Abstract 2293: ZEB1 and ZEB2 promote EMT and invasion in esophageal squamous cell carcinoma

Abstract

Abstract Introduction: Epithelial-mesenchymal transition (EMT) promotes tumor invasion and metastasis. Amongst transcription factors essential in EMT, zinc finger E-box binding proteins ZEB1 and ZEB2 (ZEB) are upregulated in several tumor types due to loss of the miR-200 family. However, the role of ZEB in esophageal squamous cell carcinoma (ESCC) remains to be elucidated. Methods: ESCC cell lines were grown. Immortalized human esophageal cells EPC2-hTERT were retrovirally co-transduced with EGFR and mutant p53 (p53R175H), early genetic lesions common in ESCC, leading to transformation as described previously (Genes Dev. 2007; 21:2788). Short hairpin RNA was expressed by lentivirus to knockdown ZEB1 and ZEB2. Epithelial cells were grown in organotypic 3-D culture, a form of tissue engineering. Laser capture microdissection (LCM) was performed to isolate RNA for gene expression profiling in the invasive cells in 3-D culture as well as microRNA determination in archived paraffin embedded primary ESCC tissue samples. Real-time RT-PCR, Western blotting and immunohistochemistry were done to determine gene expression. Senescence-associated β-galactosidase activity was determined. Results: In primary ESCC, ZEB expression was found upregulated in tumor cells at the invasive front with increased Vimentin and decreased E-cadherin expressions. Moreover, LCM documented a significant downregulation of the miR-200 family in the invasive tumor cells consistent with EMT. ZEB upregulation and concomitant loss of the miR-200 family were also found in two out of 11 ESCC cell lines, exhibiting EMT corroborated by spindle-shaped cell morphology as well as an E-cadherin to N-cadherin class switch. EGFR overexpression and p53 mutation in EPC2-hTERT cells resulted in ZEB upregulation and a significantly augmented susceptibility to EMT, where robust induction of ZEB and suppression of the miR-200 family were observed upon TGF-β treatment. In 3-D culture, the cells co-transduced with EGFR and mutant p53 markedly invaded into the underlying stroma compartment. The invasive cells had a gene signature compatible with EMT along with ZEB upregulation. Finally, knockdown of either ZEB1 or ZEB2 not only prevented TGF-β from inducing EMT, but also TGF-β-mediated massive senescence in EPC2-hTERT cells transformed by EGFR and mutant p53. Conclusions: Our data indicate that ZEB1 and ZEB2 are negatively regulated by the miR-200 family and induced upon invasion in ESCC. ZEB may regulate senescence and EMT in concert with TGF-β signaling, thus contributing to esophageal carcinogenesis and tumor progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2293.