Characterization of aberrant mTOR signaling pathway in U87MG glioblastoma cell line by quantitative phosphoproteomics

Abstract

Glioblastoma (GBM) is one of the most damaging primary brain tumors with a poor prognosis. GBM is associated with an abnormality of the mTOR signaling pathway, consisting of two distinct kinase complexes: mTORC1 and mTORC2. The protein complexes play critical roles in cellular metabolism, proliferation, cell survival, cell migration through the cytoskeletal organization, and DNA damage response in cancers. Nevertheless, the functions of mTORC2 are less unraveled. This study investigated the aberrant mTORC2 signaling pathway in glioblastoma cells using stable isotope labeling, phosphopeptide enrichment, and LC-MS/MS. We performed the quantitative phosphoproteomic analysis of U87MG grade IV glioma cells treated with the mTORC1/2 inhibitor AZD8055 and the mTORC1 inhibitor rapamycin. By comparing the level of phosphorylated peptides decreased when cells were treated by AZD8055 but not rapamycin, numerous phosphoproteins were identified as mTORC2 downstream target candidates. Interestingly, a functional analysis of phosphoproteome revealed that mTORC2 inhibition might be involved in double-strand break (DSB) repair. We further characterized the relationship between mTORC2 and BABAM1 (also known as MERIT40), a protein with an essential role in DNA damage repair. We demonstrated that pBABAM1 at Ser29 is one of the phosphorylation sites regulated by mTORC2. This phosphorylation event has been shown to promote DNA damage response, which contributes to DNA repair and cancer cell survival. Accordingly, the inactivation of mTORC2 significantly ablated pBABAM1 (Ser29), reduced DNA repair activities in the nucleus, and promoted apoptosis of the cancer cells. Furthermore, we also recognized and showed that γH2AX phosphorylation at Ser139 could potentially be downstream of mTORC2 in association with BABAM1 to repair the double-strand breaks. This investigation provided a better understanding of mTORC2 function in oncogenic DNA damage response and might lead to the development of specific mTORC2 treatments for brain cancer patients in the future.