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Journal of Experimental and Clinical Cancer Research

, 36:16

First Online: 23 January 2017Received: 17 September 2016Accepted: 13 December 2016DOI: 10.1186-s13046-016-0475-z

Cite this article as: Bozzi, F., Mogavero, A., Varinelli, L. et al. J Exp Clin Cancer Res 2017 36: 16. doi:10.1186-s13046-016-0475-z

Abstract

BackgroundStrategies aimed at obtaining a complete cytoreduction are needed to improve long-term survival for patients with colorectal cancer peritoneal carcinomatosis CRC-pc.

MethodsWe established organoid models from peritoneal metastases of two naïve CRC patients. A standard paraffin inclusion was conducted to compare their 3D structure and immunohistochemical profile with that of the corresponding surgical samples. RNA expression levels of the CRC stem cell marker LGR5 was measured by in situ hybridization. The secretome of organoids was profiled by mass spectrometry. Energy homeostasis of organoids was interfered with 4-IPP and metformin. Biochemical and metabolic changes after drug treatments were investigated by western blot and mass spectrometry. Mitochondria impairment was evaluated by electron microscopy and mitotraker staining.

ResultsThe two organoids recapitulated their corresponding clinical samples in terms of 3D structure and immmunoistochemical profile and were positive for the cancer stem cells marker LGR5. Proteomic analyses of organoids highlighted their strong dependence on energy producing pathways, which suggest that their targeting could be an effective therapeutic approach.

To test this hypothesis, we treated organoids with two drugs that target metabolism acting on AMP-activated protein kinase AMPK, the main regulator of cellular energy homeostasis, which may act as metabolic tumour suppressor in CRC. Organoids were treated with 4-IPP, an inhibitor of MIF-CD74 signalling axis which activates AMPK function, or metformin that inhibits mitochondrial respiratory chain complex I.

As a new finding we observed that treatment with 4-IPP downregulated AMPK signalling activity, reduced AKT phosphorylation and activated a JNK-mediated stress-signalling response, thus generating mitochondrial impairment and cell death. Metformin treatment enhanced AMPK activation, decreasing the activity of the anabolic factors ribosomal protein S6 and p4EBP-1 and inducing mitochondrial depolarization.

ConclusionWe provide evidence that the modulation of AMPK activity may be a strategy for targeting metabolism of CRC-pc organoids.

KeywordsOrganoids Metabolism AMPK 4-IPP Macrophage migration inhibitory factor Metformin Abbreviations3DThree-Dimensional

4-IPP4-iodo-6-phenylpyrimidine

AMPAdenosine monophosphate

ATPAdenosine triphosphate

C1Colorectal cancer peritoneal carcinomatosis organoid 1

C2Colorectal cancer peritoneal carcinomatosis organoid 2

C3Colorectal cancer peritoneal carcinomatosis organoid 3

C4Colorectal cancer peritoneal carcinomatosis organoid 4

C5Colorectal cancer peritoneal carcinomatosis organoid 5

CoACoenzyme A

CRCColorectal cancer

CRC-pcColorectal cancer peritoneal carcinomatosis

CSCsCancer Stem Cells

DAB3, 3′-diaminobenzidine

DMSODimethyl sulfoxide

FasFatty acids synthesis

FDRFalse discovery rate.

FFPEFormalin Fixed Paraffin Embedded

GC-MSIsotope-dilution gas-chromatography mass spectrometry

GOGene Ontology

HandEHaematoxylin and eosin

H2O2Hydrogen peroxide

ISHin situ Hybridization

JC-15′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide

lc-MS-MSNano-scale liquid-chromatography tandem mass spectrometry

LGR5Leucine-rich repeat-containing G-protein coupled receptor 5

MIFMacrophage Migration Inhibitory Factor

NADPHNicotinamide adenine dinucleotide phosphate

OsO4Osmium tetroxide

oxMIFOxidized Macrophage Migration Inhibitory Factor isoform

OXPHOSOxidative phosphorylation

PANTHERProtein Analysis Through Evolutionary Relationships

PDParkinson’s disease

pThrPhospho-threonine

pTyrPhospho-tyrosine

ROSReactive Oxygen Species

SDStandard deviation

SDSSodium dodecyl sulfate

STRINGSearch Tool for the Retrieval of Interacting Genes-proteins

TCATricarboxylic acid cycle

Electronic supplementary materialThe online version of this article doi:10.1186-s13046-016-0475-z contains supplementary material, which is available to authorized users.

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Autor: Fabio Bozzi - Angela Mogavero - Luca Varinelli - Antonino Belfiore - Giacomo Manenti - Claudio Caccia - Chiara C. Volpi -

Fuente: https://link.springer.com/







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