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USP32 expression and deubiquitination function. a USP32 (396 bp) and USP6 (245 bp) RT-PCR with a multitissue panel of spleen, testis, prostate, ovary, small intestine, colon, leukocyte, and thymus cDNAs. b Domain structure of partial USP32 constructs (I, II, III) and in vivo deubiquitination assay. Plasmid combinations (pGEX- USP32 and Ub-b-gal) were cotransformed into in DH5a cells. 1. Ubb-gal plasmid only. 2. Empty pGEX ? Ub-b-gal. 3. pGEX-USP32-I ?Ub-b-gal. 4. pGEX-USP32-II ? Ub-b-gal. 5. pGEX-USP32- III ? Ub-b-gal. 6. pGEX-Ubp3 ? Ub-b-gal [Ubp3 was used as a
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USP32, on chromosomal band 17q23.1-17q23.2, is a highly conserved but uncharacterized gene that gave rise during evolution to a well-known hominoid-specific proto-oncogene, USP6. We investigated the expression profile of USP32 in human tissues and examined its functions to gain insight into this novel member of the well-conserved ubiquitination sys...
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... to amplify dif- ferent-sized PCR products for USP32 and USP6 transcripts in a multitissue cDNA panel to distinguish their expression patterns. Earlier, USP6 was reported to be expressed only in testis ( Paulding et al. 2003), but we also demonstrated USP6 transcript in ovary, whereas USP32 was ubiquitously expressed in the multitissue cDNA panel (Fig. 1a). USP32 (1604 aa) is predicted to be a ubiquitin protease due to the C-terminal aspargine, cysteine, histidine, and aspartic acid residues in the peptidase domains (Fig. 1b, c) conserved in DUBs. To confirm the predicted function of USP32, an in vivo deubiquitination assay (Papa and Hochstrasser 1993) was performed. Bold letters show ...
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... be expressed only in testis ( Paulding et al. 2003), but we also demonstrated USP6 transcript in ovary, whereas USP32 was ubiquitously expressed in the multitissue cDNA panel (Fig. 1a). USP32 (1604 aa) is predicted to be a ubiquitin protease due to the C-terminal aspargine, cysteine, histidine, and aspartic acid residues in the peptidase domains (Fig. 1b, c) conserved in DUBs. To confirm the predicted function of USP32, an in vivo deubiquitination assay (Papa and Hochstrasser 1993) was performed. Bold letters show the conserved amino acids. * indicates active aspargine (738th amino acid of USP32), cysteine (743rd amino acid of USP32), histidine (1526th amino acid of USP32), and aspartic ...
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... peptidase domains was cloned as three overlapping inserts into pGEX vectors as GST fusion peptides. These constructs (I, II, and III) and Ub-b-gal plasmids were cotransformed into DH5a cells for ectopic expression. Deubiquitination assay tested the protease ability of the peptides expressed from pGEX based on ubiquitin removal from the Ub-b-gal. (Fig. 1b). Construct I (lane 3), which harbored only the first peptidase region (amino acids 733-911), and construct III (lane 5), which harbored only the second and the third peptidase regions (amino acids 1225-1318 and 1510-1565), had minimal enzymatic activity as faint b-gal bands could be visualized for darker exposures of the film (darker ...
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... are cells transfected with USP32-1-GFP (1), GFP-USP32-2 (2), and GFP-USP32-3 (3) together with the structures of the constructs (1009) USP32 silencing and its effect on proliferation rate Anti-USP32 and control shRNA vectors were stably transfected into HeLa cells. Silencing of USP32, detected with qRT-PCR (Fig. 3a), resulted in a more than 30% reduction in the proliferation rate of transfected cells (both poly-and monoclonal) at 96 h post plating detected by MTT (Fig. 3b) and growth curve assays ( Supplementary Fig. 1). Moreover, USP32-silenced HeLa cells had lower migration abilities compared to control cells as indicated by the transwell migration assay (Fig. 3c). ...
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Citations
... Aberrant expression of USP32 triggers certain diseases such as Parkinson's, Fragile X Syndrome, Chronic Kidney Disease and Cancer [11,[17][18][19]. Recent research has demonstrated the high expression of USP32 in a range of cancers [20][21][22][23][24][25][26] and its role in the initiation and progression of cancer. ...
The regulatory significance of ubiquitin-specific peptidase 32 (USP32) in tumor is significant, nevertheless, the biological roles and regulatory mechanisms of USP32 in non-small cell lung cancer (NSCLC) remain unclear. According to our research, USP32 was strongly expressed in NSCLC cell lines and tissues and was linked to a bad prognosis for NSCLC patients. Interference with USP32 resulted in a significant inhibition of NSCLC cell proliferation, migration potential, and EMT development; on the other hand, USP32 overexpression had the opposite effect. To further elucidate the mechanism of action of USP32 in NSCLC, we screened H1299 cells for interacting proteins and found that USP32 interacts with BAG3 (Bcl2-associated athanogene 3) and deubiquitinates and stabilizes BAG3 in a deubiquitinating activity-dependent manner. Functionally, restoration of BAG3 expression abrogated the antitumor effects of USP32 silencing. Furthermore, USP32 increased the phosphorylation level of the RAF/MEK/ERK signaling pathway in NSCLC cells by stabilizing BAG3. In summary, these findings imply that USP32 is critical to the development of NSCLC and could offer a theoretical framework for the clinical diagnosis and management of NSCLC patients in the future.
... It shares over 90% sequence identity with USP6, the first DUB discovered as an oncogene [6]. In 2010, Akhavantabasi et al. identified that USP32 is a membrane-bound DUB highly expressed in breast cancer (BC), and knockdown of USP32 led to a decrease in the proliferation and migration of BC cells [7]. In subsequent studies, several studies supported that USP32 functions as a universal oncogene in cancer. ...
Background
Ubiquitin-specific protease 32 (USP32) is a highly conserved gene that promotes cancer progression. However, its role in hepatocellular carcinoma (HCC) is not well understood. The aim of this project is to explore the clinical significance and functions of USP32 in HCC.
Methods
The expression of USP32 in HCC was evaluated using data from TCGA, GEO, TISCH, tissue microarray, and human HCC samples from our hospital. Survival analysis, PPI analysis and GSEA analysis were performed to evaluate USP32-related clinical significance, key molecules and enrichment pathways. Using the ssGSEA algorithm and TIMER, we investigated the relationships between USP32 and immune infiltrates in the TME. Univariate and multivariate Cox regression analyses were then used to identify key USP32-related immunomodulators and constructed a USP32-related immune prognostic model. Finally, CCK8, transwell and colony formation assays of HCC cells were performed and an HCC nude mouse model was established to verify the oncogenic role of USP32.
Results
USP32 is overexpressed in HCC and its expression is an independent predictive factor for outcomes of HCC patients. USP32 is associated with pathways related to cell behaviors and cancer signaling, and its expression is significantly correlated with the infiltration of immune cells in the TME. We also successfully constructed a USP32-related immune prognostic model using 5 genes. Wet experiments confirmed that knockdown of USP32 could repress the proliferation, colony formation and migration of HCC cells in vitro and inhibit tumor growth in vivo.
Conclusion
USP32 is highly expressed in HCC and closely correlates with the TME of HCC. It is a potential target for improving the efficacy of chemotherapy and developing new strategies for targeted therapy and immunotherapy in HCC.
... Ubiquitin molecules are also recycled into the cytoplasmic pool to exert their functions. the multi-tissue cDNA panel, USP32 was highly expressed in the leukocytes, thymus, spleen, testis, prostate, ovary, small intestine, and colon [23]. We queried USP32 in human tissues through the Human Protein Atlas website, the testis showed the greatest RNA expression of USP32 (Fig. 3). ...
... We queried USP32 in human tissues through the Human Protein Atlas website, the testis showed the greatest RNA expression of USP32 (Fig. 3). USP32 is upregulated in a variety of cancers, including small cell lung cancer [24], gastric cancer [25,26], breast cancer [23,27,28], epithelial ovarian cancer [29], glioblastoma [30], gastrointestinal stromal tumor [31], pancreatic duct adenocarcinoma [32] and acute myeloid leukemia [33]. Endogenous USP32 is found in the cytoplasm and membrane, according to the findings of a subcellular separation experiment [34] and a fluorescence protection experiment [23]. ...
... USP32 is upregulated in a variety of cancers, including small cell lung cancer [24], gastric cancer [25,26], breast cancer [23,27,28], epithelial ovarian cancer [29], glioblastoma [30], gastrointestinal stromal tumor [31], pancreatic duct adenocarcinoma [32] and acute myeloid leukemia [33]. Endogenous USP32 is found in the cytoplasm and membrane, according to the findings of a subcellular separation experiment [34] and a fluorescence protection experiment [23]. This is consistent with earlier findings that USP32 is an active membrane-bound ubiquitin protease [24]. ...
An essential protein regulatory system in cells is the ubiquitin-proteasome pathway. The substrate is modified by the ubiquitin ligase system (E1-E2-E3) in this pathway, which is a dynamic protein bidirectional modification regulation system. Deubiquitinating enzymes (DUBs) are tasked with specifically hydrolyzing ubiquitin molecules from ubiquitin-linked proteins or precursor proteins and inversely regulating protein degradation, which in turn affects protein function. The ubiquitin-specific peptidase 32 (USP32) protein level is associated with cell cycle progression, proliferation, migration, invasion, and other cellular biological processes. It is an important member of the ubiquitin-specific protease family. It is thought that USP32, a unique enzyme that controls the ubiquitin process, is closely linked to the onset and progression of many cancers, including small cell lung cancer, gastric cancer, breast cancer, epithelial ovarian cancer, glioblastoma, gastrointestinal stromal tumor, acute myeloid leukemia, and pancreatic adenocarcinoma. In this review, we focus on the multiple mechanisms of USP32 in various tumor types and show that USP32 controls the stability of many distinct proteins. Therefore, USP32 is a key and promising therapeutic target for tumor therapy, which could provide important new insights and avenues for antitumor drug development. The therapeutic importance of USP32 in cancer treatment remains to be further proven. In conclusion, there are many options for the future direction of USP32 research.
... The exceptionally high level of conservation shown in the USP32 gene, which is located at the 17q23.1-17q23.2 chromosomal unit, remains unexplained [33]. In contrast to mammalian tissue that does not include cancerous cells, USP32 has been discovered in 22% of breast cancer tumours that originate from primary breast tissue. ...
Cancer is a fatal illness that is responsible for the deaths of millions of individuals all over the globe every year. It is possible that people will be able to survive for a longer period of time as a result of early identification, staging, and individualised biomarker therapy. Proteases known as deubiquitinating enzymes are responsible for removing ubiquitin tags from proteins so that they may be broken down by the proteasome. Enzymes that deubiquitinate proteins have several purposes throughout the body. One of the numerous roles that Deubiquitinating Enzyme plays is to keep an eye on the progression of tumours. The expression of members of the deubiquitinating enzyme family was shown to be elevated in a wide range of oncocells and tissues during various stages of cancer, according to a number of investigations. Based on these findings, deubiquitinating enzymes have the potential to be exploited as a therapeutic target in cancer treatment. In this article, the functions of members of the Deubiquitinating Enzyme family, as well as ubiquitin-specific proteases and other important family members, are dissected. Our goal was to get a better understanding of the connection between the expression patterns of deubiquitinating enzymes and carcinomas so that future research might investigate the possibility of developing inhibitors and gene therapies to improve the diagnosis and prognosis of cancer.
... However, an open question is how RAB7 monoubiquitination contributes to USP32 depletion-dependent defects in EGFR degradation and tumorigenesis. Notably, our and others' work has already linked USP32 to breast and other cancers, highlighting the implications of deregulated USP32 function [19][20][21]. ...
Behind the scenes of signaling cascades initiated by activated receptors, endocytosis determines the fate of internalized proteins through degradation in lysosomes or recycling. Over the years, significant progress has been made in understanding the mechanisms of endocytosis and deregulation in disease states. Here we review the role of the EGF-SNX3-EGFR axis in breast cancers with an extended discussion on deregulated EGFR endocytosis in cancer.
... Already a decade ago, USP32 has been described as active, membrane-bound ubiquitin protease that is overexpressed in breast cancer. 7 Meanwhile, additional reports proposed an oncogenic role for USP32 in small cell lung cancer, 8 gastric cancer, 9 and epithelial ovarian cancer. 10 Silencing of USP32 reduced cancer cell proliferation and migration in these studies and suggested USP32 as potential target for future therapy, although the tumor-promoting effects of USP32 are not fully understood yet. ...
... GFP-tagged USP32 localizes to the trans-Golgi network. 7 Our immunofluorescence microscopy analysis confirmed colocalization of endogenous USP32 and the Golgi marker GM130 ( Figure S1B). USP32 expression has been correlated with tumorigenesis, 7-9 but the physiological function of the enzyme was unknown when we started our investigations. ...
The endosomal-lysosomal system is a series of organelles in the endocytic pathway that executes trafficking and degradation of proteins and lipids and mediates the internalization of nutrients and growth factors to ensure cell survival, growth, and differentiation. Here, we reveal regulatory, non-proteolytic ubiquitin signals in this complex system that are controlled by the enigmatic deubiquitinase USP32. Knockout (KO) of USP32 in primary hTERT-RPE1 cells results among others in hyperubiquitination of the Ragulator complex subunit LAMTOR1. Accumulation of LAMTOR1 ubiquitination impairs its interaction with the vacuolar H⁺-ATPase, reduces Ragulator function, and ultimately limits mTORC1 recruitment. Consistently, in USP32 KO cells, less mTOR kinase localizes to lysosomes, mTORC1 activity is decreased, and autophagy is induced. Furthermore, we demonstrate that depletion of USP32 homolog CYK-3 in Caenorhabditis elegans results in mTOR inhibition and autophagy induction. In summary, we identify a control mechanism of the mTORC1 activation cascade at lysosomes via USP32-regulated LAMTOR1 ubiquitination.
... USP32 was reported to be one protein that contributed to the chimeric Tre2 (USP6) oncogene [154]. The pro-cancer effects of USP32 have been observed in breast cancer and glioblastoma [155,156]. Additionally, USP32 promotes GC cell growth, metastasis, and chemoresistance by upregulating SMAD2, an important protein in the TGF-β signaling pathway [55]. However, it is unclear whether USP32 regulates the ubiquitination level of SMAD2. ...
Gastric cancers (GCs) are malignant tumors with a high incidence that threaten global public health. Despite advances in GC diagnosis and treatment, the prognosis remains poor. Therefore, the mechanisms underlying GC progression need to be identified to develop prognostic biomarkers and therapeutic targets. Ubiquitination, a post-translational modification that regulates the stability, activity, localization, and interactions of target proteins, can be reversed by deubiquitinases (DUBs), which can remove ubiquitin monomers or polymers from modified proteins. The dysfunction of DUBs has been closely linked to tumorigenesis in various cancer types, and targeting certain DUBs may provide a potential option for cancer therapy. Multiple DUBs have been demonstrated to function as oncogenes or tumor suppressors in GC. In this review, we summarize the DUBs involved in GC and their associated upstream regulation and downstream mechanisms and present the benefits of targeting DUBs for GC treatment, which could provide new insights for GC diagnosis and therapy.
... Overexpression of USP28 correlated with a better survival in patients with invasive ductal breast carcinoma [71]. USP28 stabilized LSD1 and conferred stem-cell-like traits to breast cancer cells [72]. USP32 was overexpressed in 50% of breast cancer cell lines and 22% of primary breast tumors compared to mammary epithelial cells [73]. ...
Breast cancer is the most frequently diagnosed cancer in women, accounting for 30% of new diagnosing female cancers. Emerging evidence suggests that ubiquitin and ubiquitination played a role in a number of breast cancer etiology and progression processes. As the primary deubiquitinases in the family, ubiquitin-specific peptidases (USPs) are thought to represent potential therapeutic targets. The role of ubiquitin and ubiquitination in breast cancer, as well as the classification and involvement of USPs are discussed in this review, such as USP1, USP4, USP7, USP9X, USP14, USP18, USP20, USP22, USP25, USP37, and USP39. The reported USPs inhibitors investigated in breast cancer were also summarized, along with the signaling pathways involved in the investigation and its study phase. Despite no USP inhibitor has yet been approved for clinical use, the biological efficacy indicated their potential in breast cancer treatment. With the improvements in phenotypic discovery, we will know more about USPs and USPs inhibitors, developing more potent and selective clinical candidates for breast cancer.
... GRID1 is also at the breakpoint of several structural translocations such as t(10;20)(q23;q13) DPM1/GRID1 in Acute Myeloid Leukemia 37 . USP32 is overexpressed in breast cancer and human small cell lung cancer and may serve as an oncogene through promoting cell proliferation and tumor metastasis 38,39 . Many AKAP proteins, including AKAP11, are also commonly mutated in breast cancer 40 . ...
Somatic mutations and changes in expression of RAD21 are common in many types of cancer. Moreover, sub-optimal levels of RAD21 expression in early development can result in cohesinopathies. Altered RAD21 levels can result directly from mutations in the RAD21 gene. However, whether DNA variants outside of the RAD21 gene could control its expression and thereby contribute to cancer and developmental disease is unknown. In this study, we searched for genomic variants that modify RAD21expression to determine their potential to contribute to development or cancer by RAD21 dysregulation. We searched 42,953,834 genomic variants for a spatial-eQTL association with the transcription of RAD21. We identified 123 significant associations (FDR < 0.05), which are local (cis) or long-distance (trans) regulators of RAD21 expression. The 123 variants co-regulate a further seven genes (AARD, AKAP11, GRID1, KCNIP4, RCN1, TRIOBP, and USP32), enriched for having Sp2 transcription factor binding sites in their promoter regions. The Sp2 transcription factor and six of the seven genes had previously been associated with cancer onset, progression, and metastasis. Our results suggest that genome-wide variation in non-coding regions impacts on RAD21 transcript levels in addition to other genes, which then could impact on oncogenesis and the process of ubiquitination. This identification of distant co-regulation of oncogenes represents a strategy for discovery of novel genetic regions influencing cancer onset and a potential for diagnostics.
... Previous studies have reported that USP32 is expressed at high level in several cancers, include breast cancer [37], small-cell lung cancer [38], and gastric carcinomas [39]. In contrast, SLC35F2 is expressed highly in bladder [40], papillary thyroid [41], lung [42], and non-small-cell lung cancer [43]. ...
... USP32 is a poorly characterized DUB family member and was identified previously as a membrane-bound protease overexpressed in breast cancers [37]. However, several studies have reported that overexpression of USP32 promotes tumorigenesis and drug resistance in different cancer types [38,39]. ...
Background: The most commonly preferred chemotherapeutic agents to treat cancers are small-molecule drugs. However, the differential sensitivity of various cancer cells to small molecules and untargeted delivery narrow the range of potential therapeutic applications. The mechanisms responsible for drug resistance in a variety of cancer cells are also largely unknown. Several deubiquitinating enzymes (DUBs) are the main determinants of drug resistance in cancer cells.
Methods: We used CRISPR-Cas9 to perform genome-scale knockout of the entire set of genes encoding ubiquitin-specific proteases (USPs) and systematically screened for DUBs resistant to the clinically evaluated anticancer compound YM155. A series of in vitro and in vivo experiments were conducted to reveal the relationship between USP32 and SLC35F2 on YM155-mediated DNA damage in cancer cells.
Results: CRISPR-based dual-screening method identified USP32 as a novel DUB that governs resistance for uptake of YM155 by destabilizing protein levels of SLC35F2, a solute-carrier protein essential for the uptake of YM155. The expression of USP32 and SLC35F2 was negatively correlated across a panel of tested cancer cell lines. YM155-resistant cancer cells in particular exhibited elevated expression of USP32 and low expression of SLC35F2.
Conclusion: Collectively, our DUB-screening strategy revealed a resistance mechanism governed by USP32 associated with YM155 resistance in breast cancers, one that presents an attractive molecular target for anti-cancer therapies. Targeted genome knockout verified that USP32 is the main determinant of SLC35F2 protein stability in vitro and in vivo, suggesting a novel way to treat tumors resistant to small-molecule drugs.
