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CRISPR/Cas9 genome-wide screening identifies LUC7L2 that promotes radioresistance via autophagy in nasopharyngeal carcinoma cells.

ABSTRACT: Radioresistance emerges as the major obstacle to nasopharyngeal carcinoma (NPC) treatment, further understanding of underlying mechanisms is necessary to overcome the radioresistance and improve the therapeutic effect. In this study, we first identified a candidate radioresistant-related gene LUC7L2 via CRISPR/Cas9 high-throughput screening and quantitative proteomic approach. Overexpression of LUC7L2 in NPC cells promoted cell viability following exposure to ionizing radiation (IR), while knockdown of LUC7L2 significantly slowed down the DNA replication and impaired cell survival, sensitized NPC-radioresistant cells to IR. Using immunoprecipitation assay, we found SQSTM1, an autophagy receptor, was a potential binding partner of LUC7L2. Down-regulation of LUC7L2 in NPC-radioresistant cells led to reduction of SQSTM1 expression and enhancement of autophagy level. Furthermore, LUC7L2 knockdown in combination with autophagy inhibitor, chloroquine (CQ), resulted in more NPC-radioresistant cell death. Besides, LUC7L2 was obviously distributed in NPC tissues, and high LUC7L2 expression correlated with shorter survival in NPC patients. Our data suggest that LUC7L2 plays a huge part in regulating radioresistance of NPC cells, and serves as a promising therapeutic target in re-sensitizing NPC to radiotherapy.

SUBMITTER: Shen L

PROVIDER: S-EPMC8671510 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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).

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CRISPR/Cas9 genome-wide screening identifies LUC7L2 that promotes radioresistance via autophagy in nasopharyngeal carcinoma cells.

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