3145 - Knockdown and Inhibition of RNA-Binding Proteins Musashi-1 and Musashi-2 Modulate DNA Repair and Radiosensitize Triple-Negative Breast Cancer
Presenter(s)
F. Troschel1, K. A. Brücksken1, F. K. Meier1, C. Meißner1, G. Poschmann2, E. Korsching3, H. Hassan4, A. A. WalyEldeen4, S. A. Ibrahim4, R. M. Samer5, M. Sicking1, M. Götte6, B. Greve1, and H. T. Eich7; 1Department of Radiation Oncology, Münster University Hospital, Münster, Germany, 2Institute of Molecular Medicine, Proteome research, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany, 3Cancer and Complex Systems Research Group, Münster University, Münster, Germany, 4Zoology Department, Faculty of Science, Cairo University, Cairo, Egypt, 5Pathology Department, Faculty of Medicine, Aswan University, Aswan, Egypt, 6Department of Gynecology and Obstetrics, Münster University Hospital, Münster, Germany, 7Department of Radiation Therapy – Radiation Oncology, University Hospital of Muenster, Muenster, Germany
Purpose/Objective(s): Given their function as RNA-binding proteins, the Musashi proteins Msi1 and Msi2 play key roles in gene expression regulation. As they are overexpressed in cancer, they have been linked to oncogenic signaling. Here, we present data on their implication in DNA repair and radioresistance, their direct binding partners and subsequent genomic and proteomic downstream targets as well as their expression in primary triple-negative breast cancer (TNBC) tissues. We also evaluate a potential inhibitor of both proteins.
Materials/Methods: We first determined RNA and protein expression of both Msi1 and Msi2 in TNBC samples and healthy tissues. We subsequently performed RNA immunoprecipitation sequencing (RIP-Seq) to assess direct binding partners in MDA-MB-231 triple-negative breast cancer cells and additionally generated RNA-Seq and mass spectrometry-based proteomic data after siRNA-based targeting of Msi1 or Msi2. We validated important targets in primary samples. The functional relevance of both Msi1 and Msi2 was determined with DNA damage arrays, longitudinal quantitative digital holographic microscopy (DHM) tracking, radiation (clonogenic assays) and chemotherapy (MTT assay) experiments. Subsequently, we assessed an inhibitor of the Msi proteins, Gossypolone, in cell lines and patient-derived primary triple-negative breast cancer samples with the same methodology.
Results: The Msi proteins are overexpressed in TNBC compared to healthy tissues. RIP-Seq helped identify 2590 RNA binding partners for Msi1 and 1214 for Msi2 with an incomplete overlap (344 genes, 28.3% of Msi2 genes) between both. Downstream effects on transcriptomic and proteomic levels included DNA repair, regulation of cell cycle and cell division pathways (p < 0.001 for all). Consequently, knockdown of Msi1 or Msi2 abrogated proliferation and sensitized cells to chemotherapy and irradiation in clonogenic assays at 2, 4, and 6 Gy. Radiation-induced DNA damage was increased after Msi1 and Msi2 knockdown and DHM indicated a decrease in cell proliferation and motility in single-cell analyses. Gossypolone showed a functional phenocopy of Msi targeting, including reduced proliferation and radiosensitization as well as suppressed Msi1 and Msi2 levels. Proteomic analyses indicated a large overlap of significantly altered pathways between the inhibition approach and Msi knockdown (27/49 gene ontology terms, 55.1%).
Conclusion: Msi proteins are overexpressed and associated with oncogenic signaling in TNBC. Msi targeting – either via knockdown or inhibition – compromised proliferation, motility, and DNA repair and sensitized TNBC cells to irradiation and chemotherapy. Given promising pre-clinical data, Msi inhibition deserves further study as a therapy-sensitizing approach.