The DNA damage response DDR is a cellular
The DNA damage response (DDR) is a cellular mechanism that protects against DNA damage induced by endogenous and exogenous factors, it includes changes in cellular processes such as 5416 regulation, DNA damage repair, apoptosis and chromatin remodeling. In recent years, the DDR has been recognized as an important innate tumor suppressor pathway , , , . DNA tumor virus infections activate and modulate DDR signaling pathways through multiple independent mechanisms , , . Recent evidence suggests that EBV oncoproteins target multiple aspects of DDR signaling, such as heightening DNA damage by increasing the cellular levels of reactive oxygen species (ROS), disrupting the mitotic checkpoint and suppressing DNA repair mechanisms .
DNA double strand breaks (DSBs) are considered to be the most lethal form of DNA damage induced by DNA-damaging agents, such as ionizing irradiation. The ability to repair DSBs is related not only to cancer susceptibility but also to sensitivity of cells to radiotherapy and chemotherapy . The DNA-dependent protein kinase (DNA-PK) is a critical component of the DSB repair machinery , , , . It is a serine/threonine kinase complex composed of a heterodimer of the catalytic subunit DNA-PKcs and the regulator subunit Ku proteins (Ku70/Ku86). In response to DSB formation, DNA-PKcs is recruited to DSBs by the DNA end-binding Ku70/86 heterodimer and is rapidly phosphorylated at multiple serine and threonine residues. DNA-PKcs phosphorylation at the Thr2609 cluster region is particularly significant because it is critical for DSB repair , . In addition to its classical role in DSB repair, recent findings demonstrate damage-independent functions of DNA-PK that affect a variety of critical cellular processes associated with malignancy , . Specifically, a recent report defines a general role for DNA-PK in metabolic regulation , , .
AMP-activated protein kinase (AMPK) is a crucial energy sensor that helps maintain cellular energy homeostasis and especially as a negative regulator of glycolysis , . It is a serine/threonine protein kinase composed of a heterotrimeric complex, including α, β and γ subunits. The α-subunit of AMPK has catalytic activity and phosphorylation of AMPKα at Thr172 is necessary for full enzyme activity , . Besides acting as an energy sensor, recent work describes a novel function for AMPK as a sensor of genomic stress and a participant in the DDR pathway , , .
In this study, we report that in NPC cells, EBV-encoded LMP1 repressed DSB repair by inhibiting DNA-PK phosphorylation and activity. Moreover, LMP1 disrupted the physical interaction between DNA-PK and AMPK and reduced the phosphorylation of AMPK by disturbing the subcellular location of the protein after DNA damage. Reactivation of AMPK markedly inhibited glycolysis and promoted apoptosis induced by DNA damage, and enhanced radiosensitivity both in vivo and in vitro. This study revealed a new mechanism of LMP1-induced radioresistance occurring through the modulation of DDR signaling pathways and provided a mechanistic rationale supporting the use of AMPK activators for facilitating NPC radiotherapy.
Materials and methods
Discussion The global burden of mortality from EBV-attributed malignancies accounts for 1.8% of all cancer deaths in 2010, in which NPC and gastric cancer accounted for 92% of all EBV-attributed cancer deaths , . Human DNA tumor viruses often impinge on the DDR in order to establish the necessary cellular milieu to replicate viral DNA. Studies have shown that EBV-encoded LMP1 can modulate the DDR by inhibiting DNA repair through different mechanisms, such as repressing the PI3-K/Akt/FOXO3a pathway or p53-mediated DNA repair and transcriptional activity , . In this study, we demonstrated that LMP1 could repress DSB repair in NPC cells, at least partly by inhibiting the phosphorylation and activity of DNA-PK, a central component of DSB repair. Corresponding with this, γH2AX, a marker for DSBs, was significantly higher in NPC tumors, especially in LMP1-positive tumors, suggesting that LMP1 impaired the ability to repair DSBs in NPC and might produce genetic instability, which is a cause of cancer.