A novel protein cPFKFB4 encoded by hsa_circ_0065394 strengthens PKM2-mediated glucose metabolic reprogramming to facilitate pancreatic cancer progression under hypoxia.

[BACKGROUND] Hypoxia is a hallmark of solid tumors and gives rise to the glucose metabolic reprogramming of cancers. Peptides and proteins encoded by circular RNAs (circRNAs) are identified as pivotal mediators in the malignant progression of tumors. Nonetheless, proteins encoded by hypoxia-related circRNAs in pancreatic cancer (PC) remain uncharacterized and poorly understood.

[METHODS] Hypoxia-responsive circRNA (hsa_circ_0065394) in PC was screened using a hypoxic culture system. The circularization mechanism of hsa_circ_0065394 was investigated by employing bioinformatics and RNA immunoprecipitation. An assessment of the expression profile and potential clinical significance of hsa_circ_0065394 in PC was conducted utilizing qRT-PCR and in situ hybridization. Dual-luciferase reporter gene assay, mass spectrometry and western blotting identified a protein cPFKFB4 encoded by hsa_circ_0065394. The biological functions of cPFKFB4 were verified by gain- and loss-of-function experiments in vivo and in vitro. From a mechanistic standpoint, how cPFKFB4 modulates the interaction between hnRNP G and hnRNP A1 as well as the regulation of PKM alternative splicing was explored by conducting mass spectrometry, co-immunoprecipitation, immunofluorescence, PKM splicing, RNA immunoprecipitation, western blotting, and rescue experiments.

[RESULTS] Hypoxia-associated hsa_circ_0065394 was noticeably up-regulated in PC tissues and tightly associated with tumor size. The biological cyclization of hsa_circ_0065394 was mediated by hnRNP L and Alu repeat sequences. Mechanistically, hsa_circ_0065394 encoded a 94 amino acid protein cPFKFB4 through a cap-independent mechanism. cPFKFB4 disrupted the hnRNP G-hnRNP A1 complex through specific binding to hnRNP G, facilitated hnRNP A1-mediated regulation of PKM alternative splicing, thereby heightening the expression of PKM2, and then strengthening glycolysis of PC cells to accelerate the proliferation and metastasis of PC under hypoxic conditions.

[CONCLUSIONS] cPFKFB4, a protein encoded by hypoxia-responsive circular RNA, facilitates the proliferation and metastasis of PC cells under hypoxic conditions by regulating PKM2-mediated glycolysis. Our findings not only offer fresh standpoints and valuable insights into the crosstalk between hypoxia, glucose metabolism reprogramming, and PC, but also provide a potential biomarker and therapeutic target for the treatment of PC.