Inhibition of mutant EGFR in lung cancer cells triggers SOX2-FOXO6-dependent survival pathways
- Massachusetts General Hospital, Harvard Medical School, United States
- Howard Hughes Medical Institute, Massachusetts General Hospital, Harvard Medical School, United States
Figures
Figure 1 with 4 supplements
SOX2 transcript is specifically induced by erlotinib in EGFR-mutant and addicted lung cancer cell lines.
(A) Cell lines were treated with an inhibitor of the driving oncogenic lesion for 24 hr (erlotinib for EGFR-mutant, lapatinib for HER2-amplified and crizotinib for ALK-translocated cells), followed …
Figure 1—figure supplement 1
Gene expression profiling after erlotinib treatment.
(A) Heat map showing the list of fourfold significantly (FDR <0.05) upregulated (green) or downregulated (red) transcripts in HCC827 cells with erlotinib treatment. (B) Time course of changes in …
Figure 1—figure supplement 2
Effect of various treatments on SOX2 expression in different cell contexts.
(A) HCC827 and PC9 cells. The erlotinib curve is the same as in Figure 1B. (B) Left panel, H1650 cells (EGFR DEL15 activating but IC50 >1 µM) and WM164 (BRAF V600E) show minimal induction following …
Figure 1—figure supplement 3
Overlap of differentially expressed genes.
Erlotinib treatment of EGFR-mutant cells was compared to MEK inhibitor treatment of BRAF-mutant melanoma cells lines and CL-387,785 (irreversible EGFR inhibitor) treatment of EGFR-mutant lung cancer …
Figure 1—figure supplement 4
Time course of SOX2 induction by quantitative immunofluorescence microscopy.
The data for the 24 hr time points are the same as in Figure 2A. p-values are shown for the comparison of mean SOX2 fluorescence of each treated population to DMSO (Student's t-test, unequal …
Figure 2 with 6 supplements
Induction of SOX2 in erlotinib-treated cells.
(A) Left, HCC827 (upper) or PC9 (lower) cells were treated with 0.1 µM erlotinib for 24 hr, followed by immunofluorescence staining using an antibody to SOX2 and DAPI. For erlotinib-treated cells …
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Figure 2—source data 1
Raw immunofluorescence data for quantitation of SOX2 staining in HCC827 cells with erlotinib treatment in Figure 2A, and SOX2+ Ki67 staining in Figure 2—figure supplement 3.
- https://doi.org/10.7554/eLife.06132.009
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Figure 2—source data 2
Raw immunofluorescence data for quantitation of SOX2 staining in PC9 cells with erlotinib treatment in Figure 2A and Figure 1—figure supplement 4.
- https://doi.org/10.7554/eLife.06132.010
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Figure 2—source data 3
Raw immunofluorescence data for quantitation of SOX2 staining in HCC827 cells recovered after retreatment (x2) with erlotinib, compared to previously untreated, in Figure 2B.
- https://doi.org/10.7554/eLife.06132.011
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Figure 2—source data 4
Raw immunofluorescence data for quantitation of SOX2 staining in HCC827 and PC9 cells with increasing dose of erlotinib in Figure 2—figure supplement 1.
- https://doi.org/10.7554/eLife.06132.012
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Figure 2—source data 5
Raw immunofluorescence data for quantitation of SOX2 staining in PC9 cells recovered after retreatment (x2) with erlotinib, compared to previously untreated cells, in Figure 2—figure supplement 4A.
- https://doi.org/10.7554/eLife.06132.013
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Figure 2—source data 6
Raw immunofluorescence data for quantitation of phospho-EGFR (pY1068) in parental and erlotinib-resistant PC9 cells in Figure 2—figure supplement 4B.
- https://doi.org/10.7554/eLife.06132.014
Figure 2—figure supplement 1
Increasing the dose of erlotinib does not significantly increase the fraction of SOX2+ cells.
HCC827 (left) and PC9 (right) cells were treated for 24 hr with the indicated dose of erlotinib, followed by immunofluorescence microscopy with antibodies to SOX2 and DAPI. The distribution of SOX2+ …
Figure 2—figure supplement 2
Stem cell markers do not colocalize with SOX2+ cells.
Immunofluorescence microscopy was carried out on erlotinib-treated HCC827 (upper panels) or PC9 (lower panels) cells using antibodies to SOX2 and various stem cell markers. CD133 (upper row of …
Figure 2—figure supplement 3
SOX2 is expressed most highly in nonproliferative cells.
Left panels, HCC827 cells were treated for 24 hr with 0.1 µM erlotinib, followed by immunofluorescence microscopy with antibodies to SOX2, Ki-67, and DAPI. The left three pairs of panels show HCC827 …
Figure 2—figure supplement 4
Stochastic induction of SOX2 by erlotinib in PC9 cells.
(A) Retreatment of PC9 cells after a period of recovery does not increase the fraction of cells capable of inducing SOX2. Left panels, images of cells stained for SOX2 (green) and DAPI (blue). Right …
Figure 2—figure supplement 5
The highest induction of SOX2 in individually isolated subclones of EGFR-mutant cells occurs in a subset of cells, as for the parental cells.
(A) Immunofluorescence analysis of colonies formed from single HCC827 cells for DAPI (blue), SOX2 (green), and Ki67 (red). (B) Similar analysis of DAPI (blue) and SOX2 (green) in PC9 cells. For …
Figure 2—figure supplement 6
KDM5A is not induced following treatment of PC9 cells with erlotinib for 24 hr.
Data are shown as mean Ct (normalized to GAPDH and untreated cells) of 3 replicates −/+ SEM.
Figure 3 with 1 supplement
SOX2 is induced by erlotinib in a subset of EGFR-mutant cells in vivo.
Nude mice were xenografted subcutaneously with PC9 cells and treated with a single oral dose of erlotinib (100 mg/kg) (or carrier) when the tumors had reached ∼500 mm3. Tumors were harvested 24 hr …
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Figure 3—source data 1
Number of SOX2+cells per field for quantitation of SOX2 staining in PC9 cell xenografts in Figure 3.
- https://doi.org/10.7554/eLife.06132.022
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Figure 3—source data 2
Raw absorbance data for quantitation of SOX2 staining in HCC827 cell xenografts in Figure 3—figure supplement 1.
- https://doi.org/10.7554/eLife.06132.023
Figure 3—figure supplement 1
Erlotinib treatment results in induction of SOX2 in vivo.
Quantitative immunohistochemistry for SOX2 was performed in FFPE sections from mice xenografted subcutaneously with HCC827 cells and treated with a single oral dose of erlotinib (red in dot plot) or …
Figure 4
SOX2 is induced by therapy targeting the resistance genotype in cell lines derived by rebiopsy of patients.
Short-term cultures of tumor cells derived from patients at the time of acquired resistance (both tumors EGFR genotype exon 19 deletion + T790M) were treated with the indicated agents for 24 hr, …
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Figure 4—source data 1
Raw immunofluorescence data for quantitation of SOX2 staining with different treatments in patient-derived tumor cells.
- https://doi.org/10.7554/eLife.06132.026
Figure 5 with 4 supplements
Induction of SOX2 protects cells from erlotinib-induced apoptosis.
(A) HCC827 cells were stably transduced with a doxycycline inducible epitope-tagged SOX2 lentiviral expression vector. Doxycycline was added for 3 hr (‘SOX2-tag’) and then removed prior to the …
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Figure 5—source data 1
Raw immunofluorescence data for quantitation of SOX2 staining in HCC827 cells with inducible SOX2 in Figure 5—figure supplement 1A.
- https://doi.org/10.7554/eLife.06132.028
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Figure 5—source data 2
Raw immunofluorescence data for quantitation of SOX2 and cleaved caspase-3 costaining in PC9 cells transfected with siCTRL or siSOX2 in Figure 5—figure supplement 2.
- https://doi.org/10.7554/eLife.06132.029
Figure 5—figure supplement 1
Induction of exogenous SOX2.
(A) Quantitative immunofluorescence analysis confirms that ectopic SOX2 expression with a short pulse of doxycycline increases the fraction of SOX2+ cells without significantly increasing the amount …
Figure 5—figure supplement 2
SOX2 expression modulates erlotinib-induced apoptosis.
Left panel, quantitative immunofluorescence analysis showing expression of SOX2 (x-axis) and cleaved caspase-3 (y-axis) in PC9 cells transfected with siCTRL (blue) or siSOX2 (red) and treated with …
Figure 5—figure supplement 3
The effect of siRNA targeting SOX2 is specific.
(A), PC9 cells were transfected with two different siRNA duplexes targeting SOX2 (or control siRNA), followed by addition of DMSO or 0.1 µM erlotinib for 24 hr and immunoblot of protein lysates with …
Figure 5—figure supplement 4
Quantitation of the effect of SOX2 knockdown on BIM levels.
Each BIM isoform (EL, L, and S) was normalized to the GAPDH loading control and untreated, siCTRL cells.
Figure 6 with 1 supplement
Erlotinib-induced SOX2 directly regulates expression of BIM and BMF.
(A) The levels of transcripts for each of the indicated BH3 domain-containing proteins was assessed by quantitative PCR at multiple time points after erlotinib treatment in uninduced PC9 cells (blue …
Figure 6—figure supplement 1
Effect of SOX2 overexpression on apoptotic regulators.
Induction of BIM, BMF, and HRK following erlotinib treatment of HCC827 cells was assessed as in Figure 6A.
Figure 7 with 4 supplements
SOX2 expression in EGFR-mutant cells is regulated by FOXO6.
(A) Putative FOXO protein binding sites within the promoter of SOX2, identified using TRANSFAC and Zhang et al., 2011. (B) HCC827 cells were transfected with control siRNA or siRNA targeting the …
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Figure 7—source data 1
Raw immunofluorescence data for quantitation of SOX2 staining with different FOXO protein knockdown in Figure 7C.
- https://doi.org/10.7554/eLife.06132.037
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Figure 7—source data 2
Raw immunofluorescence data for quantitation of SOX2 and FOXO6 costaining in HCC827 cells in Figure 7—figure supplement 3.
- https://doi.org/10.7554/eLife.06132.038
Figure 7—figure supplement 1
Recurrent FOXO binding sites in erlotinib-induced genes.
(A) MSigDB/TRANSFAC output for the 12 genes most highly upregulated by erlotinib (FDR <0.05). (B) Although binding sites for FOXF2 are also enriched, knockdown of FOXF2 does not decrease …
Figure 7—figure supplement 2
FOXO6 uniquely regulates SOX2 expression.
(A) Same lysates as Figure 7E, showing immunoblot for FOXO proteins. (B) Similar data as in Figure 7B, but shown after immunoblot of protein lysates with the indicated antibodies. Immunoblot for …
Figure 7—figure supplement 3
Distribution of FOXO6 vs SOX2 nuclear staining.
(A) HCC827 cells were left untreated or were treated with 0.1 µM erlotinib for 24 hr. Cells were stained with goat anti-SOX2 and rabbit anti-FOXO6 primary antibodies, followed by anti-goat-Alexa …
Figure 7—figure supplement 4
Assessing the role of previously identified regulators on erlotinib-induced expression of SOX2.
(A) Pre-treatment of PC9 cells with FGF10 has minimal effects on SOX2 induction by erlotinib. (B) The addition of exogenous Wnt3A has no effect on induction of SOX2 by erlotinib. (C) The …
Figure 8
Knockdown of SOX2 sensitizes HCC2935 cells to erlotinib-induced apoptosis.
(A) HCC2935 cells were transfected with siCTRL or siSOX2 48 hr prior to erlotinib addition and assayed for cytoxicity 48 hr later with Syto-60. Data are displayed as the mean of 5 replicates −/+ …
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Figure 8—source data 1
Raw immunofluorescence data for quantitation of SOX2 staining in HCC2935 cells in Figure 8B.
- https://doi.org/10.7554/eLife.06132.044
Figure 9
Model of SOX2 feedback signaling pathway.
In untreated cells, mutant EGFR drives cell survival by activating downstream signaling pathways, including PI3K and MAPK, which inhibit apoptosis through transcriptional and post-transcriptional …
Additional files
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Supplementary file 1
siRNA, primer, and probe sequences/sources used in the study.
- https://doi.org/10.7554/eLife.06132.046
