Exosomes confer chemoresistance to pancreatic cancer cells by promoting ROS detoxification and miR-155-mediated suppression of key gemcitabine-metabolising enzyme, DCK.
BACKGROUND
Chemoresistance is a significant clinical problem in pancreatic cancer (PC) and underlying molecular mechanisms still remain to be completely understood. Here we report a novel exosome-mediated mechanism of drug-induced acquired chemoresistance in PC cells.


METHODS
Differential ultracentrifugation was performed to isolate extracellular vesicles (EVs) based on their size from vehicle- or gemcitabine-treated PC cells. Extracellular vesicles size and subtypes were determined by dynamic light scattering and marker profiling, respectively. Gene expression was examined by qRT-PCR and/or immunoblot analyses, and direct targeting of DCK by miR-155 was confirmed by dual-luciferase 3'-UTR reporter assay. Flow cytometry was performed to examine the apoptosis indices and reactive oxygen species (ROS) levels in PC cells using specific dyes. Cell viability was determined using the WST-1 assay.


RESULTS
Conditioned media (CM) from gemcitabine-treated PC cells (Gem-CM) provided significant chemoprotection to subsequent gemcitabine toxicity and most of the chemoresistance conferred by Gem-CM resulted from its EVs fraction. Sub-fractionation grouped EVs into distinct subtypes based on size distribution and marker profiles, and exosome (Gem-Exo) was the only sub-fraction that imparted chemoresistance. Gene expression analyses demonstrated upregulation of SOD2 and CAT (ROS-detoxifying genes), and downregulation of DCK (gemcitabine-metabolising gene) in Gem-Exo-treated cells. SOD/CAT upregulation resulted, at least in part, from exosome-mediated transfer of their transcripts and they suppressed basal and gemcitabine-induced ROS production, and partly promoted chemoresistance. DCK downregulation occurred through exosome-delivered miR-155 and either the functional suppression of miR-155 or restoration of DCK led to marked abrogation of Gem-Exo-mediated chemoresistance.


CONCLUSIONS
Together, these findings establish a novel role of exosomes in mediating the acquired chemoresistance of PC.
Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, Mobile, AL, USA.
Girijesh Kumar
GK
Patel
Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, Mobile, AL, USA.
Mohammad Aslam
MA
Khan
Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, Mobile, AL, USA.
Arun
A
Bhardwaj
Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, Mobile, AL, USA.
Sanjeev K
SK
Srivastava
Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, Mobile, AL, USA.
Haseeb
H
Zubair
Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, Mobile, AL, USA.
Mary C
MC
Patton
Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, Mobile, AL, USA.|Department of Biochemistry and Molecular Biology, College of Medicine, University of South Alabama, Mobile, AL, USA.
Seema
S
Singh
Department of Interdisciplinary Clinical Oncology, Mitchell Cancer Institute, University of South Alabama, Mobile, AL, USA.
Moh'd
M
Khushman
Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, Mobile, AL, USA.|Department of Biochemistry and Molecular Biology, College of Medicine, University of South Alabama, Mobile, AL, USA.
Ajay P
AP
Singh
D020413:3' Untranslated Regions; D002374:Catalase / Q000235:genetics*; D045744:Cell Line, Tumor; D003841:Deoxycytidine / Q000031:analogs & derivatives / Q000494:pharmacology; D003842:Deoxycytidine Kinase / Q000235:genetics*; D019008:Drug Resistance, Neoplasm; D000067493:Dynamic Light Scattering; D055354:Exosomes / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D035683:MicroRNAs / Q000235:genetics*; D010190:Pancreatic Neoplasms / Q000235:genetics* / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism; D013482:Superoxide Dismutase / Q000235:genetics*; D000093542:Gemcitabine; D000099156:Superoxide Dismutase 2
D020413:3' Untranslated Regions; D002374:Catalase; D003841:Deoxycytidine; D003842:Deoxycytidine Kinase; D035683:MicroRNAs; D017382:Reactive Oxygen Species; D013482:Superoxide Dismutase; D000093542:Gemcitabine; D000099156:Superoxide Dismutase 2; C524942:MIRN155 microRNA, human; C577219:CAT protein, human