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 3

BTC and AREG are transcriptionally or post-transcriptionally regulated by the ZEB1/miR-200 axis.

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BTC and AREG are transcriptionally or post-transcriptionally regulated b...
(A) 3′-UTR reporter assays. 344SQ cells were transiently cotransfected with pre–miR-200s and Btc or Areg 3′-UTR luciferase reporters in which predicted miR-200 binding sites were wild-type (WT) or mutated (Mut). Results were normalized using a dual firefly/Renilla luciferase system. Mean ± SD from triplicate samples. (B) RNA polymerase II (Pol II) ChIP assays of Areg, Btc, and Hbegf promoters. Mean ± SD from triplicate samples. (C) GATA6 ChIP assays on Btc and Areg promoters. IgG was used as a negative control. (D) Promoter reporter assays. 393P cells were transiently cotransfected with Btc or Areg promoter luciferase reporters (500 ng/well in 24-well plates) and control or a GATA6 expression vector (500 ng). Mean ± SD from triplicate samples. (E) qPCR analysis (bar graph) and Western blot analysis (gels) of GATA6 expression. qPCR values, normalized on the basis of ribosomal protein L32 mRNA levels, represent the mean ± SD from triplicate samples and were expressed relative to 393P_vector cells (Vec), which were set at 1.0. Actin was included as a loading control. (F) 3′-UTR reporter assays. 344SQ cells were transiently cotransfected with pre–miR-200a and Gata6 3′-UTR luciferase reporters, in which predicted miR-200 binding sites were wild-type or mutated. Results were normalized using a dual firefly/Renilla luciferase system. ZEB1 3′-UTR was used as a positive control. Mean ± SD from triplicate samples. Results are expressed relative to the normalized luciferase activity in hRL-transfected (empty vector–transfected) cells.