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. 2014 Oct 1;74(19):5572-84.
doi: 10.1158/0008-5472.CAN-13-3724. Epub 2014 Aug 14.

EGFR blockade enriches for lung cancer stem-like cells through Notch3-dependent signaling

Rajeswara Rao Arasada  1 , Joseph M Amann  1 , Mohammad A Rahman  2 , Stacey S Huppert  3 , David P Carbone  4
Affiliations

EGFR blockade enriches for lung cancer stem-like cells through Notch3-dependent signaling

Rajeswara Rao Arasada et al. Cancer Res. .

Abstract

Mutations in the epidermal growth factor receptor (EGFR) are the most common actionable genetic abnormalities yet discovered in lung cancer. However, targeting these mutations with kinase inhibitors is not curative in advanced disease and has yet to demonstrate an impact on potentially curable, early-stage disease, with some data suggesting adverse outcomes. Here, we report that treatment of EGFR-mutated lung cancer cell lines with erlotinib, while showing robust cell death, enriches the ALDH(+) stem-like cells through EGFR-dependent activation of Notch3. In addition, we demonstrate that erlotinib treatment increases the clonogenicity of lung cancer cells in a sphere-forming assay, suggesting increased stem-like cell potential. We demonstrate that inhibition of EGFR kinase activity leads to activation of Notch transcriptional targets in a γ secretase inhibitor-sensitive manner and causes Notch activation, leading to an increase in ALDH high(+) cells. We also find a kinase-dependent physical association between the Notch3 and EGFR receptors and tyrosine phosphorylation of Notch3. This could explain the worsened survival observed in some studies of erlotinib treatment at early-stage disease, and suggests that specific dual targeting might overcome this adverse effect.

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Conflict of interest statement

Conflict of Interest: DPC has done advisory boards for Pfizer. No other relevant conflicts.

Figures

Figure 1
Figure 1
Erlotinib treatment of EGFR mutant lung cancer cells increases the fraction of ALDH+ cells. A, HCC4006 and B, HCC827 cells were treated with DMSO, 0.1 μM of erlotinib for five days and subjected to aldefluor assay to detect the ALDH+ cells. A portion of the cells was pre-incubated with the ALDH inhibitor DEAB (+DEAB) to provide a gate (ALDH negative cells) for flow cytometry.
Figure 2
Figure 2
Erlotinib treatment increases the sphere-forming ability in EGFR mutant lung cancer cells. HCC827 cells were treated with DMSO or 0.1 μM of erlotinib for five days and allowed to recover for 4 days prior to subjected to sphere-forming assay in a 96 well plate. A, Spheres were imaged and B, their total number was quantitated (p < 0.008). C, H358 cells were treated with DMSO or erlotinib (1uM) and equal number of cells were subjected to soft agar colony formation assay and D, colony number was quantitated.
Figure 3
Figure 3
EGFR mediated negative regulation of Notch3 transcriptional activity in a tyrosine kinase-dependent manner. A–B, HEK293FT cells were transiently transfected with either 12X-CSL reporter (A) or full length HES1 reporter (B) with empty vector or N3DA construct and increasing amounts of wild type or kinase inactive EGFR to determine the role of EGFR on transcriptional activity of Notch3-ICD. The data presented are the average of three assays. C, Erlotinib stimulates gene expression of Hes1. HCC827 cells were treated with erlotinib for the indicated time periods and RNA was analyzed by qRT-PCR for the expression of Hes1.
Figure 4
Figure 4
Inhibition of Notch activation by a gamma secretase inhibitor prevents erlotinib-induced ALDH+ cells. A, HCC4006 and B, HCC827 cells were treated with DMSO (i), 0.1 μM erlotinib (ii), a combination of 0.1 μM erlotinib and 1 μM PF-03084014 (iii), or 1 μM PF-03084014 alone (iv)for 7 days and subjected to aldefluor assay. C, Erlotinib stimulated Notch transcriptional gene targets Hes1 and Hey1 are sensitive to GSI. HCC4006 (top) and HCC827 (bottom) were treated with 0.1 μM of erlotinib, PF-03084014 alone or in combination and RNA was analyzed by qRT-PCR for expression of Notch target genes Hes1 and Hey1.
Figure 5
Figure 5
Depletion of Notch3 abolishes erlotinib-induced ALDH+ cells. A, HCC4006 cells were transfected with either non-targeting control (NTC-siRNA) (top) in the absence or presence of erlotinib as indicated, Notch1 siRNAs -1, -2 and -3 (middle), or Notch3 siRNAs -1, -2, and -3 (bottom). Forty-eight hours post-transfection, cells were treated with 0.1μM erlotinib for 3 days and ALDH activity was measured. As a control, NTC siRNA treated cells were also treated with DMSO (top, middle panel). A portion of the cells was pre-incubated with the ALDH inhibitor DEAB (+DEAB) to provide a gate for ALDH-negative cells (top left panel). B, HCC827 cells were transfected with non-targeting control pool (NTC-siRNA), pool of Notch3 or Notch1 siRNAs. Forty-eight hours following transfection, cells were treated with 0.1 μM erlotinib (B) for 3 days and ALDH activity was measured. As a control, NTC siRNA treated cells were also treated with DMSO (top, left panel). A similar experiment was also performed with an individual siRNA (bottom panel).
Figure 6
Figure 6
EGF-mediated association between EGFR and Notch 3 receptors in HCC2429 cells is dependent on EGFR kinase activity. A. HCC2429 cells were stimulated with EGF (25 ng/ml) for the indicated times. Cell lysate was prepared from each time point and immunoprecipitated with EGFR antibody. Western analysis of precipitated proteins was performedand probed with a Notch3 antibody (top) and an EGFR antibody (middle). Whole cell lysate was subjected to western analyses using Notch3, and β-tubulin antibodies to detect the total expression of these proteins. B, HCC4006 C, HCC827cells were treated with DMSO or 0.1 μM erlotinib for 24 hours. Cell lysate was prepared from each treatment and immunoprecipitated with EGFR antibody and blotted for Notch3, or reciprocal immunoprecipitation was performed by precipitating Notch3 and blotting for EGFR. Whole cell lysate was subjected to western analyses using Notch3, EGFR and actin antibodies to detect the total expression of these proteins. IP= Immunoprecipitation, WCL = Whole Cell Lysate.
Figure 7
Figure 7
EGF mediated tyrosine phosphorylation of the Notch3 receptor. A&B, HCC2429 cells were stimulated with EGF (25 ng/ml) for the indicated times to detect the ligand induced tyrosine phosphorylation of Notch3 receptor. A Cell lysates were immunoprecipitated with Notch3 antibody followed by western analysis using a phosphotyrosine antibody. The blot was stripped and re-probed with anti-Notch3 (bottom). B, A reciprocal immunoprecipitation was done with a phosphotyrosine antibody with a subsequent western blot probed for Notch3 (top). Equal amount of cell lysate was analyzed for Notch3 expression (middle) and tubulin (bottom).
Figure 8
Figure 8
EGFR kinase activity is necessary for the tyrosine phosphorylation of the Notch3 receptor. A, HCC4006 and B, HCC827 cells were treated with DMSO or erlotinib (0.1 μM) for indicated periods of time. Cell lysates from each time point were either precipitated with a phosphotyrosine antibody followed by a western blot probed with Notch3 antibody (top), or immunoprecipitated with Notch3 antibody followed by a western blot probed with a phosphotyrosine antibody. Whole cell lysate was also subjected to western analysis to check for of levels Notch3, EGFR-pY1173, and tubulin.
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