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N6-Methyladenosine modification mediated by METTL3 promotes DNA-PKcs expression to induce anlotinib resistance in
Yining Zhang1,2, Guohong Shen3, Dan Zhang4
1The First Clinical College of Cheeloo College of Medicine, Shandong University, Jinan, Shandong, China.
Background:
Acquired anlotinib resistance is still a key challenge in osteosarcoma treatment. Unravelling the mechanisms underlying anlotinib resistance is the key to optimising its efficacy for treating osteosarcoma. Previous studies have explored the pivotal function of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) with regard to osteosarcoma chemoresistance.
Methods:
We used bioinformatics analysis to predict DNA-PKcs and Beclin-1 interactions, confirmed through immunofluorescence (IF) and co-immunoprecipitation (co-IP). Dual-luciferase analyses and Methylated RNA immunoprecipitation (MeRIP) were implemented to detect the detected m6A modifications. RNA fluorescence in situ hybridisation (FISH)-IF co-localisation and RNA immunoprecipitation (RIP) were conducted to explore the interplay between PRKDC mRNA and the indicated proteins.
Results:
Anlotinib-treated osteosarcoma cells exhibited increased DNA-PKcs levels, and silencing DNA-PKcs augmented osteosarcoma sensitivity to anlotinib. DNA-PKcs affects anlotinib-induced autophagy by interacting with Beclin-1 and regulating its ubiquitination. Notably, PRKDC mRNA, encoding DNA-PKcs, underwent N6-Methyladenosine (m6A) modification. Methyltransferase-like 3 (METTL3) positively regulated DNA-PKcs expression. Functionally, METTL3 enhances anlotinib resistance in osteosarcoma, which is reversed by PRKDC knockdown. Mechanistically, METTL3 binds to PRKDC mRNA and facilitates m6A methylation. Additionally, m6A methylated PRKDC mRNA is identified via YTH N6-methyladenosine RNA-binding protein 1 (YTHDF1), augmenting its expression.
Conclusion:
These findings revealed that DNA-PKcs promotes anlotinib resistance by regulating protective autophagy, while METTL3 induces PRKDC m6A modification, enhancing its expression. Thus, targeting METTL3/PRKDC may be a novel strategy for improving therapeutic efficacy in human osteosarcoma.
Key Points:
DNA-PKcs knockdown heightens osteosarcoma sensitivity to anlotinib. DNA-PKcs modulates anlotinib-induced protective autophagy through interacts with Beclin-1 and regulates its ubiquitination. m6A modification of OLE_LINK82PRKDC mRNA induced by METTL3 contributes to anlotinib resistance in osteosarcoma. m6A methylation of PRKDC mRNA recognised by YTHDF1 amplifies the expression of DNA-PKcs.
Insights
DNA-PKcs promotes anlotinib resistance in osteosarcoma by regulating autophagy. METTL3 enhances this resistance by inducing m6A modification of PRKDC mRNA, increasing DNA-PKcs expression. Targeting METTL3/PRKDC may improve osteosarcoma treatment.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- Anlotinib resistance is a significant challenge in osteosarcoma treatment.
- Understanding resistance mechanisms is crucial for optimizing anlotinib efficacy.
- DNA-dependent protein kinase catalytic subunit (DNA-PKcs) has been implicated in osteosarcoma chemoresistance.
Purpose of the Study:
- To elucidate the role of DNA-PKcs in anlotinib resistance in osteosarcoma.
- To investigate the interaction between DNA-PKcs and Beclin-1 in autophagy regulation.
- To explore the impact of N6-Methyladenosine (m6A) modification on DNA-PKcs expression and anlotinib resistance.
Main Methods:
- Bioinformatics analysis, immunofluorescence, and co-immunoprecipitation to study protein interactions.
- Dual-luciferase and Methylated RNA immunoprecipitation (MeRIP) assays for m6A modification detection.
- RNA fluorescence in situ hybridisation (FISH)-IF and RNA immunoprecipitation (RIP) to analyze mRNA-protein interplay.
Main Results:
- Increased DNA-PKcs levels were observed in anlotinib-resistant osteosarcoma cells; its silencing sensitized cells to anlotinib.
- DNA-PKcs interacts with Beclin-1, regulating its ubiquitination and affecting anlotinib-induced autophagy.
- Methyltransferase-like 3 (METTL3) positively regulates DNA-PKcs expression by m6A modification of PRKDC mRNA, enhancing anlotinib resistance.
- YTH N6-methyladenosine RNA-binding protein 1 (YTHDF1) recognizes m6A methylated PRKDC mRNA, amplifying DNA-PKcs expression.
Conclusions:
- DNA-PKcs promotes anlotinib resistance via regulation of protective autophagy.
- METTL3-induced m6A modification of PRKDC mRNA enhances DNA-PKcs expression, contributing to resistance.
- Targeting the METTL3/PRKDC pathway presents a potential therapeutic strategy for overcoming anlotinib resistance in osteosarcoma.
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