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 4

p110α mediates invasion and metastasis.

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p110α mediates invasion and metastasis. (A) qPCR analysis of the mRNA le...
(A) qPCR analysis of the mRNA levels of genes in the PI3K pathway. Values were normalized on the basis of ribosomal protein L32 mRNA levels and are expressed as ratios (393P_ZEB1 to 393P_vector; 344SQ_miR-200 to 344SQ_vector). (B) Western blot analysis of KP cells. (C) qPCR assays (bar graph) and Western blot analysis (gels) of 344SQ_Pik3ca shRNA cells (shRNA #1 and #2) and 344SQ_scr shRNA cells (Scr). Controls include 344SQ_scr shRNA cells and blotting for actin. qPCR values were normalized on the basis of ribosomal protein L32 mRNA levels and represent the mean ± SD from triplicate samples. (D) Density of 344SQ_Pik3ca shRNA cells and 344SQ_scr shRNA cells in monolayer culture was measured by MTT assay at the indicated time points. Values represent the mean ± SD from triplicate samples. (E) Soft agar colony formation. Colonies were photographed (images) and counted (bar graph) after 14 days of incubation. Original magnification, ×4. Values represent the mean ± SD from triplicate wells. (F) Invasion assays. Cells were photographed (images) and quantified (bar graph). Scale bars: 100 μm. (G) Scatter plots of primary tumor volume (left scatter plot) and numbers of visible lung metastases (right scatter plot) in syngeneic mice injected in the flanks with 344SQ_Pik3ca shRNA (n = 5) or 344SQ_scr cells (n = 5). Each mouse is indicated with a dot. Mean ± SD calculated for each cohort.