Alternative splicing modulation in breast cancer using RNA oligonucleotides

• Grant number: 24/21235-0
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: August 01, 2025
• End date: January 31, 2029
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Patricia Pereira Coltri
• Grantee: Hilan Ribeiro de Morais Lopes
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Breast cancer is a growing global health problem, especially among women. For this reason, the search for regulatory and therapeutic pathways targeting this cancer has increased in recent years. Alterations in the transcription and processing of messenger RNAs (mRNAs) and microRNAs (miRNAs) are frequently observed in this type of tumor. Pre-mRNA splicing is essential for RNA processing and gene expression control. This process is responsible for joining exons and removing introns, forming mature mRNAs. Splicing is finely regulated and has implications in multiple cellular functions. The great majority of genes in mammals has multiple introns, therefore several different isoforms can be generated through alternative splicing. A large macromolecular complex of proteins and RNAs named spliceosome catalyzes splicing. In addition to several spliceosome components, splicing depends upon RNA-binding proteins (RBP), like hnRNPs A1, K, A2B1 and the splicing factor SRSF6. These RBPs targets exons and/or introns and modulate intron retention, exon skipping and other mechanisms important to control splicing outcome. These factors can interact with the pre-mRNA during its processing and modulate the production of the mature mRNA. Preliminary in silico analyses have revealed that the BCL-X, CD44 and PKM genes can undergo changes in their splicing pattern in breast cancer cells because of variations in the expression of the hnRNP A1, K and A2B1 proteins. In some conditions, the splicing pattern of these genes can generate RNA isoforms that act enhancing the tumor phenotype inhibiting cell death, facilitating epithelial-mesenchymal transition and contributing for tumor energy metabolism. Additionally, several miRNAs are encoded from intronic regions and it is possible that splicing affects their biogenesis and maturation. Altered expression of the miRNAs miR-128, miR-502, miR-181, miR-214 and miR-199b can also affect breast cancer progression. In this context, the hypothesis of this project is that controlling the activity of the hnRNP A1, K, A2B1 and SRSF6 proteins can modulate the alternative splicing of genes important for the development of breast cancer, interfering with the tumor phenotype. To this end, RNA oligonucleotides capable of binding to hnRNPs A1, K, A2B1 and SRSF6 will be used to analyze the global splicing pattern of cells, and especially the BCL-X, CD44 and PKM genes, as well as the biogenesis of the miRNAs miR-128, miR-502, miR-181, miR-214, miR-199b. Additionally, we intend to address whether the use of these oligos change protein localization and aggregation features within the cell. Finally, we will analyze how these alterations influence the tumor phenotype and whether this could lead to an important therapeutic approach targeting breast cancer cells.