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Genome-wide CRISPR/Cas9 screening identifies CARHSP1 responsible for radiation resistance in glioblastoma.

ABSTRACT: Glioblastomas (GBM) is the most common primary malignant brain tumor, and radiotherapy plays a critical role in its therapeutic management. Unfortunately, the development of radioresistance is universal. Here, we identified calcium-regulated heat-stable protein 1 (CARHSP1) as a critical driver for radioresistance utilizing genome-wide CRISPR activation screening. This is a protein with a cold-shock domain (CSD)-containing that is highly similar to cold-shock proteins. CARHSP1 mRNA level was upregulated in irradiation-resistant GBM cells and knockdown of CARHSP1 sensitized GBM cells to radiotherapy. The high expression of CARHSP1 upon radiation might mediate radioresistance by activating the inflammatory signaling pathway. More importantly, patients with high levels of CARHSP1 had poorer survival when treated with radiotherapy. Collectively, our findings suggested that targeting the CARHSP1/TNF-α inflammatory signaling activation induced by radiotherapy might directly affect radioresistance and present an attractive therapeutic target for GBM, particularly for patients with high levels of CARHSP1.

SUBMITTER: Zhu GD

PROVIDER: S-EPMC8295287 | biostudies-literature | 2021 Jul

REPOSITORIES: biostudies-literature

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Genome-wide CRISPR/Cas9 screening identifies CARHSP1 responsible for radiation resistance in glioblastoma.

Zhu Guo-Dong GD Yu Jing J Sun Zheng-Yu ZY Chen Yan Y Zheng Hong-Mei HM Lin Mei-Lan ML Ou-Yang Shi S Liu Guo-Long GL Zhang Jie-Wen JW Shao Feng-Min FM

Cell death & disease 20210721 8

Glioblastomas (GBM) is the most common primary malignant brain tumor, and radiotherapy plays a critical role in its therapeutic management. Unfortunately, the development of radioresistance is universal. Here, we identified calcium-regulated heat-stable protein 1 (CARHSP1) as a critical driver for radioresistance utilizing genome-wide CRISPR activation screening. This is a protein with a cold-shock domain (CSD)-containing that is highly similar to cold-shock proteins. CARHSP1 mRNA level was upre ...[more]

PMID: 34290231

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Project description:BackgroundMetastasis is one of the most lethal hallmarks of esophageal squamous cell carcinoma (ESCC), yet the mechanisms remain unclear due to a lack of reliable experimental models and systematic identification of key drivers. There is urgent need to develop useful therapies for this lethal disease.MethodsA genome-wide CRISPR/Cas9 screening, in combination with gene profiling of highly invasive and metastatic ESCC sublines, as well as PDX models, was performed to identify key regulators of cancer metastasis. The Gain- and loss-of-function experiments were taken to examine gene function. Protein interactome, RNA-seq, and whole genome methylation sequencing were used to investigate gene regulation and molecular mechanisms. Clinical significance was analyzed in tumor tissue microarray and TCGA databases. Homology modeling, modified ELISA, surface plasmon resonance and functional assays were performed to identify lead compound which targets MEST to suppress cancer metastasis.FindingsHigh MEST expression was associated with poor patient survival and promoted cancer invasion and metastasis in ESCC. Mechanistically, MEST activates SRCIN1/RASAL1-ERK-snail signaling by interacting with PURA. miR-449a was identified as a direct regulator of MEST, and hypermethylation of its promoter led to MEST upregulation, whereas systemically delivered miR-449a mimic could suppress tumor metastasis without overt toxicity. Furthermore, molecular docking and computational screening in a small-molecule library of 1,500,000 compounds and functional assays showed that G699-0288 targets the MEST-PURA interaction and significantly inhibits cancer metastasis.InterpretationWe identified the MEST-PURA-SRCIN1/RASAL1-ERK-snail signaling cascade as an important mechanism underlying cancer metastasis. Blockade of MEST-PURA interaction has therapeutic potential in management of cancer metastasis.FundingThis work was supported by National Key Research and Development Program of China (2021YFC2501000, 2021YFC2501900, 2017YFA0505100); National Natural Science Foundation of China (31961160727, 82073196, 81973339, 81803551); NSFC/RGC Joint Research Scheme (N_HKU727/19); Natural Science Foundation of Guangdong Province (2021A1515011158, 2021A0505030035); Key Laboratory of Guangdong Higher Education Institutes of China (2021KSYS009).

Project description:Background: Cisplatin is one of the frontline anticancer agents. However, development of cisplatin-resistance limits the therapeutic efficacy of cisplatin-based treatment. The expression of microtubule-associated serine/threonine kinase 1 (MAST1) is a primary factor driving cisplatin-resistance in cancers by rewiring the MEK pathway. However, the mechanisms responsible for MAST1 regulation in conferring drug resistance is unknown. Methods: We implemented a CRISPR/Cas9-based, genome-wide, dual screening system to identify deubiquitinating enzymes (DUBs) that govern cisplatin resistance and regulate MAST1 protein level. We analyzed K48- and K63-linked polyubiquitination of MAST1 protein and mapped the interacting domain between USP1 and MAST1 by immunoprecipitation assay. The deubiquitinating effect of USP1 on MAST1 protein was validated using rescue experiments, in vitro deubiquitination assay, immunoprecipitation assays, and half-life analysis. Furthermore, USP1-knockout A549 lung cancer cells were generated to validate the deubiquitinating activity of USP1 on MAST1 abundance. The USP1-MAST1 correlation was evaluated using bioinformatics tool and in different human clinical tissues. The potential role of USP1 in regulating MAST1-mediated cisplatin resistance was confirmed using a series of in vitro and in vivo experiments. Finally, the clinical relevance of the USP1-MAST1 axis was validated by application of small-molecule inhibitors in a lung cancer xenograft model in NSG mice. Results: The CRISPR/Cas9-based dual screening system identified USP1 as a novel deubiquitinase that interacts, stabilizes, and extends the half-life of MAST1 by preventing its K48-linked polyubiquitination. The expression analysis across human clinical tissues revealed a positive correlation between USP1 and MAST1. USP1 promotes MAST1-mediated MEK1 activation as an underlying mechanism that contributes to cisplatin-resistance in cancers. Loss of USP1 led to attenuation of MAST1-mediated cisplatin-resistance both in vitro and in vivo. The combined pharmacological inhibition of USP1 and MAST1 using small-molecule inhibitors further abrogated MAST1 level and synergistically enhanced cisplatin efficacy in a mouse xenograft model. Conclusions: Overall, our study highlights the role of USP1 in the development of cisplatin resistance and uncovers the regulatory mechanism of MAST1-mediated cisplatin resistance in cancers. Co-treatment with USP1 and MAST1 inhibitors abrogated tumor growth and synergistically enhanced cisplatin efficacy, suggesting a novel alternative combinatorial therapeutic strategy that could further improve MAST1-based therapy in patients with cisplatin-resistant tumors.

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Genome-wide CRISPR/Cas9 screening identifies CARHSP1 responsible for radiation resistance in glioblastoma.

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Genome-wide CRISPR/Cas9 screening identifies CARHSP1 responsible for radiation resistance in glioblastoma.

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