ABSTRACT
Medulloblastoma (MB) encompasses diverse subgroups, and leptomeningeal disease/metastasis (LMD) plays a substantial role in associated fatalities. Despite extensive exploration of canonical genes in MB, the molecular mechanisms underlying LMD and the involvement of the orthodenticle homeobox 2 (OTX2) gene, a key driver in aggressive MB Group 3, remain insufficiently understood. Recognizing OTX2's pivotal role, we investigated its potential as a catalyst for aggressive cellular behaviors, including migration, invasion, and metastasis. OTX2 overexpression heightened cell growth, motility, and polarization in Group 3 MB cells. Orthotopic implantation of OTX2-overexpressing cells in mice led to reduced median survival, accompanied by the development of spinal cord and brain metastases. Mechanistically, OTX2 acted as a transcriptional activator of the Mechanistic Target of Rapamycin (mTOR) gene's promoter and the mTORC2 signaling pathway, correlating with upregulated downstream genes that orchestrate cell motility and migration. Knockdown of mTOR mRNA mitigated OTX2-mediated enhancements in cell motility and polarization. Analysis of human MB tumor samples (N = 952) revealed a positive correlation between OTX2 and mTOR mRNA expression, emphasizing the clinical significance of OTX2's role in the mTORC2 pathway. Our results reveal that OTX2 governs the mTORC2 signaling pathway, instigating LMD in Group 3 MBs and offering insights into potential therapeutic avenues through mTORC2 inhibition.
Subject(s)
Gene Expression Regulation, Neoplastic , Mechanistic Target of Rapamycin Complex 2 , Medulloblastoma , Meningeal Neoplasms , Otx Transcription Factors , Animals , Female , Humans , Male , Mice , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Cerebellar Neoplasms/genetics , Cerebellar Neoplasms/pathology , Cerebellar Neoplasms/metabolism , Mechanistic Target of Rapamycin Complex 2/metabolism , Mechanistic Target of Rapamycin Complex 2/genetics , Medulloblastoma/genetics , Medulloblastoma/pathology , Medulloblastoma/metabolism , Meningeal Neoplasms/genetics , Meningeal Neoplasms/pathology , Meningeal Neoplasms/metabolism , Meningeal Neoplasms/secondary , Otx Transcription Factors/metabolism , Otx Transcription Factors/genetics , Signal TransductionABSTRACT
p53-binding protein 1 (53BP1) acts as an 'adaptor/mediator' for transducing DNA damage signals, especially following detection of DNA double-strand breaks. In an effort to broaden our understanding of the protein network surrounding 53BP1, we isolated possible 53BP1 binding partners by co-immunoprecipitation, and identified them via tandem mass spectrometric analysis. The 53BP1-associated proteins included RPA1 and RPA2, two components of the replication protein A (RPA) complex. The presence of RPA components in the immunoprecipitates was confirmed by immunoblotting, and we found that the association between 53BP1 and RPA2 was disrupted following DNA damage induced by treatment with camptothecin, a topoisomerase I inhibitor. To investigate the functional meaning of the 53BP1 and RPA interaction, we established U2OS osteosarcoma cell lines stably expressing dominant-negative fragments of 53BP1. We found that camptothecin-induced RPA2 phosphorylation was inhibited in these cells, and also following 53BP1 knockdown by siRNA transfection. On the cellular level, camptothecin-induced apoptosis was augmented in the dominant-negative cell lines, resulting in increased chemosensitivity to this drug. Taken together, these results suggest that 53BP1 is involved in DNA damage-induced RPA2 hyperphosphorylation, and inhibition of 53BP1 function may sensitize cancer cells to camptothecin treatment.
