CPT1A through succinylation regulates the CTCF/LINC01503‒91aa feedback loop to promote gemcitabine resistance in pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the most malignant solid tumors, characterized by strong invasiveness and poor clinical prognosis. Recent studies have revealed that long non-coding RNAs (lncRNAs) can serve as translational templates to produce functional microproteins, which further participate in the regulation of cellular metabolism. OBJECTIVE: To investigate the function and detailed mechanism of the novel protein 1503-91aa encoded by LINC01503 in gemcitabine-resistant PDAC, and explore the possibility of clinical translation. METHODS: The differentially expressed lncRNAs in gemcitabine-resistant PDAC cells with coding potential were screened by whole transcriptome sequencing and coding prediction from database. A series of cellular, molecular and in vivo assays were performed to characterize the novel microprotein 1503-91aa, its upstream transcriptional regulation, and its role in mediating PDAC gemcitabine resistance. A series of molecular assays clarified the interaction between 1503-91aa and carnitine palmitoyl transferase 1A (CPT1A). Finally, the clinical translational potential of the CPT1A inhibitor etomoxir in combination with gemcitabine was validated using our center's gemcitabine-resistant patient-derived xenograft (PDX) model. RESULTS: Here, we revealed that LINC01503 had coding potential in drug-resistant PDAC and encoded a novel 91-amino acid protein, which designated as 1503-91aa. Simultaneously, LINC01503's transcriptional regulation is mediated by CTCF and then 1503-91aa expression levels were significantly upregulated in PDAC. Functionally, 1503-91aa, instead of LINC01503 confers gemcitabine resistance in vitro and in vivo. Mechanistically, 1503-91aa targeted the CPT1A, and up-regulated CPT1A activity through antagonizing the association of malonyl-CoA (MCoA), the best known metabolic intermediate inhibiting CPT1A, to promote fatty acid oxidation (FAO) and thereby facilitate gemcitabine resistance. We discovered that CPT1A functions as a succinyltransferase to regulate the succinylation level of CTCF, thereby controlling its stability. This establishes a positive feedback loop that continuously sustains 1503-91aa protein levels to regulate CPT1A activity. Consequently, this induces persistent FAO in PDAC cells, ensuring a continuous energy source that facilitates gemcitabine resistance in pancreatic cancer. Notably, the combined application of the etomoxir and gemcitabine exerts a synergistic effect on PDAC. CONCLUSIONS: These findings provide novel insights into the molecular mechanisms underlying gemcitabine resistance in PDAC and highlight CTCF/LINC01503-91aa/CPT1A feedback loop as a potential prognostic biomarkers and therapeutic targets. KEY POINTS: We identified that LINC01503 is capable of encoding the protein product 1503-91aa. 1503-91aa competed with MCoA to unleash CPT1A activity for fatty acid oxidation and thereby mediating gemcitabine resistance in PDAC. Etomoxir and gemcitabine have synergistic effects in gemcitabine efficiency.