Dataset Information

Phosphoproteomic Analysis Defines BABAM1 as mTORC2 Downstream Effector Promoting DNA Damage Response in Glioblastoma Cells

ABSTRACT: Glioblastoma (GBM) is a devastating primary brain cancer with a poor prognosis. GBM is associated with an abnormal mechanistic target of rapamycin (mTOR) signaling pathway, consisting of two distinct kinase complexes: mTORC1 and mTORC2. The complexes play critical roles in cell proliferation, survival, migration, metabolism, and DNA damage response. This study investigated the aberrant mTORC2 signaling pathway in GBM cells by performing quantitative phosphoproteomic analysis of U87MG cells under different drug treatment conditions. 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 BRISC and BRCA1-A Complex Member 1 (BABAM1). We demonstrated that pBABAM1 at Ser29 is regulated by mTORC2 to initiate DNA damage response, contributing 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 that histone H2AX phosphorylation at Ser139 (γH2AX) could be controlled by mTORC2 to repair the DNA. These results provided a better understanding of mTORC2 function in oncogenic DNA damage response and might lead to specific mTORC2 treatments for brain cancer patients in the future.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Brain

DISEASE(S): Brain Cancer

SUBMITTER: Nuttiya Kalpongnukul

LAB HEAD: Nuttiya Kalpongnukul

PROVIDER: PXD033325 | Pride | 2022-11-08

REPOSITORIES: Pride

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Publications

Phosphoproteomic Analysis Defines BABAM1 as mTORC2 Downstream Effector Promoting DNA Damage Response in Glioblastoma Cells.

Kalpongnukul Nuttiya N Bootsri Rungnapa R Wongkongkathep Piriya P Kaewsapsak Pornchai P Ariyachet Chaiyaboot C Pisitkun Trairak T Chantaravisoot Naphat N

Journal of proteome research 20221031 12

Glioblastoma (GBM) is a devastating primary brain cancer with a poor prognosis. GBM is associated with an abnormal mechanistic target of rapamycin (mTOR) signaling pathway, consisting of two distinct kinase complexes: mTORC1 and mTORC2. The complexes play critical roles in cell proliferation, survival, migration, metabolism, and DNA damage response. This study investigated the aberrant mTORC2 signaling pathway in GBM cells by performing quantitative phosphoproteomic analysis of U87MG cells under ...[more]

PMID: 36315652

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Project description:Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors. Despite radical surgery and radiotherapy supported by chemotherapy, the disease still remains incurable with extremely low median survival rate of 12-15 months from the time of initial diagnosis. The main cause of treatment failure is considered to be the presence of cells that are resistant to such treatment. MicroRNAs (miRNAs) as regulators of gene expression are involved in the tumor pathogenesis, including GBM. MiR-338 is a brain specific miRNA which has been described to target pathways involved in proliferation and differentiation. In our study, miR-338-3p and -5p were differentially expressed in GBM tissue in comparison to non-tumor brain tissue. Overexpression of miR-338-3p with miRNA mimic did not show any changes in proliferation rates in GBM cell lines (A172, T98G, U87MG). On the other hand, pre-miR-338-5p notably decreased proliferation and caused cell cycle arrest. Since radiation is currently the main treatment modality in GBM, we combined overexpression of pre-miR-338-5p with radiation, which led to significantly decreased of cell proliferation, and increased cell cycle arrest and apoptosis in comparison to only irradiated cells. To better elucidate the mechanism of action, we performed gene expression profiling analysis that revealed targets of miR-338-5p being Ndfip1, Rheb, ppp2R5a. These genes have been described to be involved in DNA damage response, proliferation and cell cycle regulation. To our knowledge, this is the first study to describe role of miR-338-5p in GBM and its potential to improve sensitivity of GBM to radiation. Study was performed on three glioblastoma multiforme cell lines A172, T98G and U87MG. This experiment was performed on Affymetrix GeneChip Human Gene ST 1.0 to elucidate the targets of miRNA-338-5p. Cell lines were seeded 24 hours prior transfection. After transfection with pre-miR338-5p or negative control cell were cultured for 24 hours and harvested. RNA was isolated using MirVana miRNA Isolation Kit (Ambion, USA) and checked for RNA integrity by Bioanalyzer 2100 and purity by ratios 260/280>1.8 and 260/230>1.8 by Nanodrop2000.

Dataset Information

Phosphoproteomic Analysis Defines BABAM1 as mTORC2 Downstream Effector Promoting DNA Damage Response in Glioblastoma Cells

Publications

Phosphoproteomic Analysis Defines BABAM1 as mTORC2 Downstream Effector Promoting DNA Damage Response in Glioblastoma Cells.

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