Differential induction of apoptosis in HER2 and EGFR addicted cancers following PI3K inhibition

Abstract

Non-small cell lung cancers with activating mutations in the epidermal growth factor receptor (EGFR) are highly responsive to EGFR tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib. Such cancers are "addicted" to EGFR, and treatment with a TKI invariably leads to down-regulation of the PI3K-AKT-mTOR and MEK-ERK signaling pathways, resulting in apoptosis. Using a dual PI3K-mTOR inhibitor, NVP-BEZ235, we evaluated whether PI3K-mTOR inhibition alone induced apoptosis in these cancers. In contrast to HER2-amplified breast cancers, we found that PI3K-mTOR inhibition did not promote substantial apoptosis in the EGFR mutant lung cancers. However, blocking both PI3K-mTOR and MEK simultaneously led to apoptosis to similar levels as the EGFR TKIs, suggesting that down-regulation of these pathways may account for much of the apoptosis promoted by EGFR inhibition. In EGFR mutant lung cancers, down-regulation of both intracellular pathways converged on the BH3 family of proteins regulating apoptosis. PI3K inhibition led to down-regulation of Mcl-1, and MEK inhibition led to up-regulation of BIM. In fact, down-regulation of Mcl-1 by siRNA was sufficient to sensitize these cancers to single-agent MEK inhibitors. Surprisingly, an AKT inhibitor did not decrease Mcl-1 levels, and when combined with MEK inhibitors, failed to induce apoptosis. Importantly, we observed that the combination of PI3K-mTOR and MEK inhibitors effectively shrunk tumors in a transgenic and xenograft model of EGFR T790M-L858R cancers. These data indicate simultaneous inhibition of PI3K-mTOR and MEK signaling is an effective strategy for treating EGFR mutant lung cancers, including those with acquired resistance to EGFR TKIs.

Fig. 1.

PI3K-mTOR inhibition effectively reduces cell viability in HER2-amplified breast cancers, but combined PI3K-mTOR and MEK inhibition is necessary to effectively reduce cell viability in EGFR mutant lung cancer cells. (A) HER2-amplified BT474 cells (top) and EGFR mutant HCC827 cells (lower) and were treated with increasing doses of the PI3K-mTOR inhibitor NVP-BEZ235, the MEK inhibitor AZD6244, or the combination of both (BEZ/AZD), and total cell viability was determined after 72 h by staining cells with the nucleic acid stain, Syto60. Data are presented as the percent of viable cells versus DMSO (-) treated cells. ± S.D. Student's t-test were performed comparing BEZ with BEZ/AZD at indicated concentrations; * indicates a P value <0.001, # indicates P > 0.05 (not significant). (B) Cells were treated with either DMSO (-) or the indicated drug(s) for 6 h (TKIs 1 μM, NVP-BEZ235 0.2 μM for all cell lines, AZD6244 0.2 μM for SKBR3 cells, 1 μM for HCC827 and PC9 cells, and 2 μM for BT474 cells). Protein lysates were immunoblotted and probed with the indicated antibodies. (C) Cells were treated with the indicated drug(s) as in (B) for 16 days, and cell viability was determined by Syto60 assay. The fold difference of viable cells is presented relative to the viable cells treated with tyrosine kinase inhibitor [TKI, gefitinib (gef) for HCC827 and PC9 cells, and lapatinib (lap) for SKBR3 and BT474 cells]. BEZ versus BEZ/AZD (HCC827, P < 0.001; PC9, P < 0.001), and BEZ versus TKI (HCC827, P < 0.001; PC9, P < 0.001).

Fig. 2.

EGFR mutant cells downregulate Mcl-1 in response to PI3K-mTOR inhibition. (A) EGFR mutant cells (HCC827, H1975, and HCC827 GR6) and HER2-amplified cells (SKBR3 and BT474) were treated with either DMSO control or the indicated drug(s) (TKIs 1 μM, NVP-BEZ235 0.2 μM, AZD6244 0.2 μM for SKBR3 cells, 1 μM for HCC827 and HCC827 GR6 cells, and 2 μM for H1975 and BT474 cells) and 72 h later cells were subjected to flow cytometry to determine subG₀/G₁ population and the distribution of cycling cells, as described in Materials and Methods. Data are presented as mean ± S.D. of triplicate experiments. (TKI is CL-387,785 (CL) for H1975 cells and gefitinib/PHA-665752 (gef/PHA) for HCC827 GR6 cells). Statistical analyses comparing BEZ versus BEZ/AZD were P < 0.001 for HCC827, H1975, HCC827 GR6, and were not significant (P > 0.01) for BT474 an SKBR3. (B) Cells were treated with either DMSO (-) or drug for 30 h. Cell lysates were prepared and subjected to immunoblotting with the indicated antibodies. The SKBR3 cells were treated for 72 h with the indicated drugs because no substantial PARP cleavage was observed after 30 h with any of the drug treatments.

Fig. 3.

Loss of Mcl-1 expression sensitizes EGFR mutant cells to single-agent MEK inhibition. (A) EGFR mutant HCC827 cells were transfected with scrambled (sc) or Mcl-1 siRNA followed by treatment with either DMSO (-) or the indicated drugs for 30 h. Protein lysates were assessed with the indicated antibodies. (B) Cells were transfected and drug treated as in (A). Forty-eight hours following drug treatment, cells were assessed for subG₀/G₁ DNA content. (C) HCC827 cells were treated for 16 h with either DMSO (-) or the PI3K/mTOR dual inhibitor BEZ235 (.2 μM), the PI3K inhibitor, ZSTK474 (1 μM), or the AKT inhibitor, AKT 1/2 (1 μM), and cell lysates were prepared and subjected to immunoblotting with the indicated antibodies. (D) HCC827 cells were treated for 72 h with either DMSO (-) or indicated drugs, and cells were assessed for subG₀/G₁ DNA content. Mean + S.D. of triplicate experiments is shown. BEZ/AZD versus AKT/AZD, P < 0.001; BEZ/AZD versus ZST/AZD, P > 0.01, not significant.

Fig. 4.

The combination of BEZ/AZD is an effective treatment strategy against in EGFR mutant cancers in vivo. (A) HCC827 xenografts were treated with the indicated drug regimens (as described in SI Methods), and tumor volumes (± S.E.M.) were plotted over time. (B and C) HCC827 xenografts were treated with the indicated drug regimens. Tumors were harvested 1.5 h after treatment on Day 3. Tumor lysates were analyzed by immunoblotting with the indicated antibodies. (D) H1975 xenografts were treated with the indicated drug regimens [as described in Materials and Methods and identical to treatments in (A)], and average tumor volumes (± S.E.M.) were plotted over time. (E) (Left) Representative Magnetic Resonance Images (MRI) of T790M/L858R (TL) mice fed doxycycline to induce tumor formation (Pre-Rx) and treated with the indicated drug regimens for 3 weeks. Note: Red arrows point to tumor. (Right) Tumor volumes of TL mice treated with NVP-BEZ235 (BEZ), AZD6244 (AZD), or AZD/BEZ. The y axis at “1” is the normalized tumor volume at “Day 0” of the respected drug treatments. Tumor volumes of AZD/BEZ versus BEZ alone, P < 0.001, AZD/BEZ versus AZD, P < 0.001). (F) Tumor lysates were prepared from TL mice treated with either vehicle treatment (-) or the indicated drug treatment as in (B). Immunoblotting was performed with the indicated antibodies.