Metformin exerts anti-cancerogenic effects and reverses epithelial-to-mesenchymal transition trait in primary human intrahepatic cholangiocarcinoma cells

Sabina Di Matteo,Jessica Faccioli,Vincenzo Cardinale,Giuseppe Donato,Angelo Oddi ,Agostino Maria De Rose ,Guido Carpino,Nadine Landolina,L Nevi ,D Costantini ,Eugenio Gaudio,A M Marziani ,P.b Berloco ,Chiara Napoletano,F Giulitti ,Fabio Melandro,F Giuliante ,Gian Luca Grazi,Diletta Overi,Maria Consiglia Bragazzi ,Lorenzo Moretta,Domenico Alvaro

doi:10.1038/s41598-021-81172-0

Abstract

Intrahepatic cholangiocarcinoma (iCCA) is a highly aggressive cancer with marked resistance to chemotherapeutics without therapies. The tumour microenvironment of iCCA is enriched of Cancer-Stem-Cells expressing Epithelial-to-Mesenchymal Transition (EMT) traits, being these features associated with aggressiveness and drug resistance. Treatment with the anti-diabetic drug Metformin, has been recently associated with reduced incidence of iCCA. We aimed to evaluate the anti-cancerogenic effects of Metformin in vitro and in vivo on primary cultures of human iCCA. Our results showed that Metformin inhibited cell proliferation and induced dose- and time-dependent apoptosis of iCCA. The migration and invasion of iCCA cells in an extracellular bio-matrix was also significantly reduced upon treatments. Metformin increased the AMPK and FOXO3 and induced phosphorylation of activating FOXO3 in iCCA cells. After 12 days of treatment, a marked decrease of mesenchymal and EMT genes and an increase of epithelial genes were observed. After 2 months of treatment, in order to simulate chronic administration, Cytokeratin-19 positive cells constituted the majority of cell cultures paralleled by decreased Vimentin protein expression. Subcutaneous injection of iCCA cells previously treated with Metformin, in Balb/c-nude mice failed to induce tumour development. In conclusion, Metformin reverts the mesenchymal and EMT traits in iCCA by activating AMPK-FOXO3 related pathways suggesting it might have therapeutic implications.

Highlights

Intrahepatic cholangiocarcinoma is a highly aggressive cancer with marked resistance to chemotherapeutics without therapies
Abbreviations AMPK Protein kinase AMP-activated catalytic subunit alpha 2 (PRKAA2) cancer stem cells (CSC) Cancer stem cells Cytokeratin-19 known as KRT19 or Keratin 19 E-Cadherin known as CDH1 or Cadherin 1 Epithelial-to-Mesenchymal Transition (EMT) Epithelial mesenchymal transition FOXO3 Forkhead box O3 iCCA Intrahepatic cholangiocarcinoma mesenchymal to epithelial transition (MET) Mesenchymal epithelial transition mTORC1 Mammalian target of rapamycin ND Not detectable peribiliary glands (PBGs) Peribiliary glands propidium iodide (PI) Propidium iodide PSC Primary sclerosing cholangitis VIM Vimentin WB Western blot
The main findings of our study indicate that, in primary cultures of human Large and Small duct-type iCCA, Metformin: (1) inhibited cell proliferation and induced apoptosis in a dose- and a time-dependent manner; (2) inhibited cell migration and invasion and colony formation capacity of iCCA cells; (3) downregulated the gene expression of mesenchymal and EMT genes and upregulated epithelial genes; (4) activated AMPK and FOXO3; (5) after prolonged exposure, induced MET in Large duct-type iCCA as demonstrated by the expression of Cytokeratin-19, the down-regulation of Vimentin and changes of cell morphology; (6) abolished the in vivo capacity of Large duct-type iCCA to develop tumour after subcutaneous injection in mice

Summary

Introduction

Intrahepatic cholangiocarcinoma (iCCA) is a highly aggressive cancer with marked resistance to chemotherapeutics without therapies. The tumour microenvironment of iCCA is enriched of CancerStem-Cells expressing Epithelial-to-Mesenchymal Transition (EMT) traits, being these features associated with aggressiveness and drug resistance. Metformin increased the AMPK and FOXO3 and induced phosphorylation of activating FOXO3 in iCCA cells. Abbreviations AMPK Protein kinase AMP-activated catalytic subunit alpha 2 (PRKAA2) CSC Cancer stem cells Cytokeratin-19 known as KRT19 or Keratin 19 E-Cadherin known as CDH1 or Cadherin 1 EMT Epithelial mesenchymal transition FOXO3 Forkhead box O3 iCCA Intrahepatic cholangiocarcinoma MET Mesenchymal epithelial transition mTORC1 Mammalian target of rapamycin ND Not detectable PBGs Peribiliary glands PI Propidium iodide PSC Primary sclerosing cholangitis VIM Vimentin WB Western blot. Cholangiocarcinoma (CCA) represents the second most frequent liver cancer that develops as a highly aggressive tumour with both a poor prognosis and increasing incidence worldwide. The histological appearance might reflect a different origin i.e.from large intrahepatic bile ducts with associated peribiliary glands (PBGs) or from interlobular bile ducts, bile ductules and canals of Hering[5]

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