Dynamic regulation of ROCK in tumor cells controls CXCR4-driven adhesion events

Abstract

SummaryCXCR4 is a chemokine receptor often found aberrantly expressed on metastatic tumor cells. To investigate CXCR4 signaling in tumor cell adhesion, we stably overexpressed CXCR4 in MCF7 breast tumor cells. Cell attachment assays demonstrate that stimulation of the receptor with its ligand, CXCL12, promotes adhesion of MCF7-CXCR4 cells to both extracellular matrix and endothelial ligands. To more closely mimic the conditions experienced by a circulating tumor cell, we performed the attachment assays under shear stress conditions. We found that CXCL12-induced tumor cell attachment is much more pronounced under flow. ROCK is a serine/threonine kinase associated with adhesion and metastasis, which is regulated by CXCR4 signaling. Thus, we investigated the contribution of ROCK activity during CXC12-induced adhesion events. Our results demonstrate a biphasic regulation of ROCK in response to adhesion. During the initial attachment, inhibition of ROCK activity is required. Subsequently, re-activation of ROCK activity is required for maturation of adhesion complexes and enhanced tumor cell migration. Interestingly, CXCL12 partially reduces the level of ROCK activity generated by attachment, which supports a model in which stimulation with CXCL12 regulates tumor cell adhesion events by providing an optimal level of ROCK activity for effective migration.

The transmembrane adaptor protein NTAL signals to mast cell cytoskeleton via the small GTPase Rho

“…It is likely that the significantly larger proportion of active RhoA in NTAL À/À cells is relevant to their greater secretory responsiveness [7,8]; this is in line with the reported requirements for RhoA in secretion from mast cells [18]. Active RhoA may be necessary to initiate the secretory process (perhaps by activating a fusogen), and its immediate subsequent inactivation may be required for cell attachment [28,29] and/or F-actin disassembly [26]. The later increase in RhoA activity would then be required for the formation of newly polymerized actin serving an inhibitory function in exocytosis and promoting cell spreading and migration [29].…”

“…Active RhoA may be necessary to initiate the secretory process (perhaps by activating a fusogen), and its immediate subsequent inactivation may be required for cell attachment [28,29] and/or F-actin disassembly [26]. The later increase in RhoA activity would then be required for the formation of newly polymerized actin serving an inhibitory function in exocytosis and promoting cell spreading and migration [29]. The role of NTAL in the recovery of RhoA after its initial inhibition was supported by the differential effects of ROCK inhibition on spreading of WT and NTAL À/À cells.…”

PDZ-RhoGEF is essential for CXCR4-driven breast tumor cell motility through spatial regulation of RhoA

“…Although the MCF7-CXCR4 cell line has enhanced migratory responses to CXCL12 (Rhodes et al, 2011;Struckhoff et al, 2010), it still retains most of the epithelial characteristics and relatively low migration rates of parental MCF7 cells. We speculated that PRG depletion may be a less potent inhibitor of migration of invasive, mesenchymally transformed breast cancer cells.…”

“…Cell culture MCF7-CXCR4 cells stably overexpress the CXCR4 chemokine receptor in MCF7 cells (Mao et al, 2011;Rhodes et al, 2011;Struckhoff et al, 2010). MDA-MB-231 cells, obtained from ATCC (Manassas, VA), and Phoenix ecotropic retrovirus packaging cell lines (Garry Nolan via National Gene Vector Biorepository) were cultured in DMEM with 10% FBS and antibiotics.…”

“…CXCR4-driven tumor cell migration depends on temporal regulation of RhoA signaling (Bartolomé et al, 2004;MolinaOrtiz et al, 2009;Struckhoff et al, 2010;Tan et al, 2006;Vicente-Manzanares et al, 2002), but the molecular mechanisms by which CXCR4 regulates RhoA activity to promote motility is currently unknown. As direct activators of RhoA, RhoGEFs (guanine nucleotide exchange factors) are good candidates to signals from CXCR4 into RhoA activation during tumor cell motility.…”