Abstract
Abstract Tumor cell motility is dependent upon integrin activation, focal adhesion (FA) formation and dissolution, and cytoskeleton reorganization. The IPP complex [integrin linked kinase (ILK), PINCH1, Parvin] regulates FA formation via binding of ILK to B-integrin. ILK serves as an adaptor by binding PINCH1 and Parvin thus linking FAs and actin. Rsu1 binds to PINCH1 and also regulates stress kinase signaling. The IPP complex is elevated in fibrotic disease and many common tumors contributing to changes in adhesion and migration. However, the Rsu1 locus is deleted in subsets of gliomas and liver carcinomas and Rsu1 expression is altered by alternative splicing in other advanced tumors. To identify the functional contribution of Rsu1 to the IPP complex regulation of adhesion, migration, and survival, Rsu1 and PINCH1 were independently depleted from MCF10A mammary epithelial cells. siRNA-mediated depletion of either Rsu1 or PINCH1 altered the localization but the not the expression of FA proteins, inhibited adhesion, and blocked migration. Both Rsu1- and PINCH1-depleted cells exhibited disorganized actin stress fibers and constitutive phosphorylation of actin regulatory proteins. Reduction of either Rsu1 or PINCH1 blocked acini formation in 3D culture. However, only Rsu1 depletion blocked the phosphorylation of p38 Map kinase. Depletion of Rsu1 caused significant reduction in PINCH1, but PINCH1 depletion resulted in only a modest reduction in Rsu1 implying a function for Rsu1 in regulating the IPP complex through PINCH1 stabilization. Expression of wt-Rsu1 in Rsu1-depleted cells restored all Rsu1 functions. In contrast, reconstitution of Rsu1-depleted cells with an Rsu1 mutant that is non-binding for PINCH1 failed to induce FA formation or cell migration, suggesting that binding of Rsu1 to PINCH1 is required for Rsu1 regulation of adhesion and migration. Unexpectedly, this non-binding mutant restored both the level of PINCH1 and cell spreading as well as p38 MAP kinase phosphorylation. These data indicate that Rsu1 regulates the stability of PINCH1 by a mechanism other than direct binding and that Rsu1-PINCH1 association is not required to promote cell spreading. Rather it is required to promote formation of FAs and the turnover of FAs necessary for migration. These findings differentiate the critical functions that Rsu1, PINCH1 and IPP complex contribute to cell adhesion and migration and explain how, even when disassociated, members of this complex contribute to tumor cell migration. Supported by W81XWH-10-1-0224, Predoctoral fellowship from the CDMRP Breast Cancer Research Program. Citation Format: Reyda Gonzalez-Nieves, Mary Lou Cutler. Rsu1 regulates MCF10A mammary epithelial cell migration and acini formation by binding to the IPP complex but it promotes spreading and Map kinase activation independent of IPP. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2616. doi:10.1158/1538-7445.AM2013-2616
Published Version
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