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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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SOX2 is expressed most highly in nonproliferative cells.Left panels, HCC827 cells were treated for 24 hr with 0.1 µMerlotinib, followed by immunofluorescence microscopy with antibodies toSOX2, Ki-67, and DAPI. The left three pairs of panels show HCC827 cellsat 20× magnification, the right pair of panels shows a doublet ofcells at 60× magnification. Right panel, the distribution ofnuclear Ki-67 mean fluorescence in the SOX2− and SOX2+cells in DMSO-treated (green) and erlotinib treated (red) cells isdisplayed. p = 0.015 and p < 0.0001 for the comparison ofeach SOX2+ population to the corresponding SOX2− population(Student's t-test, unequal variances, N =31–1834, means of Ki67 fluorescence for SOX2 −/+cells are 0.13/0.1 for DMSO-treated and 0.03/0.007 forerlotinib-treated). Source data are included as Figure2—source data 1.DOI:http://dx.doi.org/10.7554/eLife.06132.017
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fig2s3: SOX2 is expressed most highly in nonproliferative cells.Left panels, HCC827 cells were treated for 24 hr with 0.1 µMerlotinib, followed by immunofluorescence microscopy with antibodies toSOX2, Ki-67, and DAPI. The left three pairs of panels show HCC827 cellsat 20× magnification, the right pair of panels shows a doublet ofcells at 60× magnification. Right panel, the distribution ofnuclear Ki-67 mean fluorescence in the SOX2− and SOX2+cells in DMSO-treated (green) and erlotinib treated (red) cells isdisplayed. p = 0.015 and p < 0.0001 for the comparison ofeach SOX2+ population to the corresponding SOX2− population(Student's t-test, unequal variances, N =31–1834, means of Ki67 fluorescence for SOX2 −/+cells are 0.13/0.1 for DMSO-treated and 0.03/0.007 forerlotinib-treated). Source data are included as Figure2—source data 1.DOI:http://dx.doi.org/10.7554/eLife.06132.017

Mentions: The level of SOX2 induction in cultured cells exposed to erlotinib showedconsiderable heterogeneity, with a subset of cells (∼20%, with someexperimental variability) expressing high levels (Figure 2A). The SOX2+ fraction was not increased by higher drugdosage, beyond that required for full inhibition of EGFR (Figure 2—figure supplement 1). Given the link betweenSOX2 expression and cellular reprogramming, we first asked whether cells with thehigh SOX2 expression represent a subset with stem cell markers. However, SOX2expression did not correlate with expression of the putative stem cell markers CD133,CD44, CD24, OCT-4, or KLF-4 (Figure 2—figuresupplement 2) nor did microarray-based expression profiling of highSOX2-sorted cells identify a stem-like signature (data not shown). Nonetheless,SOX2-expressing cells had a very low proliferative index, as measured by Ki67staining (0.5% of Ki67+/SOX2+ vs 51% Ki67+/SOX2− HCC827cells at baseline [p = 0.015]; and 0.15% Ki67+/SOX2+ vs 6.4%Ki67+/SOX2− cells following erlotinib [p < 0.0001]) (Figure 2C, Figure 2—figure supplement 3).10.7554/eLife.06132.008Figure 2.Induction of SOX2 in erlotinib-treated cells.


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)

SOX2 is expressed most highly in nonproliferative cells.Left panels, HCC827 cells were treated for 24 hr with 0.1 µMerlotinib, followed by immunofluorescence microscopy with antibodies toSOX2, Ki-67, and DAPI. The left three pairs of panels show HCC827 cellsat 20× magnification, the right pair of panels shows a doublet ofcells at 60× magnification. Right panel, the distribution ofnuclear Ki-67 mean fluorescence in the SOX2− and SOX2+cells in DMSO-treated (green) and erlotinib treated (red) cells isdisplayed. p = 0.015 and p < 0.0001 for the comparison ofeach SOX2+ population to the corresponding SOX2− population(Student's t-test, unequal variances, N =31–1834, means of Ki67 fluorescence for SOX2 −/+cells are 0.13/0.1 for DMSO-treated and 0.03/0.007 forerlotinib-treated). Source data are included as Figure2—source data 1.DOI:http://dx.doi.org/10.7554/eLife.06132.017
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Related In: Results  -  Collection

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fig2s3: SOX2 is expressed most highly in nonproliferative cells.Left panels, HCC827 cells were treated for 24 hr with 0.1 µMerlotinib, followed by immunofluorescence microscopy with antibodies toSOX2, Ki-67, and DAPI. The left three pairs of panels show HCC827 cellsat 20× magnification, the right pair of panels shows a doublet ofcells at 60× magnification. Right panel, the distribution ofnuclear Ki-67 mean fluorescence in the SOX2− and SOX2+cells in DMSO-treated (green) and erlotinib treated (red) cells isdisplayed. p = 0.015 and p < 0.0001 for the comparison ofeach SOX2+ population to the corresponding SOX2− population(Student's t-test, unequal variances, N =31–1834, means of Ki67 fluorescence for SOX2 −/+cells are 0.13/0.1 for DMSO-treated and 0.03/0.007 forerlotinib-treated). Source data are included as Figure2—source data 1.DOI:http://dx.doi.org/10.7554/eLife.06132.017
Mentions: The level of SOX2 induction in cultured cells exposed to erlotinib showedconsiderable heterogeneity, with a subset of cells (∼20%, with someexperimental variability) expressing high levels (Figure 2A). The SOX2+ fraction was not increased by higher drugdosage, beyond that required for full inhibition of EGFR (Figure 2—figure supplement 1). Given the link betweenSOX2 expression and cellular reprogramming, we first asked whether cells with thehigh SOX2 expression represent a subset with stem cell markers. However, SOX2expression did not correlate with expression of the putative stem cell markers CD133,CD44, CD24, OCT-4, or KLF-4 (Figure 2—figuresupplement 2) nor did microarray-based expression profiling of highSOX2-sorted cells identify a stem-like signature (data not shown). Nonetheless,SOX2-expressing cells had a very low proliferative index, as measured by Ki67staining (0.5% of Ki67+/SOX2+ vs 51% Ki67+/SOX2− HCC827cells at baseline [p = 0.015]; and 0.15% Ki67+/SOX2+ vs 6.4%Ki67+/SOX2− cells following erlotinib [p < 0.0001]) (Figure 2C, Figure 2—figure supplement 3).10.7554/eLife.06132.008Figure 2.Induction of SOX2 in erlotinib-treated cells.

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