p62-mediated Selective Autophagy Endows Virus-transformed Cells with Insusceptibility to DNA Damage under Oxidative Stress

Abstract

1DNA damage response (DDR) and selective autophagy both can be activated by reactive 2 oxygen/nitrogen species (ROS/RNS), and both are of paramount importance in cancer 3 development. The selective autophagy receptor and ubiquitin (Ub) sensor p62 plays a key role in 4 their crosstalk. ROS production has been well documented in latent infection of oncogenic viruses 5 including Epstein-Barr Virus (EBV). However, p62-mediated selective autophagy and its interplay 6 with DDR have not been investigated in these settings. In this study, we provide evidence that p62 7 is upregulated through the ROS-Keap1-NRF2 pathway, and considerable levels of p62-mediated 8 selective autophagy are constitutively induced, in virus-transformed cells. Inhibition of autophagy 9 results in p62 accumulation, and promotes ROS-induced DNA damage and cell death, as well as 10 downregulates the DNA repair proteins CHK1 and RAD51. In contrast, MG132-mediated 11 proteasome inhibition, which induces rigorous autophagy, promotes p62 degradation but 12 accumulation of the DNA repair proteins CHK1 and RAD51. However, small hairpin RNA (shRNA)-13 mediated p62 depletion or transient expression of p62 in B cell lines had no apparent effects on 14 the protein levels of CHK1 and RAD51. These results indicate that p62 accumulation resulting from 15 autophagy inhibition is prerequisite for p62-mediated proteasomal degradation of CHK1 and 16 RAD51. Furthermore, shRNA-mediated p62 depletion in EBV-transformed lymphoblastic cell lines 17 results in significant increases of endogenous RNF168-γH2AX damage foci and chromatin 18 ubiquitination in the nucleus, indicative of activation of RNF168-mediated DNA repair mechanisms. 19Finally, we have evaluated endogenous p62 and autophagy levels and their correlation with 20 endogenous ROS and DNA damage levels in B cell population of AIDS lymphoma patients versus 21 age-matched subjects. Our results have unveiled a pivotal role for p62-mediated selective 22 autophagy that governs DDR in the setting of oncogenic virus latent infection, and provide a novel 23 insight into virus-mediated oncogenesis. 24 3 Author Summary 1 Reactive oxygen/nitrogen species (ROS/RNS) can induce both DNA damage response (DDR) and 2 selective autophagy, which play crucial roles in cancer development. The selective autophagy 3 receptor and ubiquitin (Ub) sensor p62 links their crosstalk. However, p62-mediated selective 4 autophagy and its interplay with DDR have not been investigated in latent infection of oncogenic 5 viruses including Epstein-Barr Virus (EBV). In this study, we provide evidence that p62-mediated 6 selective autophagy is constitutively induced in virus-transformed cells, and that its inhibition 7 exacerbates ROS-induced DNA damage, and promotes proteasomal degradation of CHK1 and 8 RAD51 in a p62-dependent manner. However, rigorous autophagy induction results in 9 accumulation of DNA repair proteins CHK1 and RAD51, and p62 degradation. Furthermore, small 10 hairpin RNA (shRNA)-mediated p62 depletion or transient expression of p6...

Kaposi’s sarcoma-associated herpesvirus (KSHV) utilizes the NDP52/CALCOCO2 selective autophagy receptor to disassemble processing bodies

“…Taken together, our data and that of others support that viral KSHV enhancement of autophagy is central to latent viral tumourigenesis and the creation of a proinflammatory environment and may also play an important role in regulating the balance between latent versus lytic gene expression for KSHV and related herpesviruses (Dong et al , 2016; Park et al , 2016; Wang et al , 2019). Although this may suggest that targeting autophagic processes could be a useful strategy for treating KS, as supported by the regression of iatrogenic KS lesions after treatment with the mTORC1 complex inhibitor and autophagy inducer, rapamycin (Stallone et al , 2005), the complicated balance of autophagy regulation by KSHV suggests the need for more selective therapies.…”

How Oncogenic Viruses Exploit p62-Mediated Selective Autophagy for Cancer Development

“…Importantly, recent studies have indicated that oncogenic viruses induce p62 expression in their latency, through different mechanisms, including the Keap1-NRF2 pathway [40,41]. We have shown that EBV and HTLV1 activate p62mediate selective autophagy in their latency [42]. To further explore the mechanisms, we have accumulated evidence showing that EBV and HTLV1 induce p62, partially by NFκB that is activated downstream of LMP1 and Tax signaling pathways, respectively, and also likely by the Keap1-NRF2 pathway (to be published).…”

“…We have shown that a well-controlled autophagy-p62 interplay renders EBV-infected cells with the ability to balance pro-survival DNA damage resistance and pro-mutagenic genomic perturbation under oxidative stress [42,53], and have defined two distinct roles for the autophagy-p62 interplay in this setting: cytoplasmic p62 mediates selective autophagy, and nuclear p62 upon autophagy inhibition represses DNA repair at least by promoting proteasomemediated degradation of CHK1 and RAD51 [42]. These original findings disclose novel mechanisms underneath LMP1-mediated oxidative DDR.…”

Sequestosome 1/p62: A multitasker in the regulation of malignant tumor aggression (Review)

“…Recently, an increasing number of studies have confirmed that under conditions of various intracellular or extracellular pressures, including nutrient deficiency, and endoplasmic reticulum, oxidative and metabolic stresses, such as hypoxia and high concentrations of insulin, SQSTM1/p62 may act as either a proto-oncogene or tumor suppressor gene, promoting or inhibiting malignant tumor progression, respectively (20)(21)(22)(23). By directly binding to numerous cancer-associated genes, such as Tribbles 3, EGFR, COX-2, MMP1/2, membrane type-MMP, c-Myc, Snail, Twist (20), RAD51 recombinase, filamin A (21), ring finger protein 168 (an E3 ubiquitin-protein ligase) (22) and checkpoint kinase 1 (23), SQSTM1/p62 can mitigate genetic instability and DNA damage foci, and induce DNA repair, thereby playing a role in cancer oncogenesis, malignant progression, senescence and chemoradiotherapeutic sensitivity (20)(21)(22)(23). Since SQSTM1/p62 is at the center of a hub of various complicated signaling pathways in different cancer types and as its functions have wide implications in different cellular systems, the underlying regulatory mechanisms of its role in steering tumor progression are not entirely known and remain to be revealed in the future.…”