Epithelial-to-mesenchymal transition drives a pro-metastatic Golgi compaction process through scaffolding protein PAQR11
Xiaochao Tan, … , Yanzhuang Wang, Jonathan M. Kurie
Xiaochao Tan, … , Yanzhuang Wang, Jonathan M. Kurie
Published November 21, 2016
Citation Information: J Clin Invest. 2017;127(1):117-131. https://doi.org/10.1172/JCI88736.
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Research Article Cell biology Oncology

Epithelial-to-mesenchymal transition drives a pro-metastatic Golgi compaction process through scaffolding protein PAQR11

Abstract

Tumor cells gain metastatic capacity through a Golgi phosphoprotein 3–dependent (GOLPH3-dependent) Golgi membrane dispersal process that drives the budding and transport of secretory vesicles. Whether Golgi dispersal underlies the pro-metastatic vesicular trafficking that is associated with epithelial-to-mesenchymal transition (EMT) remains unclear. Here, we have shown that, rather than causing Golgi dispersal, EMT led to the formation of compact Golgi organelles with improved ribbon linking and cisternal stacking. Ectopic expression of the EMT-activating transcription factor ZEB1 stimulated Golgi compaction and relieved microRNA-mediated repression of the Golgi scaffolding protein PAQR11. Depletion of PAQR11 dispersed Golgi organelles and impaired anterograde vesicle transport to the plasma membrane as well as retrograde vesicle tethering to the Golgi. The N-terminal scaffolding domain of PAQR11 was associated with key regulators of Golgi compaction and vesicle transport in pull-down assays and was required to reconstitute Golgi compaction in PAQR11-deficient tumor cells. Finally, high PAQR11 levels were correlated with EMT and shorter survival in human cancers, and PAQR11 was found to be essential for tumor cell migration and metastasis in EMT-driven lung adenocarcinoma models. We conclude that EMT initiates a PAQR11-mediated Golgi compaction process that drives metastasis.

Authors

Xiaochao Tan, Priyam Banerjee, Hou-Fu Guo, Stephen Ireland, Daniela Pankova, Young-ho Ahn, Irodotos Michail Nikolaidis, Xin Liu, Yanbin Zhao, Yongming Xue, Alan R. Burns, Jonathon Roybal, Don L. Gibbons, Tomasz Zal, Chad J. Creighton, Daniel Ungar, Yanzhuang Wang, Jonathan M. Kurie

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Figure 7

ZEB1 silences miR-206 and miR-148a through distinct mechanisms.

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ZEB1 silences miR-206 and miR-148a through distinct mechanisms. (A) qPCR...
(A) qPCR analysis of miR levels expressed as a ratio (ZEB1/Vec), with 307P_vector set at 1.0. (B) The bar graph shows qPCR analysis of miR levels in 344SQ cells stably transfected with ZEB1 shRNAs (#1 or #2) or scrambled control shRNA (shCTL). Results expressed as a ratio (shZEB1/shCTL), with 344SQ_shCTL set at 1.0. (C) Reporter activities in 393P cells transiently transfected with Mir148a promoter reporters containing WT (blue) or mutated (white) E-boxes. (D) miR-148a chromatin immunoprecipitation assays on chromatin immunoprecipitated with anti-ZEB1 or IgG antibodies. PCR amplification of E1/E2 locus (148a-E1+2) or E3 locus (148a-E3) on the Mir148a promoter. Results expressed as a ratio of qPCR values (anti-ZEB1/IgG). Mir200b/c/429 locus (200b-E) and the miR-148a precursor (pre-148a) were included as positive and negative controls, respectively. Arrows indicate locations of PCR primers. (E) Reporter activities in 393P_vector cells (Vec) and 393P_ZEB1 cells (ZEB1) transiently transfected with miR-206 promoter reporters containing intact (blue or red) or mutated (white) miR binding sites. (F) miR-206 ChIP assays. Chromatin was immunoprecipitated with anti-ZEB1 (ZEB1), anti-GATA3 (GATA3), or IgG antibodies. PCR amplification of sites within the miR-206 precursor (pre-206) or a negative control region in the miR-206 promoter (1k). Arrows indicate locations of PCR primers. (G) qPCR analysis of GATA3 levels in 344SQ cells stably transfected with GATA3 shRNA (shGATA3#1 or shGATA3#2) or scrambled shRNA (shCTL). Results are expressed as a ratio (shGATA3/shCTL), with shCTL set at 1.0. (H) qPCR analysis of miR-148a levels and miR-206 levels. Results are expressed as a ratio (shGATA3/shCTL), with shCTL set at 1.0. Unless otherwise indicated, results represent the averages of triplicate samples per condition. P values determined using 2-tailed Student’s t test or ANOVA for comparisons between 2 groups or more than 2 groups, respectively. Results were replicated (n ≥ 2 experiments).