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.

Identification and characterization of stromal-like cells with CD207+/low CD1a+/low phenotype derived from histiocytic lesions – a perspective in vitro model for drug testing

“…Moreover, we have also noted that HAN-1 cells had increased mRNA expression for TWIST1, which also correlated with elevated levels of anti-apoptotic BCL-2. This profile is consistent with the results obtained by Khales et al [ 64 ], who showed that TWIST1 might promote the stemness phenotype of esophageal squamous cell carcinoma cells suppressing their sensitivity to apoptosis via up-regulation of BCL-2 and down-regulation of BAX. The HAN-1 cells were also distinguished by elevated levels of small non-coding RNAs, namely miR-17-5p, miR-34c-5p, miR-124–1-3p, miR-145-5p and miR-320a-3p that also exists as a so-called onomiRNAs particularly involved in progression and tumorigenesis of various cancers.…”

CD44 and its implication in neoplastic diseases

“…High expression of CD44 plays roles in tumor progression and stemness in esophageal squamous cell carcinoma (ESCC). 97 , 98 , 99 CD44 is a novel stem cell marker in ESCC that has been reported to be eliminated by inhibiting the canonical NOTCH pathway. 100 Moreover, CD44v9 was shown to be strongly associated with EMT and poor prognosis in patients with ESCC.…”

Tumor biomarkers for diagnosis, prognosis and targeted therapy

“… 648 Twist is expressed in mesodermal and ectodermal-derived tissues, and it has been found that Twsit1 and Twsit2 which are structurally homologous are overexpressed in multiple human cancers. 647 , 648 Twsit1 overexpression has been confirmed to be strongly associated with aggressiveness and metastasis in cancer patients, including sarcoma, glioma, melanoma, ESCC, 649 neuroblastoma, 650 cervical cancer, 651 RCC, 652 and hematological malignancies including AML, chronic myeloid leukemia (CML), ALL, CLL, lymphomas. 653 In CML patients, the increased expression of Twist is related to tumor progression, tumor staging, and drug resistance, and Twist can be applied as a biomarker to assess MRD.…”