Abstract A36: Knockdown of integrin-linked kinase reduces invasive and metastatic potential of renal cell carcinoma
Authors: Seok Han Kyung , Peter Raven , Shannon Awrey , Estelle Li , Ladan Fazli , Martin Gleave , Alan So 
Published: May 2015

Abstract
Introduction: Integrin-linked kinase (ILK) is a serine/threonine kinase implicated in the regulation of cell growth and survival, cell cycle progression, epithelial-mesenchymal transition (EMT), invasion and migration, angiogenesis. However, the role of ILK has not been evaluated in renal cell carcinoma (RCC). We investigated the role of ILK on cancer progression and metastasis and therapeutic potential of ILK inhibition in RCC.
Methods: Baseline expression of ILK was evaluated by immunofluorescence and western blotting in non-cancerous renal tubular cells (HK-2) and RCC cells (UMRC-6, UMRC-3 and Caki-1). Molecular EMT markers were examined by Western blotting. RNAi using siRNA was used to knock down ILK in vitro. After transient transfection, crystal violet assay and cell cycle analysis using FACS were performed to check the effect of ILK on tumor growth and cell cycle. Confocal microscopic evaluation was performed to check changes of stress fibers and focal adhesions stained with phalloidin-rhodamine and vinculin antibody. Scratch assay and Matrigel invasion assay were performed to evaluate changes of EMT phenotypes after knockdown of ILK. To evaluate the effects of ILK knockdown in vivo, subcataneuous xenografts and orthotopic renal xenografts were used and tumor growth was traced.
Results: ILK is less expressed in normal cells (HK-2) and low stage RCC cells (UMRC-6) but highly expressed in advanced and metastatic RCC cells (UMRC-3 and Caki-1). Caki-1, metastatic RCC cells showed higher expressions of molecular EMT markers including Snail, Zeb1 but decreased activity of GSK3β. Knockdown of ILK using si-ILK inhibited tumor proliferation but the inhibition rate was less than 10% and cell cycle progression was not significantly affected by ILK inhibition. However, ILK knockdown suppressed formation of stress fibers and focal adhesions in UMRC-3 and Caki-1 cells and also effectively impeded phenotypic EMT markers including cell migration and invasion in Caki-1 and UMRC-3 cells. In subcutaneous tumor xenografts with Caki-1-shILK or Caki-1-EV, ILK knockdown inhibited tumor growth and progression. In primery orthotopic animal model, ILK knockdown showed inhibitory effects of invasion and metastasis.
Conclusions: ILK is highly expressed in advanced RCC and its high expression is related to EMT-related protein in RCC. Knockdown of ILK inhibited molecular EMT markers and suppressed cell migration and invasion in vitro and metastasis in orthotopic tumor model. These results suggest the therapeutic potential of ILK inhibition on invasion and metastasis in advanced RCC.
Citation Format: Han Kyung Seok, Raven Peter, Awrey Shannon, Li Estelle, Fazli Ladan, Gleave Martin, So Alan. Knockdown of integrin-linked kinase reduces invasive and metastatic potential of renal cell carcinoma. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr A36.