Role of tissue transglutaminase induced integrin trafficking in kidney cancer metastasis

Integrins plays an important role in metastasis formation by orchestrating the cell adhesion and cytoskeletal dynamics together with the surrounding extracellular matrix. Tissue transglutaminase (TG2) is the ubiquitously expressed member of transglutaminase family which is able to catalyze Ca+2 dependent cross-linking by forming an intermolecular ε-(γ-glutamyl)-lysine isopeptide bond between proteins. Recent study demonstrated that regardless from its enzymatic activity, the upregulation of TG2 along with ITGβ1 and SDC4 can be a new diagnostic marker in the metastatic state of renal cell carcinoma (RCC), a notoriously radio-resistant and chemoresistant cancer. To date, integrin targeted therapeutic agents is not able to stabilize the metastatic progress of RCC therefore this study seeks to understand whether TG2 together with SDC4 may behave as a central player during the endo/exocytic cycle of β1 integrins. For this purpose, crosstalk between TG2 and cell surface receptors SDC4, ITGβ1 was investigated in RCC cell lines using co-immunoprecipitation (IP) assay followed by the measurement of cell attachment and spreading potential of TG2 downregulated RCC cells on β1 integrin substrates. In order to clarify the role of TG2-SDC4 interplay in integrin internalization and recycling a biotinylation-based IP and Flow Cytometry assay using active integrin binding antibody was performed, respectively. Results from this study showed that TG2 was associated with ITGβ1&SDC4 in all four primary and metastatic site RCC cell lines with the highest interaction affinity detected for the metastatic Caki-1 cells. Cell adhesion assays suggested that TG2 is a major component that mediates the RCC cell adhesion as the silencing of TG2 in all four RCC cell lines resulted in a marked decrease in their adhesion potential on β1 integrin substrates. The analysis of the role of TG2 and SDC4 in integrin β1 trafficking in Caki-1 cells using shRNA technology suggested that TG2 together with SDC4 contributes to the β1 integrin internalization and recycling. These findings support a role for TG2 in association with SDC4 in the development of RCC metastases through regulation of the cell adhesion and β1 integrin trafficking.