ZEB1 sensitizes lung adenocarcinoma to metastasis suppression by PI3K antagonism
Yanan Yang, Young-Ho Ahn, Yulong Chen, Xiaochao Tan, Lixia Guo, Don L. Gibbons, Christin Ungewiss, David H. Peng, Xin Liu, Steven H. Lin, Nishan Thilaganathan, Ignacio I. Wistuba, Jaime Rodriguez-Canales, Georgia McLendon, Chad J. Creighton, Jonathan M. Kurie
Yanan Yang, Young-Ho Ahn, Yulong Chen, Xiaochao Tan, Lixia Guo, Don L. Gibbons, Christin Ungewiss, David H. Peng, Xin Liu, Steven H. Lin, Nishan Thilaganathan, Ignacio I. Wistuba, Jaime Rodriguez-Canales, Georgia McLendon, Chad J. Creighton, Jonathan M. Kurie
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Research Article Oncology

ZEB1 sensitizes lung adenocarcinoma to metastasis suppression by PI3K antagonism

Abstract

Epithelial tumor cells that have undergone epithelial-to-mesenchymal transition (EMT) are typically prone to metastasis and drug resistance and contribute to a poor clinical outcome. The transcription factor ZEB1 is a known driver of EMT, and mediators of ZEB1 represent potential therapeutic targets for metastasis suppression. Here, we have shown that phosphatidylinositol 3-kinase–targeted (PI3K-targeted) therapy suppresses metastasis in a mouse model of Kras/Tp53-mutant lung adenocarcinoma that develops metastatic disease due to high expression of ZEB1. In lung adenocarcinoma cells from Kras/Tp53-mutant animals and human lung cancer cell lines, ZEB1 activated PI3K by derepressing miR-200 targets, including amphiregulin (AREG), betacellulin (BTC), and the transcription factor GATA6, which stimulated an EGFR/ERBB2 autocrine loop. Additionally, ZEB1-dependent derepression of the miR-200 and miR-183 target friend of GATA 2 (FOG2) enhanced GATA3-induced expression of the p110α catalytic subunit of PI3K. Knockdown of FOG2, p110α, and RHEB ameliorated invasive and metastatic propensities of tumor cells. Surprisingly, FOG2 was not required for mesenchymal differentiation, suggesting that mesenchymal differentiation and invasion are distinct and separable processes. Together, these results indicate that ZEB1 sensitizes lung adenocarcinoma cells to metastasis suppression by PI3K-targeted therapy and suggest that treatments to selectively modify the metastatic behavior of mesenchymal tumor cells are feasible and may be of clinical value.

Authors

Yanan Yang, Young-Ho Ahn, Yulong Chen, Xiaochao Tan, Lixia Guo, Don L. Gibbons, Christin Ungewiss, David H. Peng, Xin Liu, Steven H. Lin, Nishan Thilaganathan, Ignacio I. Wistuba, Jaime Rodriguez-Canales, Georgia McLendon, Chad J. Creighton, Jonathan M. Kurie

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Figure 6

FOG2 is a mediator of ZEB1.

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FOG2 is a mediator of ZEB1. (A) Coimmunoprecipitation of FOG2 and GATA3 ...
(A) Coimmunoprecipitation of FOG2 and GATA3 in 344SQ cells. A GFP-tagged GATA3 expression vector was generated to discriminate between exogenous GATA3 and IgG heavy chain on the basis of size. Extracts were subjected to immunoprecipitation with IgG, anti-FOG2, or anti-GATA3 antibodies. Total extracts and immunoprecipitates were then blotted with anti-FOG2 or -GATA3. (B) qPCR analysis (bar graph) and Western blot analysis (gels) of FOG2 expression. qPCR values, normalized on the basis of ribosomal protein L32 mRNA levels, represented the mean ± SD from triplicate samples and were expressed relative to 393P_vector cells (Vec), which were set at 1.0. (C) 3′-UTR reporter assays. 344SQ cells were transiently cotransfected with Fog2 3′-UTR reporters, in which the predicted miR binding sites were wild-type or mutated (mut) and control, miR-183, or miR-200c precursors. Mean ± SD from triplicate samples. (D) Western blot analysis of FOG2 expression in 344SQ cells that had been transiently transfected with indicated miRNA precursors. (E and F) Western blot analysis of FOG2 (E) and p110α (F) in 344SQ_scr shRNA cells (Scr) and 344SQ_Fog2 shRNA cells. (G) Invasion assays. Invasive 344SQ_Fog2 shRNA (#3 and #4) and 344SQ_scr shRNA cells were photographed (images) and quantified (bar graph). Mean ± SD from triplicate samples (bar graph). Scale bars: 100 εm. (H) Scatter plots of primary tumor weight (left) and numbers of visible lung metastases (right) in syngeneic mice injected with 344SQ_Fog2 shRNA (#3 and #4) or 344SQ_scr cells. Mean ± SD for each cohort. Actin was included as a loading control for Western blots.