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CRISPR-based knockout screening identifies the loss of MIEF2 to enhance oxaliplatin resistance in colorectal cancer through inhibiting the mitochondrial apoptosis pathway.

ABSTRACT: The first-line anticancer agent oxaliplatin (OXL) is the preferred drug for treating colorectal cancer (CRC); however, the development of drug resistance is common in patients treated with OXL, which considerably reduces the efficacy of OXL-based regimens. By performing genome-wide CRISPR/Cas9 library knockdown screening, we found that mitochondrial elongation factor 2 (MIEF2) was among the top candidate genes. The OXL-resistant cell lines and organoids developed in the present study showed stable but low expression of MIEF2. Reduced MIEF2 expression may enhance CRC resistance to OXL by reducing mitochondrial stability and inhibiting apoptosis by decreasing cytochrome C release. In conclusion, among the different biomarkers of OXL resistance in CRC, MIEF2 may serve as a specific biomarker of OXL responsiveness and a potential target for the development of therapies to improve chemotherapeutic effectiveness.

SUBMITTER: Xie C

PROVIDER: S-EPMC9465453 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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CRISPR-based knockout screening identifies the loss of MIEF2 to enhance oxaliplatin resistance in colorectal cancer through inhibiting the mitochondrial apoptosis pathway.

Xie Chaozheng C Li Kang K Li Ya Y Peng Xudong X Teng Biyun B He Kuan K Jin Aishun A Wang Wang W Wei Zhengqiang Z

Frontiers in oncology 20220829

The first-line anticancer agent oxaliplatin (OXL) is the preferred drug for treating colorectal cancer (CRC); however, the development of drug resistance is common in patients treated with OXL, which considerably reduces the efficacy of OXL-based regimens. By performing genome-wide CRISPR/Cas9 library knockdown screening, we found that mitochondrial elongation factor 2 (MIEF2) was among the top candidate genes. The OXL-resistant cell lines and organoids developed in the present study showed stab ...[more]

PMID: 36106106

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Project description:MicroRNAs (miRNAs) play a critical role in many biological processes and are aberrantly expressed in human cancers. Particular miRNAs function either as tumor suppressors or oncogenes and appear to have diagnostic and prognostic significance. Although numerous miRNAs are dys-regulated in colorectal cancer (CRC) only a small fraction has been characterized functionally. Using high-throughput functional screening and miRNA profiling of clinical samples the present study aims at identifying miRNAs important for the control of cellular growth and/or apoptosis in CRC. The high-throughput functional screening was carried out in six CRC cell lines transfected with a pre-miR library including 319 synthetic human pre-miRs. Phenotypic alterations were evaluated by immunostaining of cleaved cPARP (apoptosis) or MKI67 (proliferation). Additionally, TaqMan Human MicroRNA Array Set v2.0 was used to profile the expression of 667 miRNAs in 14 normal colon mucosa and 46 microsatellite stable stage II CRC patients. Among the miRNAs that induced growth arrest and apoptosis in the CRC cell lines, and at same time were dys-regulated in the clinical samples, miR-375 was selected for further analysis. Independent in vitro analysis of transient and stable transfected CRC cell lines confirmed that miR-375 reduces cell viability through the induction of apoptotic death. We identified YAP1 as a direct miR-375 target in CRC and show that HELLS and NOLC1 are down-stream targets. Knock-down of YAP1 mimicked the phenotype induced by miR-375 over-expression indicating that miR-375 most likely exerts its pro-apoptotic role through YAP1 and its anti-apoptotic down-stream targets BIRC5 and BCL2L1. Finally, in vivo analysis of mouse xenograft tumors showed that miR-375 expression significantly reduced tumor growth. We conclude that the high-throughput screening successfully identified miRNAs that induce apoptosis and/or inhibit proliferation in CRC cells. Finally, combining the functional screening with profiling of CRC tissue samples we identified clinically relevant miRNAs and miRNA targets in CRC.

Project description:BackgroundOxaliplatin resistance usually leads to therapeutic failure and poor prognosis in colorectal cancer (CRC), while the underlying mechanisms are not yet fully understood. Metabolic reprogramming is strongly linked to drug resistance, however, the role and mechanism of metabolic reprogramming in oxaliplatin resistance remain unclear. Here, we aim to explore the functions and mechanisms of purine metabolism on the oxaliplatin-induced apoptosis of CRC.MethodsAn oxaliplatin-resistant CRC cell line was generated, and untargeted metabolomics analysis was conducted. The inosine 5'-monophosphate dehydrogenase type II (IMPDH2) expression in CRC cell lines was determined by quantitative real-time polymerase chain reaction (qPCR) and western blotting analysis. The effects of IMPDH2 overexpression, knockdown and pharmacological inhibition on oxaliplatin resistance in CRC were assessed by flow cytometry analysis of cell apoptosis in vivo and in vitro.ResultsMetabolic analysis revealed that the levels of purine metabolites, especially guanosine monophosphate (GMP), were markedly elevated in oxaliplatin-resistant CRC cells. The accumulation of purine metabolites mainly arose from the upregulation of IMPDH2 expression. Gene set enrichment analysis (GSEA) indicated high IMPDH2 expression in CRC correlates with PURINE_METABOLISM and MULTIPLE-DRUG-RESISTANCE pathways. CRC cells with higher IMPDH2 expression were more resistant to oxaliplatin-induced apoptosis. Overexpression of IMPDH2 in CRC cells resulted in reduced cell death upon treatment with oxaliplatin, whereas knockdown of IMPDH2 led to increased sensitivity to oxaliplatin through influencing the activation of the Caspase 7/8/9 and PARP1 proteins on cell apoptosis. Targeted inhibition of IMPDH2 by mycophenolic acid (MPA) or mycophenolate mofetil (MMF) enhanced cell apoptosis in vitro and decreased in vivo tumour burden when combined with oxaliplatin treatment. Mechanistically, the Wnt/β-catenin signalling was hyperactivated in oxaliplatin-resistant CRC cells, and a reciprocal positive regulatory mechanism existed between Wnt/β-catenin and IMPDH2. Blocking the Wnt/β-catenin pathway could resensitize resistant cells to oxaliplatin, which could be restored by the addition of GMP.ConclusionsIMPDH2 is a predictive biomarker and therapeutic target for oxaliplatin resistance in CRC.

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CRISPR-based knockout screening identifies the loss of MIEF2 to enhance oxaliplatin resistance in colorectal cancer through inhibiting the mitochondrial apoptosis pathway.

Publications

CRISPR-based knockout screening identifies the loss of MIEF2 to enhance oxaliplatin resistance in colorectal cancer through inhibiting the mitochondrial apoptosis pathway.

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