Epithelial-mesenchymal Transition Transcription Factors Zeb1 Research Articles

Abstract 3305: Markers of epithelial-mesenchymal transition as indicators of prognosis in lymph node aspirates from lung cancer patients

Abstract Lung cancer accounts for the largest number of cancer-related deaths worldwide. Despite a new more discriminatory staging system for predicting survival, great variation in outcome within stage persists. Gene expression arrays and microRNA signatures from surgically resected primary tumors can predict survival and drug sensitivity within stage I disease better than standard staging protocols. Genes associated with epithelial mesenchymal transition (EMT) contribute to the malignant phase of tumor cell growth by controlling the switch to a metastatic phenotype. We hypothesize that the analysis of expression of EMT markers in thoracic lymph nodes may predict survival outcomes and drug responses in lung cancer patients. Endobronchial ultrasound fine needle aspiration (EBUS-FNA) of lymph nodes was performed on patients with known or suspected lung cancer to diagnose and/or stage disease. Total RNA was isolated from the FNA specimens, reverse transcribed, and analyzed for expression of a panel of genes associated with EMT using a probe-based real-time quantitative polymerase chain reaction (pqRT-PCR). Samples were analyzed for the expression of the epithelial markers cytokeratin 17 (CK17) and cytokeratin 19 (CK19) and the EMT markers Ecadherin (ECAD), Ncadherin (NCAD), and transcription factors snail, slug, ZEB1 and ZEB2. Samples from thirty-seven patients were processed for the study. Patients with metastatic disease had higher levels of snail and NCAD and lower levels of slug and ZEB2 compared to node negative and node positive patients, while patients with node negative disease had higher slug and lower levels of NCAD compared to node positive patients. ECAD did not vary by node positivity or presence of metastasis. The patients with adenocarcinoma on cytology showed higher levels of ECAD and snail, while patients diagnosed with squamous cell carcinoma had higher levels of ZEB1. In contrast, two patients with benign disease had low levels of all markers. Four patients with cytopathologically negative nodes expressed the epithelial markers CK17 and CK19, which are not typically present in lymph nodes. In limited follow-up, two patients with cytopathologically negative lymph nodes but high levels of ZEB1 had progression of disease. From this pilot study we have shown that EBUS-FNA consistently provides adequate samples from which RNA can be isolated and that genes associated with EMT can be used to identify cancer cells that have migrated to adjacent lymph nodes. We plan to enroll a total of 100 patients and follow them for 2 years to determine if this technique can identify patients at higher risk of metastatic disease. 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 3305.

Taz-Tead1 Links Cell-Cell Contact to Zeb1 Expression, Proliferation, and Dedifferentiation in Retinal Pigment Epithelial Cells

PURPOSE. The Hippo signaling pathway imposes the cell contact inhibition that establishes organ size and tissue topology from Drosophila to mammals. This pathway regulates activity of the Yap and Taz transcription factors, which link epithelial-mesenchymal transition (EMT) to cell proliferation. Here, the authors provide evidence that Taz and its coactivator, Tead1, regulate expression of the EMT transcription factor Zeb1 to control RPE cell proliferation and differentiation. METHODS. Real-time PCR was used to examine mRNA expression during RPE dedifferentiation in primary cultures of RPE cells and after knockdown of Yap and Taz by lentivirus shRNA. Immunofluorescence was used to follow subcellular localization of proteins in cells. Chromatin immunoprecipitation was used to detect Taz at the Zeb1 promoter in vivo. RESULTS. Zeb1 is overexpressed during RPE dedifferentiation, leading to cell proliferation, EMT, and repression of the RPE specification transcription factor gene Mitf. Taz-TEAD1 translocation to the nucleus coincides with loss of cell-cell contact and with onset of Zeb1 expression in the nucleus. shRNA knockdown of Taz prevented the overexpression of Zeb1 and, in turn, prevented proliferation, repression of Mitf and Mitf target genes, and EMT when RPE cells were placed in primary culture. Taz binds to the Zeb1 promoter in vivo, suggesting that it directly induces Zeb1 transcription. CONCLUSIONS. These results provide evidence of a molecular mechanism linking cell-cell contact to cell proliferation and dedifferentiation in RPE cells.

Zeb1 RepressesMitfand Regulates Pigment Synthesis, Cell Proliferation, and Epithelial Morphology

Epithelial-mesenchymal transition (EMT) is important in fibrotic responses, formation of cancer stem cells, and acquisition of a metastatic phenotype. Zeb1 represses epithelial specification genes to enforce epithelial-mesenchymal phenotypic boundaries during development, and it is one of several E-box-binding repressors whose overexpression triggers EMT. The purpose of this study was to investigate the potential role for Zeb1 in EMT leading to the dedifferentiation of retinal pigment epithelial (RPE) cells. Real-time PCR was used to examine mRNA expression during RPE dedifferentiation in primary cultures of RPE cells from Zeb1(+/-) mice and after knockdown of Zeb1 by lentivirus shRNA. Chromatin immunoprecipitation was used to detect Zeb1 at gene promoters in vivo. Zeb1 is overexpressed during RPE dedifferentiation. Heterozygous mutation or shRNA knockdown to prevent this overexpression eliminates the onset of proliferation, loss of epithelial morphology, and pigment, which characterizes RPE dedifferentiation. Zeb1 binds to the Mitf A promoter in vivo, and Zeb1 mutation or shRNA knockdown derepresses the gene. The authors link Zeb1 expression to cell-cell contact and demonstrate that forcing dedifferentiated RPE cells to adopt cell-cell only contacts via sphere formation reverses the overexpression of Zeb1 and reprograms RPE cells back to a pigmented phenotype. Overexpression of the EMT transcription factor Zeb1 has an important role in RPE dedifferentiation via its regulation of Mitf. Expression of Zeb1 and, in turn, RPE dedifferentiation, is linked to cell-cell contact, and these contacts can be used to diminish Zeb1 expression and reprogram dedifferentiated RPE cells.