Complications from metastasis are the leading cause of mortality from breast cancer. Invasive cancer cells use F-actin-rich protrusions called invadopodia to degrade extracellular matrix (ECM) barriers to migration. We have shown that integrin ?1-mediated adhesion stimulates the Arg nonreceptor tyrosine kinase to interact with the actin polymerization regulators cortactin, N-WASp, and Vav2 at nascent sites of F-actin-mediated cell edge protrusion in non-cancerous cells. Each of these proteins localizes to invadopodia and is required for invadopodial function. Indeed, we find that Arg-mediated cortactin phosphorylation triggers actin polymerization within human breast cancer cell invadopodia, leading to their stabilization and acquisition of matrix-degrading activity. We will elucidate the mechanisms by which the integrin ?1:Arg:cortactin:N-WASp:Vav2 axis controls invadopodia function during breast cancer invasion and metastasis and screen for inhibitors of key interactions between these regulators as lead compounds for drug development.
Our first aim i s to understand how Arg is localized and regulated within invadopodia. Arg uses distinct domains to bind directly to F-actin, microtubules, and integrin ?1. Our preliminary work strongly suggests that these interactions regulate localization and activation of this key regulator at invadopodia. We will use RNAi knockdown of Arg and integrin ?1, rescue with interaction-defective Arg and integrin ?1 mutants, and use quantitative immunofluorescence and matrix degradation assays to determine which of these interactions mediates Arg localization to and activity within invadopodia in invasive human breast cancer cells.
Our second aim i s to identify the interactions most critical for invadopodia function and screen for inhibitors of these interactions. In addition to binding cortactin, Arg uses a distinct domain to bind and activate N-WASp. Arg-mediated cortactin phosphorylation also promotes its binding to Vav2, a regulator of actin polymerization. We hypothesize that Arg coordinates the activation and assembly of cortactin, N-WASp, and Vav2 within invadopodia to trigger Arp2/3 complex-mediated actin polymerization. We will use a knockdown/complementation approach similar to Aim 1 to identify which interactions are most critical for invadopodial function. We will also perform high throughput small molecule screens to identify compounds that disrupt key interactions between these proteins and test their ability to block breast cancer cell invasiveness.
Our third aim i s to test how disruption of key invadopodial actin regulators affect breast cancer invasion and metastasis. Invadopodia mediate penetration of matrix barriers in vitro, but whether and how they mediate breast cancer invasiveness in vivo has not been rigorously tested. We will use scid mouse xenograft and MMTV-polyoma middle T breast cancer models to determine how disrupting these invadopodial regulators affects breast cancer cell invasion and metastasis in vivo.
Complications from metastasis of tumor cells to other organs are the leading cause of mortality from breast cancer. Metastatic cancer cells escape the tumor and enter the bloodstream by forming filamentous-actin-rich protrusions called invadopodia that digest extracellular matrix barriers to allow invasion of surrounding tissue. We have discovered an important biochemical pathway involving the integrin 1; Arg; cortactin; N-WASp; and Vav2 proteins that regulates invadopodial function. Studying how these proteins interact to promote invadopodia formation should identify novel strategies to disrupt breast cancer invasion and metastasis.
|Li, Xiaofeng; Tao, Yeqing; Murphy, James W et al. (2017) The repeat region of cortactin is intrinsically disordered in solution. Sci Rep 7:16696|
|Rosenberg, Brian J; Gil-Henn, Hava; Mader, Christopher C et al. (2017) Phosphorylated cortactin recruits Vav2 guanine nucleotide exchange factor to activate Rac3 and promote invadopodial function in invasive breast cancer cells. Mol Biol Cell 28:1347-1360|
|Miller, Megan B; Yan, Yan; Machida, Kazuya et al. (2017) Brain Region and Isoform-Specific Phosphorylation Alters Kalirin SH2 Domain Interaction Sites and Calpain Sensitivity. ACS Chem Neurosci 8:1554-1569|
|Ganesan, S; Luu, T T; Chambers, B J et al. (2017) The Abl-1 Kinase is Dispensable for NK Cell Inhibitory Signalling and is not Involved in Murine NK Cell Education. Scand J Immunol 86:135-142|
|Wetzel, Dawn M; Rhodes, Emma L; Li, Shaoguang et al. (2016) The Src kinases Hck, Fgr and Lyn activate Arg to facilitate IgG-mediated phagocytosis and Leishmania infection. J Cell Sci 129:3130-43|
|Katrancha, Sara Marie; Koleske, Anthony J (2015) SNARE Complex Dysfunction: A Unifying Hypothesis for Schizophrenia. Biol Psychiatry 78:356-8|
|Courtemanche, Naomi; Gifford, Stacey M; Simpson, Mark A et al. (2015) Abl2/Abl-related gene stabilizes actin filaments, stimulates actin branching by actin-related protein 2/3 complex, and promotes actin filament severing by cofilin. J Biol Chem 290:4038-46|
|Simpson, Mark A; Bradley, William D; Harburger, David et al. (2015) Direct interactions with the integrin ?1 cytoplasmic tail activate the Abl2/Arg kinase. J Biol Chem 290:8360-72|
|Kerrisk, Meghan E; Cingolani, Lorenzo A; Koleske, Anthony J (2014) ECM receptors in neuronal structure, synaptic plasticity, and behavior. Prog Brain Res 214:101-31|
|DePoy, Lauren M; Perszyk, Riley E; Zimmermann, Kelsey S et al. (2014) Adolescent cocaine exposure simplifies orbitofrontal cortical dendritic arbors. Front Pharmacol 5:228|
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