USP28 Data Analysis

HGNC Gene Name
ubiquitin specific peptidase 28
HGNC Gene Symbol
USP28
Identifiers
hgnc:12625 NCBIGene:57646 uniprot:Q96RU2
Orthologs
mgi:2442293 rgd:1311555
INDRA Statements
deubiquitinations all statements
Pathway Commons
Search for USP28
Number of Papers
115 retrieved on 2022-05-22

DepMap Analysis

The Dependency Map (DepMap) is a genome-wide pooled CRISPR-Cas9 knockout proliferation screen conducted in more than 700 cancer cell lines spanning many different tumor lineages. Each cell line in the DepMap contains a unique barcode, and each gene knockout is assigned a “dependency score” on a per cell-line basis which quantifies the rate of CRISPR-Cas9 guide drop. It has been found that proteins with similar DepMap scores across cell lines, a phenomenon known as co-dependent genes, have closely related biological functions. This can include activity in the same or parallel pathways or membership in the same protein complex or the same pathway.

We identified the strongest seven co-dependent genes (“Symbol”) for DUBs and ran GO enrichment analysis. We used Biogrid, IntAct, and Pathway Commons PPIDs, and the NURSA protein-protein interaction databases (PPIDs) to determine whether co-dependent genes interact with one another. The “Evidence” column contains the PPIDs in which the interaction appears as well as whether there is support for the association by an INDRA statement. As another approach to identify potential interactors, we looked at proteomics data from the Broad Institute's Cancer Cell Line Encyclopedia (CCLE) for proteins whose expression across ~375 cell lines strongly correlated with the abundance of each DUB; it has previously been observed that proteins in the same complex are frequently significantly co-expressed. The correlations and associated p-values in the CCLE proteomics dataset are provided. And, we determined whether co-dependent genes yield similar transcriptomic signatures in the Broad Institute's Connectivity Map (CMap). A CMap score greater than 90 is considered significantly similar.

DepMap Correlations

Symbol Name DepMap Correlation Evidence CCLE Correlation CCLE Z-score CCLE p-value (adj) CCLE Significant CMAP Score CMAP Type
TP53 tumor protein p53 0.642 IntAct INDRA (9) Reactome (4) 0.23 1.18 4.25e-05
TP53BP1 tumor protein p53 binding protein 1 0.58 BioGRID IntAct NURSA INDRA (8) Reactome (2) 0.34 1.77 3.25e-10
CDKN1A cyclin dependent kinase inhibitor 1A 0.518 INDRA (4) -0.20 -1.17 3.06e-03
MDM2 MDM2 proto-oncogene -0.478 Reactome (4) -0.18 -1.10 1.94e-02
ATM ATM serine/threonine kinase 0.399 INDRA (11) 0.16 0.81 5.39e-03
CHEK2 checkpoint kinase 2 0.396 INDRA (6) 0.15 0.74 1.04e-02
PPM1D protein phosphatase, Mg2+/Mn2+ dependent 1D -0.378 0.09 0.41 4.11e-01

Dependency GO Term Enrichment

Gene set enrichment analysis was done on the genes correlated with USP28using the terms from Gene Ontology and gene sets derived from the Gene Ontology Annotations database via MSigDB.

Using the biological processes and other Gene Ontology terms from well characterized DUBs as a positive control, several gene set enrichment analyses were considered. Threshold-less methods like GSEA had relatively poor results. Over-representation analysis with a threshold of of the top 7 highest absolute value Dependency Map correlations yielded the best results and is reported below.

GO Identifier GO Name GO Type p-value p-value (adj.) q-value
GO:0071479 cellular response to ionizing radiation Biological Process 1.54e-14 9.94e-12 2.58e-12
GO:0030330 DNA damage response, signal transduction by p53 class mediator Biological Process 1.84e-13 1.18e-10 1.54e-11
GO:0042770 signal transduction in response to DNA damage Biological Process 7.04e-13 4.53e-10 3.93e-11
GO:0010212 response to ionizing radiation Biological Process 1.41e-12 9.11e-10 5.92e-11
GO:0031570 DNA integrity checkpoint Biological Process 2.11e-12 1.36e-09 7.05e-11
GO:0009314 response to radiation Biological Process 3.91e-12 2.52e-09 1.09e-10
GO:0071478 cellular response to radiation Biological Process 6.47e-12 4.17e-09 1.36e-10
GO:0090399 replicative senescence Biological Process 6.48e-12 4.17e-09 1.36e-10
GO:0000075 cell cycle checkpoint Biological Process 1.41e-11 9.07e-09 2.36e-10
GO:0072395 signal transduction involved in cell cycle checkpoint Biological Process 1.64e-11 1.06e-08 2.50e-10
GO:0071158 positive regulation of cell cycle arrest Biological Process 2.78e-11 1.79e-08 3.88e-10
GO:0072331 signal transduction by p53 class mediator Biological Process 4.97e-11 3.20e-08 6.39e-10
GO:0044774 mitotic DNA integrity checkpoint Biological Process 1.08e-10 6.96e-08 1.26e-09
GO:0071156 regulation of cell cycle arrest Biological Process 1.13e-10 7.29e-08 1.26e-09
GO:0071214 cellular response to abiotic stimulus Biological Process 1.57e-10 1.01e-07 1.55e-09
GO:0071480 cellular response to gamma radiation Biological Process 1.53e-10 9.88e-08 1.55e-09
GO:1902807 negative regulation of cell cycle G1/S phase transition Biological Process 2.90e-10 1.87e-07 2.70e-09
GO:0007093 mitotic cell cycle checkpoint Biological Process 9.69e-10 6.24e-07 8.54e-09
GO:0010332 response to gamma radiation Biological Process 2.20e-09 1.41e-06 1.84e-08
GO:1902806 regulation of cell cycle G1/S phase transition Biological Process 2.56e-09 1.65e-06 2.04e-08
GO:0007050 cell cycle arrest Biological Process 6.94e-09 4.47e-06 5.28e-08
GO:1901988 negative regulation of cell cycle phase transition Biological Process 1.05e-08 6.77e-06 7.64e-08
GO:0044839 cell cycle G2/M phase transition Biological Process 1.13e-08 7.29e-06 7.90e-08
GO:0097193 intrinsic apoptotic signaling pathway Biological Process 1.59e-08 1.03e-05 1.07e-07
GO:0044843 cell cycle G1/S phase transition Biological Process 1.74e-08 1.12e-05 1.12e-07
GO:0090068 positive regulation of cell cycle process Biological Process 1.86e-08 1.20e-05 1.15e-07
GO:0045930 negative regulation of mitotic cell cycle Biological Process 3.40e-08 2.19e-05 2.03e-07
GO:0007569 cell aging Biological Process 4.58e-08 2.95e-05 2.64e-07
GO:0010948 negative regulation of cell cycle process Biological Process 4.73e-08 3.05e-05 2.64e-07
GO:0045787 positive regulation of cell cycle Biological Process 8.55e-08 5.51e-05 4.62e-07
GO:0010165 response to X-ray Biological Process 1.38e-07 8.86e-05 7.20e-07
GO:1901987 regulation of cell cycle phase transition Biological Process 2.00e-07 1.29e-04 1.02e-06
GO:1901796 regulation of signal transduction by p53 class mediator Biological Process 2.62e-07 1.69e-04 1.29e-06
GO:2001020 regulation of response to DNA damage stimulus Biological Process 5.07e-07 3.26e-04 2.42e-06
GO:0031668 cellular response to extracellular stimulus Biological Process 1.28e-06 8.24e-04 5.95e-06
GO:0002039 p53 binding Molecular Function 1.33e-06 8.56e-04 6.02e-06
GO:0072332 intrinsic apoptotic signaling pathway by p53 class mediator Biological Process 2.22e-06 1.43e-03 9.59e-06
GO:0044389 ubiquitin-like protein ligase binding Molecular Function 2.23e-06 1.44e-03 9.59e-06
GO:0034644 cellular response to UV Biological Process 2.40e-06 1.55e-03 1.01e-05
GO:2001021 negative regulation of response to DNA damage stimulus Biological Process 2.59e-06 1.67e-03 1.06e-05
GO:0007568 aging Biological Process 2.70e-06 1.74e-03 1.08e-05
GO:0071496 cellular response to external stimulus Biological Process 3.20e-06 2.06e-03 1.21e-05
GO:0006975 DNA damage induced protein phosphorylation Biological Process 3.26e-06 2.10e-03 1.21e-05
GO:0090400 stress-induced premature senescence Biological Process 3.26e-06 2.10e-03 1.21e-05
GO:0008630 intrinsic apoptotic signaling pathway in response to DNA damage Biological Process 5.35e-06 3.45e-03 1.95e-05
GO:0070482 response to oxygen levels Biological Process 6.10e-06 3.93e-03 2.13e-05
GO:0047485 protein N-terminus binding Molecular Function 6.00e-06 3.87e-03 2.13e-05
GO:0071481 cellular response to X-ray Biological Process 7.69e-06 4.95e-03 2.63e-05
GO:0071482 cellular response to light stimulus Biological Process 1.03e-05 6.63e-03 3.44e-05
GO:0009411 response to UV Biological Process 1.29e-05 8.31e-03 4.23e-05
GO:0042772 DNA damage response, signal transduction resulting in transcription Biological Process 1.58e-05 1.02e-02 5.09e-05
GO:0009267 cellular response to starvation Biological Process 1.66e-05 1.07e-02 5.23e-05
GO:0000781 chromosome, telomeric region Cellular Component 2.01e-05 1.29e-02 6.23e-05
GO:0046827 positive regulation of protein export from nucleus Biological Process 2.44e-05 1.57e-02 7.43e-05
GO:0071157 negative regulation of cell cycle arrest Biological Process 2.94e-05 1.89e-02 8.79e-05
GO:0042594 response to starvation Biological Process 3.20e-05 2.06e-02 9.39e-05
GO:0006367 transcription initiation from RNA polymerase II promoter Biological Process 3.25e-05 2.10e-02 9.39e-05
GO:0097718 disordered domain specific binding Molecular Function 5.76e-05 3.71e-02 1.61e-04
GO:0043516 regulation of DNA damage response, signal transduction by p53 class mediator Biological Process 5.76e-05 3.71e-02 1.61e-04
GO:0045935 positive regulation of nucleobase-containing compound metabolic process Biological Process 6.17e-05 3.97e-02 1.69e-04
GO:0006302 double-strand break repair Biological Process 6.65e-05 4.29e-02 1.80e-04
GO:0006352 DNA-templated transcription, initiation Biological Process 7.52e-05 4.84e-02 2.00e-04

Transcriptomics

The following table shows the significantly differentially expressed genes after knocking out USP28 using CRISPR-Cas9.

Knockout Differential Expression

Symbol Name log2-fold-change p-value p-value (adj.)
RPLP1 ribosomal protein lateral stalk subunit P1 8.92e-01 5.33e-10 4.24e-06
RPL7A ribosomal protein L7a 8.02e-01 4.53e-07 1.80e-03
PSMB1 proteasome 20S subunit beta 1 3.24e-01 7.10e-06 1.88e-02
RPS27A ribosomal protein S27a 7.82e-01 1.02e-05 2.04e-02
MT1E metallothionein 1E 5.16e-01 1.39e-05 2.21e-02
RPS12 ribosomal protein S12 8.74e-01 2.26e-05 2.86e-02
RPS26 ribosomal protein S26 3.79e-01 2.52e-05 2.86e-02
PSMD6 proteasome 26S subunit, non-ATPase 6 4.78e-01 5.07e-05 4.03e-02
ADGRG1 adhesion G protein-coupled receptor G1 7.58e-01 7.28e-05 4.82e-02

Gene Set Enrichment Analysis

There were too few differentially expressed genes to run a meaningful GSEA.

Literature Mining

INDRA was used to automatically assemble known mechanisms related to USP28 from literature and knowledge bases. The first section shows only DUB activity and the second shows all other results.

Deubiquitinase Activity

psp cbn pc bel_lc signor biogrid lincs_drug tas hprd trrust ctd vhn pe drugbank omnipath conib crog dgi | rlimsp isi tees geneways eidos trips medscan sparser reach
USP28 deubiquitinates FBXW7. 10 / 11
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In addition, the autocatalytic ubiquitylation of Fbw7 can be antagonized by the deubiquitinase Usp28 [XREF_BIBR].

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It was recently shown that the deubiquitinase Usp28 deubiquitinates and stabilizes Fbxw7 [XREF_BIBR].

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To test whether Usp28 directly deubiquitinates Fbw7, we used an in vitro assay with immunopurified SCF (Fbw7) and recombinant E1 and E2 enzymes, which leads to the accumulation of autoubiquitinated Fb[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]

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Indeed, experiments in MEFs and human tumor cells demonstrate that Usp28 directly deubiquitinates and stabilizes Fbw7.

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We discuss mechanisms that regulate ubiquitination by Fbw7, including ubiquitin-specific proteases such as USP28 that counteract Fbw7 activity and thereby stabilise oncoproteins.

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Indeed, wild-type Usp28, but not the catalytically inactive C171A mutant, promoted Fbw7 deubiquitination in vivo.

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In addition, USP28 antagonizes FBW7 mediated ubiquitination and stabilizes HIF-1alpha [XREF_BIBR, XREF_BIBR].

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Usp28 preferentially deubiquitinates Fbw7 and at intermediate levels maintains stable Fbw7 but does not allow excessive Fbw7 substrate stabilization.

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As USP28 deubiquitinates and stabilizes FBW7, allowing the FBW7 and SCF ligase complex to bind and degrade substrates containing a Cdc4 phosphodegron motif, we hypothesized that forced expression of USP28 would target BRAF for degradation.

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USP28 deubiquitinates and stabilizes FBW7 resulting in enhanced degradation of FBW7 substrates.
USP28 deubiquitinates MYC. 8 / 8
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Usp28, an ubiquitin-specific protease, binds to myc through an interaction with fbw7alpha, an f-box protein that is part of an scf-type ubiquitin ligase. Therefore, it stabilizes myc.

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Review

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An early study showed that USP28 deubiquitinates c-Myc via interacting with Fbw7alpha whereas a recent study reveals that USP37 deubiquitinates c-Myc independently of Fbw7 and c-Myc phosphorylation.

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Recent evidence has revealed that c-Myc can be deubiquitylated and regulated by USP28, USP36 and USP37.

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The resultant weakly acidic microenvironment enhances the deubiquitination of MYC by USP28, improves the stability of MYC, and activates the promoter of Slug, ultimately promoting the stem cell-like properties of breast cancer.

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The product of this is lactic acid that changes the pH of the local environment which then promotes the formation of a weakly acidic microenvironment, thus consequently enhancing the deubiquitination of MYC by the deubiquitination enzyme USP28 and improving the stability of MYC [38].

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USP28 directly deubiquitinates and stabilizes MYC.

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Identified in a retroviral shRNA library screen, USP28 has been shown to decrease MYC polyubiquitination and increase MYC stability by antagonizing the activity of the SCF FBW7 ubiquitin ligase complex [XREF_BIBR].
USP28 deubiquitinates TP53. 7 / 7
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Because 53BP1 is known to bind USP28 as well as p53, a possible scenario is that it bridges the two proteins so that USP28 can deubiquitinate p53, preventing the tumor suppressor from being targeted to the proteasome for degradation.
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While 53BP1, USP28, and p53 have not yet been demonstrated to form a ternary complex, USP28 was able to deubiquitinate p53 in vitro [9].

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While 53BP1, USP28, and p53 have not yet been demonstrated to form a ternary complex, USP28 was able to deubiquitinate p53 in vitro [XREF_BIBR].

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The nuclear p53 accumulation caused by overexpression of wild type USP28 was not due to a specific increase in p53 mRNA levels (XREF_FIG), further supporting our observation that USP28 deubiquitinates p53 for protein stabilization.

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The signalling pathway involves 53BP1 and the deubiquitylase USP28 acting in a complex to deubiquitylate and stabilise p53, which in turn controls cell fate.

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its interacting protein USP28 that can directly deubiquitinate p53 in vitro and ectopically stabilize p53 in vivo

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Intriguingly, 53BP1 mediates p53 activation independently of its DNA repair activity, but requiring its interacting protein USP28 that can directly deubiquitinate p53 in vitro and ectopically stabilize p53 in vivo.
USP28 deubiquitinates CLSPN. 3 / 3
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De-ubiquitinase USP28 inhibits ubiquitination of Claspin protein and its proteasomal degradation [XREF_BIBR].

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Claspin is a bona fide USP28 substrate, as Claspin deubiquitination by USP28 could also be reconstituted in vitro.

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USP28 deubiquitinates UCK1. 3 / 3
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We then evaluated whether USP28 could deubiquitinate UCK1.

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In vitro deubiquitination assay further confirmed that WT-USP28, but not USP28-CA, efficiently deubiquitinated UCK1, indicating that USP28 served as direct DUB for UCK1.

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Conversely, USP28 silencing in HEK293T markedly increased the ubiquitination of endogenous UCK1, implying that endogenous UCK1 was also a target of USP28.
USP28 deubiquitinates KDM1A. 3 / 3
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We found that WT-USP28, but not CI-USP28, specifically removed LSD1 ubiquitination (XREF_FIG), indicating that USP28 directly deubiquitinated LSD1.

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USP7 and USP28 inhibited LSD1 ubiquitination and stabilized LSD1 protein level [XREF_BIBR, XREF_BIBR].
USP28 leads to the deubiquitination of ZNF304. 2 / 2
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No evidence text available

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Notably, unlike wild-type USP28, a USP28 derivative containing a mutation in the PRKD1 phosphorylation site, USP28 (S899A), was unable to reduce ZNF304 ubiquitination (XREF_FIG).
USP28 deubiquitinates ATM. 1 / 1
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Mechanistically, USP28 deubiquitinates and stabilizes the specificity factors and mediators of ATM and ATR signaling by protecting them from ubiquitination-mediated proteasome degradation.
Modified USP28 leads to the deubiquitination of MYC. 1 / 1
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Overexpression of USP28 inhibited FBW7-depedent ubiquitination of c-MYC and cyclin E1, both of which are oncoproteins.
USP28 deubiquitinates JUN. 1 / 1
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At present, the list of USP28 substrates shared with Fbw7 encompasses c-Myc, cyclin E1 [89], c-Jun, NICD1 [90], and HIF1
USP28 deubiquitinates CHEK2. 1 / 1
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USP28 deubiquitinates CCNE1. 1 / 1
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. This study pointed towards USP28 as an oncogene, potentially acting via stabilisation of c-Myc and cyclin E1.
Modified USP28 leads to the deubiquitination of Cyclin. 1 / 1
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Overexpression of USP28 inhibited FBW7-depedent ubiquitination of c-MYC and cyclin E1, both of which are oncoproteins.
USP28 deubiquitinates ATR. 1 / 1
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Mechanistically, USP28 deubiquitinates and stabilizes the specificity factors and mediators of ATM and ATR signaling by protecting them from ubiquitination-mediated proteasome degradation.
USP28 deubiquitinates BRAF. 1 / 1
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Here, we demonstrate that the deubiquitinating enzyme USP28 functions through a feedback loop to destabilize RAF family members.
USP28 deubiquitinates TP53BP1. 1 / 1
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Modified USP28 leads to the deubiquitination of BCHE. 1 / 1
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Overexpression of USP28 inhibited FBW7-depedent ubiquitination of c-MYC and cyclin E1, both of which are oncoproteins.
USP28 deubiquitinates STAT3. 1 / 1
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As shown in XREF_FIG, the IP indicated that STAT3 itself was polyubiquitinated and USP28 deubiquitinated the polyubiquitination of STAT3.
USP28 deubiquitinates HIF1A. 1 / 1
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USP28 deubiquitinates KITLG. 1 / 1
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In this setting, USP28 deubiquitinates the SCF component FBW7, allowing FBW7 to act as a substrate recognition factor targeting substrates for proteosomal mediated degradation.
Modified USP28 leads to the deubiquitination of KDM1A. 1 / 1
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However, co-expression of WT-USP28, but not CI-USP28, almost completely abolished LSD1 ubiquitination (lane 3 vs. lane 4, XREF_FIG).
Modified USP28 leads to the deubiquitination of Cyclin on E1. 1 / 1
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Overexpression of USP28 inhibited FBW7-depedent ubiquitination of c-MYC and cyclin E1, both of which are oncoproteins.

Other Statements

psp cbn pc bel_lc signor biogrid lincs_drug tas hprd trrust ctd vhn pe drugbank omnipath conib crog dgi | rlimsp isi tees geneways eidos trips medscan sparser reach
USP28 affects MYC
1 | 1 1 33
USP28 activates MYC.
1 | 1 1 18
USP28 activates MYC. 10 / 21
1 | 1 1 18

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Usp28, an ubiquitin-specific protease, binds to myc through an interaction with fbw7alpha, an f-box protein that is part of an scf-type ubiquitin ligase. Therefore, it stabilizes myc.

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Independently, USP28 has been reported to modulate the activity of the Myc proto-oncogene [XREF_BIBR].

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FBXW7-185aa reduced the half-life of c-Myc by antagonizing USP28 induced c-Myc stabilization.

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A previous study in glioma implicated that the short protein FBXW7-185aa interacts with the deubiquitinating enzyme USP28, preventing USP28 from binding to FBXW7 and antagonizing USP28 induced c-Myc stabilization [XREF_BIBR].

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This peptide supposes to repress glioma malignancy through USP28 induced c-Myc stabilization.

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Since FBW7gamma lacks the USP28 interaction motif of FBW7alpha, Usp28 selectively antagonize nucleoplasmic MYC degradation while not affecting nucleolar degradation.

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Knockdown of Usp28 results in a decrease of c-MYC while overexpression of the enzyme and leads to stabilization of the transcription factor .

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Besides, knockdown of USP28 blocked the effect of c-Myc on activation of ataxia telangiectasia-mutated and ataxia telangiectasia and Rad3 related DNA damage checkpoint after irradiation.

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In this study FBXW7-185aa reduced the half-life of c-Myc by antagonizing USP28 induced c-Myc stabilization.

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USP28 has also been reported to antagonize ubiquitin-dependent degradation of the oncogene product MYC as well as JUN and Notch 105 .
USP28 inhibits MYC.
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USP28 inhibits MYC. 7 / 7
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Thereafter, qRT‐PCR assays were performed on FBXW7, SKP2, USP28, and USP36, which confirmed that Dem could interact with FBXW7 and downregulate the expression of c‐Myc.
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Furthermore, overexpressing USP28 inhibited FBXW7-185aa-induced c-Myc turnover (XREF_FIG).

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In addition, nickel and hypoxia exposure decreased USP28, a c-Myc de-ubiquitinating enzyme, contributing to a higher steady state level of c-Myc ubiquitination and promoting c-Myc degradation.

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In colon carcinoma, USP28 antagonizes the activity of the SCFFBW7 ubiquitin ligase complex to regulate the Myc stability and further promotes cell differentiation [XREF_BIBR].

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Moreover, circFBXW7 effectively inhibits glioma proliferation and cell cycle progression by antagonizing USP28 induced c-Myc stabilization [XREF_BIBR].

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In addition, nickel and hypoxia exposure decreased USP28, a c-myc de-ubiquitinating enzyme, contributing to a higher steady state level of c-myc ubiquitination and promoting c-myc degradation.

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FBXW7-185aa competitively interacts with USP28 and acts as a tumor-suppressive " decoy " that prevents USP28 binding to FBXW7alpha, thus antagonizing USP28 induced c-Myc stabilization.
USP28 increases the amount of MYC.
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Modified USP28 increases the amount of MYC. 4 / 4
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Overexpression of USP28 in BR cells enhances c-Myc expression and hence increases ASS1 transcription upon arginine deprivation, and consequently leads to cell survival.

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As shown in XREF_FIG, overexpression of USP28 in A549 prevented the loss of c-Myc, suggesting that USP28 was involved in c-Myc degradation.

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As expected, depletion of USP28 remarkably decreased MYC protein and reversed epinephrine induced increase in MYC protein expression (XREF_SUPPLEMENTARY and XREF_FIG), whereas overexpression of USP28 enhanced MYC expression (XREF_SUPPLEMENTARY).

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Intriguingly, both ectopic expression of Usp28 in wild-type MEFs (+/+ +) and complete knockout of Usp28 (-/-) strongly and equivalently increased levels of Myc, Jun, and Notch.
USP28 increases the amount of MYC. 2 / 2
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Moreover, USP28 positively regulated the protein level of c-Myc, and c-Myc negatively regulated the radiosensitivity of ECA109 and ECA109R cells.

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Importantly, USP28 WT remarkably increased protein expression of MYC, whereas USP28 Mut reversed lactate induced MYC and SLUG (XREF_FIG).
USP28 decreases the amount of MYC.
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Modified USP28 decreases the amount of MYC. 1 / 1
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These outcomes indicated that a decreased USP28 protein level caused less USP28 binding to the c, Myc, and Fbw7alpha complex, and sequentially increased the steady state ubiquitination levels of c-Myc.
USP28 decreases the amount of MYC. 1 / 1
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In human tumor cell lines, shRNA mediated depletion of Usp28 downregulates Myc levels and attenuates proliferation.

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Recently, upregulation of USP28 has been reported to be associated with poor prognosis in NSCLC patients and promote NSCLC cell proliferation XREF_BIBR.

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Functional assays demonstrated that overexpression of USP28 promoted cell proliferation and aerobic glycolysis of colorectal cancer, while USP28 inhibition could reverse these effects.

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This suggests that Usp28 in different cellular contexts is a mediator of proliferation.

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Overexpressing USP28 was found in NSCLC tumors and enhanced NSCLC cell proliferation, and low survival was related to high USP28 levels in patients.

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Overexpression of USP28 promoted NSCLC cells proliferation, and was associated with poor prognosis in NSCLC patients.

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In conclusion, our data indicated that high expression of USP28 in NSCLC promoted tumour cells proliferation, and miR-4295 may target USP28.

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The exact mechanism by which USP28 promotes NSCLC cell proliferation is undetermined yet.

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Loss of 53BP1, USP28, or TRIM37 suppresses p53 elevation and proliferation arrest triggered by centrosome loss.

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Up-regulation of USP28 promoted NSCLC cells proliferation.

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USP28 increases aerobic glycolysis and promotes cell proliferation upon stabilization of FOXC1 [50].
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Downregulating USP28 expression can inhibit the proliferation and growth of lung cancer cells [XREF_BIBR, XREF_BIBR].

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Downregulation of CCND1 and USP28 Inhibits Proliferation and Induces Apoptosis in A549 Cells.

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Moreover, 53BP1 or USP28 deletion restored NPC proliferation and brain size without correcting the upstream centrosome defects or extended mitosis.

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MTT assays showed that USP28 overexpression reduced cell proliferation in MDA-MB-231 and MCF7 cells, and these effects were partially reversed by transfection with miR-500a-5p mimics.

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CCK-8 assay shows that si-CCND1 and si-USP28 significantly inhibit A549 cell proliferation compared with si-controls (XREF_FIG, E and F).

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Additionally, according to the study by Popov, et al., depletion of USP28 in HeLa and LS174T cells can inhibit cell growth and proliferation due to its inability to enhance c-Myc stability [XREF_BIBR].

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Importantly, silencing KLHL2 or USP28 overexpression not only inhibited AML cell proliferation but also sensitized AML cells to 5 '-AZA-induced apoptosis in vitro and in vivo.
USP28 affects TP53
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USP28 activates TP53.
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USP28 activates TP53. 10 / 21
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53BP1 and USP28 mediate p53 dependent cell cycle arrest in response to centrosome loss and prolonged mitosis.

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53BP1 and USP28 somehow measure mitotic duration and activate p53 upon mitotic exit.

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53BP1 and USP28 mediate p53 activation and G1 arrest after centrosome loss or extended mitotic duration.

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Loss of 53BP1, USP28, and TRIM37 all prevented p53 stabilization and G1 arrest following centrosome loss.

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Previous reports showed that 53BP1 and USP28 activate p53, preventing the proliferation of cells that have an increased chance of mitotic errors [ xref ].
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53BP1 and USP28 somehow measure mitotic duration and activate p53 upon mitotic exit.

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Knockout of USP28 did not alter basal levels of p53 or prevent p53 stabilization in response to doxorubicin induced DNA damage (XREF_FIG and Fig.

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USP28Delta cells were used as the control because inactivation of USP28 prevents p53 activation and G1 arrest that is observed as a consequence of delayed mitosis following centrosome loss in RPE1 cells13 .

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Consistently, overexpression of the wild type USP28 but not USP28 CI in normal, unstressed cells caused ectopic nuclear p53 accumulation and cell cycle arrest uniformly across the entire population (100%, XREF_FIG; not shown).

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We conclude that USP28 and 53BP1 do not alter p53 regulation by MDM2 or modulate basal p53 stability.
USP28 decreases the amount of TP53.
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USP28 decreases the amount of TP53. 2 / 2
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Conversely, depletion of USP28 resulted in significantly increased K48 ubiquitination and simultaneously diminished p53 level, which could be fully reversed by addition of recombinant USP28 (XREF_FIG).

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Notably, USP28 knockdown cells had decreased expression of p53, p21 and p16 INK4a, suggesting that the effect of USP28 on cell proliferation was mediated by regulating the expression of p53, p21 and p16 INK4a.
USP28 inhibits TP53.
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USP28 inhibits TP53. 1 / 1
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Supporting this, tumor cells silenced for USP28 or 53BP1 have previously been shown to prevent p53 elevation and growth arrest in response to prolonged prometaphase in cancer cells with increased propensity of mitotic errors.
USP28 affects FBXW7
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USP28 inhibits FBXW7.
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The Usp28 deubiquitinase antagonizes Fbw7 mediated turnover of multiple oncoproteins, including Myc, Jun, and Notch, and promotes tumorigenesis in the intestine.

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Other proteins regulating C-MYC stability include : CIP2A, a C-MYC-interacting protein that specifically inhibits PP2A activity against C-MYC; USP28, a de-ubiquitinating enzymes that antagonizes FBW7 and promotes C-MYC stability and TRUSS, a receptor for DDB1 (damage specific DNA binding protein 1)-CUL4 (Cullin 4) E3 ligase complex.

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Fbw7 dependent substrate ubiquitination is antagonized by the Usp28 deubiquitinase.

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Steady-state levels of Fbw7 in Usp28 -/- MEFs were rescued by MG132 treatment, demonstrating that Fbw7 was degraded by the proteasome in the absence of Usp28.

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Consequently, half of the normal dose of Usp28 (in the Usp28 +/- cells) is adequate to maintain stable Fbw7 but is not sufficient to antagonize Fbw7 mediated substrate degradation.

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Although single allele deletion of Usp28 allows FBXW7 mediated substrate degradation, it has little effect on FBXW7 stability.

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For Myc, we also examined the effects of knock-down of USP28, a Ub specific protease that antagonizes Fbw7 dependent Myc destruction [XREF_BIBR].

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On the other hand, downregulation of Fbw7, induced by Usp28 loss is either not sufficient to promote embryonic lethality, associated with Fbxw7 knockout, or does not occur in lineages, in which Fbw7 i[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]

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Unexpectedly, we found that genetic deletion of Usp28 rescued the lethality of Fbw7 deficient primary fibroblasts.

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Moreover, GSK3beta and FBW7 dependent HIF-1alpha degradation can be antagonized by the USP28 (ubiquitin specific peptidase-28), suggesting that FBW7 and USP28 could reciprocally regulate cell migration and angiogenesis in a HIF-1alpha-dependent manner [XREF_BIBR].
USP28 bound to MYC inhibits FBXW7. 1 / 1
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As a ubiquitin specific protease, Usp28 binds to Myc and antagonizes the activity of Fbw7, thereby stabilizing Myc.
USP28 activates FBXW7.
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USP28 activates FBXW7. 5 / 5
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In agreement with this hypothesis, treatment of Usp28 -/- MEFs with PiB, a specific Pin1 inhibitor, attenuated degradation of ectopic Fbw7.

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Preferential targeting of Fbw7 by Usp28 shifts the default activity of this system toward Fbw7 substrate ubiquitination and provides a mechanism to maintain physiological levels of multiple proto-onco[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]

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In contrast, complete absence of Usp28 triggers autocatalytic turnover of Fbw7 and leads to Fbw7 substrate stabilization.The most likely explanation for such nonlinear pattern of regulation is that Fb[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]

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This result suggests that Usp28 preferentially stabilizes Fbw7 due to more efficient binding and is consistent with the idea that Fbw7 targets Usp28 to its substrates.We concluded that stabilization o[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]

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In addition, FBXW7 induced degradation of MYC is fine tuned by the de-ubiquitylation enzyme USP28 [XREF_BIBR], which slows down poly-ubiquitylation, as well as by the E3 ligase betaTRCP, which counteracts FBXW7 by conjugating mixed Ub chains to MYC that disfavors degradation.
USP28 decreases the amount of FBXW7.
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USP28 decreases the amount of FBXW7. 2 / 2
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Knockdown of Usp28 in human tumor cells also decreased endogenous Fbw7 levels, suggesting that regulation by Usp28 is a conserved mechanism that maintains Fbw7 stability.

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Overexpressing USP28 reduced the expression of FBXW7 and suppressed FBXW7-185aa-induced c-Myc destabilization (XREF_FIG H).
USP28 increases the amount of FBXW7.
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Modified USP28 increases the amount of FBXW7. 1 / 1
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In contrast, in the intestine and cerebellum, complete loss of Usp28 did not reduce Fbw7 levels but downregulated Fbw7 substrates.Mechanistic analysis revealed that this biphasic response to Usp28 los[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
USP28 affects STAT3
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USP28 activates STAT3.
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USP28 activates STAT3. 10 / 12
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Moreover, the CHX chase assay revealed that overexpression of USP28 prolonged the half-life of STAT3 protein in NSCLC cells, which further suggested that USP28 stabilized STAT3 protein (XREF_FIG).

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As shown in XREF_FIG, overexpression of USP28 significantly upregulated STAT3 derived luciferase activity, but another USP isoform, USP25, had no effect on STAT3 luciferase activity.

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USP28 increases the stability of STAT3.

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USP28 mediates STAT3 signaling in NSCLC cells.

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As stated before, USP28 mediated STAT3 signaling by stabilizing STAT3 protein, which suggested that USP28 was functional in NSCLC cells.

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In this study, the deubiquitinating enzyme USP28 was found to mediate STAT3 signaling in NSCLC cells.

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These results demonstrated that USP28 was functional in NSCLC cells, and promoted NSCLC cell growth by inducing STAT3 signaling.

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As shown in XREF_FIG, overexpression of USP28 upregulated STAT3 protein in a dose dependent manner, but the mRNA level of STAT3 was not changed obviously (XREF_FIG).

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XREF_BIBR In this study, we found that USP28 mediated STAT3 signaling and promoted NSCLC cell growth (XREF_FIG and XREF_FIG).

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In addition, the immunoblotting assay also revealed that overexpression of USP28 enhanced the STAT3 signaling in NSCLC cells (XREF_FIG).
USP28 increases the amount of STAT3.
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USP28 increases the amount of STAT3. 5 / 5
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The whole cell lysates also revealed that USP28 increased the protein level of STAT3 (XREF_FIG).

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In contrast, knockdown of USP28 suppressed STAT3 derived luciferase activity (XREF_FIG), and inhibited the expression of STAT3 in NSCLC cells (XREF_FIG).

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In contrast, knockdown of USP28 downregulated the protein level of STAT3 in NSCLC cells (XREF_FIG), but the mRNA level did not show significant change (XREF_FIG).

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Whole cell lysates were also prepared for immunoblotting, which showed that USP28 upregulated the expression level of STAT3 (XREF_FIG).

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In addition, the immunoblotting also showed that knockdown of USP28 downregulated STAT3 expression in vivo (XREF_FIG).
USP28-C171A increases the amount of STAT3. 1 / 1
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However, ectopic expression of USP28 C171A, a catalytically inactive mutant, could not enhance STAT3 levels, which indicated that USP28 regulated the post-translational modification of STAT3 (XREF_FIG).
USP28 affects cell growth
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USP28 activates cell growth.
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Further, we found that USP28 promoted PC cell growth by facilitating cell cycle progression and inhibiting apoptosis.

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As shown in XREF_FIG, knockdown of USP28 significantly inhibited NSCLC cell growth in both A549 and H1299 cells.

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Furthermore, USP28 regulates the expression of MYC protein, which is essential in USP28 induced cell growth in glioma cells.

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Importantly, USP28 promoted MYC expression, which was required for USP28 induced cell growth in human glioma.

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These results demonstrated that USP28 was functional in NSCLC cells, and promoted NSCLC cell growth by inducing STAT3 signaling.

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Overexpression of USP28 accelerated PC cell growth, whereas USP28 knockdown impaired PC cell growth both in vitro and in vivo.

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XREF_BIBR Previous study has reported that USP28 was overexpressed in NSCLC and overexpression of USP28 promoted NSCLC cell growth, but its mechanism was still unknown in NSCLC cells.

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Moreover, knockdown of USP28 inhibited cell growth of NSCLC cells in vitro and delayed NSCLC tumor growth in vivo.
USP28 inhibits cell growth.
| 1 4
| 1 4

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Previous study demonstrated that depletion of USP28 inhibited both cell-cycle and cell growth through regulation of MYC abundance XREF_BIBR.

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Knockdown of USP28 suppressed the cell growth of NSCLC both in vitro and in vivo.

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Knockdown of USP28 inhibits NSCLC cell growth.

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Moreover, knockdown of USP28 inhibited cell growth of NSCLC cells in vitro and delayed NSCLC tumor growth in vivo.

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Additionally, according to the study by Popov, et al., depletion of USP28 in HeLa and LS174T cells can inhibit cell growth and proliferation due to its inability to enhance c-Myc stability [XREF_BIBR].
USP28 affects KDM1A
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USP28 activates KDM1A.
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USP28 activates KDM1A. 6 / 6
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In agreement with this notion, knockdown of USP28 reduced the protein level and functional activity of LSD1, but these could be rescued by exogenous LSD1 expression (XREF_FIG).

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Overexpression of USP28 largely reversed HDAC5-knockdown-induced LSD1 protein degradation, suggesting HDAC5 positively regulates LSD1 by stabilizing USP28.

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Overexpression of USP28 largely reversed HDAC5-KD-induced LSD1 protein degradation, suggesting a role of HDAC5 as a positive regulator of LSD1 through upregulation of USP28 protein.

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Interestingly, the downregulation of LSD1 by two independent USP28 shRNAs in HCT116 cells could be restored by MG132 treatment (XREF_FIG), indicating that USP28 enhances LSD1 stabilization through a deubiquitination event.

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To test whether USP28 directly regulates protein stability of LSD1, we co-expressed LSD1 with USP28 or vector control in HEK293 cells and examined the degradation of LSD1.

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First, our data indicates that USP28 is a specific DUB for LSD1, as knockdown of USP28 reduced LSD1 stability, whereas overexpression of USP28 stabilized LSD1.
USP28 increases the amount of KDM1A.
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USP28 increases the amount of KDM1A. 3 / 3
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In this study, we found that USP28 was the major factor responsible for LSD1 stabilization in cancer cell lines and tumor samples, and that knockdown of USP28 decreased the level of LSD1 and suppressed CSC like properties in vitro and tumorigenicity in vivo.

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We therefore screened a panel of DUBs in which 23 DUBs ' cDNA plasmids were transfected into 293T cells, and found that USP15, USP21, USP22, and USP28 upregulated KDM1A levels (XREF_SUPPLEMENTARY).

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When these DUBs were co-expressed with LSD1 in HEK293 cells, we noticed that USP28 significantly increased LSD1 level, similar to that treated with MG132 (XREF_SUPPLEMENTARY).
Modified USP28 increases the amount of KDM1A. 1 / 1
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The steady-state level of LSD1 was enhanced by ectopic USP28 expression in a dose dependent manner (XREF_FIG).
USP28 inhibits KDM1A.
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USP28 inhibits KDM1A. 2 / 2
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Overexpression of USP28 largely reversed HDAC5-knockdown-induced LSD1 protein degradation, suggesting HDAC5 positively regulates LSD1 by stabilizing USP28.

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To understand whether HDAC5 may stabilize LSD1 protein through upregulation of USP28 protein stability, a rescue study was carried out in MDA-MB-231 and MCF10A-CA1a cells using concurrent transfection of HDAC5 siRNA and USP28 expression plasmids, and showed that overexpression of USP28 completely blocked the destabilization of LSD1 by HDAC5 depletion (XREF_FIG, XREF_SUPPLEMENTARY).
USP28 decreases the amount of KDM1A.
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USP28 decreases the amount of KDM1A. 1 / 1
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Our study showing that increase of LSD1 protein expression by Jade-2 siRNA and decrease of LSD1 protein expression by USP28 siRNA in MDA-MB-231 cells confirmed the roles of Jade-2 and USP28 as LSD1 ubiquitin ligase and deubiquitinase in breast cancer cells (XREF_FIG; XREF_SUPPLEMENTARY).
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Down-regulation of USP28 promoted NSCLC cells proliferation and induced cells apoptosis.

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Down-regulation of USP28 induced cell apoptosis.

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Moreover, the upregulation of CCND1 and USP28 partially reverses the impaired proliferation and promoted apoptosis rate induced by miR-3940-5p in A549 cells (XREF_FIG, E-H).

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Hence, silencing of USP28 antagonized apoptosis induced by caspase-8 depletion and also reverted accumulation and activation of p53 (XREF_FIG).

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USP28 was identified as an interaction partner of 53BP1 [125], and loss of USP28 has been reported to lead to IR-induced apoptosis in H460 cells, in a similar manner to what has been seen in Chk2, p53 and PUMA null mice.

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Moreover, FACS apoptosis analysis indicated that down-regulation of USP28 expression by siRNA increased the NSCLC cells apoptosis rate.

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Downregulation of CCND1 and USP28 Inhibits Proliferation and Induces Apoptosis in A549 Cells.
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These results indicate that CCND1 and USP28 are upregulated in A549 cells, and knockdown of CCND1 or USP28 obviously restrains cell proliferation and promotes apoptosis.

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Further, we found that USP28 promoted PC cell growth by facilitating cell cycle progression and inhibiting apoptosis.

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XREF_BIBR reported that overexpression of USP28 promotes NSCLC cell proliferation and apoptosis inhibition and is associated with poor prognosis in NSCLC patients.
USP28 affects BRAF
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USP28 inhibits BRAF.
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USP28 inhibits BRAF. 7 / 7
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Loss of USP28 enhances BRAF stabilization and confers resistance to vemurafenib in melanoma.

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Loss of USP28 mediated BRAF degradation drives resistance to RAF cancer therapies.

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The loss of USP28 promoted MAPK activation and resistance to RAF inhibitor therapy by stabilizing BRAF in cell culture and in vivo models.

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Having established that USP28 and FBW7 reduces BRAF stability, we tested the effect of USP28 and FBW7 depletion on BRAF expression.

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Both knockdown of USP28 and FBW7 significantly enhanced endogenous BRAF stability (XREF_FIG).

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Importantly, of the 59 patients harboring BRAF V600E mutations 27% (16/59) displayed a> 50% decrease in USP28 mRNA expression levels, suggesting that in tumors harboring BRAF alterations, loss of USP28 may further increase the tumorigenic potential of these tumors by stabilizing BRAF and enhancing downstream MAPK activation.

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However, we noted that loss of USP28 or FBW7 did not fully prevent BRAF degradation suggesting that BRAF degradation may occur through mechanisms independent of the USP28 and FBW7 axis.
USP28 activates BRAF.
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USP28 activates BRAF. 2 / 2
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As USP28 deubiquitinates and stabilizes FBW7, allowing the FBW7 and SCF ligase complex to bind and degrade substrates containing a Cdc4 phosphodegron motif, we hypothesized that forced expression of USP28 would target BRAF for degradation.

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As expected USP28 mediated BRAF stability led to enhanced pERK levels after vemurfenib treatment compared with wild-type USP28 cells.
USP28 decreases the amount of BRAF.
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Modified USP28 decreases the amount of BRAF. 1 / 1
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Indeed, overexpression of USP28 decreased the concentrations of ectopically expressed and endogenous BRAF levels (XREF_FIG).
USP28 affects cell cycle
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USP28 activates cell cycle.
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Our results furthermore explain how USP28 may modulate the p53 dependent cell cycle checkpoint, thereby controlling tumor cell fate decisions as previously described.

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53BP1 and USP28 mediate p53 dependent cell cycle arrest in response to centrosome loss and prolonged mitosis.

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In addition to arresting centrosome deficient cells, p53, 53BP1, and USP28 are all involved in the DNA damage response (DDR), raising the possibility that centrosome loss causes a cell cycle arrest because it somehow induces DNA damage.
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Further, we found that USP28 promoted PC cell growth by facilitating cell cycle progression and inhibiting apoptosis.

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We showed that 53BP1 and USP28 are required to trigger p53 and p21- dependent cell cycle arrest, evoking an irreversible stress response that selects against unfit cells with disturbed mitosis (XREF_FIG).

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Consistently, overexpression of the wild type USP28 but not USP28 CI in normal, unstressed cells caused ectopic nuclear p53 accumulation and cell cycle arrest uniformly across the entire population (100%, XREF_FIG; not shown).

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Similarly, western blot analyses revealed that the total p53 levels in 53BP1 -/- or USP28 -/- cells were kept low during acentrosomal cell division in the presence of mitotic delay (XREF_FIG), indicating that 53BP1 and USP28 function upstream of p53 to initiate cell cycle arrest in response to centrosome loss.
USP28 inhibits cell cycle.
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As expected, USP28 overexpression further decreased the relative expression of cell cycle related genes (CDK4, CDK6, Cyclin D1, pRB, and E2F1) induced by 5 '-AZA, whereas p15 and p27 expression was significantly enhanced.

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Conversely, USP28 silencing accelerated the cell cycle progression to G2/M phase.
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USP28 activates angiogenesis.
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USP28 Deficiency Promotes Breast and Liver Carcinogenesis as well as Tumor Angiogenesis in a HIF independent Manner.

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A study has proposed a novel mechanism by which USP28 upregulates angiogenesis by antagonizing GSK-3β (glycogen synthase kinase-3β) and FBW7-dependent degradation of HIF-1α (hypoxia-inducible factor-1α), a major regulator of angiogenesis, carcinogenesis, and various processes by which cells adapt to hypoxic conditions.

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Further, lack of USP28 promotes a more malignant state of breast cancer cells, indicated by an epithelial-to-mesenchymal (EMT) transition, elevated proliferation, migration, and angiogenesis as well as a decreased adhesion.

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USP28 also upregulates angiogenesis by antagonizing GSK-3β (glycogen synthase kinase-3β) and FBW7-dependent degradation of HIF-1α (hypoxia-inducible factor-1α), a major regulator of angiogenesis, carcinogenesis, and various processes by which the cell adapts to hypoxic conditions.

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Correction : USP28 Deficiency Promotes Breast and Liver Carcinogenesis as well as Tumor Angiogenesis in a HIF independent Manner.

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A study has proposed a novel mechanism by which USP28 upregulates angiogenesis by antagonizing GSK-3beta (glycogen synthase kinase-3beta) and FBW7 dependent degradation of HIF-1alpha (hypoxia inducible factor-1alpha), a major regulator of angiogenesis, carcinogenesis, and various processes by which cells adapt to hypoxic conditions.
USP28 inhibits angiogenesis.
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Moreover, GSK3beta and FBW7 dependent HIF-1alpha degradation can be antagonized by the USP28 (ubiquitin specific peptidase-28), suggesting that FBW7 and USP28 could reciprocally regulate cell migration and angiogenesis in a HIF-1alpha-dependent manner [XREF_BIBR].
USP28 affects MAPK
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USP28 inhibits MAPK.
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USP28 inhibits MAPK. 5 / 5
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XREF_BIBR In a proportion of melanoma patients, USP28 was deleted, and loss of USP28 enhanced MAPK activity through the stabilization of RAF family members, which suggested that USP28 was a key factor in BRAF inhibitor resistance.

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The loss of USP28 promoted MAPK activation and resistance to RAF inhibitor therapy by stabilizing BRAF in cell culture and in vivo models.

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Our results show that loss of USP28 enhances MAPK activity through the stabilization of RAF family members and is a key factor in BRAF inhibitor resistance.

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Collectively our results show that loss of USP28 enhances downstream MAPK activation through the stabilization of BRAF, leading to decreased sensitivity to combination therapies involving BRAF inhibitors dabrafenib or vemurafenib (XREF_FIG).

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Importantly, of the 59 patients harboring BRAF V600E mutations 27% (16/59) displayed a> 50% decrease in USP28 mRNA expression levels, suggesting that in tumors harboring BRAF alterations, loss of USP28 may further increase the tumorigenic potential of these tumors by stabilizing BRAF and enhancing downstream MAPK activation.
USP28 activates MAPK.
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USP28 activates MAPK. 2 / 2
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Because USP28 is frequently deleted in melanoma and USP28 depletion leads to BRAF stability and enhanced MAPK kinase in HEK293T cells, we asked if interfering with USP28 expression conferred a similar response in BRAF (V600E) melanoma cell lines.

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In line with previous results, depletion of USP28 in all the melanoma cell lines tested resulted in increased stabilization of BRAF and enhanced downstream MAPK activation (XREF_FIG).
ATG7 affects USP28
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ATG7 increases the amount of USP28.
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ATG7 increases the amount of USP28. 2 / 2
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The results showed that knockdown of ATG7 decreased human usp28 promoter driven reporter transcription activity (XREF_FIG), revealing that ATG7 promotes usp28 promoter transcription.

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ATG7 promoted USP28 protein expression, which directly bound to and deubiquinated CD44s protein and inhibited CD44s protein degradation.
Modified ATG7 increases the amount of USP28. 1 / 1
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In this study, we found that ATG7 overexpression increased USP28 protein expression.
ATG7 activates USP28.
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ATG7 activates USP28. 2 / 2
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The results showed that knockdown of ATG7 decreased human usp28 promoter driven reporter transcription activity (XREF_FIG), revealing that ATG7 promotes usp28 promoter transcription.

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To investigate the mechanisms of ATG7 upregulating USP28 protein, we first examined usp28 mRNA levels in ATG7 knockdown transfectants.
ATG7 inhibits USP28.
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ATG7 inhibits USP28. 1 / 1
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The results showed that with 5-aza-2 '-deoxycytidine treatment, the inhibition of usp28 promoter activity was reversed, USP28 protein and its downstream CD44s protein abundance were also increased by ATG7 knockdown (XREF_FIG and XREF_FIG).
ATG7 decreases the amount of USP28.
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ATG7 decreases the amount of USP28. 1 / 1
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Herein, we found that specific knockdown ATG7 resulted in usp28 promoter hypermethylation as comparison to scramble nonsense transfectants in human BC cells, and increased USP28 expression with 5-aza-2-deoxycytidine treatment, strongly indicating that the promoter hypermethylation was responsible for USP28 transcription downregulation due to ATG7 knockdown.
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Valproic acid increases the amount of USP28. 6 / 6
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USP28 affects Ubiquitin
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Additionally, a deubiquitinase, USP28, has been reported to antagonize ubiquitin dependent proteasomal degradation of c-Myc.

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Interestingly, USP28 can also antagonize the ubiquitin dependent degradation of two additional oncogenic proteins, c-Jun and Notch1, expanding the substrate list that is shared by both USP28 and FBW7.

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USP28 has also been reported to antagonize ubiquitin-dependent degradation of the oncogene product MYC as well as JUN and Notch 105 .

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We transfected HeLa cells with FLAG tagged Myc or Fbw7 together with HA tagged ubiquitin and increasing amounts of Usp28 and recovered ubiquitinated proteins by immunoprecipitation.

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Interestingly, USP28 can also antagonize the ubiquitin-dependent degradation of two additional oncogenic proteins, c-Jun and Notch1, expanding the substrate list that is shared by both USP28 and FBW7.

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Here, using murine genetic models, we determined that USP28 antagonizes the ubiquitin dependent degradation of c-MYC, a known USP28 substrate, as well as 2 additional oncogenic factors, c-JUN and NOTCH1, in the intestine.
LDHA affects USP28
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LDHA activates USP28.
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LDHA activates USP28. 5 / 5
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LDHA generating lactate enhances the USP28 signaling.

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Chronic stress induced epinephrine enhances LDHA dependent metabolic activity, which increases lactate and augments USP28 that serves to stabilize the MYC protein.

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Also, high glucose consistently triggered HK2 and LDHA expression and stimulated the USP28 and MYC axis in breast cancer cells (XREF_SUPPLEMENTARY).

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Silencing of LDHA significantly reversed induction of USP28 and MYC by epinephrine (XREF_FIG), while silencing of HK2 displayed no change on the effect of epinephrine (XREF_SUPPLEMENTARY).

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Following treatment with the LDHA inhibitor sodium oxamate, we found a reduction in USP28 stabilization induced by LDHA in a dose dependent manner (XREF_FIG and XREF_SUPPLEMENTARY).
LDHA decreases the amount of USP28.
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LDHA decreases the amount of USP28. 1 / 1
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Moreover, the reduction in expression of USP28 caused by LDHA knockdown was reversed by MG132 (XREF_FIG).
CASP8 affects USP28
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CASP8 inhibits USP28.
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CASP8 inhibits USP28. 5 / 5
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Furthermore, Caspase-8 cleaves and inactivates USP28 to overcome the p53-dependent G2/M checkpoint in cancer cells [18].
| PMC

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In the nucleus, caspase-8 cleaves and inactivates the ubiquitin specific peptidase 28 (USP28), preventing USP28 from de-ubiquitinating and stabilizing wildtype p53.

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Furthermore, Caspase-8 cleaves and inactivates USP28 to overcome the p53-dependent G2/M checkpoint in cancer cells [ xref ].
| PMC

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In the nucleus, caspase-8 cleaves and inactivates the ubiquitin specific peptidase 28 (USP28), preventing USP28 from de-ubiquitinating and stabilizing wildtype p53.
| PMC

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We further provide evidence that tumor cells expressing high and nuclear levels of caspase-8 have defective p53-dependent apoptosis because caspase-8 cleaves and inactivates USP28 in cells with delayed or compromised mitosis ( xref ).
CASP8 activates USP28.
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CASP8 activates USP28. 1 / 1
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Accordingly, recombinant caspase-8 produced a robust and concentration dependent cleavage of recombinant USP28 in vitro and in whole cell lysates (XREF_SUPPLEMENTARY and XREF_SUPPLEMENTARY).
USP28 affects CLSPN
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USP28 activates CLSPN.
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USP28 activates CLSPN. 4 / 4
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These results suggest that USP28 specifically enhances Claspin protein stabilization through a de-ubiquitination event.

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The specificity of DUBs, however, is poorly understood, and it is currently unknown how Usp28 targets Claspin but not Plk1.

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Additionally, in this system, USP28 only targets Claspin but not PLK1 (both of which are subject to ubiquitination by the same E3 ligase).

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In conclusion, when faced with DNA damage, HNF-1beta-overexpressing cells conferred them with cell survival activity that is mediated through the USP28 mediated Claspin stabilization and then persistent Chk1 activation.
USP28 inhibits CLSPN.
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USP28 inhibits CLSPN. 1 / 1
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We examined whether USP28 knock-down (TOV21G cells transfected with si-USP28) reduces the levels of Claspin protein.
USP28 decreases the amount of CLSPN.
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USP28 decreases the amount of CLSPN. 1 / 1
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Knockdown of endogenous USP28 suppressed the Claspin expression and p-Chk1 activation and cell viability.
Nickel(2+) affects USP28
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Nickel(2+) decreases the amount of USP28.
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Nickel(2+) decreases the amount of USP28. 4 / 4
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Although further studies did not show that hypoxia signaling impaired the association of USP28 with Fbw7alpha and c-Myc, cellular USP28 protein levels were decreased by Ni ions and hypoxia in A549 cells.

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However, USP28 protein expression was decreased by Ni ions and hypoxia in the A549 cells (XREF_FIG).

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Here, it has been demonstrated that Ni ions and hypoxia increased H3K9m2 levels at the USP28 promoter and repressed USP28 gene expression.

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Our study demonstrated that Nickel and hypoxia exposure increased c-myc T58 phosphorylation and decreased USP28 protein levels in cancer cells, which both lead to enhanced c-myc ubiquitination and proteasomal degradation.
Nickel(2+) increases the amount of USP28.
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Nickel(2+) increases the amount of USP28. 1 / 1
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As shown in XREF_FIG, knockdown of both HIF-1alpha and HIF-2alpha attenuated the decrease of USP28 protein level induced by Ni ions or hypoxia.
USP28 affects proteolysis
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USP28 activates proteolysis.
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Overexpression of USP28 largely reversed HDAC5-knockdown-induced LSD1 protein degradation, suggesting HDAC5 positively regulates LSD1 by stabilizing USP28.

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Overexpression of USP28 largely reversed HDAC5-KD-induced LSD1 protein degradation, suggesting a role of HDAC5 as a positive regulator of LSD1 through upregulation of USP28 protein.

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ATG7 promoted USP28 protein expression, which directly bound to and deubiquinated CD44s protein and inhibited CD44s protein degradation.
Modified USP28 activates proteolysis. 1 / 1
| 1

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Overexpression of USP28 largely reversed HDAC5-KD induced LSD1 protein degradation, suggesting a role of HDAC5 as a positive regulator of LSD1 through upregulation of USP28 protein.
USP28 inhibits proteolysis.
| 1
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Overexpression of USP28 largely reversed HDAC5-knockdown-induced LSD1 protein degradation, suggesting HDAC5 positively regulates LSD1 by stabilizing USP28.
USP28 affects USP28
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USP28 decreases the amount of USP28.
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USP28 decreases the amount of USP28. 2 / 2
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In addition, Ni ions and hypoxia increased the levels of the gene silencing mark dimethylated H3 lysine 9 at USP28 promoter region, which suppressed USP28 gene expression, further depleting the cell of this c-Myc salvaging enzyme.

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Ni (II) and hypoxia increased the levels of the gene silencing mark H3K9me2 (substrate of iron dependent dioxygenase) at USP28 promoter region, which suppressed USP28 gene expression.
USP28 activates USP28.
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USP28 activates USP28. 2 / 2
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These findings suggested that ATM mediated phosphorylation of USP28 promotes dissociation of USP28 from KLHL2, destabilizing UCK1.

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Nickel ions or hypoxia could impact the de-ubiquitination process by directly inhibiting USP28 activity or indirectly decreasing USP28 activity through either the dissociation of USP28 from the c, Myc, and Fbw7alpha complex or by a reduction of cellular USP28 levels.
USP28 inhibits USP28.
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USP28 inhibits USP28. 1 / 1
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Thus, complete loss of Usp28 promotes Pin1 dependent degradation of Fbw7 by the proteasome, most likely via enhanced autocatalytic ubiquitination.Destabilization of Fbw7 in Usp28 -/- cells suggested t[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]

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Functional assays demonstrated that overexpression of USP28 promoted cell proliferation and aerobic glycolysis of colorectal cancer, while USP28 inhibition could reverse these effects.

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USP28 increases aerobic glycolysis and promotes cell proliferation upon stabilization of FOXC1 [50].
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USP28 promotes aerobic glycolysis of colorectal cancer by increasing stability of FOXC1.

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In conclusion, USP28 enhanced cell viability and aerobic glycolysis of colorectal cancer by stabilizing FOXC1, suggesting that USP28-FOXC1 might be a novel therapeutic avenue for colorectal cancer.
USP28 affects HIF1A
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USP28 inhibits HIF1A. 4 / 4
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In conclusion, knockdown of USP28 enhanced the radiosensitivity of EC cells by destabilizing c-Myc and enhancing the accumulation of HIF-1alpha.

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In their following study, they found that GSK3beta regulated cell growth, migration and angiogenesis via Fbw7 and USP28 dependent degradation of HIF1alpha [9].

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A study has proposed a novel mechanism by which USP28 upregulates angiogenesis by antagonizing GSK-3beta (glycogen synthase kinase-3beta) and FBW7 dependent degradation of HIF-1alpha (hypoxia inducible factor-1alpha), a major regulator of angiogenesis, carcinogenesis, and various processes by which cells adapt to hypoxic conditions.

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GSK-3beta regulates cell growth, migration, and angiogenesis via Fbw7 and USP28 dependent degradation of HIF-1alpha.
HDAC5 affects USP28
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HDAC5 activates USP28.
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HDAC5 activates USP28. 3 / 3
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We further demonstrated that HDAC5 promoted the protein stability of USP28, a bona fide deubiquitinase of LSD1.

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Alternatively, histone deacetylase 5 (HDAC5) promotes USP28 stability and positively regulates the protein abundance of the Lysine-specific histone demethylase 1A (LSD1) [19].
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Both LSD1 and histone deacetylases (HDACs) facilitate breast cancer proliferation, and interestingly, HDAC5 could promote the stability of USP28.
HDAC5 deubiquitinates USP28.
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Modified HDAC5 leads to the deubiquitination of USP28. 1 / 1
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In vitro pull-down assay using His tag recombinant LSD1 protein incubated with USP28-FLAG protein indicated a direct interaction of HDAC5 and USP28 (XREF_SUPPLEMENTARY), and HDAC5 overexpression significantly attenuated USP28 polyubiquitination (XREF_SUPPLEMENTARY).
TET1 affects USP28
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TET1 increases the amount of USP28.
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TET1 increases the amount of USP28. 2 / 2
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Moreover, upregulated TET1 directly demethylates USP28 promoter, thereby enhancing USP28 transcription and expression, which binds to CD44s protein, and remove the ubiquitin group from the ubiquitinated CD44s protein, resulting in stabilization of CD44s protein to mediate stem like property of human BC cells.

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Collectively, our results strongly demonstrate that ATG7 overexpression mediated TET1 upregulation contributes to usp28 promoter demethylation, thereby increasing USP28 expression, consequently resulting in CD44s protein accumulation, and finally enhancing BC stem sphere formation, invasion, and lung metastatic activity.
Modified TET1 increases the amount of USP28. 1 / 1
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TET1 overexpression markedly promoted USP28 and CD44a protein expression, and rescued cancer stem like properties of T24T (shATG7) cells (XREF_FIG and XREF_FIG).
TET1 decreases the amount of USP28.
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TET1 decreases the amount of USP28. 1 / 1
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Moreover, ATG7 inhibition stabilized AUF1 protein and thereby reduced tet1 mRNA stability and expression, which was able to demethylate usp28 promoter, reduced USP28 expression, finally promoting CD44s degradation.
FBXW7 affects USP28
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FBXW7 activates USP28.
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FBXW7 activates USP28. 2 / 2
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Furthermore, expression of dominant negative Fbw7 or Fbw7 targeting shRNAs enhanced proliferation of Usp28 +/- MEFs, suggesting that their proliferative defect is, at least in part, mediated by the ac[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]

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This result suggests that Usp28 preferentially stabilizes Fbw7 due to more efficient binding and is consistent with the idea that Fbw7 targets Usp28 to its substrates.We concluded that stabilization o[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
FBXW7 inhibits USP28.
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FBXW7 inhibits USP28. 1 / 1
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Furthermore, expression of dominant negative Fbw7 or Fbw7 targeting shRNAs enhanced proliferation of Usp28 +/- MEFs, suggesting that their proliferative defect is, at least in part, mediated by the ac[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
FBXW7-S205A inhibits USP28. 1 / 1
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Stable expression of the S205A mutant Fbw7, but not of the wild-type protein, strongly downregulated Fbw7 substrates and attenuated proliferation of the Usp28 -/- MEFs.
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Trichostatin A increases the amount of USP28. 3 / 3
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Knockdown of USP28 directly increases the expression of differentiation genes but indirectly suppresses the expression of pluripotent molecules.

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Usp28 deficiency promoted tumor cell differentiation accompanied by decreased proliferation, which suggests that USP28 acts similarly in intestinal homeostasis and colorectal cancer models.

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Knockdown of USP28 induces differentiation and suppresses self-renewal in CSCs derived from MMTV-Wnt1 mice.
USP28 affects Neoplasms
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In a mouse model of colorectal cancer, in contrast to the FBW7-deletion phenotype, intestine-specific deletion of USP28 reduced intestinal tumors.

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In SCC tumors, the inhibition of USP28 reduces tumor growth and increases cell death [17].
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Importantly, in mice carrying the tumors, USP28 deletion reduced tumor size and prolonged lifespan18.
USP28 affects FOXC1
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USP28 activates FOXC1. 3 / 3
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Moreover, proteasome inhibitor, MG132, could rescue USP28 silence induced degradation of FOXC1.

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In conclusion, USP28 enhanced cell viability and aerobic glycolysis of colorectal cancer by stabilizing FOXC1, suggesting that USP28-FOXC1 might be a novel therapeutic avenue for colorectal cancer.

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Protein expression of Forkhead Box C1 (FOXC1) was increased by USP28 over-expression, whereas knockdown of USP28 aggravated cycloheximide (CHX; protein synthesis inhibitor) stimulated decrease of FOXC1.
Lipopolysaccharide, E coli O55-B5 increases the amount of USP28.
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Lipopolysaccharide, E coli O55-B5 decreases the amount of USP28.
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