RPTOR blockade suppresses brain metastases of NSCLC by interfering the ceramide metabolism via hijacking YY1 binding.

Abstract

Background: Ceramide metabolism is crucial in the progress of brain metastasis (BM). However, it remains unexplored whether targeting ceramide metabolism may arrest BM. Methods: RNA sequencing was applied to screen different genes in primary and metastatic foci and whole-exome sequencing (WES) to seek crucial abnormal pathway in BM+ and BM-patients. Cellular arrays were applied to analyze the permeability of blood-brain barrier (BBB) and the activation or inhibition of pathway. Database and Co-Immunoprecipitation (Co-IP) assay were adopted to verify the protein-protein interaction. Xenograft and zebrafish model were further employed to verify the cellular results. Results: RNA sequencing and WES reported the involvement of RPTOR and ceramide metabolism in BM progress. RPTORwas significantly upregulated in BM foci and increased the permeability of BBB, while RPTOR deficiency attenuated the cell invasiveness and protected extracellular matrix. Exogenous RPTOR boosted the SPHK2/S1P/STAT3 cascades by binding YY1, in which YY1 bound to the regions of SPHK2 promoter (at -353 ~ -365 nt), further promoting the expression of SPHK2. The latter was rescued by YY1 RNAi. Xenograft and zebrafish model showed that RPTOR blockade suppressed BM of non-small cell lung cancer (NSCLC) and impaired the SPHK2/S1P/STAT3 pathway. Conclusion: RPTOR is a key driver gene in the brain metastasis of lung cancer, which signifies that RPTORblockade may serve as a promising therapeutic candidate for clinical application.