Autophagy suppression in DNA damaged cells occurs through a newly identified p53-proteasome-LC3 axis.

Macroautophagy is thought to have a critical role in shaping and refining cellular proteostasis in eukaryotic cells recovering from DNA damage. Here, we report a mechanism by which autophagy is suppressed in cells exposed to bacterial toxin-, chemical-, or radiation-mediated sources of genotoxicity. Autophagy suppression is directly linked to cellular responses to DNA damage, and specifically the stabilization of the tumor suppressor p53, which is both required and sufficient for regulating the ubiquitination and proteasome-dependent reduction in cellular pools of microtubule-associated protein 1 light chain 3 (LC3A/B), a key precursor of autophagosome biogenesis and maturation, in both epithelial cells and an ex vivo organoid model. Our data indicate that suppression of autophagy, through a newly identified p53-proteasome-LC3 axis, is a conserved cellular response to multiple sources of genotoxicity. Such a mechanism could potentially be important for realigning proteostasis in cells undergoing DNA damage repair.

Pathogenesis of Human Papillomaviruses Requires the ATR/p62 Autophagy-Related Pathway.

High-risk human papillomaviruses (HPVs) constitutively activate the ataxia telangiectasia and Rad3-related (ATR) DNA damage response pathway, and this is required for viral replication. In fibroblasts, activated ATR regulates transcription of inflammatory genes through its negative effects on the autophagosome cargo protein p62. In addition, suppression of p62 results in increased levels of the transcription factor GATA4, leading to cellular senescence. In contrast, in HPV-positive keratinocytes, we observed that activation of ATR resulted in increased levels of phosphorylated p62, which in turn lead to reduced levels of GATA4. Knockdown of ATR in HPV-positive cells resulted in decreased p62 phosphorylation and increased GATA4 levels. Transcriptome sequencing (RNA-seq) analysis of HPV-positive cells identified inflammatory genes and interferon factors as negative transcriptional targets of ATR. Furthermore, knockdown of p62 or overexpression of GATA4 in HPV-positive cells leads to inhibition of viral replication. These findings identify a novel role of the ATR/p62 signaling pathway in HPV-positive cells.IMPORTANCE High-risk human papillomaviruses (HPVs) infect epithelial cells and induce viral genome amplification upon differentiation. HPV proteins activate the ATR DNA damage repair pathway, and this is required for HPV genome amplification. In the present study, we show that HPV-induced ATR activation also leads to suppression of expression of inflammatory response genes. This suppression results from HPV-induced phosphorylation of the autophagosome cargo protein p62 which regulates the levels of the transcription factor GATA4. Activation of p62 in normal fibroblasts results in senescence, but this is not seen in HPV-positive keratinocytes. Importantly, knockdown of p62 or overexpression of GATA4 in HPV-positive cells abrogates viral replication. This study demonstrates that activation of ATR in HPV-positive cells triggers a p62-directed pathway inducing suppression of inflammatory gene expression independent of DNA repair and facilitating HPV replication.

Topoisomerase IIß-binding protein 1 activates expression of E2F1 and p73 in HPV-positive cells for genome amplification upon epithelial differentiation.

High-risk human papillomaviruses (HPVs) constitutively activate ataxia telangiectasia mutated (ATM) and ataxia telangiectasia- and Rad3-related (ATR) DNA damage repair pathways for viral genome amplification. HPVs activate these pathways through the immune regulator STAT-5. For the ATR pathway, STAT-5 increases expression of the topoisomerase IIß-binding protein 1 (TopBP1), a scaffold protein that binds ATR and recruits it to sites of DNA damage. TopBP1 also acts as a transcriptional regulator, and we investigated how this activity influenced the HPV life cycle. We determined that TopBP1 levels are increased in cervical intraepithelial neoplasias as well as cervical carcinomas, consistent with studies in HPV-positive cell lines. Suppression of TopBP1 by shRNAs impairs HPV genome amplification and activation of the ATR pathway but does not affect the total levels of ATR and CHK1. In contrast, knockdown reduces the expression of other DNA damage factors such as RAD51 and Mre11 but not BRCA2 or NBS1. Interestingly, TopBP1 positively regulates the expression of E2F1, a TopBP1-binding partner, and p73 in HPV-positive cells in contrast to its effects in other cell types. TopBP1 transcriptional activity is regulated by AKT, and treatment with AKT inhibitors suppresses expression of E2F1 and p73 without interfering with ATR signaling. Importantly, the levels of p73 are elevated in HPV-positive cells and its knockdown impairs HPV genome amplification. This demonstrates that p73, like p63 and p53, is an important regulator of the HPV life cycle that is controlled by the transcriptional activating properties of the multifunctional TopBP1 protein.