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.
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.
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.
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]
We discuss mechanisms that regulate ubiquitination by Fbw7, including ubiquitin-specific proteases such as USP28 that counteract Fbw7 activity and thereby stabilise oncoproteins.
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.
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.
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.
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.
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].
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].
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.
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.
The signalling pathway involves 53BP1 and the deubiquitylase USP28 acting in a complex to deubiquitylate and stabilise p53, which in turn controls cell fate.
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.
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.
Conversely, USP28 silencing in HEK293T markedly increased the ubiquitination of endogenous UCK1, implying that endogenous UCK1 was also a target of USP28.
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).
Mechanistically, USP28 deubiquitinates and stabilizes the specificity factors and mediators of ATM and ATR signaling by protecting them from ubiquitination-mediated proteasome degradation.
Mechanistically, USP28 deubiquitinates and stabilizes the specificity factors and mediators of ATM and ATR signaling by protecting them from ubiquitination-mediated proteasome degradation.
In this setting, USP28 deubiquitinates the SCF component FBW7, allowing FBW7 to act as a substrate recognition factor targeting substrates for proteosomal mediated degradation.
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.
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].
Since FBW7gamma lacks the USP28 interaction motif of FBW7alpha, Usp28 selectively antagonize nucleoplasmic MYC degradation while not affecting nucleolar degradation.
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.
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.
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.
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].
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.
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.
Overexpression of USP28 in BR cells enhances c-Myc expression and hence increases ASS1 transcription upon arginine deprivation, and consequently leads to cell survival.
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).
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.
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.
Recently, upregulation of USP28 has been reported to be associated with poor prognosis in NSCLC patients and promote NSCLC cell proliferation XREF_BIBR.
Functional assays demonstrated that overexpression of USP28 promoted cell proliferation and aerobic glycolysis of colorectal cancer, while USP28 inhibition could reverse these effects.
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.
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].
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.
Previous reports showed that 53BP1 and USP28 activate p53, preventing the proliferation of cells that have an increased chance of mitotic errors [ xref ].
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 .
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).
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).
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.
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.
The Usp28 deubiquitinase antagonizes Fbw7 mediated turnover of multiple oncoproteins, including Myc, Jun, and Notch, and promotes tumorigenesis in the intestine.
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.
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.
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.
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]
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].
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]
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]
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]
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.
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.
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]
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).
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.
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).
In contrast, knockdown of USP28 suppressed STAT3 derived luciferase activity (XREF_FIG), and inhibited the expression of STAT3 in NSCLC cells (XREF_FIG).
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).
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).
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.
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].
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).
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.
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.
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.
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.
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.
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).
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).
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).
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).
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).
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.
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.
XREF_BIBR reported that overexpression of USP28 promotes NSCLC cell proliferation and apoptosis inhibition and is associated with poor prognosis in NSCLC patients.
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.
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.
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.
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.
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).
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).
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.
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.
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.
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.
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.
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.
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].
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.
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.
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).
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.
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.
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).
The results showed that knockdown of ATG7 decreased human usp28 promoter driven reporter transcription activity (XREF_FIG), revealing that ATG7 promotes usp28 promoter transcription.
The results showed that knockdown of ATG7 decreased human usp28 promoter driven reporter transcription activity (XREF_FIG), revealing that ATG7 promotes usp28 promoter transcription.
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).
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.
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.
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.
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.
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.
Chronic stress induced epinephrine enhances LDHA dependent metabolic activity, which increases lactate and augments USP28 that serves to stabilize the MYC protein.
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).
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).
In the nucleus, caspase-8 cleaves and inactivates the ubiquitin specific peptidase 28 (USP28), preventing USP28 from de-ubiquitinating and stabilizing wildtype p53.
In the nucleus, caspase-8 cleaves and inactivates the ubiquitin specific peptidase 28 (USP28), preventing USP28 from de-ubiquitinating and stabilizing wildtype p53.
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 ).
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).
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.
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.
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.
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.
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.
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.
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.
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.
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]
Functional assays demonstrated that overexpression of USP28 promoted cell proliferation and aerobic glycolysis of colorectal cancer, while USP28 inhibition could reverse these effects.
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.
In their following study, they found that GSK3beta regulated cell growth, migration and angiogenesis via Fbw7 and USP28 dependent degradation of HIF1alpha [9].
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.
Alternatively, histone deacetylase 5 (HDAC5) promotes USP28 stability and positively regulates the protein abundance of the Lysine-specific histone demethylase 1A (LSD1) [19].
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).
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.
TET1 overexpression markedly promoted USP28 and CD44a protein expression, and rescued cancer stem like properties of T24T (shATG7) cells (XREF_FIG and XREF_FIG).
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.
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]
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]
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]
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.
Usp28 deficiency promoted tumor cell differentiation accompanied by decreased proliferation, which suggests that USP28 acts similarly in intestinal homeostasis and colorectal cancer models.
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.
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.
Knockdown of USP28 resulted in LSD1 destabilization, leading to the suppression of cancer stem cell (CSC)-like characteristics invitro and inhibition of tumorigenicity invivo, which can be rescued by ectopic LSD1 expression.
Although our results indicate that LSD1 is a major target of USP28 in breast cancer, it does not rule out the possibility that USP28 promotes CSC like traits through both LSD1 stabilization and inhibition of Myc degradation.
Hence, induced expression of SENP1 under hypoxia promotes desumoylation and activation of USP28, which then can contribute to further stabilisation of HIFs by their deubiquitynation activity (XREF_FIG).
Hence, induced expression of SENP1 under hypoxia promotes desumoylation and activation of USP28, which then can contribute to further stabilisation of HIFs by their deubiquitynation activity ( xref ).
Here, we report that USP28 is a SUMOylated protein in normoxia with moderate deubiquitinating activity towards HIF-1α in vitro, while hypoxia and HIF-1α activate USP28 through SENP1-mediated USP28 deSUMOylation to further accumulate HIF-1α protein in cells.
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.
Here, we report that USP28 is a SUMOylated protein in normoxia with moderate deubiquitinating activity towards HIF-1alpha in vitro, while hypoxia and HIF-1alpha activate USP28 through SENP1 mediated USP28 deSUMOylation to further accumulate HIF-1alpha protein in cells.
Here, we report that USP28 is a SUMOylated protein in normoxia with moderate deubiquitinating activity towards HIF-1α in vitro, while hypoxia and HIF-1α activate USP28 through SENP1-mediated USP28 deSUMOylation to further accumulate HIF-1α protein in cells.
Additional mechanism suggests that histone deacetylases 5 (HDAC5) promotes stability of USP28, thus enhancing stability of LSD1.Overexpression of USP28, HDAC5, and LSD1 is positively correlated in breast tumors.
Collectively these results suggest that USP28 expression is regulated by MAPK activity and may function through a feedback loop to negatively regulate ERK signaling.
Consistent with this, the expression of WT or ubiquitin binding mutant USP28 rescued p21 and MDM2 induction defects in USP28Delta cells, whereas expression of USP28 C171A did not (XREF_SUPPLEMENTARY B).
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).
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.
Pharmacodynamics studies demonstrated that depletion of USP28 led to a robust retention of ERK phosphorylation in tumors treated with vemurafenib (XREF_FIG).
It is important to note that in both cell lines tested vemurafenib treatment enhanced the expression USP28, indicative of a USP28 feedback loop in both these BRAF mutant cell lines (XREF_FIG).
As USP28 knockdown inhibited the ability of vemurafenib to attenuate ERK phosphorylation, we reasoned that in these cell lines BIM (Bcl-2-interacting mediator of cell death) accumulation would be down-regulated.
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].
Transwell assays showed that USP28 overexpression reduced both cell migration and invasion in MDA-MB-231 and MCF7 cells, and these effects were partially reversed by transfection with miR-500a-5p mimics.
In contrast, in a detailed study on the function of USP28 in breast cancer XREF_BIBR, Richter et al. found that USP28 deficiency in breast cancer cells enhances conversion toward a more aggressive phenotype by promoting EMT, proliferation, migration, angiogenesis, and decreased adhesion.
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.
Different from the interaction of USP28 with LSD1, USP28 mediated p53BP1 stabilization only occurs after DNA damage and no effect is found in the absence of DNA damage, suggesting that the interaction between USP28 with p53BP1 is regulated by DNA damaging.
MiR-500a-5p was transferred from CAFs to the cancer cells, and subsequently promoted proliferation and metastasis by binding to ubiquitin specific peptidase 28 (USP28).
USP28 was shown to bind c-Myc through an interaction with FBW7alpha and antagonize its E3 ligase activities in the nucleus, leading to Myc stabilization and tumor cell proliferation.
USP28 antagonizes FBW7, an F-box protein and an important component of the SKP1-CUL1-F-BOX (SCF)-type E3 ubiquitin ligase that targets key transcriptional factors to ubiquitin-directed proteasome degradation.
The APC and C-Cdh 1 target Claspin, for instance, is protected from APC/C mediated down-regulation by USP28 [XREF_BIBR], and also 53BP1 and Chk2 were reported to be stabilized by USP28 upon DNA damage [XREF_BIBR].
The APC and C-Cdh 1 target Claspin, for instance, is protected from APC/C mediated down-regulation by USP28 [XREF_BIBR], and also 53BP1 and Chk2 were reported to be stabilized by USP28 upon DNA damage [XREF_BIBR].
This leads to a switch to lactate production, and the adjusted pH then directs USP28 mediated deubiquitination and stabilization of MYC, thereby promoting stem like traits in breast cancer.
Chronic stress induced epinephrine activated LDHA to generate lactate, and the adjusted pH directed USP28 mediated deubiquitination and stabilization of MYC.
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.
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.
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.
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].
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 antagonizes FBW7, an F-box protein and an important component of the SKP1-CUL1-F-BOX (SCF)-type E3 ubiquitin ligase that targets key transcriptional factors to ubiquitin-directed proteasome degradation.
Furthermore, USP28 is mutated in human cancer cells and is reported to antagonize the tumour suppressor F-box and WD40 domain-containing protein 7 (FBW7) 31 , highlighting the potential for USP28 inhibitors in various tumours, especially colorectal cancer 104 .
Overexpression of USP28 in BR cells enhances c-Myc expression and hence increases ASS1 transcription upon arginine deprivation, and consequently leads to cell survival.
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.
Mechanistically, as well as its role in DNA repair, 53BP1 promotes p53 signalling, by coordinating ubiquitin specific peptidase 28 (USP28)-mediated p53 deubiquitination.
Given that p53 activity is quenched via MDM2 dependent p53 ubiquitination, it is tempting to speculate that a 53BP1 dependent targeting of USP28 into p53 protein complexes might counteract such events.
However, in contrast to the results in KRAS positive CRC cell lines, in WI-38 cells KRAS failed to increase ZNF304 protein levels (XREF_FIG) or significantly stimulate USP28 and PRKD1 transcription (XREF_FIG), explaining at least in part why KRAS expression does not result in INK4-ARF silencing.
Disrupting USP28 destabilizes LSD1 protein, which decreases breast cancer stem cell like characteristics in vitro and suppresses tumorigenicity in vivo [XREF_BIBR].
Although our results indicate that LSD1 is a major target of USP28 in breast cancer, it does not rule out the possibility that USP28 promotes CSC like traits through both LSD1 stabilization and inhibition of Myc degradation.
And that vitamin C reversed the induction of stem-like phenotype by inhibition of stress-induced LDH-A , and suppressing the lactic acid generation and the USP28 / MYC / SLUG pathway in BCSCs .
The combination of these results stresses the need for further investigation to determine the precise mechanism of rigosertib specifically targeting USP28 mutant cells.
There were two scenarios that resulted in an increase in ubiquitinated c-Myc levels in A549 cells : the first involved an increased phosphorylation of c-Myc at T58; and the second involved a decrease in USP28 protein levels partially mediated by HIF-1 and HIF-2.
We observed that hairpins B, C, and D efficiently suppressed ectopically expressed and endogenous USP28 levels in 293T cells; however, hairpins C and D consistently achieved a greater knockdown efficiency than hairpins A and B and were therefore used for the remainder of the experiments.
Using an ATP based cell viability assay (Cell Titer-Glo), we screened a small library of 316 FDA approved chemical compounds and identified the PLK1 inhibitor rigosertib as a compound that selectively impairs the viability of USP28 depleted cells (XREF_FIG and Table S3).
This early disassembly of the MRN complex could be prevented by altering the ubiquitination site of Nbs1 or by expressing a deubiquitinase, Usp28, which sufficiently restored homologous recombination repair and ATM, a major checkpoint kinase against DNA DSBs, activation abilities in RTS and RecQL4-depleted cells.
Previous reports showed that 53BP1 and USP28 activate p53 , preventing the proliferation of cells that have an increased chance of mitotic errors [ 104 ] .
This early disassembly of the MRN complex could be prevented by altering the ubiquitination site of Nbs1 or by expressing a deubiquitinase, Usp28, which sufficiently restored homologous recombination repair and ATM, a major checkpoint kinase against DNA DSBs, activation abilities in RTS and RecQL4-depleted cells.
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.
miR-363 directly inhibits a ubiquitin specific protease, USP28 to promote proteasome mediated degradation of Myc in human hepatocellular carcinoma [XREF_BIBR].
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]
Transwell assays showed that USP28 overexpression reduced both cell migration and invasion in MDA-MB-231 and MCF7 cells, and these effects were partially reversed by transfection with miR-500a-5p mimics.
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.
However, in contrast to the results in KRAS positive CRC cell lines, in WI-38 cells KRAS failed to increase ZNF304 protein levels (XREF_FIG) or significantly stimulate USP28 and PRKD1 transcription (XREF_FIG), explaining at least in part why KRAS expression does not result in INK4-ARF silencing.
USP28 antagonizes FBW7, an F-box protein and an important component of the SKP1-CUL1-F-BOX (SCF)-type E3 ubiquitin ligase that targets key transcriptional factors to ubiquitin-directed proteasome degradation.
Ectopic USP28 expression promoted proliferation of SW1783 glioma cells both in vitro and in vivo, and conferred enhanced tumorigenicity in a nude mouse model.
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.
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.
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.
In addition, overexpression of miR-216b suppressed the substrates ' expression of USP28, for example, c-Myc, and miR-216b overexpression also inhibited Cyclin E expression as well as upregulating p27 expression, both of which were the downstream signals of c-Myc.
USP28-mediated stabilisation of FOXM1 significantly promoted nucleus β-catenin trans-activation, which in turn led to the activation of the Wnt/β-catenin pathway.
This early disassembly of the MRN complex could be prevented by altering the ubiquitination site of Nbs1 or by expressing a deubiquitinase, Usp28, which sufficiently restored homologous recombination repair and ATM, a major checkpoint kinase against DNA DSBs, activation abilities in RTS and RecQL4-depleted cells.
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.
In addition, overexpression of miR-216b suppressed the substrates ' expression of USP28, for example, c-Myc, and miR-216b overexpression also inhibited Cyclin E expression as well as upregulating p27 expression, both of which were the downstream signals of c-Myc.
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.
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.
This early disassembly of the MRN complex could be prevented by altering the ubiquitination site of Nbs1 or by expressing a deubiquitinase, Usp28, which sufficiently restored homologous recombination repair and ATM, a major checkpoint kinase against DNA DSBs, activation abilities in RTS and RecQL4-depleted cells.
The results showed that knockdown of ATG7 decreased human usp28 promoter driven reporter transcription activity (XREF_FIG), revealing that ATG7 promotes usp28 promoter transcription.
Overexpression of USP28 in BR cells enhances c-Myc expression and hence increases ASS1 transcription upon arginine deprivation, and consequently leads to cell survival.
Conversely, USP28 silencing in HEK293T markedly increased the ubiquitination of endogenous UCK1, implying that endogenous UCK1 was also a target of USP28.
Moreover, purified USP28 was found to directly deubiquitinate p53 in vitro (XREF_FIG), raising potentially a direct role of USP28 in stabilizing p53 in vivo.
Importantly, knockout of the essential centriole component SAS6 also triggered a 53BP1, USP28, and p53 dependent G1 arrest, demonstrating that the cell cycle arrest was likely to be caused by centrosome loss and not by the loss of Plk4 activity per se [XREF_BIBR].
Moreover, expression of Pin1 correlated with Fbw7 substrate abundance in murine tissues; it decreased upon Usp28 loss in the intestine but increased in the lung, suggesting that tissue specific regula[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
Levels of NICD were only partially rescued by proteasome inhibition, suggesting that additional mechanisms contribute to the downregulation of NICD in Usp28 +/- cells.
In addition , this inhibition of miR-4295 expression not only increased USP28 3 ' - UTR activity but also promoted USP28 and CD44 protein expression ( Fig. 5B & 5C ) .
As expected, AUF1 knockdown led to increased expression of TET1, USP28, and CD44s protein (XREF_FIG and XREF_FIG), as well as promotion of cancer stem like cell sphere formation (XREF_FIG and XREF_FIG).
There were two scenarios that resulted in an increase in ubiquitinated c-Myc levels in A549 cells : the first involved an increased phosphorylation of c-Myc at T58; and the second involved a decrease in USP28 protein levels partially mediated by HIF-1 and HIF-2.
The non-amplified and cyclin E1 hi group had significantly increased USP28, while the amplified and cyclin E1 hi cancers had significantly lower FBXW7 expression consistent with a role for both in stabilizing cyclin E1.
Collectively, these data demonstrate that BRAF ubiquitination and stability is directly regulated through an interplay between the FBW7 and SCF ubiquitin ligase complex and the deubiquitinating enzyme USP28 leading to enhanced MAPK activity.
The non-amplified and cyclin E1 hi group had significantly increased USP28, while the amplified and cyclin E1 hi cancers had significantly lower FBXW7 expression consistent with a role for both in stabilizing cyclin E1.
Since 5 '-AZA treatment can activate ATM in AML cells and the ATM pathway can prevent the apoptosis induction in 5 '-AZA resistant cells XREF_BIBR, we sought to determine whether ATM modulated the effect of USP28 on the UCK1 deubiquitination in response to 5 '-AZA.
Overexpression of USP28 in BR cells enhances c-Myc expression and hence increases ASS1 transcription upon arginine deprivation, and consequently leads to cell survival.