Limits...
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.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.

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

Assessing the role of previously identified regulators onerlotinib-induced expression of SOX2.(A) Pre-treatment of PC9 cells with FGF10 has minimaleffects on SOX2 induction by erlotinib. (B) The addition ofexogenous Wnt3A has no effect on induction of SOX2 by erlotinib.(C) The beta-catenin pathway does not regulate SOX2expression. HCC827 cells were stably transduced with inducible lentiviralconstructs expressing a dominant negative TCF4 transgene (DN TCF4) or theconstitutively activated S33Y variant of Beta-Catenin (S33Y B-Cat), andwith a lentiviral TOP FLASH reporter. Left, the expected activity of eachtransgene was confirmed by TOP FLASH luciferase assay in the absence orpresence of the GSK3 inhibitor/Beta-Catenin activator BIO (and−/+ erlotinib). Representative wells after luciferaseimaging are shown above the graph. Right, the effect of each transgene onthe levels of SOX2 induction by erlotinib compared to control (GUS) cellsis minimal. (D) Knockdown of TTF1 (NKX2.1) with siRNA hasminimal effects on the degree of SOX2 induction by erlotinib.DOI:http://dx.doi.org/10.7554/eLife.06132.042
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4384750&req=5

fig7s4: Assessing the role of previously identified regulators onerlotinib-induced expression of SOX2.(A) Pre-treatment of PC9 cells with FGF10 has minimaleffects on SOX2 induction by erlotinib. (B) The addition ofexogenous Wnt3A has no effect on induction of SOX2 by erlotinib.(C) The beta-catenin pathway does not regulate SOX2expression. HCC827 cells were stably transduced with inducible lentiviralconstructs expressing a dominant negative TCF4 transgene (DN TCF4) or theconstitutively activated S33Y variant of Beta-Catenin (S33Y B-Cat), andwith a lentiviral TOP FLASH reporter. Left, the expected activity of eachtransgene was confirmed by TOP FLASH luciferase assay in the absence orpresence of the GSK3 inhibitor/Beta-Catenin activator BIO (and−/+ erlotinib). Representative wells after luciferaseimaging are shown above the graph. Right, the effect of each transgene onthe levels of SOX2 induction by erlotinib compared to control (GUS) cellsis minimal. (D) Knockdown of TTF1 (NKX2.1) with siRNA hasminimal effects on the degree of SOX2 induction by erlotinib.DOI:http://dx.doi.org/10.7554/eLife.06132.042

Mentions: The regulation of SOX2 expression by FOXO6, whose activation is normally repressed byEGFR signaling, is consistent with the critical role played by FOXO proteins asintegrators of cellular signaling pathways. The best studied isoform, FOXO1, ishighly expressed in ES cells and has been implicated in the maintenance ofpluripotency through activation of SOX2 transcription (Zhang et al., 2011). All FOXO proteins bind to similar DNAsequences, with isoform-specific activity presumably conferred by cellular andpromoter context (Furuyama et al., 2000).Indeed, all four FOXO isoforms are expressed in EGFR-mutant lung cancer cells,transcriptionally induced following EGFR inhibition and phosphorylated at homologousSerine residues, yet only FOXO6 regulates SOX2 in these cells. Given variability inknockdown efficacy (Figure 7B, column 6), wecannot exclude some contribution from the other FOXO family members on SOX2expression, but FOXO6 has the dominant effect in the cells tested. Activation ofFOXO6 may occur through both AKT dependent and independent pathways (Jacobs et al., 2003; van der Heide et al., 2005), and indeed we observed thattreatment with PI3K inhibitors alone is insufficient for induction of SOX2 (Figure 1B). Other pathways that have beenimplicated in SOX2 regulation in the developing lung, including FGF10,WNT/beta-Catenin signaling, and TTF1 (Que et al.,2007; Gontan et al., 2008; Hashimoto et al., 2012), had relatively modesteffects on its induction following erlotinib treatment in EGFR-mutant cancer cells(Figure 7—figure supplement 4),pointing to FOXO6 as the dominant pathway in this model of oncogene-dependentsignaling.


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)

Assessing the role of previously identified regulators onerlotinib-induced expression of SOX2.(A) Pre-treatment of PC9 cells with FGF10 has minimaleffects on SOX2 induction by erlotinib. (B) The addition ofexogenous Wnt3A has no effect on induction of SOX2 by erlotinib.(C) The beta-catenin pathway does not regulate SOX2expression. HCC827 cells were stably transduced with inducible lentiviralconstructs expressing a dominant negative TCF4 transgene (DN TCF4) or theconstitutively activated S33Y variant of Beta-Catenin (S33Y B-Cat), andwith a lentiviral TOP FLASH reporter. Left, the expected activity of eachtransgene was confirmed by TOP FLASH luciferase assay in the absence orpresence of the GSK3 inhibitor/Beta-Catenin activator BIO (and−/+ erlotinib). Representative wells after luciferaseimaging are shown above the graph. Right, the effect of each transgene onthe levels of SOX2 induction by erlotinib compared to control (GUS) cellsis minimal. (D) Knockdown of TTF1 (NKX2.1) with siRNA hasminimal effects on the degree of SOX2 induction by erlotinib.DOI:http://dx.doi.org/10.7554/eLife.06132.042
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4384750&req=5

fig7s4: Assessing the role of previously identified regulators onerlotinib-induced expression of SOX2.(A) Pre-treatment of PC9 cells with FGF10 has minimaleffects on SOX2 induction by erlotinib. (B) The addition ofexogenous Wnt3A has no effect on induction of SOX2 by erlotinib.(C) The beta-catenin pathway does not regulate SOX2expression. HCC827 cells were stably transduced with inducible lentiviralconstructs expressing a dominant negative TCF4 transgene (DN TCF4) or theconstitutively activated S33Y variant of Beta-Catenin (S33Y B-Cat), andwith a lentiviral TOP FLASH reporter. Left, the expected activity of eachtransgene was confirmed by TOP FLASH luciferase assay in the absence orpresence of the GSK3 inhibitor/Beta-Catenin activator BIO (and−/+ erlotinib). Representative wells after luciferaseimaging are shown above the graph. Right, the effect of each transgene onthe levels of SOX2 induction by erlotinib compared to control (GUS) cellsis minimal. (D) Knockdown of TTF1 (NKX2.1) with siRNA hasminimal effects on the degree of SOX2 induction by erlotinib.DOI:http://dx.doi.org/10.7554/eLife.06132.042
Mentions: The regulation of SOX2 expression by FOXO6, whose activation is normally repressed byEGFR signaling, is consistent with the critical role played by FOXO proteins asintegrators of cellular signaling pathways. The best studied isoform, FOXO1, ishighly expressed in ES cells and has been implicated in the maintenance ofpluripotency through activation of SOX2 transcription (Zhang et al., 2011). All FOXO proteins bind to similar DNAsequences, with isoform-specific activity presumably conferred by cellular andpromoter context (Furuyama et al., 2000).Indeed, all four FOXO isoforms are expressed in EGFR-mutant lung cancer cells,transcriptionally induced following EGFR inhibition and phosphorylated at homologousSerine residues, yet only FOXO6 regulates SOX2 in these cells. Given variability inknockdown efficacy (Figure 7B, column 6), wecannot exclude some contribution from the other FOXO family members on SOX2expression, but FOXO6 has the dominant effect in the cells tested. Activation ofFOXO6 may occur through both AKT dependent and independent pathways (Jacobs et al., 2003; van der Heide et al., 2005), and indeed we observed thattreatment with PI3K inhibitors alone is insufficient for induction of SOX2 (Figure 1B). Other pathways that have beenimplicated in SOX2 regulation in the developing lung, including FGF10,WNT/beta-Catenin signaling, and TTF1 (Que et al.,2007; Gontan et al., 2008; Hashimoto et al., 2012), had relatively modesteffects on its induction following erlotinib treatment in EGFR-mutant cancer cells(Figure 7—figure supplement 4),pointing to FOXO6 as the dominant pathway in this model of oncogene-dependentsignaling.

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.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.

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