Impact of RSUME Actions on Biomolecular Modifications in Physio-Pathological Processes.

The small RWD domain-containing protein called RSUME or RWDD3 was cloned from pituitary tumor cells with increasing tumorigenic and angiogenic proficiency. RSUME expression is induced under hypoxia or heat shock and is upregulated, at several pathophysiological stages, in tissues like pituitary, kidney, heart, pancreas, or adrenal gland. To date, several factors with essential roles in endocrine-related cancer appear to be modulated by RWDD3. RSUME regulates, through its post-translational (PTM) modification, pituitary tumor transforming gene (PTTG) protein stability in pituitary tumors. Interestingly, in these tumors, another PTM, the regulation of EGFR levels by USP8, plays a pathogenic role. Furthermore, RSUME suppresses ubiquitin conjugation to hypoxia-inducible factor (HIF) by blocking VHL E3-ubiquitin ligase activity, contributing to the development of von Hippel-Lindau disease. RSUME enhances protein SUMOylation of specific targets involved in inflammation such as IkB and the glucocorticoid receptor. For many of its actions, RSUME associates with regulatory proteins of ubiquitin and SUMO cascades, such as the E2-SUMO conjugase Ubc9 or the E3 ubiquitin ligase VHL. New evidence about RSUME involvement in inflammatory and hypoxic conditions, such as cardiac tissue response to ischemia and neuropathic pain, and its role in several developmental processes, is discussed as well. Given the modulation of PTMs by RSUME in neuroendocrine tumors, we focus on its interactors and its mode of action. Insights into functional implications and molecular mechanisms of RSUME action on biomolecular modifications of key factors of pituitary adenomas and renal cell carcinoma provide renewed information about new targets to treat these pathologies.

Mass Spectrometry-Based Metabolic Fingerprinting Contributes to Unveil the Role of RSUME in Renal Cell Carcinoma Cell Metabolism.

Clear cell renal cell carcinoma (ccRCC) is a heterogeneous disease with 50-80% patients exhibiting mutations in the von Hippel-Lindau (VHL) gene. RSUME (RWD domain (termed after three major RWD-containing proteins: RING finger-containing proteins, WD-repeat-containing proteins, and yeast DEAD (DEXD)-like helicases)-containing protein small ubiquitin-related modifier (SUMO) enhancer) acts as a negative regulator of VHL function in normoxia. A discovery-based metabolomics approach was developed by means of ultraperformance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (MS) for fingerprinting the endometabolome of a human ccRCC cell line 786-O and three other transformed cell systems (n = 102) with different expressions of RSUME and VHL. Cross-validated orthogonal projection to latent structures discriminant analysis models were built on positive, negative, and a combination of positive- and negative-ion mode MS data sets. Discriminant feature panels selected by an iterative multivariate classification allowed differentiating cells with different expressions of RSUME and VHL. Fifteen identified discriminant metabolites with level 1, including glutathione, butyrylcarnitine, and acetylcarnitine, contributed to understand the role of RSUME in ccRCC. Altered pathways associated with the RSUME expression were validated by biological and bioinformatics analyses. Combined results showed that in the absence of VHL, RSUME is involved in the downregulation of the antioxidant defense system, whereas in the presence of VHL, it acts in rerouting energy-related pathways, negatively modulating the lipid utilization, and positively modulating the fatty acid synthesis, which may promote deposition in droplets.

Modifications in the cellular proteome and their clinical application.

Post-translational modifications (PTMs) are covalent modifications in proteins during or after their synthesis. Among them, the best known are phosphorylation, methylation, acetylation, and also cleavage or binding of small peptides (ubiquitination, SUMOylation and NEDDylation). Often the protein is modified in multiple sites and these modifications are coordinated generating a PTMs crosstalk. Altered patterns of PTMs have been related to several pathologies. Currently, advances in mass spectrometry have made it possible to study multiple PTMs simultaneously. Oncology is one of the disciplines that incorporated these technologies for the need to better characterize tumors. In cancer, several alterations related to the ubiquitinlike PTMs have been described, such as SUMOylation. In particular, the interaction between different PTMs with SUMOylation has been studied in the context of the von Hippel Lindau (VHL) multitumoral syndrome, generating new putative biomarkers for the evolution of these tumors. RSUME or RWDD3, an enhancer of SUMOylation that acts on VHL and HIF proteins, shows a correlation with malignant parameters in this type of tumors, such as angiogenesis. Regulators of PTMs are becoming relevant as biomarkers in cancer.

Las modificaciones postraduccionales (PTMs por sus siglas en inglés) son modificaciones covalentes en las proteínas durante o posteriormente a su síntesis. Las más conocidas son fosforilación, metilación y acetilación, también clivajes o unión de pequeños péptidos (ubiquitinación, SUMOilación y NEDDilación). Frecuentemente la proteína es modificada en múltiples sitios y estas modificaciones se coordinan generando una interacción de PTMs. Patrones alterados de PTMs han sido relacionados con varias enfermedades. En la actualidad los avances en la espectrometría de masas han hecho posible estudiar en simultáneo múltiples PTMs. La oncología es una de las disciplinas que ha incorporado estas tecnologías por su necesidad de caracterizar a los tumores. En cáncer se han descripto varias alteraciones relacionadas a las PTMs del tipo ubiquitina como la SUMOilación. En particular la interacción entre distintas PTMs con la SUMOilación ha sido estudiada en el contexto de la enfermedad multitumoral de von Hippel Lindau, generando posibles nuevos biomarcadores para la evolución de estos tumores. RSUME o RWDD3, un enhancer de SUMOilación que actúa sobre las proteínas VHL y HIF, ha mostrado una correlación con parámetros malignos en este tipo de tumores, como la angiogénesis. Los reguladores de las PTMs están cobrando relevancia como biomarcadores en el cáncer.

Protein stabilization by RSUME accounts for PTTG pituitary tumor abundance and oncogenicity.

Increased levels of the proto-oncogene pituitary tumor-transforming gene 1 (PTTG) have been repeatedly reported in several human solid tumors, especially in endocrine-related tumors such as pituitary adenomas. Securin PTTG has a critical role in pituitary tumorigenesis. However, the cause of upregulation has not been found yet, despite analyses made at the gene, promoter and mRNA level that show that no mutations, epigenetic modifications or other mechanisms that deregulate its expression may explain its overexpression and action as an oncogene. We describe that high PTTG protein levels are induced by the RWD-containing sumoylation enhancer (RWDD3 or RSUME), a protein originally identified in the same pituitary tumor cell line in which PTTG was also cloned. We demonstrate that PTTG and RSUME have a positive expression correlation in human pituitary adenomas. RSUME increases PTTG protein in pituitary tumor cell lines, prolongs the half-life of PTTG protein and regulates the PTTG induction by estradiol. As a consequence, RSUME enhances PTTG transcription factor and securin activities. PTTG hyperactivity on the cell cycle resulted in recurrent and unequal divisions without cytokinesis, and the consequential appearance of aneuploidies and multinucleated cells in the tumor. RSUME knockdown diminishes securin PTTG and reduces its tumorigenic potential in a xenograft mouse model. Taken together, our findings show that PTTG high protein steady state levels account for PTTG tumor abundance and demonstrate a critical role of RSUME in this process in pituitary tumor cells.

Relationship between RSUME and HIF-1α/VEGF-A with invasion of pituitary adenoma.

The RWD-containing sumoylation enhancer (RSUME) can stabilize hypoxia-inducible factor-1α (HIF-1α) which promotes vascular endothelial growth factor-A (VEGF-A) expression. RSUME plays an important role in promoting the invasion of pituitary adenoma. In this study, we compared the mRNA and protein levels of RSUME, HIF-1α, and VEGF-A in pituitary adenoma tissue and analyzed the correlation. We found that the expression levels of RSUME, HIF-1α, and VEGF-A in invasive pituitary adenoma were significantly higher than in noninvasive pituitary adenoma. Moreover, a positive correlation was found between RSUME and HIF-1α/VEGF pathways. RSUME and HIF-1α were treated with hypoxia-mimicking CoCl2 and transfected into AtT-20 and GT1.1 cell lines to determine the relationship between them. It was found that RSUME effects post-transcriptional expression of HIF-1α regulated VEGF-A secretion. Reducing RSUME expression using siRNA transfection resulted in a decrease of the invasion inhibition rate of AtT-20 cells, as determined using Transwell and MTT assays. Together, we found that RSUME silencing can inhibit the invasion of pituitary adenoma cells.

RSUME promotes apoptosis of pituitary adenoma cell AtT-20 in mice / 基础医学与临床

Objective To explore the expression and influence of RWD structure small ubiquitin modified enhan-cer (RSUME) and inhibitor of nuclear factor kappa B-alpha (IκB-α) and nuclear factor kappa B-1 (NF-κB1) in pituitary adenomas of mice.Methods Real-time fluorescent quantitative PCR ( q-PCR) was used to detect the level RSUME, IκB-α, NF-κB1 mRNA and Western blot was used to detect RSUME and the nucleoprotein of IκB-α, NF-κB1 changes in the level of protein , flow cytometry was used to detect cell apoptosis .Results In protein level, RSUME and IκB-αexpression reduced ( P<0.05 ) , but NF-κB1 raised after transfection ( P<0.05); The level of RSUME mRNA was obviously lower after transfection than before (P<0.05).The level of IκB-αand NF-κB1 mRNA was not significantly changed; Flow cytometry confirmed cell apoptosis rate increased significantly after transfection.Conclusions RSUME can promote apoptosis of pituitary adenoma cells in mice by NF-κB.

The relationship between RSUME and small ubiquitin related modifiers of HIF-1α with invasive pituitary adenomas / 中国神经精神疾病杂志

Objective AIM:To study whether the RWD containing sumoylation enhancer (RSUME) enhanced small ubiquitin related modifiers (SUMO) to competitively inhibit Ubiquitin B (UBB)-mediated degradation of hypoxia hypoxia-inducible factor 1α.(HIF-1α) and the invasive pituitary adenomas.Methods The expression of protein and mRNA levels of RSUME,SUMO-1,UBB and HIF-1α were detected by using immunohistochemistry,western blot and qPCR in 38 cases of non-invasive pituitary adenoma,38 cases of invasive pituitary adenomas and 10 cases of normal pituitary capsule.The expression of SUMO-1 was analyzed in different types of pituitary adenomas.Results The protein,and mRNA levels of RSUME,SUMO-HIF-1αwere significantly higher in the invasive pituitary adenomas than in the non-invasive pituitary adenomas and normal pituitary capsule (P＜0.01).The protein and mRNA levels of RSUME,SUMO-HIF-1αwas higher in non-invasive pituitary adenomas than in normal pituitary capsule(P＜0.01).However,the protein levels of UBB-HIF-1α were significantly lower in the invasive pituitary adenomas than in the non-invasive pituitary adenomas and normal pituitary capsule (P＜0.01).The protein of UBB-HIF-1α were lower in non-invasive pituitary adenomas than in normal pituitary capsule (P＜0.01).The expression levels of SUMO were not significantly different among different types of pituitary adenomas (P＞0.05).Conclusion RSUM may increase pituitary adenomas angiogenesis and promote tumor invasion through enhancement of SUMO of HIF-1α which competitively inhibits ubiquitination of HIF-1 α.

RSUME is implicated in tumorigenesis and metastasis of pancreatic neuroendocrine tumors.

The factors triggering pancreatic neuroendocrine tumor (PanNET) progression are largely unknown. Here we investigated the role and mechanisms of the sumoylation enhancing protein RSUME in PanNET tumorigenesis. Immunohistochemical studies showed that RSUME is strongly expressed in normal human pancreas, in particular in ß-cells. RSUME expression is reduced in insulinomas and is nearly absent in other types of PanNETs suggesting a role in PanNET tumorigenesis. In human pancreatic neuroendocrine BON1 cells, RSUME stimulates hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor-A (VEGF-A), which are key components of tumor neovascularisation. In contrast, RSUME suppresses nuclear factor-κB (NF-κB) and its target interleukin-8 (IL-8). Correspondingly, PanNET cells with RSUME knockdown showed decreased HIF-1α activity and increased NF-κB and IL-8 production leading to a moderate reduction of VEGF-A release as reduced HIF-1α/VEGF-A production is partly compensated by NF-κB/IL-8-induced VEGF-A. Notably, RSUME stabilizes the tumor suppressor PTEN, which is frequently lost in PanNETs and whose absence is associated with metastasis formation. In vivo orthotopic transplantation of PanNET cells with or without RSUME expression into nude mice showed that PanNETs without RSUME have reduced PTEN expression, grow faster and form multiple liver metastases. In sum, RSUME differentially regulates key components of PanNET formation suggesting that the observed loss of RSUME in advanced PanNETs is critically involved in PanNET tumorigenesis, particularly in metastasis formation.

Correlation between RSUME sumoylation and HIF-1α/VEGF pathway in gliomas / 西安交通大学学报(医学版)

Objective To explore the expressions and correlation of RWD containing sumoylation enhancer (RSUME),small ubiquitin-like modifier (SUMO-1),hypoxia inducible factor-1α(HIF-1α)and vascular endothelial growth factor (VEGF)in gliomas of different pathologic grade.Methods We investigated the expression levels of RSUME mRNA,HIF-1α mRNA and VEGF mRNA with reverse transcription-polymerase chain reaction (RT-PCR),and investigated the immunohistochemical staining to determine the expressions of SUMO-1,HIF-1α and VEGF in 63 cases of human gliomas of different pathologic grade and 9 cases of normal brain tissues.We studied its correlation with the pathologic grade and the relationship between the expression of RSUME promoter sumoylation and HIF-1α/VEGF pathway in gliomas.Results There were significant differences (P <0.01)in the expressions of RSUME mRNA,HIF-1αmRNA and VEGF mRNA in glioma tissues.With the increasing degree of pathologic grade in tumor specimens,the expression levels of RSUME mRNA,HIF-1α mRNA and VEGF mRNA increased markedly (P <0.01 ).There was a positive correlation of the expression levels of RSUME mRNA with HIF-1αmRNA and VEGF mRNA.There were significant differences (P <0.01 )in the expressions of SUMO-1,HIF-1αand VEGF in glioma tissues by immunohistochemical staining.With the ascending of pathologic grade of tumor specimens,the expression levels of SUMO-1,HIF-1α and VEGF increased markedly (P < 0.01 ).There was a positive correlation between the expression level of SUMO-1 and HIF-1α(r =0.857,P <0.01).The Kaplan-Meier analysis and Log-rank test showed significant differences in progress free survival (PFS)between the RSUME high-expression and low-expression groups (χ2 =36.032,P <0.01).Conclusion RSUME may enhance HIF-1α/VEGF pathway through sumoylation in gliomas.It implicates that RSUME is related to angiogenesis in gliomas and can promote tumor invasion and progression,indicating that RSUME can be a novel target in gliomas treatment.

Research Progress in molecular mechanisms between RSUME and invasive pituitary adenoma / 中国肿瘤临床

Pituitary adenoma, a benign intracranial tumor, is the third most common brain tumor, second only to glioma and me-ningioma. Pituitary adenoma has been defined as a benign tumor, but some pituitary adenomas can invade the surrounding tissue. This tumor is difficult to resect and can easily recur after surgery. RWD-containing sumoylation enhancer (RSUME) can stabilize the activity of hypoxia-inducible factor-1α and inhibitor kappaB by the small ubiquitin-related modifiers. This phenomenon indicates the impor-tance of RSUME in pituitary adenoma because it promotes the invasiveness of the tumor. However, the correlation between RSUME and the invasion of pituitary adenoma remains unclear. In this study, the roles of RSUME on HIF-1α/VEGF signaling pathway and IκB/NF-κB compomers in the invasiveness of pituitary adenoma were reviewed.