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PRKDC Induces Chemoresistance in Osteosarcoma by Recruiting GDE2 to Stabilize GNAS and Activate AKT

ABSTRACT: Chemoresistance is one of the major causes of poor prognosis in osteosarcoma. Thus far, the therapeutic alternative for osteosarcoma is quite limited, thereby increasing sensitivity of the currently used drugs is an effective way to improve outcome of patients. In this study, through a Kinome-wide CRISPR screen, we identified PRKDC as a critical determinant of Doxorubicin (DOX) sensitivity in osteosarcoma. Clinical sample analysis and functional experiments demonstrated that PRKDC is hyperactivated in osteosarcoma and that knockdown of PRKDC significantly increased sensitivity of osteosarcoma to DOX. Mechanistically, PRKDC could recruit and bind with GDE2 to augment the stability of GNAS. The increased GNAS protein subsequently activate Akt S473 and T308 phosphorylation and mediated DOX sensitivity. We also showed that the PRKDC inhibitor AZD7648 and DOX could synergize with each other and intensely suppressed the growth of osteosarcoma in mice xenograft models and human organoids. In conclusion, these results revealed a novel PRKDC-GDE2-GNAS-Akt regulatory axis in DOX sensitivity and provided targetable candidates for improving therapeutic efficacy of DOX in osteosarcoma.

ORGANISM(S): Homo sapiens

PROVIDER: GSE268415 | GEO | 2024/06/14

REPOSITORIES: GEO

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Project description:GNAS, a gene encoding G-protein stimulating alpha subunit, is frequently mutated in intraductal papillary mucinous neoplasms (IPMNs), which is an indolent and slow-growing pancreatic neoplasm that secretes abundant mucin. GNAS mutation is not observed in conventional ductal adenocarcinomas of the pancreas. To determine the functional significance of GNAS mutation in pancreatic ductal cells, we examined in vitro phenotypes and gene expression profiles of cells of pancreatic ductal lineage, HPDE, PK-8, PCI-35, and MIA PaCa-2, with exogenous expression of either wild-type or mutated (R201H) GNAS. We found that exogenous GNAS upregulated intracellular cyclic-adenine monophosphate, particularly in the mutated GNAS transfectants. Exogenous GNAS induced no obvious cell-growth promotion, but induced suppression in some cells. The exogenous GNAS upregulated MUC2 and MUC5AC in HPDE and PK-8, and the latter was most sensitive to exogenous GNAS, exhibiting drastic alteration of the global gene expression that is consistent with that of IPMN. Hence, PK-8 expressing exogenous mutated GNAS may be an ideal in vitro model of IPMN. On the other hand, exogenous GNAS downregulated expression of mucin genes and produced modest alteration of gene expression profiles in PCI-35 and MIA PaCa-2, indicating lower sensitivity to exogenous GNAS. Furthermore, we showed diverse and cell-type specific mucin expression pathways with complicated interactions between signaling pathways of the G-protein coupled receptor (GPCR), the mitogen-activated protein kinase (MAPK), and the phosphatidylinositol 3 kinase (PI3K), in which the GPCR pathway appeared to be dominant in some and the MAPK pathway in others. In conclusion, mutated GNAS found in IPMNs may extensively alter gene expression profiles, including expression of mucin genes, with the interaction with MAPK and PI3K pathways in pancreatic ductal-lineage cells, which may determine the characteristic phenotype of the neoplasm. Cells of pancreatic cancer cell lines, PK-8, PCI-35,and MIA PaCa-2, were seeded at 4 M-CM-^W 10^5 cells/well in 6-well plates and incubated for 24 hours at 37M-BM-0C in 5% CO2 with humid atmosphere. Then the cells were transfected with either pcDNA 3.1/V5-His vector or pcDNA3.1-GNAS(R201H)-V5-His vector using Lipofectamine 2000 reagent (Life Technologies) according to the manufacturerM-bM-^@M-^Ys recommendations. The cells were incubated for 24 hours and collected by dissociation using trypsin. Total RNAs were isolated using the RNeasy Mini kit (Qiagen, Hilden, Germany). Serial analysis of gene expression (SAGE) library was constructed using a SOLiD SAGE Kit (Life Technologies) according to the manufactureM-bM-^@M-^Ys instruction. The constructed libraries were analysed by means of the massively parallel sequencing method using SOLiD 4 System (Life Technologies). The SAGE analysis was performed for samples obtained from single transfection experiment.

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PRKDC Induces Chemoresistance in Osteosarcoma by Recruiting GDE2 to Stabilize GNAS and Activate AKT

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