GABARAPL1 Inhibits EMT Signaling through SMAD-Tageted Negative Feedback

SIMPLE SUMMARY: Epithelial–mesenchymal transition (EMT) is involved in metastasis formation, chemoresistance, apoptosis resistance, and acquisition of stem cell properties, making this process an attractive target in cancer. However, direct targeting of EMT remains challenging. Autophagy—an intracellular mechanism—has been noted to be involved in the regulation of EMT—mainly by its involvement in the degradation of EMT actors, explaining why understanding of how autophagy could regulate EMT might be promising in the development of new cancer therapies. Here, we found that GABARAPL1—an autophagy-related gene—was increased in human NSCLC mesenchymal tumors compared to epithelial tumors, and induction of EMT in an A549 lung cancer cell line by TGF-β/TNF-α cytokines also led to an increase in GABARAPL1 expression. This regulation could involve the EMT-related transcription factors of the SMAD family. To understand the role of GABARAPL1 in EMT regulation in lung cancer cells, A549 KO GABARAPL1 were designed and used to investigate whether GABARAPL1 could inhibit EMT via its involvement in SMAD degradation. The results indicate that GABARAPL1-mediated autophagic degradation could intervene as a negative EMT-regulatory loop. 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.