Quaking (QKI) proteins belong to the signal transduction and activation of RNA (STAR) family of RNA-binding proteins that have multiple functions in RNA biology. Here, we show that QKI-5 is dramatically decreased in metastatic lung adenocarcinoma (LUAD). QKI-5 overexpression inhibits TGF-b-induced epithelialmesenchymal transition (EMT) and invasion, whereas QKI-5 knockdown has the opposite effect. QKI-5 overexpression and silencing suppresses and promotes TGF-b-stimulated metastasis in vivo, respectively. QKI-5 inhibits TGF-b-induced EMT and invasion in a TGFbR1-dependent manner. KLF6 knockdown increases TGFbR1 expression and promotes TGF-b-induced EMT, which is partly abrogated by QKI-5 overexpression. Mechanistically, QKI-5 directly interacts with the TGFbR1 3 0 UTR and causes post-transcriptional degradation of TGFbR1 mRNA, thereby inhibiting TGF-b-induced SMAD3 phosphorylation and TGF-b/SMAD signaling. QKI-5 is positively regulated by KLF6 at the transcriptional level. In LUAD tissues, KLF6 is lowly expressed and positively correlated with QKI-5 expression, while TGFbR1 expression is up-regulated and inversely correlated with QKI-5 expression. We reveal a novel mechanism by which KLF6 transcriptionally regulates QKI-5 and suggest that targeting the KLF6/QKI-5/TGFbR1 axis is a promising targeting strategy for metastatic LUAD.

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Mammalian target of rapamycin (mTOR) kinase functions as a central regulator of cell growth and metabolism, and its complexes mTORC1 and mTORC2 phosphorylate distinct substrates1-3. Dysregulation of mTOR signaling is commonly implicated in human diseases, including cancer4,5. Despite three decades of active research in mTOR, much remains to be determined1. In fact, how mTOR kinase is directly activated and recognize distinct substrates are still unknown. Here, we demonstrate that prolyl 4-hydroxylase alpha-2 (P4HA2) can hydroxylate mTOR protein and thereby activating mTOR signal. By performing co-immunoprecipitation, GST pull-down assays combined with mass spectrometry-based proteomic analysis in 293T cells, we found that P4HA2 binds directly to mTOR and hydroxylates one highly conserved proline 2341 (P2341) within a kinase domain of mTOR. Moreover, we discover that the hydroxylation of P2341 strengthens mTOR stability and allows mTOR to accurately recognize its substrates such as S6K and AKT. Our study reveals an undiscovered hydroxylation-regulatory mechanism by which P4HA2 directly activates mTOR kinase, providing insights for therapeutically targeting mTOR kinase-driven cancers.