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Background: Although mechanistic target of rapamycin (mTOR) inhibitors, such as temsirolimus, show promise in treating bladder cancer, acquired resistance often hampers efficacy. This study evaluates mechanisms leading to resistance. Methods: Cell growth, proliferation, cell cycle phases, and cell cycle regulating proteins were compared in temsirolimus resistant (res) and sensitive (parental—par) RT112 and UMUC3 bladder cancer cells. To evaluate invasive behavior, adhesion to vascular endothelium or to immobilized extracellular matrix proteins and chemotactic activity were examined. Integrin α and β subtypes were analyzed and blocking was done to evaluate physiologic integrin relevance. Results: Growth of RT112res could no longer be restrained by temsirolimus and was even enhanced in UMUC3res, accompanied by accumulation in the S- and G2/M-phase. Proteins of the cdk-cyclin and Akt-mTOR axis increased, whereas p19, p27, p53, and p73 decreased in resistant cells treated with low-dosed temsirolimus. Chemotactic activity of RT112res/UMUC3res was elevated following temsirolimus re-exposure, along with significant integrin α2, α3, and β1 alterations. Blocking revealed a functional switch of the integrins, driving the resistant cells from being adhesive to being highly motile. Conclusion: Temsirolimus resistance is associated with reactivation of bladder cancer growth and invasive behavior. The α2, α3, and β1 integrins could be attractive treatment targets to hinder temsirolimus resistance.
Although the mechanistic target of rapamycin (mTOR) inhibitor, everolimus, has improved the outcome of patients with renal cell carcinoma (RCC), improvement is temporary due to the development of drug resistance. Since many patients encountering resistance turn to alternative/complementary treatment options, an investigation was initiated to evaluate whether the natural compound, sulforaphane (SFN), influences growth and invasive activity of everolimus-resistant (RCCres) compared to everolimus-sensitive (RCCpar) RCC cell lines in vitro. RCC cells were exposed to different concentrations of SFN and cell growth, cell proliferation, apoptosis, cell cycle, cell cycle regulating proteins, the mTOR-akt signaling axis, adhesion to human vascular endothelium and immobilized collagen, chemotactic activity, and influence on surface integrin receptor expression were investigated. SFN caused a significant reduction in both RCCres and RCCpar cell growth and proliferation, which correlated with an elevation in G2/M- and S-phase cells. SFN induced a marked decrease in the cell cycle activating proteins cdk1 and cyclin B and siRNA knock-down of cdk1 and cyclin B resulted in significantly diminished RCC cell growth. SFN also modulated adhesion and chemotaxis, which was associated with reduced expression of the integrin subtypes α5, α6, and β4. Distinct differences were seen in RCCres adhesion and chemotaxis (diminished by SFN) and RCCpar adhesion (enhanced by SFN) and chemotaxis (not influenced by SFN). Functional blocking of integrin subtypes demonstrated divergent action on RCC binding and invasion, depending on RCC cell sensitivity to everolimus. Therefore, SFN administration could hold potential for treating RCC patients with established resistance towards everolimus.