Abstract
The outlook for patients with advanced renal cell cancer (RCC) has been improved by targeted agents including inhibitors of the PI3 kinase (PI3K)-AKT-mTOR axis, although treatment resistance is a major problem. Here, we aimed to understand how RCC cells acquire resistance to PI3K-mTOR inhibition. We used the RCC4 cell line to generate a model of in vitro resistance by continuous culture in PI3K-mTOR kinase inhibitor NVP-BEZ235 (BEZ235, Dactolisib). Resistant cells were cross-resistant to mTOR inhibitor AZD2014. Sensitivity was regained after 4 months drug withdrawal, and resistance was partially suppressed by HDAC inhibition, supporting an epigenetic mechanism. BEZ235-resistant cells up-regulated and/or activated numerous proteins including MET, ABL, Notch, IGF-1R, INSR and MEK/ERK. However, resistance was not reversed by inhibiting or depleting these pathways, suggesting that many induced changes were passengers not drivers of resistance. BEZ235 blocked phosphorylation of mTOR targets S6 and 4E-BP1 in parental cells, but 4E-BP1 remained phosphorylated in resistant cells, suggesting BEZ235-refractory mTORC1 activity. Consistent with this, resistant cells over-expressed mTORC1 component RAPTOR at the mRNA and protein level. Furthermore, BEZ235 resistance was suppressed by RAPTOR depletion, or allosteric mTORC1 inhibitor rapamycin. These data reveal that RAPTOR up-regulation contributes to PI3K-mTOR inhibitor resistance, and suggest that RAPTOR expression should be included in the pharmacodynamic assessment of mTOR kinase inhibitor trials.
Highlights
renal cell cancer (RCC) cells induced to be resistant to PI3 kinase (PI3K)-mammalian target of rapamycin (mTOR) inhibitor BEZ235 are cross-resistant to AZD2014
Our initial evaluation of mTOR inhibition in RCC cells showed that rapamycin but not BEZ235 enhanced S473 AKT phosphorylation, likely due to relief by rapamycin of negative feedback between S6 kinase (S6K) and insulin receptor substrate-1 (IRS-1), as we reported previously in RCC cells [36]
We used the approach of exposing RCC cells to dual PI3K-mTOR inhibitor BEZ235
Summary
Treatment of metastatic renal cell cancer (RCC) has been transformed by introduction of targeted agents, including multi-targeted inhibitors of VEGF receptor and other tyrosine kinases, and inhibitors of the mammalian target of rapamycin (mTOR) [1]. MTOR is a serine threonine kinase that exists in two protein complexes: mTOR complex 1 (mTORC1) and 2. RAPTOR up-regulation mediates resistance to PI3K-mTOR inhibition. VMM from the National Institute for Health Research (NIHR; www.nihr.ac.uk) Oxford Biomedical Research Centre (award A93024). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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