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Updated: Jul 31, 2025
Pooled CRISPR-Based Genetic Screens in Mammalian Cells
Published on: September 4, 2019
Genome-wide CRISPR/Cas9 screening identifies a targetable MEST-PURA interaction in cancer metastasis
Wen Wen Xu1, Long Liao1, Wei Dai2
1Key Laboratory of Biological Targeting Diagnosis, Therapy and Rehabilitation of Guangdong Higher Education Institutes and Key Laboratory of Protein Modification and Degradation, The Fifth Affiliated Hospital of Guangzhou Medical University and School of Basic Medical Sciences, Guangzhou Medical University, Guangzhou, China; MOE Key Laboratory of Tumor Molecular Biology, National Engineering Research Center of Genetic Medicine, College of Life Science and Technology, Jinan University, Guangzhou, China.
Background:
Metastasis 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.
Methods:
A 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.
Findings:
High 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.
Interpretation:
We 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.
Funding:
This 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).
Insights
High MEST expression drives esophageal cancer metastasis by activating specific signaling pathways. Targeting the MEST-PURA interaction with compounds like G699-0288 shows promise for new anti-metastasis therapies.
Area of Science:
- Oncology
- Molecular Biology
- Cancer Research
Background:
- Metastasis is a major cause of mortality in esophageal squamous cell carcinoma (ESCC).
- Understanding ESCC metastasis mechanisms requires effective models and driver identification.
- Novel therapeutic strategies are urgently needed for this aggressive cancer.
Purpose of the Study:
- To identify key regulators of cancer metastasis in ESCC.
- To elucidate the molecular mechanisms driving ESCC invasion and metastasis.
- To discover potential therapeutic targets and compounds for inhibiting ESCC metastasis.
Main Methods:
- Genome-wide CRISPR/Cas9 screening and gene profiling of ESCC sublines and PDX models.
- Gain- and loss-of-function experiments, interactome, RNA-seq, and methylation sequencing.
- Clinical data analysis (TCGA), compound screening, and functional assays to identify MEST inhibitors.
Main Results:
- High MEST expression correlates with poor survival and promotes ESCC invasion and metastasis.
- MEST activates the SRCIN1/RASAL1-ERK-snail signaling pathway via PURA interaction.
- miR-449a regulates MEST; G699-0288 inhibits MEST-PURA interaction and metastasis.
Conclusions:
- The MEST-PURA-SRCIN1/RASAL1-ERK-snail cascade is a critical mechanism in ESCC metastasis.
- Blocking the MEST-PURA interaction offers a potential therapeutic strategy for managing cancer metastasis.
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