Abstract B52: Calcium-sensitized tissue transglutaminase translocates to the plasma membranes and inhibits neuroblastoma cell migration

Tissue-transglutaminase (TG2) is a dual function G-protein with Ca2+-dependent transglutaminase activity and GTP-binding/hydrolyzing activity. TG2 is physiologically involved in cell differentiation, cell death, migration, invasion of cancer cells. In the extracellular matrix (ECM), TG2 mediates cell- ECM interactions through fibronectin and integrins and promotes cell attachment, migration and invasion. When present on the cytoplasmic side of the plasma membrane TG2 activates phospholipase C and RhoA-ROCK-2 signaling pathways that promote cell differentiation. While mechanism of TG2-induced cell differentiation is well characterized, the mechanism underlying TG2-mediated cell migration is not known. In present study, we explored the mechanism of how TG2 may be involved in cell migration by using wild type neuroblastoma cells (SH-SY5Y) which do not express TG2, neuroblastoma cells expressing exogenous TG2 (SHYTG2), and a transamidation-deficient TG2 containing SH-SY5Y cells (SHYmutant). Our cell migration assays (scratch assay and collagen-coated transwell assay) and cell invasion assay (matrigel-coated trasnwell assay) indicate that TG2 expressing cells exhibit higher migrating and invasive properties. To further investigate the role of TG2 in cell migration, we utilized a natural compound, resveratrol. Resveratrol (3,5,4′-trihydroxystilbene) is a polyphenolic and phytoalexin compound present in grapes and blueberries and has shown promising results in the treatment of neuroblastoma. In our studies, resveratrol (1 μM -10 μM) significantly prevented migration and invasion of TG2-expressing cells. In migrating cells, resveratrol increases the immunoreactivity of TG2 without affecting the total TG2 protein level. In these cells, resveratrol increases calcium levels, and depletion of intracellular calcium by a calcium chelator, BAPTA, attenuate resveratrol-enhanced TG2 immunoreactivity. To further analyze resveratrol-induced cellular distribution and conformational states of TG2, we performed native gel electrophoresis and detected an additional TG2 protein band with slower migration in resveratrol-treated migrating cells. This TG2 form is non-phosphorylated, exclusively present in plasma membrane fractions and sensitive to intracellular Ca2+ concentrations suggesting a calcium requirement in TG2-regulated cell migration. The observed less mobility of transamidation-defective SH-SY5Y cells (SHYmutant) and the reduced migration of SHYTG2 cells in the presence of a TG2 transamidation inhibitor, monodensylcadevarine, in scratch assays further support the requirement of TG2 in cell migration. Our mechanistic studies suggest that resveratrol induces conformational changes in TG2 (compact to open structure form), and Ca2+-mediated TG2 association to plasma membrane is responsible for the inhibitory effects of resveratrol on cell migration. Together, present study indicates that TG2 plays a key role in neuroblastoma cell migration. Although higher concentrations of resveratrol (∼100 μM) induces cell death in cancer cells, the current study suggest that lower physiologically relevant dose of resveratrol (∼1 μM) prevent the migration and invasion of cancer cells from the primary site. Since retinoic acid treatment increases TG2 protein level and resveratrol inhibits the migration of TG2-expressing cells, this study also proposes the use of resveratrol with retinoic acid for the treatment of neuroblatoma. Citation Format: Ambrish Kumar, Jianjun Hu, Holly A. LaVoie, Kenneth B. Walsh, Donald J. DiPette, Ugra S. Singh. Calcium-sensitized tissue transglutaminase translocates to the plasma membranes and inhibits neuroblastoma cell migration. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B52.