title: Identification of genes associated with pancreatic cancer metastasis by CRISPR/Cas9 screening
Creators: name: Zhang, Shuman
Publisher: name: Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU)
Year: 0
Subjects: subject: DDC Classification::5 Naturwissenschaften und Mathematik :: 57 Biowissenschaften; Biologie :: 570 Biowissenschaften; Biologie

description: Background Pancreatic cancer is a common malignant tumor of the digestive system. It is highly aggressive, rapidly progressing, easily metastatic, and extremely difficult to treat. 80 % of patients with pancreatic cancer are already locally advanced or metastatic at the time of diagnosis with a poor prognosis. This research is expected to identify the factors associated with metastasis that can provide an opportunity to control the progression of pancreatic cancer. Methods A mouse ACF CRISPR knockout library of 3,466 sgRNAs directed against 915 genes was transfected into a mouse PDAC cell line (TB 32047) to select the genes required or impeded for cell migration by Transwell screening. After accelerated and impaired migration screening, the next generation sequence (NGS) showed candidate genes for further validation. Knockout PRKAR1A, STK11 and Hkdc1 in pancreatic cancer cell lines using CRISPR/Cas9 technology. Wound healing assay, orthotopic pancreatic tumor mouse models and CAM assay assessed cell migration and metastasis changes after PRKAR1A, STK11 and Hkdc1 knockout. RNA sequencing was performed to characterize PRKAR1A, STK11, and Hkdc1 knockouts and to analyze pathway enrichment. Results CRISPR/Cas9 screening identified sgRNAs that led to enhanced or inhibited migration. PRKAR1A and STK11 were the top candidate genes whose knockout promoted migration, while knockout Hkdc1 inhibited cell migration in vitro. Interestingly, loss of PRKAR1A leads to enhanced activity of cAMP-dependent protein kinase (PKA). Treatment with an inhibitor (H89) against PKA effectively reduced the migration of pancreatic cancer cells. STK11 plays as a tumor suppressor in pancreatic cancer. Interestingly, the STK11 mutant is closely associated with enhanced phosphodiesterase (PDE) expression especially PDE4D, PDE4B, or PDE10A. PDEs could function as a transcriptional biomarker that identifies the STK11 mutation in PDAC. Hkdc1 is a novel hexokinase. However, the absence of Hkdc1 did not affect hexokinase activity. Then in vivo assay indicated that the loss of PRKAR1A or STK11 led to increased liver metastasis in mice, while the Hkdc1 knockout had the opposite effect. Mechanistic analyzes revealed that inhibition of PRKAR1A or STK11 led to vessel morphogenesis, such as Sox17, Ephb3, CD55, Ccn2, Itga7, Itgb3, and the immune effector process, such as IL11 and IL33. The expression of Hkdc1 showed a negative correlation with Klf4. Conclusion In conclusion, in-depth analyzes of which gene can regulate pancreatic cancer metastasis by CRISPR/Cas9 screening can provide an essential basis for heterogeneity, staging diagnosis and prognostic judgment of pancreatic cancer, and individualized treatment. Vascular morphogenesis and immune effector process pathways are essential for cell migration and metastasis in PRKAR1A, STK11, and Hkdc1 loss cells. It can help to adopt different therapeutic strategies for different mutation tumors to rationalize the efficacy and reduce ineffective treatment, ultimately prolonging patients' survival and improving their quality of life.