Page 225 - Haematologica March 2020

CDCA7 promotes lymphoma invasion
A recent report described the association of CDCA7 overexpression in the most aggressive breast cancer sub- type with metastatic relapse and that CDCA7 mediates breast cancer migration through transcriptional upregula- tion of the EZH2 epigenetic modifier.44 However, CDCA7 knockdown did not substantially modify EZH2 mRNA levels in lymphoma cells (Online Supplementary Figure S9), indicating that CDCA7 regulates migration and invasion through distinct mechanisms in breast cancer and lym- phoma.
During cell migration, tubulin and actomyosin cytoskeletons are located in opposite ends of the cells, and cell migration involves their constant and coordinated remodeling.11,16 We propose that CDCA7 is required for the dynamic remodeling of both cytoskeletons and that its absence (as in knockdown cells) elicits depolarization and stabilizes cortical actin filaments, thus preventing the high dynamism of tubulin and actomyosin cytoskeletons required for cell migration. Supporting this hypothesis, we have shown that tubulin and F-actin are grouped in oppo- site poles of most control lymphoma cells and that both redistribute around the cell in CDCA7-silenced lym- phoma cells. Similarly, the polarized distribution of TPM3 and p-MLC observed in most control cells is lost upon CDCA7 knockdown.
In contrast, the dotted distribution of α-actinin was not
lost in CDCA7-silenced cells. It should be noted that
besides binding to actin filaments, α-actinin associates
with a number of signaling molecules, ion channels, tran-
Figure 8. The inhibition of myosin activation or actin polymerization reestablish the migration capacity in CDCA7-silenced cells. Toledo cells were transduced with the indicated short hairpin (sh) RNA, and seeded on the upper surface of the fibronectin-coated polycarbonate membrane of transwell chambers in the presence of 20 mM fasudil, 25 mM blebbistatin, or 0.1 mg/mL cytochalasin D; 10% fetal bovine serum was used in the lower chamber as a chemoattractant. Quantification of the relative migration capacity is shown as the mean + stan- dard error of mean of three independent transductions. **P<0.01; ns, non-sig- nificant (one-tailed t-test).
re-established the migratory capacity of silenced cells. These results suggest that CDCA7 silencing might induce ROCK activation in lymphoma cells.
We have shown that CDCA7 is critically involved in the anchorage-independent growth of lymphoid tumors and in lymphomagenesis.18 While CDCA7 is also expressed in normal diploid fibroblasts, its silencing in these cells did not inhibit their anchorage-dependent proliferation.18 Hence, given the essential role of CDCA7 in lymphoma progression and invasion, treatments that inhibit its expression or its activity represent an attractive strategy for controlling lymphoma growth, invasion, and metasta- tic dissemination.
Acknowledgments
The authors thank D. Trono for plasmids; the Spanish Ministerio de Economía y Competitividad for grants SAF2017- 88881-R and SAF2017-88885-R (MINECO/AEI/FEDER, UE) to MRC and TIV, respectively; the Comunidad de Madrid for European Social Fund (ESF) cofinanced AORTASANA-CM; B2017/BMD-3676 programme to MRC; and Instituto de Salud Carlos III (CIBERNED) for funding to TIV. The cost of this pub- lication was paid in part with FEDER funds.
scription factors, and transmembrane receptors, including 45
integrins. It therefore seems likely that α-actinin dissoci- ates from F-actin in CDCA7-silenced cells, remaining associated with integrins or other transmembrane recep- tors. Indeed, our data support the notion that α-actinin is associated with active integrins in both control- and CDCA7-knockdown cells. Among the four α-actinin iso- forms identified, non-muscular cells only express ACTN1 and ACTN4.46 The staining of lymphoma cells with a monoclonal antibody specific to ACTN1 was markedly increased in CDCA7-silenced cells relative to control cells, raising the possibility that CDCA7 knockdown upregulat- ed ACTN1 expression. However, the staining of these cells with a polyclonal antibody common to ACTN1 and ACTN4 showed no substantial differences between con- trol and silenced cells, and ACTN1 immunoblot analysis revealed similar protein levels in both cell populations. Together these results support the notion that instead of regulating ACTN1 levels, CDCA7 regulates, by unknown mechanisms, ACTN1 conformation or its association with other proteins, thus increasing the exposure of the epitope recognized by the monoclonal antibody. We propose a model whereby CDCA7 is required for the dynamic asso- ciation/dissociation of integrin-bound α-actinin to/from F- actin. The association of α-actinin to the actomyosin cytoskeleton would mask the epitope recognized by the monoclonal antibody. The absence of CDCA7 would hamper the association of integrin-bound α-actinin to this cytoskeleton, exposing the epitope and, more importantly, altering the cytoskeleton dynamics required for efficient migration. Forced CDCA7 downregulation would also hinder migration through the stabilization of F-actin and myosin activation. Of note, both processes can be activat- ed by ROCK33 and we show herein that ROCK inhibition
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