Crispr Screen to identify miRNAs associated with sphingosine kinase 2 and response to therapies in oral cancer

• Grant number: 23/09624-9
• Support Opportunities: Scholarships in Brazil - Support Program for Fixating Young Doctors
• Start date: August 01, 2023
• End date: June 30, 2025
• Field of knowledge: Health Sciences - Dentistry
• Agreement: CNPq
• Principal Investigator: Andréia Machado Leopoldino
• Grantee: Gabriel da Silva
• Host Institution: Faculdade de Ciências Farmacêuticas de Ribeirão Preto (FCFRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Associated research grant: 23/01454-7 - Crispr screen to identify miRNAs associated with sphingosine kinase 2 and response to therapies in Oral Cancer, AP.R

Abstract

Oral cancer is a malignant neoplasm that can affect the oral cavity and lips, with oral squamous cell carcinoma (OSCC) being the most common subtype. OSCC is highly aggressive and frequently metastasizes to cervical lymph nodes. Despite advances in therapy and increased knowledge about factors associated with OSCC, the survival rate for patients with OSCC has not improved substantially in recent years. Thus, the study of mechanisms associated with the development and progression of OSCC is still necessary. Sphingosine kinase 2 (SK2) is one of the enzymes responsible for the formation of phosphorylated sphingolipids and has been associated with tumorigenesis. The role of SK2 in cancer is debatable, as it appears to have opposite effects (tumor suppressor or oncogenic) depending on the expression level and cellular context. Our research group identified that SK2 overexpression can affect several important processes in cancer. For example, induce malignant transformation of normal oral keratinocytes, enhance tumor growth in vivo, and confer tumor stem cell characteristics and resistance to genotoxic agents. In addition, through sequencing we identified 133 microRNAs differentially expressed in the strain with overexpression of SK2 (NOK-SK2), and many of these miRNAs are related to tumorigenesis, such as miR-205-5p and mir-296-5p. Through functional assays, we observed that the inhibition of miR-296-5p in NOK-SK2 cells reduces the clonogenic capacity and the formation of spheroids and alters the expression of proteins involved in the epithelial-mesenchymal transition. Taken together, these results suggest that the deregulation of these more than 100 miRNAs may play a relevant role in the tumorigenic role of SK2 overexpression. Currently, a relevant technique to study genes essential for tumorigenesis is the CRISPR-based genetic screening (CRISPR screen). In the CRISPR screen system, pools of cells with various genomic modifications are generated that can be tracked by sgRNA sequences (single-guide RNAs) integrated into the DNA of target cells. As part of this project, a CRISPR screen focusing on microRNAs (1,594 targets) will be performed in cells (with and without SK2 alteration) grown in 3D culture and in a xenograft model. Cells with the genomic modifications competed with each other based on the competence conferred by the miRNA deletion. Making it possible to identify in the remaining cells after selective pressure which miRNAs whose deletion made the cell more or less able to grow in 3D culture and in vivo. Thus, we hope that the large-scale screening of miRNAs will generate important results regarding the role of miRNAs in the tumorigenic effects of KS2, and also make it possible to identify miRNAs that can be exploited as therapeutic targets. Furthermore, the use of modulators for the miRNAs identified in the CRISPR screen combined with SK2 modulation may result in a form of therapy to be explored in the future

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

MILAN, THAIS MORE; SILVA, GABRIEL; SOUSA, LUCAS OLIVEIRA; LEOPOLDINO, ANDREIA MACHADO. Sphingosine Kinase 2 Controls the Aggressive Phenotype of Oral Squamous Cell Carcinoma by Regulating miR-205 and miR-296 through p53. American Journal of Pathology, v. 195, n. 2, p. 13-pg., 2025-01-24. (16/19103-2, 23/09624-9, 22/10740-0)