Abstract
We previously reported the identification of the diagnostic and prognostic miRNA signatures in plasma samples of lung cancer patients detected by low dose computed tomography (LDCT) screening. Interestingly we also found miRNA profiles able to predict lung cancer development and its aggressiveness up to 2 years before lung cancer detection by LDCT. Circulating miRNAs associated with risk of aggressive disease onset showed to be concordantly regulated in corresponding normal lung tissues, rather than in tumor tissue. Recently, several papers suggest that miRNAs released by the microenvironment can also have a role in targeting tumor cells promoting their growth and invasiveness, and may act as hormones by functionally activating Toll-like receptors.
In an effort to examine the components of the lung that are most involved in the generation of plasma miRNA signatures we looked at the expression of our 24 miRNAs in several cell components including fibroblasts, hematopoietic, endothelial, normal and tumor epithelial cells and in their respective culture medium. Our preliminary results indicated that every single miRNA is released differently and independently from the level of expression in respective cells across cell types and that tumor cells are less involved in the release of miRNAs. We also observed that several miRNAs are expressed and released from non-epithelial components only, whereas other miRNAs are expressed in all cellular components but released from specific cell types only.
Among the 24 miRNA composing the signatures, we focused on two miRNAs (mir-486-5p, mir-660) that are strongly down-modulated in plasma samples of patients with an aggressive form of lung cancer. To evaluate if they have also a functional role in lung tumorigenesis, their activity was assessed in lung cancer (A549) and normal human bronchial epithelial (HBEC-KT1) cellular models using miRNA mimics or inhibitors. A new method for in vitro functional studies was settled-up. A549 and HBEC cells were infected with a lentivirus carrying the gene encoding Green Fluorescent Protein (GFP) (A549GFP and HBECGFP) and their biological features were tested using a fluorescence microplate reader. By using this methodology, it was possible to assess the biological endpoints by measuring the emission of fluorescent signal without interrupting cellular growth or migration. Our experiments using mir-486-5p and mir-660 mimics on A549GFP show an impairment of migratory and invasive capacity of these cells. Moreover, inhibition of miRNAs in HBECGFP resulted in an increased number of migrated and invaded cells compared to control.
To evaluate the biological effect of these two miRNAs, in an in vivo model we injected A549 transiently transfected with mir-486 and mir-660 subcutaneously in both flanks of 4 to 6 weeks old female nude mice. Xenograft growth was monitored weekly by caliper measurement and data were shown as tumour growth rate or as Kaplan-Meyer curve (time required to reach tumor volume of 250 mm3 as endpoint). We demonstrated that miRNAs transiently over-expressed in A549 cells subcutaneously implanted in mice significantly reduce tumor growth and increase mice survival.
Our preliminary findings on the origin of miRNAs support the conclusion that plasma miRNAs are heterogeneous and contributed by different cellular components, and not specifically by tumor cells, a result in keeping with the predicting value of the miRNA test up to two years before clinical detection of lung cancer. Moreover, functional studies on mir-486-5p and mir-660, strongly down-modulated in plasma of patients, suggested that may act as tumor suppressor miRNAs. Further analyses are in progress to identify the specific pathways in which they are involved. Nonetheless these preliminary results set the basis for a possible therapeutic use of miRNAs in lung cancer.
Citation Format: Orazio Fortunato, Mattia Boeri, Carla Verri, Massimo Moro, Francesca Andriani, Luca Roz, Ugo Pastorino, Gabriella Sozzi. Origin and functional role of plasma circulating miRNAs. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr A19.
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