YKT6 expression, exosome release, and survival in non-small cell lung cancer

// Marc Ruiz-Martinez 1 , Alfons Navarro 1 , Ramón M. Marrades 2 , Nuria Viñolas 3 , Sandra Santasusagna 1 , Carmen Muñoz 1 , Josep Ramírez 4 , Laureano Molins 5 , Mariano Monzo 1 1 Molecular Oncology and Embryology Laboratory, Department of Human Anatomy and Embryology, School of Medicine, University of Barcelona, IDIBAPS, Barcelona, Spain 2 Department of Pneumology, Institut Clínic del Tórax (ICT), Hospital Clinic de Barcelona, University of Barcelona, IDIBAPS, CIBER de Enfermedades Respiratorias (CIBERES), Barcelona, Spain 3 Department of Medical Oncology, Institut Clinic Malalties Hemato-Oncològiques (ICMHO), Hospital Clinic de Barcelona, University of Barcelona, IDIBAPS, Barcelona, Spain 4 Department of Pathology, Centro de Diagnóstico Biomédico (CDB), Hospital Clinic de Barcelona, University of Barcelona, IDIBAPS, CIBERES, Barcelona, Spain 5 Department of Thoracic Surgery, Institut Clínic del Tórax (ICT), Hospital Clinic de Barcelona, University of Barcelona, Barcelona, Spain Correspondence to: Mariano Monzo, email: mmonzo@ub.edu Keywords: YKT6, exosomes, NSCLC, miR-134, miR-135b Received: February 11, 2016      Accepted: May 19, 2016      Published: June 06, 2016 ABSTRACT Background: Cancer-derived exosomes are involved in metastasis. YKT6 is a SNARE protein that participates in the regulation of exosome production and release, but its role in non-small cell lung cancer (NSCLC) has not been examined. Materials and Methods: Ultracentrifugation-purified exosomes from the A549 cell line were studied by CRYO-TEM, nanoparticle tracking analysis and western blot (TSG101 marker). YKT6 was inhibited using a DsiRNA and selected pre-microRNAs. MicroRNAs targeting YKT6 were validated by Renilla/Luciferase assay and western blot. YKT6 expression and its prognostic impact were analyzed in 98 tissue specimens from resected NSCLC patients. Results: Membranous nanosized vesicles (mode size: 128nm) with TSG101 protein were purified from A549 cells. YKT6 inhibition reduced exosome release by 80.9%. We validated miR-134 and miR-135b as miRNAs targeting YKT6, and transfection with the pre-miRNAs also produced a significant reduction in exosome release. The analysis of YKT6 in tumor samples showed that patients with high levels had shorter disease-free and overall survival. Conclusions: YKT6 is a key molecule in the regulation of exosome release in lung cancer cells and is in turn precisely regulated by miR-134 and miR-135b. Moreover, YKT6 levels impact prognosis of resected NSCLC patients.