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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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The effect of siRNA targeting SOX2 is specific.(A), PC9 cells were transfected with two different siRNAduplexes targeting SOX2 (or control siRNA), followed by addition of DMSOor 0.1 µM erlotinib for 24 hr and immunoblot of protein lysateswith the indicated antibodies. (B), the degree of knockdownof SOX2 was quantitatively assessed by qPCR. Data are shown as mean Ct(normalized to GAPDH and untreated siCTRL cells) of 3 replicates−/+ SEM.DOI:http://dx.doi.org/10.7554/eLife.06132.032
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fig5s3: The effect of siRNA targeting SOX2 is specific.(A), PC9 cells were transfected with two different siRNAduplexes targeting SOX2 (or control siRNA), followed by addition of DMSOor 0.1 µM erlotinib for 24 hr and immunoblot of protein lysateswith the indicated antibodies. (B), the degree of knockdownof SOX2 was quantitatively assessed by qPCR. Data are shown as mean Ct(normalized to GAPDH and untreated siCTRL cells) of 3 replicates−/+ SEM.DOI:http://dx.doi.org/10.7554/eLife.06132.032

Mentions: To determine the functional significance of endogenous SOX2 induction, we next usedsiRNAs to block its induction in erlotinib-treated cells. While both HCC827 and PC9cells are highly sensitive to EGFR inhibition at baseline, SOX2 knockdown furtherincreased erlotinib-induced apoptosis, as determined by PARP and caspase-3 cleavageassays and by cell enumeration (Figure 5B,Dand Figure 5—figure supplement 2).The apoptotic effect of the most potent siRNA was rescued by expression of an ectopicsiRNA-resistant SOX2 construct (Figure 5C) andindividual siRNAs-induced apoptosis in proportion to the degree of knockdown (Figure 5—figure supplement 3),confirming the specificity of the effect. As with expression of exogenous SOX2,endogenous SOX2 suppression itself did not have a consistent effect on EGFRsignaling, as measured by phosphorylation of EGFR, AKT, or ERK (Figure 5B).


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)

The effect of siRNA targeting SOX2 is specific.(A), PC9 cells were transfected with two different siRNAduplexes targeting SOX2 (or control siRNA), followed by addition of DMSOor 0.1 µM erlotinib for 24 hr and immunoblot of protein lysateswith the indicated antibodies. (B), the degree of knockdownof SOX2 was quantitatively assessed by qPCR. Data are shown as mean Ct(normalized to GAPDH and untreated siCTRL cells) of 3 replicates−/+ SEM.DOI:http://dx.doi.org/10.7554/eLife.06132.032
© Copyright Policy
Related In: Results  -  Collection

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

fig5s3: The effect of siRNA targeting SOX2 is specific.(A), PC9 cells were transfected with two different siRNAduplexes targeting SOX2 (or control siRNA), followed by addition of DMSOor 0.1 µM erlotinib for 24 hr and immunoblot of protein lysateswith the indicated antibodies. (B), the degree of knockdownof SOX2 was quantitatively assessed by qPCR. Data are shown as mean Ct(normalized to GAPDH and untreated siCTRL cells) of 3 replicates−/+ SEM.DOI:http://dx.doi.org/10.7554/eLife.06132.032
Mentions: To determine the functional significance of endogenous SOX2 induction, we next usedsiRNAs to block its induction in erlotinib-treated cells. While both HCC827 and PC9cells are highly sensitive to EGFR inhibition at baseline, SOX2 knockdown furtherincreased erlotinib-induced apoptosis, as determined by PARP and caspase-3 cleavageassays and by cell enumeration (Figure 5B,Dand Figure 5—figure supplement 2).The apoptotic effect of the most potent siRNA was rescued by expression of an ectopicsiRNA-resistant SOX2 construct (Figure 5C) andindividual siRNAs-induced apoptosis in proportion to the degree of knockdown (Figure 5—figure supplement 3),confirming the specificity of the effect. As with expression of exogenous SOX2,endogenous SOX2 suppression itself did not have a consistent effect on EGFRsignaling, as measured by phosphorylation of EGFR, AKT, or ERK (Figure 5B).

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