The m6A demethylase ALKBH5-mediated upregulation of DDIT4-AS1 maintains pancreatic cancer stemness and suppresses chemosensitivity by activating the mTOR pathway.

BACKGROUND: Chemoresistance is a major factor contributing to the poor prognosis of patients with pancreatic cancer, and cancer stemness is one of the most crucial factors associated with chemoresistance and a very promising direction for cancer treatment. However, the exact molecular mechanisms of cancer stemness have not been completely elucidated. METHODS: m6A-RNA immunoprecipitation and sequencing were used to screen m6A-related mRNAs and lncRNAs. qRT-PCR and FISH were utilized to analyse DDIT4-AS1 expression. Spheroid formation, colony formation, Western blot and flow cytometry assays were performed to analyse the cancer stemness and chemosensitivity of PDAC cells. Xenograft experiments were conducted to analyse the tumour formation ratio and growth in vivo. RNA sequencing, Western blot and bioinformatics analyses were used to identify the downstream pathway of DDIT4-AS1. IP, RIP and RNA pulldown assays were performed to test the interaction between DDIT4-AS1, DDIT4 and UPF1. Patient-derived xenograft (PDX) mouse models were generated to evaluate chemosensitivities to GEM. RESULTS: DDIT4-AS1 was identified as one of the downstream targets of ALKBH5, and recruitment of HuR onto m6A-modified sites is essential for DDIT4-AS1 stabilization. DDIT4-AS1 was upregulated in PDAC and positively correlated with a poor prognosis. DDIT4-AS1 silencing inhibited stemness and enhanced chemosensitivity to GEM (Gemcitabine). Mechanistically, DDIT4-AS1 promoted the phosphorylation of UPF1 by preventing the binding of SMG5 and PP2A to UPF1, which decreased the stability of the DDIT4 mRNA and activated the mTOR pathway. Furthermore, suppression of DDIT4-AS1 in a PDX-derived model enhanced the antitumour effects of GEM on PDAC. CONCLUSIONS: The ALKBH5-mediated m6A modification led to DDIT4-AS1 overexpression in PDAC, and DDIT-AS1 increased cancer stemness and suppressed chemosensitivity to GEM by destabilizing DDIT4 and activating the mTOR pathway. Approaches targeting DDIT4-AS1 and its pathway may be an effective strategy for the treatment of chemoresistance in PDAC.