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. 2021 May;12(10):1536-1548.
doi: 10.1111/1759-7714.13937. Epub 2021 Mar 25.

Zinc-finger E-box-binding homeobox 1 (ZEB1) plays a crucial role in the maintenance of lung cancer stem cells resistant to gefitinib

Fariz Nurwidya  1   2 , Fumiyuki Takahashi  1   2 , Wira Winardi  1   2 , Ken Tajima  1   2 , Yoichiro Mitsuishi  1   2 , Akiko Murakami  1   2 , Isao Kobayashi  1   2 , Takeshi Nara  3   4 , Muneaki Hashimoto  3   5 , Motoyasu Kato  1   2 , Moulid Hidayat  1   2 , Kentaro Suina  1   2   6 , Daisuke Hayakawa  1   2   7 , Tetsuhiko Asao  1   2 , Ryo Ko  1   2 , Takehito Shukuya  1   2 , Toshifumi Yae  1   2 , Naoko Shimada  1   2   7 , Yasuko Yoshioka  1   2 , Shinichi Sasaki  1   2 , Kazuhisa Takahashi  1   2   7
Affiliations

Zinc-finger E-box-binding homeobox 1 (ZEB1) plays a crucial role in the maintenance of lung cancer stem cells resistant to gefitinib

Fariz Nurwidya et al. Thorac Cancer. 2021 May.

Abstract

Background: Zinc-finger E-box-binding homeobox 1 (ZEB1) is an important regulator of epithelial-mesenchymal transition (EMT) and is involved in the maintenance of cancer stem cells (CSCs) via miR-200c and BMI1 pathway. Recent studies revealed that ZEB1 contributes to the EMT-mediated acquired resistance to gefitinib in EGFR-mutant non-small cell lung cancer (NSCLC). However, the precise role of ZEB1 in the maintenance of lung CSCs that lead to acquired resistance to gefitinib remains unclear.

Methods: PC9 and HCC827 NSCLC cell lines were treated with high concentrations of gefitinib, and surviving cells were referred to as "gefitinib-resistant persisters" (GRPs). ZEB1 knockdown or overexpression was performed to determine the biological significance of ZEB1 in the CSC features of GRPs, and animal models were studied for in vivo validation. Expression of ZEB1, BMI1, and ALDH1A1 was analyzed by immunohistochemistry in tumor specimens from NSCLC patients with acquired resistance to gefitinib.

Results: GRPs had characteristic features of mesenchymal and CSC phenotypes with high expression of ZEB1 and BMI1, and decreased miR-200c, in vitro and in vivo. ZEB1 silencing attenuated the suppression of miR-200c, resulting in the reduction in BMI1 and reversed the mesenchymal and CSC features of GRPs. Furthermore, ZEB1 overexpression induced EMT and increased the levels of CD133- and BMI1-positive GRPs in vitro and gefitinib resistance in vivo. Finally, ZEB1, BMI1, and ALDH1A1 were highly expressed in tumor specimens from EGFR-mutant NSCLC patients with gefitinib resistance.

Conclusions: ZEB1 plays an important role in gefitinib-resistant lung CSCs with EMT features via regulation of miR-200c and BMI1.

Keywords: ZEB1; cancer stem cells; epithelial-mesenchymal transition; gefitinib resistance; lung cancer.

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Conflict of interest statement

Kazuhisa Takahashi and Fumiyuki Takahashi received research funding from Chugai Pharm, Ono Pharma, Taiho Pharm, Nippon Boehringer Ingelheim, AstraZeneca, Pfizer, MSD, and Lilly Japan, outside the submitted work. The remaining authors declare that they have no conflicts of interest relevant to the subject of this manuscript.

Figures

FIGURE 1
FIGURE 1
Gefitinib‐resistant persisters (GRPs) of PC9 and HCC827 show gene signatures reminiscent of epithelial‐mesenchymal transition (EMT) and cancer stem cells (CSCs). (a) The mRNA expression of E‐cadherin, fibronectin, vimentin, and N‐cadherin is evaluated by quantitative real‐time PCR (qPCR). (b) The mRNA expression of stem cell‐related factors is evaluated by qPCR. (c) The mRNA expression of zinc‐finger E‐box‐binding homeobox 1 (ZEB1) and BMI1, and miRNA expression of miR‐200c is evaluated by qPCR. Data are normalized to beta actin (ACTB) expression. All values are average of triplicate experiments, with error bars indicating SEM (*p < 0.05; **p < 0.01)
FIGURE 2
FIGURE 2
PC9‐GRP tumors show increased tumor growth, and high gene expression of zinc‐finger E‐box‐binding homeobox 1 (ZEB1), low miR‐200c, high BMI1, and gene expression profiles reminiscent of epithelial‐mesenchymal transition (EMT) and cancer stem cells (CSCs). (a) Comparison of tumor size (volume) between PC9 and PC9‐GRPs in vivo. (b) Quantitative real‐time PCR (qPCR) analysis of expression of ZEB1, miR‐200c, BMI1, CD133, ALDH1A1, and vimentin. Data are normalized to expression of beta actin (ACTB) and represent mean ± SEM for at least three tumors (*p < 0.05; **p < 0.01). (c) Double‐staining fluorescence immunohistochemistry for thyroid transcription factor 1 (TTF1) to distinguish cancer tissue from noncancer tissue and evaluate the expression of ZEB1 and BMI1 in PC9‐GRPs and PCR9‐parent tumors. Scale bars indicate 200 μm. GRP, gefitinib‐resistant persisters
FIGURE 3
FIGURE 3
Stable knockdown of zinc‐finger E‐box‐binding homeobox 1 (ZEB1) reverses epithelial‐mesenchymal transition (EMT) and cancer stem cells (CSC) features in PC9‐ and HCC827‐GRPs. (a) Quantitative real‐time PCR (qPCR) analysis for confirming knockdown efficiency of ZEB1. (b) Microscopic images for ZEB1‐silenced PC9‐ and HCC827‐GRPs displaying epithelial‐like morphology versus shControl cells (scale bar indicate 200 μm). (c) qPCR analysis of mRNA expression of ZEB1 and BMI1, and miRNA expression of miR‐200c. (d) qPCR analysis of mRNA expression of vimentin and E‐cadherin. (e) qPCR analysis of mRNA expression of stem cell‐related factors. Data are normalized to beta actin (ACTB) expression. All values are average of triplicate experiments with error bars indicating SEM (ns, nonsignificant; *p < 0.05; **p < 0.01). (f) Western blotting analysis of ZEB1, CD133, and BMI1. GRP, gefitinib‐resistant persisters
FIGURE 4
FIGURE 4
Knockdown of zinc‐finger E‐box‐binding homeobox 1 (ZEB1) reverses the in vitro self‐renewal and in vivo cancer stem cells (CSCs) phenotype of PC9‐GRPs. (a) Silencing of ZEB1 in PC9‐GRPs reduces the number of spheres than shControl (**p < 0.01). (b) Fluorescence immunohistochemistry analysis of spheres using antibodies against ZEB1, Oct4, or CD133. Cell nuclei are stained with DAPI (blue). Images are obtained on an Axioplan 2 imaging system with AxioVision software, scale bars indicate 200 μm. (c) Tumor volume analysis of PC9‐GRPs transduced with shControl or shZEB1. ZEB1‐silenced gefitinib‐resistant persisters (GRPs) showed significantly reduced tumor growth than shControl GRPs (*p < 0.05). (d) Quantitative real‐time PCR analysis of mRNA expression of ZEB1, BMI1, CD133, ALDH1A1 and vimentin. Data are normalized to beta actin (ACTB) expression and represent mean ± SEM for at least three tumors (*p < 0.05; **p < 0.01). (e) Double‐staining fluorescence immunohistochemistry with thyroid transcription factor 1 (TTF1) to analyze protein expression of ZEB1 and BMI1. Scale bars indicate 200 μm
FIGURE 5
FIGURE 5
Overexpression of zinc‐finger E‐box‐binding homeobox 1 (ZEB1) increases self‐renewal ability and cancer stem cell (CSC) marker‐positive cells in PC9‐ and HCC827‐GRPs. (a) Quantitative real‐time PCR and western blotting analysis for confirming overexpression of ZEB1. (b) Sphere formation assay in PC9 and HCC827 cells overexpressing ZEB1 or mock (**p < 0.01, scale bars indicate 200 μm). (c) Quantification of half maximal inhibitory concentration (IC50) values for ZEB1‐overexpressing PC9 and HCC827 cells or mock treated with gradient concentrations of gefitinib. (d) Quantification of BMI1‐ and CD133‐positive PC9 and HCC827 cells overexpressing ZEB1 or mock treated with gefitinib. The number of CD133 and BMI1‐positive cells are counted and compared with DAPI numbers from each field. Data are shown as mean of positive cells percentage from five fields in each experiment. **p < 0.01, scale bars indicate 200 μm. GRP, gefitinib‐resistant persisters
FIGURE 6
FIGURE 6
Overexpression of zinc‐finger E‐box‐binding homeobox 1 (ZEB1) increases tumor growth and induces resistance to gefitinib in vivo. (a) Comparison of tumor size for PC9‐mock and PC9‐ZEB1 tumors treated with vehicle or gefitinib (10 mg/ml or 20 mg/ml). (b) Quantification of antitumor activity of gefitinib is calculated for individual tumors as the percentage of tumor growth inhibition, according to the following formula: 100 – [(Vx/Vc) × 100], where Vx is the tumor volume for treated mice and Vc is tumor volume in the control group at a given time, x
FIGURE 7
FIGURE 7
Specimens from lung cancer patients with acquired resistance to epidermal growth factor receptor‐tyrosine kinase inhibitors (EGFR‐TKIs) show increased expression of zinc‐finger E‐box‐binding homeobox 1 (ZEB1), BMI1, and ALDH1A1 proteins. Representative images of fluorescence immunohistochemistry staining of tumor samples from lung cancer patients pretreatment and after recurrence. Double staining using antibodies specific to either EGFR‐19del or EGFR‐L858R is performed to confirm the tumor area in the specimens. Scale bars indicate 200 μm. del, deletion
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