MicroRNA-27a-3p Modulates FoxO1 to Induce Tumour-Like Phenotypes in Bile Ducts - European Medical Journal

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MicroRNA-27a-3p Modulates FoxO1 to Induce Tumour-Like Phenotypes in Bile Ducts

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Hepatology
EMJ Hepatology 9.1 Feature Image
Authors:
Patricia Munoz-Garrido, ¹† Lea Duwe, ¹† Colm O'Rourke,1 Letizia Satriano,1 Monika Lewinska,1 Juan LaFuente-Barquero,1 Andrzej Taranta,1 Awaisa Ghazal,1 Dan Høgdall,1,2 Jens Marquardt,3 Matthias Matter,4 Jesus Banales,5 *Jesper Andersen1

1. Biotech Research & Innovation Centre (BRIC), Department of Health and Medical Sciences, University of Copenhagen, Denmark
2. Department of Oncology, Herlev and Gentofte Hospital, Copenhagen University Hospital, Herlev, Denmark
3. Department of Medicine I, UKSH-Campus Lübeck, Germany
4. Department of Pathology, Institute of Pathology and Medical Genetics, University Hospital of Basel, Switzerland
5. Biodonostia Health Research Institute, Department of Liver Diseases, San Sebastian, Spain
*Correspondence to [email protected]

†Equal author contribution

Disclosure:

The laboratory of Andersen is funded by a competitive grant from the Novo Nordisk Foundation (#14040). Munoz-Garrido, O’Rourke, and LaFuente-Barquero were awarded postdoctoral fellowships from the European Union (EU) Marie Curie programme (MiRCHOL, EpiTarget, and EpiCC, respectively). Munoz-Garrido was awarded a Sheila Sherlock postdoctoral fellowship from the European Association for the Study of the Liver (EASL) and Young Investigator Bursary for International Liver Congress (ILC) 2018. Taranta was awarded an individual postdoctoral fellowship from the Lundbeck Foundation. Duwe was awarded a PhD project grant from the Danish Cancer Research Foundation and Young Investigator Bursary for ILC 2021. This project was supported by the EU’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 801481.

Acknowledgements:

The authors would like to thank the patients and their families for inclusion of samples and clinical data.

Citation:
EMJ Hepatol. ;9[1]:32-33. Abstract Review No. AR.3..
Keywords:
Cholangiocarcinoma (CCA), epigenomics, FOXO1, microRNA (miR), transcription factor.

Each article is made available under the terms of the Creative Commons Attribution-Non Commercial 4.0 License.

BACKGROUND AND AIMS

Cholangiocarcinoma (CCA) is a clinically1,2 and molecularly heterogeneous disease.3,4 The ability of microRNAs (miRs) to target multiple signalling pathways links them to tumour heterogeneity.5-7 Here, the authors analysed the miR landscape of CCA and non-malignant livers to define their roles in orchestrating gene regulation and promoting CCA development.

METHOD

The authors performed miR sequencing (miRseq) of 190 human samples (99 intrahepatic CCAs, 28 extrahepatic CCAs, and 63 matched adjacent livers), including matched transcriptome data from 184 samples, to study miRs and regulation of target genes in CCA. High-throughput screening (HTS) using a library of 2,700 miR mimics was performed to characterise their proliferative impact in three primary normal human cholangiocyte cultures. After miRseq-HTS data integration, the authors performed target prediction and pathway over-representation analyses. The main target was confirmed by luciferase reporter assay and characterised by quantitative PCR, immunoblotting, and in situ hybridisation in tissue microarrays. To identify the biological role, the authors generated miR-CRISPR knock-out lines and performed proliferation and wound-healing assays.

RESULTS

MiRseq revealed 398 differentially expressed miRs (388 upregulated, 10 downregulated) in CCA compared with non-malignant tissues. Unsupervised hierarchical clustering identified three tumour subgroups, differing significantly based on tumour location, overall survival, tumour microenvironment (i.e., macrophage, hepatic stellate, and endothelial cell content), and immune infiltrates (dendritic cells, CD4+, and CD8+ T cells).

In HTS, overexpression of 224 miRs increased the normal human cholangiocyte proliferation rate, of which 35 miRs were upregulated in CCA. Pathway over-representation analysis showed FoxO signaling as the major affected pathway.

Further, miR-27a-3p and FoxO1 interaction revealed a strong negative correlation in the CCA cohort, which was independently validated in an external cohort (TCGA-CHOL). This interaction was confirmed in vitro by luciferase reporter assay. In situ hybridisation showed that miR-27a-3p is highly expressed in tumour cells and vascular smooth muscle. MiR-27a-3p knock-out CCA cells showed decreased proliferation and migration, highlighting miR-27a-3p as an oncogenic dependency in CCA.

CONCLUSION

MiRs are highly deregulated in CCA. MiR-27a-3p targets FoxO1, contributing to altered FoxO signalling and tumour phenotypes (Figure 1).

Figure 1: miR-27a-3p regulation of FOXO1 in cholangiocarcinoma.
CCA: cholangiocarcinoma; eCCA: extrahepatic cholangiocarcinoma; iCCA: intrahepatic cholangiocarcinoma; KO: knock out; miR-seq: microRNA sequencing.

References
Valle JW et al. Biliary tract cancer. Lancet. 2021;397(10272):428-44. Banales JM et al. Cholangiocarcinoma 2020: the next horizon in mechanisms and management. Nat Rev Gastroenterol Hepatol. 2020;17(9):557-88. Nepal C et al. Genomic perturbations reveal distinct regulatory networks in intrahepatic cholangiocarcinoma. Hepatology. 2018;68(3):949-63. Farshidfar F et al. Integrative genomic analysis of cholangiocarcinoma identifies distinct IDH-mutant molecular profiles. Cell Rep. 2017;19(13):2878-80. Lozano E et al. Causes of hOCT1-dependent cholangiocarcinoma resistance to sorafenib and sensitization by tumor-selective gene therapy. Hepatology. 2019;70(4):1246-61. Oishi N et al. Transcriptomic profiling reveals hepatic stem-like gene signatures and interplay of miR-200c and epithelial-mesenchymal transition in intrahepatic cholangiocarcinoma. Hepatology. 2012;56(5):1792-803. Lampis A et al. MIR21 drives resistance to heat shock protein 90 inhibition in cholangiocarcinoma. Gastroenterology. 2018;154(4):1066-79.e5.

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