U-PAR expression in cancer associated fibroblast: new acquisitions in multiple myeloma progression

S Ciavarella,A Iacobazzi,F Margheri,S Tommasi,A Rana,G Fibbi,A Chillà,A Guarini,B Pilato,F Merchionne,A Laurenzana,R Lacalamita,M Del Rosso,C Minoia,S Serratì,S De Summa

doi:10.1186/s12885-017-3183-y

Abstract

BackgroundMultiple Myeloma (MM) is a B-cell malignancy in which clonal plasma cells progressively expand within the bone marrow (BM) as effect of complex interactions with extracellular matrix and a number of microenvironmental cells. Among these, cancer-associated fibroblasts (CAF) mediate crucial reciprocal signals with MM cells and are associated to aggressive disease and poor prognosis. A large body of evidence emphasizes the role of the urokinase plasminogen activator (u-PA) and its receptor u-PAR in potentiating the invasion capacity of tumor plasma cells, but little is known about their role in the biology of MM CAF. In this study, we investigated the u-PA/u-PAR axis in MM-associated fibroblasts and explore additional mechanisms of tumor/stroma interplay in MM progression.MethodsCAF were purified from total BM stromal fraction of 64 patients including monoclonal gammopathy of undetermined significance, asymptomatic and symptomatic MM, as well as MM in post-treatment remission. Flow cytometry, Real Time PCR and immunofluorescence were performed to investigate the u-PA/u-PAR system in relation to the level of activation of CAF at different stages of the disease. Moreover, proliferation and invasion assays coupled with silencing experiments were used to prove, at functional level, the function of u-PAR in CAF.ResultsWe found higher activation level, along with increased expression of pro-invasive molecules, including u-PA, u-PAR and metalloproteinases, in CAF from patients with symptomatic MM compared to the others stages of the disease. Consistently, CAF from active MM as well as U266 cell line under the influence of medium conditioned by active MM CAF, display higher proliferative rate and invasion potential, which were significantly restrained by u-PAR gene expression inhibition.ConclusionsOur data suggest that the stimulation of u-PA/u-PAR system contributes to the activated phenotype and function of CAF during MM progression, providing a biological rationale for future targeted therapies against MM.

Highlights

Multiple Myeloma (MM) is a B-cell malignancy in which clonal plasma cells progressively expand within the bone marrow (BM) as effect of complex interactions with extracellular matrix and a number of microenvironmental cells
Patients and cell cultures BM aspirates were obtained for diagnostic purpose from 64 subjects including 18 monoclonal gammopathy of undetermined significance (MGUS), 14 asymptomatic MM, 20 symptomatic active MM, and 12 MM in remission phase. sMM patients had a median age of 72 years, they were newly diagnosed and treatment-naïve
Frequency and activation state of cancer-associated fibroblasts (CAF) vary among MM progression As depicted in Fig. 1a, flow cytometry analysis demonstrated that CD45− cells co-expressing fibroblast specific protein 1 (FSP-1) and alpha smooth muscle actin (α-SMA), as major phenotypic markers of CAF, are significantly increased in the BM of patients with sMM (75 ± 18%)

Summary

Introduction

Multiple Myeloma (MM) is a B-cell malignancy in which clonal plasma cells progressively expand within the bone marrow (BM) as effect of complex interactions with extracellular matrix and a number of microenvironmental cells. CAF typically display enhanced expression of structural proteins as alpha-smooth muscle actin (α-SMA), vimentin and fibroblast specific protein 1 (FSP-1), and secrete high amounts of cytokines and growth factors directly involved in MM pathogenesis, such as interleukin 6 (IL-6), vascular endothelial growth factor (VEGF), transforming growth factor-beta (TGF-β), fibroblast growth factor-2 (FGF-2), and hepatocyte growth factor (HGF). They gain improved capacity of extracellular matrix (ECM) degradation by over-producing metalloproteinases (MMPs) and other enzymes [4], which favours the expansion of PC clone within the BM [5]. Despite most research focused on the role of this system in cancer cells, very little is known about its function in CAF as pivotal controllers of MM progression

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