LOXL1-AS1 contributes to medulloblastoma metastasis: A potential target for mesenchymal stem cell exosome-delivered gene therapy

Abstract Introduction: Medulloblastoma (MB) is the most common malignant tumor in children. MB-related treatment failures and deaths mostly result from metastasis. One of the key genetic drivers in MB is MYCN, whose aberrant expressions are associated with tumor aggressiveness and poor prognosis. Long non-coding (lnc)RNAs have been implied as potential targets for cancer treatment. More insights into the role of lncRNAs in MYCN-mediated MB progression would have both prognostic and therapeutic significance in metastatic MB. Cell-derived vesicles such as exosome can encapsulate siRNAs to improve their bioavailability and targeted delivery to brain tumors. Mesenchymal stem cell (MSC) is a readily available and modifiable source to produce functional exosomes. Objective: To identify the downstream lncRNAs of MYCN that contributes to MB metastasis, and to develop a novel gene therapy for MB by MSC exosome-delivered siRNAs to target these prometastatic lncRNAs. Methods: An RNA sequencing analysis was used to identify LOXL1-AS1 as a MYCN targeting gene in MYCN-overexpressing MB cells. The association of LOXL1-AS1 and MYCN was validated in MB cell lines by RT-qPCR and Western blot assay, and in tumor tissues by in-situ hybridization and immunohistochemistry. The anti-tumor effect of LOXL1-AS1 knockdown in MYCN-overexpressing MB cells was assessed in vitro by migration and sphere formation assays, and in an orthotopic xenograft mouse model by intracranial injection. To generate therapeutic exosomes, MSCs were transfected with exogenous LOXL1-AS1 siRNAs and then subjected to exosome characterization of size distribution and marker expression. The effect of exosome treatment on MB cells was evaluated using RT-qPCR and wound healing assay. Results: Elevated LOXL1-AS1 was found in MYCN-overexpressed MB cells compared to parental cells, and this association was confirmed in clinical tissue samples. LOXL1-AS1 expression was correlated with poor prognosis in SHH-α and SHH-γ MB subtypes of patients. Knockdown of LOXL1-AS1 significantly reduced cell migration and cancer stemness properties of MB cells in vitro, and suppressed orthotopic tumor growth, metastasis, and enhanced survival in xenografted mice. Exogenous siRNAs were successfully packaged into MSC exosomes by cell transfection. Treatment of MB cells with LOXL1-AS1 siRNA-loaded exosomes effectively reduced LOXL1-AS1 expression and inhibited cell migration compared to those treated with unmodified exosomes. Conclusion: Our study has proven the contributive role of LOXL1-AS1 in promoting cancer progression and metastasis of MYCN-associated MB, and provided in vitro evidence to support the feasibility of LOXL1-AS1 gene therapy through siRNA-loaded MSC exosomes. In vivo biodistribution and therapeutic efficacy assessment of MSC exosome-delivered LOXL1-AS1 siRNAs are being investigated in animal models. Citation Format: Anh Duy Do, Chia-Ling Hsieh, Tai-Tong Wong, Shian-Ying Sung. LOXL1-AS1 contributes to medulloblastoma metastasis: A potential target for mesenchymal stem cell exosome-delivered gene therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5369.