The Notch ligand Jagged2 promotes lung adenocarcinoma metastasis through a miR-200–dependent pathway in mice
Yanan Yang, Young-Ho Ahn, Don L. Gibbons, Yi Zang, Wei Lin, Nishan Thilaganathan, Cristina A. Alvarez, Daniel C. Moreira, Chad J. Creighton, Philip A. Gregory, Gregory J. Goodall, Jonathan M. Kurie
Yanan Yang, Young-Ho Ahn, Don L. Gibbons, Yi Zang, Wei Lin, Nishan Thilaganathan, Cristina A. Alvarez, Daniel C. Moreira, Chad J. Creighton, Philip A. Gregory, Gregory J. Goodall, Jonathan M. Kurie
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Research Article Oncology

The Notch ligand Jagged2 promotes lung adenocarcinoma metastasis through a miR-200–dependent pathway in mice

Abstract

Epithelial tumor cells transit to a mesenchymal state in response to extracellular cues, in a process known as epithelial-to-mesenchymal transition (EMT). The precise nature of these cues has not been fully defined, an important issue given that EMT is an early event in tumor metastasis. Here, we have found that a population of metastasis-prone mouse lung adenocarcinoma cells expresses Notch and Notch ligands and that the Notch ligand Jagged2 promotes metastasis. Mechanistically, Jagged2 was found to promote metastasis by increasing the expression of GATA-binding (Gata) factors, which suppressed expression of the microRNA-200 (miR-200) family of microRNAs that target the transcriptional repressors that drive EMT and thereby induced EMT. Reciprocally, miR-200 inhibited expression of Gata3, which reversed EMT and abrogated metastasis, suggesting that Gata3 and miR-200 are mutually inhibitory and have opposing effects on EMT and metastasis. Consistent with this, high levels of Gata3 expression correlated with EMT in primary tumors from 2 cohorts of lung adenocarcinoma patients. These findings reveal what we believe to be a novel Jagged2/miR-200–dependent pathway that mediates lung adenocarcinoma EMT and metastasis in mice and may have implications for the treatment of human epithelial tumors.

Authors

Yanan Yang, Young-Ho Ahn, Don L. Gibbons, Yi Zang, Wei Lin, Nishan Thilaganathan, Cristina A. Alvarez, Daniel C. Moreira, Chad J. Creighton, Philip A. Gregory, Gregory J. Goodall, Jonathan M. Kurie

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Figure 6

Gata3 directly suppresses miR-200 transcription and induces EMT.

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Gata3 directly suppresses miR-200 transcription and induces EMT. (A) ChI...
(A) ChIP assays examined Gata3 binding to the miR-200b promoter in human H322 lung cancer cells. A 239-bp PCR product (arrow) was generated from the Gata3 IP samples (Gata3), using primers for a proximal promoter segment (PCR #1), but not from upstream regulatory sequences (PCR #2), which are illustrated graphically above the gel. Putative binding sites for GATA3 (red star) and other transcription factors (colored ovals and rectangles) predicted by TRANSFAC (BIOBASE Biological Databases). (B) MCF-7 cells cotransfected with reporters containing the proximal miR-200 promoter (–321 to +15 bp from the transcription start site) or empty luciferase vector (pGL3) and vectors containing Gata3 (WT), inactive Gata3 mutant (mut), or empty vector (vec). Values were expressed as the mean (± SD) of triplicate wells. The differences between groups were estimated by 1-way ANOVA (P < 0.01). (C) Images of Gata3-depleted or control 344SQ transfectants. Scale bar: 100 μm. (D) Images of invaded Gata3-depleted (black bar) and control (white bar) 344SQ transfectants, which were counted and expressed as the mean values (± SD) of replicate (triplicate) wells (bar graph). Scale bar: 100 μm. (E) Quantitative RT-PCR analysis of the indicated genes (green) in Gata3-depleted and control shRNA transfectants treated for 48 hours with (black bars) or without (white bars) TGF-β (1 ng/ml). Values represent the mean (± SD) of triplicate samples. (F) Western blotting of control and Gata3-depleted cells treated with medium or TGF-β (1 ng/ml) for 48 hours. Band density was quantified by densitometry, normalized to that of medium-treated control cells (which are set at 1.0), and is indicated below each blot.