YAP1-dependent HC-driven cholangiocarcinogenesis, and DNMT1 re-expression restored ICC development following TEAD repression. Co-expression of DNMT1 with AKT was sufficient to induce tumor development including ICC. DNMT1 was detected in a subset of HCs and dysplastic BECs in cholestatic human livers prone to ICC development.ConclusionWe identified a novel NOTCH-YAP1/TEAD-DNMT1 axis essential for HC-to-BEC/ICC conversion, which may be relevant in cholestasis-to-ICC pathogenesis in the clinic.

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Project description:β-Catenin, the downstream effector of the Wnt signaling, plays important roles in hepatic development, regeneration, and tumorigenesis. However, its role at hepatocyte adherens junctions (AJ) is relatively poorly understood, chiefly due to spontaneous compensation by γ-catenin. We simultaneously ablated β- and γ-catenin expression in mouse liver by interbreeding β-catenin-γ-catenin double-floxed mice and Alb-Cre transgenic mice. Double knockout mice show failure to thrive, impaired hepatocyte differentiation, cholemia, ductular reaction, progressive cholestasis, inflammation, fibrosis, and tumorigenesis, which was associated with deregulation of tight junctions (TJ) and bile acid transporters, leading to early morbidity and mortality, a phenotype reminiscent of progressive familial intrahepatic cholestasis (PFIC). To address the mechanism, we specifically and temporally eliminated both catenins from hepatocytes using adeno-associated virus 8 carrying Cre-recombinase under the thyroid-binding globulin promoter (AAV8-TBG-Cre). This led to a time-dependent breach of the blood-biliary barrier associated with sequential disruption of AJ and TJ verified by ultrastructural imaging and intravital microscopy, which revealed unique paracellular leaks around individual hepatocytes, allowing mixing of blood and bile and leakage of blood from one sinusoid to another. Molecular analysis identified sequential losses of E-cadherin, occludin, claudin-3, and claudin-5 due to enhanced proteasomal degradation, and of claudin-2, a β-catenin transcriptional target, which was also validated in vitro.ConclusionWe report partially redundant function of catenins at AJ in regulating TJ and contributing to the blood-biliary barrier. Furthermore, concomitant hepatic loss of β- and γ-catenin disrupts structural and functional integrity of AJ and TJ via transcriptional and posttranslational mechanisms. Mice with dual catenin loss develop progressive intrahepatic cholestasis, providing a unique model to study diseases such as PFIC. (Hepatology 2018;67:2320-2337).

Dataset Information

Epigenetic deregulation of MLF1 drives intrahepatic cholangiocarcinoma progression through EGFR/AKT and Wnt/β-catenin signaling.

Background

Methods

Results

Conclusions

Publications

Epigenetic deregulation of MLF1 drives intrahepatic cholangiocarcinoma progression through EGFR/AKT and Wnt/β-catenin signaling.

Similar Datasets

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