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Crispr screen to identify miRNAs associated with sphingosine kinase 2 and response to therapies in Oral Cancer

Abstract

Oral squamous cell carcinoma (OSCC) is the most common subtype of head and neck cancer, with late diagnosis in advanced stages, often metastasizing to cervical lymph nodes, and with a high rate of disease recurrence. Despite advances in therapy and increased knowledge about risk factors associated with OSCC, the survival rate for patients has not improved substantially in the last few years. Thus, the study of the mechanisms associated with the development and progression of OSCC is still needed. Sphingosine kinase 2 (SPHK2) is one of the enzymes responsible for the formation of phosphorylated sphingolipids and has been associated with tumorigenesis. The role of SPHK2 in cancer is not consensus, as it appears to have opposite effects (tumor suppressor or oncogenic) depending on the expression level and cellular context. Our research group has identified that overexpression of SPHK2 can affect several important processes in oral carcinogenesis and resistance to therapies (such as cisplatin and paclitaxel). In addition, with large-scale RNA sequencing, we identified several differentially expressed microRNAs in keratinocyte cell lines overexpressing SPHK2 (NOK-SPHK2), and many of these miRNAs have been related to oral cancer. Taken together, these results suggest that the miRNAs may play a relevant role in the tumorigenic role of sphingolipids. Currently, a relevant technique to study genes essential for tumorigenesis is CRISPR-based gene screening (CRISPR screen). In the CRISPR screen system, pools of cells with various genomic modifications are generated that can be screened by sgRNA sequences (single guide RNAs) integrated into the DNA of the target cells. In this project, CRISPR screen focusing on microRNAs (1,594 targets) will be performed in cells (with and without SPHK2 alteration) grown in 3D culture and in a xenograft tumor model in mice. A selected drug combination will be used in the treatment of tumor-bearing mice, which has been shown to be more efficient in inhibiting the growth of CEO xenograft tumors than monotherapy in previous work by our group. Thus, we will be able to identify in the remaining cells (after the selective pressure) which miRNAs whose deletion made the cell more or less fit for growth in 3D culture and in vivo. Thus, we expect that the large-scale screening of miRNAs will yield important results regarding the role of miRNAs in the tumorigenic effects of SPHK2/sphingolipids and, furthermore, make it possible to identify miRNAs that can be exploited as therapeutic targets. Furthermore, the use of modulators for the miRNAs identified by CRISPR screen combined with modulation of SPHK2 may be explored in the future as a therapeutic strategy. (AU)

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