Cortactin Is a New Player in Aggressiveness and Diffusion of Chronic Lymphocytic Leukaemia
Authors: Martini Veronica , Frezzato Federica , Trimarco Valentina , Pizzi Marco , Chiodin Giorgia , Severin Filippo , Scomazzon Edoardo , Guzzardo Vincenza , Saraggi Deborah , Raggi Flavia , Martinello Leonardo , Facco Monica , Visentin Andrea , Semenzato Gianpietro , Trentin Livio 
Published: October 2019

Abstract
INTRODUCTION
Cortactin is an actin-binding protein involved in several cell functions, i.e. the assembly and the organization of cytoskeleton. Its overexpression was observed in several human cancers and experimental data support the role of cortactin in metastatic capability through the regulation of cell motility and the release of matrix metalloproteinase-9 (MMP-9). The activity of this protein is regulated by its phosphorylation in Tyr residues by Src kinase family. We previously demonstrated that in leukemic cells from B-CLL patients the Src kinase Lyn is overexpressed, activated and involved in the resistance to apoptosis. Recently, we found that cortactin is overexpressed in patients with B-CLL. Here we investigated the involvement of cortactin in the release of MMP-9 and, therefore, in the progression of B-CLL.
METHODS
Blood samples were collected from 10 controls and 34 B-CLL patients. Informed consent was obtained according to the Declaration of Helsinki. Untouched peripheral blood B cells were purified using the RosetteSep for human B cells isolation kit. The samples that were used had at least 95% of normal CD19+ or neoplastic CD5+/CD19+ cells, as assessed by flow-cytometry (FC). The purified B cells (2×106 cells/ml) were cultured in RPMI medium with or without CXCL12 (100ng/ml) and Ibrutinib (5μM) for the evaluation of MMP-9 production using ELISA. MMP-9 release by neoplastic B cells was also investigated after cortactin silencing in 4 patients which expressed high level of cortactin. The protein was silenced by SMARTpool siRNA collection (Dharmacon, Thermo Scientific), according to the manufacturer's instructions. Immunohistochemical (IHC) staining was performed on formalin-fixed, paraffin-embedded tissue sections using a fully automated platform.
RESULTS
By IHC and FC we confirmed the increased expression of cortactin in patients with respect to controls, moreover we defined three groups of patients according to different expression levels of Cortactin. We found that the release of MMP-9 by neoplastic B cells correlated to the expression of cortactin after 5 and 24-hrs culture. To investigate whether cortactin was involved in MMP-9 secretion in B-CLL, a cortactin-targeted siRNA silencing system was used to knockdown this protein in 4 patients with high cortactin expression. We found that following cortactin knockdown, leukemic cells showed a defect in MMP-9 secretion, as assessed by ELISA test. This protease also showed a decreased gelatinolytic activity in culture medium, supporting the hypothesis that cortactin is involved in the regulation of MMP-9 secretion in B-CLL malignant cells. We also demonstrated that the incubation of leukemic cells with PP2 or Btk decreased Tyr phosphorylation level of cortactin and shut down the release of MMP-9 in culture medium, also following CXCL12 triggering. Interestingly, we found that cortactin levels were significantly reduced following 3 months of Ibrutinib treatment and remained lower till the last clinical control, at 6 months of treatment. Consistent with these data we demonstrated i) a significant reduction in MMP-9 levels after in vivotreatment with Ibrutinib ii) a strong correlation between Cortactin expression and MMP-9 levels in CLL patients before and following Ibrutinib therapy. Finally, we also observed a redistribution of lymphocytes from the tissues to the periphery and a consequent reduction of lymphadenopathy in 4 cases where the levels of cortactin were higher than those observed in the patient who did not show enlarged lymph nodes at the time of diagnosis.
CONCLUSIONS
The overexpression of cortactin in neoplastic B cells and the correlation between cortactin levels, activity and MMP-9 release suggest a role of this protein in metastatic invasion and in the B-CLL aggressiveness. In addition, cortactin might represent a biomarker for diagnosis and prognosis and a target for new therapeutic strategies.

Disclosures
No relevant conflicts of interest to declare.