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.
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.
Entities:
Keywords:
EGFR mutations; SOX2; human; human biology; lung cancer; medicine; mouse; resistance; targeted cancer therapy
The invariable development of drug resistance presents a critical challenge to the
success of targeted cancer therapies (Jänne et
al., 2005; O'Hare et al.,
2006; Poulikakos and Rosen, 2011).
Several mechanisms leading to such acquired resistance have been identified in patients
with EGFR-mutant non-small cell lung cancer (NSCLC) treated with small
molecule EGFR inhibitors such as erlotinib. Following dramatic initial tumor shrinkage,
tumor regrowth is most frequently associated with the emergence of a secondary genetic
change, the T790M ‘gatekeeper’ mutation within the EGFR kinase domain,
which restores ATP binding in the presence of drug (Pao et al., 2004; Kobayashi et al.,
2005; Yun et al., 2007). In other
cases, amplification of related receptor tyrosine kinases (e.g., MET)
or mutational activation of downstream kinases (e.g., BRAF, PIK3CA) may
bypass the effect of EGFR inhibition (Engelman et al.,
2007; Sequist et al., 2011; Ohashi et al., 2012). Different drug resistance
mechanisms may coexist within different metastatic lesions of individual patients.
Recently, clinical trials involving rebiopsy of tumor lesions at the earliest sign of
drug resistance have also revealed phenotypic conversions that may contribute to drug
resistance, including activation of epithelial-to-mesenchymal transition (EMT) and the
remarkable trans-differentiation of lung cancers from adenocarcinoma to small cell
histologies (Thomson et al., 2005; Witta et al., 2006; Sequist et al., 2011). While some well-defined resistance
mechanisms, such as the T790M-EGFR gatekeeper mutation and MET
amplification, may be addressed using second line targeted drugs, the plasticity of
cancer cell adaptation to disrupted oncogenic signaling poses a major challenge to the
long-term success of these promising therapies.In vitro modeling of acquired resistance to EGFR inhibitors has raised the possibility
that a transient so-called ‘drug-tolerant’ state may precede the
development of mutationally defined, heritable drug resistance (Sharma et al., 2010). By analogy with bacterial models of
antibiotic resistance, such an intermediate state may be unstable, but enable treated
cells to survive in the presence of drug long enough to acquire mutations that
ultimately confer sustained drug resistance (Balaban et
al., 2004). In PC9 mutant, EGFR-addicted lung cancer cells, EGFR inhibition
triggers apoptosis in the vast majority of cells in vitro, uncovering approximately 0.3%
that are drug tolerant, quiescent, and expressing the stem cell marker CD133 and the
histone H3K4 demethylase KDM5A (Sharma et al.,
2010). These drug tolerant cells readily revert to a drug-sensitive state
following removal of the EGFR inhibitor, and their emergence in vitro is suppressed by
treatment with an EGFR inhibitor combined with inhibitors of either histone deacetylases
(HDACs) or the IGF-1 receptor. While this intermediate resistance mechanism remains to
be validated in the clinical setting, it raises the possibility of suppressing
pre-conditions that favor the acquisition of drug resistance, in order to circumvent the
challenge of treating multiple established drug-resistant pathways.Beyond the selection of cancer cell populations with transient drug-resistant
phenotypes, recent studies of targeted cancer drugs have defined more rapid signaling
feedback loops that modulate the cellular response to growth factor inhibition. For
instance, acute loss of ERK signaling triggered by RAF or MEK inhibitors in
BRAF mutant melanoma cells relieves ERK-dependent inhibition of RAS
and CRAF, whose activation through ErbB receptor signaling may lead to paradoxical
proliferative signals (Pratilas et al., 2009;
Paraiso et al., 2010; Lito et al., 2012). Similarly, in BRAF mutant
colorectal cancers, feedback activation of EGFR-dependent signaling attenuates the
consequences of mutant BRAF inhibition, suppressing the apoptotic effect (Corcoran et al., 2012; Prahallad et al., 2012). In addition to signaling feedback loops,
transcriptional outputs that generally limit cell proliferation have also been
implicated following disruption of EGFR activity, including the expression of
transcriptional repressors, regulators of mRNA stability and microRNAs (Kobayashi et al., 2006; Amit et al., 2007; Avraham et al.,
2010).Here, we screened for early, unique transcriptional changes following erlotinib
treatment in mutant EGFR-addicted cells, identifying highly specific induction of SOX2,
a master transcriptional regulator required for embryonic stem cell maintenance. SOX2
represses the expression of pro-apoptotic molecules that mediate death following
oncogene withdrawal in these cells. The induction of SOX2 results from the activation of
FOXO6, a forkhead family transcription factor, following EGFR inhibition. Knockdown or
ectopic expression of SOX2 modulates the degree of apoptosis observed following oncogene
withdrawal and promotes drug resistance, pointing to a novel homeostatic mechanism that
may contribute to cellular adaptation to the withdrawal of growth factor signaling,
which underlies most approaches to targeted cancer therapy.
Results
SOX2 is specifically induced in EGFR-mutated lung cancer cells
following treatment with the EGFR inhibitor erlotinib
To interrogate the transcriptional response to EGFR inhibition, we used HCC827 lung
cancer cells, harboring an amplified mutated EGFR allele (in-frame
deletion of 15 nucleotides in exon 19) and displaying exquisite sensitivity to the
EGFR inhibitor erlotinib. Cell cultures were treated in triplicate with 1 µM
erlotinib for 6 hr, followed by mRNA isolation and whole transcriptome analysis
(Affymetrix U133 Plus 2.0 expression arrays) (Rothenberg, 2015). A total of 35 genes showed
>fourfold change in expression (FDR <0.05), including 22 downregulated
and 13 upregulated transcripts (represented by 48 unique probe sets; Figure 1—figure supplement 1A). Among
induced transcripts, SOX2 was unique in the specificity and rapidity of its induction
following EGFR inhibition (Figure 1, Figure 1—figure supplement 1B). Thus,
SOX2 was strongly induced in three mutant EGFR-addicted lung cancer cell lines
(HCC827, PC9, H3255) following treatment with physiologically relevant concentrations
of erlotinib (0.1 µM), but not when these cells were treated with comparably
effective doses of cytotoxic chemotherapy (Figure
1A,B and Figure 1—figure
supplement 2A). SOX2 was also not induced in other oncogene-dependent
models, such as ALK-translocated lung cancer cells treated with
crizotinib, HER2-amplified breast cancer cells exposed to lapatinib
or BRAF-mutant melanoma cells treated with AZD6244 (Figure 1A and Figure 1—figure supplement 2B). Consistent with its dependence on
suppression of mutant EGFR signaling in the context of EGFR
‘addiction’, SOX2 was not induced following erlotinib treatment of
H1975 cells, which harbor both an EGFR activating mutation and the T790M gatekeeper
mutation that confers resistant to erlotinib; or in H1650 cells with mutated EGFR
that are relatively resistant to the effects of EGFR inhibition in part through
genetic loss of PTEN (Figure
1—figure supplement 2B) (Sos et
al., 2009). However, treatment of H1975 cells with the L858R/T790M
mutation-selective inhibitor WZ4002 resulted in SOX2 induction (Figure 1—figure supplement 2B, right) (Zhou et al., 2009). In cells that show
erlotinib-mediated induction of SOX2, siRNA-mediated knockdown of EGFR also led to
strong induction of SOX2 (in the absence of erlotinib), confirming the specificity of
the drug effect (Figure 1C). Simultaneous
treatment of cells with actinomycin D and erlotinib suppressed the induction of SOX2,
consistent with a primary effect of EGFR inhibition in increasing SOX2 transcript
levels (Figure 1—figure supplement 2C).
Figure 1—figure supplement 1.
Gene expression profiling after erlotinib treatment.
(A) Heat map showing the list of fourfold significantly (FDR
<0.05) upregulated (green) or downregulated (red) transcripts in
HCC827 cells with erlotinib treatment. (B) Time course of
changes in transcript levels for erlotinib-responsive genes.
DOI:
http://dx.doi.org/10.7554/eLife.06132.004
Figure 1.
SOX2 transcript is specifically induced by erlotinib in EGFR-mutant
and addicted lung cancer cell lines.
(A) Cell lines were treated with an inhibitor of the driving
oncogenic lesion for 24 hr (erlotinib for EGFR-mutant,
lapatinib for HER2-amplified and crizotinib for
ALK-translocated cells), followed by isolation of
total RNA and quantitative PCR for SOX2 transcript. (B) PC9
and HCC827 cells were treated with different agents, followed by
quantitative PCR for SOX2. The IC50 for PC9 of erlotinib, taxol, AZD6244,
and GDC0941 is 0.05, 0.005, 5, and >10 μM; for HCC827, 0.1,
0.01, >10, and 1 µM (data not shown). (C) PC9
cells were transfected with control siRNA or siRNA targeting EGFR. 48 hr
after transfection, the levels of SOX2 and EGFR were determined by qPCR.
(D) PC9 and HCC827 cells were treated continuously with
0.1 µM erlotinib for 9 days, with fresh media/drug added every 3
days. SOX2 level at each time point was analyzed by qPCR (left) or
immunoblot (right). All qPCR data are displayed as mean Ct value
(normalized to GAPDH and untreated cells) of 3–6 replicates
−/+ SEM, with data in (C) normalized to
untreated siCTRL cells.
DOI:
http://dx.doi.org/10.7554/eLife.06132.003
(A) Heat map showing the list of fourfold significantly (FDR
<0.05) upregulated (green) or downregulated (red) transcripts in
HCC827 cells with erlotinib treatment. (B) Time course of
changes in transcript levels for erlotinib-responsive genes.
DOI:
http://dx.doi.org/10.7554/eLife.06132.004
(A) HCC827 and PC9 cells. The erlotinib curve is the same as
in Figure 1B. (B)
Left panel, H1650 cells (EGFR DEL15 activating but IC50 >1
µM) and WM164 (BRAF V600E) show minimal induction following
treatment with erlotinib or the MEK inhibitor AZD6244, respectively. Data
for HCC827 cells are from Figure
1A. Right panel, H1975 cells, possessing an
EGFR activating L858R mutation and a T790M
erlotinib-resistance gatekeeper mutation, do not induce SOX2 with
erlotinib treatment (1 µM) but do with the EGFR/T790M selective
inhibitor WZ4002 (1 µM). (C) Effect of actinomycin D
on induction of SOX2 transcript in HCC827 cells treated with
erlotinib.
DOI:
http://dx.doi.org/10.7554/eLife.06132.005
Erlotinib treatment of EGFR-mutant cells was compared to MEK inhibitor
treatment of BRAF-mutant melanoma cells lines and
CL-387,785 (irreversible EGFR inhibitor) treatment of
EGFR-mutant lung cancer cells.
DOI:
http://dx.doi.org/10.7554/eLife.06132.006
The data for the 24 hr time points are the same as in Figure 2A. p-values are shown for the
comparison of mean SOX2 fluorescence of each treated population to DMSO
(Student's t-test, unequal variances, N =
341–3485, % SOX2+ is shown). Source data are included as
Figure
2—source data 2.
DOI:
http://dx.doi.org/10.7554/eLife.06132.007
Figure 1—figure supplement 2.
Effect of various treatments on SOX2 expression in different cell
contexts.
(A) HCC827 and PC9 cells. The erlotinib curve is the same as
in Figure 1B. (B)
Left panel, H1650 cells (EGFR DEL15 activating but IC50 >1
µM) and WM164 (BRAF V600E) show minimal induction following
treatment with erlotinib or the MEK inhibitor AZD6244, respectively. Data
for HCC827 cells are from Figure
1A. Right panel, H1975 cells, possessing an
EGFR activating L858R mutation and a T790M
erlotinib-resistance gatekeeper mutation, do not induce SOX2 with
erlotinib treatment (1 µM) but do with the EGFR/T790M selective
inhibitor WZ4002 (1 µM). (C) Effect of actinomycin D
on induction of SOX2 transcript in HCC827 cells treated with
erlotinib.
DOI:
http://dx.doi.org/10.7554/eLife.06132.005
SOX2 transcript is specifically induced by erlotinib in EGFR-mutant
and addicted lung cancer cell lines.
(A) Cell lines were treated with an inhibitor of the driving
oncogenic lesion for 24 hr (erlotinib for EGFR-mutant,
lapatinib for HER2-amplified and crizotinib for
ALK-translocated cells), followed by isolation of
total RNA and quantitative PCR for SOX2 transcript. (B) PC9
and HCC827 cells were treated with different agents, followed by
quantitative PCR for SOX2. The IC50 for PC9 of erlotinib, taxol, AZD6244,
and GDC0941 is 0.05, 0.005, 5, and >10 μM; for HCC827, 0.1,
0.01, >10, and 1 µM (data not shown). (C) PC9
cells were transfected with control siRNA or siRNA targeting EGFR. 48 hr
after transfection, the levels of SOX2 and EGFR were determined by qPCR.
(D) PC9 and HCC827 cells were treated continuously with
0.1 µM erlotinib for 9 days, with fresh media/drug added every 3
days. SOX2 level at each time point was analyzed by qPCR (left) or
immunoblot (right). All qPCR data are displayed as mean Ct value
(normalized to GAPDH and untreated cells) of 3–6 replicates
−/+ SEM, with data in (C) normalized to
untreated siCTRL cells.DOI:
http://dx.doi.org/10.7554/eLife.06132.003
Gene expression profiling after erlotinib treatment.
(A) Heat map showing the list of fourfold significantly (FDR
<0.05) upregulated (green) or downregulated (red) transcripts in
HCC827 cells with erlotinib treatment. (B) Time course of
changes in transcript levels for erlotinib-responsive genes.DOI:
http://dx.doi.org/10.7554/eLife.06132.004
Effect of various treatments on SOX2 expression in different cell
contexts.
(A) HCC827 and PC9 cells. The erlotinib curve is the same as
in Figure 1B. (B)
Left panel, H1650 cells (EGFR DEL15 activating but IC50 >1
µM) and WM164 (BRAF V600E) show minimal induction following
treatment with erlotinib or the MEK inhibitor AZD6244, respectively. Data
for HCC827 cells are from Figure
1A. Right panel, H1975 cells, possessing an
EGFR activating L858R mutation and a T790M
erlotinib-resistance gatekeeper mutation, do not induce SOX2 with
erlotinib treatment (1 µM) but do with the EGFR/T790M selective
inhibitor WZ4002 (1 µM). (C) Effect of actinomycin D
on induction of SOX2 transcript in HCC827 cells treated with
erlotinib.DOI:
http://dx.doi.org/10.7554/eLife.06132.005
Overlap of differentially expressed genes.
Erlotinib treatment of EGFR-mutant cells was compared to MEK inhibitor
treatment of BRAF-mutant melanoma cells lines and
CL-387,785 (irreversible EGFR inhibitor) treatment of
EGFR-mutant lung cancer cells.DOI:
http://dx.doi.org/10.7554/eLife.06132.006
Time course of SOX2 induction by quantitative immunofluorescence
microscopy.
The data for the 24 hr time points are the same as in Figure 2A. p-values are shown for the
comparison of mean SOX2 fluorescence of each treated population to DMSO
(Student's t-test, unequal variances, N =
341–3485, % SOX2+ is shown). Source data are included as
Figure
2—source data 2.
Figure 2.
Induction of SOX2 in erlotinib-treated cells.
(A) Left, HCC827 (upper) or PC9 (lower) cells were treated
with 0.1 µM erlotinib for 24 hr, followed by immunofluorescence
staining using an antibody to SOX2 and DAPI. For erlotinib-treated cells
(middle and right pairs of images), the heterogeneity in induced SOX2
levels per cell in each population is indicated by dashed outlines
indicating DAPI+ nuclei lacking SOX2, white arrows for nuclei with
low (but detectable) SOX2 and green arrows for nuclei with high SOX2.
Right, distribution of SOX2 fluorescence in each sample. Mean
fluorescence counts for each cell were quantitated and normalized to
exposure time as described in ‘Materials and methods’. p
< 0.0001 for the comparison of erlotinib-treated cells to DMSO
(Student's t-test, unequal variances, N =
1219–3485, means are 0.005/0.04 for untreated/treated HCC827 and
0.001/0.008 for untreated/treated PC9, % SOX2+ is shown). Source
data are included as Figure 2—source data 1, 2.
(B) Induction of SOX2 in erlotinib-retreated cells.
HCC827 cells were treated with 1.0 µM erlotinib for 24 hr (75%
cell killing), followed by removal of drug, replating of cells after 7
days of recovery and retreatment with the same concentration of
erlotinib. This protocol was repeated, and then cells were treated a
third time with erlotinib for 24 hr and analyzed by immunofluorescence
microscopy using antibodies to SOX2 and DAPI. The increase in
SOX2-positive cells was highly significant for both erlotinib pretreated
and untreated cells, but no enrichment was observed as a consequence of
pretreatment p < 0.0001 for the comparison of erlotinib-treated
cells to DMSO (Student's t-test, unequal
variances, N = 1106–2143, means are 0.005/0.17 for
untreated/treated-parental, 0.007/0.2 for untreated/treated-pretreated, %
SOX2+ is shown). Source data are included as Figure
2—source data 3. (C) Immunofluorescence
analysis of single colonies formed from single clones of HCC827 cells
stained for DAPI (blue), SOX2 (green), and Ki67 (red).
DOI:
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http://dx.doi.org/10.7554/eLife.06132.009
DOI:
http://dx.doi.org/10.7554/eLife.06132.010
DOI:
http://dx.doi.org/10.7554/eLife.06132.011
DOI:
http://dx.doi.org/10.7554/eLife.06132.012
DOI:
http://dx.doi.org/10.7554/eLife.06132.013
DOI:
http://dx.doi.org/10.7554/eLife.06132.014
HCC827 (left) and PC9 (right) cells were treated for 24 hr with the
indicated dose of erlotinib, followed by immunofluorescence microscopy
with antibodies to SOX2 and DAPI. The distribution of SOX2+ cells
is shown. N = 607–1169 for HCC827 cells, 2746–6818
for PC9, % SOX2+ is shown. Note: increased cell death at the
highest dose of erlotinib precludes accurate determination of
SOX2+ PC9 cells. Source data are included as Figure
2—source data 4.
DOI:
http://dx.doi.org/10.7554/eLife.06132.015
Immunofluorescence microscopy was carried out on erlotinib-treated HCC827
(upper panels) or PC9 (lower panels) cells using antibodies to SOX2 and
various stem cell markers. CD133 (upper row of panels) could be detected
in a rare population of PC9 cells (but not HCC827 cells) that was clearly
mutually exclusive from SOX2+ cells, while CD44 and MYC were
expressed in the majority of cells, irrespective of SOX2 expression.
Neither membrane localization of CD24 (rightmost panel, middle rows) nor
expression of OCT4 and KLF4 (data not shown) could be detected in either
cell line.
DOI:
http://dx.doi.org/10.7554/eLife.06132.016
Left panels, HCC827 cells were treated for 24 hr with 0.1 µM
erlotinib, followed by immunofluorescence microscopy with antibodies to
SOX2, Ki-67, and DAPI. The left three pairs of panels show HCC827 cells
at 20× magnification, the right pair of panels shows a doublet of
cells at 60× magnification. Right panel, the distribution of
nuclear Ki-67 mean fluorescence in the SOX2− and SOX2+
cells in DMSO-treated (green) and erlotinib treated (red) cells is
displayed. p = 0.015 and p < 0.0001 for the comparison of
each 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 for
erlotinib-treated). Source data are included as Figure
2—source data 1.
DOI:
http://dx.doi.org/10.7554/eLife.06132.017
(A) Retreatment of PC9 cells after a period of recovery does
not 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 for
untreated/treated-parental and 0.001/0.03 for
untreated/treated-pretreated, % SOX2+ is shown). Note: the
apparent decrease in SOX2+ fraction with retreatment is likely the
result of partial selection for erlotinib resistance with serial
retreatment, as shown in (B) below. Source data are included
as Figure
2—source data 5. (B) Induction of SOX2 in
cells with acquired resistance to erlotinib. PC9 cells were made
resistant to erlotinib by continuous culture in the presence of 0.1
µM drug for 30 days. Upper panel, qPCR for SOX2 expression was
performed on lysates prepared from cells at the indicated time points. On
day 30, resistant cells were replated in the absence of drug and then
retreated with increasing concentrations of erlotinib on day 31. Data are
shown 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, parental
and resistant PC9 cells were treated with increasing concentrations of
erlotinib for 6 hr, followed by quantitative immunofluorescence analysis
for pEGFR. N = 2073–6297 cells. Source data are included as
Figure
2—source data 6. Lower panels, representative images
show strong decrease in pEGFR (red) with 1 µM erlotinib in
parental (left) but not resistant (right) cells.
DOI:
http://dx.doi.org/10.7554/eLife.06132.018
(A) Immunofluorescence analysis of colonies formed from
single HCC827 cells for DAPI (blue), SOX2 (green), and Ki67 (red).
(B) Similar analysis of DAPI (blue) and SOX2 (green) in
PC9 cells. For HCC827 cells, because the cloning efficiency is extremely
low (<1%), a single cell suspension of HCC827 cells was plated at
low density and allowed to form discrete colonies. PC9 cells were cloned
by limiting dilution, and individual subclones were expanded prior to
analysis.
DOI:
http://dx.doi.org/10.7554/eLife.06132.019
Data are shown as mean Ct (normalized to GAPDH and untreated cells) of 3
replicates −/+ SEM.
DOI:
http://dx.doi.org/10.7554/eLife.06132.020
DOI:
http://dx.doi.org/10.7554/eLife.06132.007Other transcripts induced or repressed following erlotinib treatment of mutant
EGFR-addicted cells were not selective to EGFR signaling. Downregulated genes
included known direct transcriptional targets of ERK signaling (CCND1, FOSL1,
EGR1, IER3, IL-8) and shared feedback inhibitors of receptor tyrosine
kinase (RTK) signaling (DUSP6) (Amit et al., 2007). This gene set overlaps with genes known to be
differentially expressed following treatment of BRAF-mutant melanoma
cells with a MEK inhibitor and exposure of EGFR-mutant lung cancer
cells to an irreversible EGFR inhibitor (Figure
1—figure supplement 3) (Kobayashi
et al., 2006; Pratilas et al.,
2009). In addition to SOX2, the 12 other transcripts induced by EGFR
inhibition included genes encoding metabolizing enzymes (CYP1B1,
CYP1A1) that are normally induced by treatment with a variety of small
chemical entities, genes that we also found to be equally well induced by inhibition
of downstream signaling pathways (MEK/MAPK inhibition using AZD6244 and PI3K/mTOR
inhibition using BEZ235), one transcript (CCNG2) previously
described in another EGFR model (Kobayashi et al.,
2006) and a long noncoding RNA (NEAT1). Taken all
together, these results indicate that suppression of EGFR signaling in mutant
EGFR-addicted lung cancer cells is highly specific in triggering transcriptional
induction of SOX2.
Figure 1—figure supplement 3.
Overlap of differentially expressed genes.
Erlotinib treatment of EGFR-mutant cells was compared to MEK inhibitor
treatment of BRAF-mutant melanoma cells lines and
CL-387,785 (irreversible EGFR inhibitor) treatment of
EGFR-mutant lung cancer cells.
DOI:
http://dx.doi.org/10.7554/eLife.06132.006
Induction of SOX2 in erlotinib-treated cells
SOX2 encodes a master transcriptional regulator, implicated in stem cell maintenance
and iPS cell generation. It is also required for upper aerodigestive tract
development, and is known to be amplified in a subset of esophageal and squamous lung
cancers, although it has not been previously implicated in lung adenocarcinomas,
including the subset driven by mutant EGFR (Ellis
et al., 2004; Gontan et al., 2008;
Bass et al., 2009). Remarkably, SOX2
expression following exposure of HCC827 cells to erlotinib was transient, peaking at
24 hr after exposure to therapeutic levels of the drug (Figure 1D). Thereafter, SOX2 expression returned to basal levels
despite continued erlotinib treatment in surviving cells (Figure 1D, Figure
1—figure supplement 4).
Figure 1—figure supplement 4.
Time course of SOX2 induction by quantitative immunofluorescence
microscopy.
The data for the 24 hr time points are the same as in Figure 2A. p-values are shown for the
comparison of mean SOX2 fluorescence of each treated population to DMSO
(Student's t-test, unequal variances, N =
341–3485, % SOX2+ is shown). Source data are included as
Figure
2—source data 2.
DOI:
http://dx.doi.org/10.7554/eLife.06132.007
The level of SOX2 induction in cultured cells exposed to erlotinib showed
considerable heterogeneity, with a subset of cells (∼20%, with some
experimental variability) expressing high levels (Figure 2A). The SOX2+ fraction was not increased by higher drug
dosage, beyond that required for full inhibition of EGFR (Figure 2—figure supplement 1). Given the link between
SOX2 expression and cellular reprogramming, we first asked whether cells with the
high SOX2 expression represent a subset with stem cell markers. However, SOX2
expression did not correlate with expression of the putative stem cell markers CD133,
CD44, CD24, OCT-4, or KLF-4 (Figure 2—figure
supplement 2) nor did microarray-based expression profiling of high
SOX2-sorted cells identify a stem-like signature (data not shown). Nonetheless,
SOX2-expressing cells had a very low proliferative index, as measured by Ki67
staining (0.5% of Ki67+/SOX2+ vs 51% Ki67+/SOX2− HCC827
cells 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).
Figure 2—figure supplement 1.
Increasing the dose of erlotinib does not significantly increase the
fraction of SOX2+ cells.
HCC827 (left) and PC9 (right) cells were treated for 24 hr with the
indicated dose of erlotinib, followed by immunofluorescence microscopy
with antibodies to SOX2 and DAPI. The distribution of SOX2+ cells
is shown. N = 607–1169 for HCC827 cells, 2746–6818
for PC9, % SOX2+ is shown. Note: increased cell death at the
highest dose of erlotinib precludes accurate determination of
SOX2+ PC9 cells. Source data are included as Figure
2—source data 4.
DOI:
http://dx.doi.org/10.7554/eLife.06132.015
Figure 2—figure supplement 2.
Stem cell markers do not colocalize with SOX2+ cells.
Immunofluorescence microscopy was carried out on erlotinib-treated HCC827
(upper panels) or PC9 (lower panels) cells using antibodies to SOX2 and
various stem cell markers. CD133 (upper row of panels) could be detected
in a rare population of PC9 cells (but not HCC827 cells) that was clearly
mutually exclusive from SOX2+ cells, while CD44 and MYC were
expressed in the majority of cells, irrespective of SOX2 expression.
Neither membrane localization of CD24 (rightmost panel, middle rows) nor
expression of OCT4 and KLF4 (data not shown) could be detected in either
cell line.
DOI:
http://dx.doi.org/10.7554/eLife.06132.016
Figure 2—figure supplement 3.
SOX2 is expressed most highly in nonproliferative cells.
Left panels, HCC827 cells were treated for 24 hr with 0.1 µM
erlotinib, followed by immunofluorescence microscopy with antibodies to
SOX2, Ki-67, and DAPI. The left three pairs of panels show HCC827 cells
at 20× magnification, the right pair of panels shows a doublet of
cells at 60× magnification. Right panel, the distribution of
nuclear Ki-67 mean fluorescence in the SOX2− and SOX2+
cells in DMSO-treated (green) and erlotinib treated (red) cells is
displayed. p = 0.015 and p < 0.0001 for the comparison of
each 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 for
erlotinib-treated). Source data are included as Figure
2—source data 1.
DOI:
http://dx.doi.org/10.7554/eLife.06132.017
Induction of SOX2 in erlotinib-treated cells.
(A) Left, HCC827 (upper) or PC9 (lower) cells were treated
with 0.1 µM erlotinib for 24 hr, followed by immunofluorescence
staining using an antibody to SOX2 and DAPI. For erlotinib-treated cells
(middle and right pairs of images), the heterogeneity in induced SOX2
levels per cell in each population is indicated by dashed outlines
indicating DAPI+ nuclei lacking SOX2, white arrows for nuclei with
low (but detectable) SOX2 and green arrows for nuclei with high SOX2.
Right, distribution of SOX2 fluorescence in each sample. Mean
fluorescence counts for each cell were quantitated and normalized to
exposure time as described in ‘Materials and methods’. p
< 0.0001 for the comparison of erlotinib-treated cells to DMSO
(Student's t-test, unequal variances, N =
1219–3485, means are 0.005/0.04 for untreated/treated HCC827 and
0.001/0.008 for untreated/treated PC9, % SOX2+ is shown). Source
data are included as Figure 2—source data 1, 2.
(B) Induction of SOX2 in erlotinib-retreated cells.
HCC827 cells were treated with 1.0 µM erlotinib for 24 hr (75%
cell killing), followed by removal of drug, replating of cells after 7
days of recovery and retreatment with the same concentration of
erlotinib. This protocol was repeated, and then cells were treated a
third time with erlotinib for 24 hr and analyzed by immunofluorescence
microscopy using antibodies to SOX2 and DAPI. The increase in
SOX2-positive cells was highly significant for both erlotinib pretreated
and untreated cells, but no enrichment was observed as a consequence of
pretreatment p < 0.0001 for the comparison of erlotinib-treated
cells to DMSO (Student's t-test, unequal
variances, N = 1106–2143, means are 0.005/0.17 for
untreated/treated-parental, 0.007/0.2 for untreated/treated-pretreated, %
SOX2+ is shown). Source data are included as Figure
2—source data 3. (C) Immunofluorescence
analysis of single colonies formed from single clones of HCC827 cells
stained for DAPI (blue), SOX2 (green), and Ki67 (red).DOI:
http://dx.doi.org/10.7554/eLife.06132.008
Raw immunofluorescence data for quantitation of SOX2
staining in HCC827 cells with erlotinib treatment in Figure 2A, and SOX2+
Ki67 staining in Figure
2—figure supplement 3.
DOI:
http://dx.doi.org/10.7554/eLife.06132.009
Raw immunofluorescence data for quantitation of SOX2
staining in PC9 cells with erlotinib treatment in Figure 2A and Figure 1—figure supplement
4.
DOI:
http://dx.doi.org/10.7554/eLife.06132.010
Raw immunofluorescence data for quantitation of SOX2
staining in HCC827 cells recovered after retreatment (x2) with
erlotinib, compared to previously untreated, in Figure 2B.
DOI:
http://dx.doi.org/10.7554/eLife.06132.011
Raw immunofluorescence data for quantitation of SOX2
staining in HCC827 and PC9 cells with increasing dose of
erlotinib in Figure
2—figure supplement 1.
DOI:
http://dx.doi.org/10.7554/eLife.06132.012
Raw immunofluorescence data for quantitation of SOX2
staining in PC9 cells recovered after retreatment (x2) with
erlotinib, compared to previously untreated cells, in Figure 2—figure supplement
4A.
DOI:
http://dx.doi.org/10.7554/eLife.06132.013
Raw immunofluorescence data for quantitation of phospho-EGFR
(pY1068) in parental and erlotinib-resistant PC9 cells in Figure 2—figure supplement
4B.
DOI:
http://dx.doi.org/10.7554/eLife.06132.014
Increasing the dose of erlotinib does not significantly increase the
fraction of SOX2+ cells.
HCC827 (left) and PC9 (right) cells were treated for 24 hr with the
indicated dose of erlotinib, followed by immunofluorescence microscopy
with antibodies to SOX2 and DAPI. The distribution of SOX2+ cells
is shown. N = 607–1169 for HCC827 cells, 2746–6818
for PC9, % SOX2+ is shown. Note: increased cell death at the
highest dose of erlotinib precludes accurate determination of
SOX2+ PC9 cells. Source data are included as Figure
2—source data 4.DOI:
http://dx.doi.org/10.7554/eLife.06132.015
Stem cell markers do not colocalize with SOX2+ cells.
Immunofluorescence microscopy was carried out on erlotinib-treated HCC827
(upper panels) or PC9 (lower panels) cells using antibodies to SOX2 and
various stem cell markers. CD133 (upper row of panels) could be detected
in a rare population of PC9 cells (but not HCC827 cells) that was clearly
mutually exclusive from SOX2+ cells, while CD44 and MYC were
expressed in the majority of cells, irrespective of SOX2 expression.
Neither membrane localization of CD24 (rightmost panel, middle rows) nor
expression of OCT4 and KLF4 (data not shown) could be detected in either
cell line.DOI:
http://dx.doi.org/10.7554/eLife.06132.016
SOX2 is expressed most highly in nonproliferative cells.
Left panels, HCC827 cells were treated for 24 hr with 0.1 µM
erlotinib, followed by immunofluorescence microscopy with antibodies to
SOX2, Ki-67, and DAPI. The left three pairs of panels show HCC827 cells
at 20× magnification, the right pair of panels shows a doublet of
cells at 60× magnification. Right panel, the distribution of
nuclear Ki-67 mean fluorescence in the SOX2− and SOX2+
cells in DMSO-treated (green) and erlotinib treated (red) cells is
displayed. p = 0.015 and p < 0.0001 for the comparison of
each 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 for
erlotinib-treated). Source data are included as Figure
2—source data 1.DOI:
http://dx.doi.org/10.7554/eLife.06132.017
Stochastic induction of SOX2 by erlotinib in PC9 cells.
(A) Retreatment of PC9 cells after a period of recovery does
not 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 for
untreated/treated-parental and 0.001/0.03 for
untreated/treated-pretreated, % SOX2+ is shown). Note: the
apparent decrease in SOX2+ fraction with retreatment is likely the
result of partial selection for erlotinib resistance with serial
retreatment, as shown in (B) below. Source data are included
as Figure
2—source data 5. (B) Induction of SOX2 in
cells with acquired resistance to erlotinib. PC9 cells were made
resistant to erlotinib by continuous culture in the presence of 0.1
µM drug for 30 days. Upper panel, qPCR for SOX2 expression was
performed on lysates prepared from cells at the indicated time points. On
day 30, resistant cells were replated in the absence of drug and then
retreated with increasing concentrations of erlotinib on day 31. Data are
shown 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, parental
and resistant PC9 cells were treated with increasing concentrations of
erlotinib for 6 hr, followed by quantitative immunofluorescence analysis
for pEGFR. N = 2073–6297 cells. Source data are included as
Figure
2—source data 6. Lower panels, representative images
show strong decrease in pEGFR (red) with 1 µM erlotinib in
parental (left) but not resistant (right) cells.DOI:
http://dx.doi.org/10.7554/eLife.06132.018
The highest induction of SOX2 in individually isolated subclones of
EGFR-mutant cells occurs in a subset of cells, as for
the parental cells.
(A) Immunofluorescence analysis of colonies formed from
single HCC827 cells for DAPI (blue), SOX2 (green), and Ki67 (red).
(B) Similar analysis of DAPI (blue) and SOX2 (green) in
PC9 cells. For HCC827 cells, because the cloning efficiency is extremely
low (<1%), a single cell suspension of HCC827 cells was plated at
low density and allowed to form discrete colonies. PC9 cells were cloned
by limiting dilution, and individual subclones were expanded prior to
analysis.DOI:
http://dx.doi.org/10.7554/eLife.06132.019
KDM5A is not induced following treatment of PC9 cells with erlotinib
for 24 hr.
Data are shown as mean Ct (normalized to GAPDH and untreated cells) of 3
replicates −/+ SEM.DOI:
http://dx.doi.org/10.7554/eLife.06132.020To test whether the heterogeneous induction of SOX2 following EGFR inhibition
represents a stochastic event or a heritable property shared by a subset of the
parental population, we treated cells sequentially with pulses of erlotinib.
Retreatment of cells after a period of recovery produced similar heterogeneity of
SOX2+ cells as the initial treatment, pointing to the absence of enrichment
for highly inducible cells (Figure 2B and
Figure 2—figure supplement 4A). In
addition, SOX2 could still be induced in cells made resistant to erlotinib, but only
at the much higher doses of drug required to fully inhibit EGFR in resistant cells
(Figure 2—figure supplement 4B).
Finally, cloning of 5–6 individual HCC827 and PC9 cells consistently generated
mixed populations, including high level SOX2 inducers together with non-expressing
cells, demonstrating that this heterogeneity is likely stochastic, rather than
heritable (Figure 2C and Figure 2—figure supplement 5). Thus, erlotinib
treatment of EGFR-mutant cells results in transient and
heterogeneous induction of SOX2, with a stochastic distribution, integrally tied to
inhibition of EGFR, in which the cells with the highest expression have a low
proliferative index.
Figure 2—figure supplement 4.
Stochastic induction of SOX2 by erlotinib in PC9 cells.
(A) Retreatment of PC9 cells after a period of recovery does
not 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 for
untreated/treated-parental and 0.001/0.03 for
untreated/treated-pretreated, % SOX2+ is shown). Note: the
apparent decrease in SOX2+ fraction with retreatment is likely the
result of partial selection for erlotinib resistance with serial
retreatment, as shown in (B) below. Source data are included
as Figure
2—source data 5. (B) Induction of SOX2 in
cells with acquired resistance to erlotinib. PC9 cells were made
resistant to erlotinib by continuous culture in the presence of 0.1
µM drug for 30 days. Upper panel, qPCR for SOX2 expression was
performed on lysates prepared from cells at the indicated time points. On
day 30, resistant cells were replated in the absence of drug and then
retreated with increasing concentrations of erlotinib on day 31. Data are
shown 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, parental
and resistant PC9 cells were treated with increasing concentrations of
erlotinib for 6 hr, followed by quantitative immunofluorescence analysis
for pEGFR. N = 2073–6297 cells. Source data are included as
Figure
2—source data 6. Lower panels, representative images
show strong decrease in pEGFR (red) with 1 µM erlotinib in
parental (left) but not resistant (right) cells.
DOI:
http://dx.doi.org/10.7554/eLife.06132.018
Figure 2—figure supplement 5.
The highest induction of SOX2 in individually isolated subclones of
EGFR-mutant cells occurs in a subset of cells, as for
the parental cells.
(A) Immunofluorescence analysis of colonies formed from
single HCC827 cells for DAPI (blue), SOX2 (green), and Ki67 (red).
(B) Similar analysis of DAPI (blue) and SOX2 (green) in
PC9 cells. For HCC827 cells, because the cloning efficiency is extremely
low (<1%), a single cell suspension of HCC827 cells was plated at
low density and allowed to form discrete colonies. PC9 cells were cloned
by limiting dilution, and individual subclones were expanded prior to
analysis.
DOI:
http://dx.doi.org/10.7554/eLife.06132.019
Induction of SOX2 in EGFR-mutant tumors following erlotinib treatment
To test the physiological significance of SOX2 induction following withdrawal of
mutant EGFR signaling, we first made use of mouse tumor models. The effectiveness of
EGFR inhibitors in treating patients with EGFR-mutant NSCLC is well modeled in mouse
xenograft assays, where oral administration of erlotinib for a few days is sufficient
to cause massive regression of established tumors. We generated PC9 cell-derived
subcutaneous tumors in nude mice and treated these with a single oral dose of 100
mg/kg erlotinib when the tumors had reached approximately 500 mm3,
harvesting tumors 24 hr after treatment. Immunohistochemical (IHC) analysis revealed
minimal SOX2 expression in mock-treated xenografts (mean 0.2 SOX2+ nuclei per
field), but clearly increased (and heterogeneous) SOX2 positive cells after a single
dose of erlotinib (mean: 7.4 SOX2+ nuclei per field, N = 147–151
fields, p < 0.0001) (Figure 3). Similar
studies in HCC827 cell-derived xenografts revealed low (but detectable) levels of
SOX2 expression in mock-treated tumors; again, a single oral dose of erlotinib
increased both the number of SOX2-positive cells and the level of SOX2 expression per
nucleus (Figure 3—figure supplement
1). Thus, in a physiological setting that mimics the initial therapeutic
response to EGFR inhibitors in EGFR-mutant NSCLC, treated cancer
cells rapidly induce SOX2.
Figure 3.
SOX2 is induced by erlotinib in a subset of EGFR-mutant cells in
vivo.
Nude mice were xenografted subcutaneously with PC9 cells and treated with
a single oral dose of erlotinib (100 mg/kg) (or carrier) when the tumors
had reached ∼500 mm3. Tumors were harvested 24 hr after
treatment, and immunohistochemistry for SOX2 was carried out on
formalin-fixed, paraffin embedded tumor specimens. The panels show
automated scoring of SOX2+ nuclei (brown) as described in
‘Materials and methods’. The actual IHC images for the
areas indicated by rectangles are shown magnified to the right. p
< 0.0001 for the comparison erlotinib-treated vs control
(Student's t-test, unequal variances, N =
147–151 fields from 4 xenografts, mean SOX2+
nuclei/10× field −/+ SEM is shown for each
treatment). Source data are included as Figure
3—source data 1.
DOI:
http://dx.doi.org/10.7554/eLife.06132.021
DOI:
http://dx.doi.org/10.7554/eLife.06132.022
DOI:
http://dx.doi.org/10.7554/eLife.06132.023
Quantitative immunohistochemistry for SOX2 was performed in FFPE sections
from mice xenografted subcutaneously with HCC827 cells and treated with a
single oral dose of erlotinib (red in dot plot) or carrier (green).
Because HCC827 cells possess a degree of basal SOX2 in vivo, the average
mean fluorescence of tumor cells from each xenograft was used to
determine significance. p < 0.0001 for the comparison of
erlotinib-treated vs control (Analysis of Variance to control for the
variation among individual mice, N = 12,893–16,140 cells).
Source data are included as Figure 3—source data 2.
DOI:
http://dx.doi.org/10.7554/eLife.06132.024
Figure 3—figure supplement 1.
Erlotinib treatment results in induction of SOX2 in vivo.
Quantitative immunohistochemistry for SOX2 was performed in FFPE sections
from mice xenografted subcutaneously with HCC827 cells and treated with a
single oral dose of erlotinib (red in dot plot) or carrier (green).
Because HCC827 cells possess a degree of basal SOX2 in vivo, the average
mean fluorescence of tumor cells from each xenograft was used to
determine significance. p < 0.0001 for the comparison of
erlotinib-treated vs control (Analysis of Variance to control for the
variation among individual mice, N = 12,893–16,140 cells).
Source data are included as Figure 3—source data 2.
DOI:
http://dx.doi.org/10.7554/eLife.06132.024
SOX2 is induced by erlotinib in a subset of EGFR-mutant cells in
vivo.
Nude mice were xenografted subcutaneously with PC9 cells and treated with
a single oral dose of erlotinib (100 mg/kg) (or carrier) when the tumors
had reached ∼500 mm3. Tumors were harvested 24 hr after
treatment, and immunohistochemistry for SOX2 was carried out on
formalin-fixed, paraffin embedded tumor specimens. The panels show
automated scoring of SOX2+ nuclei (brown) as described in
‘Materials and methods’. The actual IHC images for the
areas indicated by rectangles are shown magnified to the right. p
< 0.0001 for the comparison erlotinib-treated vs control
(Student's t-test, unequal variances, N =
147–151 fields from 4 xenografts, mean SOX2+
nuclei/10× field −/+ SEM is shown for each
treatment). Source data are included as Figure
3—source data 1.DOI:
http://dx.doi.org/10.7554/eLife.06132.021
Number of SOX2+cells per field for quantitation of
SOX2 staining in PC9 cell xenografts in Figure 3.
DOI:
http://dx.doi.org/10.7554/eLife.06132.022
Raw absorbance data for quantitation of SOX2 staining in
HCC827 cell xenografts in Figure 3—figure supplement 1.
DOI:
http://dx.doi.org/10.7554/eLife.06132.023
Erlotinib treatment results in induction of SOX2 in vivo.
Quantitative immunohistochemistry for SOX2 was performed in FFPE sections
from mice xenografted subcutaneously with HCC827 cells and treated with a
single oral dose of erlotinib (red in dot plot) or carrier (green).
Because HCC827 cells possess a degree of basal SOX2 in vivo, the average
mean fluorescence of tumor cells from each xenograft was used to
determine significance. p < 0.0001 for the comparison of
erlotinib-treated vs control (Analysis of Variance to control for the
variation among individual mice, N = 12,893–16,140 cells).
Source data are included as Figure 3—source data 2.DOI:
http://dx.doi.org/10.7554/eLife.06132.024
Induction of SOX2 in patient-derived EGFR-mutant tumor cells
While established cancer cell lines, such as PC9 and HCC827, recapitulate the
phenomenon of oncogene addiction, patient-derived cell lines directly cultured from
biopsies may be more representative of heterogeneous primary cultures (Crystal et al., 2014). Such biopsies are
typically obtained at the time of disease progression, where defining drug resistance
mechanisms may shape further therapy. We therefore analyzed short-term cultures of
EGFR-mutant cells derived by re-biopsy of two patients who had initially responded to
erlotinib therapy but subsequently developed progressive disease due to the
acquisition of a T790M gatekeeper mutation. SOX2 induction was absent following
treatment with erlotinib, which was ineffective in inhibiting EGFR in these resistant
patient-derived cells (Figure 4). However, the
novel ‘third line’ irreversible, EGFR-mutant-specific inhibitor WZ4002
demonstrated potent EGFR inhibition in these cells, along with induction of SOX2
(Figure 4). Thus, SOX2 induction is
consistently observed following acute withdrawal of EGFR signals in cancer models as
well as in patient-derived cells that exhibit oncogene dependence on the EGFR
pathway.
Figure 4.
SOX2 is induced by therapy targeting the resistance genotype in cell
lines derived by rebiopsy of patients.
Short-term cultures of tumor cells derived from patients at the time of
acquired resistance (both tumors EGFR genotype exon 19
deletion + T790M) were treated with the indicated agents for 24 hr,
followed by isolation of total RNA and qPCR for SOX2 transcript (left
panels) or quantitative immunofluorescence analysis after staining with
antibodies to SOX2 (middle panels). The effect of each treatment on
downstream signaling was determined by immunoblot analysis with the
indicated antibodies (right panels). For qPCR, data are displayed as the
mean of 4 replicates −/+ SEM. For histograms, p-values are
shown for the comparison of each treatment to DMSO (Student's
t-test, unequal variances, N = 229–1808,
means are 0.001/0.0014/0.0054 for DMSO/erlotinib/WZ4002-treated MGH134 and
0.003/0.003/0.011 for DMSO/erlotinib/WZ4002-treated MGH141, % SOX2+
is shown). Source data are included as Figure 4—source
data 1.
DOI:
http://dx.doi.org/10.7554/eLife.06132.025
DOI:
http://dx.doi.org/10.7554/eLife.06132.026
SOX2 is induced by therapy targeting the resistance genotype in cell
lines derived by rebiopsy of patients.
Short-term cultures of tumor cells derived from patients at the time of
acquired resistance (both tumors EGFR genotype exon 19
deletion + T790M) were treated with the indicated agents for 24 hr,
followed by isolation of total RNA and qPCR for SOX2 transcript (left
panels) or quantitative immunofluorescence analysis after staining with
antibodies to SOX2 (middle panels). The effect of each treatment on
downstream signaling was determined by immunoblot analysis with the
indicated antibodies (right panels). For qPCR, data are displayed as the
mean of 4 replicates −/+ SEM. For histograms, p-values are
shown for the comparison of each treatment to DMSO (Student's
t-test, unequal variances, N = 229–1808,
means are 0.001/0.0014/0.0054 for DMSO/erlotinib/WZ4002-treated MGH134 and
0.003/0.003/0.011 for DMSO/erlotinib/WZ4002-treated MGH141, % SOX2+
is shown). Source data are included as Figure 4—source
data 1.DOI:
http://dx.doi.org/10.7554/eLife.06132.025
Raw immunofluorescence data for quantitation of SOX2 staining
with different treatments in patient-derived tumor cells.
DOI:
http://dx.doi.org/10.7554/eLife.06132.026
Regulation of anti-apoptotic signals by SOX2
To examine the consequence of ectopic SOX2 expression, we generated HCC827 cells with
a lentiviral-driven, doxycycline-inducible construct. Careful titration of
doxycycline-permitted induction of ectopic SOX2 to physiologic levels in the absence
of erlotinib, comparable at the single cell level to endogenous SOX2 induction in the
presence of erlotinib, though in a larger fraction of cells (Figure 5A and Figure
5—figure supplement 1A). As expected, treatment of control HCC827
cells with erlotinib led to the inhibition of EGFR signaling, as measured by
decreased phosphorylation of EGFR, AKT, and ERK, and caused dramatic apoptosis, as
determined by increased PARP and caspase-3 cleavage (Figure 5A, lanes 1–2). In contrast, expression of ectopic SOX2
significantly decreased erlotinib-mediated apoptosis, resulting in decreased PARP and
caspase-3 cleavage (Figure 5A, lane 2 vs 4).
Exogenous SOX2 itself did not alter basal or erlotinib-inhibited phosphorylation of
EGFR, AKT, or ERK (Figure 5A, lanes 3–4).
Figure 5.
Induction of SOX2 protects cells from erlotinib-induced
apoptosis.
(A) HCC827 cells were stably transduced with a doxycycline
inducible epitope-tagged SOX2 lentiviral expression vector. Doxycycline
was added for 3 hr (‘SOX2-tag’) and then removed prior to
the addition of DMSO or 0.1 µM erlotinib for 24 hr, followed by
immunoblot of protein lysates with the indicated antibodies. Exogenous
SOX2 migrates more slowly than the endogenous protein due to the presence
of the tag. (B) HCC827 (left) or PC9 (right) cells were
transfected with control siRNA or siRNA targeting SOX2. 24-hr after
transfection, DMSO or 0.1 µM erlotinib was added. The effect of
SOX2 knockdown was assessed by immunoblot analysis of protein lysates
with the indicated antibodies after overnight treatment. (C)
PC9 cells were stably transduced with a tagged SOX2 lentiviral vector in
which silent mutations were introduced into the target site for the most
potent siRNA against SOX2. Cells were transfected with the indicated
siRNAs, treated with doxycycline followed by erlotinib as in
(A), and protein lysates were analyzed by immunoblot with
the indicated antibodies after overnight treatment. The increased PARP
and caspase-3 cleavage observed when erlotinib treatment is combined with
siRNA targeting SOX2 (lane 4) is suppressed by siRNA-resistant, exogenous
SOX2 (lane 6). (D) PC9 and HCC827 cells were transfected in
96-well plates and treated 24 hr later with a dilution series of
erlotinib, followed by Syto-60 assay. Data are displayed as the mean of
3–5 replicates −/+ SEM. p = 0.001 (PC9) and
0.04 (HCC827) for the comparison of mean IC50 for siCTRL vs siSOX2
(Student's t-test, unequal variances).
(E) Preventing SOX2 induction using siRNA decreases the
development of acquired erlotinib resistance. PC9 cells were transfected
with control siRNA or siRNA targeting SOX2, followed by treatment after
24 hr with 1.0 µM erlotinib. Erlotinib-containing medium was
renewed every 3 days, and plates were fixed and stained with Crystal
Violet at the indicated times. The left panels demonstrate the absence of
toxicity following transfection with siRNA targeting SOX2 in the absence
of erlotinib. Middle panels demonstrate cell loss after 3 days of
treatment, due to erlotinib-induced apoptosis; at higher magnification,
more control cells remain attached than cells transfected with siRNA
against SOX2. Right panels show colonies of proliferating cells after 2
weeks of continuous erlotinib treatment. p < 0.0001for the number
of cells per 20× field (N = 33 fields per sample) or the
number of colonies per plate (N = 9 plates per sample from three
independent experiments), for siRNA targeting SOX2 vs control cells
(Student's t-test, unequal variances).
DOI:
http://dx.doi.org/10.7554/eLife.06132.027
DOI:
http://dx.doi.org/10.7554/eLife.06132.028
DOI:
http://dx.doi.org/10.7554/eLife.06132.029
(A) Quantitative immunofluorescence analysis confirms that
ectopic SOX2 expression with a short pulse of doxycycline increases the
fraction of SOX2+ cells without significantly increasing the
amount of SOX2 per cell. The same conditions were used as in Figure 5A p < 0.0001, for the
comparison of erlotinib-treated to DMSO (Student's
t-test, unequal variances, N = 830–1115
cells, means of SOX2 fluorescence are 0.0007/0.03 for untreated/treated
cells without exogenous SOX2 and 0.03/0.06 for untreated/treated cells
with exogenous SOX2, % SOX2+ is shown). Source data are included
as Figure
5—source data 1. (B) Left, HCC827 cells
were stably transduced with a tetracycline-inducible SOX2 expression
vector (without an epitope tag). Cells were treated with carrier (lanes
1–4) or 0.1 µg/ml doxycycline (lanes 5–8) for 3 hr
prior to addition of 0.1 µM erlotinib, with doxycycline left in
the cell culture media during erlotinib treatment to ensure strong
induction of exogenous SOX2. Protein lysates were prepared at 3-hr
intervals, and immunoblot analysis was carried out with the indicated
antibodies. The increase in the intensity of the SOX2 band in lane
1–4 with time is due to induction of endogenous SOX2 by erlotinib;
in lanes 5–8, strong induction of exogenous SOX2 with increasing
time in doxycycline masks the endogenous protein. Right, the levels of
each BIM isoform were quantitated using ImageJ software and normalized to
GAPDH and time zero, confirming suppression of their induction by ectopic
SOX2. (C) Physiologic levels of ectopic SOX2 decrease BIM
induction by erlotinib. Cells were treated with erlotinib for 12 or 24
hr, −/+ doxycycline to induce tagged SOX2, followed by
immunoblot of lysates. Here, doxycycline was added for 3 hr and then
removed prior to addition of erlotinib, to maintain ectopic SOX2
induction at physiologic levels during erlotinib treatment. Therefore,
ectopic SOX2 is (slightly) apparent at time zero when erlotinib is added
(lane 4), peaks 12 hr later (lane 5) and has decreased at 24 hr (lane 6).
However, decreased BIM levels, together with decreased cleaved
PARP/caspase-3 (lanes 5–6 vs 2–3), are apparent throughout.
The use of a lower percentage acrylamide gel does not permit BIM L and S
to be distinguished from BIM EL.
DOI:
http://dx.doi.org/10.7554/eLife.06132.030
Left panel, quantitative immunofluorescence analysis showing expression
of SOX2 (x-axis) and cleaved caspase-3 (y-axis) in PC9 cells transfected
with siCTRL (blue) or siSOX2 (red) and treated with erlotinib (N =
2452–3792). Knockout of SOX2 results in decreased SOX2 expression
and increased cleaved caspase-3. Right panels, representative
immunofluorescence images from erlotinib-treated cells showing DAPI
(blue), SOX2 (red), and cleaved-caspase-3 (green) staining. Source data
are included as Figure 5—source data 2.
DOI:
http://dx.doi.org/10.7554/eLife.06132.031
(A), PC9 cells were transfected with two different siRNA
duplexes targeting SOX2 (or control siRNA), followed by addition of DMSO
or 0.1 µM erlotinib for 24 hr and immunoblot of protein lysates
with the indicated antibodies. (B), the degree of knockdown
of 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
Each BIM isoform (EL, L, and S) was normalized to the GAPDH loading
control and untreated, siCTRL cells.
DOI:
http://dx.doi.org/10.7554/eLife.06132.033
Figure 5—figure supplement 1.
Induction of exogenous SOX2.
(A) Quantitative immunofluorescence analysis confirms that
ectopic SOX2 expression with a short pulse of doxycycline increases the
fraction of SOX2+ cells without significantly increasing the
amount of SOX2 per cell. The same conditions were used as in Figure 5A p < 0.0001, for the
comparison of erlotinib-treated to DMSO (Student's
t-test, unequal variances, N = 830–1115
cells, means of SOX2 fluorescence are 0.0007/0.03 for untreated/treated
cells without exogenous SOX2 and 0.03/0.06 for untreated/treated cells
with exogenous SOX2, % SOX2+ is shown). Source data are included
as Figure
5—source data 1. (B) Left, HCC827 cells
were stably transduced with a tetracycline-inducible SOX2 expression
vector (without an epitope tag). Cells were treated with carrier (lanes
1–4) or 0.1 µg/ml doxycycline (lanes 5–8) for 3 hr
prior to addition of 0.1 µM erlotinib, with doxycycline left in
the cell culture media during erlotinib treatment to ensure strong
induction of exogenous SOX2. Protein lysates were prepared at 3-hr
intervals, and immunoblot analysis was carried out with the indicated
antibodies. The increase in the intensity of the SOX2 band in lane
1–4 with time is due to induction of endogenous SOX2 by erlotinib;
in lanes 5–8, strong induction of exogenous SOX2 with increasing
time in doxycycline masks the endogenous protein. Right, the levels of
each BIM isoform were quantitated using ImageJ software and normalized to
GAPDH and time zero, confirming suppression of their induction by ectopic
SOX2. (C) Physiologic levels of ectopic SOX2 decrease BIM
induction by erlotinib. Cells were treated with erlotinib for 12 or 24
hr, −/+ doxycycline to induce tagged SOX2, followed by
immunoblot of lysates. Here, doxycycline was added for 3 hr and then
removed prior to addition of erlotinib, to maintain ectopic SOX2
induction at physiologic levels during erlotinib treatment. Therefore,
ectopic SOX2 is (slightly) apparent at time zero when erlotinib is added
(lane 4), peaks 12 hr later (lane 5) and has decreased at 24 hr (lane 6).
However, decreased BIM levels, together with decreased cleaved
PARP/caspase-3 (lanes 5–6 vs 2–3), are apparent throughout.
The use of a lower percentage acrylamide gel does not permit BIM L and S
to be distinguished from BIM EL.
DOI:
http://dx.doi.org/10.7554/eLife.06132.030
Induction of SOX2 protects cells from erlotinib-induced
apoptosis.
(A) HCC827 cells were stably transduced with a doxycycline
inducible epitope-tagged SOX2 lentiviral expression vector. Doxycycline
was added for 3 hr (‘SOX2-tag’) and then removed prior to
the addition of DMSO or 0.1 µM erlotinib for 24 hr, followed by
immunoblot of protein lysates with the indicated antibodies. Exogenous
SOX2 migrates more slowly than the endogenous protein due to the presence
of the tag. (B) HCC827 (left) or PC9 (right) cells were
transfected with control siRNA or siRNA targeting SOX2. 24-hr after
transfection, DMSO or 0.1 µM erlotinib was added. The effect of
SOX2 knockdown was assessed by immunoblot analysis of protein lysates
with the indicated antibodies after overnight treatment. (C)
PC9 cells were stably transduced with a tagged SOX2 lentiviral vector in
which silent mutations were introduced into the target site for the most
potent siRNA against SOX2. Cells were transfected with the indicated
siRNAs, treated with doxycycline followed by erlotinib as in
(A), and protein lysates were analyzed by immunoblot with
the indicated antibodies after overnight treatment. The increased PARP
and caspase-3 cleavage observed when erlotinib treatment is combined with
siRNA targeting SOX2 (lane 4) is suppressed by siRNA-resistant, exogenous
SOX2 (lane 6). (D) PC9 and HCC827 cells were transfected in
96-well plates and treated 24 hr later with a dilution series of
erlotinib, followed by Syto-60 assay. Data are displayed as the mean of
3–5 replicates −/+ SEM. p = 0.001 (PC9) and
0.04 (HCC827) for the comparison of mean IC50 for siCTRL vs siSOX2
(Student's t-test, unequal variances).
(E) Preventing SOX2 induction using siRNA decreases the
development of acquired erlotinib resistance. PC9 cells were transfected
with control siRNA or siRNA targeting SOX2, followed by treatment after
24 hr with 1.0 µM erlotinib. Erlotinib-containing medium was
renewed every 3 days, and plates were fixed and stained with Crystal
Violet at the indicated times. The left panels demonstrate the absence of
toxicity following transfection with siRNA targeting SOX2 in the absence
of erlotinib. Middle panels demonstrate cell loss after 3 days of
treatment, due to erlotinib-induced apoptosis; at higher magnification,
more control cells remain attached than cells transfected with siRNA
against SOX2. Right panels show colonies of proliferating cells after 2
weeks of continuous erlotinib treatment. p < 0.0001for the number
of cells per 20× field (N = 33 fields per sample) or the
number of colonies per plate (N = 9 plates per sample from three
independent experiments), for siRNA targeting SOX2 vs control cells
(Student's t-test, unequal variances).DOI:
http://dx.doi.org/10.7554/eLife.06132.027
Raw immunofluorescence data for quantitation of SOX2
staining in HCC827 cells with inducible SOX2 in Figure 5—figure supplement
1A.
DOI:
http://dx.doi.org/10.7554/eLife.06132.028
Raw immunofluorescence data for quantitation of SOX2 and
cleaved caspase-3 costaining in PC9 cells transfected with
siCTRL or siSOX2 in Figure
5—figure supplement 2.
DOI:
http://dx.doi.org/10.7554/eLife.06132.029
Induction of exogenous SOX2.
(A) Quantitative immunofluorescence analysis confirms that
ectopic SOX2 expression with a short pulse of doxycycline increases the
fraction of SOX2+ cells without significantly increasing the
amount of SOX2 per cell. The same conditions were used as in Figure 5A p < 0.0001, for the
comparison of erlotinib-treated to DMSO (Student's
t-test, unequal variances, N = 830–1115
cells, means of SOX2 fluorescence are 0.0007/0.03 for untreated/treated
cells without exogenous SOX2 and 0.03/0.06 for untreated/treated cells
with exogenous SOX2, % SOX2+ is shown). Source data are included
as Figure
5—source data 1. (B) Left, HCC827 cells
were stably transduced with a tetracycline-inducible SOX2 expression
vector (without an epitope tag). Cells were treated with carrier (lanes
1–4) or 0.1 µg/ml doxycycline (lanes 5–8) for 3 hr
prior to addition of 0.1 µM erlotinib, with doxycycline left in
the cell culture media during erlotinib treatment to ensure strong
induction of exogenous SOX2. Protein lysates were prepared at 3-hr
intervals, and immunoblot analysis was carried out with the indicated
antibodies. The increase in the intensity of the SOX2 band in lane
1–4 with time is due to induction of endogenous SOX2 by erlotinib;
in lanes 5–8, strong induction of exogenous SOX2 with increasing
time in doxycycline masks the endogenous protein. Right, the levels of
each BIM isoform were quantitated using ImageJ software and normalized to
GAPDH and time zero, confirming suppression of their induction by ectopic
SOX2. (C) Physiologic levels of ectopic SOX2 decrease BIM
induction by erlotinib. Cells were treated with erlotinib for 12 or 24
hr, −/+ doxycycline to induce tagged SOX2, followed by
immunoblot of lysates. Here, doxycycline was added for 3 hr and then
removed prior to addition of erlotinib, to maintain ectopic SOX2
induction at physiologic levels during erlotinib treatment. Therefore,
ectopic SOX2 is (slightly) apparent at time zero when erlotinib is added
(lane 4), peaks 12 hr later (lane 5) and has decreased at 24 hr (lane 6).
However, decreased BIM levels, together with decreased cleaved
PARP/caspase-3 (lanes 5–6 vs 2–3), are apparent throughout.
The use of a lower percentage acrylamide gel does not permit BIM L and S
to be distinguished from BIM EL.DOI:
http://dx.doi.org/10.7554/eLife.06132.030
Left panel, quantitative immunofluorescence analysis showing expression
of SOX2 (x-axis) and cleaved caspase-3 (y-axis) in PC9 cells transfected
with siCTRL (blue) or siSOX2 (red) and treated with erlotinib (N =
2452–3792). Knockout of SOX2 results in decreased SOX2 expression
and increased cleaved caspase-3. Right panels, representative
immunofluorescence images from erlotinib-treated cells showing DAPI
(blue), SOX2 (red), and cleaved-caspase-3 (green) staining. Source data
are included as Figure 5—source data 2.DOI:
http://dx.doi.org/10.7554/eLife.06132.031
The effect of siRNA targeting SOX2 is specific.
(A), PC9 cells were transfected with two different siRNA
duplexes targeting SOX2 (or control siRNA), followed by addition of DMSO
or 0.1 µM erlotinib for 24 hr and immunoblot of protein lysates
with the indicated antibodies. (B), the degree of knockdown
of 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
Quantitation of the effect of SOX2 knockdown on BIM levels.
Each BIM isoform (EL, L, and S) was normalized to the GAPDH loading
control and untreated, siCTRL cells.DOI:
http://dx.doi.org/10.7554/eLife.06132.033To determine the functional significance of endogenous SOX2 induction, we next used
siRNAs to block its induction in erlotinib-treated cells. While both HCC827 and PC9
cells are highly sensitive to EGFR inhibition at baseline, SOX2 knockdown further
increased erlotinib-induced apoptosis, as determined by PARP and caspase-3 cleavage
assays and by cell enumeration (Figure 5B,D
and Figure 5—figure supplement 2).
The apoptotic effect of the most potent siRNA was rescued by expression of an ectopic
siRNA-resistant SOX2 construct (Figure 5C) and
individual 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 EGFR
signaling, as measured by phosphorylation of EGFR, AKT, or ERK (Figure 5B).
Left panel, quantitative immunofluorescence analysis showing expression
of SOX2 (x-axis) and cleaved caspase-3 (y-axis) in PC9 cells transfected
with siCTRL (blue) or siSOX2 (red) and treated with erlotinib (N =
2452–3792). Knockout of SOX2 results in decreased SOX2 expression
and increased cleaved caspase-3. Right panels, representative
immunofluorescence images from erlotinib-treated cells showing DAPI
(blue), SOX2 (red), and cleaved-caspase-3 (green) staining. Source data
are included as Figure 5—source data 2.
DOI:
http://dx.doi.org/10.7554/eLife.06132.031
Figure 5—figure supplement 3.
The effect of siRNA targeting SOX2 is specific.
(A), PC9 cells were transfected with two different siRNA
duplexes targeting SOX2 (or control siRNA), followed by addition of DMSO
or 0.1 µM erlotinib for 24 hr and immunoblot of protein lysates
with the indicated antibodies. (B), the degree of knockdown
of 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
Given the effect of transient SOX2 induction in acutely modulating cell survival
following erlotinib treatment of EGFR-mutant cells, we tested whether this translates
into a longer term impact on acquired drug resistance. We used the well-characterized
PC9 cell model of EGFR-mutant NSCLC to transfect siRNAs against SOX2 (the same duplex
validated by rescue in Figure 5C) or control,
followed by continuous exposure to 1.0 µM erlotinib (Figure 5E). As expected, SOX2 knockdown alone had no significant
toxicity, while erlotinib treatment led to massive cell death at 3 days (Figure 5E, left and middle images). At high
magnification, many more individual surviving cells remained in control transfected
cells than after transfection with siRNA targeting SOX2 (Figure 5E, middle insets). With continued incubation in
erlotinib, drug-resistant colonies emerged in control transfected cultures at higher
frequency than in those treated with siRNA targeting SOX2 (Figure 5E, right). Consistent with the short-term duration of
siRNA effectiveness, these results suggest that preventing the short-term induction
of SOX2 allows fewer cells to survive the initial exposure to erlotinib, preventing
an adaptation response that ultimately delays the emergence of erlotinib-resistant
colonies.
Erlotinib-induced SOX2 directly regulates expression of BIM and
BMF
The mechanisms underlying the cell death response of cancer cells following
withdrawal of oncogene-addicting signals are complex but appear to result in part
from activation of the pro-apoptotic proteins BIM and PUMA (Costa et al., 2007; Gong et
al., 2007; Bean et al., 2013). To
search for additional targets that mediate SOX2's anti-apoptotic effect, we
screened for altered expression of multiple pro-apoptotic and anti-apoptotic proteins
in cells with overexpression of ectopic SOX2, following treatment with erlotinib. In
both HCC827 and PC9 EGFR-mutant lung cancer cells, three of 14 BH3-domain containing
proteins tested showed strongly reduced mRNA induction by erlotinib in the presence
of ectopic SOX2: BIM, BMF, and HRK (but not PUMA) (Figure 6A and Figure 6—figure
supplement 1). All three of these are known to induce apoptosis to
different degrees, depending on the type of apoptotic stimulus. The effect of ectopic
SOX2 on BIM protein levels was confirmed using two different expression strategies
(Figure 5—figure supplement 1B,C).
Ectopic expression of SOX2, both at high and physiological levels, attenuated the
erlotinib-induced induction of SOX2. In contrast, SOX2 knockdown coincident with
erlotinib treatment led to an overall increase in BIM levels, although this effect
was attenuated when averaged across a cell population with heterogeneous induction of
endogenous SOX2 (Figure 5—figure supplement
4).
Figure 6.
Erlotinib-induced SOX2 directly regulates expression of
BIM and BMF.
(A) The levels of transcripts for each of the indicated BH3
domain-containing proteins was assessed by quantitative PCR at multiple
time points after erlotinib treatment in uninduced PC9 cells (blue lines)
and in cells in which expression of SOX2 was induced with doxycycline
(red lines), which was not removed prior to erlotinib addition in order
to further increase SOX2 levels, as shown in Figure 5—figure supplement 1B. The y-axis
maximum for all graphs is set to 4 except for HRK (y maximum = 11)
and BMF (y maximum = 110). Data are displayed as mean Ct of 4
replicates (normalized to untreated cells and GAPDH) −/+
SEM. (B) Upper panel, ChIP seq demonstrates SOX2 binding to
BIM and BMF. HCC827 cells were
treated with 0.1 µM erlotinib for 24 hr, followed by chromatin
immunoprecipitation (ChIP) using anti-SOX2 antibody and ChIP Seq as
described in ‘Materials and methods’. ChIP seq signal
tracks are displayed. Lower panel, HCC827 cells were left untreated or
were treated with 0.1 µM erlotinib for 24 hr, followed by
chromatin immunoprecipitation using anti-SOX2 antibody or IgG as a
negative control, and qPCR with control primers or primers within the
peaks indicated by the gray boxes in the ChIP seq tracks. Data are
displayed as mean Ct value (normalized to input chromatin) of 4
replicates −/+ standard error, p-values for the comparison
of untreated vs erlotinib treated cells are shown (Student's
t-test, unequal variances). (C)
Knockdown of BIM and BMF decreases apoptosis. Left panel, PC9 cells were
transfected with siRNA constructs targeting BIM and BMF (alone or
together) or a control siRNA. 24 hr after transfection, cells were
treated with DMSO or 0.1 µM erlotinib. 24 hr after treatment,
protein lysates were prepared, and immunoblot was performed with the
indicated antibodies. Right panel, the efficiency of knockdown of BMF was
confirmed by qPCR.
DOI:
http://dx.doi.org/10.7554/eLife.06132.034
Induction of BIM, BMF, and HRK following erlotinib treatment of HCC827
cells was assessed as in Figure
6A.
DOI:
http://dx.doi.org/10.7554/eLife.06132.035
Figure 6—figure supplement 1.
Effect of SOX2 overexpression on apoptotic regulators.
Induction of BIM, BMF, and HRK following erlotinib treatment of HCC827
cells was assessed as in Figure
6A.
DOI:
http://dx.doi.org/10.7554/eLife.06132.035
Figure 5—figure supplement 4.
Quantitation of the effect of SOX2 knockdown on BIM levels.
Each BIM isoform (EL, L, and S) was normalized to the GAPDH loading
control and untreated, siCTRL cells.
DOI:
http://dx.doi.org/10.7554/eLife.06132.033
Erlotinib-induced SOX2 directly regulates expression of
BIM and BMF.
(A) The levels of transcripts for each of the indicated BH3
domain-containing proteins was assessed by quantitative PCR at multiple
time points after erlotinib treatment in uninduced PC9 cells (blue lines)
and in cells in which expression of SOX2 was induced with doxycycline
(red lines), which was not removed prior to erlotinib addition in order
to further increase SOX2 levels, as shown in Figure 5—figure supplement 1B. The y-axis
maximum for all graphs is set to 4 except for HRK (y maximum = 11)
and BMF (y maximum = 110). Data are displayed as mean Ct of 4
replicates (normalized to untreated cells and GAPDH) −/+
SEM. (B) Upper panel, ChIP seq demonstrates SOX2 binding to
BIM and BMF. HCC827 cells were
treated with 0.1 µM erlotinib for 24 hr, followed by chromatin
immunoprecipitation (ChIP) using anti-SOX2 antibody and ChIP Seq as
described in ‘Materials and methods’. ChIP seq signal
tracks are displayed. Lower panel, HCC827 cells were left untreated or
were treated with 0.1 µM erlotinib for 24 hr, followed by
chromatin immunoprecipitation using anti-SOX2 antibody or IgG as a
negative control, and qPCR with control primers or primers within the
peaks indicated by the gray boxes in the ChIP seq tracks. Data are
displayed as mean Ct value (normalized to input chromatin) of 4
replicates −/+ standard error, p-values for the comparison
of untreated vs erlotinib treated cells are shown (Student's
t-test, unequal variances). (C)
Knockdown of BIM and BMF decreases apoptosis. Left panel, PC9 cells were
transfected with siRNA constructs targeting BIM and BMF (alone or
together) or a control siRNA. 24 hr after transfection, cells were
treated with DMSO or 0.1 µM erlotinib. 24 hr after treatment,
protein lysates were prepared, and immunoblot was performed with the
indicated antibodies. Right panel, the efficiency of knockdown of BMF was
confirmed by qPCR.DOI:
http://dx.doi.org/10.7554/eLife.06132.034
Effect of SOX2 overexpression on apoptotic regulators.
Induction of BIM, BMF, and HRK following erlotinib treatment of HCC827
cells was assessed as in Figure
6A.DOI:
http://dx.doi.org/10.7554/eLife.06132.035To determine whether BIM and BMF are endogenous SOX2 target genes, we carried out
chromatin immunoprecipitation sequencing (ChIP seq) experiments, using
erlotinib-treated HCC827 cells. Strong SOX2-binding peaks were present within
∼2 kB of the transcriptional start sites (TSS) of both BIM
and BMF genes (for BMF, the peak spans the TSS; for
BIM, it is located within the first intron) (Figure 6B, upper). Importantly, ChIP qPCR demonstrated
significant enhancement of SOX2 binding with erlotinib treatment at both peaks
compared to untreated cells, suggesting that binding is functionally significant
(Figure 6B, lower).Consistent with their functional roles, knockdown of either BIM or BMF (but not HRK)
decreased erlotinib-induced apoptosis, with combined BIM and BMF knockdown displaying
an additive effect (Figure 6C). The role of
BMF could be even more important, since BMF knockdown increases BIM levels, which may
blunt the anti-apoptotic effect of BMF loss (Figure
6C, left). Together, these results suggest that SOX2 induction following
erlotinib exposure in EGFR-mutant cells suppresses transcriptional induction of the
BH3-only BIM and BMF genes, which contribute to
apoptosis following oncogene withdrawal.
Induction of SOX2 following EGFR inhibition is regulated by FOXO6
To search for mediators of SOX2 induction, we explored the Molecular Signatures and
TRANSFAC databases for transcription factor target sequences within the promoters of
the 12 highest erlotinib-induced genes (Wingender
et al., 2000; Subramanian et al.,
2005). Several binding motifs for FOXO proteins were highly significantly
enriched (q-value = 0.003 or less): for SOX2, multiple sites were present
within 2 kb of the transcriptional start site (Figure
7A and Figure 7—figure supplement
1). Expression of all of the FOXO family members was detectable at baseline
in HCC827 cells and erlotinib treatment (8 hr) was associated with a
1.6–4.4-fold induction (Figure 7B), as
well as with loss of the AKT-mediated inhibitory N-terminal threonine phosphorylation
of the FOXO proteins (Figure 7—figure
supplement 2A).
Figure 7.
SOX2 expression in EGFR-mutant cells is regulated by FOXO6.
(A) Putative FOXO protein binding sites within the promoter
of SOX2, identified using TRANSFAC and Zhang et al., 2011. (B) HCC827 cells were
transfected with control siRNA or siRNA targeting the indicated FOXO
proteins (alone or FOXOs 1, 3a and 4 in combination). 72-hr after
transfection, DMSO or 0.1 µM erlotinib was added for 8 hr, and the
levels of SOX2 and FOXO mRNAs were determined by qPCR. Data are shown as
mean Ct (normalized to GAPDH and untreated siCTRL cells) of 3 replicates
−/+ SEM. Only knockdown of FOXO6 results in significantly
decreased induction of SOX2 mRNA by erlotinib compared to siCTRL cells. p
< 0.0001, other siFOXOs are without significant decrease
(Student's t-test, unequal variance). Although
siRNA pools targeting FOXOs 3a and 4 also decrease FOXO1, the lack of a
SOX2 effect with specific FOXO1 knockdown argues against their role in
regulation of SOX2. (C) The effect of FOXO6 knockdown on
induction of SOX2 in HCC827 cells is shown by immunofluorescence after
staining of cells with SOX2 and DAPI (upper panels) and quantitated for
knockdown of all of the FOXO isoforms (lower panel). Only knockdown of
FOXO6 significantly decreases induction of SOX2 by erlotinib compared to
siCTRL cells. p < 0.0001, other siFOXOs are without significant
decrease (N = 766–1027 cells, Student's
t-test, unequal variances). Source data are included
as Figure
7—source data 1. Knockdown efficiency is demonstrated in
Figure 7B. (D)
Multiple different siRNAs effectively targeting FOXO6 block
erlotinib-mediated induction of SOX2. HCC827 (upper) or PC9 (lower) cells
were transfected with control siRNA or four different siRNA duplexes
targeting FOXO6, treated with 0.1 µM erlotinib for 24 hr and mRNA
was analyzed by qPCR. Data are shown as mean Ct (normalized to ACTB and
untreated siCTRL cells) of 4 replicates −/+ SEM. p <
0.0001 for the comparison of each FOXO6 siRNA to siCTRL for both SOX2 and
FOXO6 (Student's t-test, unequal variances). The
effect of each siRNA on the levels of the other FOXO isoforms is shown in
Figure 7—figure supplement
2C. (E) Knockdown of FOXO6 has minimal effects on
other downstream components of EGFR signaling. HCC827 cells were
transfected with siRNA targeting FOXO6 and treated with 0.1 µM
erlotinib overnight followed by immunoblot analysis of protein lysates
with the indicated antibodies. Knockdown of FOXO6 is demonstrated in
Figure 7—figure supplement
2A.
DOI:
http://dx.doi.org/10.7554/eLife.06132.036
DOI:
http://dx.doi.org/10.7554/eLife.06132.037
DOI:
http://dx.doi.org/10.7554/eLife.06132.038
(A) MSigDB/TRANSFAC output for the 12 genes most highly
upregulated by erlotinib (FDR <0.05). (B) Although
binding sites for FOXF2 are also enriched, knockdown of FOXF2 does not
decrease erlotinib-mediated induction of SOX2.
DOI:
http://dx.doi.org/10.7554/eLife.06132.039
(A) Same lysates as Figure
7E, showing immunoblot for FOXO proteins. (B)
Similar data as in Figure 7B, but
shown after immunoblot of protein lysates with the indicated antibodies.
Immunoblot for FOXO4 was consistently unable to detect a band of the
correct size (65 kD). (C) Effect of individual siRNA
duplexes targeting FOXO6 on the levels of other FOXO isoforms. Data are
presented as in Figure 7D.
Individual siFOXO6 siRNA duplexes −01 and −02 do not alter
the levels of FOXOs 1, 3a or 4.
DOI:
http://dx.doi.org/10.7554/eLife.06132.040
(A) HCC827 cells were left untreated or were treated with
0.1 µM erlotinib for 24 hr. Cells were stained with goat anti-SOX2
and rabbit anti-FOXO6 primary antibodies, followed by anti-goat-Alexa
Fluor 488 (green) and anti-rabbit-Alexa Fluor 647 (red) secondary
antibodies (and DAPI in blue). FOXO6 appears to colocalize (yellow in the
leftmost panels) with SOX2 in cells with the highest expression of the
latter, especially in erlotinib-treated cells (arrows). (B)
Quantitative immunofluorescence analysis demonstrates a positive
correlation between FOXO6 and SOX2 nuclear fluorescence in individual
cells (Correlation coefficient R untreated/treated = 0.7/0.5, N
= 1700/835 cells). Source data are included as Figure
7—source data 2.
DOI:
http://dx.doi.org/10.7554/eLife.06132.041
(A) Pre-treatment of PC9 cells with FGF10 has minimal
effects on SOX2 induction by erlotinib. (B) The addition of
exogenous Wnt3A has no effect on induction of SOX2 by erlotinib.
(C) The beta-catenin pathway does not regulate SOX2
expression. HCC827 cells were stably transduced with inducible lentiviral
constructs expressing a dominant negative TCF4 transgene (DN TCF4) or the
constitutively activated S33Y variant of Beta-Catenin (S33Y B-Cat), and
with a lentiviral TOP FLASH reporter. Left, the expected activity of each
transgene was confirmed by TOP FLASH luciferase assay in the absence or
presence of the GSK3 inhibitor/Beta-Catenin activator BIO (and
−/+ erlotinib). Representative wells after luciferase
imaging are shown above the graph. Right, the effect of each transgene on
the levels of SOX2 induction by erlotinib compared to control (GUS) cells
is minimal. (D) Knockdown of TTF1 (NKX2.1) with siRNA has
minimal effects on the degree of SOX2 induction by erlotinib.
DOI:
http://dx.doi.org/10.7554/eLife.06132.042
Figure 7—figure supplement 1.
Recurrent FOXO binding sites in erlotinib-induced genes.
(A) MSigDB/TRANSFAC output for the 12 genes most highly
upregulated by erlotinib (FDR <0.05). (B) Although
binding sites for FOXF2 are also enriched, knockdown of FOXF2 does not
decrease erlotinib-mediated induction of SOX2.
DOI:
http://dx.doi.org/10.7554/eLife.06132.039
Figure 7—figure supplement 2.
FOXO6 uniquely regulates SOX2 expression.
(A) Same lysates as Figure
7E, showing immunoblot for FOXO proteins. (B)
Similar data as in Figure 7B, but
shown after immunoblot of protein lysates with the indicated antibodies.
Immunoblot for FOXO4 was consistently unable to detect a band of the
correct size (65 kD). (C) Effect of individual siRNA
duplexes targeting FOXO6 on the levels of other FOXO isoforms. Data are
presented as in Figure 7D.
Individual siFOXO6 siRNA duplexes −01 and −02 do not alter
the levels of FOXOs 1, 3a or 4.
DOI:
http://dx.doi.org/10.7554/eLife.06132.040
SOX2 expression in EGFR-mutant cells is regulated by FOXO6.
(A) Putative FOXO protein binding sites within the promoter
of SOX2, identified using TRANSFAC and Zhang et al., 2011. (B) HCC827 cells were
transfected with control siRNA or siRNA targeting the indicated FOXO
proteins (alone or FOXOs 1, 3a and 4 in combination). 72-hr after
transfection, DMSO or 0.1 µM erlotinib was added for 8 hr, and the
levels of SOX2 and FOXO mRNAs were determined by qPCR. Data are shown as
mean Ct (normalized to GAPDH and untreated siCTRL cells) of 3 replicates
−/+ SEM. Only knockdown of FOXO6 results in significantly
decreased induction of SOX2 mRNA by erlotinib compared to siCTRL cells. p
< 0.0001, other siFOXOs are without significant decrease
(Student's t-test, unequal variance). Although
siRNA pools targeting FOXOs 3a and 4 also decrease FOXO1, the lack of a
SOX2 effect with specific FOXO1 knockdown argues against their role in
regulation of SOX2. (C) The effect of FOXO6 knockdown on
induction of SOX2 in HCC827 cells is shown by immunofluorescence after
staining of cells with SOX2 and DAPI (upper panels) and quantitated for
knockdown of all of the FOXO isoforms (lower panel). Only knockdown of
FOXO6 significantly decreases induction of SOX2 by erlotinib compared to
siCTRL cells. p < 0.0001, other siFOXOs are without significant
decrease (N = 766–1027 cells, Student's
t-test, unequal variances). Source data are included
as Figure
7—source data 1. Knockdown efficiency is demonstrated in
Figure 7B. (D)
Multiple different siRNAs effectively targeting FOXO6 block
erlotinib-mediated induction of SOX2. HCC827 (upper) or PC9 (lower) cells
were transfected with control siRNA or four different siRNA duplexes
targeting FOXO6, treated with 0.1 µM erlotinib for 24 hr and mRNA
was analyzed by qPCR. Data are shown as mean Ct (normalized to ACTB and
untreated siCTRL cells) of 4 replicates −/+ SEM. p <
0.0001 for the comparison of each FOXO6 siRNA to siCTRL for both SOX2 and
FOXO6 (Student's t-test, unequal variances). The
effect of each siRNA on the levels of the other FOXO isoforms is shown in
Figure 7—figure supplement
2C. (E) Knockdown of FOXO6 has minimal effects on
other downstream components of EGFR signaling. HCC827 cells were
transfected with siRNA targeting FOXO6 and treated with 0.1 µM
erlotinib overnight followed by immunoblot analysis of protein lysates
with the indicated antibodies. Knockdown of FOXO6 is demonstrated in
Figure 7—figure supplement
2A.DOI:
http://dx.doi.org/10.7554/eLife.06132.036
Raw immunofluorescence data for quantitation of SOX2
staining with different FOXO protein knockdown in Figure 7C.
DOI:
http://dx.doi.org/10.7554/eLife.06132.037
Raw immunofluorescence data for quantitation of SOX2 and
FOXO6 costaining in HCC827 cells in Figure 7—figure supplement
3.
DOI:
http://dx.doi.org/10.7554/eLife.06132.038
Recurrent FOXO binding sites in erlotinib-induced genes.
(A) MSigDB/TRANSFAC output for the 12 genes most highly
upregulated by erlotinib (FDR <0.05). (B) Although
binding sites for FOXF2 are also enriched, knockdown of FOXF2 does not
decrease erlotinib-mediated induction of SOX2.DOI:
http://dx.doi.org/10.7554/eLife.06132.039
FOXO6 uniquely regulates SOX2 expression.
(A) Same lysates as Figure
7E, showing immunoblot for FOXO proteins. (B)
Similar data as in Figure 7B, but
shown after immunoblot of protein lysates with the indicated antibodies.
Immunoblot for FOXO4 was consistently unable to detect a band of the
correct size (65 kD). (C) Effect of individual siRNA
duplexes targeting FOXO6 on the levels of other FOXO isoforms. Data are
presented as in Figure 7D.
Individual siFOXO6 siRNA duplexes −01 and −02 do not alter
the levels of FOXOs 1, 3a or 4.DOI:
http://dx.doi.org/10.7554/eLife.06132.040
Distribution of FOXO6 vs SOX2 nuclear staining.
(A) HCC827 cells were left untreated or were treated with
0.1 µM erlotinib for 24 hr. Cells were stained with goat anti-SOX2
and rabbit anti-FOXO6 primary antibodies, followed by anti-goat-Alexa
Fluor 488 (green) and anti-rabbit-Alexa Fluor 647 (red) secondary
antibodies (and DAPI in blue). FOXO6 appears to colocalize (yellow in the
leftmost panels) with SOX2 in cells with the highest expression of the
latter, especially in erlotinib-treated cells (arrows). (B)
Quantitative immunofluorescence analysis demonstrates a positive
correlation between FOXO6 and SOX2 nuclear fluorescence in individual
cells (Correlation coefficient R untreated/treated = 0.7/0.5, N
= 1700/835 cells). Source data are included as Figure
7—source data 2.DOI:
http://dx.doi.org/10.7554/eLife.06132.041
Assessing the role of previously identified regulators on
erlotinib-induced expression of SOX2.
(A) Pre-treatment of PC9 cells with FGF10 has minimal
effects on SOX2 induction by erlotinib. (B) The addition of
exogenous Wnt3A has no effect on induction of SOX2 by erlotinib.
(C) The beta-catenin pathway does not regulate SOX2
expression. HCC827 cells were stably transduced with inducible lentiviral
constructs expressing a dominant negative TCF4 transgene (DN TCF4) or the
constitutively activated S33Y variant of Beta-Catenin (S33Y B-Cat), and
with a lentiviral TOP FLASH reporter. Left, the expected activity of each
transgene was confirmed by TOP FLASH luciferase assay in the absence or
presence of the GSK3 inhibitor/Beta-Catenin activator BIO (and
−/+ erlotinib). Representative wells after luciferase
imaging are shown above the graph. Right, the effect of each transgene on
the levels of SOX2 induction by erlotinib compared to control (GUS) cells
is minimal. (D) Knockdown of TTF1 (NKX2.1) with siRNA has
minimal effects on the degree of SOX2 induction by erlotinib.DOI:
http://dx.doi.org/10.7554/eLife.06132.042Given the evidence of erlotinib-mediated FOXO activation and its potential regulation
of SOX2, we tested the consequence of siRNA-mediated knockdown of each gene family
member, alone and in combination. Knockdown of FOXO6 using pooled siRNA constructs,
but not the other FOXO proteins (individually or simultaneously), dramatically
reduced erlotinib-mediated induction of SOX2 (Figure
7B,C and Figure 7—figure
supplement 2B). The effect of FOXO6 knockdown on SOX2 was evident using
multiple individual siRNAs targeting FOXO6 in both HCC827 and PC9 cells (Figure 7D; effect on other FOXO isoforms is shown
in Figure 7—figure supplement 2C).
Although some individual siRNAs targeting FOXO6 had off target effects on other
FOXOs, direct targeting of FOXOs 1, 3a, and 4 had no effect on SOX2 expression,
further supporting the specificity of the FOXO6 effect (Figure 7B). The effect of FOXO6 knockdown on SOX2 was not
associated with any consistent effect on other aspects of EGFR signaling, although a
moderate decrease in phospho-EGFR was observed in some experiments without
significant differences in phospho-AKT or phospho-ERK (Figure 7E). Notably, FOXO6 expression was also heterogeneous and
partially colocalized with SOX2 expression among populations of both untreated and
treated cells (Figure 7—figure supplement
3). FOXO6 differs from other FOXO proteins in that even the inactive
protein is localized in the nucleus (Jacobs et al.,
2003; van der Heide et al., 2005).
However, it shares the FOXO protein AKT-dependent inhibitory phosphorylation, whose
suppression following repression of mutant EGFR signaling may in part explain the
erlotinib-mediated FOXO6 activation.
Figure 7—figure supplement 3.
Distribution of FOXO6 vs SOX2 nuclear staining.
(A) HCC827 cells were left untreated or were treated with
0.1 µM erlotinib for 24 hr. Cells were stained with goat anti-SOX2
and rabbit anti-FOXO6 primary antibodies, followed by anti-goat-Alexa
Fluor 488 (green) and anti-rabbit-Alexa Fluor 647 (red) secondary
antibodies (and DAPI in blue). FOXO6 appears to colocalize (yellow in the
leftmost panels) with SOX2 in cells with the highest expression of the
latter, especially in erlotinib-treated cells (arrows). (B)
Quantitative immunofluorescence analysis demonstrates a positive
correlation between FOXO6 and SOX2 nuclear fluorescence in individual
cells (Correlation coefficient R untreated/treated = 0.7/0.5, N
= 1700/835 cells). Source data are included as Figure
7—source data 2.
DOI:
http://dx.doi.org/10.7554/eLife.06132.041
Erlotinib resistance in SOX2-expressing EGFR-mutant cells
Virtually all EGFR-mutant lung cancer cell lines established from patients who have
not been treated with erlotinib are highly sensitive to this drug, although a few
cells lines appear to be intrinsically resistant. The HCC2935 human lung cancer cell
line 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 T790M
gatekeeper mutation within EGFR and no amplification of the MET bypass signaling
pathway (Zheng et al., 2011). Notably, SOX2
expression at baseline is detectable in 90% of HCC2935 cells (compared to 3% of
HCC827 and <1% PC9 cells), and its expression per cell is further increased
upon EGFR inhibition (Figure 8B). To test
whether increased SOX2 contributes to decreased erlotinib sensitivity in HCC2935
cells, we knocked down SOX2 using siRNA. A striking increase in erlotinib
cytotoxicity was evident following SOX2 suppression (IC50 0.8 µM for siCTRL
cells with no further toxicity up to 10 µM, IC50 0.1 µM for siSOX2
cells), associated with higher levels of BIM and increased PARP and caspase-3
cleavage (Figure 8A,C). Thus, increased
baseline SOX2 contributes to erlotinib resistance in these EGFR-mutant cells.
Figure 8.
Knockdown of SOX2 sensitizes HCC2935 cells to erlotinib-induced
apoptosis.
(A) HCC2935 cells were transfected with siCTRL or siSOX2 48 hr
prior to erlotinib addition and assayed for cytoxicity 48 hr later with
Syto-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 for
the comparison of mean IC50 for siCTRL vs siSOX2 (Student's
t-test, unequal variances). (B) Upper
panels, images of untreated and erlotinib-treated HCC2935 cells,
demonstrating SOX2 expression in the majority of cells. Lower panels, the
distribution of SOX2 in HCC2935 was determined by quantitative
immunofluorescence microscopy. p < 0.0001 for the comparison of mean
SOX2 fluorescence in untreated vs treated cells (Student's
t-test, unequal variances, N = 3342/1181, means
for SOX2 fluorescence are 0.17/0.24 for untreated/treated cells, %
SOX2+ is shown). Source data is included as Figure 8—source
data 1. (C) HCC2935 cells were transfected with
control siRNA or siRNA targeting SOX2. 48 hr after transfection, DMSO or 1.0
µM erlotinib was added. The effect of SOX2 knockdown was assessed by
immunoblot analysis of protein lysates with the indicated antibodies after
overnight treatment.
DOI:
http://dx.doi.org/10.7554/eLife.06132.043
DOI:
http://dx.doi.org/10.7554/eLife.06132.044
Knockdown of SOX2 sensitizes HCC2935 cells to erlotinib-induced
apoptosis.
(A) HCC2935 cells were transfected with siCTRL or siSOX2 48 hr
prior to erlotinib addition and assayed for cytoxicity 48 hr later with
Syto-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 for
the comparison of mean IC50 for siCTRL vs siSOX2 (Student's
t-test, unequal variances). (B) Upper
panels, images of untreated and erlotinib-treated HCC2935 cells,
demonstrating SOX2 expression in the majority of cells. Lower panels, the
distribution of SOX2 in HCC2935 was determined by quantitative
immunofluorescence microscopy. p < 0.0001 for the comparison of mean
SOX2 fluorescence in untreated vs treated cells (Student's
t-test, unequal variances, N = 3342/1181, means
for SOX2 fluorescence are 0.17/0.24 for untreated/treated cells, %
SOX2+ is shown). Source data is included as Figure 8—source
data 1. (C) HCC2935 cells were transfected with
control siRNA or siRNA targeting SOX2. 48 hr after transfection, DMSO or 1.0
µM erlotinib was added. The effect of SOX2 knockdown was assessed by
immunoblot analysis of protein lysates with the indicated antibodies after
overnight treatment.DOI:
http://dx.doi.org/10.7554/eLife.06132.043
Raw immunofluorescence data for quantitation of SOX2 staining
in HCC2935 cells in Figure
8B.
DOI:
http://dx.doi.org/10.7554/eLife.06132.044
Discussion
The dramatic responsiveness of EGFR-mutated NSCLC to small molecule
inhibitors of the EGFR kinase has provided a paradigm for the targeted therapy of
epithelial cancers and has established a new standard of care for a genetically defined
subset of patients with lung cancer. However, the invariable development of drug
resistance greatly limits the effectiveness of this therapy, despite efforts to
circumvent acquired genetic abnormalities, such as the recurrent T790M-EGFR gatekeeper
mutation or MET amplification. Understanding how immediate signaling
feedback loops modulate the cellular response to targeted inhibitors and how cellular
heterogeneity may lead to transient drug tolerant states may thus provide important
therapeutic opportunities.
FOXO6 regulates SOX2 expression
The regulation of SOX2 expression by FOXO6, whose activation is normally repressed by
EGFR signaling, is consistent with the critical role played by FOXO proteins as
integrators of cellular signaling pathways. The best studied isoform, FOXO1, is
highly expressed in ES cells and has been implicated in the maintenance of
pluripotency through activation of SOX2 transcription (Zhang et al., 2011). All FOXO proteins bind to similar DNA
sequences, with isoform-specific activity presumably conferred by cellular and
promoter 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 homologous
Serine residues, yet only FOXO6 regulates SOX2 in these cells. Given variability in
knockdown efficacy (Figure 7B, column 6), we
cannot exclude some contribution from the other FOXO family members on SOX2
expression, but FOXO6 has the dominant effect in the cells tested. Activation of
FOXO6 may occur through both AKT dependent and independent pathways (Jacobs et al., 2003; van der Heide et al., 2005), and indeed we observed that
treatment with PI3K inhibitors alone is insufficient for induction of SOX2 (Figure 1B). Other pathways that have been
implicated 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 modest
effects 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-dependent
signaling.
Figure 7—figure supplement 4.
Assessing the role of previously identified regulators on
erlotinib-induced expression of SOX2.
(A) Pre-treatment of PC9 cells with FGF10 has minimal
effects on SOX2 induction by erlotinib. (B) The addition of
exogenous Wnt3A has no effect on induction of SOX2 by erlotinib.
(C) The beta-catenin pathway does not regulate SOX2
expression. HCC827 cells were stably transduced with inducible lentiviral
constructs expressing a dominant negative TCF4 transgene (DN TCF4) or the
constitutively activated S33Y variant of Beta-Catenin (S33Y B-Cat), and
with a lentiviral TOP FLASH reporter. Left, the expected activity of each
transgene was confirmed by TOP FLASH luciferase assay in the absence or
presence of the GSK3 inhibitor/Beta-Catenin activator BIO (and
−/+ erlotinib). Representative wells after luciferase
imaging are shown above the graph. Right, the effect of each transgene on
the levels of SOX2 induction by erlotinib compared to control (GUS) cells
is minimal. (D) Knockdown of TTF1 (NKX2.1) with siRNA has
minimal effects on the degree of SOX2 induction by erlotinib.
DOI:
http://dx.doi.org/10.7554/eLife.06132.042
SOX2 regulates apoptosis through BIM and BMF
Our study extends the pro-apoptotic signals implicated in withdrawal of mutant EGFR
signaling to include BMF, in addition to BIM and PUMA (Gong et al., 2007; Bean et
al., 2013). In contrast to the latter, BMF binds with significant affinity
to a subset of BCL-2 family members (BCL-2, BCL-xL, and BCL-w) (Chen et al., 2005; Kuwana et
al., 2005), yet it clearly contributes to the apoptotic response to
erlotinib (Figure 6). The direct binding by
SOX2 of the BIM and BMF genes is consistent with
the ability of the four reprogramming factors (SOX2, OCT-4, KLF-4, and c-MYC) to bind
to the promoters of several anti-apoptotic genes (including BMF) which are induced
early during the reprogramming process (Kim et al.,
2008; Soufi et al., 2012).
Interestingly, control of SOX2 expression and its modulation of apoptosis may differ
in EGFR-mutant lung cancer, compared with other forms of NSCLC. In a recent study of
lung cancers with wild-type EGFR, the activation of EGFR upregulated SOX2, thereby
decreasing apoptosis through BCL2L1, a phenomenon that was notably absent in
EGFR-mutant lung cancers (Chou et al., 2013).
A similar pathway was reported in a prostate cancer cell line, also with wild-type
EGFR (Rybak and Tang, 2013). Thus, an
EGFR-SOX2-BCL2L1 pathway may be implicated in cancer cells with wild-type EGFR,
whereas the EGFR-FOXO6-SOX2-BIM/BMF pathway we describe is specific for cells that
are dependent on mutant EGFR signals for their survival.
Heterogeneity of SOX2 induction and implications for targeted therapy
The heterogeneous induction of SOX2 within a clonally derived cell population could
reflect the existence of an intrinsic subpopulation with heritable traits, as
recently proposed in medulloblastoma and skin carcinoma (Boumahdi et al., 2014; Vanner
et al., 2014). Alternatively, it could result from stochastic variation
between cells in the activity of cellular signaling pathways. We favor the latter
model, given the absent coexpression of SOX2 with putative stem cell markers, the
failure to enrich for SOX2 positive cells following repeated erlotinib treatments,
and the regeneration of heterogeneous SOX2 inducibility following single cell cloning
experiments. A stochastic cell killing model for TRAIL-induced apoptosis of HeLa
cells was recently described, in which naturally occurring cell-to-cell variation in
the levels or activity of upstream signaling proteins leads to differential induction
of mitochondrial membrane permeability and apoptosis only in some cells (Spencer et al., 2009).Critically, the ability of siRNA targeting SOX2 to substantially decrease the number
and rate at which resistant subclones of EGFR-mutant cells emerge following
continuous erlotinib treatment suggests that, despite its heterogeneous, transient,
and stochastic expression, SOX2 contributes to the emergence of stably acquired
resistance (Figure 5E). In these respects, our
observations are reminiscent of transient drug-tolerant persister cells (DTPs), also
observed following erlotinib treatment (Sharma et
al., 2010). However, there are also significant differences between the
mechanisms underlying DTPs and those observed here. DTPs emerge following longer
erlotinib exposure at much higher concentrations, and they constitute a lower
percentage of cells within the population. Their expression of the stem cell marker
CD133 and the chromatin remodeling protein KDM5A was not reproduced by SOX2+
cells (Figure 2—figure
supplements 2, 6). Furthermore, DTPs are detectable in many cancer cell
lines following treatment with multiple cytotoxic and targeted agents, whereas
induction of SOX2 appears to be strictly limited to targeted EGFR inhibition in cells
addicted to mutant EGFR signaling. SOX2 induction may thus be a specific and early
signaling response to EGFR withdrawal, which contributes to increased cell survival,
thus enhancing the likelihood of epigenetic events leading to DTPs and ultimately to
stable genetic mechanisms of acquired drug resistance. Our results with HCC2935 cells
(Figure 8) further suggest that in a subset
of cases, high basal SOX2 may even blunt the initial signaling response to EGFR
inhibitors.In summary, the rewiring of cellular signaling pathways driven by a dominant
mutationally activated kinase underlies oncogene addiction in cancer, providing
powerful opportunities for targeted therapy. At the same time, feedback pathways that
attenuate these signaling readouts may play a major role in enhancing the development
of drug resistance. The EGFR-FOXO6-SOX2 signaling pathway regulating expression of
the BIM and BMF apoptotic factors thus identifies a feedback loop that may attenuate
the effectiveness of anti-EGFR therapy in cancer and contribute toward the ultimate
development of drug resistance (Figure 9). The
contribution of key embryonic regulators such as SOX2 points to the conservation of
critical developmental pathways in cancer cells, which modulate their response to the
disruption of oncogenic signals.
Figure 9.
Model of SOX2 feedback signaling pathway.
In untreated cells, mutant EGFR drives cell survival by activating
downstream signaling pathways, including PI3K and MAPK, which inhibit
apoptosis through transcriptional and post-transcriptional effects on
BH3-domain proteins, including pro-apoptotic BIM and BMF. In most cells (red
lines), erlotinib treatment results in EGFR inhibition, inhibition of
downstream signaling and increased pro-apoptotic proteins, leading to
apoptosis. The high SOX2 induced by erlotinib through activation of FOXO6 in
some cells (blue lines) counteracts the pro-apoptotic effects of EGFR
inhibition, sufficiently decreasing the levels of BIM and BMF to delay the
apoptotic response.
DOI:
http://dx.doi.org/10.7554/eLife.06132.045
Model of SOX2 feedback signaling pathway.
In untreated cells, mutant EGFR drives cell survival by activating
downstream signaling pathways, including PI3K and MAPK, which inhibit
apoptosis through transcriptional and post-transcriptional effects on
BH3-domain proteins, including pro-apoptotic BIM and BMF. In most cells (red
lines), erlotinib treatment results in EGFR inhibition, inhibition of
downstream signaling and increased pro-apoptotic proteins, leading to
apoptosis. The high SOX2 induced by erlotinib through activation of FOXO6 in
some cells (blue lines) counteracts the pro-apoptotic effects of EGFR
inhibition, sufficiently decreasing the levels of BIM and BMF to delay the
apoptotic response.DOI:
http://dx.doi.org/10.7554/eLife.06132.045
Materials and methods
Cell lines and reagents
All cell lines were grown in RPMI (GIBCO) with 10% FBS and were obtained from the
ATCC or the Massachusetts General Hospital Center for Molecular Therapeutics, which
performs routine cell line authentication testing by SNP and STR analysis. Erlotinib,
AZD6244, BEZ235, crizotonib, lapatinib, WZ4002 (Selleckchem, Houston, TX), and BIO
(Sigma, St. Louis, MO) were dissolved in DMSO.
Microarray analysis
HCC827 cells were plated in triplicates and treated the following day for 6 hr with
DMSO, erlotinib, AZD6244, and BEZ235 (each 1 µM) or for 0, 3, 6, 12, and 24 hr
with erlotinib (single samples for each time point). Total mRNA was isolated using
the RNeasy Mini Kit (Qiagen). Generation of cRNA, hybridization to GeneChip Human
Genome U133 Plus 2.0 mRNA expression arrays and array scanning were done according to
the manufacturer's standard protocols (Affymetrix, Inc.). Raw Affymetrix CEL
files were converted to a single value for each probe set using Robust Multi-array
Average (RMA) and normalized using quantile normalization. Quality control was
performed using the distribution analysis, correlation and principle variance
components analysis functions in Jmp Genomics (SAS Institute). Individual genes with
statistically significantly altered expression after treatment (compared to untreated
cells) were identified using ANOVA after model adjustment for multiple hypothesis
testing across LSMeans differences using the False Discovery Method of Benjamini and
Hochberg (FDR) with Alpha set to 0.05. The complete data set is available at the NCBI
Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/), Accession GSE51212.
Quantitative PCR
For RT-qPCR, 1 µg mRNA was converted to cDNA using the First-strand cDNA
Synthesis Kit (GE Healthcare, Pittsburgh, PA). cDNA was analyzed on a 7500 Real Time
PCR System (Applied Biosystems) using TaqMan Gene Expression Master Mix and TaqMan
gene expression assays (with GAPDH or ACTB as control, individual assays listed in
Supplementary file
1—Thermo Fisher Scientific, Grand Island, NY). For ChIP-qPCR,
immunoprecipitated chromatin (see below) was analyzed on the same system, using
paired DNA PCR primers (Supplementary file 1) and Power SYBR Green PCR Master Mix (Applied
Biosystems).
Immunoblot analysis and antibodies
Immunoblotting was performed using standard methods. After treatment with the
indicated drugs, cells were washed with cold PBS and lysed in buffer containing 20 mM
Tris pH 7.5, 150 mM NaCl, 100 mM MgCl2, 1% Nonidet P-40 and 10% glycerol
supplemented with HALT protease and phosphatase inhibitor cocktail (Thermo Fisher
Scientific) using a Q800R sonicator (Qsonica, Newtown, CT). Lysates were centrifuged
at 16,000×g for 5 min at 4°C. Protein concentrations
were determined by BCA assay (Thermo Fisher Scientific). Proteins were resolved by
SDS-PAGE and transferred to a polyvinylidenes difluoride membranes (Biorad) using the
Transblot Turbo Transfer System (Biorad, Hercules, CA). Immunoblotting was performed
per each antibody manufacturer's specifications. Antibodies used were pEGFR
(Y1068), EGFR, pAKT (S473), AKT, pERK (T202/Y204), ERK, cleaved PARP, cleaved
caspase-3, SOX2, BIM, phospho-FOXO1 (T24)/FoxO3a (T32), FOXO1, FOXO3 (Cell Signaling
Technology, Beverly, MA), GAPDH (EMD Millipore, Billerica, MA), Ki67 (Epitomics,
Burlingame, CA), phospho-FOXO6 (S184) (Abcam, Cambridge, MA), FOXO6 (Proteintech,
Chicago, IL), CD133, GKLF, OCT4, MYC (Santa Cruz, Dallas, TX), CD44 and CD24 (BD
Biosciences, San Jose, CA), and TY1 (Diagenode, Denville, NJ).
Chromatin immunoprecipitation
ChIP assays were carried out using approximately 5–10 × 106
HCC827 cells, following the procedures described previously (Mikkelsen et al., 2007; Ku et
al., 2008). In brief, chromatin from formaldehyde-fixed cells was
fragmented to a size range of 200–700 bases with a Branson 250 sonifier.
Solubilized chromatin was immunoprecipitated overnight with goat anti-SOX2 antibody
or goat IgG as a negative control (both antibodies are from R&D Systems,
Minneapolis, MN). Antibody–chromatin complexes were pulled down with protein
G-Dynabeads (Thermo Fisher Scientific), washed, and then eluted. After crosslink
reversal, RNase A, and proteinase K treatment, immunoprecipitated DNA was extracted
with the Agencourt AMPure XP PCR Purification Kit (Beckman Coulter, Brea, CA). ChIP
DNA was quantified with Qubit (Thermo Fisher Scientific).
ChIP seq
5 ng purified DNA (immunoprecipiated chromatin and input controls) were used to
prepare Illumina compatible sequencing libraries for sequencing using the MiSeq
Desktop Sequencer (Illumina, San Diego, CA). ChIP seq reads were aligned to the hg19
reference genome using BWA (Li and Durbin,
2009). Aligned reads were extended to 200 bp to approximate fragment sizes,
and then 25-bp resolution density maps were derived by counting the number of
fragments overlapping each position, using IGV tools (Robinson et al., 2011). The density maps were normalized to 5
million reads, and IGV was used to visualize ChIP seq coverage maps (Thorvaldsdottir et al., 2013).
For immunofluorescence (IF) analysis, cells plated in chamber slides were fixed with
4% formaldehyde, permeabilized/blocked with 5% normal goat serum/0.3% Triton X-100
and then incubated overnight at 4°C with antibody to SOX2 (either
rabbit—Cell Signaling Technology or goat—R&D systems). SOX2
staining was visualized with appropriate Alexa Fluor conjugated secondary antibodies
(Jackson Immunoresearch, West Grove, PA). Ki-67 costaining was performed with
antibody to Ki-67 conjugated to Alexa Fluor 488 (Epitomics). Nuclei were visualized
with DAPI. Immunohistochemistry (IHC) of SOX2 on formalin-fixed, paraffin embedded
tumor tissue was performed according to the antibody manufacturer's suggested
protocol by the Specialized Histopathology Laboratory of the Massachusetts General
Hospital using the SignalStain Boost IHC detection reagent (Cell Signaling
Technology). All IF/IHC samples were imaged and quantitated using the Vectra
Automated Multispectral Imaging System (PerkinElmer, Waltham, MA). Images were
initially scanned at 4× magnification and then multiple high-powered fields
were automatically acquired. The emission spectra of each fluorophore/IHC stain was
computationally unmixed by preparing matched single stained samples. Unmixed images
were segmented to identify individual cell nuclei based on DAPI or hematoxylin
signal, and the mean nuclear signal for each fluorophore/IHC stain was calculated
using inForm Advanced Image Analysis Software (PerkinElmer). For some samples, in
addition to analyzing the mean signal for each stain on every cell analyzed,
individual nuclei were scored as positive or negative for SOX2 using the scoring
function in inForm to set a fixed threshold for SOX2 signal based on the presence or
absence of any degree of SOX2 staining. For fluorescence images, when samples to be
compared were acquired using different exposure times, data were normalized to
exposure time using the normalized counts setting/function in inForm.
Toxicity assays
Cells were plated in multiple replicate wells at 2500 cells per well in 96-well
format, treated 24 hr after plating and analyzed 72 hr later. Wells were fixed with
4% formaldehyde and stained with Syto60 red fluorescence nucleic acid stain (Thermo
Fisher Scientific) for 1 hr at room temperature. After washing each well three times
with water, the fluorescence of each well was analyzed using a Spectramax M5 plate
reader (Molecular Devices, Sunnyvale, CA) with excitation 630 nm, emission 697 nm,
and cutoff 695 nm, background corrected by subtracting the mean signal from empty
wells and normalized to the mean value of untreated wells.
siRNA
All Dharmacon ON-TARGET plus siRNA pools were purchased from Thermo Fisher
Scientific. Sequences are included in Supplementary file 1. Cells were plated at
12,500–25,000 per ml in 96-well plates (0.2 ml), 8-chamber slides (0.5 ml),
6-well plates (3 ml), or 60 mm plates (6 ml) in antibiotic-free medium and
transfected the following day with each siRNA (12.5 nM final concentration) with the
Dharmafect I transfection reagent (2 µl/200 μl for PC9 and 4
µl/200 μl for HCC827 and HCC2935) according to the
manufacturer's standard protocol. Media were changed 6–24 hr after
transfection, and isolation of mRNA and protein lysates and immunofluorescence
analysis was carried out 48–96 hr after transfection (and treatment with
erlotinib).
Lentiviral-inducible expression
Lentiviral-inducible expression constructs containing SOX2 (wild type and epitope
tagged) or WNT pathway mutants under the control of a doxycycline-inducible promoter
or control vector containing the GUS gene were constructed by subcloning each ORF
into the pInducer 20 lentiviral vector (Meerbrey et
al., 2011) using the Gateway cloning system (Thermo Fisher Scientific).
This vector also contains a neomycin resistance cassette. Lentiviral expression
constructs were cotransfected into 293T cells with the HIV-1 packaging construct
pCMVdeltaR8.91 and the VSV-G envelope construct pMD.G using TransIT-LT-1 transfection
reagent (Thermo Fisher Scientific). Viral supernatants were collected at 48 and 72 hr
post transfection in DMEM with 30% FCS, filtered through 0.45 μm syringe
filters to remove cell debris and stored in aliquots at −80°C.
Transductions were carried out in the presence of 8 μg/ml Polybrene (Sigma) by
spinoculation at 1200×g and at 32°C for 60 min in a
Sorvall Legend RT table-top centrifuge. Viral supernatant was exchanged for fresh
media 24 hr after spinoculation. To generate stable cDNA expressing cell lines, cells
were selected in G418. Tetracycline-reduced FBS (Clontech, Mountain View, CA) was
substituted for all media for cells transduced with the pInducer 20 vectors. To
induce expression of SOX2 or WNT pathway constructs, doxycycline (Sigma) at 0.1
µg/ml was added to all cultures at least 3 hr prior to addition of
erlotinib.
Luciferase reporter assay
Stable cell lines expressing the TOP-FLASH reporter were generated by transducing
cells with 7TFP recombinant lentiviruses (Fuerer
and Nusse, 2010), and luciferase assay was performed as previously
described (Singh et al., 2012). Briefly,
Rediject D-Luciferin Ultra (Perkin Elmer) was added in 0.2 ml fresh media
(1–200 dilution) to each well of cells in a 96-well plate and incubated for 15
min at 37°C. Luciferase activity was imaged with the IVIS Lumina II In Vivo
Imaging System (Perkin Elmer). The radiance of each well was determined using Living
Image 4.2 software (Perkin Elmer), background corrected by subtracting the mean
signal from empty wells and normalized both to the relative cell number of each well
as determined by Syto60 assay and the resulting normalized mean value of untreated
wells.
Xenograft tumor studies
All mouse studies were carried out according to Institutional IUCAC guidelines. Mice
were treated by oral gavage with a single 100 mg/kg dose of erlotinib when
subcutaneous tumors had reached ∼500 mm3 in sizes
(∼21–28 days). PC9 and HCC827 xenograft tumors were harvested 21 hr
after erlotinib treatment.
Statistical analyses
All statistical analyses, including number of replicates and statistical method used,
are included in the relevant figure legends.eLife posts the editorial decision letter and author response on a selection of the
published articles (subject to the approval of the authors). An edited version of the
letter sent to the authors after peer review is shown, indicating the substantive
concerns or comments; minor concerns are not usually shown. Reviewers have the
opportunity to discuss the decision before the letter is sent (see review
process). Similarly, the author response typically shows only responses
to the major concerns raised by the reviewers.Thank you for choosing to send your work entitled “Inhibition of mutant EGFR in
lung cancer cells triggers SOX2-FOXO6 dependent survival pathways” for
consideration at eLife. Your full submission has been evaluated by
Charles Sawyers (Senior editor) and 3 peer reviewers, one of whom is a member of our
Board of Reviewing Editors, and the decision was reached after discussions between the
reviewers.The comments of the reviewers are listed below. A critical issue noted by the reviewers
was that your study fails to make a direct connection between SOX2 and drug resistance.
As a result, we are returning your manuscript. The current manuscript will not be
considered further for publication. However, if new data are obtained that directly
address this criticism we would be pleased to consider a new manuscript for publication.
Please note that a new submission would be subject to full editorial review.Targeted cancer therapies can lead to the development of drug resistance. The mechanisms
that cause drug resistance need to be understood if we are to improve the efficacy of
cancer treatment. This study, which seeks to understand resistance to EGFR-targeted
therapy, is therefore significant. The authors explored gene expression changes induced
by erlotinib that might contribute to the development of resistance. The authors report
that a FOXO6 - Sox2 pathway represses expression of the pro-apoptotic proteins BIM and
BMF to in a sub-set of cells confer survival. The authors propose that this survival
pathway provides the tumor cells with the opportunity to accumulate mutations that drive
drug resistance. This conclusion, which is not tested, is interesting and potentially
important for the design of cancer therapies.Specific points:1) The authors do not establish whether their observations regarding SOX2 are relevant
to any important clinical observation or outcomes. The paper is introduced with a
discussion of resistance, but the reported study does not address resistance. Does
modulation of SOX2 have effects on drug resistance?2) To further relate the findings to drug resistance, the authors should determine what
occurs following a repeat challenge with erlotinib. The authors show that SOX2 induction
is transient. Does the proportion of cells that can increase SOX2 following erlotinib
exposure increase in the surviving cells (i.e. does erlotinib select for cells with this
phenotype)? If not, then these data may be describing some form “fractional
kill” or stochastic resistance, akin to that shown in Spencer et al., Nature,
2009. This would also be interesting, but not really related to the clinically observed
problem of acquired resistance to erlotinib and other similar inhibitors.3) The data presented show that SOX2 expression is not only heterogeneous, but also very
rare. In Figure 2, for example, it appears that
< 20% (maybe less than 10%) of cells express SOX2 after erlotinib exposure. Thus,
it is unclear why removing SOX2 from ∼10-20% of cells would have the pronounced
effects on erlotinib-mediated death that are shown in Figure 4A-B. The dose of erlotinib used in Figure 2 is very low. Will the proportion of cells with SOX2 induction
increase as a function of erlotinib dose?4) At least 70% of patients who acquire resistance to EGFR inhibition do so on the basis
of identifiable new mutations in EGFR or MET. I know the authors discuss a sort-of
resistant state that SOX2. The authors should examine whether modulation of SOX2 affects
selection for mutants in vivo.5) BIM is identified as a key player in SOX2 signaling based on SOX2 over-expression
assays (Figure 5A). However, SOX2 siRNA causes
very small effects on BIM protein expression (Figure
7D). Since erlotinib causes major loss of ERK activity (Figure 7D), this would be anticipated to cause BIM stabilization.
One interpretation of Figure 7D is therefore that
SOX2 is not very important for BIM protein expression in this system. Figure 4C using SOX2 over-expression would appear to
support this conclusion (although in that experiment, it is not clear why SOX2 is not
induced by erlotinib in the control cells, but oddly is induced in the SOX2
over-expressing cells, which appear not to express SOX2 at time = 0).6) BIM and BMF are identified as SOX2 target genes. Evidence to support this conclusion
is required (e.g. ChIP assays).7) The conclusion that FOXO6, but not other FOXO family members, mediates effects of
erlotinib on SOX2 expression is not fully established. First, redundancy of FOXO
isoforms is not considered, this is especially a problem since the efficiency of
FOXO1/3a/4 knockdown is not presented. Second, ChIP assays are required to test the
chromatin interactions of these FOXO proteins in the cells employed in this study. The
connection to FOXO6 would be strengthened if it were shown that FOXO6 function was
heterogeneous and matched SOX2 expression.8) There are a number of technical concerns with the analysis presented. First, the
siRNA studies are poorly described, for example, the siRNA are not defined. Second,
important controls are missing. Only one siRNA is presented for each knockdown and no
rescue studies were performed. Evidence that the siRNA caused a knockdown is missing;
for example in Figure 3. In other cases the
extent of knockdown appears limited (e.g. FOXO6 in Figure 3C). Since the study is highly dependent on the use of siRNA
approaches, these questions limit the impact of the study.[Editors' note: further revisions were requested prior to acceptance, as
described below.]Thank you for choosing to send your work, “Inhibition of mutant EGFR in lung
cancer cells triggers SOX2-FOXO6 dependent survival pathways”, for further
consideration by eLife. Your submission has been assessed by Detlef
Weigel and Charles Sawyers in consultation with a member of the Board of Reviewing
Editors. We consider that the manuscript has been improved by the inclusion of new data
and revisions to the text. However, there are a number of points in the original review
that were not adequately addressed, specifically:5) Effect of SOX2 on BIM protein expression. Figure
4C (now Figure 5–figure supplement
1B) shows that the large increase in SOX2 expression does not appear to cause
major changes in BIM expression. Moreover, why is BIM expression in lane 5 slightly
greater than in lane 1? These data do not make a strong case for a role of SOX2 in the
regulation of BIM protein expression. This is consistent with the modest effects of SOX2
knockdown to increase BIM protein expression (Figure
5–figure supplement 4). The authors need to clarify the experimental
basis for their conclusion that BIM protein contributes to the effects of SOX2.7) The conclusion that FOXO6 mediates the effects of erlotinib on SOX2 expression is not
well supported by the data, because it appears that the RNAi data presented is
confounded by off-target effects. Thus, siFOXO6 causes decreased expression of all other
FOXO mRNA tested (Figure 7B) and siFOXO1/3a/4
causes decreased FOXO6 mRNA expression (Figure
7B). These data do not allow the authors to distinguish between the roles of
different FOXO proteins (and raise questions concerning what other genes might be
affected). The authors do present additional data to address this point (Figure 7D) by using more than the single siRNAs
employed elsewhere; but no controls for off-target effects on other FOXO proteins are
presented.1) The authors do not establish whether their observations regarding SOX2 are
relevant to any important clinical observation or outcomes. The paper is introduced
with a discussion of resistance, but the reported study does not address resistance.
Does modulation of SOX2 have effects on drug resistance?We now show new data in Figure 5E, demonstrating
that transient siRNA-mediated knockdown of SOX2, coincident with erlotinib exposure,
decreases the subsequent development of stable, acquired resistance to erlotinib. While
continuous culture of control PC9 cells in erlotinib for two weeks leads to the
emergence of erlotinib-resistant clones, the number of resistant colonies is
significantly decreased when SOX2 induction by erlotinib is prevented with siRNA.2) To further relate the findings to drug resistance, the authors should
determine what occurs following a repeat challenge with erlotinib. The authors show
that SOX2 induction is transient. Does the proportion of cells that can increase SOX2
following erlotinib exposure increase in the surviving cells (i.e. does erlotinib
select for cells with this phenotype)? If not, then these data may be describing some
form “fractional kill” or stochastic resistance, akin to that shown in
Spencer et al., Nature, 2009. This would also be interesting, but not really related
to the clinically observed problem of acquired resistance to erlotinib and other
similar inhibitors.We undertook the requested retreatment experiments and we show in the new Figure 2B for HCC827 cells (and in Figure 2–figure supplement 4A for PC9
cells) that re-challenge with erlotinib after a period of recovery from two prior
treatments (75-90% cell killing for each treatment), does not increase the proportion of
cells with SOX2 inducible expression. Instead, SOX2 expression returns to basal levels
in the absence of drug, and re-treatment re-induces SOX2 to similar levels and with
similar cell distribution as with the prior treatment. These data are consistent with a
stochastic model of SOX2 induction. As noted by the reviewer, a similar stochastic cell
killing model was described in Spencer et al, Nature, 2009 with respect to TRAIL-induced
apoptosis. However, this in no way suggests absence of clinical significance, since (as
shown in Point #1 above), the transient expression of SOX2 itself enhances the
subsequent emergence of stably resistant colonies. Progressively increasing fractions of
SOX2 inducible cells following each erlotinib treatment are not expected in this
model.We also now show in the new Figure 2–figure
supplement 4B, that in PC9 cells that have developed acquired resistance
following long-term, continuous erlotinib treatment, stochastic
induction of SOX2 still occurs but only at the much higher doses of erlotinib required
to inhibit EGFR in these resistant cells.Taken together, these data support the heterogeneous, transient and stochastic induction
of SOX2 following erlotinib treatment as a precursor to the development of stable
acquired drug resistance.3) The data presented show that SOX2 expression is not only heterogeneous, but
also very rare. In
, for example,
it appears that < 20% (maybe less than 10%) of cells express SOX2 after
erlotinib exposure. Thus, it is unclear why removing SOX2 from ∼10-20% of
cells would have the pronounced effects on erlotinib-mediated death that are shown
in
. The dose of
erlotinib used in
is very low. Will the proportion of cells with SOX2 induction increase as a
function of erlotinib dose?In our experiments, the fraction of SOX2+ cells varies from 17-24% for HCC827
cells and 18-32% for PC9 cells (see Figure 2 and
Figure 2–figure supplement 1). Given
the threshold for immunofluorescence detection, it is likely that our immunofluorescence
assay underestimates the number of cells with SOX2, and as such we
propose a heterogeneous range of expression, rather than a truly rare and unique subset
of the population. It is also difficult to determine precisely what level of SOX2
expression in an individual cell is required for a measurable effect on cell survival.
We note that unlike the single cell data in Figure
2A, the new Figure 5B shows an
immunoblot assay using total cell lysates, which obscures the contribution of individual
cells to the apoptotic effect of SOX2 knockdown.As suggested by the reviewers, we also increased the dose of erlotinib beyond that
initially tested. We show in the new Figure
2–figure supplement 1, that further increasing the dose of erlotinib
beyond that required for full inhibition of EGFR signaling to 1.0μM does not
substantially increase the fraction of SOX2+ HCC827 cells (Increased cell death
in PC9 cells at the highest concentration precludes acute SOX2 induction analysis).4) At least 70% of patients who acquire resistance to EGFR inhibition do so on
the basis of identifiable new mutations in EGFR or MET. I know the authors discuss a
sort-of resistant state that SOX2. The authors should examine whether modulation of
SOX2 affects selection for mutants in vivo.The reviewers bring up the important point that stably acquired resistance to EGFR
inhibitors can take many forms, from secondary gatekeeper mutations within EGFR, to
induction of MET or other “bypass tracks”, to epithelial-mesenchymal
transition (EMT) and even lineage switching from non-small cell to small cell histology.
All of these complex mechanisms are noted in patients after prolonged treatment with
erlotinib, but only few of these are well recapitulated using the standard cell line
models, which tend to favor the gatekeeper mutation mechanism (even MET amplification is
very rare in vitro). There are ample previous publications in this area. Given its
complexity, we feel that defining the precise mechanism of long-term resistance in
various PC9 and HCC827 clones, and the extent to which the various frequencies might be
altered in the rare colonies that do ultimately emerge despite SOX2 suppression, would
be beyond the scope of this manuscript.5) BIM is identified as a key player in SOX2 signaling based on SOX2
over-expression assays (). However, SOX2 siRNA causes very small effects on BIM
protein expression (). Since erlotinib causes major loss of ERK activity
(),
this would be anticipated to cause BIM stabilization. One interpretation of
is therefore that SOX2 is not very important for BIM protein expression in this
system.
using SOX2 over-expression would appear to support this conclusion (although in
that experiment, it is not clear why SOX2 is not induced by erlotinib in the control
cells, but oddly is induced in the SOX2 over-expressing cells, which appear not to
express SOX2 at time = 0).The reviewers correctly point out that the activity of BIM is controlled at several
levels—both transcriptional and post-translational (in the latter case, primarily
by ERK-dependent phosphorylation/stabilization). Our data suggest transcriptional
regulation by SOX2 as an additional mechanism that contributes to BIM regulation, but it
is clearly not the only one (for example, the loss of MAPK pathway activity following
erlotinib treatment likely leads to stabilization of the pool of previously transcribed
BIM, that would be unaffected by SOX2 modulation of new BIM transcription). However, the
effect of manipulating SOX2 levels on the degree of apoptosis clearly demonstrates that
in certain cellular contexts, SOX2 can have an important, functional role.We have replaced Figure 4C with the blot in Figure 5A (previously a supplemental figure in the
original manuscript), so as to remove any confusion about how the experiment was
performed. In this experiment, doxycycline was added for two hours to induce exogenous
SOX2 and then removed prior to the addition of erlotinib for 24 hours, followed by
preparation of lysates and immunoblot with the indicated antibodies. Here, both
induction of physiologic levels of exogenous, epitope-tagged (and slower migrating) SOX2
by doxycycline, and induction of endogenous SOX2 by erlotinib, can be clearly
appreciated, still resulting in decreased apoptotic PARP and Caspase-3 cleavage. We have
moved the original Figure 4C to Figure 5–figure supplement 1B and clarified
how the experiment was done in the accompanying legend.6) BIM and BMF are identified as SOX2 target
genes. Evidence to support this conclusion is required (e.g. ChIP
assays).As requested by the reviewers we have performed ChIP assays. In the new Figure 6B, we use CHIP seq and ChIP qPCR to
demonstrate erlotinib-induced binding of endogenous SOX2 to upstream regions of both the
BIM and BMF genes.7) The conclusion that FOXO6, but not other FOXO family members, mediates
effects of erlotinib on SOX2 expression is not fully established. First, redundancy
of FOXO isoforms is not considered, this is especially a problem since the efficiency
of FOXO1/3a/4 knockdown is not presented. Second, ChIP assays are required to test
the chromatin interactions of these FOXO proteins in the cells employed in this
study. The connection to FOXO6 would be strengthened if it were shown that FOXO6
function was heterogeneous and matched SOX2 expression.The efficiency of knockdown for all FOXO siRNAs in Figure 3 is shown in the new Figure 7B
using qPCR. For all FOXO family members where good quality antibodies are available, we
have also added Western blot assays (new Figure
7–figure supplement 2B). We agree with the reviewer’s comment
about the redundancy of FOXO isoforms, and this is shown in multiple FOXO knockdown
experiments (Figure 7B). However, the triple
knockdown of FOXO 1, FOXO3a and FOXO4 does not decrease SOX2 induction like single FOXO6
knockdown.To further support the role of FOXO6 alone, we use four different siRNAs to confirm that
its down regulation suppresses SOX2 induction by erlotinib in both HCC827 and PC9 cells
(Figure 7D).As requested by the reviewers, we show in the new Figure 7–figure supplement 3 that FOXO6 expression is heterogeneous,
and correlated with SOX2 expression.We were unfortunately unable to demonstrate binding of FOXO6 to the promoter of SOX2 by
chromatin immunoprecipitation, most likely due to the poor quality of available
antibodies, since ChIP seq tracks for FOXO6-bound chromatin were inadequate across the
genome.8) There are a number of technical concerns with the analysis presented. First,
the siRNA studies are poorly described, for example, the siRNA are not defined.
Second, important controls are missing. Only one siRNA is presented for each
knockdown and no rescue studies were performed. Evidence that the siRNA caused a
knockdown is missing; for example in
. In other cases
the extent of knockdown appears limited (e.g. FOXO6 in
). Since the
study is highly dependent on the use of siRNA approaches, these questions limit the
impact of the study.We apologize for these omissions. siRNA target sequences are now clearly defined in the
Methods sections of the manuscript. The new Figure
5C demonstrates dramatic rescue of the apoptotic effect of the most potent
siRNA targeting SOX2 by a siRNA resistant, exogenous SOX2.Figure 7C also now demonstrates that four
independent siRNA that effectively target FOXO6 decrease erlotinib-induced induction of
SOX2 (shown above under point 7).The degree of FOXO6 knockdown in Figure 3C at the
protein level is 50% (despite 80-90% knockdown at the mRNA level), which may be due
technical issues related to the antibodies used (see Discussion about ChIP for FOXO6
above), or to the relatively long half-life of the protein (we waited up to 96 hours
after siRNA transfection to assay for knockdown). Consistent with some residual FOXO6
protein remaining after knockdown, we observed significant, though incomplete
suppression of SOX2 induction by erlotinib (see Figure
7D).[Editors' note: further revisions were requested prior to acceptance, as
described below.]We consider that the manuscript has been improved by the inclusion of new data
and revisions to the text. However, there are a number of points in the original
review that were not adequately addressed, specifically:5) Effect of SOX2 on BIM protein expression.
(now
) shows that the large increase in SOX2
expression does not appear to cause major changes in BIM expression. Moreover, why is
BIM expression in lane 5 slightly greater than in lane 1? These data do not make a
strong case for a role of SOX2 in the regulation of BIM protein expression. This is
consistent with the modest effects of SOX2 knockdown to increase BIM protein
expression (). The
authors need to clarify the experimental basis for their conclusion that BIM protein
contributes to the effects of SOX2.The critical point about BIM is its relative induction compared to
baseline, rather than a comparison of basal levels across two panels. As we
show quantitatively in Figure 5–figure
supplement 1B (together with the relevant protein bands from the referenced
supplementary figure), the levels of each BIM isoform are clearly increased by 12 hours
of treatment with erlotinib in the absence of ectopic SOX2 (blue lines). The
erlotinib-mediated increase in BIM is clearly suppressed over the same
time period by the simultaneous addition of erlotinib and ectopic SOX2 induction (red
lines). The slightly increased basal BIM levels at time zero in lane 5 versus lane 1
noted by the reviewer are most likely due to experimental variation and are not relevant
to the apoptotic effects observed.This quantitation is now included with the revised Figure 5–figure supplement 1B. In addition, we in the revised Figure 5–figure supplement 1C a different
experiment using tagged, ectopic SOX2 and extend the time of treatment to 24 hours.
Here, the levels of ectopic SOX2 induction (lanes 5-6) are comparable to endogenous SOX2
induction by erlotinib (lane 2-3); decreased BIM levels are clearly apparent (the use of
a lower percentage acrylamide gel does not permit BIM L and S to be distinguished from
EL).To address the question of large changes in SOX2 expression leading to smaller changes
in BIM expression, we would not expect a linear relationship between arbitrary
“levels” of SOX2 versus BIM. What is important here is the functional
consequence of BIM and BMF induction (and of preventing their induction by siRNA or
ectopic SOX2), which we clearly demonstrate.As to the major data relating to SOX2 and BIM, we note the following: in our manuscript,
we claim that the transient induction of SOX2, following acute suppression of mutant
EGFR signaling, reduces induction of two pro-apoptotic proteins, BIM
and BMF, thereby reducing cell death and setting the stage for acquired drug resistance
to EGFR inhibitors. This is based on several lines of evidence:(1) Potent suppression of BIM and BMF by ectopic SOX2 (Figure 5–figure supplement 1B, Figure
6A and its associated figure supplement 1, together with the new data
above).(2) Increased BIM following SOX2 knockdown in the presence of erlotinib (Figure 5–figure supplement 4).(3) Direct, erlotinib-dependent binding of endogenous SOX2 to both BIM and BMF
regulatory regions by ChIP assays (Figures 6A and
B).(4) The functional consequences of this SOX2-mediated effect is demonstrated by the
cooperative suppression of erlotinib-induced apoptosis, when both BIM and BMF induction
are prevented using siRNA (Figure 6C).7) The conclusion that FOXO6 mediates the effects of erlotinib on SOX2
expression is not well supported by the data, because it appears that the RNAi data
presented is confounded by off-target effects. Thus, siFOXO6 causes decreased
expression of all other FOXO mRNA tested () and siFOXO1/3a/4 causes decreased
FOXO6 mRNA expression (). These data do not allow the authors to distinguish
between the roles of different FOXO proteins (and raise questions concerning what
other genes might be affected). The authors do present additional data to address
this point () by using more than the single siRNAs employed elsewhere;
but no controls for off-target effects on other FOXO proteins are
presented.Our critical finding is that only knockdown of FOXO6 significantly
decreases SOX2 induction by erlotinib. Given the reviewer’s concern about
possible nonspecific effects of the siRNA pool targeting FOXO6, we have now added data
on different FOXO isoform levels for each of the four individual siRNAs that comprised
the FOXO6 pool (see the revised Figure 7–figure
supplement 2C). Two different siRNA duplexes (-01 and -02) are uniquely
targeting FOXO6, without effect on other FOXO family members. Two other siRNAs within
the pool (-03 and -04) do have some off-target effect on FOXOs1, 3a and 4. Note in the
new figure that with 24 hours of erlotinib treatment, FOXO6 is induced by erlotinib to a
much greater degree than the other FOXOs in both HCC827 and PC9 cells, hence these
off-target effects are less impressive than the on-target effect on FOXO6. This new data
suggests that the nonspecific effects of the FOXO6 siRNA pool on other FOXOs are likely
due to duplexes -03 and -04.In addition to two independent siRNAs (-01, -02) being highly specific
in knocking down FOXO6 and reducing SOX2 expression, it is key to also note that
we have tested the other FOXO family members directly for their effect on SOX2
expression (). siRNAs that effectively knock down either
FOXO1, FOXO3a or FOXO4 have no significant effect on SOX2. Hence, the off target effects
of FOXO siRNAs -03 and -04 on other FOXOs are not responsible for suppression of SOX2
expression.In addition to addressing the reviewers’ concern regarding siRNA off-target
effects, the role for FOXO6 is further supported by our demonstration of FOXO6
co-localization with SOX2 (Figure 7–figure
supplement 3), as requested by the reviewers of our initial submission. With
respect to other pathways, we used multiple approaches to test several other (e.g.
non-FOXO) pathways implicated in SOX2 regulation, including FGF, WNT and NKX2.1 (Figure 7–figure supplement 4), and none of
these affected SOX2 induction by erlotinib. Therefore, our conclusion, that FOXO6 is the
primary mediator of erlotinib’s effect on SOX2 expression, is
well supported by the data.We thank the reviewer/editors for this opportunity to address the remaining concerns
from our resubmitted manuscript. Together with the extensive additional data and edits
provided in our revised manuscript, we provide multiple lines of evidence that SOX2
induction contributes to upfront and acquired resistance to erlotinib in EGFR-mutant
lung cancer cells. These include:(1) Enhancement of acute erlotinib-induced apoptosis and suppression of long-term
acquired resistance, when SOX2 induction is prevented with siRNA (an effect that is
completely rescued by a SOX2 construct that is resistant to siRNA targeting).(2) Detailed analysis of the heterogeneous, transient and stochastic nature of SOX2
induction in multiple EGFR-mutant cell lines, xenografts and patient-derived models.(3) A proposed mechanism for both mutant EGFR’s regulation of SOX2 through FOXO6,
and SOX2’s regulation of apoptosis through BIM and BMF.(4) A demonstration of the functional importance of high basal SOX2 for upfront
erlotinib resistance, in addition to acquired drug resistance.The critical importance of acute and transient signaling feedback pathways for the
development of resistance to targeted anti-cancer therapies has been established for
several oncogene-addicted cancers (primarily mutant BRAF-driven melanoma and colorectal
cancer). We show that similar pathways are involved in EGFR-mutant lung
cancer. These studies, by their very nature, may be different from well-characterized
developmental roles of FOXO and SOX genes, but their adaptation in cancer cells and
their contribution to acquired resistance to signaling inhibitors is of significant
biological interest and high clinical relevance. Given the scope of our observations and
the readily addressed points raised by this secondary review, we are most grateful for
your consideration.
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Authors: Lin Chen; Simon N Willis; Andrew Wei; Brian J Smith; Jamie I Fletcher; Mark G Hinds; Peter M Colman; Catherine L Day; Jerry M Adams; David C S Huang Journal: Mol Cell Date: 2005-02-04 Impact factor: 17.970
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Authors: Akira Ooki; Maria Del Carmen Rodriguez Pena; Luigi Marchionni; Wikum Dinalankara; Asma Begum; Noah M Hahn; Christopher J VandenBussche; Zeshaan A Rasheed; Shifeng Mao; George J Netto; David Sidransky; Mohammad O Hoque Journal: Cancer Res Date: 2017-11-27 Impact factor: 12.701
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