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 2

ZEB1 activates p110α through an EGFR/ERBB2 autocrine loop.

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ZEB1 activates p110α through an EGFR/ERBB2 autocrine loop. (A) ELISA of ...
(A) ELISA of PIP3 levels (picomoles) catalyzed in vitro by p110 isozymes immunoprecipitated from epithelial (307P, 393P, 412P, and 531P1) and mesenchymal (344P, 344SQ, 531P2, and 531LN2) KP cells. (B) ELISA. p110α-specific enzymatic activity in KP cells. Mean ± SD from triplicate samples. (C) Array-based detection of phosphorylated RTKs (p-RTK) in cell lysates. Duplicate phosphorylated EGFR (p-EGFR) and ERBB2 (p-ERBB2) are indicated (arrows); positive control proteins are in the top left, top right, and lower right corners. Densitometric analysis (bar graphs) of p-EGFR and p-ERBB2 expressed as mean values of duplicate spots in each transfectant relative to those of 393P_vector cells, which were set at 1.0. (D) Western blot analysis using phospho-specific (p-) and total protein antibodies. Controls include blotting for actin. (E) qPCR analysis of mRNA levels of ERBB ligands. Mean ± SD from triplicate samples. *P < 0.01 compared with 393P_vector; #P < 0.01 compared with 344SQ_vector. (F) ELISA. β-cellulin (BTC) and amphiregulin (AREG) levels in conditioned media. Mean ± SD from triplicate samples. (G and H) Western blot analysis using phospho-specific and total protein antibodies (G) and ELISA of p110α enzymatic activity (H). 393P_ZEB1 cells were treated with neutralizing antibodies against AREG and/or BTC for 24 hours. IgG was used as a negative control. Mean ± SD from triplicate samples. (I) Western blot analysis. Cells were treated with drug or vehicle (0) for 24 hours. Controls include blotting for actin. (J) ELISA. p110α enzymatic activity in 393P_ZEB1 cells treated with the indicated doses of drug or vehicle (0) for 24 hours. Mean ± SD from triplicate samples.