Pyrroline-5-carboxylate reductase 1 interacts with Nudix hydrolase 5 to drive bladder cancer malignant progression via activation of the APOE/JAK2/STAT3 signalling axis.

BACKGROUND: Bladder cancer (BCa) is one of the most common urological malignancies, with a steadily increasing global incidence. Pyrroline-5-carboxylate reductase 1 (PYCR1) is a metabolic regulator with noncanonical oncogenic functions; however, its protein interaction network and downstream mechanisms in BCa remain incompletely understood. METHODS: PYCR1 expression and its clinical significance were evaluated using the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets and tissue microarrays. Cell counting kit-8, colony formation, EdU, 3D Transwell experiments and xenograft models were used to investigate the biological functions of PYCR1. RNA sequencing, Western blot and quantitative polymerase chain reaction (qPCR) were performed to identify its downstream targets. Mass spectrometry combined with co-immunoprecipitation identified Nudix hydrolase 5 (NUDT5) as a PYCR1-interacting protein. Nuclear-cytoplasmic fractionation and adenosine triphosphate (ATP) quantification were used to measure nuclear ATP levels. RESULTS: PYCR1 was upregulated in BCa and promoted tumour proliferation and migration in vitro and in vivo. Mechanistically, PYCR1 interacted with NUDT5 and promoted NUDT5-dependent nuclear ATP generation. Wild-type NUDT5 restored Apolipoprotein E (APOE) expression in PYCR1-knockdown cells, whereas a catalytically inactive NUDT5 mutant failed to do so. RNA sequencing, gene set enrichment analysis, Western blot, and qPCR demonstrated that PYCR1 activated the APOE/JAK2/STAT3 signalling axis. Transcription factor prediction and dual-luciferase reporter assays identified SP1 as a transcriptional regulator of APOE. Moreover, pharmacological inhibition of NUDT5 with TH5427 suppressed BCa progression and JAK2/STAT3 activation in vitro and inhibited xenograft growth in vivo. CONCLUSIONS: PYCR1 interacts with NUDT5 to increase nuclear ATP levels, thereby sustaining SP1-dependent APOE transcription and activating JAK2/STAT3 signalling, ultimately promoting BCa progression. TH5427 may represent a promising therapeutic candidate for BCa.