TWIST1 activates cancer stem cell marker genes to promote epithelial-mesenchymal transition and tumorigenesis in esophageal squamous cell carcinoma

Abstract

Background: Esophageal squamous cell carcinoma (ESCC) is one of the deadliest cancers worldwide. Overexpression of EMT master transcription factors can promote differentiated cells to undergo cancer reprogramming processes and acquire a stem cell-like status.

Methods: The KYSE-30 and YM-1 ESCC cell lines were transduced with retroviruses expressing TWIST1 or GFP and analyzed by quantitative reverse transcription PCR (qRT-PCR), chromatin immunoprecipitation (ChIP), and immunostaining to investigate the correlation between TWIST1 and stemness markers expression. Cells expressing TWIST1 were characterized for mRNA candidates by qRT-PCR and for protein candidates by Flow cytometry and Immunocytochemistry. TWIST1-ESCC cells were also evaluated for apoptosis and drug resistance.

Results: Here we identify a role for TWIST1 in the establishment of ESCC cancer stem cell (CSC)-like phenotype, facilitating the transformation of non-CSCs to CSCs. We provide evidence that TWIST1 expression correlates with the expression of CSC markers in ESCC cell lines. ChIP assay results demonstrated that TWIST1 regulates CSC markers, including CD44, SALL4, NANOG, MEIS1, GDF3, and SOX2, through binding to the E-box sequences in their promoters. TWIST1 promoted EMT through E-cadherin downregulation and vimentin upregulation. Moreover, TWIST1 expression repressed apoptosis in ESCC cells through upregulation of Bcl-2 and downregulation of the Bax protein, and increased ABCG2 and ABCC4 transporters expression, which may lead to drug resistance.

Conclusions: These findings support a critical role for TWIST1 in CSC-like generation, EMT progression, and inhibition of apoptosis in ESCC. Thus, TWIST1 represents a therapeutic target for the suppression of esophageal cell transformation to CSCs and ESCC malignancy.

Keywords: Cancer stem cell; Epithelial-to-mesenchymal transition; Esophageal squamous cell carcinoma; TWIST1.

Fig. 1

TWIST1 expression levels in the ESCC cell lines. A Fluorescence and inverted microscopy images of TWIST1 and GFP expression in KYSE-30 and YM-1 cells after retroviral transduction. B qRT-PCR analysis of the TWIST1 expression in KYSE-30 and YM-1 cells. C, D Western blot analysis of the TWIST1 nuclear and cytoplasmic protein expression in KYSE-30 cells expressing GFP + TWIST1 or GFP as control. The β-actin protein was used as loading control. Blots were cropped for clarity. For the original Western blot image, see Fig. S1

Fig. 2

The effect of TWIST1 on the expression of CD44 surface marker in KYSE-30 cells. A, B, C Representative flow cytometry diagrams for the CD44 expression. D, E Mean fluorescence intensity (MFI) levels of the CD44 expression in KYSE-30 cells expressing GFP + TWIST1 or GFP as control

Fig. 3

The effect of TWIST1 overexpression on the induction of stemness genes in KYSE-30 and YM-1 ESCC cells. A, B Expression levels of stem cell and ABC transporter genes were determined by qRT-PCR in KYSE-30 and YM-1 cells expressing GFP + TWIST1 or GFP as control. The error bars indicate standard error of the mean; * P < 0.01

Fig. 4

TWIST1 regulates the expression of E-cadherin, vimentin, β-catenin, Bcl2 and Bax proteins in ESCC cell lines. Representative images of ICC staining (in brown) for (A, B) E-cadherin, (C, D) vimentin, (E, F) β-catenin, (G, H) Bcl2, (I, J) Bax in KYSE-30 (upper panel) or YM-1 (lower panel) cells expressing GFP + TWIST1 or GFP as a control. Nuclei are stained with hematoxylin. The blue color indicates negative immunostaining. K Bar charts showing the percentage of positive cells for E-cadherin, vimentin, β-catenin, Bcl2, and Bax proteins to compare the TWIST1-expressing cells with the control cells. Twenty randomly chosen fields was counted at 40X magnification (≥ 150 cells) for each sample and the values of positive cells (%) were averaged. * P < 0.01. Scale bar: 100 μm

Fig. 5

TWIST1 regulates SOX2, CD44, GDF3, NANOG, MEIS1, and SALL4 gene expression through binding to their promoter region. Schematic representation of (A) SOX2, (B) CD44, (C) GDF3, (D) NANOG, (E) MEIS1, and (F) SALL4 promoter sequences indicating putative TWIST1-binding sites within a 2 kb region upstream of the TSS (+ 1). Arrows denote the canonical E-box consensus elements (CANNTG). The horizontal bars indicate the amplified promoter regions by PCR. G ChIP assays identified binding of the TWIST1 protein to the SOX2, CD44, GDF3, NANOG, MEIS1, and SALL4 proximal promoter regions. ChIP was performed in KYSE-30 cells expressing GFP + TWIST1, and was analyzed by PCR using promoter-specific primers. GAPDH was used as an internal reference gene for normalization. Anti-histone H3 antibody (ab1791, Abcam) was used as a positive control in ChIP. ChIP and PCR negative control samples consisted of no-antibody and no-chromatin, respectively. Genomic DNA from KYSE-30 cells expressing GFP + TWIST1 was used as a positive control for the input DNA. Gels were cropped for clarity. For the original gel images, see Fig. S2. H Band intensities of SOX2, CD44, GDF3, NANOG, MEIS1, and SALL4 PCR products for primer sets 1 and 2 were quantified using the Image J software, and the values were normalized to their corresponding control inputs, and the average relative band intensities are shown as bar graph. The results of triplicate experiments are shown as the mean ± S.D. (* p < 0.01, Student’s t-test). IP, immunoprecipitation; P1, Primer set 1; P2, Primer set 2

Fig. 6

Analysis of Protein–protein interaction (PPI) network. TWIST1-protein interaction networks were generated using the STRING and GeneMANIA online tools

Fig. 7

Proposed regulatory role of TWIST1 in promoting CSCs formation in ESCC. TWIST1 acts as a transcriptional regulator and triggers non-CSCs to reacquire CSC phenotypes through self-renewal and reprogramming processes (A), as well as through EMT (B)