ZEB1 drives prometastatic actin cytoskeletal remodeling by downregulating miR-34a expression
Young-Ho Ahn, Don L. Gibbons, Deepavali Chakravarti, Chad J. Creighton, Zain H. Rizvi, Henry P. Adams, Alexander Pertsemlidis, Philip A. Gregory, Josephine A. Wright, Gregory J. Goodall, Elsa R. Flores, Jonathan M. Kurie
Young-Ho Ahn, Don L. Gibbons, Deepavali Chakravarti, Chad J. Creighton, Zain H. Rizvi, Henry P. Adams, Alexander Pertsemlidis, Philip A. Gregory, Josephine A. Wright, Gregory J. Goodall, Elsa R. Flores, Jonathan M. Kurie
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Research Article Oncology

ZEB1 drives prometastatic actin cytoskeletal remodeling by downregulating miR-34a expression

Abstract

Metastatic cancer is extremely difficult to treat, and the presence of metastases greatly reduces a cancer patient’s likelihood of long-term survival. The ZEB1 transcriptional repressor promotes metastasis through downregulation of microRNAs (miRs) that are strong inducers of epithelial differentiation and inhibitors of stem cell factors. Given that each miR can target multiple genes with diverse functions, we posited that the prometastatic network controlled by ZEB1 extends beyond these processes. We tested this hypothesis using a mouse model of human lung adenocarcinoma metastasis driven by ZEB1, human lung carcinoma cells, and human breast carcinoma cells. Transcriptional profiling studies revealed that ZEB1 controls the expression of numerous oncogenic and tumor-suppressive miRs, including miR-34a. Ectopic expression of miR-34a decreased tumor cell invasion and metastasis, inhibited the formation of promigratory cytoskeletal structures, suppressed activation of the RHO GTPase family, and regulated a gene expression signature enriched in cytoskeletal functions and predictive of outcome in human lung adenocarcinomas. We identified several miR-34a target genes, including Arhgap1, which encodes a RHO GTPase activating protein that was required for tumor cell invasion. These findings demonstrate that ZEB1 drives prometastatic actin cytoskeletal remodeling by downregulating miR-34a expression and provide a compelling rationale to develop miR-34a as a therapeutic agent in lung cancer patients.

Authors

Young-Ho Ahn, Don L. Gibbons, Deepavali Chakravarti, Chad J. Creighton, Zain H. Rizvi, Henry P. Adams, Alexander Pertsemlidis, Philip A. Gregory, Josephine A. Wright, Gregory J. Goodall, Elsa R. Flores, Jonathan M. Kurie

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Figure 6

miR-34a regulates actin cytoskeletal remodeling and RHO family GTPase activity.

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miR-34a regulates actin cytoskeletal remodeling and RHO family GTPase ac...
(A–C) Cells imaged at leading edge of scratch-wounded confluent cultures. (A) Filopodia (arrows) formed in 344SQ_vector, but not 344SQ_miR-34a, cells. Brackets denote lamellipodia. (B) Focal adhesions in anti-vinculin–stained cultures, counted per defined surface area of confluent cells (circles) using ImageJ. Data are mean ± SD (n = 10). Red, phalloidin; green, vinculin. (C) Cells were outlined (white lines), and their surface areas were measured using ImageJ. Data are mean ± SD (n = 20 [344SQ_vector]; 12 [344SQ_miR-34a]). (D) Attached cells were quantified 1, 2, or 3 hours after seeding by optical densitometry (595 nm) of cells stained with crystal violet. Data are mean ± SD (n = 3). *P < 0.01. (E) Cells seeded in soft agar were stained with nitrotetrazolium blue 3 weeks after seeding, and colonies larger than 100 μm in diameter were counted. Data are mean ± SD (n = 3). (F) Attached cells were quantified by optical densitometry 3 hours after seeding and expressed relative to miR-34a levels from Figure 2A. Correlation (R and P, 1-tailed Pearson’s correlation test) is indicated. (G) MDA-MB-231 cells transiently transfected with control, miR-34a, or miR-200b precursors and imaged under fluorescence (blue, DAPI; red, phalloidin; green, anti–ZO-1). As a comparison, miR-200b–transfected cells demonstrated mesenchymal-to-epithelial transition. (H and I) Western blot analysis of GTP-bound (CDC42-GTP) and total (CDC42) RHO family GTPases. Phospho-ERK1/2 (H) and phospho-SMAD3 (I) were included as positive controls for EGF- and TGF-β–induced signaling, respectively. Scale bars: 50 μm (A, B, and G); 100 μm (C); 500 μm (E). See complete unedited blots in the supplemental material.