Identification of Ras suppressor-1 (RSU-1) as a potential breast cancer metastasis biomarker using a three-dimensional<i>in vitro</i>approach

Abstract

// Vasiliki Gkretsi 1 , Andreas Stylianou 1 , Maria Louca 1 , Triantafyllos Stylianopoulos 1 1 Cancer Biophysics Laboratory, Department of Mechanical and Manufacturing Engineering, University of Cyprus, Nicosia, Cyprus Correspondence to: Triantafyllos Stylianopoulos, email: tstylian@ucy.ac.cy Keywords: Ras suppressor-1, extracellular matrix stiffness, invasion, tumor spheroids, atomic force microscopy Received: July 16, 2016&nbsp;&nbsp;&nbsp;&nbsp; Accepted: February 20, 2017&nbsp;&nbsp;&nbsp;&nbsp; Published: March 09, 2017 ABSTRACT Breast cancer (BC) is the most common malignant disease in women, with most patients dying from metastasis to distant organs, making discovery of novel metastasis biomarkers and therapeutic targets imperative. Extracellular matrix (ECM)-related adhesion proteins as well as tumor matrix stiffness are important determinants for metastasis. As traditional two-dimensional culture does not take into account ECM stiffness, we employed 3-dimensional collagen I gels of increasing concentration and stiffness to embed BC cells of different invasiveness (MCF-7, MDA-MB-231 and MDA-MB-231-LM2) or tumor spheroids. We tested the expression of cell-ECM adhesion proteins and found that Ras Suppressor-1 (RSU-1) is significantly upregulated in increased stiffness conditions. Interestingly, RSU-1 siRNA-mediated silencing inhibited Urokinase Plasminogen Activator, and metalloproteinase-13, whereas tumor spheroids formed from RSU-1-depleted cells lost their invasive capacity in all cell lines and stiffness conditions. Kaplan-Meier survival plot analysis corroborated our findings showing that high RSU-1 expression is associated with poor prognosis for distant metastasis-free and remission-free survival in BC patients. Taken together, our results indicate the important role of RSU-1 in BC metastasis and set the foundations for its validation as potential BC metastasis marker.