PS1320 CDCA7 REGULATES LYMPHOMA CELLS MIGRATION, INVASIVENESS AND THEIR MICROTUBULE AND ACTOMYOSIN CYTOSKELETONS DYNAMICS
Authors: Martín‐Cortázar C. , Jiménez‐P. R. , Chiodo Y. , Bernabé M. , Cayuela M.L. , Iglesias T. , Campanero M.R. 
Published: June 2019

Background:

Metastasis is the major cause of death from cancer. Indeed, lymphoid neoplasm metastases are extremely difficult to treat due to their heterogeneity and their frequent affectation of the central nervous system. To acquire metastatic capacity, cells require increasing their migration and invasion capacities. In spite of the relevance of these processes, the mechanisms and genes involved in their regulation are poorly understood.

Immortality is essential, but insufficient for tumorigenesis. Certainly, some non‐tumor cells, such as lymphoblastoid B‐cell lines (LCLs), are able to divide indefinitely. To identify genes involved in tumor traits beyond immortalization, our group took the innovative approach of comparing gene expression profiles of B‐cell lymphomas with immortal LCLs. Among >1,600 genes differentially expressed in lymphoma cells, we focused in CDCA7 as one of the most significantly up‐regulated genes.

CDCA7 had been identified as a C‐MYC target gene whose mRNA was overexpressed in human tumors, particularly in chronic leukemia. Several studies based in CDCA7 overexpression suggested however that CDCA7 exhibited a limited in vivo transformation capacity. Moreover, its forced expression inhibited C‐MYC‐induced transformation. In contrast, we showed recently that CDCA7 knock‐down in lymphoma and leukemia cells sharply decreased their tumorigenic capacity without affecting their proliferation in liquid culture.

Aims:

The goal of this work was to investigate the potential role of CDCA7 in lymphoma invasion and migration.

Methods:

DG‐75, BL‐2, and Toledo lymphoma cell lines were transduced with lentivirus encoding control or CDCA7‐specific shRNA. We used subcutaneous xenografts in NOD‐SCID immunodeficient mice and in the yolk sac of zebrafish embryos to analyze the invasion capacity of lymphoma cells in vivo. We also used transwell permeable plates coated with matrigel or fibronectin to assess the invasion or migration capacity of these cells in vitro. Flow cytometry and quantitative PCR (qPCR) were used to determine the expression of potential mediators of cell migration and invasion, and immunofluorescence confocal imaging was employed to determine the cellular distribution of cytoskeleton components.

Results:

In the mouse xenograft model, we observed that CDCA7 silencing decreased tumor growth. Subsequent immunohistochemical analysis of the tumors revealed that, contrary to control cells, CDCA7‐silenced cells barely infiltrated and disorganized the neighbor tissues. Moreover, CDCA7 knockdown markedly decreased DG‐75 and Toledo cells invasion in the zebrafish model. Migration and invasion in vitro assays with lentivirally knocked‐down DG‐75, BL‐2, and Toledo lymphoma cells showed that CDCA7 mediates invasion and migration through matrigel‐ and fibronectin‐coated transwells, respectively. As qPCR and flow cytometry analyses revealed that CDCA7 does not regulate the expression of MMP2 and MMP9 or the expression of the integrin α4β1 (the main fibronectin receptor of these cells), we next investigated whether CDCA7 regulated cytoskeleton dynamics. Confocal immunofluorescence imaging analysis revealed that CDCA7 knock‐down markedly increased actin polymerization and myosin light chain phosphorylation, while impairing actin and tubulin cytoskeletons polarization.

Summary/Conclusion:

Our results strongly suggest that CDCA7 is a critical mediator of lymphoma cells invasion and migration, likely by regulating actomyosin and tubulin cytoskeleton dynamics.