ROBO3 promotes growth and metastasis of pancreatic carcinoma

Highlights

• ROBO3 expression is up-regulated in pancreatic cancer.

• miR-383 as a suppressor of ROBO3, and revealed its expression.

• We revealed an interaction between Wnt inhibitor, SFRP, and ROBO3 in pancreatic cancer.

• ROBO3 may contribute to the progression of pancreatic cancer by sequestering Wnt inhibitor SFRP, which in turn leads to increased Wnt /β-catenin pathway activity.

Abstract

Pancreatic carcinoma is a highly lethal malignancy with an extremely poor prognosis. Recent genome-wide studies have implicated axon guidance pathways, including the SLIT/ROBO pathway, in pancreatic tumor development and progression. Here we showed that ROBO3 expression is up-regulated in pancreatic cancer tissue samples and cell lines. Over-expression of ROBO3 promotes pancreatic cancer cell growth, invasion and metastasis in vitro and in mouse xenograft tumor models. We identified miR-383 as a suppressor of ROBO3, and revealed its expression to be inversely correlated with ROBO3. Over-expression of ROBO3 activates Wnt pathway components, β-catenin and GSK-3, and the expression of markers indicating an EMT. By means of immunoprecipitation, we revealed an interaction between Wnt inhibitor SFRP and ROBO3 in pancreatic cancer cell lines. Our work suggests that ROBO3 may contribute to the progression of pancreatic cancer by sequestering Wnt inhibitor SFRP, which in turn leads to increased Wnt/β-catenin pathway activity. We also confirmed that ROBO3 increases with clinical grade and miR-383 expression is inversely correlated to that of ROBO3.

Introduction

Pancreatic cancer is the eighth leading cause of cancer deaths worldwide and has a very poor prognosis, with an overall 5-year survival rate of <5% [1]. Pancreatic cancer is characterized by early metastasis [2] and the majority of patients present with local regional spread and/or distant metastasis. Its diagnosis at an advanced stage is a major obstacle to long-term survival [3]. The highly aggressive nature of pancreatic cancer may be partly attributed to the chemotherapy-resistant characteristics of specific subgroups of pancreatic cancer cells, namely, those with an endothelial to mesenchymal transition (EMT) phenotype and cancer stem cells [4]. The EMT is an orchestrated series of events leading to altered cell–cell and cell–extracellular matrix interactions, which ultimately confers upon cells migratory and invasive properties. The loss of cell–cell adhesion occurs because of the inhibition of epithelial characteristics, such as E-cadherin, and the acquisition of mesenchymal features, such as N-cadherin, Twist, Snail1 and Snail2 expression [5]. Numerous molecules, signaling pathways, transcription factors and microRNAs have been described that induce EMT pathways in pancreatic cancer in vivo or in vitro [6].

Pancreatic tumors, of which more than 90% are pancreatic ductal adenocarcinomas (PDAC), make up a heterogeneous set of diseases encompassing cancer tissues of endocrine and exocrine pancreas [7]. Previous studies have characterized multiple cellular signaling pathways which may explain the major features of pancreatic tumorigenesis [8], including the Hedgehog, transforming growth factor-β (TGF-β), Notch1, KRAS, and Wnt signaling pathways.

The SLIT/ROBO pathway has also been implicated in tumor development and progression [9], including pancreatic cancer [10], [11]. Three Slits (SLIT1, SLIT2 and SLIT3) and four ROBO receptors (ROBO1–ROBO4) have been characterized in mammals [12]. Slits are secreted glycoproteins and the main ligands for Roundabout receptors (ROBOs) [13]. ROBO receptors are members of the immunoglobulin superfamily of cell adhesion molecules (ICAMs) and are capable of homophilic and heterophilic interactions, suggesting that they have Slit-independent functions [14]. ROBO3 is a known inhibitor of the ROBO2 signaling pathway [15], although a positive interaction between the two receptors in regulating eagle expression in serotonergic neuron differentiation has also been described [16]. The molecular mechanism by which dys-regulated SLIT/ROBO signaling promotes tumor progression is unclear. Both a direct ROBO-mediated intracellular signaling pathway [17] and a crosstalk of the HGF tyrosine kinase receptor MET pathway (through CDC42) and Wnt/β-catenin signaling activity (through β-catenin) [18] have been described.

MicroRNAs (miRNAs) are a class of small, non-coding endogenous RNAs, 21–30 nucleotides in length, that direct the translational repression or mRNA destabilization (or both) of protein-coding genes [19]. A number of miRNAs have been implicated in pancreatic cancer [20], many of which are putatively involved in regulating the EMT process and maintenance of pancreatic cancer stem cells, including the miR-200 family, miR-203, miR-21, miR-34, miR-365, miR-144, and let-7 [20].