Page 217 - Haematologica March 2020

taining endopeptidases, is the most common group of extracellular matrix (ECM) proteases involved in tumor invasion and metastasis.4 MMP-2 and MMP-9, in particu- lar, are highly expressed in metastatic cancer cells and con- tribute to the progression of tumors and formation of metastases.5 Ex vivo studies suggest that carcinoma cells may also use a protease-independent scheme of invasion, whereby cells either squeeze through existing interstices in the ECM or displace ECM components.6
To invade surrounding tissues and vessels, cells must
acquire the ability to migrate. Indeed, cell migration is
required for the initial scattering of cells, egress from the
primary tumor, basement membrane penetration, intrava-
sation, and extravasation. Single carcinoma cells can
migrate in mesenchymal or amoeboid manners.7
Mesenchymal migration involves formation of protru-
sions and their adhesion to the substrate at the cell front,
and loss of adhesion at the opposite end. During direction-
al cell migration, actin polymerization drives protrusion
formation, whereas the tension generated by non-muscle
myosin II (NM-II) retracts the rear end of the cell.8 The
adhesion of the cell to the ECM at the protrusion end is as
important as its dissociation at the opposite end of the
cell.9 The interaction with the substrate is mediated main-
ly by integrins, which have binding-motifs for ECM pro-
teins. The connection between integrins and the actin
Helicase, lymphoid-specific (HELLS) SNF2 family member and is required for nucleosome remodeling by HELLS and for DNA methylation maintenance.20,21 AKT-mediated phosphorylation of CDCA7 promotes its nuclear exclu- sion and sequestration to the cytoplasm.22 CDCA7 mRNA was found to be deregulated in several tumor types, including lymphoid tumors,23 and we recently showed that CDCA7 protein is also overexpressed in lymphoid tumors and that its silencing markedly impairs lymphoid tumor growth without inhibiting the proliferation of nor- mal cells.18
As lymphoid tumors can be highly invasive,2,3 we inves- tigated the potential role of CDCA7 in lymphoid tumor invasion. Here we show that CDCA7 is critical for inva- sion and migration of lymphoma cells and for the reorgan- ization of the tubulin and actomyosin cytoskeletons.
Methods
Details of the Methods can be found in the Online Supplementary Appendix.
Lentivirus production, cell transduction, and immunoblotting
Lentiviral particles were produced and cells were trans- duced, as described elsehwere,24 employing vectors encod- ing either a non-targeting short hairpin (sh)RNA or CDCA7-targeting shRNA, sh-25 and sh-83. Cell lysates were prepared, resolved in sodium dodecylsulfate poly- acrylamide gel electrophoresis, transferred to nitrocellu- lose membranes, and probed as described previously.25
In vitro and in vivo migration and invasion assays
In vitro transwell migration and invasion assays were carried out in Boyden chambers using filters (3-mm pore size) coated with fibronectin or a matrigel solution. In vivo invasion assays were performed using zebrafish embryos and subcutaneous xengrafts in mice. All animal proce- dures were approved by the CSIC Ethics Committee (ref. 054/2014 and 634/2017) and by the Madrid Regional
authorities (ref. PROEX 31/14 and 215/17).
Results
CDCA7 silencing inhibits lymphoma invasion of adja- cent tissues
Subcutaneous inoculation of lymphoma cells in immunodeficient mice gives rise to the formation of solid tumors26,27 whose growth is impaired upon CDCA7 knock- down.18 To further investigate the role of CDCA7 in lym- phomagenesis, we screened for histological differences between control- and CDCA7-silenced tumors. We there- fore transduced DG-75 Burkitt lymphoma cells with lentivirus encoding CDCA7-specific shRNA (sh-25 and sh- 83) to knock down CDCA7 expression. Immunoblot analysis showed that both shRNA markedly decreased CDCA7 levels in these cells relative to the levels in control cells transduced with a non-targeting shRNA (sh-Ctl) (Figure 1A). As previously reported,18 CDCA7 silencing decreased tumor growth (Online Supplementary Table S1). Hematoxylin-eosin staining of tumor sections revealed that 100% of tumors formed by control-transduced cells contained muscle or adipose tissue (Figure 1B, C and
cytoskeleton is mediated by actin-binding proteins such as
talin, vinculin and α-actinin.10 NM-II molecules are actin-
binding proteins comprised of two heavy chains that have
ATPase activity, two regulatory light chains that regulate
NM-II activity, and two essential light chains that stabilize
the heavy chain structure.11 A major factor that determines
cell migration is the cell’s intrinsic contractility capacity,10
which is modulated through the coordinated regulation of
myosin activity and actin polymerization.9 Myosin activi-
ty is exquisitely regulated through phosphorylation by
signaling complexes and scaffold proteins to finely tune
migration.10 In particular, phosphorylation of Ser19 in the
regulatory light chain induces the ATPase activity of NM- II.11
The mechanisms of invasion by lymphoma cells are poorly understood, but various reports suggest that the capacity of normal blood T lymphocytes and the lym- phoma T-cell line SupT1 to move through three-dimen- sional collagen lattices does not require ECM degradation.7,12 Instead, the motile capacity of these cells might be critical for migration through these gels.7,13 While various migration modes have been described in normal non-lymphoid cells,14 normal lymphocytes appear to migrate mainly in an amoeboid fashion. Amoeboid migra- tion is characterized by weak adhesion to the substrate9,10 and rapid cycles of actin polymerization and actomyosin contraction at the front and rear edges, respectively.15
Cell migration also involves the reorganization of the microtubule cytoskeleton. Microtubules are organized around the microtubule-organizing center and, similar to the actin filaments, are polarized.16 The microtubule-orga- nizing center is reoriented between the nucleus and the leading edge in migrating cells and contributes to direc- tional cell migration.17 The mechanisms that regulate the reorganization of the tubulin cytoskeleton in migrating lymphocytes do, however, remain unknown.
We recently showed that CDCA7 is a critical mediator of lymphomagenesis.18 CDCA7 was identified as a MYC- target gene.19 Its encoded protein associates with the
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CDCA7 promotes lymphoma invasion
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