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


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.
Molecular Oncology and Embryology Laboratory, Department of Human Anatomy and Embryology, School of Medicine, University of Barcelona, IDIBAPS, Barcelona, Spain.
Marc
M
Ruiz-Martinez
Molecular Oncology and Embryology Laboratory, Department of Human Anatomy and Embryology, School of Medicine, University of Barcelona, IDIBAPS, Barcelona, Spain.
Alfons
A
Navarro
Department of Pneumology, Institut Clínic del Tórax (ICT), Hospital Clinic de Barcelona, University of Barcelona, IDIBAPS, CIBER de Enfermedades Respiratorias (CIBERES), Barcelona, Spain.
Ramón M
RM
Marrades
Department of Medical Oncology, Institut Clinic Malalties Hemato-Oncològiques (ICMHO), Hospital Clinic de Barcelona, University of Barcelona, IDIBAPS, Barcelona, Spain.
Nuria
N
Viñolas
Molecular Oncology and Embryology Laboratory, Department of Human Anatomy and Embryology, School of Medicine, University of Barcelona, IDIBAPS, Barcelona, Spain.
Sandra
S
Santasusagna
Molecular Oncology and Embryology Laboratory, Department of Human Anatomy and Embryology, School of Medicine, University of Barcelona, IDIBAPS, Barcelona, Spain.
Carmen
C
Muñoz
Department of Pathology, Centro de Diagnóstico Biomédico (CDB), Hospital Clinic de Barcelona, University of Barcelona, IDIBAPS, CIBERES, Barcelona, Spain.
Josep
J
Ramírez
Department of Thoracic Surgery, Institut Clínic del Tórax (ICT), Hospital Clinic de Barcelona, University of Barcelona, Barcelona, Spain.
Laureano
L
Molins
Molecular Oncology and Embryology Laboratory, Department of Human Anatomy and Embryology, School of Medicine, University of Barcelona, IDIBAPS, Barcelona, Spain.
Mariano
M
Monzo
D000072283:A549 Cells; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002289:Carcinoma, Non-Small-Cell Lung / Q000175:diagnosis / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology; D049109:Cell Proliferation / Q000235:genetics; D002470:Cell Survival / Q000235:genetics; D055354:Exosomes / Q000378:metabolism*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008175:Lung Neoplasms / Q000175:diagnosis / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology; D008297:Male; D035683:MicroRNAs / Q000235:genetics; D008875:Middle Aged; D011379:Prognosis; D050683:R-SNARE Proteins / Q000235:genetics*
D035683:MicroRNAs; D050683:R-SNARE Proteins; C491482:YKT6 protein, human
NSCLC; YKT6; exosomes; miR-134; miR-135b