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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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Stochastic induction of SOX2 by erlotinib in PC9 cells.(A) Retreatment of PC9 cells after a period of recovery doesnot increase the fraction of cells capable of inducing SOX2. Left panels,images of cells stained for SOX2 (green) and DAPI (blue). Right panels, p< 0.0001 for the comparison of erlotinib-treated cells to DMSO(Student's t-test, unequal variances, N =3834–7951 cells, means of SOX2 fluorescence are 0.002/0.04 foruntreated/treated-parental and 0.001/0.03 foruntreated/treated-pretreated, % SOX2+ is shown). Note: theapparent decrease in SOX2+ fraction with retreatment is likely theresult of partial selection for erlotinib resistance with serialretreatment, as shown in (B) below. Source data are includedas Figure2—source data 5. (B) Induction of SOX2 incells with acquired resistance to erlotinib. PC9 cells were maderesistant to erlotinib by continuous culture in the presence of 0.1µM drug for 30 days. Upper panel, qPCR for SOX2 expression wasperformed on lysates prepared from cells at the indicated time points. Onday 30, resistant cells were replated in the absence of drug and thenretreated with increasing concentrations of erlotinib on day 31. Data areshown as mean Ct of 4 replicates (normalized to ACTB and untreated cells)−/+ SEM. The first six data points are the same as in Figure 1D. Middle panels, parentaland resistant PC9 cells were treated with increasing concentrations oferlotinib for 6 hr, followed by quantitative immunofluorescence analysisfor pEGFR. N = 2073–6297 cells. Source data are included asFigure2—source data 6. Lower panels, representative imagesshow strong decrease in pEGFR (red) with 1 µM erlotinib inparental (left) but not resistant (right) cells.DOI:http://dx.doi.org/10.7554/eLife.06132.018
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fig2s4: Stochastic induction of SOX2 by erlotinib in PC9 cells.(A) Retreatment of PC9 cells after a period of recovery doesnot increase the fraction of cells capable of inducing SOX2. Left panels,images of cells stained for SOX2 (green) and DAPI (blue). Right panels, p< 0.0001 for the comparison of erlotinib-treated cells to DMSO(Student's t-test, unequal variances, N =3834–7951 cells, means of SOX2 fluorescence are 0.002/0.04 foruntreated/treated-parental and 0.001/0.03 foruntreated/treated-pretreated, % SOX2+ is shown). Note: theapparent decrease in SOX2+ fraction with retreatment is likely theresult of partial selection for erlotinib resistance with serialretreatment, as shown in (B) below. Source data are includedas Figure2—source data 5. (B) Induction of SOX2 incells with acquired resistance to erlotinib. PC9 cells were maderesistant to erlotinib by continuous culture in the presence of 0.1µM drug for 30 days. Upper panel, qPCR for SOX2 expression wasperformed on lysates prepared from cells at the indicated time points. Onday 30, resistant cells were replated in the absence of drug and thenretreated with increasing concentrations of erlotinib on day 31. Data areshown as mean Ct of 4 replicates (normalized to ACTB and untreated cells)−/+ SEM. The first six data points are the same as in Figure 1D. Middle panels, parentaland resistant PC9 cells were treated with increasing concentrations oferlotinib for 6 hr, followed by quantitative immunofluorescence analysisfor pEGFR. N = 2073–6297 cells. Source data are included asFigure2—source data 6. Lower panels, representative imagesshow strong decrease in pEGFR (red) with 1 µM erlotinib inparental (left) but not resistant (right) cells.DOI:http://dx.doi.org/10.7554/eLife.06132.018

Mentions: 10.7554/eLife.06132.013Figure 2—source data 5.Raw immunofluorescence data for quantitation of SOX2staining in PC9 cells recovered after retreatment (x2) witherlotinib, compared to previously untreated cells, in Figure 2—figure supplement4A.


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)

Stochastic induction of SOX2 by erlotinib in PC9 cells.(A) Retreatment of PC9 cells after a period of recovery doesnot increase the fraction of cells capable of inducing SOX2. Left panels,images of cells stained for SOX2 (green) and DAPI (blue). Right panels, p< 0.0001 for the comparison of erlotinib-treated cells to DMSO(Student's t-test, unequal variances, N =3834–7951 cells, means of SOX2 fluorescence are 0.002/0.04 foruntreated/treated-parental and 0.001/0.03 foruntreated/treated-pretreated, % SOX2+ is shown). Note: theapparent decrease in SOX2+ fraction with retreatment is likely theresult of partial selection for erlotinib resistance with serialretreatment, as shown in (B) below. Source data are includedas Figure2—source data 5. (B) Induction of SOX2 incells with acquired resistance to erlotinib. PC9 cells were maderesistant to erlotinib by continuous culture in the presence of 0.1µM drug for 30 days. Upper panel, qPCR for SOX2 expression wasperformed on lysates prepared from cells at the indicated time points. Onday 30, resistant cells were replated in the absence of drug and thenretreated with increasing concentrations of erlotinib on day 31. Data areshown as mean Ct of 4 replicates (normalized to ACTB and untreated cells)−/+ SEM. The first six data points are the same as in Figure 1D. Middle panels, parentaland resistant PC9 cells were treated with increasing concentrations oferlotinib for 6 hr, followed by quantitative immunofluorescence analysisfor pEGFR. N = 2073–6297 cells. Source data are included asFigure2—source data 6. Lower panels, representative imagesshow strong decrease in pEGFR (red) with 1 µM erlotinib inparental (left) but not resistant (right) cells.DOI:http://dx.doi.org/10.7554/eLife.06132.018
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fig2s4: Stochastic induction of SOX2 by erlotinib in PC9 cells.(A) Retreatment of PC9 cells after a period of recovery doesnot increase the fraction of cells capable of inducing SOX2. Left panels,images of cells stained for SOX2 (green) and DAPI (blue). Right panels, p< 0.0001 for the comparison of erlotinib-treated cells to DMSO(Student's t-test, unequal variances, N =3834–7951 cells, means of SOX2 fluorescence are 0.002/0.04 foruntreated/treated-parental and 0.001/0.03 foruntreated/treated-pretreated, % SOX2+ is shown). Note: theapparent decrease in SOX2+ fraction with retreatment is likely theresult of partial selection for erlotinib resistance with serialretreatment, as shown in (B) below. Source data are includedas Figure2—source data 5. (B) Induction of SOX2 incells with acquired resistance to erlotinib. PC9 cells were maderesistant to erlotinib by continuous culture in the presence of 0.1µM drug for 30 days. Upper panel, qPCR for SOX2 expression wasperformed on lysates prepared from cells at the indicated time points. Onday 30, resistant cells were replated in the absence of drug and thenretreated with increasing concentrations of erlotinib on day 31. Data areshown as mean Ct of 4 replicates (normalized to ACTB and untreated cells)−/+ SEM. The first six data points are the same as in Figure 1D. Middle panels, parentaland resistant PC9 cells were treated with increasing concentrations oferlotinib for 6 hr, followed by quantitative immunofluorescence analysisfor pEGFR. N = 2073–6297 cells. Source data are included asFigure2—source data 6. Lower panels, representative imagesshow strong decrease in pEGFR (red) with 1 µM erlotinib inparental (left) but not resistant (right) cells.DOI:http://dx.doi.org/10.7554/eLife.06132.018
Mentions: 10.7554/eLife.06132.013Figure 2—source data 5.Raw immunofluorescence data for quantitation of SOX2staining in PC9 cells recovered after retreatment (x2) witherlotinib, compared to previously untreated cells, in Figure 2—figure supplement4A.

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