Deep Sequencing Analysis Reveals Distinctive Non-Coding RNAs When Comparing Tumor Multidrug-Resistant Cells and Extracellular Vesicles with Drug-Sensitive Counterparts.

Diana Sousa,Raquel T Lima,M Helena Vasconcelos,Rune Matthiesen

doi:10.3390/cancers12010200

Abstract

Multidrug resistance (MDR) is one of the main limitations of cancer treatment. The overexpression of drug-efflux pumps, such as P-glycoprotein (P-gp), is a major cause of MDR. Importantly, different studies have shown that extracellular vesicles (EVs) participate in the communication between MDR cells and drug-sensitive counterparts, promoting dissemination of the MDR phenotype. In the present work, we aimed to identify RNA species present in MDR cells and in EVs released by those cells, which may be associated with the MDR phenotype. The RNA content from two pairs (leukemia and lung cancer) of MDR (P-gp overexpressing) cells and their drug-sensitive counterparts, as well as from their EVs, was analyzed by deep sequencing. Our results showed distinctive transcripts for MDR cells and their EVs, when compared with their drug-sensitive counterparts. Remarkably, two pseudogenes (a novel pseudogene and RNA 5.8S ribosomal pseudogene 2) were found to be increased in EVs released by MDR cells in both leukemia and lung cancer models. Moreover, six miRs (miR-204-5p, miR-139-5p, miR-29c-5p, miR-551b-3p, miR-29b-2-5p, and miR-204-3p) exhibited altered levels in lung cancer MDR cells and their EVs. This study provides insights into the contribution of EVs to MDR.

Highlights

Cancer multidrug resistance (MDR) is a particular case of drug resistance, in which tumor cells fail to respond to more than one drug with different molecular structures and mechanisms of action [1,2].MDR is the result of a network of altered cellular mechanisms, being one of the major challenges of Cancers 2020, 12, 200; doi:10.3390/cancers12010200 www.mdpi.com/journal/cancersCancers 2020, 12, 200 cancer treatment
To confirm that extracellular vesicles (EVs) released by MDR cells are able to transfer a drug-resistant phenotype to drug-sensitive cells, as previously described by others [22,23,24], NCI-H460 cells were co-cultured with EVs isolated from the NCI-H460/R (MDR) cells and treated with doxorubicin
We argue that the two pseudogenes found to be increased in EVs released by MDR cells

Summary

Introduction

Cancer multidrug resistance (MDR) is a particular case of drug resistance, in which tumor cells fail to respond to more than one drug with different molecular structures and mechanisms of action [1,2].MDR is the result of a network of altered cellular mechanisms, being one of the major challenges of Cancers 2020, 12, 200; doi:10.3390/cancers12010200 www.mdpi.com/journal/cancersCancers 2020, 12, 200 cancer treatment. Several studies have associated high levels of P-gp with drug resistance, MDR, in a variety of tumor types [3]. Another important cellular alteration which was more recently described is the release of extracellular vesicles (EVs) by MDR cells [4]. Exosomes are developed by the endocytic pathway and microvesicles are produced by budding of the plasma membrane. In the past, these two classes of EVs were distinguished on the basis of their size [6]

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