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Neuronal injury induces JNK phosphorylation of DLK, which reduces DLK ubiquitination and creates a positive feedback loop to enhance JNK signaling and increase apoptosis., Neurons are highly polarized cells that often project axons a considerable distance. To respond to axonal damage, neurons must transmit a retrograde signal to the nucleus to enable a transcriptional stress response. Here we describe a mechanism by which this signal is propagated through injury-induced stabilization of dual leucine zipper-bearing kinase (DLK/MAP3K12). After neuronal insult, specific sites throughout the length of DLK underwent phosphorylation by c-Jun N-terminal kinases (JNKs), which have been shown to be downstream targets of DLK pathway activity. These phosphorylation events resulted in increased DLK abundance via reduction of DLK ubiquitination, which was mediated by the E3 ubiquitin ligase PHR1 and the de-ubiquitinating enzyme USP9X. Abundance of DLK in turn controlled the levels of downstream JNK signaling and apoptosis. Through this feedback mechanism, the ubiquitin–proteasome system is able to provide an additional layer of regulation of retrograde stress signaling to generate a global cellular response to localized external insults.

The core LATS kinases of the Hippo tumor suppressor pathway phosphorylate and inhibit the downstream transcriptional co-activators YAP and TAZ, which are implicated in various cancers. Recent studies have identified various E3 ubiquitin ligases that negatively regulate the Hippo pathway via ubiquitination, yet few deubiquitinating enzymes (DUB) have been implicated. In this study, we report the DUB USP9X is an important regulator of the core kinases of this pathway. USP9X interacted strongly with LATS kinase and to a lesser extent with WW45, KIBRA, and Angiomotin, and LATS co-migrated exclusively with USP9X during gel filtration chromatography analysis. Knockdown of USP9X significantly downregulated and destabilized LATS and resulted in enhanced nuclear translocation of YAP and TAZ, accompanied with activation of their target genes. In the absence of USP9X, cells exhibited an epithelial-to-mesenchymal transition phenotype, acquired anchorage-independent growth in soft agar, and led to enlarged, disorganized, three-dimensional acini. YAP/TAZ target gene activation in response to USP9X knockdown was suppressed by knockdown of YAP, TAZ, and TEAD2. Deletion of USP9X in mouse embryonic fibroblasts resulted in significant downregulation of LATS. Furthermore, USP9X protein expression correlated positively with LATS but negatively with YAP/TAZ in pancreatic cancer tissues as well as pancreatic and breast cancer cell lines. Overall, these results strongly indicate that USP9X potentiates LATS kinase to suppress tumor growth. Cancer Res; 77(18); 4921–33. ©2017 AACR.

Das Glioblastom ist bis heute eine Erkrankung mit infauster Prognose. Trotz aller Fortschritte der Strahlentherapie, der Chirurgie und der Chemotheraphie bleibt das Glioblastom eine unheilbare Erkrankung. Die Radiotheraphie zählt zusammen mit der Chirurgie zu den wichtigsten therapeutischen Verfahren in der Behandlung des Glioblastoms. Die Erforschung neuer Methoden zur Sensibilisierung des Tumors gegenüber Bestrahlung hat eine hohe Relevanz. Ein wichtiger Mechanismus, der zu Strahlenresistenz von Tumoren und T-Zell-Leukämien beiträgt, ist die Verhinderung der Apoptose durch Stabilisierung bzw. Überexpression anti-apoptotisch wirksamer Proteine wie Bcl-2, Bcl-xL und Mcl-1. Mcl-1 ist im Vergleich zu den anderen antiapoptotichen Proteinen der Bcl-2 Proteinfamilie ein kurzlebiges Protein mit einer kurzen Halbwertszeit, das in vielen Glioblastomen überexprimiert wird. Mcl-1 wird post-translational unter anderem durch E3-Ligasen und Deubiquitinasen reguliert. Die Deubiquitinase USP9x kann durch Abspaltung von Polyubiquitinketten Mcl-1 stabilisieren und dadurch Apoptose verhindern. In dieser Arbeit sollte überprüft werden, welche Rolle USP9x für die Mcl-1-Stabilität in Glioblastomzellen und die Entwicklung von Radioresistenzen spielt. Für unsere Versuche verwendeten wir die zwei Glioblastomzelllinien A172 und U373. In beiden wurde die Expression antiapoptotischer Proteine und USP9x, sowie die Apoptoseinduktion nach Bestrahlung untersucht. Nach Herunterregulierung von USP9x prüften wir dessen Rolle für die Mcl-1-Stabilität, die Induktion der Apoptose sowie das klonogene Überleben nach Bestrahlung. Anhand der Caspasenaktivierung, der DNA-Fragmentierung und des Zusammenbruchs des mitochondrialen Membranpotentials zeigten wir, dass die A172-Zellen nach Bestrahlung weniger Apoptose induzieren als die U373-Zellen. Beide Zelllinien induzieren jedoch weniger Apoptose nach Bestrahlung als Jurkat-Zellen. Beide Zelllinien wiesen hohe Mcl-1 und USP9x-Mengen auf. Nach Bestrahlung blieben die Mcl-1-Level in den A172-Zellen konstant, in den U373-Zellen nahmen sie kontinuierlich ab. In den A172-Zellen hatte die USP9x-Menge Einfluss auf die Mcl-1-Abundanz. In den U373-Zellen hatten erniedrigte USP9x-Level keinerlei Auswirkung auf die Mcl-1-Menge. Dies deutet auf eine USP9x-vermittelte Mcl-1-Stabilisierung in A172-Zellen hin. In beiden Zelllinien hatte sowohl eine Herunterregulierung von Mcl-1 als auch eine Herunterregulierung von USP9x eine Induktion der Apoptose zur Folge, welche bei den A172 stärker ausfiel als bei den U373-Zellen. Wahrscheinlich wird in den A172-Zellen die Induktion der Apoptose stark über Mcl-1 reguliert. In den U373-Zellen konnten wir eine hohe Abundanz des antiapoptotischen Bcl-2 nachweisen, welches eventuell die Relevanz von Mcl-1 bei der Apoptoseresistenz in diesen Zellen in den Hintergrund stellt. Unabhängig von der Art des Zelltodes führte eine Herunterregulierung von USP9x zu einer starken Abnahme des klonogenen Überlebens von A172-Zellen nach Bestrahlung. Aus unseren Ergebnissen schlossen wir, dass USP9x einen Radioresistenzfaktor in Glioblastomzelllinien darstellen kann. Auch in Glioblastomzellen scheint ein Zusammenhang zwischen der USP9x-Menge und der Mcl-1-Abundanz zu bestehen. Wahrscheinlich führen dabei hohe USP9x-Mengen in A172-Zellen zu einer Stabilisierung von Mcl-1. Eine Herunterregulierung von USP9x hatte in beiden Zelllinien keinen vermehrten Zelltod durch Apoptose zur Folge. Jedoch führte unabhängig von der Art des Zelltodes eine Herunterregulierung von USP9x zu einer Senkung des klonogenen Überlebens von A172-Zellen. Es existieren also weitere, Mcl-1-unabhängige Wege, über welche die Deubiquitinase USP9x eine Radioresistenz in Glioblastomen erzeugt. Eine USP9x-vermittelte Apoptoseresistenz durch Mcl-1-Stabilisierung, wie sie von Trivingo et al. für Jurkat-T-Zell-Leukämiezellen beschrieben wurde, konnte in bestrahlten Glioblastomzellen jedoch nicht gezeigt werden.

SummaryPosttranslational modification of proteins by the small molecule ubiquitin is a key regulatory event, and the enzymes catalyzing these modifications have been the focus of many studies. Deubiquitinating enzymes, which mediate the removal and processing of ubiquitin, may be functionally as important but are less well understood. Here, we present an inventory of the deubiquitinating enzymes encoded in the human genome. In addition, we review the literature concerning these enzymes, with particular emphasis on their function, specificity, and the regulation of their activity.

// Bo Yang 1, 3 ,* , Shiming Zhang 1, * , Zhihao Wang 1 , Chunxu Yang 1 , Wen Ouyang 1 , Fuxiang Zhou 1 , Yunfeng Zhou 1, 2 , Conghua Xie 1, 2 1 Department of Radiation and Medical Oncology, Zhongnan Hospital, Wuhan University, Wuchang District, Wuhan, 430071, China 2 Hubei Key Laboratory of Tumor Biological Behaviors, Zhongnan Hospital, Wuhan University, Wuchang District, Wuhan, 430071, China 3 Department of Oncology, Wuhan General Hospital of Guangzhou Command PLA, Wuchang District, Wuhan, 430070, China * These authors have contributed equally to this work Correspondence to: Conghua Xie, email: chxie_65@whu.edu.cn Keywords: high grade gliomas, USP9X, β-catenin, deubiquitination, prognosis Received: May 12, 2016 Accepted: October 14, 2016 Published: October 22, 2016 ABSTRACT β-catenin is a crucial signal transduction molecule in the Wnt/β-catenin signal pathway, and increased β-catenin expression has consistently been found in high grade gliomas. However, the mechanisms responsible for β-catenin overexpression have remained elusive. Here we show that the deubiquitinase USP9X stabilizes β-catenin and thereby promotes high grade glioma cell growth. USP9X binds β-catenin and removes the Lys 48-linked polyubiquitin chains that normally mark β-catenin for proteasomal degradation. Increased USP9X expression correlates with increased β-catenin protein in high grade glioma tissues. Moreover, patients with high grade glioma overexpressing USP9X have a poor prognosis. Knockdown of USP9X suppresses cell proliferation, inhibits G1/S phase conversion, and induces apoptosis in U251 and A172 cells. Interestingly, c-Myc and cyclinD1, which are important downstream target genes in the Wnt/β-catenin signal pathway, also show decreased expression in cells with siRNA-mediated down-regulation of USP9X. Down-regulation of USP9X also consistently inhibits the tumorigenicity of primary glioma cells in vivo. In summary, these results indicate that USP9X stabilizes β-catenin and activates Wnt/β-catenin signal pathway to promote glioma cell proliferation and survival. USP9X could also potentially be a novel therapeutic target for high grade gliomas.

Peroxin 5 (PEX5), the peroxisomal protein shuttling receptor, binds newly synthesized peroxisomal matrix proteins in the cytosol and promotes their translocation across the organelle membrane. During the translocation step, PEX5 itself becomes inserted into the peroxisomal docking/translocation machinery. PEX5 is then monoubiquitinated at a conserved cysteine residue and extracted back into the cytosol in an ATP-dependent manner. We have previously shown that the ubiquitin-PEX5 thioester conjugate (Ub-PEX5) released into the cytosol can be efficiently disrupted by physiological concentrations of glutathione, raising the possibility that a fraction of Ub-PEX5 is nonenzymatically deubiquitinated in vivo. However, data suggesting that Ub-PEX5 is also a target of a deubiquitinase were also obtained in that work. Here, we used an unbiased biochemical approach to identify this enzyme. Our results suggest that ubiquitin-specific protease 9X (USP9X) is by far the most active deubiquitinase acting on Ub-PEX5, both in female rat liver and HeLa cells. We also show that USP9X is an elongated monomeric protein with the capacity to hydrolyze thioester, isopeptide, and peptide bonds. The strategy described here will be useful in identifying deubiquitinases acting on other ubiquitin conjugates.

α-Synuclein is central to the pathogenesis of Parkinson disease (PD). Mutations as well as accumulation of α-synuclein promote the death of dopaminergic neurons and the formation of Lewy bodies. α-Synuclein is monoubiquitinated by SIAH, but the regulation and roles of monoubiquitination in α-synuclein biology are poorly understood. We now report that the deubiquitinase USP9X interacts in vivo with and deubiquitinates α-synuclein. USP9X levels are significantly lower in cytosolic fractions of PD substantia nigra and Diffuse Lewy Body disease (DLBD) cortices compared to controls. This was associated to lower deubiquitinase activity toward monoubiquitinated α-synuclein in DLBD cortical extracts. A fraction of USP9X seems to be aggregated in PD and DLBD, as USP9X immunoreactivity is detected in Lewy bodies. Knockdown of USP9X expression promotes accumulation of monoubiquitinated α-synuclein species and enhances the formation of toxic α-synuclein inclusions upon proteolytic inhibition. On the other hand, by manipulating USP9X expression levels in the absence of proteolytic impairment, we demonstrate that monoubiquitination controls the partition of α-synuclein between different protein degradation systems. Deubiquitinated α-synuclein is mostly degraded by autophagy, while monoubiquitinated α-synuclein is preferentially degraded by the proteasome. Moreover, monoubiquitination promotes the degradation of α-synuclein, whereas deubiquitination leads to its accumulation, suggesting that the degradation of deubiquitinated α-synuclein by the autophagy pathway is less efficient than the proteasomal one. Lower levels of cytosolic USP9X and deubiquitinase activity in α-synucleinopathies may contribute to the accumulation and aggregation of monoubiquitinated α-synuclein in Lewy bodies. Our data indicate that monoubiquitination is a key determinant of α-synuclein fate.

The overexpressed ErbB2/HER2 receptor is a clinically validated cancer target whose surface localization and internalization mechanisms remain poorly understood. Downregulation of the overexpressed 185-kDa ErbB2 receptor is rapidly (2–6 hours) induced by the HSP90 chaperone inhibitor geldanamycin (GA), whereas its downregulation and lysosomal degradation are more slowly (24 hours) induced by the proteasome inhibitor bortezomib/PS341. In PS341-treated SK-BR-3 cells, overexpressed ErbB2 coprecipitates with the E3 ubiquitin ligase c-Cbl and also with the deubiquitinating enzyme USP9x; moreover, siRNA downregulation of USP9x enhances PS341-induced ErbB2 downregulation. Because polyubiquitin linkages via lysine 48 (K48) or 63 (K63) can differentially address proteins for 26S proteasomal degradation or endosome trafficking to the lysosome, multiple reaction monitoring (MRM)/mass spectrometry (MS) and polyubiquitin linkage–specific antibodies were used to quantitatively track K48-linked and K63-linked ErbB2 polyubiquitination following either GA or PS341 treatment of SK-BR-3 cells. MRM/MS revealed that unlike the rapid, modest (4-fold to 8-fold), and synchronous GA induction of K48 and K63 polyubiquitinated ErbB2, PS341 produces a dramatic (20-fold to 40-fold) sequential increase in polyubiquitinated ErbB2 consistent with K48 polyubiquitination followed by K63 editing. Fluorescence microscopic imaging confirmed that PS341, but not GA, induces colocalization of K48-linked and K63-linked polyubiquitin with perinuclear lysosome-sequestered ErbB2. Thus, ErbB2 surface overexpression and recycling seem to depend on its polyubiquitination and deubiquitination; as well, the contrasting effects of PS341 and GA on ErbB2 receptor localization, polyubiquitination, and degradation point to alternate cytoplasmic trafficking likely regulated by different K48 and K63 polyubiquitin editing mechanisms. Cancer Res; 70(9); 3709–17. ©2010 AACR.

The Hippo pathway has been identified as a key barrier for tumorigenesis, acting through downregulation of YAP/TAZ activity. Elevated YAP/TAZ activity has been documented in many human cancers. Ubiquitylation has been shown to play a key role in regulating YAP/TAZ activity through downregulation of a number of Hippo pathway components. Several ubiquitin ligase complexes have been implicated in this process, however, little is known about the deubiquitylating enzymes that counteract these activities to regulate YAP/TAZ. Here we identify the deubiquitylating enzyme USP9x as a regulator of YAP/TAZ activity. We demonstrate that USPx regulates ubiquitin-mediated turnover of the YAP inhibitor, Angiomotin. USP9x acts to deubiquitylate Angiomotin at lysine 496, resulting in stabilization of Angiomotin and lower YAP/TAZ activity. USP9x mRNA levels were reduced in several cancers. Clinically, USP9x mRNA levels were reduced in several cancers with low USPx expression correlating with poor prognosis in renal clear cell carcinoma. Our data indicate that USP9x may be a useful biomarker for renal clear cell carcinoma.

Defective centrosome duplication is implicated in microcephaly and primordial dwarfism as well as various ciliopathies and cancers. Yet, how the centrosome biogenesis is regulated remains poorly understood. Here we report that the X-linked deubiquitinase USP9X is physically associated with centriolar satellite protein CEP131, thereby stabilizing CEP131 through its deubiquitinase activity. We demonstrate that USP9X is an integral component of centrosome and is required for centrosome biogenesis. Loss-of-function of USP9X impairs centrosome duplication and gain-of-function of USP9X promotes centrosome amplification and chromosome instability. Significantly, USP9X is overexpressed in breast carcinomas, and its level of expression is correlated with that of CEP131 and higher histologic grades of breast cancer. Indeed, USP9X, through regulation of CEP131 abundance, promotes breast carcinogenesis. Our experiments identify USP9X as an important regulator of centrosome biogenesis and uncover a critical role for USP9X/CEP131 in breast carcinogenesis, supporting the pursuit of USP9X/CEP131 as potential targets for breast cancer intervention., USP9X is a deubiquitinating enzyme with many known substrates and functions; it has been linked to cancer but the mechanisms remain unclear. Here Li et al. report that USP9X stabilizes the centrosomal protein CEP131 leading to centrosome amplification and breast cancer development.

Squamous cell carcinoma is a malignancy of squamous epithelia and affects multiple organs of the body. Investigating the underlying signalling pathways and the molecular mechanisms is essential to gain a profound understanding of the disease pathology, aetiology and to uncover potential drug targets. The deubiquitylating enzyme, USP9X, has been associated with epithelial cancers of breasts, colon, lung, liver, pancreas, mouth and the skin. The current study investigated the functional and molecular role of USP9X in head and neck squamous carcinoma cell lines (HNSCC) and skin keratinocytes. Also, the role of USP9X in DNA damage response of keratinocytes was investigated. USP9X protein expression was altered in four HNSCC cell lines, derived from tongue (SCC15 and CAL27) and pharynx (Fadu and Detroit 562) by treating them with siRNAs (loss of function) or by transfecting them with plasmids (gain of function). CyQuant and Flow cytometry analyses revealed that USP9X promoted proliferation and regulated cell cycle of these cell lines. RT-qPCR analysis of target gene expression revealed that USP9X also regulated Notch activity in these cells. However, at least in one cell line (FaDu), USP9X’s regulation of proliferation did not appear to operate through the Notch pathway but possibly occurs via mTOR pathway. Immunoblot analysis revealed a decrease in downstream targets of mTOR complex 1 namely, total ribosomal protein (S6) and its phosphorylated form (pS6), when USP9X was depleted from FaDu cells. In contrast, in immortalised but non- tumourigenic HaCaT human keratinocytes, USP9X depletion led to an increase in cell proliferation and again, USP9X levels directly correlated with Notch pathway activity.

The anticancer properties of cruciferous vegetables are well known and attributed to an abundance of isothiocyanates such as benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC). While many potential targets of isothiocyanates have been proposed, a full understanding of the mechanisms underlying their anticancer activity has remained elusive. Here we report that BITC and PEITC effectively inhibit deubiquitinating enzymes (DUB), including the enzymes USP9x and UCH37, which are associated with tumorigenesis, at physiologically relevant concentrations and time scales. USP9x protects the antiapoptotic protein Mcl-1 from degradation, and cells dependent on Mcl-1 were especially sensitive to BITC and PEITC. 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 isothiocyanates also increased ubiquitination of the oncogenic fusion protein Bcr-Abl, resulting in degradation under low isothiocyanate concentrations and aggregation under high isothiocyanate concentrations. USP9x inhibition paralleled the decrease in Bcr-Abl levels induced by isothiocyanate treatment, and USP9x silencing was sufficient to decrease Bcr-Abl levels, further suggesting that Bcr-Abl is a USP9x substrate. Overall, our findings suggest that USP9x targeting is critical to the mechanism underpinning the well-established anticancer activity of isothiocyanate. We propose that the isothiocyanate-induced inhibition of DUBs may also explain how isothiocyanates affect inflammatory and DNA repair processes, thus offering a unifying theme in understanding the function and useful application of isothiocyanates to treat cancer as well as a variety of other pathologic conditions. Cancer Res; 75(23); 5130–42. ©2015 AACR.

In this issue of Blood, Peterson et al demonstrate that inhibition of both Usp9x and Usp24 results in efficient degradation of Mcl-1, induction of apoptosis, and inhibition of tumor growth in B-cell malignancies.

Repair of DNA alkylation damage is critical for genomic stability and involves multiple conserved enzymatic pathways. Alkylation damage resistance, which is critical in cancer chemotherapy, depends on the overexpression of alkylation repair proteins. However, the mechanisms responsible for this upregulation are unknown. Here, we show that an OTU domain deubiquitinase, OTUD4, is a positive regulator of ALKBH2 and ALKBH3, two DNA demethylases critical for alkylation repair. Remarkably, we find that OTUD4 catalytic activity is completely dispensable for this function. Rather, OTUD4 is a scaffold for USP7 and USP9X, two deubiquitinases that act directly on the AlkB proteins. Moreover, we show that loss of OTUD4, USP7, or USP9X in tumor cells makes them significantly more sensitive to alkylating agents. Taken together, this work reveals a novel, noncanonical mechanism by which an OTU family deubiquitinase regulates its substrates, and provides multiple new targets for alkylation chemotherapy sensitization of tumors.

The discovery that heterozygous disruption of Becn1 in mice leads to spontaneous tumor development was the first direct link arguing for a predominantly tumor-suppressor function of autophagy. However, the mechanisms by which BECN1 restrains tumorigenesis and whether autophagy-independent functions of BECN1 contribute to its tumor-restraining potential remained unexplored. We recently described a novel function of BECN1—regulation of oncogene MCL1 protein levels. Our results show that BECN1 regulates MCL1 levels in an inverse-reciprocal manner, whereby changes in the levels of one of the 2 proteins inversely affects the proteasomal degradation of the other. Importantly, this mechanism is independent of autophagy and of the physical interaction between BECN1 and MCL1. In vitro and in vivo analysis using several models, including patient-derived melanoma cells and tissue samples from patients with melanocytic lesions at different stages, showed that the identified mechanism of inverse coregulation between BECN1 and MCL1 significantly contributes to their opposing roles in tumorigenesis.

Glioblastoma is a very aggressive form of brain tumor with limited therapeutic options. Usually, glioblastoma is treated with ionizing radiation (IR) and chemotherapy after surgical removal. However, radiotherapy is frequently unsuccessful, among others owing to resistance mechanisms the tumor cells have developed. Antiapoptotic B-cell leukemia (Bcl)-2 family members can contribute to radioresistance by interfering with apoptosis induction in response to IR. Bcl-2 and the closely related Bcl-xL and Mcl-1 are often overexpressed in glioblastoma cells. In contrast to Bcl-2 and Bcl-xL, Mcl-1 is a short-lived protein whose stability is closely regulated by ubiquitylation-dependent proteasomal degradation. Although ubiquitin ligases facilitate degradation, the deubiquitylating enzyme ubiquitin-specific protease 9x (USP9x) interferes with degradation by removing polyubiquitin chains from Mcl-1, thereby stabilizing this protein. Thus, an inability to downregulate Mcl-1 by enhanced USP9x activity might contribute to radioresistance. Here we analyzed the impact of USP9x on Mcl-1 levels and radiosensitivity in glioblastoma cells. Correlating Mcl-1 and USP9x expressions were significantly higher in human glioblastoma than in astrocytoma. Downregulation of Mcl-1 correlated with apoptosis induction in established glioblastoma cell lines. Although Mcl-1 knockdown by siRNA increased apoptosis induction after irradiation in all glioblastoma cell lines, USP9x knockdown significantly improved radiation-induced apoptosis in one of four cell lines and slightly increased apoptosis in another cell line. In the latter two cell lines, USP9x knockdown also increased radiation-induced clonogenic death. The massive downregulation of Mcl-1 and apoptosis induction in A172 cells transfected with USP9x siRNA shows that the deubiquitinase regulates cell survival by regulating Mcl-1 levels. In contrast, USP9x regulated radiosensitivity in Ln229 cells without affecting Mcl-1 levels. We conclude that USP9x can control survival and radiosensitivity in glioblastoma cells by Mcl-1-dependent and Mcl-1-independent mechanisms.

Although previous studies attributed a pro-survival role to USP9X in human cancer, how USP9X affects B-cell acute lymphoblastic leukemia (B-ALL) remains unclear. Here, we found that USP9X is overexpressed in B-ALL cell lines and human patients. We investigated the role of USP9X in B-ALL and found that USP9X knockdown significantly reduced leukemic cell growth and increased spontaneous apoptosis, thereby improving survival in immunodeficient mice. These effects are partially mediated by the intrinsic apoptotic pathway, as we found that USP9X-knockdown leukemic cells displayed MCL1 down-regulation, with decreased BCL-2/BCL-XL levels and increased BAX levels. In addition, we demonstrated that USP9X inhibition negatively regulates mTORC1 activity toward its substrate S6K1. Clinically, USP9X inhibition sensitized glucocorticoid-resistant ALL cells to prednisolone; this observation reveals a potential avenue for improving the treatment of drug-resistant relapses. Collectively, our findings suggest that the combination of USP9X targeting and glucocorticoids treatment has attractive utility in B-ALL. This approach represents a potential strategy for promising combination therapies for lymphoid malignancies.

BIX-01294, an euchromatic histone-lysine N-methyltransferase 2 (EHMT2) inhibitor, has been reported to induce apoptosis in human neuroblastoma cells and inhibit the proliferation of bladder cancer cells. However, the definite mechanism of the apoptosis mediated by BIX-01294 in bladder cancer cells remains unclear. In the present study, we found that BIX-01294 induced caspase-dependent apoptosis in human bladder cancer cells. Moreover, our data show BIX-01294 stimulates endoplasmic reticulum stress (ER stress) and up-regulated expression of PMAIP1 through DDIT3 up-regulation. Furthermore, down-regulation of the deubiquitinase USP9X by BIX-01294 results in downstream reduction of MCL1 expression, leading to apoptosis eventually. Thus, our findings demonstrate PMAIP1-USP9X-MCL1 axis may contribute to BIX-01294-induced apoptosis in human bladder cancer cells.

Hintergrund: Antiapoptotische Bcl-2-Proteine sind bedeutend in der Tumorentstehung und in der Entwicklung von Therapieresistenzen. Das antiapoptotische Mcl-1 spielt in vielen Tumoren eine wichtige Rolle dabei [Skvara et al., 2005]. Die Regulierung des Mcl-1-Levels durch proteasomale Degradierung ist bedeutend für dessen schnellen Umsatz. Mcl-1 ist im Vergleich zu den anderen antiapoptotischen Bcl-2-Proteinen ein kurzlebiges Protein, dessen Level nach Hemmung der Translation schnell abnimmt [Quinn et al., 2011]. Weitere Studien verdeutlichten, dass Mcl-1 durch die Deubiquitinase USP9X, die den proteasomalen Abbau des antiapoptotischen Proteins verhindert, stabilisiert wird. In diesen Studien bewirkte das Herunterregulieren von USP9X vermehrten Zelltod [Schwickart et al., 2010; Trivigno et al., 2012]. Ein möglicher Zusammenhang zwischen der Stabilisierung von Mcl-1 durch die Deubiquitinase USP9X sollte nun auch in Prostatazellen untersucht werden. Methoden: In den zwei etablierten Prostatazelllinien LNCaP und PC3 wurden die Level verschiedener Proteine der Bcl-2-Familie und der Deubiquitinase USP9X mittels Western Blot untersucht. Ebenfalls mittels Western Blot wurde die Aktivierung der Caspasen nach Bestrahlung gemessen. Die Initiierung der Apoptose nach Bestrahlung wurde anhand der durchflusszytometrischen Messung des mitochondrialen Membranpotentials mittels TMRE-Färbung und der Fragmentierung der DNA mittels Propidiumiodidfärbung nach Nicolettimethode analysiert. Eine Messung des klonogenen Überlebens erfolgte mittels Klonogenitätstest. Durch Lipofektion von siRNA wurde die Aktivität der Deubiquitinase USP9X herunterreguliert. Schließlich erfolgte per Western Blot die Messung der Mcl-1- und USP9X-Abundanzen auch in Patientengewebeproben der Prostata unterschiedlicher Malignität. Ergebnisse und Diskussion: In den LNCaP- und PC3-Zellen zeigte sich in der durchflusszytometrischen Untersuchung nur ein geringer Anteil an Zellen mit zusammengebrochenem mitochondrialen Membranpotential sowie an Zellen mit fragmentierter DNA. Bei den LNCaP-Zellen wurde keine, bei den PC3-Zellen nur eine geringe Caspasenaktivierung nach Bestrahlung beobachtet. Auch im Klonogenitätstest zeigten die Zelllinien ein radioresistentes Verhalten. Die Messung der Mengen pro- und antiapoptotischer Bcl-2-Proteine ergab Unterschiede in der Stabilität des antiapoptotischen Bcl-2-Proteins Mcl-1, welches in den LNCaP-Zellen stabil nach Bestrahlung war hingegen in den PC3-Zellen abnahm. Gleichzeitig zeigte sich in den PC3-Zellen eine Heraufregulierung des proapoptotischen BH3-only Proteins Noxa. Die Menge der Deubiquitinase USP9X war in beiden Zelllinien nach Bestrahlung stabil. Durch die Herunterregulierung von USP9X kam es in beiden Zelllinien zu einer Reduktion der Mcl-1-Menge und zu einem signifikanten Anstieg der Zellen mit zusammengebrochenem mitochondrialen Membranpotential bzw. der Zellen mit fragmentierter DNA. Ebenso erniedrigten sich die Überlebensfraktionen der Zellen im Klonogenitätstest signifikant. Dieser Effekt fiel in den LNCaP-Zellen schon ab geringeren Bestrahlungsdosen auf. Schließlich zeigte sich für die Mcl-1-Proteinmenge in Patientenproben der Prostata eine angedeutete Korrelation erhöhter Werte mit ansteigender Malignität des Gewebes. Dies konnte für USP9X nicht beobachtet werden. Zusammenfassend zeigten unsere Ergebnisse eine Radiosensibilisierung der Prostatazelllinien LNCaP und PC3 durch Herunterregulierung von USP9X. In den LNCaP-Zellen kam es durch die USP9X-Herunterregulierung zu einer Destabilisierung der Mcl-1-Menge. Die PC3-Zellen zeigten auch bei nicht veränderter USP9X-Aktivität abnehmende Mcl-1-Mengen nach Bestrahlung. Möglicherweise werden durch die Herunterregulierung der Deubiquitinase in diesen Zellen andere Substrate beeinflusst. Auch der Einfluss des BH3-only-Proteins Noxa könnte in den PC3-Zellen Einfluss auf die Mcl-1-Stabilität haben.

Protein ubiquitination, a major post-translational modification in eukaryotes, requires an adequate pool of free ubiquitin. Cells maintain this pool by two pathways, both involving deubiquitinases (DUBs): recycling of ubiquitin from ubiquitin conjugates and processing of ubiquitin precursors synthesized de novo. Although many advances have been made in recent years regarding ubiquitin recycling, our knowledge on ubiquitin precursor processing is still limited, and questions such as when are these precursors processed and which DUBs are involved remain largely unanswered. Here we provide data suggesting that two of the four mammalian ubiquitin precursors, UBA < inf > 52 < /inf > and UBA < inf > 80 < /inf > , are processed mostly post-translationally whereas the other two, UBB and UBC, probably undergo a combination of co-and post-translational processing. Using an unbiased biochemical approach we found that UCHL < inf > 3 < /inf > , USP < inf > 9 < /inf > X, USP < inf > 7 < /inf > , USP < inf > 5 < /inf > and Otulin/Gumby/FAM < inf > 105 < /inf > b are by far the most active DUBs acting on these precursors. The identification of these DUBs together with their properties suggests that each ubiquitin precursor can be processed in at least two different manners, explaining the robustness of the ubiquitin de novo synthesis pathway. We are grateful to Dr. Cheryl Arrowsmith (University of Toronto, Canada) for providing the plasmids pET28a-LIC-USP5 (Addgene plasmid 25299) and pET28a-LIC-USP5(C335A). We thank Dr. João M. Cabral (IBMC, University of Porto, Portugal) for critically reading the manuscript. This work was supported by national funds through FCT - Fundação para a Ciência e a Tecnologia/MEC – Ministério da Educação e Ciência and when applicable co-funded by Fundo de Desenvolvimento Regional (FEDER) funds within the partnership agreement PT2020 related with the research unit number 4293; by Project “NORTE-07-0124-FEDER-000003 -Cell homeotasis tissue organization and organism biology”, co-funded by Programa Operacional Regional do Norte (ON.2—O Novo Norte), under the Quadro de Referência Estratégico Nacional (QREN), through FEDER and by FCT; by Portuguese National Mass Spectrometry Network (RNEM) through the project REDE/1504/REM/2005; and by Química Orgânica, Produtos Naturais e Agroalimentares (QOPNA) research unit funds provided by FCT, European Union, QREN, FEDER and Operational Competitiveness Programme (COMPETE) under the projects PEst-C/QUI/UI0062/2013 and FCOMP-01-0124-FEDER-037296. C.P.G. and M.P.P. were supported by FCT, COMPETE and Fundo Social Europeu. A.V.M. was supported by the project FCOMP-01-0124-FEDER-027627-EXPL/BEX-BCM/0320/2012 financed by national funds from FCT/Ministério da Educação e Ciência (PIDDAC) and co-funded by FEDER through COMPETE—Programa Operacional Factores de Competitividade (POFC).

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and deadly malignancies. Recently, the deubiquitinating protease USP9X has been shown to behave as an oncogene in a number of neoplasms, including those of breast, brain, colon, esophagus and lung, as well as KRAS wild-type PDAC. However, other studies suggest that USP9X may function as a tumor-suppressor in a murine PDAC model when USP9X expression is depleted during early pancreatic development. To address the conflicting findings surrounding the role of USP9X in PDAC, we examined the effects of knocking down USP9X in five human PDAC cell lines (BxPC3, Capan1, CD18, Hs766T, and S2-013). We demonstrate that knocking down USP9X in each of the PDAC cell lines reduces their anchorage-dependent growth. Using an inducible shRNA system to knock down USP9X in both BxPC3 and Capan1 cells, we also determined that USP9X is necessary for the anchorage-independent growth. In addition, knockdown of USP9X alters the cell cycle profile of BxPC3 cells and increases their invasive capacity. Finally, we show that an inhibitor of deubiquitinating proteases, WP1130, induces significant cytotoxicity in each of the five PDAC cell lines tested. Overall, our work and the work of others indicate that the function and role of USP9X is highly context-dependent. Although USP9X may function as a tumor-suppressor during the establishment of PDAC, data presented here argue that USP9X promotes cell growth in advanced PDAC cells when PDAC is typically diagnosed. Hence, USP9X may be a promising therapeutic target for the treatment of advanced PDAC.

Mcl-1 is a unique Bcl-2 family member that plays crucial roles in apoptosis. Apoptosis-unrelated functions of Mcl-1 are however emerging, further justifying its tight regulation. Here we unravel a novel mechanism of Mcl-1 regulation mediated by the haplo-insufficient tumour suppressor Beclin 1. Beclin 1 negatively modulates Mcl-1 stability in a reciprocal manner whereby depletion of one leads to the stabilization of the other. This co-regulation is independent of autophagy and of their physical interaction. Both Beclin 1 and Mcl-1 are deubiquitinated and thus stabilized by binding to a common deubiquitinase, USP9X. Beclin 1 and Mcl-1 negatively modulate the proteasomal degradation of each other through competitive displacement of USP9X. The analysis of patient-derived melanoma cells and tissue samples shows that the levels of Beclin 1 decrease, while Mcl-1 levels subsequently increase during melanoma progression in a significant inter-dependent manner. The identified inverse co-regulation of Beclin 1 and Mcl-1 represents a mechanism of functional counteraction in cancer.

Background: In the ubiquitin-proteasome pathway, the ubiquitinated substrates either undergo degradation by the proteasome or stabilization through the action of the deubiquitinating enzyme. We have previously found that the deubiquitinating enzyme Fam is colocalized with AF-6, one of the effectors of the Ras small GTPase, at cell-cell contact sites in epithelial cells and interacts with AF-6 in vivo and in vitro. Fam has deubiquitinating activity in vitro and prevents the ubiquitination of AF-6 in intact cells. The degradation of β-catenin, which accumulates at the cell-cell contact sites as a cadherin/catenin complex, is thought to be regulated by the ubiquitin-proteasome pathway. These observations prompted us to examine the possible Fam regulation of the stabilization of β-catenin. Results: We found that Fam interacted with β-catenin both in vivo and in vitro. The Fam- binding site of β-catenin mapped to the region close to the APC or Axin- binding site of β-catenin. Overexpression of Fam in mouse L cells resulted in an elevation of β-catenin levels and in an elongation of the half-life of β-catenin. In these L cells, Fam was colocalized with β-catenin at the dot- like structures in the cytoplasm. Conclusion: These results indicate that Fam interacts with and stabilizes β-catenin in vivo, presumably through the deubiquitination of β-catenin.