DNA damage response (DDR) and selective autophagy both can be activated by reactive oxygen/nitrogen species (ROS/RNS), and both are of paramount importance in cancer development. The selective autophagy receptor and ubiquitin (Ub) sensor p62 plays a key role in their crosstalk. ROS production has been well documented in latent infection of oncogenic viruses including Epstein-Barr Virus (EBV). However, p62-mediated selective autophagy and its interplay with DDR have not been investigated in these settings. In this study, we provide evidence that considerable levels of p62-mediated selective autophagy are spontaneously induced, and correlate with ROS-Keap1-NRF2 pathway activity, in virus-transformed cells. Inhibition of autophagy results in p62 accumulation in the nucleus, and promotes ROS-induced DNA damage and cell death, as well as downregulates the DNA repair proteins CHK1 and RAD51. In contrast, MG132-mediated proteasome inhibition, which induces rigorous autophagy, promotes p62 degradation but accumulation of the DNA repair proteins CHK1 and RAD51. However, pretreatment with an autophagy inhibitor offsets the effects of MG132 on CHK1 and RAD51 levels. These findings imply that p62 accumulation in the nucleus in response to autophagy inhibition promotes proteasome-mediated CHK1 and RAD51 protein instability. This claim is further supported by the findings that transient expression of a p62 mutant, which is constitutively localized in the nucleus, in B cell lines with low endogenous p62 levels recaptures the effects of autophagy inhibition on CHK1 and RAD51 protein stability. These results indicate that proteasomal degradation of RAD51 and CHK1 is dependent on p62 accumulation in the nucleus. However, small hairpin RNA (shRNA)-mediated p62 depletion in EBV-transformed lymphoblastic cell lines (LCLs) had no apparent effects on the protein levels of CHK1 and RAD51, likely due to the constitutive localization of p62 in the cytoplasm and incomplete knockdown is insufficient to manifest its nuclear effects on these proteins. Rather, shRNA-mediated p62 depletion in EBV-transformed LCLs results in significant increases of endogenous RNF168-γH2AX damage foci and chromatin ubiquitination, indicative of activation of RNF168-mediated DNA repair mechanisms. Our results have unveiled a pivotal role for p62-mediated selective autophagy that governs DDR in the setting of oncogenic virus latent infection, and provide a novel insight into virus-mediated oncogenesis.
As a ubiquitin sensor and a signal-transducing adaptor, p62 is crucial for NF-κB activation, which involves the ubiquitin machinery in diverse contexts. However, whether p62 is required for EBV LMP1 activation of NF-κB is an open question.
Non-small-cell lung carcinoma (NSCLC) is the major type of lung cancer, which is among the leading causes of cancer-related deaths worldwide. LIMD1 was previously identified as a tumor suppressor in lung cancer, but their detailed interaction in this setting remains unclear. In this study, we have carried out multiple genome-wide bioinformatic analyses for a comprehensive understanding of LIMD1 in NSCLC, using various online algorithm platforms that have been built for mega databases derived from both clinical and cell line samples. Our results indicate that LIMD1 expression level is significantly downregulated at both mRNA and protein levels in both lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC), with a considerable contribution from its promoter methylation rather than its gene mutations. The Limd1 gene undergoes mutation only at a low rate in NSCLC (0.712%). We have further identified LIMD1-associated molecular signatures in NSCLC, including its natural antisense long non-coding RNA LIMD1-AS1 and a pool of membrane trafficking regulators. We have also identified a subgroup of tumor-infiltrating lymphocytes, especially neutrophils, whose tumor infiltration levels significantly correlate with LIMD1 level in both LUAD and LUSC. However, a significant correlation of LIMD1 with a subset of immune regulatory molecules, such as IL6R and TAP1, was only found in LUAD. Regarding the clinical outcomes, LIMD1 expression level only significantly correlates with the survival of LUAD (p<0.01) but not with that of LUSC (p>0.1) patients. These findings indicate that LIMD1 plays a survival role in LUAD patients at least by acting as an immune regulatory protein. To further understand the mechanisms underlying the tumor-suppressing function of LIMD1 in NSCLC, we show that LIMD1 downregulation remarkably correlates with the deregulation of multiple pathways that play decisive roles in the oncogenesis of NSCLC, especially those mediated by EGFR, KRAS, PIK3CA, Keap1, and p63, in both LUAD and LUSC, and those mediated by p53 and CDKN2A only in LUAD. This study has disclosed that LIMD1 can serve as a survival prognostic marker for LUAD patients and provides mechanistic insights into the interaction of LIMD1 with NSCLC, which provide valuable information for clinical applications.
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...
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