In1-ghrelin splicing variant is associated with reduced disease-free survival of breast cancer patients and increases malignancy of breast cancer cells lines

OXFORD UNIVERSITY: This is a pre-copyedited, author-produced version of an article accepted for publication in Carcinogenesis following peer review. The version of record David Rincón-Fernández, Michael D Culler, Natia Tsomaia, Gema Moreno-Bueno, Raúl M Luque, Manuel D Gahete, Justo P Castaño; In1-ghrelin splicing variant is associated with reduced disease-free survival of breast cancer patients and increases malignancy of breast cancer cells lines, Carcinogenesis, Volume 39, Issue 3, 8 March 2018, Pages 447–457, https://doi.org/10.1093/carcin/bgx146Ghrelin gene generates several variants that regulate multiple pathophysiological functions, including tumor-related processes. In1-ghrelin is a splicing variant that was previously shown to be overexpressed in breast cancer (BCa), where it correlated with proliferation markers; however, its possible association with clinical outcome of BCa patients and underlying mechanisms are still unknown. To address this issue, expression levels and clinical associations of In1-ghrelin were analyzed in a cohort of 117 BCa samples. Additionally, a battery of cellular and molecular assays was implemented using two BCa cell lines (MCF-7 and MDA-MB-231), wherein the role of In1-ghrelin on proliferation, migration, dedifferentiation and signaling pathways was explored. The results generated revealed that high expression of In1-ghrelin in BCa samples was associated with lymph node metastasis and reduced disease-free survival. Indeed, In1-ghrelin overexpression stimulated proliferation and migration in MCF-7 and MDA-MB-231 cells. Similar results were found by treating MDA-MB-231 and MCF-7 with In1- ghrelin-derived peptides. Conversely, In1-ghrelin silencing decreased proliferation and migration capacities of MDA-MB-231. Furthermore, In1-ghrelin (but not ghrelin) overexpression increased the capacity to form mammospheres in both cell lines. These effects could be associated with activation of MAPK-ERK, Jag1/Notch, Wnt/β-catenin and/or TGF-β1 pathways. Altogether, our data indicate that In1-ghrelin could play relevant functional roles in the regulation of BCa development and progression and may provide insights to identify novel biomarkers and new therapeutic approaches for this pathology.BIO-0139, CTS-1406, PI-639-2012, PI-0541-2013 (Junta de Andalucia), BFU2013-43282-R, BFU2016-80360-R (MINECO), PI13-00651, PI16/00264 (Proyectos de Investigación en Salud FIS, funded by Instituto de Salud Carlos III), GETNE Grant 2014, Merck Serono Grant 2013 and CIBERobn (to RML and JPC); PI13/00132, RETICC RD12/0036/0007, CIBERonc and S2010/BMD-2303 (to GMB