Calpain 2 and Src dependence distinguishes mesenchymal and amoeboid modes of tumour cell invasion: a link to integrin function

Abstract

Cancer cells can invade three-dimensional matrices by distinct mechanisms, recently defined by their dependence on extracellular proteases, including matrix metalloproteinases. Upon treatment with protease inhibitors, some tumour cells undergo a 'mesenchymal to amoeboid' transition that allows invasion in the absence of pericellular proteolysis and matrix degradation. We show here that in HT1080 cells, this transition is associated with weakened integrin-dependent adhesion, consistently reduced cell surface expression of the a2b1 integrin collagen receptor and impaired signalling downstream, as judged by reduced autophosphorylation of focal adhesion kinase (FAK). On examining cancer cells that use defined invasion strategies, we show that distinct from mesenchymal invasion, amoeboid invasion is independent of intracellular calpain 2 proteolytic activity that is usually needed for turnover of integrin-linked adhesions during two-dimensional planar migration. Moreover, an inhibitor of Rho/ROCK signalling, which specifically impairs amoeboid-like invasion, restores cell surface expression of a2b1 integrin, downstream FAK autophosphorylation and calpain 2 sensitivity -features of mesenchymal invasion. These findings link weakened integrin function to a lack of requirement for calpain 2-mediated integrin adhesion turnover during amoeboid invasion. In keeping with the need for integrin adhesion turnover, mesenchymal invasion is uniquely sensitive to Src inhibitors. Thus, the need for a major pathway that controls integrin adhesion turnover defines and distinguishes cancer cell invasion strategies.

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

“…One of the defining features of mesenchymal cell motility is the requirement for regulated focal adhesion formation and disassembly Carragher et al, 2006). Mounting evidence suggests that the expression profile of tropomyosin isoforms is an important determinant of focal adhesion structure and function (Gupton et al, 2005;Zheng et al, 2008;Bach et al, 2009;Drappatz et al, 2009;O'Neill, 2009), and we have shown that elevated Tm5NM1 expression induces focal adhesion stabilization (Bach et al, 2009).…”

“…As Src kinase is a major regulator of focal adhesion stability and mesenchymal motility (Fincham and Frame, 1998;Webb et al, 2004;Carragher et al, 2006), and we have demonstrated that total Src expression is decreased in cells with elevated Tm5NM1 (Bach et al, 2009), we next tested whether Src kinase activity is suppressed. Using antibodies to the activated form of Src kinase, we find significantly decreased activated Src, relative to total Src protein in cells with elevated Tm5NM1 (Figures 3g and h).…”

“…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).…”

“…Rapid expansion and contraction allow amoeboid cells to squeeze through obstructing matrix, and this is highly dependant on actomyosin contractility (Sahai and Marshall, 2003;Sanz-Moreno et al, 2008;Friedl and Wolf, 2010). 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.…”

“…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.…”