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 5

p110α and RHEB are downstream mediators of ZEB1.

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p110α and RHEB are downstream mediators of ZEB1. (A) Invasion assays. In...
(A) Invasion assays. Invasive 344SQ_Rheb shRNA cells (#3 and #5) and 344SQ_scr shRNA cells were photographed (images) and quantified (bar graph) after 24 hours of incubation. Mean ± SD from triplicate samples. Scale bars: 100 μm. (B) Scatter plots of primary tumor weight and numbers of visible lung metastases in syngeneic mice injected with 344SQ_Rheb shRNA or 344SQ_scr cells. Mean ± SD for each cohort. (C) RNA polymerase II ChIP assays of Pik3ca (top) and Rheb (bottom) promoters. (D and E) Luciferase assays of PIK3CA (D) and Rheb (E) promoter activity. 344SQ cells were cotransfected with GATA3 expression and reporter plasmids. Results were normalized using a dual firefly/Renilla luciferase system. Mean ± SD from triplicate samples. Results are expressed relative to the normalized luciferase activity in pGL3 basic vector–transfected cells. (F and G) GATA3 ChIP assays on the PIK3CA (F) and RHEB (G) promoters in 293T cells and H322 cells, respectively. Controls include whole-cell lysates (input), ChIP using IgG, and amplification of an upstream region in the PIK3CA promoter (PCR #1) and intron 7 of RHEB (PCR #2) containing no predicted GATA-binding sites. Sizes of the PCR products generated from regions containing predicted GATA-binding sites in PIK3CA promoter (PCR #2 and PCR #3) and RHEB promoter (PCR #1) are indicated.