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  • GABARAPL1 Inhibits EMT Signaling through SMAD-Tageted Negative Feedback.

GABARAPL1 Inhibits EMT Signaling through SMAD-Tageted Negative Feedback.

Biology (2021-10-24)
Marine Jacquet, Eric Hervouet, Timothée Baudu, Michaël Herfs, Chloé Parratte, Jean-Paul Feugeas, Valérie Perez, Célia Reynders, Marie Ancion, Marc Vigneron, Aurélie Baguet, Michaël Guittaut, Annick Fraichard, Gilles Despouy
ABSTRACT

The pathway of selective autophagy, leading to a targeted elimination of specific intracellular components, is mediated by the ATG8 proteins, and has been previously suggested to be involved in the regulation of the Epithelial-mesenchymal transition (EMT) during cancer's etiology. However, the molecular factors and steps of selective autophagy occurring during EMT remain unclear. We therefore analyzed a cohort of lung adenocarcinoma tumors using transcriptome analysis and immunohistochemistry, and found that the expression of ATG8 genes is correlated with that of EMT-related genes, and that GABARAPL1 protein levels are increased in EMT+ tumors compared to EMT- ones. Similarly, the induction of EMT in the A549 lung adenocarcinoma cell line using TGF-β/TNF-α led to a high increase in GABARAPL1 expression mediated by the EMT-related transcription factors of the SMAD family, whereas the other ATG8 genes were less modified. To determine the role of GABARAPL1 during EMT, we used the CRISPR/Cas9 technology in A549 and ACHN kidney adenocarcinoma cell lines to deplete GABARAPL1. We then observed that GABARAPL1 knockout induced EMT linked to a defect of GABARAPL1-mediated degradation of the SMAD proteins. These findings suggest that, during EMT, GABARAPL1 might intervene in an EMT-regulatory loop. Indeed, induction of EMT led to an increase in GABARAPL1 levels through the activation of the SMAD signaling pathway, and then GABARAPL1 induced the autophagy-selective degradation of SMAD proteins, leading to EMT inhibition.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
ANTI-GABARAP (N-TERM) antibody produced in rabbit, IgG fraction of antiserum, buffered aqueous solution
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Phalloidin–Tetramethylrhodamine B isothiocyanate, sequence from Amanita phalloides(synthetic: peptide sequence)
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Protease Inhibitor Cocktail, for use with mammalian cell and tissue extracts, DMSO solution
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Anti-Actin (20-33) antibody produced in rabbit, IgG fraction of antiserum, buffered aqueous solution
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Duolink® In Situ Detection Reagents Red
Sigma-Aldrich
Earle′s Balanced Salts, With sodium bicarbonate, without phenol red, liquid, sterile-filtered, suitable for cell culture