Resistance to the mTOR Inhibitor Temsirolimus Alters Adhesion and Migration Behavior of Renal Cell Carcinoma Cells through an Integrin α5– and Integrin β3–Dependent Mechanism

Abstract

Inhibitors of the mammalian target of rapamycin (mTOR) have improved the treatment of renal cell carcinoma (RCC). However, chronic drug exposure may trigger resistance, limiting the utility of these agents. The metastatic behavior of RCC cells, susceptible (RCCpar) or resistant (RCCres) to the mTOR inhibitor temsirolimus, was investigated. Adhesion to vascular endothelium or immobilized collagen and fibronectin was quantified. Chemotactic motility was evaluated with a modified Boyden chamber assay. Integrin α and β subtype receptors were analyzed by flow cytometry and Western blot analysis. The physiological relevance of the integrins was then determined by blocking studies and small interfering RNA knockdown. Adhesion to endothelial cells and to fibronectin (not to collagen) and chemotaxis were enhanced in RCCres compared to RCCpar. RCCres detached from fibronectin and motile activity further increased under retreatment with low-dosed temsirolimus. α5 integrin was diminished inside the cell and at the cell surface, whereas the β3 subtype was reduced intracellularly but elevated at the plasma membrane. In RCCpar, blocking α5 surface receptors enhanced RCC-collagen but reduced RCC-fibronectin interaction, whereas the opposite was true for RCCres. Chemotaxis of RCCpar but not of RCCres was strongly diminished by the α5 antibody. Blocking β3 significantly lowered chemotaxis with stronger effects on RCCres, compared to RCCpar. Importantly, β3 knockdown reduced chemotaxis of RCCpar but upregulated the motile behavior of RCCres. Temsirolimus resistance is characterized by quantitative alterations of integrin α5 and β3 expression, coupled to functional changes of the integrin molecules, and forces a switch from RCC adhesion to RCC migration.