We previously showed that MMP-9 contributes to CLL pathology by regulating cell survival and migration and that, when present at high levels, MMP-9 induces cell arrest. To further explore the latter function, we studied whether MMP-9 influences the gene-expression profile in CLL. Microarray analyses rendered 131 differentially expressed genes in MEC-1 cells stably transfected with MMP-9 (MMP-9-cells) versus cells transfected with empty vector (Mock-cells). Ten out of twelve selected genes were also differentially expressed in MEC-1 cells expressing the catalytically inactive MMP-9MutE mutant (MMP-9MutE-cells). Incubation of primary CLL cells with MMP-9 or MMP-9MutE also regulated gene and protein expression, including CD99, CD226, CD52, and CD274. Because CD99 is involved in leukocyte transendothelial migration, we selected CD99 for functional and mechanistic studies. The link between MMP-9 and CD99 was reinforced with MMP-9 gene silencing studies, which resulted in CD99 upregulation. CD99 gene silencing significantly reduced CLL cell adhesion, chemotaxis and transendothelial migration, while CD99 overexpression increased cell migration. Mechanistic analyses indicated that MMP-9 downregulated CD99 via binding to α4β1 integrin and subsequent inactivation of the Sp1 transcription factor. This MMP-9-induced mechanism is active in CLL lymphoid tissues, since CD99 expression and Sp1 phosphorylation was lower in bone marrow-derived CLL cells than in their peripheral blood counterparts. Our study establishes a new gene regulatory function for MMP-9 in CLL. It also identifies CD99 as an MMP-9 target and a novel contributor to CLL cell adhesion, migration and arrest. CD99 thus constitutes a new therapeutic target in CLL, complementary to MMP-9.
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The authors thank Dr. Dolors Colomer for some of the paired CLL samples; Dr. Miguel A. Vega for expert help and advice with the microarray data analyses; Dr. Pedro Lastres for help with the flow cytometry analyses; and Guillermo Padilla for expert help with bioinformatics analyses.
This work was supported by Grant SAF2015-69180R and Red Temática de Investigación Cooperativa en Cáncer Grant RD12/0036/0061 from the Ministry of Economy and Competitivity (Spain) (to AGP); S2010/BMD-2314 (to AGP) from the Comunidad de Madrid/European Union; and by the Concerted Research Actions (KU Leuven C1 Grant C16/17/010) and the Research Foundation of Flanders (FWO-Vlaanderen, to EUB, PEVdS, and GO).
NAM and EB performed most of the research, designed experiments, and analyzed data; RUC performed research and analyzed data; AS, AGG, and CPS performed and analyzed some experiments; EUB designed and prepared cell transfectants and analyzed data; GO and PEVdS prepared and characterized the recombinant MMP-9 variants and critically reviewed the manuscript; JAGM contributed patient samples, with clinical, biological, and cytogenetic data; AGP designed and supervised research, had full access to the data, and wrote the paper. All authors reviewed and approved the final version of the manuscript.
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Aguilera-Montilla, N., Bailón, E., Uceda-Castro, R. et al. MMP-9 affects gene expression in chronic lymphocytic leukemia revealing CD99 as an MMP-9 target and a novel partner in malignant cell migration/arrest. Oncogene 38, 4605–4619 (2019). https://doi.org/10.1038/s41388-019-0744-3
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