Sa1787 Regulation of Chemotherapy Resistance Through Epithelial-Mesenchymal Transition by Zinc Finger E-Box-Binding Proteins

Abstract

Introduction: Zinc finger E-box-binding (ZEB) proteins ZEB1 and ZEB2 are transcription factors essential in transforming growth factor (TGF)-β-mediated epithelial-to-mesenchymal transition (EMT) and cancer stem cell functions. We have demonstrated recently that ZEBs are upregulated in esophageal squamous cell carcinoma (ESCC) cells with mesenchymal characteristics and increased malignant potential such as anchorage independent growth, invasiveness and tumorigenicity upon xenograft transplantation (Cancer Res. 2011;71:683647). However, the functional roles of ZEBs in chemotherapeutic resistance remain to be elucidated. Methods: Primary tumor tissues from patients who underwent surgery with or without neoadjuvant chemotherapy were analyzed. ESCC cell lines and In Vitro transformed human esophageal cells EPC2T were treated with either 5-fluorouracil or cisplatin, and subjected to WST-1 colorimetric cell proliferation assays, phase contrast imaging, flow cytometry, quantitative RT-PCR andWestern blotting analyses. Short hairpin RNA was stably transduced by lentivirus to knockdown ZEB1 and ZEB2 in a regulatable manner (Tet-On system). Results: 70% of primary tumor tissues from patients who received neoadjuvant chemotherapy (n=10) contained ZEB1 positive ESCC cells, reminiscent of EMT. By contrast, ZEB1 positive ESCC cells were found in 33% of patients without neoadjuvant chemotherapy (n=51). EMT was present in a subset of cultured ESCC cells. The percentage of mesenchymal cells correlated with downregulation of E-cadherin and upregulation of N-cadherin, Vimentin and ZEBs. Moreover, flow cytometry revealed that the mesenchymal ESCC cells express a high level of CD44, a cancer stem cell marker, implicated in invasion, metastasis and chemoresistance. Interestingly, chemotherapeutic drug sensitivity was reduced as a function of the frequency of mesenchymal subset of the cells when four different cell lines were compared. In two cell lines (TE1 and TE5) with moderate levels of mesenchymal cells, the cells surviving 5-fluorouracil treatment were predominantly mesenchymal. In HCE7 cells displaying fully mesenchymal characteristics, knockdown of either ZEB1 or ZEB2 increased 5-fluorouracil sensitivity. Moreover, TGF-β induced EMT in EPC2T cells, resulted in a significantly increased cell viability rate upon cisplatin treatment and that was antagonized by knockdown of either ZEB1 or ZEB2. Conclusions: These data demonstrate that ZEB1 and ZEB2 confer chemotherapy resistance through EMT-mediated enrichment of a unique subset of ESCC cells defined by a CD44 upregulation. Neoadjuvant therapy may induce ZEB1 positive cells, thus having broader implications upon EMT and cancer therapy.