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Biologic and Materials Sciences and Division of Prosthodontics

Geng Lab - Current Research

Cell Signaling in Cell Adhesion and Stem Cell Research

1. Cell Adhesion Research

Adhesion and migration of mammalian cells are of crucial importance in a number of biological events, such as fertilization, embryogenesis, pattern, tissue and organ formation, and in a variety of physiological and pathological processes, including lymphocyte trafficking, leukocyte recruitment, hemostasis, wound healing, tumor angiogenesis and cancer metastasis. All these cellular interactions are precisely regulated by temporal and spatial presentation of various cell adhesion molecules and chemotactical molecules displaying appropriate specificity and affinity for proper development and functioning of the organism. My research has been focused on cell adhesion and migration of leukocytes, platelets, endothelial cells and tumor cells for their functional roles in inflammation, thrombosis and cancer growth and metastasis in the past.

1.1. Leukocyte-Endothelial Cell Interactions

The leukocyte-endothelial cell interactions are mediated by at least four families of cell adhesion molecules. They are selectins (CD62), selectin ligands, integrins and IgG superfamily of cell adhesion molecules. The selectin family of cell adhesion molecules interacts with their cognate ligands; and these interactions are generally believed to mediate initial attachment, rolling and weak adhesion of leukocytes on the activated endothelial cells. The integrin family of cell adhesion molecules interacts with the cell adhesion molecules of immunoglobulin superfamily; and these interactions are mainly responsible for firm adhesion and signal transduction, which can then trigger diapedesis of leukocytes for transendothelial migration. Eventually, the emigrated leukocytes are guided by increasing concentrations of various soluble chemoattractants to move to their destinations, such as the site of infection or tissue injury.

During my postdoctoral training in Dr. Rodger P. McEver’s lab, we discovered that P-selectin is a cell adhesion molecule for human neutrophils (Geng, et al., Nature 343:757, 1990; Geng, et al., J. Biol. Chem. 266:22313, 1991; Geng, et al., J. Biol. Chem. 267:27739, 1992). Using P- and E-selectin affinity chromatography and monoclonal antibody approach, we showed that a sialoglycoprotein, called PSGL-1 (P-selectin glycoprotein ligand-1; CD162) functions as an important human leukocyte ligand for P-selectin (Ma, et al., J. Biol. Chem. 269:27739, 1994) and E-selectin (Asa, et al., J. Biol. Chem. 270:11662, 1995). It is now generally recognized that PSGL-1 functions as a high affinity ligand for all three selectins.

Along this line of investigation, we found that P-selectin increased adhesion of human neutrophils to fibrinogen, which acts in concert with platelet activating factor (PAF) or interleukine-8 (IL-8) for further enhancing the activation of αMβ2 (Ma et al., Blood 104:2549, 2004; Xu et al., Cell Adhesion & Migration 1:115, 2007). We further reported that P-selectin-/- mice manifested impaired leukocyte adhesion, which was ‘rescued’ by soluble P-selectin. Mechanistically, the cytoplasmic domain of PSGL-1 formed a constitutive complex with Nef-associated factor 1 (Naf1). Upon P-selectin binding, Src kinases phosphorylated the Y552PPM motif of Naf1, which recruited the p85/p110δ heterodimer of phosphoinositide-3 kinase (PI3K) and activated leukocyte integrins. Inhibition of this signal transduction pathway diminished leukocyte adhesion to capillary venules and suppressed peritoneal infiltration of leukocytes. Our results thus demonstrate the functional importance of a novel PSGL-1 signaling mechanism (Wang et al., Nat. Immunol. 8:882, 2007).

1.2. Tumor Cell-Endothelial Cell Interactions

The Slit family of guidance cues binds to Roundabout (Robo) receptors for modulation of neuronal, leukocytic and endothelial migration. We found Slit2 expression in a large number of solid tumors and Robo1 expression in vascular endothelial cells. Recombinant Slit2 protein attracted endothelial cells and promoted tube formation in a Robo1- and phosphatidylinositol kinase-dependent manner. Neutralization of Robo1 reduced the microvessel density and the tumor mass of human malignant melanoma A375 cells in vivo. These findings demonstrate the angiogenic function of Slit-Robo signaling (Wang et al., Cancer Cell 4:19, 2003; Wang et al., Cancer Science 99:510, 2008; Yang et al. Biochem. Biophys. Res. Comm. 396:571, 2010).

In addition, we found that ectopic expression of Slit2 and Robo1 or recombinant Slit2 treatment of Robo1-expressing colorectal epithelial carcinoma cells recruited an ubiquitin ligase Hakai for E-cadherin ubiquitination and lysosomal degradation, epithelial-mesenchymal transition (EMT), and tumor growth and liver metastasis, which were rescued by knockdown of Hakai. In contrast, knockdown of endogenous Robo1 or specific blockade of Slit2 binding to Robo1 prevented E-cadherin degradation and reversed EMT, resulting in diminished tumor growth and liver metastasis. Ectopic expression of Robo1 also triggered a malignant transformation in Slit2-positive human embryonic kidney 293 cells. Importantly, the expression of Slit2 and Robo1 was significantly associated with an increased metastatic risk and poorer overall survival in colorectal carcinoma patients. We conclude that engagement of Robo1 by Slit2 induces malignant transformation through Hakai-mediated E-cadherin ubiquitination and lysosomal degradation during colorectal epithelial cell carcinogenesis (Zhou et al., Cell Res. 21:609, 2011).

2. Stem Cell Research

The intestine is particularly susceptible to chemoradiation due to a continuous requirement for tissue maintenance by actively cycling intestinal stem cells (ISCs). ISC death is a major side effect of chemoradiotherapy that disrupts intestinal homeostasis and causes a paucity of intestinal tissue. How to reduce devastating and sometimes lethal intestinal injury caused by intensive chemoradiotherapy for treating late-staged metastatic cancer is clinically challenging. Identification of novel therapeutics that protect ISC survival and function during chemoradiotherapy could greatly increase the range of treatment options, while decreasing catastrophic tissue and organ damage caused by chemoradiotherapy that leads to ultimate death of cancer patients. Using lethal doses of chemoradiation as an acute tissue/organ injury and repair model, we discovered that induction of endogenous ISCs by administering recombinant R-spondin 1 (Rspo1) plus Slit2 mitigated gut impairment and protected 30-70% animals from death, without concomitantly decreasing tumor sensitivity to chemotherapy (Zhou et al., Nature 501:107, 2013). We believe that understanding how to protect cancer patients from chemoradiation may lead to therapeutic eradication of metastatic cancers with currently available conventional chemoradiotherapy.