mTOR inhibition induces compensatory, therapeutically targetable MEK activation in renal cell carcinoma

Abstract

Rapamycin derivatives allosterically targeting mTOR are currently FDA approved to treat advanced renal cell carcinoma (RCC), and catalytic inhibitors of mTOR/PI3K are now in clinical trials for treating various solid tumors. We sought to investigate the relative efficacy of allosteric versus catalytic mTOR inhibition, evaluate the crosstalk between the mTOR and MEK/ERK pathways, as well as the therapeutic potential of dual mTOR and MEK inhibition in RCC. Pharmacologic (rapamycin and BEZ235) and genetic manipulation of the mTOR pathway were evaluated by in vitro assays as monotherapy as well as in combination with MEK inhibition (GSK1120212). Catalytic mTOR inhibition with BEZ235 decreased proliferation and increased apoptosis better than allosteric mTOR inhibition with rapamycin. While mTOR inhibition upregulated MEK/ERK signaling, concurrent inhibition of both pathways had enhanced therapeutic efficacy. Finally, primary RCC tumors could be classified into subgroups [(I) MEK activated, (II) Dual MEK and mTOR activated, (III) Not activated, and (IV) mTOR activated] based on their relative activation of the PI3K/mTOR and MEK pathways. Patients with mTOR only activated tumors had the worst prognosis. In summary, dual targeting of the mTOR and MEK pathways in RCC can enhance therapeutic efficacy and primary RCC can be subclassified based on their relative levels of mTOR and MEK activation with potential therapeutic implications.

Figure 1. Novel renal cell carcinoma cell lines lack VHL and overexpress HIF.

(A) Photomicrographs of H&E stains (left panels) and bright field images (right panels) of UNC-R1 and UNC-R2 PDX derived cell lines. (B) Whole cell extracts from UNC-R1 and UNC-R2s were immunoblotted with the indicated antibodies. RCC4 2-1 (VHL null) and RCC4 3–14 (VHL positive) were included as controls.

Figure 2. Catalytic mTOR inhibitors block mTORC1 signaling more fully than allosteric mTOR inhibition.

The indicated cell lines were treated with the allosteric and catalytic mTOR inhibitors (rapamycin and BEZ235 respectively) at the indicated concentrations for 24 hrs. Whole cell extracts were then immunoblotted with the indicated antibodies.

Figure 3. Catalytic mTOR inhibition attenuates proliferation and induces apoptosis better than allosteric mTOR inhibition.

(A) The indicated cell lines were assessed for viability on the indicated days using CellTiter-Glo. Statistical significance was determined by comparing rapamycin and BEZ235 treated groups. (B) The indicated cell lines were treated with rapamycin and BEZ235 for 48 hours and immunoblotted with the indicated antibodies. (C) The indicated cell lines were treated with rapamycin and BEZ235 for 48 hours and assessed for apoptosis by flow cytometry analysis of the Annexin V+/PI – fraction. (D) 786-0 cells were stably infected with shRNAs targeting Raptor (mTORC1) or Rictor (mTORC2) and confirmed for knock-down by western blot. (E) Whole cell extracts from 786-0 shNS, shRaptor, and shRictor cells were immunoblotted with the indicated antibodies.

Figure 4. Combined mTOR and MEK inhibition attenuates cellular proliferation and increases the apoptotic response.

(A) The indicated cells were treated for 24 hrs. with rapamycin or BEZ235 and immunoblotted with the indicated antibodies. (B) 786-0 and RCC4 cells were treated increasing doses of GSK212 for 24 hrs. and immunoblotted with the indicated antibodies. (C) 786-0 and RCC4 cells were treated for 24 hrs with rapamycin and BEZ235 in the presence of Edu. Edu incorporation was assessed by flow cytometry. (D) 786-0 and RCC4 cells were treated with the indicated compounds for 24 hrs. Whole cell extracts were immunoblotted for the cell cycle related proteins indicated. (E) 786-0 and RCC4 cells were treated with indicated drugs and assessed for viability on day 4 using CellTiter-Glo 4. (F) 786-0 and RCC4 cells were plated, allowed to attach, and treated with the indicated drug(s). Photographs of wells containing 786-0 (day 11) and RCC4 (day 17) cells fixed with 4% PFA and stained with 0.1% crystal violet. (G) 786-0 and RCC4 cells were treated with the indicated compounds for 24 hrs. Whole cell extracts were immunoblotted with the indicated antibodies.

Figure 5. Subclasses of RCC can be defined by MEK and mTOR pathway activation.

(A) TCGA KIRC RPPA data was log2 transformed, median centered. Tumors were then hierarchically clustered and the indicated subgroups were determined based on expression patterns of the indicated phosphoproteins. Mutational data for mTOR pathway related genes were annotated in the upper tracks. (B) Scatter plot of TCGA KIRC tumors based on expression of pS6 and pERK. Each dot indicates a tumor. The MEK-PI3K/mTOR subclasses defined in (A) are indicated by color. (C) Patients harboring tumors within each MEK-PI3K/mTOR subclass were evaluated for differences in overall survival by the Log Rank test and shown as a Kaplan-Meier plot of overall survival.