Hypoxia Stimulates SUMOylation-Dependent Stabilization of KDM5B.
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ABSTRACT:
SUBMITTER: Zhou B
PROVIDER: S-EPMC8719622 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BACKGROUND:We have previously described CLDN6 as a tumor suppressor gene in breast cancer. Here, a new finding is that CLDN6 was upregulated under hypoxia, a commonly recognized factor that promotes tumor metastasis. In this study, we aim to explain this confusing finding and delineate the role of CLDN6 in the breast cancer metastasis induced by hypoxia. METHODS:RNAi and ChIP assays were used to confirm that CLDN6 is transcriptional regulated by HIF-1?. mRNA seq and KEGG analysis were performed to define the downstream pathways of CLDN6. The roles of the CLDN6/SENP1/HIF-1? signaling on tumor metastasis were evaluated by function experiments and clinical samples. Finally, the possible transcription factor of SENP1 was suspected and then validated by ChIP assay. RESULTS:We demonstrated a previously unrecognized negative feedback loop exists between CLDN6 and HIF-1?. CLDN6 was transcriptionally up-regulated by HIF-1? under hypoxia. On the other hand, in cytoplasm CLDN6 combines and retains ?-catenin, a transcription factor of SENP1, causing ?-catenin degradation and preventing its nuclear translocation. This process reduced SENP1 expression and prevented the deSUMOylation of HIF-1?, ultimately leading to HIF-1? degradation and breast cancer metastasis suppression. CONCLUSIONS:Our data provide a molecular mechanistic insight indicating that CLDN6 loss may lead to elevated HIF-1?-driven breast cancer metastasis in a SUMOylation-dependent manner.
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Project description:Hypoxia-inducible factor ? proteins (HIF-?s) are regulated oxygen dependently and transactivate numerous genes essential for cellular adaptation to hypoxia. NEDD8, a member of the ubiquitin-like family, covalently binds to its substrate proteins, and thus, regulates their stabilities and functions. In the present study, we examined the possibility that the HIF signaling is regulated by the neddylation. HIF-1? expression and activity were inhibited by knocking down APPBP1 E1 enzyme for NEDD8 conjugation but enhanced by ectopically expressing NEDD8. HIF-1? and HIF-2? were identified to be covalently modified by NEDD8. NEDD8 stabilized HIF-1? even in normoxia and further increased its level in hypoxia, which also occurred in von Hippel-Lindau (VHL) protein- or p53-null cell lines. The HIF-1?-stabilizing effect of NEDD8 was diminished by antioxidants and mitochondrial respiratory chain blockers. This suggests that the NEDD8 effect is concerned with reactive oxygen species driven from mitochondria rather than with the prolyl hydroxylase (PHD)/VHL-dependent oxygen-sensing system. Based on these findings, we propose that NEDD8 is an ancillary player to regulate the stability of HIF-1?. Furthermore, given the positive role played by HIF-?s in cancer promotion, the NEDD8 conjugation process could be a potential target for cancer therapy.
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Project description:Hypoxia-inducible factor 1 (HIF-1) activates the transcription of genes encoding proteins that enable cells to adapt to reduced O2 availability. Proteins encoded by HIF-1 target genes play a central role in mediating physiological processes that are dysregulated in cancer and heart disease. These diseases are also characterized by increased production of cyclic adenosine monophosphate (cAMP), the allosteric activator of cAMP-dependent protein kinase A (PKA). Using glutathione S-transferase pull-down, coimmunoprecipitation, and mass spectrometry analyses, we demonstrated that PKA interacts with HIF-1α in HeLa cervical carcinoma cells and rat cardiomyocytes. PKA phosphorylated Thr(63) and Ser(692) on HIF-1α in vitro and enhanced HIF transcriptional activity and target gene expression in HeLa cells and rat cardiomyocytes. PKA inhibited the proteasomal degradation of HIF-1α in an O2-independent manner that required the phosphorylation of Thr(63) and Ser(692) and was not affected by prolyl hydroxylation. PKA also stimulated the binding of the coactivator p300 to HIF-1α to enhance its transcriptional activity and counteracted the inhibitory effect of asparaginyl hydroxylation on the association of p300 with HIF-1α. Furthermore, increased cAMP concentrations enhanced the expression of HIF target genes encoding CD39 and CD73, which are enzymes that convert extracellular adenosine 5'-triphosphate to adenosine, a molecule that enhances tumor immunosuppression and reduces heart rate and contractility. These data link stimuli that promote cAMP signaling, HIF-1α-dependent changes in gene expression, and increased adenosine, all of which contribute to the pathophysiology of cancer and heart disease.
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Project description:BackgroundThe Slug-E-cadherin axis plays a critical role in non-small-cell lung cancers (NSCLCs) where aberrant upregulation of Slug promotes cancer metastasis. Now, the post-translational modifications of Slug and their regulation mechanisms still remain unclear in lung cancer. Hence, exploring the protein linkage map of Slug is of great interest for investigating the scenario of how Slug protein is regulated in lung cancer metastasis.MethodsThe Slug associated proteins, Ubc9 and SUMO-1, were identified using yeast two-hybrid screening; and in vitro SUMOylation assays combined with immunoprecipitation and immunoblotting were performed to explore the detail events and regulations of Slug SUMOylation. The functional effects of SUMOylation on Slug proteins were examined by EMSA, reporter assay, ChIP assay, RT-PCR, migration and invasion assays in vitro, tail vein metastatic analysis in vivo, and also evaluated the association with clinical outcome of NSCLC patients.ResultsSlug protein could interact with Ubc9 and SUMO-1 and be SUMOylated in cells. Amino acids 130-212 and 33-129 of Slug are responsible for its binding to Ubc9 and protein inhibitor of activated STAT (PIAS)y, respectively. SUMOylation could enhance the transcriptional repression activity of Slug via recruiting more HDAC1, resulting in reduced expression of downstream Slug target genes and enhanced lung cancer metastasis. In addition, hypoxia could increase Slug SUMOylation through attenuating the interactions of Slug with SENP1 and SENP2. Finally, high expression Slug and Ubc9 levels were associated with poor overall survival among NSCLC patients.ConclusionsUbc9/PIASy-mediated Slug SUMOylation and subsequent HDAC1 recruitment may play a crucial role in hypoxia-induced lung cancer progression, and these processes may serve as therapeutic targets for NSCLC.
| S-EPMC6322271 | biostudies-literature
Project description:Chemosensitivity is one of the key factors affecting the therapeutic effect on cancer, but the clinical application of corresponding drugs is rare. Hypoxia, a common feature of many solid tumors, including hepatocellular carcinoma (HCC), has been associated with resistance to chemotherapy in part through the activation of the Sonic Hedgehog (SHh) pathway. Hypoxia has also been associated with the increased SUMOylation of multiple proteins, including GLI family proteins, which are key mediators of SHh signaling, and has become a promising target to develop drug-resistant drugs for cancer treatment. However, there are few target drugs to abrogate chemotherapy resistance. Saikosaponin-d (Ssd), one of the main bioactive components of Radix bupleuri, has been reported to exert multiple biological effects, including anticancer activity. Here, we first found that Ssd inhibits the malignant phenotype of HCC cells while increasing their sensitivity to the herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) drug system under hypoxia in vitro and in vivo. Furthermore, we had explored that GLI family activation and extensive protein SUMOylation were characteristics of HCC cells, and hypoxia could activate the SHh pathway and promote epithelial-mesenchymal transition (EMT), invasion, and chemosensitivity in HCC cells. SUMOylation is required for hypoxia-dependent activation of GLI proteins. Finally, we found that Ssd could reverse the effects promoted by hypoxia, specifically active sentrin/small ubiquitin-like modifier (SUMO)-specific protease 5 (SENP5), a SUMO-specific protease, in a time- and dose-dependent manner while inhibiting the expression of SUMO1 and GLI proteins. Together, these findings confirm the important role of Ssd in the chemoresistance of liver cancer, provide some data support for further understanding the molecular mechanisms of Ssd inhibition of malignant transformation of HCC cells, and provide a new perspective for the application of traditional Chinese medicine in the chemical resistance of liver cancer.
| S-EPMC6764240 | biostudies-literature
Project description:BackgroundChronic obstructive pulmonary disease (COPD) characterized by the airway and lung inflammation, is a leading cause of morbidity and mortality worldwide, especially among smokers over 40 years of age and individuals exposed to biomass smoke. Although the detailed mechanisms of this disease remain elusive, there is feasible evidence that protein posttranslational modifications (PTMs) may play a role in its pathoetiology. We thus conducted studies to dissect the effect of cigarette smoke extracts (CSE) on the change of SUMOylated substrates in human bronchial epithelial cells (HBEs).MethodsSamples were collected in HBEs with or without 24 h of CSE insult and then subjected to Western-blot and LC-MS/MS analysis. Subsequently, bioinformatic tools were used to analyze the data. The effect of SUMOylation on cytochrome P450 1A1 (CYP1A1) was evaluated by flow cytometry.ResultsIt was noted that CSE stimulated HBEs to undergo a SUMOylation turnover as evidenced by the changes of SUMOylated substrates and SUMOylation levels for a particular substrate. The SUMOylated proteins are relevant to the regulation of biological processes, molecular function and cellular components. Particularly, CSE stimulated a significant increase of SUMOylated CYP1A1, a critical enzyme involved in the induction of oxidative stress.ConclusionsOur data provide a protein SUMOylation profile for better understanding of the mechanisms underlying COPD and support that smoking induces oxidative stress in HBEs, which may predispose to the development of COPD in clinical settings.
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Project description:Tumors experience temporal and spatial fluctuations in oxygenation. Hypoxia inducible transcription factors (HIF-α) respond to low levels of oxygen and induce re-supply oxygen. HIF-α stabilization is typically facultative, induced by hypoxia and reduced by normoxia. In some cancers, HIF-α stabilization becomes constitutive under normoxia. We develop a mathematical model that predicts how fluctuating oxygenation affects HIF-α stabilization and impacts net cell proliferation by balancing the base growth rate, the proliferative cost of HIF-α expression, and the mortality from not expressing HIF-α during hypoxia. We compare optimal net cell proliferation rate between facultative and constitutive HIF-α regulation in environments with different oxygen profiles. We find that that facultative HIF-α regulation promotes greater net cell proliferation than constitutive regulation with stochastic or slow periodicity in oxygenation. However, cell fitness is nearly identical for both HIF-α regulation strategies under rapid periodic oxygenation fluctuations. The model thus indicates that cells constitutively expressing HIF-α may be at a selective advantage when the cost of expression is low. In cancer, this condition is known as pseudohypoxia or the "Warburg Effect". We conclude that rapid and regular cycling of oxygenation levels selects for pseudohypoxia, and that this is consistent with the ecological theory of optimal defense.
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Project description:Autophagy, a self-catabolic process, has been found to be involved in abrogating the proliferation and metastasis of breast cancer. SLC9A3R1 (solute carrier family 9, subfamily A [NHE3, cation proton antiporter 3], member 3 regulator 1), a multifunctional scaffold protein, is involved in suppressing breast cancer cells proliferation and the SLC9A3R1-related signaling pathway regulates the activation of autophagy processes. However, the precise regulatory mechanism and signaling pathway of SLC9A3R1 in the regulation of autophagy processes in breast cancer cells remains unknown. Here, we report that the stability of BECN1, the major component of the autophagic core lipid kinase complex, is augmented in SLC9A3R1-overexpressing breast cancer MDA-MB-231 cells, subsequently stimulating autophagy by attenuating the interaction between BECN1 and BCL2. Initially, we found that SLC9A3R1 partially stimulated autophagy through the PTEN-PI3K-AKT1 signaling cascade in MDA-MB-231 cells. SLC9A3R1 then attenuated the interaction between BECN1 and BCL2 to stimulate the autophagic core lipid kinase complex. Further findings revealed that SLC9A3R1 bound to BECN1 and subsequently blocked ubiquitin-dependent BECN1 degradation. And the deletion of the C-terminal domain of SLC9A3R1 resulted in significantly reduced binding to BECN1. Moreover, the lack of C-terminal of SLC9A3R1 neither reduced the ubiquitination of BECN1 nor induced autophagy in breast cancer cells. The decrease in BECN1 degradation induced by SLC9A3R1 resulted in the activity of autophagy stimulation in breast cancer cells. These findings indicate that the SLC9A3R1-BECN1 signaling pathway participates in the activation of autophagy processes in breast cancer cells.
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Project description:PARP-1 is an abundant nuclear enzyme that regulates gene expression, although the underlying mechanisms are unclear. We examined the interplay between PARP-1, histone 3 lysine 4 trimethylation (H3K4me3), and linker histone H1 in the chromatin-dependent control of transcription. We show that PARP-1 is required for a series of molecular outcomes at the promoters of PARP-1-regulated genes, leading to a permissive chromatin environment that allows loading of the RNA Pol II machinery. PARP-1 does so by (1) preventing demethylation of H3K4me3 through the PARylation, inhibition, and exclusion of the histone demethylase KDM5B; and (2) promoting the exclusion of H1 and the opening of promoter chromatin. Upon depletion of PARP-1, these outcomes do not occur efficiently. Interestingly, cellular signaling pathways can use the regulated depletion of PARP-1 to modulate these chromatin-related molecular outcomes. Collectively, our results help to elucidate the roles of PARP-1 in the regulation of chromatin structure and transcription.
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Project description:DNA-protein cross-links (DPCs) are highly toxic DNA lesions consisting of proteins covalently attached to chromosomal DNA. Unrepaired DPCs physically block DNA replication and transcription. Three DPC repair pathways have been identified in Arabidopsis (Arabidopsis thaliana) to date: the endonucleolytic cleavage of DNA by the structure-specific endonuclease MUS81; proteolytic degradation of the crosslinked protein by the metalloprotease WSS1A; and cleavage of the cross-link phosphodiester bonds by the tyrosyl phosphodiesterases TDP1 and TDP2. Here we describe the evolutionary conserved STRUCTURAL MAINTENANCE OF CHROMOSOMEs SMC5/6 complex as a crucial component involved in DPC repair. We identified multiple alleles of the SMC5/6 complex core subunit gene SMC6B via a forward-directed genetic screen designed to identify the factors involved in the repair of DPCs induced by the cytidine analog zebularine. We monitored plant growth and cell death in response to DPC-inducing chemicals, which revealed that the SMC5/6 complex is essential for the repair of several types of DPCs. Genetic interaction and sensitivity assays showed that the SMC5/6 complex works in parallel to the endonucleolytic and proteolytic pathways. The repair of zebularine-induced DPCs was associated with SMC5/6-dependent SUMOylation of the damage sites. Thus, we present the SMC5/6 complex as an important factor in plant DPC repair.
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