Crosstalk Between SOX2 and TGF-Β Signaling Regulates EGFR-TKI Tolerance and Lung Cancer Dissemination

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Author Manuscript Published OnlineFirst on August 19, 2020; DOI: 10.1158/0008-5472.CAN-19-3228 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Crosstalk between SOX2 and TGF-β signaling regulates

EGFR-TKI tolerance and lung cancer dissemination

Ming-Han Kuo1,10, An-Chun Lee1,10, Shih-Hsin Hsiao2,10, Sey-En Lin3,4, Yu-Fan

Chiu1, Li-Hao Yang1, Chia-Cherng Yu5, Shih-Hwa Chiou6,7, Hsien-Neng

Huang8, Jen-Chung Ko9*, Yu-Ting Chou1*

1Institute of Biotechnology, National Tsing Hua University, Hsinchu, Taiwan; 2Division of Pulmonary Medicine, Department of Internal Medicine, Taipei Medical University Hospital, Taipei, Taiwan; 3Department of Pathology, Taipei Medical University Hospital, Taipei, Taiwan; 4Department of Pathology, Taipei Municipal Wan Fang Hospital, Taipei, Taiwan; 5Department of Medical Research, National Taiwan University Hospital, Taipei, Taiwan; 6Institute of Pharmacology, National Yang-Ming University, Taipei, Taiwan; 7Department of Medical Research, Taipei Veterans General Hospital, Taipei, Taiwan; 8Department of pathology, National Taiwan University Hospital, Hsin-Chu Branch, Hsinchu, Taiwan; 9Department of Internal Medicine, National Taiwan University Hospital, Hsin-Chu Branch, Hsinchu, Taiwan;10Co-first authors

*Corresponding authors Jen-Chung Ko, National Taiwan University Hospital, Hsin-Chu Branch, No. 25, Lane 442, Sec. 1, Jingguo Rd., Hsinchu 30013, Taiwan. E-mail: [email protected] Yu-Ting Chou, National Tsing Hua University, No. 101, Sec. 2, Kuang-Fu Rd., Hsinchu 30013, Taiwan. E-mail: [email protected]

Running title: Interplay of SOX2 and TGF- on EGFR–TKI tolerance

Conflicts of interest The authors disclose no potential conflicts of interest. This work was supported by National Tsing Hua University and Ministry of Science and Technology, Executive Yuan, Taiwan.

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 19, 2020; DOI: 10.1158/0008-5472.CAN-19-3228 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Abstract

Regulation of the stemness factor SOX2 by cytokine stimuli controls

self-renewal and differentiation in cells. Activating mutations in epidermal

growth factor receptor (EGFR) are proven therapeutic targets for tyrosine

kinase inhibitors (TKI) in lung adenocarcinoma, but acquired resistance to TKI

inevitably occurs. The mechanism by which stemness and differentiation

signaling emerge in lung cancers to affect TKI tolerance and lung cancer

dissemination has yet to be elucidated. Here we report that crosstalk between

SOX2 and TGF-β signaling affects lung cancer cell plasticity and TKI tolerance.

TKI treatment favored selection of lung cancer cells displaying mesenchymal

morphology with deficient SOX2 expression, whereas SOX2 expression

promoted TKI sensitivity and inhibited the mesenchymal phenotype.

Preselection of EGFR-mutant lung cancer cells with the mesenchymal

phenotype diminished SOX2 expression and TKI sensitivity, whereas SOX2

silencing induced vimentin but suppressed BCL2L11 expression and promoted

TKI tolerance. TGF-β stimulation downregulated SOX2 and induced

epithelial-to-mesenchymal transdifferentiation accompanied by increased TKI

tolerance, which can interfere with ectopic SOX2 expression. SOX2-positive

lung cancer cells exhibited a lower dissemination capacity than their

SOX2-negative counterparts. Tumors expressing low SOX2 and high vimentin 2

signature were associated with worse survival outcomes in patients with EGFR

mutations. These findings provide insights into how cancer cell plasticity

regulated by SOX2 and TGF-β signaling affects EGFR-TKI tolerance and lung

cancer dissemination.

Significance

Findings suggest the potential of SOX2 as a prognostic marker in

EGFR-mutant lung cancer, as SOX2-mediated cell plasticity regulated by

TGF-β stimulation and epigenetic control affects EGFR-TKI tolerance and

cancer dissemination.

Introduction

SOX2 belongs to the SOX (Sry-related HMG Box) family of proteins and

is an important transcription factor that regulates self-renewal in embryonic

stem cells (ESCs). Its downregulation in response to cytokine stimulation

determines the timing and degree of differentiation (1). Responding to

respiratory tract injuries, SOX2 signaling initiates the proliferation and

differentiation of lung progenitor cells to maintain tissue homeostasis (2,3).

SOX2, in conjunction with OCT4, KLF4, and MYC, can reverse the

mesenchymal morphology of fibroblasts and reprogram them into induced

pluripotent stem cells (iPSCs) (4,5). Moreover, inhibition of TGF- signaling

facilitates the SOX2-mediated reprogramming process of fibroblasts, whereas

activation of TGF- blocks the reprogramming process (6,7).

Cancer cell plasticity is characterized as a phenotypic switch between

the mesenchymal and epithelial states. Said switch is accompanied by

signaling pathway alterations and has been proposed to both generate

heterogeneity and mediates tumor progression (8,9). SOX2 serves as a nodal

epigenetic regulator in determining the mesenchymal-to-epithelial

transdifferentiation (MET) of lung cancer cells (9). Additionally, SOX2 promotes

epidermal growth factor receptor (EGFR) expression via a positive feedback

manner in lung cancer (10).

Activating mutations (exon 19 deletions and L858R mutation) in EGFR are

predictive markers and therapeutic targets of TKI in the treatment of patients

with lung cancer bearing mutations in EGFR (11-16). Despite initial positive

responses to treatment, patients eventually develop resistance to EGFR–TKI.

The mutations EGFR–T790M and EGFR–C797S confer acquired resistance to

gefitinib and osimertinib, respectively (17-21). However, the mutation EGFR–

T790M correlates negatively with distant metastasis and confers better patient

survival (22-25), suggesting that an EGFR-mutation-independent

TKI-resistance mechanism promotes a worse survival outcome. EMT, a

reverse process of MET and pre-invasive status in cancer plasticity, has been

linked to EGFR–TKI resistance (26-29). Neuroendocrine transformation of

adenocarcinoma has also been detected in lung tumors after EGFR–TKI

treatment (19). A drug-tolerant state is essential for the development of

acquired resistance to EGFR–TKI (30), and deficient expression of BCL2L11

has been linked to EGFR–TKI tolerance in lung cancer (31).

Since SOX2 signaling is highly expressed in both stem cells and lung

cancer cells, this study aims to test whether the mechanism behind

SOX2-regulated stem cell differentiation and reprogramming is shared by lung

cancer cells to affect TKI tolerance and cancer dissemination. We further

characterized how crosstalk between SOX2 stemness and TGF- cytokine

signaling generates lung cancer cell plasticity with distinct TKI tolerance and

dissemination patterns.

Materials and Methods

Cell culture

HCC827 and H1975 cells were obtained from Dr. Jeff Wang (Development

Center for Biotechnology, Taiwan) and Dr. Wayne Chang (National Health

Research Institutes, Taiwan), respectively (9,10). Human fibroblasts and

fibroblast-derived iPSCs were described previously (32). HCC827GR and

H1975AZDR cells were established in our laboratory by exposure of HCC827

and H1975 to stepwise increased concentrations of gefitinib and osimertinib,

respectively (29). All lung cancer cells were tested positive for human origin

carrying specific EGFR mutations by EGFR sequencing analysis

(Supplementary Fig. S1A-S1C). All lung cancer cells were cultured in

RPMI-1640 medium containing L-glutamine (4 mM), sodium pyruvate (1 mM),

HEPES (10 mM), and FBS (10%).

Quantitative real-time PCR (qPCR) and Chromatin

immunoprecipitation-qPCR (ChIP-qPCR)

The qPCR and ChIP-qPCR assays were performed as described previously

(9). Primer sequences and probes used in qPCR are listed in Supplementary

Table S1. Detailed materials for the ChIP-qPCR are described in

Supplementary Table S2 and S3.

Chemicals and reagents

EGFR–TKIs (gefitinib, erlotinib, afatinib and osimertinib) were purchased from

Cayman Chem (Ann Arbor, MI, USA). Recombinant human TGF-β was

obtained from Sino biological (Beijing, China). Trichostatin A (TSA) were

obtained from Sigma (St. Louis, MO, USA). Romidepsin and SB-431542 were

purchased from MedChemExpress (Monmouth Junction, NJ, USA). shRNA

clones were ordered from the National RNAi Core Facility, Academia Sinica,

Taiwan. Detailed information of shRNA clones is listed in Supplementary Table

S4.

Electrical Cell Substrate Impedance Sensing (ECIS) Assay

The ECIS assay was performed as described previously (29).

Tissue samples and public domain data analysis

The specimens used in IHC were obtained from surgery or biopsy at Taipei

Medical University Hospital. Approval for this study was granted by the

Institutional Review Board protocol number CRC-04-11-05. The public gene

expression profiling data sets used in this study were analyzed as described

previously (9). The gene expression profiling data of the xenograft mouse

model were derived from the report of Bivona et al. (33). The sources of these

gene expression profiling data sets are listed in Supplementary Table S5.

Immunohistochemistry (IHC)

Score of immunoreactivity pattern of all tissues from patients were examined at

the Department of Pathology, Taipei Medical University Hospital, Taiwan. The

Allred scoring system was used to give the staining scores for the expression

of SOX2 or Vimentin based on the intensity of the staining (on a scale of 0 to 3)

(11). The characteristics of patients are listed in Supplementary Table S6.

Statistics

Overall survival curves were estimated by the Kaplan–Meier method. The

differences of overall survival between the high and low gene expressing

groups were compared by log-rank test. All statistical analyses were performed

using SPSS software, version 16 (SPSS, Inc., Chicago, IL, USA). A P-value <

0.05 was considered to reach a statistically significant difference.

Results

SOX2 downregulation and VIM upregulation during stem cell

differentiation and EGFR–TKI tolerance development

To study the effect of SOX2 expression on the mesenchymal phenotype during

stem cell differentiation and reprogramming, we monitored SOX2 and Vimentin

(VIM, a mesenchymal marker) expression in ESCs, fibroblasts, and iPSCs. We

observed that during the differentiation of ESCs to fibroblasts, SOX2 was

downregulated and VIM was upregulated (Fig. 1A). TGF- signaling

antagonizes the SOX2-mediated reprogramming process of fibroblasts (6,7).

Gene expression profiling analysis revealed that TGF-β receptors (TGFBR1

and TGFBR2) and ligands (TGFB1, TGFB2, and TGFB3) were upregulated

during ESC differentiation to fibroblasts (Supplementary Fig. S2A-S2B). In

contrast, during the reprogramming of fibroblasts toward iPSCs, SOX2 and

VIM expression levels were reversed, accompanied by decreased expression

of TGF-β receptors and ligands (Fig. 1A and Supplementary Fig. S2A-S2B).

We previously demonstrated that SOX2 regulates EGFR signaling and

maintains the epithelial feature in lung cancer cells (9,10). To verify whether

the interplay between SOX2 and VIM expression also exists during the

development of EGFR–TKI tolerance in lung cancer cells, we treated

EGFR-mutant HCC827 (delE746_A750) and H1975 (L858R/T790M) lung

adenocarcinoma cells with incremental concentrations of gefitinib and

osimertinib, respectively, for 6 months. The surviving cells were pooled,

propagated, and then further named HCC827GR and H1975AZDR. According

to phase-contrast imaging, HCC827GR and H1975AZDR cells displayed a

spindle-like phenotype, which was significantly different from that of the

parental HCC827 and H1975 cells (Fig. 1B). Clonogenic analysis confirmed

that HCC827GR cells were more tolerant to gefitinib than their parental cells

(Fig. 1C, upper). The same results were obtained regarding H1975AZDR cells,

which were more tolerant to osimertinib than their parental cells (Fig. 1C,

lower). HCC827GR and H1975AZDR cells, although tolerant to EGFR–TKI,

did not acquire the EGFR mutations T790M and C797S, respectively

(Supplementary Fig. S1A-S1C). We examined SOX2 expression in the paired

EGFR–TKI-sensitive (HCC827 and H1975) and -tolerant (HCC827GR and

H1975AZDR) cells. qPCR and immunoblotting assays revealed that SOX2

expression was downregulated in both HCC827GR and H1975AZDR cells (Fig.

1D). Gene set enrichment analysis revealed that the EMT pathway was

enriched in EGFR–TKI-tolerant cells (Supplementary Fig. S3A-S3B). We

confirmed that VIM was upregulated, and E-cadherin (E-cad) was

downregulated, in HCC827GR and H1975AZDR as compared with their

parental cells (Fig. 1E and Supplementary Fig. S4A-S4B). qPCR assays

revealed that TGFBR1/2 and TGFB1/2/3 levels were upregulated in both

HCC827GR and H1975AZDR (Supplementary Fig. S5A-S5B). Epigenetic

modification of H3K27ac and H3K4me3, which mark the active enhancer and

promoter, respectively, is highly involved in stem cell differentiation. ChIP-seq

analysis indicated that the SOX2 locus in both ESCs and iPSCs exhibited

strong H3K27ac and H3K4me3 signals, which were absent in fibroblasts

(Supplementary Fig. S6A-S6B). ChIP-qPCR revealed that H3K27ac and

H3K4me3 signals were remarkably higher along the SOX2 locus in HCC827

compared with its TKI-tolerant counterpart, HCC827GR (Fig. 1F and

Supplementary Fig. S6C). In contrast, the TGFBR1 and TGFBR2 loci in

fibroblasts exhibited strong H3K27ac signals, which were absent in ESCs and

iPSCs (Supplementary Fig. S6D). These data suggest the involvement of

epigenetic silencing of SOX2 during stem cell differentiation and EGFR–TKI

tolerance development.

SOX2 downregulation in lung cancer cells with intrinsic EGFR–TKI

tolerance

To verify whether EGFR–TKI-tolerant cells are present within the

heterogeneous population of cancer cells, we selected single clones exhibiting

the mesenchymal phenotype from EGFR–TKI treatment-naïve lung cancer

cells. Two single-cell clones (M-1 and M-2) that displayed the mesenchymal

phenotype were isolated from two 96-well plates of HCC827 cells (Fig. 2A).

qPCR analysis revealed that SOX2 and E-cad were downregulated in M-1 and

M-2, whereas VIM, TGFBR1/2, and TGFB1/2/3 levels were upregulated

relative to HCC827 (Fig. 2B and Supplementary Fig. S7A-S7B). The

clonogenic assay revealed that M-1 and M-2 were more tolerant to EGFR–TKI,

including gefitinib, erlotinib, afatinib, and osimertinib, in comparison with

parental HCC827 (Fig. 2C and Supplementary Fig. S8). As mentioned

previously, H3K27ac and H3K4me3 signals at the SOX2 locus were lower in

fibroblasts and EGFR–TKI-tolerant lung cancer cells compared with ESCs and

EGFR–TKI-sensitive lung cancer cells, respectively. It was also observed that

H3K27ac and H3K4me3 signals at the SOX2 locus were lower in EGFR–TKI

treatment-naïve M-1 and M-2 cells than in the parental HCC827 (Fig. 2D;

Supplementary Fig. S9). Trichostatin A (TSA), a pan-HDAC inhibitor, and

romidepsin, a HDAC1/2 specific inhibitor, induce stem cell differentiation, cell

cycle arrest, and cancer heterogeneity (9,34,35). Accordingly, the effect of TSA

and romidepsin on SOX2 and VIM expression was examined in EGFR-mutant

HCC827 cells. We observed that treatment with TSA or romidepsin induced

SOX2 downregulation and increased VIM expression in HCC827 cells (Fig.

2E). Cells pretreated with TSA or romidepsin were further subjected to the

gefitinib tolerance assay, and it was found that TSA or romidepsin pretreatment

enhanced gefitinib tolerance in HCC827 (Fig. 2F). Moreover, overexpression

of SOX2 rendered romidepsin-pretreated cells more sensitive to EGFR–TKI

(Supplementary Fig. S10A-S10B). ChIP-seq analysis indicated that the

TGFB1, TGFB2, TGFB3, TGFBR1 and TGFBR2 loci possess potential

HDAC1 binding sites (Supplementary Fig. S11A). We observed that both

romidepsin treatment and HDAC1 silencing could induce TGFBR1/2 and

TGFB1/2/3 expression in lung cancer cells (Supplementary Fig. S11B,

S12A-S12B). These results indicate the potential involvement of SOX2

downregulation and TGF-β signaling in EGFR–TKI tolerance.

SOX2 expression inhibits EMT and promotes EGFR–TKI sensitivity

Because BCL2L11 downregulation is associated with EGFR–TKI

tolerance (31), we further evaluated BCL2L11 expression in TKI-sensitive and

-tolerant cells. qPCR analysis demonstrated that compared with H1975 and

HCC827 cells, BCL2L11 expression was reduced in H1975AZDR and

HCC827GR cells (Supplementary Fig. S13A). Gene expression profiling and

qPCR analysis showed that BCL2L11 expression was downregulated during

ESC differentiation to fibroblasts and upregulated during SOX2-mediated

reprogramming of fibroblasts to iPSCs (Supplementary Fig. S13B).

Knockdown of BCL2L11 enriched the culture for cells harboring lower SOX2

expression accompanied by higher tolerance to EGFR–TKI compared with

parental HCC827 cells, while these BCL2L11-silenced cells displayed a

slow-growing phenotype (Supplementary Fig. S13C-S13F). Overexpression of

BCL2L11 increased sensitivity to EGFR–TKI in HCC827GR and H1975AZDR

(Supplementary Fig. S13G-S13H). Kaplan–Meier survival analysis revealed

that low BCL2L11 expression predicted a low recurrence-free survival rate in

patients with non-small cell lung cancer (NSCLC) (Supplementary Fig. S14A).

To study the effect of SOX2 expression on EGFR–TKI tolerance, BCL2L11

expression, and EMT, the SOX2 gene was knocked down in HCC827. qPCR

analysis revealed that SOX2 silencing decreased BCL2L11 expression

(Supplementary Fig. S14B). ChIP-qPCR analysis revealed that the amount of

SOX2 bound to the BCL2L11 promoter was decreased in EGFR–TKI tolerant

cells compared with their parental cells (Supplementary Fig. S14C). Moreover,

SOX2 knockdown induced VIM expression (Fig. 3A, left and middle). HCC827

cells in which SOX2 had been silenced were pooled and subjected to EGFR–

TKI treatment. The clonogenic assay determined that SOX2 knockdown

enriched EGFR–TKI tolerant cells (Fig. 3A, right). To test whether SOX2

expression prevents EMT and affects EGFR–TKI sensitivity in EGFR-mutant

lung cancer cells, we overexpressed SOX2 in HCC827, followed by EGFR–

TKI treatment. qPCR assays showed that VIM was downregulated upon SOX2

overexpression in cells (Fig. 3B, left). Clonogenic analysis revealed that SOX2

upregulation increased EGFR–TKI sensitivity in HCC827 (Fig. 3B, middle and

right). Additionally, in order to study the short-term effect of EGFR–TKI

treatment on SOX2 expression, we monitored SOX2 expression in lung cancer

cells with mutated EGFR under EGFR–TKI treatment for one day. qPCR

analysis revealed that SOX2 expression was induced by osimertinib in

HCC827 but not in SOX2-negative EGFR–TKI-tolerant HCC827GR

(Supplementary Fig. S15A-S15C). This indicates the presence of crosstalk

between the EGFR mutant and SOX2 signaling in TKI-sensitive cells. To gain

further insight into the role of SOX2 in EMT and EGFR–TKI tolerance,

HCC827 cells were exposed to gefitinib for 2 weeks. qPCR assays revealed

that gefitinib treatment selected cells harboring low SOX2 and high VIM

expression (Fig. 3C). These cells were further pooled and were named

HCC827GRs. To validate the role of SOX2 expression in the prevention of

EMT and EGFR–TKI tolerance, we expressed SOX2 ectopically in

HCC827GRs. qPCR assays demonstrated that SOX2 expression inhibited

VIM but induced E-cad expression in HCC827GRs (Fig. 3D, left;

Supplementary Fig. S16A-S16B). Clonogenic assays revealed that SOX2

expression decreased the tolerance of HCC827GRs under gefitinib treatment

(Fig. 3D, right). Altogether, these data support the conclusion that SOX2

expression inhibits the mesenchymal phenotype and decreases EGFR–TKI

tolerance in lung cancer cells with mutated EGFR.

TGF-β stimulation downregulates SOX2 and induces EMT with increased

EGFR–TKI tolerance in lung cancer cells

Knowing that TGF-β inhibits SOX2-mediated reprogramming of fibroblasts

(36), the effect of TGF-β stimulation on SOX2 expression and EGFR–TKI

tolerance was tested. qPCR analysis determined that SOX2 was

downregulated by TGF-β in HCC827 and H1975 in a time-dependent manner

(Fig. 4A-B, left). Moreover, we observed that TGF-β stimulation, while inducing

a mesenchymal phenotype, inhibited BCL2L11 expression in HCC827 cells

(Supplementary Fig. S17, S18A-B). ChIP-qPCR analysis indicated that less

SOX2 bound to the BCL2L11 promoter upon TGF-β stimulation

(Supplementary Fig. S18C). The results of the clonogenic analysis revealed

that pretreatment with TGF-β enhanced the colony growth of HCC827 and

H1975 under EGFR–TKI treatment (Fig. 4A-B, right). Cotreatment of HCC827

with a TGF-β inhibitor blocked TGF-β-mediated downregulation of SOX2 and

restored EGFR-TKI sensitivity (Supplementary Fig. S18B-D). ChIP-qPCR

assays also established that the H3K27ac and H3K4me3 signals at the SOX2

locus were lower in TGF-β treated HCC827 compared with the control cells

(Fig. 4C; Supplementary Fig. S18E). Furthermore, the clonogenic assay

revealed that SOX2 overexpression antagonized TGF-β induced colony

formation in HCC827 under gefitinib treatment (Fig. 4D). Correlation analysis

showed that SOX2 was negatively associated with TGFBR1 and TGFBR2

expression in NSCLC (Supplementary Fig. S19A-S19B). Kaplan–Meier

survival analysis further revealed that tumors harboring the

SOX2-low/TGFBR1-high or SOX2-low/TGFBR2-high signature predicted a

worse survival rate in NSCLC patients (Supplementary Fig. S19C-S19D).

These findings indicate that SOX2 downregulation by TGF-β stimulation

promotes EMT and increases EGFR–TKI tolerance, and SOX2

overexpression can interfere with both events.

Decreased barrier function and enhanced invasiveness in

SOX2-downregulated/TKI-tolerant cells

Alteration in barrier function (Rb) is involved in cellular differentiation (37).

To understand the effect of EGFR–TKI selection on cell plasticity, analysis of

electric cell–substrate impedance sensing (ECIS) was used to determine

cellular electric resistance (impedance) and cell–cell contact-mediated Rb in

HCC827 and HCC827GR cells. We found that, after seeding, the levels of both

impedance and Rb surged in HCC827 but not in HCC827GR cells (Fig. 5A).

These data confirmed that the loss of cell–cell contact adhesion occurred in

HCC827GR. Since the loss of cell–cell contact adhesion is the key event for

cancer cell dissemination, we measured the migration and invasion

capabilities of HCC827 and HCC827GR. Cell-tracking assays proved that

HCC827GR cells exhibited better migration ability than HCC827 cells (Fig. 5B).

Moreover, transwell migration and invasion assays revealed that HCC827GR

and H1975AZDR were more migratory and invasive than their EGFR–

TKI-sensitive counterparts, HCC827 and H1975 (Fig. 5C). Our findings

indicate that EGFR–TKI treatment selects cells with the property of a

decreased barrier, promoting a more invasive cellular behavior.

SOX2 mediates cell proliferation in lung cancer with mutated EGFR

To gain additional insight into the effect of EGFR–TKI selection on cell

proliferation, we performed the alamarBlue cell proliferation assay in

HCC827GR and HCC827 cells. We found that SOX2-positive HCC827

proliferated faster than SOX2-negative HCC827GR (Fig. 6A). Correspondingly,

the long-term clonogenic assay showed that SOX2-positive HCC827 and

H1975 grew faster than their SOX2-negative counterparts, HCC827GR and

H1975AZDR (Fig. 6A). To study the role of SOX2 in cell proliferation of

EGFR-mutant lung cancer cells, a knockdown procedure was performed on

SOX2 in HCC827 cells, followed by clonogenic assays and cell cycle analysis.

Clonogenic assays displayed that SOX2 silencing attenuated cell growth (Fig.

6B). Cell cycle analysis revealed that SOX2 knockdown decreased the S

phase of the cell cycle (Fig. 6C). Furthermore, we found that accompanied by

loss of SOX2 expression, HCC827GR cells harbored lower EGFR expression

than their EGFR–TKI sensitive parental cells (Fig. 6D). ChIP-qPCR analysis

indicated that the amount of SOX2 bound to the EGFR promoter was

decreased in HCC827GR and TGF--treated HCC827 compared with the

parental HCC827 cells (Fig. 6E; Supplementary Fig. S20A). SOX2

overexpression in HCC827GR promoted cell growth and enhanced EGFR

expression (Fig. 6F). Additionally, knockdown of SOX2 in HCC827 cells

decreased EGFR expression (Supplementary Fig. S20B). These findings

support the conclusion that SOX2 regulates cell growth in lung cancer cells

with mutated EGFR.

SOX2 and VIM as prognostic markers in lung adenocarcinomas with

mutated EGFR

The aforementioned data indicate that SOX2 downregulation induces

EMT and promotes cancer cell dissemination. We examined SOX2 and VIM

expression in lung tumors derived from EGFR–TKI-resistant xenograft mouse

models. In the HCC827 xenograft model, erlotinib selection enriched the cells

harboring high VIM and low SOX2 expression in relapsed tumors (Fig. 7A).

Primary lung cancer cells derived from patients who developed EGFR–

TKI-acquired resistance (MGH119-R and MGH126) were compared with cells

from an EGFR–TKI treatment-naïve patient (MGH119). It was found that SOX2

was downregulated, while VIM was upregulated, in cancers with EGFR–

TKI-acquired resistance (Fig 7B). Correlation analysis showed that SOX2 was

negatively associated with VIM expression in NSCLC (Supplementary Fig.

S21A). In addition, VIM and SOX2 expression was analyzed in primary

EGFR-mutant lung tumors through immunohistochemistry staining. Because

EMT contributes to cancer cell dissemination, we examined whether there was

a correlation between VIM expression and metastasis. We found that primary

EGFR-mutant lung cancer harboring high VIM expression tended to develop

metastases to lymph nodes and distant organs (Fig. 7C). A Kaplan–Meier

survival analysis of these patients was conducted to determine the prognostic

significance of SOX2 and VIM expression. The results of this analysis showed

a positive correlation between SOX2 and a good survival rate in patients,

whereas SOX2-low/VIM-high signature was associated with a worse prognosis

(Supplementary Fig. S21B and Fig. 7D). Subsequently, patients were stratified

into treatment with or without EGFR-TKI and a Kaplan–Meier survival analysis

further revealed that tumors harboring the SOX2-low/VIM-high signature

predicted a worse survival rate in patients treated with EGFR–TKI

(Supplementary Fig. S22A). Moreover, in the xenograft model, not only SOX2

but also BCL2L11 was downregulated in TKI-resistant tumors (Supplementary

Fig. S22B). Immunohistochemistry staining of two paired lung tumors before

and after TKI treatment confirmed that SOX2 expression was downregulated

in TKI-resistant patients (Supplementary Fig. S22C). These data support the

critical role of SOX2 in lung tumor progression.

Discussion

The role of SOX2-mediated cell plasticity in EGFR–TKI tolerance and

cancer dissemination is poorly understood. In this study, it was observed that

EGFR–TKI selection enriched the culture for cells harboring low SOX2

expression with decreased H3K27ac and H3K4me3 signals on their promotors,

whereas the suppression of SOX2 expression by TGF-β stimulation or

epigenetic modifiers promoted TKI tolerance and cancer dissemination. These

findings provide important insights into how the cell fate factor SOX2 is

regulated by TGF-β cytokine stimulation and epigenetic modification to affect

EGFR–TKI tolerance and the property of dissemination in lung cancer cells

with EGFR mutations (Supplementary Fig. S23A- S23B).

The SOX family of proteins regulates fate specification and differentiation

of stem/progenitor cells (38,39). The loss of SOX10 in rare cells is associated

with the development of BRAF inhibitor resistance and heterogeneity in

melanoma (40,41). Accumulating data indicate that SOX2 mediates

self-renewal in ESCs and adult progenitor cells (1). The downregulation of

SOX2 initiates differentiation with a morphological change and pathway switch

(1). We observed that SOX2 promoted proliferation of EGFR-mutant lung

cancer cells, and SOX2 silencing initiated EMT with increased EGFR–TKI

tolerance. Expression of SOX2 together with OCT4, KLF4, and MYC can

reprogram fibroblasts into iPSCs, and this reprogramming event can be

inhibited by TGF-β stimulation (6,7). We observed that the stimulation of

TGF-β downregulated SOX2 expression while increasing EGFR–TKI tolerance.

Ectopic SOX2 expression can interfere with these processes. These data

suggest that crosstalk between SOX2 and TGF-β signaling not only regulates

stem cell pluripotency, but also mediates cancer cell plasticity and EGFR–TKI

tolerance in lung cancer.

Slow-growing persister cells tend to develop a drug-tolerant state with

better survival ability in both bacteria and cancer cells (42,43). Hata et al.

reported that clones with acquired resistance, such as those with

EGFR-T790M, are derived from slow-growing persister cells, which show high

survival and partially resistant state under EGFR–TKI selection. We observed

that HCC827GR and H1975AZDR, although they exhibit the characteristic of

slow-grow, do not harbor EGFR-T790M and -C797S mutation, respectively,

but are tolerant to EGFR–TKIs. In this paper, we found that these persister

cells are regulated by SOX2 expression and that SOX2 deficient status

provides the cells with the features of slow growth and EGFR–TKI tolerance.

This SOX2-mediated inhibitor-tolerant state may contribute to the generation

of acquired resistant clones, such as those with EGFR-T790M mutation or

MET amplification.

It has been shown that the loss of BCL2L11 expression is associated with

EMT and EGFR–TKI tolerance (31). We found that SOX2 regulates BCL2L11

and Vimentin expression during EGFR–TKI tolerance development and ESC

differentiation/iPSC reprogramming. Knockdown of BCL2L11 increased

EGFR–TKI tolerance but decreased the growth rate of lung cancer cells,

indicating the dual roles of BCL2L11 in regulating survival and proliferation. In

addition to affecting BCL2L11 expression, we observed that the knockdown of

SOX2, while decreasing cell proliferation, induced the mesenchymal

phenotype. In contrast, SOX2 expression inhibited mesenchymal marker

expression, enhanced EGFR expression, promoted proliferation, and

increased EGFR–TKI sensitivity in lung cancer cells. We found that SOX2

bound to BCL2L11 and EGFR promoters in EGFR–TKI-sensitive cells. Positive

SOX2–EGFR feedback is essential for self-renewal in neural progenitors, and

for proliferation in lung cancer cells (10,44). It has been observed that SOX2 is

highly expressed in murine lung tumors driven by the activating

EGFR-mutation, and that the knockdown of SOX2 in HCC827 enhances cell

death under EGFR–TKI treatment (45). This phenomenon could be due to the

fact that SOX2 is essential for the growth of EGFR-mutant cells and the

simultaneous inhibition of SOX2 and EGFR by RNA interference and TKI

treatment, respectively, caused more severe cell death. In this study, we

observed that SOX2 silencing endowed cells with the characteristics of slow

growth and TKI-tolerance. The upregulation of SOX2 by acute EGFR–TKI

treatment has been implicated in EGFR–TKI tolerance (46). It was also

observed that a 4-hour treatment of sensitive, and not tolerant, cells with

EGFR–TKI induced SOX2 expression (Supplementary Fig. S15C). However,

EGFR–TKI selection enriched the cells harboring low SOX2 expression

accompanied by the EMT feature. These data suggest the presence of

crosstalk between EGFR and SOX2 signaling in TKI-sensitive cells and further

purport that the loss of SOX2 expression by stimulation of differentiation

factors, such as TGF-, switches off SOX2–EGFR signaling but induces EMT,

accompanied by decreased BCL2L11 pro-apoptotic signaling, thus increasing

EGFR–TKI tolerance.

Cancer cell plasticity plays a crucial role in lung tumor progression, a

phenomenon which is attributed in part to epigenetic regulation (9). H3K27ac

and H3K4me3 histone modifications play critical roles in stem cell fate

determination, and the breadth of H3K27ac and H3K4me3 has been linked to

cell identity and transcriptional consistency (47,48). We observed that

H3K27ac and H3K4me3 signals at the SOX2 locus in ESCs and lung cancer

cells were diminished during differentiation and EGFR–TKI selection,

respectively. We observed that HDAC1 inhibition by inhibitors or shRNAs can

induce TGF- signaling and downregulate SOX2 expression accompanied by

enhanced EGFR–TKI tolerance in lung cancer cells. Additionally, we observed

that TGF-β stimulation decreased H3K27ac and H3K4me3 signals at the

SOX2 locus and induced the mesenchymal feature and EGFR–TKI tolerance.

Treatment-naïve lung cancer cells exhibiting the mesenchymal feature display

low H3K27ac and H3K4me3 signals at the SOX2 locus and contain intrinsic

EGFR–TKI tolerance. These data indicate that cytokine stimulation and

epigenetic modification on SOX2 determine the epithelial feature and EGFR–

TKI-tolerance in EGFR-mutant lung cancer cells. Because blocking TGF-β

signaling with TGF-β inhibitors can prevent SOX2 downregulation and reduce

the EGFR–TKI tolerant state, cotreatment with TGF-β inhibitors may be

beneficial to EGFR–TKI therapy. However, the dual function of TGF-β and

SOX2 in cancer proliferation and invasion and their pleiotropic activities might

pose a challenge for the development of TGF-β inhibitors as an anti-persister

therapy (49). Recently, it has been reported that the TGF-β/EMT-induced drug

tolerant state is dependent on a druggable GPX4 pathway, making

anti-persister therapy more promising (50).

Together, our data indicate that switching SOX2 on and off generates

cancer cell plasticity. This cellular property is under cytokine stimulation and

epigenetic control and endows the cells with divergent tumor dissemination

and EGFR–TKI tolerance abilities. These results demonstrate that the

interplay between SOX2 expression and TGF-β signaling affects EGFR–TKI

treatment and cancer dissemination. Moreover, our findings suggest that

SOX2 and EMT markers in EGFR-mutant lung tumors serve as prognostic

markers of cancer progression.

Acknowledgements

Y.T. Chou received MOST109-2320-B-007-003-MY3. J.C. Ko received

MOST109-2314-B-002-175. This work was supported in part by the National

Tsing Hua University-National Taiwan University Hospital Hsin-Chu Branch

Joint Research Grant.

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Figure legends

Figure 1. SOX2 is silenced in EGFR–TKI selected lung cancer cells.

A, RNA-seq (upper, GSE73211) and qPCR (lower) analysis to assess SOX2

and VIM expression in ESCs (HUES), fibroblasts, and reprogramed iPSCs. **,

P< 0.01.

B, Representative phase contrast images of HCC827 versus HCC827GR, and

H1975 versus H1975AZDR cells. Scale bar: 100m.

C, Clonogenic analysis of HCC827 versus HCC827GR (upper), and H1975

versus H1975AZDR (lower) cells treated with indicated concentrations of

gefitinib and osimertinib, respectively, for 10 days. Photographs represent the

growth of cells stained by crystal violet.

D, qPCR (upper) and immunoblotting (lower) assays to assess SOX2

expression in HCC827 versus HCC827GR (left), and H1975 versus

H1975AZDR (right). **, P< 0.01; ***, P< 0.001.

E, qPCR analysis to assess E-cad and VIM expression in HCC827 versus

HCC827GR (left), and H1975 versus H1975AZDR (right) cells. *, P< 0.05; **,

P< 0.01; ***, P< 0.001.

F, ChIP-qPCR analysis to assess the H3K27ac signal at the SOX2 locus in

HCC827 and HCC827GR cells. **, P< 0.01; ***, P< 0.001.

Figure 2. Loss of SOX2 expression in EGFR-mutant lung cancer cells

with intrinsic TKI resistance.

A, Phase contrast images of HCC827 and its single-cell derivatives, M-1 and

M-2. Scale bar: 100 m

B, qPCR analysis of VIM (left), E-cad (middle), and SOX2 (right) expression in

HCC827, M-1, and M-2 cells. *, P< 0.05; **, P< 0.01; ***, P< 0.001.

C, Clonogenic analysis of HCC827, M-1, and M-2 cells treated with indicated

concentrations of gefitinib (left) and osimertinib (right) for 7 days.

D, ChIP-qPCR assays to assess the H3K27ac signal at SOX2 locus in

HCC827, M-1, and M-2 cells. *, P< 0.05; **, P< 0.01; ***, P< 0.001; N.S., not

significant.

E, qPCR analysis to access SOX2 and VIM expression in HCC827 cells

treated with TSA (500 nM, left) or romidepsin (1 nM, right) for 2 and 7days,

respectively. *, P< 0.05; **, P< 0.01.

F, Clonogenic analysis of HCC827 cells pretreated with TSA (500 nM, left) or

romidepsin (1 nM, right) for 2 and 7days, respectively, followed by gefitinib

treatment (1 μM) for 14 days. *, P< 0.05.

Figure 3. Effect of SOX2 expression on EGFR–TKI tolerance.

A, qPCR analysis to assess SOX2 (left) and VIM (middle) expression in

HCC827 cells transduced with the lentiviral vector encoding shRNA against

SOX2 or scrambled control (SC) shRNA. shSOX2#1 and shSOX2#2 target

different regions in SOX2 mRNA. Clonogenic analysis (right) of HCC827-SC

and HCC827-shSOX2 under the treatment of gefitinib (1 μM) for 14 days. *, P<

0.05; **, P< 0.01; ***, P< 0.001.

B, qPCR analysis (left) to assess SOX2 and VIM expression in HCC827 cells

transduced with the lentiviral vector encoding SOX2 cDNA (HCC827-SOX2) or

empty control (HCC827-Ctrl). Clonogenic analysis (middle and right) of

HCC827-Ctrl and HCC827-SOX2 cells under the treatment of gefitinib (100 nM)

for 14 days. **, P< 0.01; ***, P< 0.001.

C, qPCR analysis to assess SOX2 (left) and VIM (right) expression in survived

HCC827 cells after gefitinib (100 nM) treatment for 14 days. **, P< 0.01.

D, qPCR analysis (left) of SOX2 and VIM expression in HCC827GRs

transduced with the lentiviral vector encoding SOX2 cDNA

(HCC827GRs-SOX2) or empty control vector (HCC827GRs-Ctrl).

HCC827GRs cells were derived from survived HCC827 cells under gefitinib

(100 nM) treatment for 14 days. Clonogenic analysis (right) of

HCC827GRs-Ctrl and HCC827GRs-SOX2 cells under gefitinib (100 nM)

treatment for 14 days. **, P< 0.01; ***, P< 0.001.

Figure 4. The interplay between SOX2 expression and TGF-β stimulation

affects EGFR–TKI tolerance.

A, qPCR analysis (left) of SOX2 expression in HCC827 treated with TGF-β (1

ng/mL) for the indicated time periods. Clonogenic analysis (right) to assess the

effect of TGF-β on gefitinib resistance in HCC827. HCC827 cells were

pretreated with or without TGF-β (1 ng/mL) for 72 hr, followed by gefitinib

treatment (1 μM) for 14 days. *, P< 0.05; **, P< 0.01.

B, qPCR analysis (left) of SOX2 expression in H1975 treated with TGF-β (1

ng/mL) for the indicated time periods. Clonogenic analysis (right) of H1975

cells pretreated with TGF-β (1 ng/mL) for 72 hr, followed by osimertinib

treatment (1 μM) for 14 days. *, P< 0.05; **, P< 0.01.

C, ChIP-qPCR assays to assess the H3K27ac signal at SOX2 locus in

TGF-β-treated HCC827 or control HCC827 cells. *, P< 0.05; **, P< 0.01.

D, Clonogenic analysis to assess the effects of SOX2 expression and TGF-β

stimulation on gefitinib resistance. HCC827 cells were first transduced with the

lentiviral vector encoding SOX2 cDNA (SOX2) or empty control vector.

HCC827-SOX2 or control cells were pretreated with TGF-β (1 ng/mL) for 72 hr,

followed by gefitinib treatment (1 μM) for 14 days. ***, P< 0.001; N.S., not

significant.

Figure 5. Increased invasive ability in SOX2-low EGFR–TKI tolerant lung

cancer cells.

A, ECIS analysis to measure the changes of impedance (left) and Rb (right) in

HCC827 (gefitinib sensitive) and HCC827GR (gefitinib tolerant) cells.

B, Cell tracking analysis to measure the trajectory (left) and relative migration

distance (right) of HCC827 and HCC827GR cells. **, P< 0.01.

C, Transwell migration and invasion assays of HCC827 versus HCC827GR

(left), and H1975 versus H1975AZDR (right). **, P< 0.01; ***, P< 0.001.

Figure 6. Effect of SOX2 on cell growth.

A, AlamarBlue proliferation analysis (left) of HCC827 (SOX2-positive) and

HCC827GR (SOX2-negative) cells. Clonogenic assay (right) of HCC827

versus HCC827GR, and H1975 versus H1975AZDR. **, P< 0.01; ***, P<

0.001.

B, qPCR analysis (left) of SOX2 expression in HCC827 cells transduced with

shSOX2 (shSOX2#1) or scrambled control (SC) shRNA. Clonogenic analysis

(right) of HCC827 cells transduced with shSOX2 (shSOX2#1) or scrambled

control (SC) shRNA. **, P< 0.01; ***, P< 0.001.

C, Cell cycle analysis of HCC827 cells transduced with lentiviral vectors

encoding scramble control shRNA (SC, left) or shRNA against SOX2

(shSOX2#1, right).

D, qPCR analysis of SOX2 (left) and EGFR (right) expression in HCC827 and

HCC827GR cells. **, P< 0.01; ***, P< 0.001.

E, ChIP-qPCR analysis to assess the SOX2 signal at the EGFR locus in

HCC827 and HCC827GR cells. *, P< 0.05.

F, qPCR analysis of SOX2 (left) and EGFR (middle) expression in

HCC827GR-SOX2 and HCC827GR-Ctrl. Clonogenic analysis (right) of

HCC827GR cells transduced with the lentiviral vector encoding SOX2 cDNA

(SOX2) or empty control vector (Ctrl). **, P< 0.01; ***, P< 0.001.

Figure 7. Correlation analysis of SOX2 and EMT in primary lung tumors.

A, Gene expression profiling analysis to access SOX2 and VIM expression

levels in erlotinib-resistant xenograft tumors versus vehicle-treated control

tumors in mice injected with HCC827 cells. The data were analyzed from

Bivona database.

B, List of EGFR–TKI resistant status (upper), and gene expression analysis of

SOX2 (lower left) and VIM (lower right) in EGFR-mutant lung cancer cells

derived from the EGFR–TKI naïve tumor (MGH119) and acquired EGFR–TKI

resistant tumors (MGH119-R and MGH126) from GSE64766 database.

MGH119 and MGH119-R were obtained from the same patient.

C, Chi-Square analysis to assess the correlation between VIM expression

versus metastasis to lymph node and distant organs in EGFR-mutant NSCLC.

D, Kaplan Meier analysis to assess the correlation of SOX2 (left) and VIM

(middle) expression with the overall survival of NSCLC patients harboring

EGFR mutations. The overall survival analysis was stratified by

SOX2-high/VIM-low and SOX2-low/VIM-high signatures for Kaplan−Meier

analysis in patients (right). Different groups were compared using log-rank

test.

Crosstalk between SOX2 and TGF-β signaling regulates EGFR-TKI tolerance and lung cancer dissemination

Ming-Han Kuo, An-Chun Lee, Shih-Hsin Hsiao, et al.

Cancer Res Published OnlineFirst August 19, 2020.

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