P113 isoform encoded by CUX1 circular RNA drives tumor progression via facilitating ZRF1/BRD4 transactivation

Feng Yang,Shuang Cai,Xiaojing Wang,Jianqun Wang,Boling Yuan,Guo Chen,Dan Li,Liduan Zheng,Anpei Hu,Haiyang Gao,Yi Zhou,Qilan Li,Yanhua Guo,Shikai Jin,Qiangsong Tong

doi:10.1186/s12943-021-01421-8

Feng Yang, Shuang Cai + Show 13 more

Open Access

https://doi.org/10.1186/s12943-021-01421-8

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Abstract

BackgroundMetabolic reprogramming sustains tumorigenesis and aggressiveness of neuroblastoma (NB), the most common extracranial malignancy in childhood, while underlying mechanisms and therapeutic approaches still remain elusive.MethodsCircular RNAs (circRNAs) were validated by Sanger sequencing. Co-immunoprecipitation, mass spectrometry, chromatin immunoprecipitation (ChIP) sequencing, and RNA sequencing assays were applied to explore protein interaction and target genes. Gene expression regulation was observed by ChIP, dual-luciferase reporter, real-time quantitative RT-PCR, and western blot assays. Gain- and loss-of-function studies were performed to observe the impacts of circRNA-encoded protein and its partners on the lipid metabolism, mitochondrial activity, growth, invasion, and metastasis of NB cells.ResultsA novel 113-amino acid protein (p113) of CUT-like homeobox 1 (CUX1) was identified in NB cells treated by serum deprivation. Further validating studies revealed that nuclear p113 was encoded by circRNA of CUX1, and promoted the lipid metabolic reprogramming, mitochondrial activity, proliferation, invasion, and metastasis of NB cells. Mechanistically, p113 interacted with Zuotin-related factor 1 (ZRF1) and bromodomain protein 4 (BRD4) to form a transcriptional regulatory complex, and mediated the transactivation of ZRF1/BRD4 in upregulating ALDH3A1, NDUFA1, and NDUFAF5 essential for conversion of fatty aldehydes into fatty acids, fatty acid β-oxidation, and mitochondrial complex I activity. Administration of an inhibitory peptide blocking p113-ZRF1 interaction suppressed the tumorigenesis and aggressiveness of NB cells. In clinical NB cases, high expression of p113, ZRF1, or BRD4 was associated with poor survival of patients.ConclusionsThese results indicate that p113 isoform encoded by CUX1 circular RNA drives tumor progression via facilitating ZRF1/BRD4 transactivation.

Highlights

Metabolic reprogramming sustains tumorigenesis and aggressiveness of neuroblastoma (NB), the most common extracranial malignancy in childhood, while underlying mechanisms and therapeutic approaches still remain elusive
Scale bar: 10 μm. e and f Heatmap (e) and quantification (f ) of metabolite profiling assay indicating the fatty acid levels in SH-SY5Y and BE(2)-C cells stably transfected with mock, ecircCUX1, ecircCUX1 Mut, p113, sh-Scb, or sh-ecircCUX1 (n = 3). g Seahorse extracellular flux assay showing the oxygen consumption rate (OCR) levels in SH-SY5Y and BE(2)-C cells stably transfected with mock, ecircCUX1, ecircCUX1 Mut, p113, sh-Scb, or sh-ecircCUX1, and those treated with bovine serum albumin (BSA) or oleic acid (OLE, 200 μmol·L− 1, n = 4). h Relative NAD+/Nicotinamide adenine dinucleotide (NADH) ratio and ATP levels in SH-SY5Y and BE(2)-C cells stably transfected with mock, ecircCUX1, ecircCUX1 Mut, p113, sh-Scb, or sh-ecircCUX1, and those treated with BSA or OLE (200 μmol·L− 1, n = 5)
Validating western blot assay indicated that expression of major CUT-like homeobox 1 (CUX1) isoforms [p200, p110, or CDP/CUT alternatively spliced product (CASP)] and Transcription factor 3 (TCF3) remained unaffected, while a new CUX1 isoform with a molecular weight of 13 kDa was unexpectedly upregulated in a time-dependent manner upon serum deprivation (SD) treatment (Fig. 1b)

Summary

Introduction

Metabolic reprogramming sustains tumorigenesis and aggressiveness of neuroblastoma (NB), the most common extracranial malignancy in childhood, while underlying mechanisms and therapeutic approaches still remain elusive. Neuroblastoma (NB), a solid malignancy featured by rapid progression and high mortality, accounts for more than 15% of tumor-related deaths in pediatric population [1]. For high-risk NB patients, the clinical outcome still remains unfavorable in despite of multimodal therapeutics [1]. Beside aerobic glycolysis and glutaminolysis, tumor cells obtain energies from lipid metabolism for thriving in challenging environments, including uptake of fatty acids, de novo lipid synthesis [2], and fatty acid β-oxidation (FAO) [3]. Transcriptional regulators of lipid metabolic reprogramming in NB still remain largely elusive

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