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Inhibition of mutant EGFR in lung cancer cells triggers SOX2-FOXO6-dependent survival pathways.

Rothenberg SM, Concannon K, Cullen S, Boulay G, Turke AB, Faber AC, Lockerman EL, Rivera MN, Engelman JA, Maheswaran S, Haber DA - Elife (2015)

Bottom Line: Treatment of EGFR-mutant lung cancer with erlotinib results in dramatic tumor regression but it is invariably followed by drug resistance.In characterizing early transcriptional changes following drug treatment of mutant EGFR-addicted cells, we identified the stem cell transcriptional regulator SOX2 as being rapidly and specifically induced, both in vitro and in vivo.Together, these observations point to a physiological feedback mechanism that attenuates oncogene addiction-mediated cell death associated with the withdrawal of growth factor signaling and may therefore contribute to the development of resistance.

View Article: PubMed Central - PubMed

Affiliation: Cancer Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, United States.

ABSTRACT
Treatment of EGFR-mutant lung cancer with erlotinib results in dramatic tumor regression but it is invariably followed by drug resistance. In characterizing early transcriptional changes following drug treatment of mutant EGFR-addicted cells, we identified the stem cell transcriptional regulator SOX2 as being rapidly and specifically induced, both in vitro and in vivo. Suppression of SOX2 sensitizes cells to erlotinib-mediated apoptosis, ultimately decreasing the emergence of acquired resistance, whereas its ectopic expression reduces drug-induced cell death. We show that erlotinib relieves EGFR-dependent suppression of FOXO6, leading to its induction of SOX2, which in turn represses the pro-apoptotic BH3-only genes BIM and BMF. Together, these observations point to a physiological feedback mechanism that attenuates oncogene addiction-mediated cell death associated with the withdrawal of growth factor signaling and may therefore contribute to the development of resistance.

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Knockdown of SOX2 sensitizes HCC2935 cells to erlotinib-inducedapoptosis.(A) HCC2935 cells were transfected with siCTRL or siSOX2 48 hrprior to erlotinib addition and assayed for cytoxicity 48 hr later withSyto-60. Data are displayed as the mean of 5 replicates −/+SEM. The IC50 is 0.8 µM for siCTRL and 0.1 µM for siSOX2 cells(calculated by four parameter logistic sigmoidal fit). p = 0.003 forthe comparison of mean IC50 for siCTRL vs siSOX2 (Student'st-test, unequal variances). (B) Upperpanels, images of untreated and erlotinib-treated HCC2935 cells,demonstrating SOX2 expression in the majority of cells. Lower panels, thedistribution of SOX2 in HCC2935 was determined by quantitativeimmunofluorescence microscopy. p < 0.0001 for the comparison of meanSOX2 fluorescence in untreated vs treated cells (Student'st-test, unequal variances, N = 3342/1181, meansfor SOX2 fluorescence are 0.17/0.24 for untreated/treated cells, %SOX2+ is shown). Source data is included as Figure 8—sourcedata 1. (C) HCC2935 cells were transfected withcontrol siRNA or siRNA targeting SOX2. 48 hr after transfection, DMSO or 1.0µM erlotinib was added. The effect of SOX2 knockdown was assessed byimmunoblot analysis of protein lysates with the indicated antibodies afterovernight treatment.DOI:http://dx.doi.org/10.7554/eLife.06132.04310.7554/eLife.06132.044Figure 8—source data 1.Raw immunofluorescence data for quantitation of SOX2 stainingin HCC2935 cells in Figure8B.DOI:http://dx.doi.org/10.7554/eLife.06132.044
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fig8: Knockdown of SOX2 sensitizes HCC2935 cells to erlotinib-inducedapoptosis.(A) HCC2935 cells were transfected with siCTRL or siSOX2 48 hrprior to erlotinib addition and assayed for cytoxicity 48 hr later withSyto-60. Data are displayed as the mean of 5 replicates −/+SEM. The IC50 is 0.8 µM for siCTRL and 0.1 µM for siSOX2 cells(calculated by four parameter logistic sigmoidal fit). p = 0.003 forthe comparison of mean IC50 for siCTRL vs siSOX2 (Student'st-test, unequal variances). (B) Upperpanels, images of untreated and erlotinib-treated HCC2935 cells,demonstrating SOX2 expression in the majority of cells. Lower panels, thedistribution of SOX2 in HCC2935 was determined by quantitativeimmunofluorescence microscopy. p < 0.0001 for the comparison of meanSOX2 fluorescence in untreated vs treated cells (Student'st-test, unequal variances, N = 3342/1181, meansfor SOX2 fluorescence are 0.17/0.24 for untreated/treated cells, %SOX2+ is shown). Source data is included as Figure 8—sourcedata 1. (C) HCC2935 cells were transfected withcontrol siRNA or siRNA targeting SOX2. 48 hr after transfection, DMSO or 1.0µM erlotinib was added. The effect of SOX2 knockdown was assessed byimmunoblot analysis of protein lysates with the indicated antibodies afterovernight treatment.DOI:http://dx.doi.org/10.7554/eLife.06132.04310.7554/eLife.06132.044Figure 8—source data 1.Raw immunofluorescence data for quantitation of SOX2 stainingin HCC2935 cells in Figure8B.DOI:http://dx.doi.org/10.7554/eLife.06132.044

Mentions: Virtually all EGFR-mutant lung cancer cell lines established from patients who havenot been treated with erlotinib are highly sensitive to this drug, although a fewcells lines appear to be intrinsically resistant. The HCC2935 human lung cancer cellline is remarkable for harboring a characteristic oncogene-addicting EGFR mutation(exon 19 deletion) yet having unexplained resistance to erlotinib (Figure 8A), including absence of the common T790Mgatekeeper mutation within EGFR and no amplification of the MET bypass signalingpathway (Zheng et al., 2011). Notably, SOX2expression at baseline is detectable in 90% of HCC2935 cells (compared to 3% ofHCC827 and <1% PC9 cells), and its expression per cell is further increasedupon EGFR inhibition (Figure 8B). To testwhether increased SOX2 contributes to decreased erlotinib sensitivity in HCC2935cells, we knocked down SOX2 using siRNA. A striking increase in erlotinibcytotoxicity was evident following SOX2 suppression (IC50 0.8 µM for siCTRLcells with no further toxicity up to 10 µM, IC50 0.1 µM for siSOX2cells), associated with higher levels of BIM and increased PARP and caspase-3cleavage (Figure 8A,C). Thus, increasedbaseline SOX2 contributes to erlotinib resistance in these EGFR-mutant cells.10.7554/eLife.06132.043Figure 8.Knockdown of SOX2 sensitizes HCC2935 cells to erlotinib-inducedapoptosis.


Inhibition of mutant EGFR in lung cancer cells triggers SOX2-FOXO6-dependent survival pathways.

Rothenberg SM, Concannon K, Cullen S, Boulay G, Turke AB, Faber AC, Lockerman EL, Rivera MN, Engelman JA, Maheswaran S, Haber DA - Elife (2015)

Knockdown of SOX2 sensitizes HCC2935 cells to erlotinib-inducedapoptosis.(A) HCC2935 cells were transfected with siCTRL or siSOX2 48 hrprior to erlotinib addition and assayed for cytoxicity 48 hr later withSyto-60. Data are displayed as the mean of 5 replicates −/+SEM. The IC50 is 0.8 µM for siCTRL and 0.1 µM for siSOX2 cells(calculated by four parameter logistic sigmoidal fit). p = 0.003 forthe comparison of mean IC50 for siCTRL vs siSOX2 (Student'st-test, unequal variances). (B) Upperpanels, images of untreated and erlotinib-treated HCC2935 cells,demonstrating SOX2 expression in the majority of cells. Lower panels, thedistribution of SOX2 in HCC2935 was determined by quantitativeimmunofluorescence microscopy. p < 0.0001 for the comparison of meanSOX2 fluorescence in untreated vs treated cells (Student'st-test, unequal variances, N = 3342/1181, meansfor SOX2 fluorescence are 0.17/0.24 for untreated/treated cells, %SOX2+ is shown). Source data is included as Figure 8—sourcedata 1. (C) HCC2935 cells were transfected withcontrol siRNA or siRNA targeting SOX2. 48 hr after transfection, DMSO or 1.0µM erlotinib was added. The effect of SOX2 knockdown was assessed byimmunoblot analysis of protein lysates with the indicated antibodies afterovernight treatment.DOI:http://dx.doi.org/10.7554/eLife.06132.04310.7554/eLife.06132.044Figure 8—source data 1.Raw immunofluorescence data for quantitation of SOX2 stainingin HCC2935 cells in Figure8B.DOI:http://dx.doi.org/10.7554/eLife.06132.044
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fig8: Knockdown of SOX2 sensitizes HCC2935 cells to erlotinib-inducedapoptosis.(A) HCC2935 cells were transfected with siCTRL or siSOX2 48 hrprior to erlotinib addition and assayed for cytoxicity 48 hr later withSyto-60. Data are displayed as the mean of 5 replicates −/+SEM. The IC50 is 0.8 µM for siCTRL and 0.1 µM for siSOX2 cells(calculated by four parameter logistic sigmoidal fit). p = 0.003 forthe comparison of mean IC50 for siCTRL vs siSOX2 (Student'st-test, unequal variances). (B) Upperpanels, images of untreated and erlotinib-treated HCC2935 cells,demonstrating SOX2 expression in the majority of cells. Lower panels, thedistribution of SOX2 in HCC2935 was determined by quantitativeimmunofluorescence microscopy. p < 0.0001 for the comparison of meanSOX2 fluorescence in untreated vs treated cells (Student'st-test, unequal variances, N = 3342/1181, meansfor SOX2 fluorescence are 0.17/0.24 for untreated/treated cells, %SOX2+ is shown). Source data is included as Figure 8—sourcedata 1. (C) HCC2935 cells were transfected withcontrol siRNA or siRNA targeting SOX2. 48 hr after transfection, DMSO or 1.0µM erlotinib was added. The effect of SOX2 knockdown was assessed byimmunoblot analysis of protein lysates with the indicated antibodies afterovernight treatment.DOI:http://dx.doi.org/10.7554/eLife.06132.04310.7554/eLife.06132.044Figure 8—source data 1.Raw immunofluorescence data for quantitation of SOX2 stainingin HCC2935 cells in Figure8B.DOI:http://dx.doi.org/10.7554/eLife.06132.044
Mentions: Virtually all EGFR-mutant lung cancer cell lines established from patients who havenot been treated with erlotinib are highly sensitive to this drug, although a fewcells lines appear to be intrinsically resistant. The HCC2935 human lung cancer cellline is remarkable for harboring a characteristic oncogene-addicting EGFR mutation(exon 19 deletion) yet having unexplained resistance to erlotinib (Figure 8A), including absence of the common T790Mgatekeeper mutation within EGFR and no amplification of the MET bypass signalingpathway (Zheng et al., 2011). Notably, SOX2expression at baseline is detectable in 90% of HCC2935 cells (compared to 3% ofHCC827 and <1% PC9 cells), and its expression per cell is further increasedupon EGFR inhibition (Figure 8B). To testwhether increased SOX2 contributes to decreased erlotinib sensitivity in HCC2935cells, we knocked down SOX2 using siRNA. A striking increase in erlotinibcytotoxicity was evident following SOX2 suppression (IC50 0.8 µM for siCTRLcells with no further toxicity up to 10 µM, IC50 0.1 µM for siSOX2cells), associated with higher levels of BIM and increased PARP and caspase-3cleavage (Figure 8A,C). Thus, increasedbaseline SOX2 contributes to erlotinib resistance in these EGFR-mutant cells.10.7554/eLife.06132.043Figure 8.Knockdown of SOX2 sensitizes HCC2935 cells to erlotinib-inducedapoptosis.

Bottom Line: Treatment of EGFR-mutant lung cancer with erlotinib results in dramatic tumor regression but it is invariably followed by drug resistance.In characterizing early transcriptional changes following drug treatment of mutant EGFR-addicted cells, we identified the stem cell transcriptional regulator SOX2 as being rapidly and specifically induced, both in vitro and in vivo.Together, these observations point to a physiological feedback mechanism that attenuates oncogene addiction-mediated cell death associated with the withdrawal of growth factor signaling and may therefore contribute to the development of resistance.

View Article: PubMed Central - PubMed

Affiliation: Cancer Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, United States.

ABSTRACT
Treatment of EGFR-mutant lung cancer with erlotinib results in dramatic tumor regression but it is invariably followed by drug resistance. In characterizing early transcriptional changes following drug treatment of mutant EGFR-addicted cells, we identified the stem cell transcriptional regulator SOX2 as being rapidly and specifically induced, both in vitro and in vivo. Suppression of SOX2 sensitizes cells to erlotinib-mediated apoptosis, ultimately decreasing the emergence of acquired resistance, whereas its ectopic expression reduces drug-induced cell death. We show that erlotinib relieves EGFR-dependent suppression of FOXO6, leading to its induction of SOX2, which in turn represses the pro-apoptotic BH3-only genes BIM and BMF. Together, these observations point to a physiological feedback mechanism that attenuates oncogene addiction-mediated cell death associated with the withdrawal of growth factor signaling and may therefore contribute to the development of resistance.

Show MeSH
Related in: MedlinePlus