Smoking Induced Extracellular Vesicles Release and Their Distinct Properties in Non-Small Cell Lung Cancer

Abstract

Purpose : Smoking is a strong relative risk factor for lung cancer. Extracellular vesicles (EVs), particularly exosomes, have been implicated in cancers. In this study, we characterized smoking induced extracellular vesicles in smokers with non-small cell lung cancer (NSCLC). Methods : EVs were isolated from bronchoalveolar lavage (BAL) from smokers and NSCLC patients. EV microRNAs (miRNAs) were analyzed by using a TaqMan microRNA assays. Vesicle mRNAs and long non-coding RNAs (lncRNAs) were measured with quantitative RT-PCR. Tumor associated antigens were examined by Western Blot. Results : Higher levels of local site EVs are found in the lung of smokers and NSCLC patients. Further, over 90% of lung EVs are round vesicles of approximately 50-200 nm, ie., exosomes. There are 21 EV miRNAs up regulated, while 10 miRNAs under regulated, in smokers when compared to controls (relative fold > 2, p < 0.05). These miRNAs were further observed to be dysregulated in NSCLC patients when compared to smokers. Bioinformatic analysis demonstrated that Proteoglycans, Fatty acid biosynthesis, ErbB, Hippo, TGF-beta, Wnt, Rap1, AMPK and Ras pathways were the most prominent pathways enriched in NSCLC EV miRNA signatures. In addition, messenger RNA transcripts including EGFR, KRAS, ALK, MET, LKB1, BRAF, PIK3CA, RET, and ROS1 were significantly higher expressed in lung EVs in smokers and NSCLC patients compared to controls. Long non-coding RNAs, including MALAT1, HOTAIR, HOTTIP, AGAP2-AS1, ATB, TCF7, FOXD2-AS1, HOXA11-AS, PCAF1, and BCAR4, were over expressed in EVs from smokers and NSCLC patients. Furthermore, protein levels of tumor associated antigens including BAGE, PD-L1, MAGE-3, and AKAP4 were significantly dysregulated in EVs of smokers and NSCLC patients compared to healthy controls. Conclusions : In conclusion, these data demonstrated an intrinsic relationship of smoking dysregulated EVs and EVs contained RNA, proteins which may involve in the development of NSCLC.

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“…Studies examining the effect of smoking and EV release have shown an increase in EV number and sizes. For example, one study that assessed the bronchoalveolar lavage from smokers with nonsmall cell lung cancer showed an increase in EV particle release, which subsequently led to an increase in EV microRNA expression compared to healthy controls [47]. Another study using…”

“…Studies examining the effect of smoking and EV release have shown an increase in EV number and sizes. For example, one study that assessed the bronchoalveolar lavage from smokers with non-small cell lung cancer showed an increase in EV particle release, which subsequently led to an increase in EV microRNA expression compared to healthy controls [47]. Another study using airway epithelial cells exposed to cigarette smoke extract also showed an increase in EV particle release that was prevented by scavenging thiol anti-oxidants N-acetyl-L-cysteine or glutathione [48].…”

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“…Further, protein levels of tumor-associated antigens, including BAGE, PD-L1, MAGE-3, and AKAP4, were also significantly dysregulated in EVs of smokers and NSCLC. This study concluded that an intrinsic relationship exists between smoking and dysregulated EV secretion and cargo, the contents of which may contribute to the development of NSCLC [129].…”

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“…Moreover, they have identified this lncRNA in the blood and have suggested its potential role as circulating marker [ 131 ]. Numerous reports have shown that exosomal lncHOTAIR could be a sensitive liquid biomarker of different types of cancers, such as glioblastoma multiform, breast cancer and non-small-cell lung cancer (NSCLC) [ 132 , 133 , 134 ]. However, the potential use of EXOs for HOTAIR detection in melanoma cells has not yet been explored.…”