Involvement of Rac and Rho signaling in cancer cell motility in 3D substrates

Abstract

The motility of cancer cells in 3D matrices is of two types: mesenchymal motility, in which the cells are elongated and amoeboid motility, in which the cells are round. Amoeboid motility is driven by an actomyosin-based contractile force, which is regulated by the Rho/ROCK pathway. However, the molecular mechanisms underlying the motility of elongated cells remain unknown. Here, we show that the motility of elongated cells is regulated by Rac signaling through the WAVE2/Arp2/3-dependent formation of elongated pseudopodia and cell-substrate adhesion in 3D substrates. The involvement of Rac signaling in cell motility was different in cell lines that displayed an elongated morphology in 3D substrates. In U87MG glioblastoma cells, most of which exhibit mesenchymal motility, inhibition of Rac signaling blocked the invasion of these cells in 3D substrates. In HT1080 fibrosarcoma cells, which display mixed cell motility involving both elongated and rounded cells, inhibition of Rac1 signaling not only blocked mesenchymal motility but also caused a mesenchymalamoeboid transition. Additionally, Rac1 and RhoA signaling regulated the mesenchymal and amoeboid motility in these cells, respectively, and the inhibition of both pathways dramatically decreased cell invasion. Hence, we could conclude that Rac1 and RhoA signaling simultaneously regulate cell invasion in 3D matrices.

The actin-associating protein Tm5NM1 blocks mesenchymal motility without transition to amoeboid motility

“…To confirm that the observed effects of Tm5NM1 overexpression can be generalized to cells of different derivation and origin, we employed the U-87 MG glioblastoma cell line, as this cell type is well established to migrate using a mesenchymal motility mode (Friedl, 2004;Yamazaki et al, 2009). Importantly, Tm5NM1 significantly inhibited U-87 MG cell migration in 3D collagen gels accompanied by conversion to a less elongated morphology analogous to the Tm5NM1 phenotype stimulated in the B35 cells (Supplementary Figure 1).…”

“…Notably, the Tm5NM1 cells have a distinct morphology (classified in the present study as elliptical) from either classic mesenchymal or amoeboid phenotypes. Others have reported mixed populations of cells including those that similarly cannot be classed morphologically as either mesenchymal or amoeboid Yamazaki et al, 2009). It is therefore of considerable interest to determine whether such cells may be undergoing a stationary phase.…”

“…Mesenchymal migration is characterized by cell elongation and branching polarized protrusions in the direction of migration. The chief properties that define mesenchymal migration include activation of the small GTPase Rac (Sanz-Moreno et al, 2008;Yamazaki et al, 2009), targeted expression of matrix metalloproteases (Sahai and Marshall, 2003;Wolf et al, 2003) and the regulated formation and disassembly of integrin receptor-mediated extracellular matrix adhesions mediated by the activity of Src kinase Carragher et al, 2006). In contrast, amoeboid migration is characterized by rounded cell morphology and is associated with non-apoptotic plasma membrane blebbing (Sahai and Marshall, 2003).…”

“…The migration of amoeboid cells is facilitated by the RhoA GTPase effector molecule Rho-associated kinase (ROCK), independent of matrix metalloprotease activity and less dependant on integrin receptor adhesion to the extracellular matrix (Friedl andWolf, 2003, 2010;Carragher et al, 2006). Many cells are competent to undergo transition between mesenchymal and amoeboid migration modes (Carragher et al, 2006;Yamazaki et al, 2009), and manipulating the principle molecular regulators can induce cells to switch between these modes. Thus, blocking mesenchymal migration by treatment with protease inhibitors (PIs) or blocking Rac activity (Sanz-Moreno et al, 2008) causes cells to switch to an amoeboid motility mode.…”

“…Notably, cell migration speeds are unaffected following mesenchymal to amoeboid transition . Conversely, RhoA/Rho-associated kinase inhibition inhibits amoeboid migration by switching cells from an amoeboid to a mesenchymal mode (Yamazaki et al, 2009).…”