Realgar transforming solution-induced differentiation of NB4 cell by the degradation of PML/RARα partially through the ubiquitin-proteasome pathway.

PML/retinoic acid receptor alpha (RARα), as a hallmark of acute promyeloid leukemia (APL), is directly related to the outcome of clinical APL remedy. It is reported that arsenicals can effectively degrade PML/RARα, such as arsenic trioxide and realgar. However, the high toxicity or insolubility have hampered their clinical applications. Realgar transforming solution (RTS) was produced from realgar by bioleaching process in our lab. Previous studies demonstrated that RTS had a significant anti-cancer ability on chronic myeloid leukemia through oncoprotein degradation. The capacity of RTS on treating APL is what is focused on in this study. The results showed that RTS had a noticeable sensitivity in NB4 cell, and RTS remarkably down-regulated PML/RARα expression and induced cell differentiation. Further, RTS could accumulate PML/RARα into the nuclear bodies and then execute degradation, which could be reversed by proteasome inhibitor MG132. The results also exhibited that the reduction of RTS-induced PML/RARα expression accompanied by the elevation of ubiquitin and SUMO-1 protein expression. Finally, PML and SUMO-1 had been demonstrated to be co-localized after RTS treatment by immunofluorescence co-localization assay and immunoprecipitation assay. In conclusion, these results suggested that RTS-induced cell differentiation may attribute to the PML/RARα degradation partially through the ubiquitin-proteasome pathway.

Distinct temporal roles for the promyelocytic leukaemia (PML) protein in the sequential regulation of intracellular host immunity to HSV-1 infection.

Detection of viral nucleic acids plays a critical role in the induction of intracellular host immune defences. However, the temporal recruitment of immune regulators to infecting viral genomes remains poorly defined due to the technical difficulties associated with low genome copy-number detection. Here we utilize 5-Ethynyl-2'-deoxyuridine (EdU) labelling of herpes simplex virus 1 (HSV-1) DNA in combination with click chemistry to examine the sequential recruitment of host immune regulators to infecting viral genomes under low multiplicity of infection conditions. Following viral genome entry into the nucleus, PML-nuclear bodies (PML-NBs) rapidly entrapped viral DNA (vDNA) leading to a block in viral replication in the absence of the viral PML-NB antagonist ICP0. This pre-existing intrinsic host defence to infection occurred independently of the vDNA pathogen sensor IFI16 (Interferon Gamma Inducible Protein 16) and the induction of interferon stimulated gene (ISG) expression, demonstrating that vDNA entry into the nucleus alone is not sufficient to induce a robust innate immune response. Saturation of this pre-existing intrinsic host defence during HSV-1 ICP0-null mutant infection led to the stable recruitment of PML and IFI16 into vDNA complexes associated with ICP4, and led to the induction of ISG expression. This induced innate immune response occurred in a PML-, IFI16-, and Janus-Associated Kinase (JAK)-dependent manner and was restricted by phosphonoacetic acid, demonstrating that vDNA polymerase activity is required for the robust induction of ISG expression during HSV-1 infection. Our data identifies dual roles for PML in the sequential regulation of intrinsic and innate immunity to HSV-1 infection that are dependent on viral genome delivery to the nucleus and the onset of vDNA replication, respectively. These intracellular host defences are counteracted by ICP0, which targets PML for degradation from the outset of nuclear infection to promote vDNA release from PML-NBs and the onset of HSV-1 lytic replication.

Respiratory syncytial virus induces NRF2 degradation through a promyelocytic leukemia protein - ring finger protein 4 dependent pathway.

Respiratory syncytial virus (RSV) is the most important cause of viral acute respiratory tract infections and hospitalizations in children, for which no vaccine or specific treatments are available. RSV causes airway mucosa inflammation and cellular oxidative damage by triggering production of reactive oxygen species and by inhibiting at the same time expression of antioxidant enzymes, via degradation of the transcription factor NF-E2-related factor 2 (NRF2). RSV infection induces NRF2 deacetylation, ubiquitination, and degradation through a proteasome-dependent pathway. Although degradation via KEAP1 is the most common mechanism, silencing KEAP1 expression did not rescue NRF2 levels during RSV infection. We found that RSV-induced NRF2 degradation occurs in an SUMO-specific E3 ubiquitin ligase - RING finger protein 4 (RNF4)-dependent manner. NRF2 is progressively SUMOylated in RSV infection and either blocking SUMOylation or silencing RNF4 expression rescued both NRF2 nuclear levels and transcriptional activity. RNF4 associates with promyelocytic leukemia - nuclear bodies (PML-NBs). RSV infection induces the expression of PML and PML-NBs formation in an interferon (INF)-dependent manner and also induces NRF2 - PMN-NBs association. Inhibition of PML-NB formation by blocking IFN pathway or silencing PML expression resulted in a significant reduction of RSV-associated NRF2 degradation and increased antioxidant enzyme expression, identifying the RNF4-PML pathway as a key regulator of antioxidant defenses in the course of viral infection.

Site-specific inhibition of the small ubiquitin-like modifier (SUMO)-conjugating enzyme Ubc9 selectively impairs SUMO chain formation.

Posttranslational modifications by small ubiquitin-like modifiers (SUMOs) regulate many cellular processes, including genome integrity, gene expression, and ribosome biogenesis. The E2-conjugating enzyme Ubc9 catalyzes the conjugation of SUMOs to ϵ-amino groups of lysine residues in target proteins. Attachment of SUMO moieties to internal lysines in Ubc9 itself can further lead to the formation of polymeric SUMO chains. Mono- and poly-SUMOylations of target proteins provide docking sites for distinct adapter and effector proteins important for regulating discrete SUMO-regulated pathways. However, molecular tools to dissect pathways depending on either mono- or poly-SUMOylation are largely missing. Using a protein-engineering approach, we generated high-affinity SUMO2 variants by phage display that bind the back side binding site of Ubc9 and function as SUMO-based Ubc9 inhibitors (SUBINs). Importantly, we found that distinct SUBINs primarily inhibit poly-SUMO chain formation, whereas mono-SUMOylation was not impaired. Proof-of-principle experiments demonstrated that in a cellular context, SUBINs largely prevent heat shock-triggered poly-SUMOylation. Moreover, SUBINs abrogated arsenic-induced degradation of promyelocytic leukemia protein. We propose that the availability of the new chain-selective SUMO inhibitors reported here will enable a thorough investigation of poly-SUMO-mediated cellular processes, such as DNA damage responses and cell cycle progression.

Epigallocatechin-3-gallate promotes all-trans retinoic acid-induced maturation of acute promyelocytic leukemia cells via PTEN.

Acute promyelocytic leukemia (APL) is a distinctive subtype of acute myeloid leukemia (AML) in which the hybrid protein promyelocytic leukemia protein/retinoic acid receptor α (PML/RARα) acts as a transcriptional repressor impairing the expression of genes that are critical to myeloid cell mutation. We aimed at explaining the molecular mechanism of green tea polyphenol epigallocatechin-3-gallate (EGCG) enhancement of ATRA-induced APL cell line differentiation. Tumor suppressor phosphatase and tensin homolog (PTEN) was found downregulated in NB4 cells and rescued by proteases inhibitor MG132. A significant increase of PTEN levels was found in NB4, HL-60 and THP-1 cells upon ATRA combined with EGCG treatment, paralleled by increased myeloid differentiation marker CD11b. EGCG in synergy with ATRA promote degradation of PML/RARα and restores PML expression, and increase the level of nuclear PTEN. Pretreatment of PTEN inhibitor SF1670 enhances the PI3K signaling pathway and represses NB4 cell differentiation. Moreover, the induction of PTEN attenuated the Akt phosphorylation levels, pretreatment of PI3K inhibitor LY294002 in NB4 cells, significantly augmented the cell differentiation and increased the expression of PTEN. These results therefore indicate that EGCG targets PML/RARα oncoprotein for degradation and potentiates differentiation of promyelocytic leukemia cells in combination with ATRA via PTEN.

Dual regulation of Stat1 and Stat3 by the tumor suppressor protein PML contributes to interferon α-mediated inhibition of angiogenesis.

IFNs are effective in inhibiting angiogenesis in preclinical models and in treating several angioproliferative disorders. However, the detailed mechanisms of IFNα-mediated anti-angiogenesis are not completely understood. Stat1/2/3 and PML are IFNα downstream effectors and are pivotal regulators of angiogenesis. Here, we investigated PML's role in the regulation of Stat1/2/3 activity. In Pml knock-out (KO) mice, ablation of Pml largely reduces IFNα angiostatic ability in Matrigel plug assays. This suggested an essential role for PML in IFNα's anti-angiogenic function. We also demonstrated that PML shared a large cohort of regulatory genes with Stat1 and Stat3, indicating an important role of PML in regulating Stat1 and Stat3 activity. Using molecular tools and primary endothelial cells, we demonstrated that PML positively regulates Stat1 and Stat2 isgylation, a ubiquitination-like protein modification. Accordingly, manipulation of the isgylation system by knocking down USP18 altered IFNα-PML axis-mediated inhibition of endothelial cell migration and network formation. Furthermore, PML promotes turnover of nuclear Stat3, and knockdown of PML mitigates the effect of LLL12, a selective Stat3 inhibitor, on IFNα-mediated anti-angiogenic activity. Taken together, we elucidated an unappreciated mechanism in which PML, an IFNα-inducible effector, possess potent angiostatic activity, doing so in part by forming a positive feedforward loop with Stat1/2 and a negative feedback loop with Stat3. The interplay between PML, Stat1/Stat2, and Stat3 contributes to IFNα-mediated inhibition of angiogenesis, and disruption of this network results in aberrant IFNα signaling and altered angiostatic activity.

Arsenite inhibits the function of CD133+ CD13+ liver cancer stem cells by reducing PML and Oct4 protein expression.

Cancer stem cells (CSCs) can form new tumors and contribute to post-operative recurrence and metastasis. We showed that CD133+CD13+ hepatocytes isolated from HuH7 cells and primary HCC cells display biochemical and functional characteristics typical of CSCs, suggesting that CD133+CD13+ hepatocytes in primary HCC tumors function as CSCs. We also found that arsenite treatment reduced the viability and stemness of CD133+CD13+ hepatocytes, enhanced the sensitivity of HuH7 cells to pirarubicin, and reduced the tumorigenicity of CD133+CD13+ hepatocytes xenografts in mice. The effects of sodium arsenite treatment in CD133+CD13+ hepatocytes were mediated by the post-transcriptional suppression of PML expression and the inhibition of Oct4, Sox2, and Klf4 expression at the transcriptional level. Incomplete rescue of Oct4 expression in arsenic-treated cells ectopically expressing an siRNA-resistant PML transcript suggested that OCT4 regulation in liver CSCs involves other factors in addition to PML. Our findings provide evidence of a specific role for PML in regulating Oct4 levels in liver CSCs and highlight the clinical importance of arsenic for improving the efficacy of other chemotherapeutic agents and the prevention of post-operative HCC recurrence and metastasis.

Stratification and therapeutic potential of PML in metastatic breast cancer.

Patient stratification has been instrumental for the success of targeted therapies in breast cancer. However, the molecular basis of metastatic breast cancer and its therapeutic vulnerabilities remain poorly understood. Here we show that PML is a novel target in aggressive breast cancer. The acquisition of aggressiveness and metastatic features in breast tumours is accompanied by the elevated PML expression and enhanced sensitivity to its inhibition. Interestingly, we find that STAT3 is responsible, at least in part, for the transcriptional upregulation of PML in breast cancer. Moreover, PML targeting hampers breast cancer initiation and metastatic seeding. Mechanistically, this biological activity relies on the regulation of the stem cell gene SOX9 through interaction of PML with its promoter region. Altogether, we identify a novel pathway sustaining breast cancer aggressiveness that can be therapeutically exploited in combination with PML-based stratification.