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 USP9X from literature and knowledge bases.
The first section shows only DUB activity and the second shows all other results.
Deubiquitinase Activity
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USP9X deubiquitinates and stabilizes MCL1 in distinct human cancers including human follicular lymphomas, diffuse large B cell lymphomas, glioblastoma, colon and lung cancers 19.
USP9X deubiquitylates poly-ubiquitylated MCL1, protecting it from proteasomal degradation, thus increasing its stability and thereby promoting cell survival.
Two DUBs USP9X and USP13 deubiquitinate and stabilise MCL1, and hypomorphic mutations in both have been linked to neurodevelopmental disorders and neurodegenerative disease [XREF_BIBR, XREF_BIBR].
The overexpression of USP9X stabilizes the MCL1 protein in human lymphomas, and the depletion of USP9X increases MCL1 ubiquitination, which leads to MM cell apoptosis [31].
FAM and USP9X is required for TGFbeta induced migration of MDA-MB-231 breast cancer cells; mechanistically, FAM and USP9X inhibits the monoubiquitination of SMAD4, a modification that blocks the association of SMAD4 with phospho-SMAD2 [XREF_BIBR] (XREF_FIG).
Usp9x promotes TGFbeta pathway signalling by deubiquitylating Smad4, allowing it to complex with phosphorylated receptor Smads and then shuttle into the nucleus to execute transcriptional responses to TGFbeta family ligands [XREF_BIBR].
Taken together, these results demonstrate that USP9x selectively binds to SMAD4 in competition with TIF1gamma and deubiquitinates SMAD4, promoting nuclear SMAD4 retention, SMAD3 and SMAD4 complex formation and target gene expression.
Previously, we found that ERG undergoes ubiquitination and then is deubiquitinated by USP9X in prostate cancer cells to prevent its proteasomal degradation.
A role for ERG ubiquitination in prostate cancer cells was also demonstrated by Wang et al. who showed that the enzyme USP9X, which is highly expressed in ERG positive prostate tumours, mediates ERG deubiquitination and thus its stabilization XREF_BIBR.
Indeed, previous reports have shown Usp9x deubiquitinates and stabilizes ERG, and our previously described DUB inhibitor (WP1130) demonstrated anti-tumour efficacy in ERG driven prostate cancer XREF_BIBR.
Here, we show that ubiquitin specific peptidase 9, X linked (USP9X), a deubiquitinase enzyme, binds ERG in VCaP prostate cancer cells expressing TMPRSS2-ERG and deubiquitinates ERG in vitro.
we show that ubiquitin-specific peptidase 9, X-linked (USP9X), a deubiquitinase enzyme, binds ERG in VCaP prostate cancer cells expressing TMPRSS2-ERG and deubiquitinates ERG in vitro.
Wang et al. demonstrated that ubiquitin specific peptidase 9, X linked (USP9X), a deubiquitinase enzyme, binds ERG in VCaP prostate cancer cells expressing TMPRSS2-ERG and deubiquitinates ERG in vitro XREF_BIBR.
We recently found that USP9X deubiquitinates α-synuclein, and that this process determines the partition of α-synuclein between the proteasomal and autophagy pathways.
We recently found that USP9X deubiquitinates alpha-synuclein, and that this process determines the partition of alpha-synuclein between the proteasomal and autophagy pathways.
Usp9x also deubiquitylates mono-ubiquitylated alpha-synuclein raising the possibility it may play a role in the progression of neurodegenerative diseases such as Parkinson 's disease XREF_BIBR.
Moreover, USP9X can increase MAPT phosphorylation via a second mechanism, by deubiquitinating the protein alpha-synuclein (SNCA) [XREF_BIBR], which functions as a connecting mediator between the glycogen synthase kinase 3beta (GSK3B) and MAPT and has been shown to stimulate MAPT phosphorylation via GSK3B in vitro [XREF_BIBR].
In the brain tissues of PD patients, USP9X colocalises with alpha-synuclein inclusions, and in vitro studies show a functional interaction; whilst monoubiquitylated alpha-synuclein is degraded by the proteasome, USP9X deubiquitylation of alpha-synuclein directs its degradation by the less efficient autophagy pathway [XREF_BIBR].
In addition, USP9X in LNCaP cells was not co-immunoprecipitated with IRS-2 (XREF_SUPPLEMENTARY), suggesting that USP9X does not deubiquitinate IRS-2 in LNCaP cells.
In addition, the interaction between USP9X and IRS-2 was not observed in LNCaP cells (XREF_SUPPLEMENTARY), suggesting that USP9X in LNCaP does not deubiquitinate and stabilize IRS-2 because of the absence of interaction between them.
Knockdown of USP9X dramatically reduced IRS-2 protein level, increased IRS-2 ubiquitination, and promoted the proteasomal degradation of IRS-2 in PC3 cells, suggesting that USP9X also deubiquitinates IRS-2 to prevent its proteasomal degradation.
In the Wnt pathway, FAM deubiquitinates betacatenin, preventing its degradation in mammalian cells, but the effect of FAM activity on Wnt signaling was not noted.
Since YAP1 plays a key role in human cancer development, it is possible that USP9X promotes deubiquitination and stabilization of YAP1 in human cancers.
Mechanistically, it was found that hsa_circ_0024093 could regulate the expression of USP9X, which further induced YAP1 deubiquitination to stabilize YAP1 protein.
Together these data show that USP9X deubiquitinates ZBTB38, both in basal conditions, and in conditions where ZBTB38 ubiquitination is augmented by RBBP6.
By poly-ubiquitination assays, we observed that USP9X causes deubiquitination of ZBTB38, even in cells over-expressing RBBP6 and conversely that inactivation of USP9X amplifies the polyubiquitination induced by RBBP6 over-expression (XREF_SUPPLEMENTARY).
Moreover, USP9X deubiquitinates and stabilizes SMURF1, a member of the NEDD4 family of ubiquitin ligases; silencing USP9X expression in MDA-MB-231 breast cancer cells destabilized SMURF1 and inhibited SMURF1 dependent cell migration [XREF_BIBR].
In another study, in breast cancer cells ubiquitination of Smurf1 could be reversed by the deubiquitinating enzyme USP9X through Smurf1 WW domain binding, which improved Smurf1 's stability.
To test whether USP9X de-ubiquitinates TDRD3 in cells, we transfected HeLa cells with either control or USP9X specific siRNA, and measured TDRD3 ubiquitination.
We observed that when the cellular levels of USP9X were reduced by siRNA, the TDRD3 ubiquitination levels markedly increased (XREF_FIG, upper panel -- compare lane 2 to lane 5) and inhibition of the proteasome greatly augmented this difference (XREF_FIG, upper panel -- compare lane 3 to lane 6), suggesting that USP9X is necessary to suppress TDRD3 ubiquitination.
We report here that Usp9x deubiquitinates and stabilizes PRC2, acting as a gatekeeper to the switch in H3K27me3 deposition patterns during mouse development.
Interestingly, recent findings further demonstrate that EPS15 de-ubiquitination by USP9X affects EGFR internalization and its trafficking to lysosomes.
As USP9X interacts with and deubiquitylates the MD3 domain (aa 715-988) of STIL and removal of the MD3 domain destabilized STIL, USP9X controls STIL levels via the MD3 antidegron of STIL.
To test whether USP9X deubiquitylates the STIL MD3 domain, we incubated immunoprecipitated full-length STIL or the MD3 domain of STIL with recombinant GST, GST-USP7, or GST fused to the catalytic domain of USP9X (USP9X CD).
Usp9x knockdown in melanoma increased SOX2 ubiquitination, leading to its depletion, and enhanced apoptotic effects of BRAF inhibitor and MEK inhibitors.
We identified that Usp9x deubiquitinates SOX2 and can increaseSOX2 levels, but additional studies are needed to confirm the specific ubiquitin sites among the 16 lysine residues in SOX2 that could be putative ubiquitin acceptors.
Because USP9X deubiquitinates NPHP5 (XREF_FIG), we hypothesize that NPHP5 is prone to ubiquitination when its association with USP9X is compromised in G2/M.
NPHP5 directly binds to a deubiquitinating enzyme USP9X/FAM and two E3 ubiquitin ligases BBS11/TRIM32 and MARCH7/axotrophin. NPHP5 undergoes K63 ubiquitination in a cell cycle dependent manner and K48/K63 ubiquitination upon USP9X depletion or inhibition.
USP9X can also regulate and stabilize CEP131, a centriolar satellite protein, ultimately promoting breast carcinogenesis, indicating that USP9X is a significant regulator of centrosome biogenesis and revealing a critical role for the USP9X/CEP131 axis in breast carcinogenesis
USP9x in turn stimulates JAG1 activity through two mechanisms: (1) through TRB3 deubiquitination and stabilization, and (2) through deubiquitination and activation of Mind Bomb 1, an E3 ligase required for JAG1 ubiquitination-mediated endocytosis and Notch activation.
Further, in human breast cancer cell lines, USP9X deubiquitinates and stabilizes MIB1, an activator of ligand dependent canonical NOTCH1 signaling that is important for cardiac development.
Therefore, we postulated that nitrosylated USP9X deubiquitinates and stabilizes MIB1 in valvular interstitial cells, which potentiates the ability of a ligand producing cell to activate NOTCH on a neighboring cell.
Engel et al. [XREF_BIBR] demonstrate that USP9X is the mitotic deubiquitinase of the X linked inhibitor of apoptosis protein (XIAP) and that deubiquitylation and stabilization of XIAP by USP9X lead to increased resistance toward mitotic spindle poisons.
These studies revealed a mitosis specific function for the ubiquitin specific protease 9X (USP9X), which we show to deubiquitylate and stabilize XIAP in order to promote mitotic survival.
USP8 and USP9X deubiquitinate ITCH to induce ubiquitination and degradation of the anti-apoptotic protein c-FLIP, leading to apoptosis in glioblastoma [XREF_BIBR], or to anoikis in pancreatic ductal adenocarcinoma [XREF_BIBR].
Smad4 is monoubiquitinated in lysine 519 in vivo, a modification that inhibits smad4 by impeding association with phospho-smad2. Fam reverts this negative modification, re-empowering smad4 function;control of smad4 is a good way to regulate bone formation. Fam and ectodermin/tif1gamma (ecto) were reported to respectively regulate the de-ubiquitination and ubiquitination of smad4.
Smad4 is monoubiquitinated in lysine 519 in vivo, a modification that inhibits smad4 by impeding association with phospho-smad2. Fam reverts this negative modification, re-empowering smad4 function;control of smad4 is a good way to regulate bone formation. Fam and ectodermin/tif1gamma (ecto) were reported to respectively regulate the de-ubiquitination and ubiquitination of smad4.
Smad4 is monoubiquitinated in lysine 519 in vivo, a modification that inhibits smad4 by impeding association with phospho-smad2. Fam reverts this negative modification, re-empowering smad4 function;control of smad4 is a good way to regulate bone formation. Fam and ectodermin/tif1gamma (ecto) were reported to respectively regulate the de-ubiquitination and ubiquitination of smad4.
We hypothesize that USP9X deubiquitinates Cdc20 directly; however, we can not rule out the possibility that USP9X loss enhances Cdc20 ubiquitination and degradation indirectly.
In summary, the data presented here show that USP9X antagonizes Cdc20 auto-ubiquitination and degradation to limit the turnover of MCC complexes by APC/C.
An investigation of the polyubiquitination of AMPK-RKs showed that the deubiquitinating enzyme USP9X (ubiquitin specific protease-9) modulates the deubiquitination of NUAK1 (AMPK related kinase 5) and MARK4 (microtubule-affinity-regulating kinase 4) in cells XREF_BIBR.
The USP9X mediated BCL9 deubiquitination promotes formation of the beta, catenin, BCL9, and PYGO complex, increasing the transcriptional activity of the Wnt and beta-catenin target genes.
Finally, the ubiquitin specific protease 9X (USP9X) interacts with BCL10 in activated T cells and silencing of USP9X augments BCL10 ubiquitination and impairs NF-kappaB signaling in T cells.
Mechanistically, USP9X interacts with Bcl10 of the Carma1-Bcl10-Malt1 (CBM) complex and removes the TCR-induced ubiquitin chain from Bcl10, which facilitates the association of Carma1 with Bcl0-Malt1.
It was recently shown that silencing Fam by siRNA abrogated ionomycin induced decrease in Epsin ubiquitylation, indicating that Fam plays a role in regulating levels of Epsin ubiquitylation [15].
However, in vitro data on direct deubiquitylation of Epsin by Fam are lacking, and the ~ 50% identity between Faf and Fam suggests different specificities for substrates [49]; indeed, the only reporte[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
Through biochemical experiments, we discover that USP9X reduces BMAL1 ubiquitination, enhances its stability, and increases its protein level, leading to the elevated transcriptional activity.
Here, we use affinity purification and mass spectrometry analysis to identify probable ubiquitin carboxyl-terminal hydrolase FAF-X (USP9X) as an interacting protein of the core clock protein aryl hydrocarbon receptor nuclear translocator-like protein 1 (ARNTL or BMAL1).
An investigation of the polyubiquitination of AMPK-RKs showed that the deubiquitinating enzyme USP9X (ubiquitin specific protease-9) modulates the deubiquitination of NUAK1 (AMPK related kinase 5) and MARK4 (microtubule-affinity-regulating kinase 4) in cells XREF_BIBR.
The following findings were observed : (1) Expression of USP9X was down-regulated in the kidney tissue of db/db diabetic mice; (2) overexpression of USP9X suppressed high glucose (HG)-induced expressions of EMT markers and extra cellular matrix (ECM) in NRK-52E cells; (3) depletion of USP9X further aggravated EMT process and ECM production in NRK-52E cells; (4) USP9X deubiquitinated Cx43 and suppressed its degradation to regulate EMT process; (5) USP9X deubiquitinated Cx43 by directly binding to the C-terminal Tyr 286 of Cx43.
Exogenous expression and short interfering RNA depletion experiments demonstrate that MARCH7 can be stabilized by both USP9X and USP7, which deubiquitylate MARCH7 in the cytosol and nucleus, respectively.
we report for the first time that USP9X is a deubiquitinase of Angiomotin-like 2 (AMOTL2) and that AMOTL2 mono-ubiquitination is required for YAP inhibition.
Thus, it appears that the direct effect of limiting deubiquitylation of AMOT by USP9x offsets the reduced potential for ubiquitylation due to lower E3 ligase levels.
The USP9X mediated BCL9 deubiquitination promotes formation of the beta, catenin, BCL9, and PYGO complex, increasing the transcriptional activity of the Wnt and beta-catenin target genes.
Indeed, ubiquitylation of XIAP was substantially increased upon silencing or chemical inhibition of USP9X (Figs XREF_FIG E and XREF_FIG A) in mitotic cells, while forced expression of USP9X attenuated XIAP ubiquitylation (Fig XREF_FIG A).
In this report, we find that Usp9x, a deubiquitinating enzyme, stably associates with the SMN complex via directly interacting with SMN.| Usp9x deubiquitinates SMN that is mostly mono- and di-ubiquitinated.
Interestingly, Beclin1 and the Bcl-2 family member, MCL-1, compete for their interaction with USP9X, which contributes to their stabilization by protecting them from proteasomal degradation in HEK293T cells
Conclusions: In summary, our data demonstrated that the USP9X-TTK axis may play a critical role in NSCLC, and could be considered as a potential therapeutic target.
Usp9x deubiquitinates and stabilizes the TF, ERG, in prostate, and we previously published that DUB inhibitor (WP1130) has anti-tumor activity in ERG fusion driven prostate cancer [XREF_BIBR, XREF_BIBR, XREF_BIBR].
USP9x in turn stimulates JAG1 activity through two mechanisms: (1) through TRB3 deubiquitination and stabilization, and (2) through deubiquitination and activation of Mind Bomb 1, an E3 ligase required for JAG1 ubiquitination-mediated endocytosis and Notch activation.
FAM and Usp9x deubiquitinates the monoubiquitinated SMAD4 (which is exported from the nucleus (XREF_FIG)) and thereby enables SMAD2 and SMAD4 complex formation.
Our findings indicate that USP9X-mediated deubiquitylation of DVL2 is required for canonical WNT activation, while increased DVL2 ubiquitylation is associated with localization to actin-rich projections and activation of the planar cell polarity (PCP) pathway.
?Collectively, our experiments identified USP9X as an integral component of the centrosome where it functions to stabilize PCM1 and CEP55 and promote centrosome biogenesis.
Agonist stimulation of Notch leads to the recruitment of USP9X, a deubiquitinating enzyme, which deubiquitinates epsin and enables it to promote Notch internalization.
Taken together, USP9X reduced Nrf2 ubiquitination level and promoted Nrf2-ARE pathway activation to prevent the accumulation of extracellular matrix, eventually alleviated the pathological process of diabetic renal fibrosis.
[XREF_BIBR, XREF_BIBR, XREF_BIBR - XREF_BIBR] We postulated the physical interaction between the PRICKLEs and USP9X might indicate that PRICKLE was a USP9X substrate, and that USP9X deubiquitinates and stabilizes both PRICKLE1 and PRICKLE2.
On the basis of our findings that NPHP5 is deubiquitinated by USP9X, K48 ubiquitinated by MARCH7 and K63 ubiquitinated by BBS11, we asked whether simultaneous ablation of MARCH7 and/or BBS11 might override the effects of USP9X loss on NPHP5.
On the other hand, the reduction of IRS-1 protein level was relatively small, suggesting that deubiquitination of IRS-1 by USP9X is not so active as for IRS-2.
Importantly, USP9x depletion augmented and maintained the FN induced integrin alpha5beta1 complex ubiquitination and led to a slower migration rate, suggesting that USP9x contributes to deubiquitinati[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
Because USP9X is a DUB and the SAC signal relies on inhibition of the APC/C ubiquitin ligase, USP9X might antagonize APC/C dependent ubiquitination to help maintain the SAC.
?Collectively, our experiments identified USP9X as an integral component of the centrosome where it functions to stabilize PCM1 and CEP55 and promote centrosome biogenesis.
USP9x in turn stimulates JAG1 activity through two mechanisms: (1) through TRB3 deubiquitination and stabilization, and (2) through deubiquitination and activation of Mind Bomb 1, an E3 ligase required for JAG1 ubiquitination-mediated endocytosis and Notch activation.
In Oral Squamous Cell Carcinoma (OSCC) cells, the high expression of USP9X increases the deubiquitination of PD-L1 and reduces its degradation, resulting in protein accumulation in these cells.
[XREF_BIBR, XREF_BIBR, XREF_BIBR - XREF_BIBR] We postulated the physical interaction between the PRICKLEs and USP9X might indicate that PRICKLE was a USP9X substrate, and that USP9X deubiquitinates and stabilizes both PRICKLE1 and PRICKLE2.
Usp9X becomes competent to deubiquitinate ZAP70 through TCR-dependent phosphorylation and enhancement of its catalytic activity and association with the LAT signalosome.
In addition, USP9X can also function as a tumor suppressor. USP9X strongly interacts with LATS, a core kinase in the Hippo pathway. Increased USP9X expression significantly up-regulates and stabilizes LATS and leads to a decrease in the transport of YAP/TAZ into the nucleus as well as inhibiting of their target genes.
Recent reports have suggested also that USP9X enhances Mcl-1 stability by preventing its proteasomal destruction through de-ubiquitination [XREF_BIBR].
We therefore investigated the effect of Usp9X knockdown in Mcl1 -/- MEFs to rule out the possibility that USP9X mediates its mitosis specific effects by stabilizing MCL1.
For example, by removing K48 linked polyubiquitin chains from MCL1, USP9X inhibits the proteasomal degradation of MCL1, a pro survival BCL2 family member that is overexpressed in multiple cancer types and contributes to chemoresistance and tumor recurrence [XREF_BIBR].
Together, our results indicate that radiation induced activation of USP9x inhibits Mcl-1 degradation and apoptosis resulting in increased radioresistance.
USP9x expression correlated moderately but significantly with Mcl-1 expression in glioblastoma, supporting our hypothesis that USP9x stabilizes Mcl-1 in glioblastoma cells.
Importantly, examination of USP9X targets MCL1 and beta-catenin by western blot analysis did not reveal a change in their protein levels when USP9X was knocked down in DAOY cells.
Furthermore, the chemical inhibition of USP9X increased the sensitivity of the human lung carcinoma lines A549 and H1299 to an anti-apoptotic inhibitor (ABT-737, targets BCL-xl, but not MCL1).
In Jurkat T lymphoma and K562 chronic myelogenous cells, USP9X is enzymatically activated in response to ionising radiation and causes MCL1 stabilisation, in turn inhibiting apoptosis and resulting in radioresistance [XREF_BIBR].
We have shown previously that USP9X inhibition by WP1130 reduces MCL-1 levels, promotes apoptosis and increases tumor cell sensitivity to chemotherapy [XREF_BIBR].
While Usp9X is known to modulate Mcl-1 expression, our observed effects of Usp9X manipulation on Bag3 and Bcl-2 have not been previously described and may suggest that Usp9X also interacts with Bag3 as well as Bcl-2.
XREF_BIBR More recently, in non small cell lung carcinoma, inhibition of USP9X by either siRNA knockdown or via a small molecule inhibitor WP1130 decreased MCL1 expression and sensitized cells to radiation therapy.
Furthermore, knockdown of Usp9X or Bag3 depleted endogenous Mcl-1 protein levels and in turn enhanced apoptosis induced through Bcl-2 and Bcl-xL inhibition [XREF_BIBR].
Similar distinction was also observed for Mcl-1, one observed substrate of Usp9x, where Usp9x KD increased Mcl-1 levels in 8041 cells but decreased Mcl-1 in MIAPACA2 cells.
These isothiocyanates increased Mcl-1 ubiquitination and either isothiocyanate treatment, or RNAi mediated silencing of USP9x decreased Mcl-1 levels, consistent with the notion that USP9x is a primary target of isothiocyanate activity.
These ITCs increased Mcl-1 ubiquitination and either ITC treatment or RNAi mediated silencing of USP9x decreased Mcl-1 levels, consistent with the notion that USP9x is a primary target of ITC activity.
Mechanistically, Usp9X knockdown caused concomitant suppression of Bag3, Mcl-1 and Bcl-2 expression, which remarkably recapitulates the effects of CP-d and n-ATF 5-S1 on these molecules.
Additionally, Schwickart et al. demonstrated that in hematologic malignancies 51, the expression of deubiquitinase USP9X expression correlates with that of Mcl-1, and USP9X binds to Mcl-1, stabilizes it and thereby promotes Mcl-1 overexpression and cell survival.
Although USP9X depletion did not cause an obvious change in Mcl-1 degradation during mitosis, it did increase the loss of other mitotic APC/C substrates, in particular cyclin A, but also cyclin B and NEK2A (XREF_FIG A).
Therefore, the indirectly supported, USP9X mediated degradation of MCL-1 can be regarded as a novel autophagy independent, oncosuppressive function of BECLIN 1 [XREF_BIBR].
Indeed, knock-down of USP9X reduced the half-life of Mcl-1 and increased its conjugation to Lys48 linked poly-ubiquitin chains [XREF_BIBR] that generally target proteins for proteasomal degradation.
Indeed, knockdown of Usp9X increased mitotic cell death in Mcl1 -/- cells to a similar extent as in WT MEFs, thus indicating independence of MCL1 (Fig XREF_FIG A and B).
The USP9X inhibitor WP1130 or depletion of USP9X by its specific shRNAs induces MCL-1 degradation in cells and increases tumor cell sensitivity to chemo and radiotherapies 39.
Moreover, knock-down of Usp9X or Bag3 depleted endogenous Mcl-1 protein levels and in turn enhanced apoptosis induced through Bcl-2 and Bcl-xL inhibition.
Usp9x siRNA transfection downregulated Mcl-1 levels and increased the activated levels of apoptosis related proteins (caspase-9, caspase-3 and PARP) (XREF_FIG).
So, knockdown of USP9X not only inhibited the proliferation of glioma cells in vitro, but also suppressed the tumorigenicity of primary glioma cells in vivo.
In breast cancer, USP9X deubiquitinates and stabilizes YAP to promote breast cancer cell proliferation and chemoresistance to therapeutic drugs XREF_BIBR.
FAF enhanced the proliferation of human gingival fibroblasts, human skin fibroblasts, and human umbilical vein endothelial cells in subconfluent and confluent cells, suggesting that FAF might be functioning as a competence factor.
In contrast, in immortalized but non tumorigenic HaCaT keratinocytes, USP9X depletion led to increase in cell proliferation, maintaining direct regulation of Notch activity.
These data demonstrate that loss of USP9X decreases the proliferation of ReNcell VM cells but does not alter their morphology, cell death or differentiation.
When FRT tagged USP9X was introduced into USP9X -/- cells, the cell proliferation rate nearly recovered to that of WT cells, whereas overexpression of USP9X in WT cells slightly promoted cell proliferation as well.
Depletion of USP9X led to decreased cell proliferation (XREF_FIG), while depletion of both USP9X and YAP1 did not cause a further decrease in cell proliferation compared with cells depleted of YAP1 alone.
Recent studies have demonstrated that FAM134B inhibits colon cancer cells proliferation, migration, wound healing, colony formation and tumour formation in vivo [21,39] .
As inhibition of USP9X resulted in caspase 3 and caspase 8 activation and increased the rates of apoptosis, USP9X might promote cell survival by inhibiting apoptosis in glioma cells.
In contrast, USP9x knockdown slightly increased apoptosis in IR resistant A172 cells and significantly in Ln229 cells and reduced clonogenic survival after irradiation only on these two cell lines.
Inhibition of USP9X induces apoptosis in FLT3-ITD-positive AML cells cooperatively by inhibiting the mutant kinase through aggresomal translocation and inducing oxidative stress.
Although both Usp9X and Mcl-1 knockdown elicited some features of apoptosis, broad spectrum caspase inhibition was ineffective in preventing knockdown induced MPNST cell death suggesting that caspase independent death pathways were also activated.
Although USP9x knockdown reduced Mcl-1 levels and increased apoptosis in A172 cells, USP9x regulated radiosensitivity independently of Mcl-1 in Ln229 cells.
Considering our observations with respect to Bcl-2 family members and IAPs, Usp9X appears to block death-receptor-mediated apoptosis most likely at the level of initiator- as well as effector caspases, involving a combination of factors.
Whole-genome transcriptome analysis revealed that FAM induced up-regulation of apoptosis related genes and genes that encode for mediators of oxidative stress resistance and heat shock proteins.
These results show that expression of USP9X is upregulated in hepatoma cells SMMC7721 and HepG2, and that downregulating USP9X by siRNA may induce cell apoptosis, inhibit cell growth and cell migration in the HCC SMMC7721 and HepG2 cell lines.
Expression levels of ARF-BP1 and Mule and Usp9x appears to be critical for the maintenance of proper cellular balance of anti- and pro apoptotic proteins, and contributes to cell sensitivity to apoptosis and is linked to tumor formation [XREF_BIBR, XREF_BIBR].
By evaluating signaling in U251 cells treated with a Wnt ligand, we found that USP9X knockdown partly blocked the activation of Wnt and beta- catenin pathway by regulating the stability of beta-catenin.
In summary, these results indicate that USP9X stabilizes beta-catenin and activates Wnt and beta-catenin signal pathway to promote glioma cell proliferation and survival.
The results of the western blot suggested that knockdown of USP9X decreased beta-catenin protein, but the results of RT-PCR suggested that beta-catenin mRNA expression levels did not change (XREF_SUPPLEMENTARY).
Previous studies have suggested that USP9X is co-immunoprecipitated with beta-catenin and inhibits the degradation of beta-catenin through the deubiquitination of beta-catenin in mouse lymphocyte cells [XREF_BIBR].
Down-regulation of USP9X also consistently inhibits the tumorigenicity of primary glioma cells in vivo.In summary, these results indicate that USP9X stabilizes beta-catenin and activates Wnt and beta-catenin signal pathway to promote glioma cell proliferation and survival.
USP9X has also been reported to increase the levels of beta-catenin, a transcription factor linked to the growth of brain tumors XREF_BIBR, XREF_BIBR, and overexpression of USP9X has been reported to enhance the half-life and expression of beta-catenin in mouse L cells and MCF7 cells, respectively XREF_BIBR, XREF_BIBR.
However, in contrast to previous studies, which indicated Usp9x positively regulates beta-catenin protein levels, significantly increased pbeta-catenin33/37/41 and Tyr654 pbeta-catenin levels observed in Usp9x -/Y neocortices implied a novel regulatory mechanism in NPs.
Overexpression of Dvl, ATDC, and deubiquitinating enzyme Fam, which also affects the degradation of beta-catenin, has been reported to elevate beta-catenin level.
In MDCK cells, overexpression of the USP9X catalytic domain increased the steady-state levels of beta-catenin, presumably by its ability to deubiquitylate and hence rescue from proteasomal degradation.
However, in established human pancreatic tumor cells, Usp9x supports tumor cell survival and the malignant phenotype, illustrating wide distinctions in function in murine tumor cell models and bona fide human pancreatic cancer while also highlighting the potential for Usp9x inhibitors to be used in the treatment of human PDAC.
The hypothesis that UBP43 is an anti-neoplastic target is consistent with recent work reported with the deubiquitinase USP9X, which promoted cancer cell survival by regulating stability of a pro survival BCL2 family member.
On the contrary, USP9X acts as an oncogene and stabilizes MCL1, a critical antiapoptotic member of the BCL-2 family, and thereby promotes cell survival of multiple myeloma XREF_BIBR.
Additionally, Schwickart et al. demonstrated that in hematologic malignancies 51, the expression of deubiquitinase USP9X expression correlates with that of Mcl-1, and USP9X binds to Mcl-1, stabilizes it and thereby promotes Mcl-1 overexpression and cell survival.
As inhibition of USP9X resulted in caspase 3 and caspase 8 activation and increased the rates of apoptosis, USP9X might promote cell survival by inhibiting apoptosis in glioma cells.
A recent study reported that USP9X activates SMAD4 by deubiquitination at K519, resulting in the activation of SMAD2/3, and promotion of TGFbeta pathway, which will induce cell apoptosis.
USP9X was previously reported to regulate Smad4 transcriptional activity positively, thus we hypothesized that decreased USP9X expression would attenuate Smad4 function and TGF-beta responsiveness in HCC cell lines [XREF_BIBR].
Since USP9X was previously reported to positively regulate SMAD4 transcriptional activity 17 and SMAD4 is commonly mutated in PDA 4, we hypothesized that Usp9x loss would attenuate Smad4 function or TGFbeta responsiveness in PDA cell lines.
Smad4 is monoubiquitinated in lysine 519 in vivo, a modification that inhibits smad4 by impeding association with phospho-smad2. Fam reverts this negative modification, re-empowering smad4 function;control of smad4 is a good way to regulate bone formation. Fam and ectodermin/tif1gamma (ecto) were reported to respectively regulate the de-ubiquitination and ubiquitination of smad4.
Sall4 shows an interaction with Usp9x (XREF_FIG B), an essential component of the TGF-beta and BMP signaling pathway, which activates Smad4 by removing a monoubiquitin group.
Loss of the USP9x homologue Fat facets in Drosophila inhibits the activity of the Smad4 homologue Medea through ubiquitination of Medea on K738 (equivalent to K519 in human Smad4) (Stinchfield et al.,[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
Smad4 is monoubiquitinated in lysine 519 in vivo, a modification that inhibits smad4 by impeding association with phospho-smad2. Fam reverts this negative modification, re-empowering smad4 function;control of smad4 is a good way to regulate bone formation. Fam and ectodermin/tif1gamma (ecto) were reported to respectively regulate the de-ubiquitination and ubiquitination of smad4.
In order to demonstrate USP9X negatively regulates pVHL in clear cell renal cell carcinoma (ccRCC) which is a VHL disease associated neoplasm, USP9X was knocked down in 786-0 cells stably expressing HA tagged pVHL.
As knockdown of USP9X in HEK293T cells significantly increased pVHL levels, both mRNA levels and protein half-life of pVHL were evaluated in HEK293T cells to further investigate the mechanism by which USP9X regulates pVHL.
This study shows that inhibition of USP9X function by either shRNA or a chemical inhibitor significantly enhances pVHL levels and suppresses tumor cell proliferation.
To investigate whether and how other substrates of USP9X might affect the cellular function of USP9X promoted CEP131 stabilization, we analysed by WB analysis the expression of IPO5, PRMT5 and PPM1B, which were also identified as interactors of USP9X (XREF_SUPPLEMENTARY), and the results indicate that the protein abundance of these proteins was essentially unchanged upon USP9X knockdown (XREF_SUPPLEMENTARY).
Since centrosome dysregulation associated mitotic defects could result in genome instability and cell apoptosis XREF_BIBR XREF_BIBR XREF_BIBR, we examined whether USP9X promoted CEP131 stabilization plays a role in genome stability and cell death.
In vitro deubiquitination assays revealed that, while USP9X was able to remove ubiquitins of CEP131 and K504R, but not that of CEP131 and K254R (XREF_FIG), favouring the argument that USP9X targets K254 of CEP131 for deubiquitination, although polyubiquitin chains conjugated onto CEP131 and K254R and CEP131 and K504R were both dramatically reduced (XREF_FIG).
Combining the findings that mildly disrupted centrosomal localization of PCM1 has minimal effect on CEP131 recruitment and the observations that USP9X directly interacts with CEP131 and opposes its polyubiquitin linkages in vitro, we get the conclusion that the effect of USP9X on CEP131 stabilization is attributed to the interplay between these two molecules but not through USP9X targeting other substrates like PCM1, and USP9X regulated PCM1 stabilization on centrosome activity, if it does so, seems to be independent of USP9X promoted CEP131 stabilization.
On the other hand, Li and colleagues demonstrated that USP9X, a well studied protein with oncogenic potential (Schwickart et al., 2010), promotes CEP131 stabilization through deubiquitination (Li et a[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
To gain molecular insights into the functional connection between USP9X and CEP131, we examined whether USP9X promoted CEP131 stabilization is dependent on the enzymatic activity of USP9X.
USP9X might also act as a regulator of the TGF-beta pathway, another signaling circuitry of great relevance to cancer, as witnessed by the fact that loss of USP9X abolishes multiple TGF-beta gene responses XREF_BIBR.
FAM and USP9x activity is required for Smad4 mediated TGFbeta signaling, because it re-enables Smad4 to form complexes with R-Smads and signal in the nucleus.
FAM knockdown also blocks TGFbeta mediated induction of a synthetic Smad promoter fused to luciferase (CAGA12-lux, Figure 1 E), in line with the notion that FAM is a critical factor for Smad signaling[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
Since USP9X was previously reported to positively regulate SMAD4 transcriptional activity 17 and SMAD4 is commonly mutated in PDA 4, we hypothesized that Usp9x loss would attenuate Smad4 function or TGFbeta responsiveness in PDA cell lines.
Where indicated, control cells were supplemented with 5 muM SB431542 (Tocris) in the medium to quench autocrine TGFbeta signaling.For FAM knockdown, the sequences of the siRNA were as follows : # 1 : [MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
The observations that knockdown of USP9x decreases the cellular level of AGS3 (XREF_FIG) and overexpression of USP9x increases the staining intensity of AGS3 (XREF_FIG) imply that USP9x can regulate the level of AGS3.
These data show that ectopic expression of USP9x at a high level increases the staining signal of AGS3, and that this effect of USP9x requires its deubiquitinating activity.
To test whether the ability of USP9x to enhance AGS3 staining depends on its deubiquitinating activity, we repeated the experiment using the UCH domain of USP9x alone, which is the catalytic domain responsible for the deubiquitinase activity of USP9x XREF_BIBR, XREF_BIBR.
Relative to the full-length USP9x, which increased the AGS3 staining primarily in cells expressing a high level of transfected USP9x-HA (~ 30% of these cells), the UCH domain led to enhanced AGS3 staining in almost all transfected cells displaying a moderate-to-high level of overexpression (XREF_FIG).
Taken together, our study suggests that USP9x can modulate the level of a subpopulation of AGS3, and this modulation plays a role in regulating the structure of the late Golgi compartments.
The observations that knockdown of USP9x decreases the cellular level of AGS3 (XREF_FIG) and overexpression of USP9x increases the staining intensity of AGS3 (XREF_FIG) imply that USP9x can regulate the level of AGS3.
Collectively, our study supports a model in which USP9x can modulate the level of AGS3 (or a pool of it), and that this modulation plays a role in regulating the structure and/or function of the late Golgi compartments.
Under this scenario, it would be worthwhile to investigate whether USP9x modulates the level of AGS3 associated with the Golgi membrane considering our findings that knockdown of AGS3 or USP9x both affect the late Golgi compartments (XREF_FIG).
In contrast, overexpression of either USP9x or its deubiquitinating domain UCH increases the amount of AGS3, whereas expression of the mutant UCH domain that lacks deubiquitinating activity does not have the same effect.
It is thus possible that USP9X promotes or prevents H 2 O 2 -triggered cell death by acting on different targets at varying levels of oxidative damage.
Finally, the Usp9X pharmacological inhibitor WP1130 significantly reduced human MPNST growth and induced tumor cell death in an in vivo xenograft model.
Since centrosome dysregulation associated mitotic defects could result in genome instability and cell apoptosis XREF_BIBR XREF_BIBR XREF_BIBR, we examined whether USP9X promoted CEP131 stabilization plays a role in genome stability and cell death.
Interestingly, different from Peterson 's finding that USP9X is an oncogene in B-cell malignancies [XREF_BIBR], we found that knockdown of USP9X did not induce cell death of T-ALL cells, indicating that the role of USP9X is cell type dependent.
Moreover, USP9x silencing resulted in significantly increased cell death in BaF3 and p210 cells (XREF_SUPPLEMENTARY), suggesting that the ITC induced inhibition of USP9x can, at least partially, account for the reduced viability and increased cell death observed upon ITC treatment.
Indeed, knockdown of Usp9X increased mitotic cell death in Mcl1 -/- cells to a similar extent as in WT MEFs, thus indicating independence of MCL1 (Fig XREF_FIG A and B).
In brief, USP9X promoted the migration and invasion of PANC-1 cells probably by provoking epithelial-mesenchymal transition, and also inhibited apoptosis.
G9 mediated inhibition of Usp9x in pancreatic cancer cells not only reduced the in vitro 3D growth and invasion but also inhibited tumor growth of human pancreatic cancer MIAPACA2 cells in vivo.
Importantly, reintroduction of RNF115 in USP9X depleted cells partially rescued the reduced proliferation, migration, and invasion of breast cancer cells by USP9X knockdown.
The invasided cell numbers were reduced after the overexpression of FAM3D-AS1, suggesting that FAM3D-AS1 can inhibit the cell invasion ( xref E), Thus, these results revealed that FAM3D-AS1 possessed the function of inhibition of cell proliferation and invasion.