We propose to test the hypothesis that when ligand binds chemokine receptors, a """"""""dynamic chemosynapse"""""""" forms, comprised of receptor and adaptor proteins that serve to enable polarization, activation, amplification, and oscillation of small GTPases and kinases involved in organization of the actin cytoskeleton, and chemotaxis. Moreover, we propose that interference of chemokine receptor association with these adaptors will ablate CXCR2- and CXCR4-mediated chemotaxis, intravasation, extravasation, and metastasis. There are three specific aims. I) To test the hypothesis that a major role of AP-2 in mediating CXCR2/CXCR4 chemotaxis and metastasis is to orchestrate polarization and amplification of intracellular signals. We will separately interfere with AP-2 mediated receptor trafficking versus polarization of intracellular signals to distinguish the importance of these two functions of AP-2 in CXCR2/CXCR4 mediated chemotaxis and metastasis. 2) To characterize the role of IQGAP1 and VASP in linking the CXCR2 and CXCR4 chemosynapse to the actin cytoskeleton. We will characterize the interacting domains of IQGAP1 and VASP with CXCR2 and CXCR4 and determine the effects of altering these interactions on the functional responses to chemokine (including extravasation, intravasation, metastasis) of leukocytes, endothelial cells and breast cancer cells. 3) To test the hypothesis that Src-family kinase activation at the CXCR2 and CXCR4 `chemosynapse'drives chemotaxis that is Dock2 dependent and largely PI3K independent. We will determine the role of the Src-p130Cas-CrkL-Dock2 pathway in PI3K independent CXC2/CXCR4 chemotaxis. We will study these processes in dHL-60, dU937, HMEC-1 CXCR2 expressing cells and in the breast cancer cell lines naturally expressing CXCR4 (highly invasive MDA-MB-231, DU4475, BT-549 and non-invasive MCF-7, MDA-MB- 453 and MCF-10A cells). Microfluidic devices and real time intravital microscopy using 2 photon imaging will be used to track chemotaxis in vitro and intravasation/extravasation /metastasis in vivo, respectively. Characterization of the functional significance of this interaction between chemokine receptors and adaptor proteins that comprise the chemosynapse will unveil important new therapeutic targets for the treatment of malignancies. Characterization of the mechanism for PI3K independent chemotaxis will provide valuable information for design of therapies for sepsis, arthritis, and metastasis.
This study aims to test the hypothesis that receptors involved in regulating the motility of immune cells and tumor cells must interact with a number of proteins in the cytoplasm of the cell to stimulate proper organization of the cytoskeleton. In this study we propose to disrupt these protein/protein interactions to develop a new way of blocking tumor cell metastasis and chronic inflammation. We also will interrupt specific intracellular signals initiated by these protein/protein interactions to point the way to new targets for drug therapy for chronic inflammation and metastasis.
|Sai, Jiqing; Rogers, Matthew; Hockemeyer, Kathryn et al. (2016) Study of Chemotaxis and Cell-Cell Interactions in Cancer with Microfluidic Devices. Methods Enzymol 570:19-45|
|Richmond, Ann; Yang, Jinming (2016) The role of NF-kB in modulating antitumor immunity. Oncoimmunology 5:e1005522|
|Johnson, Douglas B; Estrada, Monica V; Salgado, Roberto et al. (2016) Melanoma-specific MHC-II expression represents a tumour-autonomous phenotype and predicts response to anti-PD-1/PD-L1 therapy. Nat Commun 7:10582|
|Vilgelm, Anna E; Johnson, C Andrew; Prasad, Nripesh et al. (2016) Connecting the Dots: Therapy-Induced Senescence and a Tumor-Suppressive Immune Microenvironment. J Natl Cancer Inst 108:djv406|
|Duvall-Noelle, N; Karwandyar, A; Richmond, A et al. (2016) LASP-1: a nuclear hub for the UHRF1-DNMT1-G9a-Snail1 complex. Oncogene 35:1122-33|
|Vilgelm, Anna; Richmond, Ann (2015) Combined therapies that induce senescence and stabilize p53 block melanoma growth and prompt antitumor immune responses. Oncoimmunology 4:e1009299|
|Su, Yingjun; Richmond, Ann (2015) Chemokine Regulation of Neutrophil Infiltration of Skin Wounds. Adv Wound Care (New Rochelle) 4:631-640|
|Liu, Yan; Hawkins, Oriana E; Vilgelm, Anna E et al. (2015) Combining an Aurora Kinase Inhibitor and a Death Receptor Ligand/Agonist Antibody Triggers Apoptosis in Melanoma Cells and Prevents Tumor Growth in Preclinical Mouse Models. Clin Cancer Res 21:5338-48|
|Vilgelm, Anna E; Pawlikowski, Jeff S; Liu, Yan et al. (2015) Mdm2 and aurora kinase a inhibitors synergize to block melanoma growth by driving apoptosis and immune clearance of tumor cells. Cancer Res 75:181-93|
|Singh, Vandana; Raghuwanshi, Sandeep K; Smith, Nikia et al. (2014) G Protein-coupled receptor kinase-6 interacts with activator of G protein signaling-3 to regulate CXCR2-mediated cellular functions. J Immunol 192:2186-94|
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