mTOR-independent 4E-BP1 phosphorylation is associated with cancer resistance to mTOR kinase inhibitors

Abstract

ATP-competitive mTOR kinase inhibitors (mTorKIs) are a new generation of mTOR-targeted agents with more potent anticancer activity than rapamycin in several tumor models. However, the sensitivity and resistance of cancer cells to mTorKIs remain poorly understood. In this study, we tested mTorKIs against a large panel of colorectal cancer (CRC) cell lines, and found that mTorKIs displayed broader anti-CRC activity than rapamycin, including CRC cells with K-Ras or B-Raf mutations, suggesting that these mTorKIs are particularly useful for CRCs resistant to EGFR inhibitors. Unexpectedly, we found that 40% CRC cell lines were intrinsically drug resistant. Moreover, we discovered an mTOR-independent 4E‑BP1 phosphorylation that was correlated with mTorKI resistance. Altogether, our findings provide compelling preclinical support for testing mTorKIs in human CRC clinical trials. They further reveal the existence of significant intrinsic mTorKI drug resistance in cancer cells and suggest that 4E-BP1 phosphorylation is a predictive biomarker for mTorKI sensitivity and resistance.

Figure 1

Effect of BEZ235, PP242 and WYE354 on mTOR signaling in drug-sensitive and -resistant CRC cell lines. (A) Three most mTorKI-sensitive CRC cell lines (SW480, LOVO205 and CACO-2) were treated with rapamycin (Rapa), BEZ235, PP242 and WYE354 for 12 h. The effect of mTOR inhibitors on mTOR signaling was analyzed by substrate phosphorylation of mTORC1 and mTORC2 by western blot. (B) Three most mTorKI-resistant CRC cell lines (SW620, COLO205 and HCT116) were treated with rapamycin, BEZ235, PP242 and WYE354 for 12 h. The effect of mTOR inhibitors on mTOR signaling was analyzed by substrate phosphorylation of mTORC1 and mTORC2 by western blot.

Figure 2

Differential anticancer effect of mTOR kinase inhibitors (mTorKIs) toward SW480 and SW620 cells. (A) SW480 and SW620 cells were cultured in soft agar in the absence or presence of different mTOR inhibitors. The anchorage-independent cell growth was analyzed by the ability of these cells to form colonies. A representative result is shown for SW480 cells. (B) Quantification of the soft-agar assay results are expressed as the ratio of colonies in treated vs. control cells. Data represent mean ± SD from three independent triplicate experiments. *p < 0.01, vs. control. (C) Two mTorKI-sensitive CRC cell lines (SW480 and CACO-2) were treated with high dose of mTorKIs for 72 h, and apoptotic cells were quantified. Indomethecin (Indo) was used as a positive control. Data represent means ± SD from three independent triplicate experiments. (D) Two mTorKI-resistant CRC cell lines (SW620 and HCT116) were treated with a high dose of mTorKIs for 72 h, and apoptotic cells were quantified. Indomethecin (Indo) was used as a positive control. Data represent means ± SD from three independent triplicate experiments.

Figure 3

mTorKIs inhibit 4E-BP1 phosphorylation in SW480 but not SW620 xenograft tumors. (A) Xenograft tumor mouse models were orally administered with BEZ235 at 45 mg/kg/day or PP242 at 60 mg/kg/day once daily for 28 d. Upper parts show representative tumors (as indicated by arrow-heads) in treated and control xenograft mice. Lower part shows tumor volume measurements (means ± SD; n = 8; *p < 0.01, vs. control). (B) Xenograft tumor mouse models derived from SW480 and SW620 were orally administered with BEZ235 at 45 mg/kg/day or PP242 at 60 mg/kg/day, once daily for 28 d. On day 28, CRC tumors were removed and analyzed for inhibition of PI3K-mTOR signaling by western blot. Four tumor samples from control group and 5 samples from each mTorKIs treatment group were shown here, with each lane representing an individual tumor sample.

Figure 4

The kinetics of mTOR inhibition by mTorKIs in SW480 and SW620 cells. SW480 and SW620 cells were treated with mTOR inhibitors rapamycin (A), BEZ235 (B), PP242 (C) and WYE354 (D) for different times. The phosphorylation and total level of different mTOR substrates was determined by western blot.

Figure 5

mTOR-independent 4E-BP1 phosphorylation in SW620 cells. (A) BEZ235 inhibits mTOR kinase activity toward 4E-BP1 in both SW480 and SW620 cells. SW480 and SW620 cells were treated with 100 nM BEZ235 or drug vehicle control (DMSO) for 6 h. mTOR was immunoprecipitation and assayed for in vitro kinase activity toward bacterial recombinant S6K1 and GST-4E-BP1 by western blot. (B) siRNA-mediated knockdown of mTORC1 inhibits 4E-BP1 phosphorylation in SW480 but not SW620 cells. SW480 and SW620 cells were transfected with siRNA targeting mTOR, raptor and rictor, respectively, for 48 h. Inhibition of mTOR signaling was analyzed by western blot.