Single Molecule Studies of Abl2/Arg Breast Cancer Invasion Control Switch

Yale University, New Haven CT

Investigators

Abstract

? DESCRIPTION (provided by applicant): Single molecule studies of Abl2/Arg breast cancer invasion control switch Approximately 230,000 women are diagnosed with breast cancer every year, leading to over 40,000 deaths. Over 75% of newly diagnosed breast cancers exhibit invasion into the tissue surrounding the tumor, which subsequently spreads to distant organs to form metastases. We have shown that breast cancer cells deficient in the Abl2/Arg non-receptor tyrosine kinase have decreased ability to degrade and invade through extracellular matrix because they cannot form protrusions called invadopodia. Furthermore, invadopodia formation requires proper signaling by the ?1 integrin and EGF cell surface receptors (EGFR). I provide strong evidence that Arg can directly interact with ?1 integrin receptor and that this interactionis necessary to mediate EGF-stimulated invadopodia formation. Cell surface receptor dysfunction is commonly found in cancer cells and is a hallmark of disease progression. Understanding how ?1 integrin and EGFR recruit Arg to the membrane for activation is critical to understand how a key molecular switch of breast cancer invasion is turned on. In this proposal, I will test the hypothesis that ?1 integrin and EGF receptors change Arg membrane recruitment frequency, duration, and spatial patterning to regulate invadopodia-mediated cancer cell invasion. My first aim is to determine the roles of ?1 integrin and EGF receptors in Arg recruitment. In this aim, I will use Total Internal Reflection Fluorescence (TIRF) microscopy to track membrane recruitment of single Arg molecules and various Arg mutants in response to different activation patterns of ?1 integrin and EGF receptors. These experiments will reveal how these receptors affect Arg recruitment frequency and duration on the membrane. The Arg mutants will also test which domains are necessary for proper recruitment. My second aim is to study how surface receptors change Arg spatial distribution on the cell membrane. In this aim, I will use Photoactivatible Light Microscopy (PALM) techniques to obtain superresolution images of Arg on the cell membrane superimposed on ?1 integrin or EGF receptors or markers of invadopodia precursors. I will test the hypothesis that activation of cell surface receptors recruit Arg to the cell membrane and then directs it towards sites of invadopodia formation. These experiments will be able to test how Arg localization patterns change over the course of invadopodia development. My third aim is to understand how changes in Arg recruitment affect cancer cell invasion. In addition to its kinase domain, Arg contains many domains that regulate the kinase activity and mediate interactions with other proteins. Cancer cells lacking Arg are deficient in forming functional invadopodia and exhibit less invasive behavior. In this aim, I will test the hypothesis that the Arg domains that are important for proper recruitment to the cell membrane are also critical for functional cancer cell invasion. I will use in vitro matrix degradation and transwell assays to assess the ability of Arg mutants to form invadopodia and invade matrix.