Implication of Netrin-1 Gain of Expression in Canine Nodal Lymphoma

SIMPLE SUMMARY: Canine lymphomas represent one of the most frequent groups of neoplasia, for which prognosis may be poor. Treatments are based on polychemotherapy, with variable responses. As in human lymphomas, more and more targeted therapies are studied and developed. Therapy to restore apoptosis in neoplastic cells is one of them. Netrin-1 is a ligand of dependence receptors. When bound to its receptor, a positive signaling is triggered. When unbound, apoptosis is induced. In some human cancers, neoplastic cells can lose the ability to induce apoptosis by overexpressing netrin-1, or by decreasing the receptor expression. We hypothesized a similar pathway in canine lymphomas. We observed increased expression of netrin-1, particularly in high-grade nodal lymphomas. In vitro evaluation of an anti-netrin-1 antibody is encouraging as apoptosis is restored in a T-cell lymphoma cell line. Netrin-1 appears thus as a possible survival factor in dog lymphomas. This study suggests it can be a promising tool for a targeted therapy in lymphoma management in dogs. ABSTRACT: Netrin-1 is a member of the laminin superfamily, and is known to interact with specific receptors, called dependence receptors. While upon netrin-1 binding these receptors initiate positive signaling, in absence of netrin-1, these receptors trigger apoptosis. Tumor cells can avoid apoptosis by inactivating these receptors or by gaining ligand expression. The aim of the present study was to investigate the expression of netrin-1, the ligand of dependence receptors, in canine healthy lymph nodes (LN), and in lymphomas and to evaluate efficiency of a netrin-1 interfering compound in cell cultures from canine lymphoma. Thirty-two control LN and 169 lymphomas were analyzed through immunohistochemistry. Netrin-1 was expressed in the nucleoli of lymphoid and non-lymphoid cells in controls. Acquisition of a cytoplasmic expression was present in B-cell lymphomas (23.1 % in low-grade and 50.6% in high-grade) and T-cell lymphomas (50.0 % in low-grade and 78.8 % in high-grade), with a significant difference between the high- and low-grade in B-cell lymphomas. Through flow cytometry, we showed a significant increase in netrin-1 expression in either high-grade B-cell and T-cell lymphomas (19 and 5, respectively) compared with healthy LN (5), likewise an RT-qPCR analysis demonstrated a significant increase in netrin-1 expression level in 14 samples of lymphomas compared with eight samples of healthy LN. A T-cell aggressive canine lymphoma cell line and four primary canine nodal lymphomas cell cultures were treated with a netrin-1 interfering antibody. Apoptosis by measuring caspase 3 activity was significantly increased in the cell line and viability was decreased in three of the four primary cell cultures. Together, these data suggest that netrin-1 expression is increased in lymphoma, and more specifically in high-grade lymphomas, and that netrin-1 can act as a survival factor for the neoplastic cells, and so be a therapeutic target.