Date Published: November 18, 2009
Publisher: Public Library of Science
Author(s): Alexander E. Finlayson, Kevin W. Freeman, Juha Klefstrom. http://doi.org/10.1371/journal.pone.0007833
Abstract: Invasion through the extracellular matrix (ECM) is important for wound healing, immunological responses and metastasis. We established an invasion-based cell motility screen using Boyden chambers overlaid with Matrigel to select for pro-invasive genes. By this method we identified antisense to MARCKS related protein (MRP), whose family member MARCKS is a target of miR-21, a microRNA involved in tumor growth, invasion and metastasis in multiple human cancers. We confirmed that targeted knockdown of MRP, in both EpRas mammary epithelial cells and PC3 prostate cancer cells, promoted in vitro cell migration that was blocked by trifluoperazine. Additionally, we observed increased immunofluoresence of E-cadherin, β-catenin and APC at sites of cell-cell contact in EpRas cells with MRP knockdown suggesting formation of adherens junctions. By wound healing assay we observed that reduced MRP supported collective cell migration, a type of cell movement where adherens junctions are maintained. However, destabilized adherens junctions, like those seen in EpRas cells, are frequently important for oncogenic signaling. Consequently, knockdown of MRP in EpRas caused loss of tumorigenesis in vivo, and reduced Wnt3a induced TCF reporter signaling in vitro. Together our data suggest that reducing MRP expression promotes formation of adherens junctions in EpRas cells, allowing collective cell migration, but interferes with oncogenic β-catenin signaling and tumorigenesis.
Partial Text: Invasion of cells through the extracellular matrix (ECM) is a complicated process involving migration by two alternating modes of action with either cellular deformation allowing movement through the ECM or by degradation of the ECM using factors such as MMPs , . An established approach for assaying cellular movement through the ECM is using Boyden chambers overlaid with Matrigel, a basement membrane extract composed of extracellular matrix. Though Matrigel lacks many of the important attributes of endogenous basement membrane, such as less physical cross-linking , it is frequently used to identify chemical factors that either promote or inhibit invasion of a particular cell type. Since movement of cells through the extracellular matrix (ECM) is necessary for normal physiological processes, such as wound healing and movement of immune cells, in addition to pathological processes, such as metastasis , we decided to establish an in vitro invasion-based cell motility genetic screen.
Our data suggest that knockdown of MRP promotes adherens junction formation. A possible route that MRP could regulate adherens junction formation is through its regulation of calmodulin (CaM) via the scaffolding protein IQGAP. MRP is known to bind to Ca2+/CaM with nanomolar affinity, but does not bind to CaM , . IQGAP, which is regulated by Ca2+/CaM is important for controlling cell migration and adherens junction formation , , making it a possible downstream effector of MRP. Alternatively, in Drosophila a novel pathway to adherens junction formation was identified. It involved localization of a synaptotagmin-like protein Btsz to PIP2. Btsz binds PIP2 and increases adherens junction formation through stabilization of E-cadherin. In this system decreasing PIP2 levels blocked adherens junction formation . Interestingly, MARCKS family members regulate accessibility to PIP2 and any reduction in their expression could promote adherens junction formation through this pathway. Whatever may be the case identifying how MRP regulates adherens junction formation is an important next step.