Estudio del efecto de la NEDDilación sobre la expresión de MMP-9 en un modelo de Leucemia Linfática Crónica

Abstract

Chronic Lymphocytic Leukemia (CLL) is a malignant disease caused by the accumulation of mature B lymphocytes due to an inherent defect in apoptosis. This effect is produced by the constitutive activation of different signaling pathways. MMP-9 plays an important role in the survival, angiogenesis and metastasis by the activation and regulation of survival signaling pathways. MMP-9 promoter contains several binding sites for AP-1 and NF-kB transcription factors. Previous studies from our group described that an inhibition of neddylation with MLN4924 in CLL cells represses the expression of MMP9 in parallel with an increase in apoptosis. Therefore, we focused our study on the effect of this drug on the expression of MMP-9 and how it is regulated by NF-kB and AP-1 pathways in MEC-1, a cell line of Chronic Lymphocytic Leukemia. To achieve this aim, we compared the effect of MLN4924, LY294002 (PI3K inhibitor) and IL-1ß, Forskolin and Bortezomib (proteasome inhibitor) in MEC-1 and in B-CLL cells. We also determined cell death, mRNA and protein expression by flow cytometry, RT-qPCR and Western Blot, respectively. A dose-dependent effect on cell death by apoptosis was determined upon treatment with MLN4924, but not with LY294002. However, MMP-9 and genes regulated by NF-kB (BCL-2, MCL-1, TANK, XIAP, BCL-XL) were overexpressed at mRNA level with the increment of MLN4924. On the other hand, IL-1ß has an effect on the expression of Bcl-2 and not on MMP-9. Moreover, we observed the upregulation of MMP-9 and NURR1 on MEC-1 and B-CLL cells after the treatment with Forskolin. Furthermore, Bortezomib has an effect dose-dependent on MMP-9 overexpression. Altogether, our results demonstrate that the regulation of MMP-9 by MLN4924 in MEC-1 cells differs from that in primary B-CLL cells; MLN4924 induces MMP-9 in MEC-1 cells, as it does with NURR1. Nonetheless, the co-culture with HS-5 cells induced MMP-9 expression in MEC-1 cells, but the same did not happen with the NURR1 expression. However, the treatment wih Bortezomib and MLN4924 reversed the induction of MMP9 by co-culture with HS-5 cells. Our data suggest a common regulatory pathway for MMP-9 and NURR1 in MEC-1 cells.Máster en Biología Molecular y Biomedicin