Valosin-containing Protein Expression Research Articles

NRF3 suppresses the metastasis of triple-negative breast cancer cells by inhibiting ERK activation in a ROS-dependent manner.

Our previous study demonstrated that NRF3 (NFE2L3, Nuclear Factor-erythroid 2-related factor 3) could suppress cell metastasis and proliferation in breast cancer. In this study, we investigated the mechanisms underlying its function in breast cancer. In the present study, NRF3 expression and its clinical characteristics in breast cancer were analyzed using public datasets and clinical specimens. After breast cancer cells were overexpressed NRF3, FACS was used to detect the intracellular ROS levels. The migration and invasion activities of NRF3-ectopic expressed breast cancer cells were determined by transwell assay. To validate the role of ROS/ERK axis in the inhibitory effect of NRF3 in cell metastasis, ROS scavenger NAC was also included. We found that NRF3 mRNA was highly expressed, while NRF3 protein was extremely lowly expressed in breast cancer tissues compared with their normal counterparts, and low level NRF3 was associated with poorer prognosis in patients with triple negative breast cancer (TNBC). More interestingly, overexpression of NRF3 protein significantly increased cellular ROS production and dramatically decreased p-ERK level and cell migration in TNBC cells. Mechanistically, NRF3 protein was found to be mutually regulated by valosin-containing protein (VCP). Strikingly, VCP-knockdown dramatically increased NRF3 protein expression, but NRF3-knockin also decreased VCP expression in return. Moreover, antioxidant NAC treatment effectively increased the level of p-ERK and VCP expression, as well as cell migration and invasion abilities of TNBC cells. NRF3, a tumor suppressor downregulated by VCP, could attenuate cell metastasis in TNBC cells by increasing cellular ROS accumulation and subsequently inhibiting the ERK phosphorylation.

Stool Protein Mass Spectrometry Identifies Biomarkers for Early Detection of Diffuse-type Gastric Cancer.

There is a high unmet need for early detection approaches for diffuse gastric cancer (DGC). We examined whether the stool proteome of mouse models of gastric cancer (GC) and individuals with hereditary diffuse gastric cancer (HDGC) have utility as biomarkers for early detection. Proteomic mass spectrometry of the stool of a genetically engineered mouse model driven by oncogenic KrasG12D and loss of p53 and Cdh1 in gastric parietal cells [known as Triple Conditional (TCON) mice] identified differentially abundant proteins compared with littermate controls. Immunoblot assays validated a panel of proteins, including actinin alpha 4 (ACTN4), N-acylsphingosine amidohydrolase 2 (ASAH2), dipeptidyl peptidase 4 (DPP4), and valosin-containing protein (VCP), as enriched in TCON stool compared with littermate control stool. Immunofluorescence analysis of these proteins in TCON stomach sections revealed increased protein expression compared with littermate controls. Proteomic mass spectrometry of stool obtained from patients with HDGC with CDH1 mutations identified increased expression of ASAH2, DPP4, VCP, lactotransferrin (LTF), and tropomyosin-2 relative to stool from healthy sex- and age-matched donors. Chemical inhibition of ASAH2 using C6 urea ceramide was toxic to GC cell lines and GC patient-derived organoids. This toxicity was reversed by adding downstream products of the S1P synthesis pathway, which suggested a dependency on ASAH2 activity in GC. An exploratory analysis of the HDGC stool microbiome identified features that correlated with patient tumors. Herein, we provide evidence supporting the potential of analyzing stool biomarkers for the early detection of DGC. Prevention Relevance: This study highlights a novel panel of stool protein biomarkers that correlate with the presence of DGC and has potential use as early detection to improve clinical outcomes.

Nanoparticle-Mediated Therapy with miR-198 Sensitizes Pancreatic Cancer to Gemcitabine Treatment through Downregulation of VCP-Mediated Autophagy.

Pancreatic ductal adenocarcinoma (PDAC) remains an extremely aggressive disease characterized by rapidly acquired multi-drug resistance, including to first-line chemotherapeutic agent gemcitabine. Autophagy is a process that is often exploited by cancer and is one of several intrinsic factors associated with resistance to gemcitabine. We have previously found that miR-198 acts as a tumor suppressor in PDAC through the targeting of factors including Valosin-containing protein (VCP). VCP has been reported to play an important role in autophagic flux. In this study, we investigated whether the repression of VCP through miR-198 administration disrupts the autophagy process and sensitizes PDAC cells to gemcitabine treatment in vitro. Moreover, we used LGA-PEI (LPNP) nanoparticles to effectively administer miR-198 to tumors in vivo, inducing tumor sensitization to gemcitabine and leading to a significant reduction in tumor burden and metastases and a concomitant downregulation of VCP expression and autophagy maturation. Our results indicate a potential therapeutic strategy for targeting gemcitabine resistant PDAC and establishes the use of LPNPs for effective therapeutic delivery of nucleic acids in vitro and in vivo.

Dissecting Sperm Mitochondrial G-Quadruplex Structures Using a Fluorescent Probe Biomarker to Monitor and Regulate Fertilization Capability.

To monitor the levels of mitochondrial DNA G-quadruplexes (mtDNA G4s) in spermatozoa and to explore the possibility using mtDNA G4s as a reliable marker in patients with multiple clinical insemination failures, a novel chemical TPE-mTO probe engineered in our previous work was used on both samples from the mice sperm and from patients with fertilization failure. Expression of valosin-containing protein and the zona-free hamster egg assay were used to evaluate mitophagy and human sperm penetration. RNA-sequencing was used to explore expression changes of key genes affected by mtDNA G4s. Results showed that the probe can track mtDNA G4s in spermatozoa easily and quickly with fewer backgrounds. Significantly increased mtDNA G4s were also found in patients with fertilization failure, using the flow-cytometry-based TPE-mTO probe detection method. A sperm-hamster egg penetration experiment showed that abnormal fertilization caused by increased mtDNA G4s can be effectively restored by a mitophagy inducer. This study provides a novel method for monitoring etiological biomarkers in patients with clinical infertility and treatment for patients with abnormal fertilization caused by mtDNA G4 dysfunction.

The mechanism of VCP-mediated metastasis of osteosarcoma based on cell autophagy and the EMT pathway.

Study of the molecular mechanisms of metastasis is still the research focus for osteosarcoma (OS) prevention. This study investigates the mechanism of valosin-containing protein (VCP) promoting OS metastasis in vitro through autophagy and epithelial-mesenchymal transition (EMT). Different cell lines of osteosarcoma (143B and MG63) were adopted in this study. The level of VCP expression in osteosarcoma cells was changed, and the level of autophagy and the progression of the epithelial-mesenchymal transition (EMT) were observed. Then autophagy and EMT in OS cells were changed artificially, and proliferation and migration ability were observed. The expression of LC3II/I was decreased, but the insolubilized P62 protein expression was increased in the VCP inhibiting group and the autophagy inhibitor treatment group. Simultaneously, E-cadherin protein expression increased while N-cadherin protein expression decreased in the VCP inhibiting group but increased in the TGF-β1 treatment group. In addition, suppressing VCP can cause a decrease in Transforming Growth Factor β1 (TGF-β1), smad2, smad3, phosphorylated smad2 (p-smad2), and phosphorylated smad3 (p-smad3). Autophagy inhibitors and agonists have no significant effect on the migration and invasion of OS cells but can significantly affect the ability of cells to resist anoikis. EMT inhibitors and agonists have a proportional effect on the migration and invasion of OS cells. VCP is likely to promote the migration and invasion of OS cells by inducing EMT, possibly via TGF-β1/smad2/3 signaling pathway. In this process, VCP-mediated autophagy may contribute to successful distant metastasis of tumor cells indirectly.

MiR-198 sensitizes pancreatic cancer to gemcitabine treatment through downregulation of VCP-mediated autophagy maturation.

e16290 Background: The mechanism of human pancreatic ductal adenocarcinoma (PDAC) resistance to nucleoside analog and first-line chemotherapy drug gemcitabine is not clearly understood, but some studies have associated it with increased autophagy. In cancer biology, autophagy plays dual roles as it can promote tumor suppression during early stages, but in established tumors, it plays a crucial role in tumor growth by enhancing survival under metabolic and therapeutic stress. We have previously found that miR-198 acts as a tumor suppressor in PDAC through the targeting of a network of tumorigenic factors, including the Valosin-containing protein (VCP), which has been reported to play an important role in autophagy. In this study, we investigate whether the repression of VCP through miR-198 replacement disrupts the autophagy process and sensitizes PDAC cells to gemcitabine treatment. Methods: MIAPaCa2 cells with forced overexpression of mesothelin (MSLN) and AsPC-1 cell lines and, CDX and PDX mouse models were used for gemcitabine sensitization studies. For miR-198 replacement, a miR-198 expression vector-loaded lactic co-glycolic-acid-modified polyethylenimine polyplex (LPNP-p198) was used. Autophagy disruption experiments were run over miR-198 and/or VCP overexpressing cell lines. Nude mice were used for subcutaneous and orthotopic CDX models and SCID/Beige were used for the subcutaneous PDX model. Results: Cell lines were treated with LPNP-p198 in combination with gemcitabine and cell growth was significantly inhibited when compared to gemcitabine alone. Additionally, it was determined that miR-198 disrupts the autophagy maturation process and, that it is then restored by overexpression of VCP. LPNP-p198 can effectively enter tumor cells and induce tumor sensitization in vivo, resulting in an 80-90% reduction of tumor burden and metastatic spread in the LPNP-p198 plus gemcitabine group when compared to controls, with the concomitant downregulation of VCP expression in the tumor tissue. In addition, this group had the fewest mice with jaundice, ascites, and metastases to the abdominal cavity, spleen, liver, and kidney. Finally, to address the potential of the observed effect in the context of the heterogenic nature of PDAC, we confirmed our findings in a PDX mouse model, where a marked reduction in tumor burden was observed in the LPNP-p198 plus gemcitabine group compared to controls. Conclusions: Our findings indicate that miR-198 replacement disrupts the autophagy maturation process and sensitizes PDAC cells to gemcitabine through VCP repression, indicating a potential therapeutic strategy for targeting gemcitabine-resistant PDAC and, establishing the use of LPNPs as a prototype for effective nucleic acid delivery in vitro and in vivo, with potential to be used from bench to clinic.

VCP interaction with HMGB1 promotes hepatocellular carcinoma progression by activating the PI3K/AKT/mTOR pathway

BackgroundHepatocellular carcinoma (HCC) is the most common pathological type of liver cancer. Valosin-containing protein (VCP) is a member of the AAA-ATPase family associated with multiple molecular functions and involved in tumor metastasis and prognosis. However, the role of VCP in HCC progression is still unclear.MethodsWe examined the expression of VCP in HCC using the RNA sequencing and microarray data from public databases and measured it in clinical samples and cell lines by western blot, and immunohistochemistry (IHC). We also evaluated the correlation between VCP and clinical features. The VCP-interacting proteins were identified by co-immunoprecipitation combined with mass spectrometry (CoIP/MS). The underlying molecular mechanisms were investigated using in vitro and in vivo models of HCC.ResultsWe found that VCP expression is significantly increased in tumor tissues and is associated with advanced TNM stages and poorer prognosis in HCC patients. In vitro analyses revealed that VCP overexpression promoted HCC cell proliferation, migration, and invasion via PI3K/AKT/mTOR pathway activation. Conversely, VCP knockdown resulted in the reverse phenotypes. In vivo studies indicated that up-regulated VCP expression accelerated tumor growth in a subcutaneous HCC model. The D1 domain of VCP and A box of HMGB1 were identified as the critical regions for their interaction, and D1 area was required for the tumor-promoting effects induced by VCP expression. VCP enhanced the protein stability of HMGB1 by decreasing its degradation via ubiquitin–proteasome process. Inhibition of HMGB1 markedly attenuated VCP-mediated HCC progression and downstream activation of PI3K/AKT/mTOR signals.ConclusionCollectively, these findings demonstrate that VCP is a potential prognostic biomarker in HCC and exhibits oncogenic roles via PI3K/AKT/mTOR pathway activation. HMGB1 played an essential role in VCP-mediated HCC progression, indicating that VCP and HMGB1 are potential therapeutic targets in human HCC.

Flavivirus recruits the valosin-containing protein–NPL4 complex to induce stress granule disassembly for efficient viral genome replication

Flaviviruses are human pathogens that can cause severe diseases, such as dengue fever and Japanese encephalitis, which can lead to death. Valosin-containing protein (VCP)/p97, a cellular ATPase associated with diverse cellular activities (AAA-ATPase), is reported to have multiple roles in flavivirus replication. Nevertheless, the importance of each role still has not been addressed. In this study, the functions of 17 VCP mutants that are reportedly unable to interact with the VCP cofactors were validated using the short-interfering RNA rescue experiments. Our findings of this study suggested that VCP exerts its functions in replication of the Japanese encephalitis virus by interacting with the VCP cofactor nuclear protein localization 4 (NPL4). We show that the depletion of NPL4 impaired the early stage of viral genome replication. In addition, we demonstrate that the direct interaction between NPL4 and viral nonstructural protein (NS4B) is critical for the translocation of NS4B to the sites of viral replication. Finally, we found that Japanese encephalitis virus and dengue virus promoted stress granule formation only in VCP inhibitor-treated cells and the expression of NS4B or VCP attenuated stress granule formation mediated by protein kinase R, which is generally known to be activated by type I interferon and viral genome RNA. These results suggest that the NS4B-mediated recruitment of VCP to the virus replication site inhibits cellular stress responses and consequently facilitates viral protein synthesis in the flavivirus-infected cells.

Functional Inhibition of Valosin-Containing Protein Induces Cardiac Dilation and Dysfunction in a New Dominant-Negative Transgenic Mouse Model.

Valosin-containing protein (VCP) was found to play a vital protective role against cardiac stresses. Genetic mutations of VCP are associated with human dilated cardiomyopathy. However, the essential role of VCP in the heart during the physiological condition remains unknown since the VCP knockout in mice is embryonically lethal. We generated a cardiac-specific dominant-negative VCP transgenic (DN-VCP TG) mouse to determine the effects of impaired VCP activity on the heart. Using echocardiography, we showed that cardiac-specific overexpression of DN-VCP induced a remarkable cardiac dilation and progressively declined cardiac function during the aging transition. Mechanistically, DN-VCP did not affect the endogenous VCP (EN-VCP) expression but significantly reduced cardiac ATPase activity in the DN-VCP TG mouse hearts, indicating a functional inhibition. DN-VCP significantly impaired the aging-related cytoplasmic/nuclear shuffling of EN-VCP and its co-factors in the heart tissues and interrupted the balance of the VCP-cofactors interaction between the activating co-factors, ubiquitin fusion degradation protein 1 (UFD-1)/nuclear protein localization protein 4 (NPL-4) complex, and its inhibiting co-factor P47, leading to the binding preference with the inhibitory co-factor, resulting in functional repression of VCP. This DN-VCP TG mouse provides a unique functional-inactivation model for investigating VCP in the heart in physiological and pathological conditions.

Sexually dimorphic expression of a chicken sex chromosome gene (VCP) reflects differences in gonadal development between males and females

The chicken has a Z-W sex chromosome system, in which the males are the homogametic sex (ZZ) and the females the heterogametic sex (ZW). The smaller W chromosome is generally considered to be a highly degraded copy of the Z chromosome that retains around 28–30 homologous protein-coding genes’ These Z-W homologues are thought to have important, but undefined, roles in development, and here we explore the role of one of these genes, VCP (Valosin Containing Protein) in gonadogenesis. We established RNA expression levels of both Z and W VCP homologues, the levels of VCP protein, and the cellular localization of VCP protein in male and female embryonic gonads during development. We also assessed the effects of female-to-male sex-reversal on VCP expression in developing gonads. The results showed that both VCP RNA and protein are expressed at higher levels in female than male gonads, and the expression levels of VCP protein and VCP-Z transcript, but not VCP-W transcript, are decreased in female-to-male sex reversed gonads. In addition, the spatial expression of VCP protein differs between male and female embryonic gonads: in testes, VCP protein is mainly confined to the medullary sex cords, while in ovaries, VCP protein is expressed throughout the medulla and at higher levels in the cortex. The results suggest that sexually dimorphic expression of chicken VCP reflects differences in gonadal morphology between sexes.

Valosin-containing Protein in Psoriasis: A Clinical and Immunohistochemical Study.

Psoriasis is a chronic immune-mediated inflammatory skin disease, affects about 2% to 3% of the world population. Valosin-containing protein (VCP) is one of the newly discovered markers that is highly expressed in neoplasms and hyperproliferative lesions. This work aimed to study the role of VCP in psoriasis vulgaris by immunohistochemical study and correlate its expression with the available clinicopathologic data. This prospective case-control study was conducted on 25 patients with psoriasis vulgaris and 25 age-matched and sex-matched healthy individuals as a control group. Skin biopsies were taken under local anesthesia from cases and controls. VCP immunoreactivity showed that epidermal VCP expression had a significant stepwise increase (P=0.002) from control to lesional psoriatic sections. Epidermal VCP H-score was significantly associated with the progressive course (P=0.037). Similarly, VCP in the dermis showed a significant expression in lesional psoriatic skin (P≤0.001). Higher VCP in the dermis in cases with a history of joint affection (P<0.05) was detected. We concluded that VCP is a promising marker for follow-up and monitoring of psoriatic patients and may play a role as a therapeutic target.

Targeting Novel Sodium Iodide Symporter Interactors ADP-Ribosylation Factor 4 and Valosin-Containing Protein Enhances Radioiodine Uptake.

The sodium iodide symporter (NIS) is required for iodide uptake, which facilitates thyroid hormone biosynthesis. NIS has been exploited for over 75 years in ablative radioiodine (RAI) treatment of thyroid cancer, where its ability to transport radioisotopes depends on its localization to the plasma membrane. The advent of NIS-based in vivo imaging and theranostic strategies in other malignancies and disease modalities has recently increased the clinical importance of NIS. However, NIS trafficking remains ill-defined. Here, we used tandem mass spectrometry followed by coimmunoprecipitation and proximity ligation assays to identify and validate two key nodes-ADP-ribosylation factor 4 (ARF4) and valosin-containing protein (VCP)-controlling NIS trafficking. Using cell-surface biotinylation assays and highly inclined and laminated optical sheet microscopy, we demonstrated that ARF4 enhanced NIS vesicular trafficking from the Golgi to the plasma membrane, whereas VCP-a principal component of endoplasmic reticulum (ER)-associated degradation-governed NIS proteolysis. Gene expression analysis indicated VCP expression was particularly induced in aggressive thyroid cancers and in patients who had poorer outcomes following RAI treatment. Two repurposed FDA-approved VCP inhibitors abrogated VCP-mediated repression of NIS function, resulting in significantly increased NIS at the cell-surface and markedly increased RAI uptake in mouse and human thyroid models. Collectively, these discoveries delineate NIS trafficking and highlight the new possibility of systemically enhancing RAI therapy in patients using FDA-approved drugs. SIGNIFICANCE: These findings show that ARF4 and VCP are involved in NIS trafficking to the plasma membrane and highlight the possible therapeutic role of VCP inhibitors in enhancing radioiodine effectiveness in radioiodine-refractory thyroid cancer.

Targeting valosin-containing protein enhances the efficacy of radiation therapy in esophageal squamous cell carcinoma.

Overcoming resistance to radiation is a great challenge in cancer therapy. Here, we highlight that targeting valosin‐containing protein (VCP) improves radiation sensitivity in esophageal squamous cell carcinoma (ESCC) cell lines and show the potential of using VCP as a prognosis marker in locally advanced ESCC treated with radiation therapy. Esophageal squamous cell carcinoma cell lines with high VCP expression were treated with VCP inhibitor combined with radiotherapy. Cell proliferation, colony formation, cell death, and endoplasmic reticulum (ER) stress signaling were evaluated. Moreover, patients with newly diagnosed locally advanced ESCC who were treated with radiotherapy were analyzed. Immunohistochemistry was used to detect the expression of VCP. The correlation between overall survival and VCP was investigated. Esophageal squamous cell carcinoma cells treated with VCP inhibitor and radiotherapy showed attenuated cell proliferation and colony formation and enhanced apoptosis. Further investigation showed this combined strategy activated the ER stress signaling involved in unfolded protein response, and inhibited the ER‐associated degradation (ERAD) pathway. Clinical analysis revealed a significant survival benefit in the low VCP expression group. Targeting VCP resulted in antitumor activity and enhanced the efficacy of radiation therapy in ESCC cells in vitro. Valosin‐containing protein is a promising and novel target. In patients with locally advanced ESCC who received radiotherapy, VCP can be considered as a useful prognostic indicator of overall survival. Valosin‐containing protein inhibitors could be developed for use as effective cancer therapies, in combination with radiation therapy.

Valosin-containing protein promotes metastasis of osteosarcoma through autophagy induction and anoikis inhibition via the ERK/NF-κβ/beclin-1 signaling pathway.

Valosin-containing protein (VCP) promotes the development of metastasis in osteosarcoma (OS) via the PI3K/Akt signaling pathway. However, inhibition of the PI3K/Akt pathway does not completely reverse VCP-mediated invasion and migration of OS, suggesting that VCP-mediated OS invasion and migration involves additional mechanisms. In the present study, a positive correlation between the expression of VCP and cell autophagy was observed among OS tissues. Inhibiting VCP may decrease the survival of malignant cells; however, an autophagy stimulator may compensate for VCP inhibition and promote malignant cell survival. Altering the level of autophagy did not affect cell invasiveness or migration. ERK, NF-κβ and beclin-1 protein expression levels were markedly decreased following VCP inhibition. These findings indicated that VCP may induce autophagy and enhance anoikis resistance without affecting cell invasiveness or migration. Via anoikis resistance, VCP may promote metastasis in OS. Therefore, targeting of the ERK/NF-κβ/beclin-1 signaling pathway may be an effective therapeutic strategy for the management of OS.

Upregulation of valosin-containing protein (VCP) is associated with poor prognosis and promotes tumor progression of orbital B-cell lymphoma.

ObjectiveThis study aimed to investigate the relationship between VCP expression and the prognosis of orbital B-cell lymphoma patients and the influence of downregulation of VCP on the apoptosis and invasion abilities of lymphoma cells.MethodsWe recruited 66 orbital B-cell lymphoma patients. VCP expression in 66 samples of orbital B-cell lymphoma was determined by immunohistochemistry using monoclonal VCP antibodies. Based on VCP-expression levels detected by immunohistochemistry, we chose ten cases of orbital tumor paraffin tissue from the patients. Total RNA was extracted and differences in VCP gene-expression levels compared among patients using quantitative reverse-transcription (qRT) PCR. We used siRNA to knock down VCP in the lymphoma cell lines Raji and SUDHL4. qRT-PCR and Western blot were applied to detect VCP mRNA and protein expression, respectively. SUDHL48 assays were applied to investigate cell proliferation. Hoechst 33258 staining and flow-cytometry analysis were applied to investigate cell apoptosis. Transwell assays were applied to investigate invasive ability. Survival analysis was used to evaluate prognostic values.ResultsExpression levels of VCP were correlated with the stage, tumor grade, and recurrence rate of patients. VCP mRNA-expression levels were consistent with VCP-expression levels in orbital B-cell lymphoma tissue. Moreover, survival analysis revealed that lower VCP-expression levels were correlated with longer overall survival of orbital B-cell lymphoma patients. Down-regulation of VCP with siRNA did not inhibit cell proliferation. However, it dramatically increased apoptosis and suppressed the invasion of B-cell lymphoma cells.ConclusionVCP expression played an important role in the progression of orbital B-cell lymphoma. VCP could be a useful marker for predicting the prognosis of orbital B-cell lymphoma patients. VCP may be a potential therapeutic target for orbital B-cell lymphoma.

Valosin-containing protein is associated with maintenance of meiotic arrest in mouse oocytes†.

Valosin-containing protein (VCP) is a member of the highly conserved AAA (ATPase associated with a variety of cellular activities) superfamily. A previous study has shown that targeted deletion of Vcp in mice results in early embryonic lethality. The aim of the present study was to analyze the expression and localization of VCP and its function in meiotic arrest of mouse oocytes. Vcp mRNA and protein were expressed in multiple mouse tissues. In the ovary, VCP protein was mainly expressed in oocytes and granulosa cells. After ovulation and fertilization, Vcp mRNA and protein were detected in oocytes and preimplantation embryos. Furthermore, VCP protein was localized in both the cytoplasm and nucleus of germinal vesicle (GV)-stage oocytes and preimplantation embryos. Moreover, knockdown of Vcp in GV-stage oocytes led to a significantly increased rate of germinal vesicle breakdown (GVBD). In addition, inhibition of VCP protein improved the GVBD rate in mouse GV-stage oocytes. When VCP inhibition was reversed, the final GVBD rate returned to normal. These results provide the first evidence for a novel function of VCP in meiotic arrest of mouse oocytes.

Valosin-containing protein VCP/p97 is essential for the intracellular development of Leishmania and its survival under heat stress.

Valosin-containing protein (VCP)/p97/Cdc48 is one of the best-characterised type II cytosolic AAA+ ATPases most known for their role in ubiquitin-dependent protein quality control. Here, we provide functional insights into the role of the Leishmania VCP/p97 homologue (LiVCP) in the parasite intracellular development. We demonstrate that although LiVCP is an essential gene, Leishmania infantum promastigotes can grow with less VCP. In contrast, growth of axenic and intracellular amastigotes is dramatically affected upon decreased LiVCP levels in heterozygous and temperature sensitive (ts) LiVCP mutants or the expression of dominant negative mutants known to specifically target the second conserved VCP ATPase domain, a major contributor of the VCP overall ATPase activity. Interestingly, these VCP mutants are also unable to survive heat stress, and a ts VCP mutant is defective in amastigote growth. Consistent with LiVCP's essential function in amastigotes, LiVCP messenger ribonucleic acid undergoes 3'Untranslated Region (UTR)-mediated developmental regulation, resulting in higher VCP expression in amastigotes. Furthermore, we show that parasite mutant lines expressing lower VCP levels or dominant negative VCP forms exhibit high accumulation of polyubiquitinated proteins and increased sensitivity to proteotoxic stress, supporting the ubiquitin-selective chaperone function of LiVCP. Together, these results emphasise the crucial role LiVCP plays under heat stress and during the parasite intracellular development.

Interleukin-6 induced overexpression of valosin-containing protein (VCP)/p97 is associated with androgen-independent prostate cancer (AIPC) progression.

Though Androgen deprivation therapy (ADT) is effective initially, numerous patients become resistant to it and develop castration resistant PCa (CRPC). Cytokines promotes ligand independent activation of AR. Interleukin-6 (IL-6) levels are elevated in CRPC patients and regulate AR activity. However, progression to CRPC is not fully understood. In this study, we analyzed differential protein expression in LNCaP cells treated with IL-6 using proteomics. Results revealed altered expression of 27 proteins and Valosin-containing protein (VCP)/p97 plays a predominant role in co-regulation of altered proteins. Interestingly, IL-6 induced VCP expression through Pim-1 via STAT3 is AR independent there by suggesting a role for VCP in CRPC. Transfection of LNCaP cells for VCP overexpression showed an increased cell proliferation, migration, and invasion where as its inhibition by NMS-873 showed the reverse effect causing cell death. Mechanistic studies demonstrate that cell death occurs due to apoptosis by endoplasmic reticulum (ER) stress, elevated cell cycle inhibitors p21, p27kip1, and active PARP and reduced Bcl-2. VCP promotes cell invasion and migration by altering E-cadherin and Vimentin levels inversely triggering EMT of PCa cells. VCP immunostaining revealed no staining in BPH but strong staining in PCa. This study determines VCP may play an important role in progression to CRPC and it can be a favorable target with to develop new therapies to treat ADT resistant prostate cancer.

F199. O-GLCNAC DYSREGULATION IN SCHIZOPHRENIA CORTEX

BackgroundO-linked β-linked N-acetylglucosamine (O-GlcNAc) is a post-translational glycosylation modification with ubiquitous functions in cell biology. The attachment of GlcNAc to serine or threonine (S/T) residues transiently adorns thousands of nuclear, cytosolic, and mitochondrial proteins and modulates protein function and localization via dynamic cooperation with kinase and phosphatase enzymes. O-GlcNAc transferase (OGT) exists in complex with S/T phosphatase subunits PP1β and PP1γ and, along with the activity of O-GlcNAcase (OGA), facilitates rapid cycling between O-GlcNAcylation and phosphorylation states to serve as an “on/off switch” for substrate activation. Altered levels of OGT, OGA, and/or O-GlcNAc have been shown to influence many pathways pertinent to schizophrenia (SZ) pathophysiology. Notably, elevated O-GlcNAc and enhanced O-GlcNAcylation mediate glucose tolerance and insulin resistance which can lead to diabetes, an illness often found comorbid with SZ. Elevated O-GlcNAcylation can also produce mitochondrial abnormalities consistent with those identified in SZ. In an exploratory study of glycosylation enzyme transcript expression, our lab found OGT mRNA levels 253% higher in SZ than non-psychiatrically ill comparison (COMP) subjects (p < 0.0001). Based on this evidence, we hypothesized that OGT protein levels or the ratio of OGT:OGA enzymes are elevated in SZ brain.MethodsExpression of OGT and OGA were measured using western blots of superior temporal gyrus (STG; Brodmann Area 22) homogenates from sex- and age-matched pairs of SZ and COMP subjects (N = 17). Standard immunoblotting methods and commercially available antibodies were used to detect the targets of interest and protein levels were normalized to intralane valosin containing protein (VCP) expression; VCP expression has previously been found to be unchanged in SZ STG.ResultsIn the current study, we found OGA protein levels reduced 18% in SZ (p < 0.01) and SZ subjects demonstrate a trend toward increased ratios of OGT:OGA (p = 0.05). OGT was not different between groups (p = 0.77).DiscussionOur current results partially support our original hypothesis that an altered ratio of OGT:OGA may contribute to abnormalities of O-GlcNAcylation and consequent cellular metabolic abnormalities in SZ. A trend toward increased OGT:OGA along with decreased expression of OGA would produce the same functional outcome as the originally predicted OGT increase: upregulation of protein O-GlcNAcylation. Given that the mRNA study used samples of dorsolateral prefrontal cortex (DLPFC) while our protein-level measures were from STG, it is not inconceivable that potential O-GlcNAc dysregulation could arise from upregulated OGT in one brain region, but downregulated OGA in another. To elaborate on these findings, we will investigate OGT and OGA expression in the DLPFC and will assess total O-GlcNAcylation in both brain regions to determine functional consequences of altered enzyme expression.

The valosin-containing protein is a novel repressor of cardiomyocyte hypertrophy induced by pressure overload.

SummaryHypertension‐induced left ventricular hypertrophy (LVH) is an independent risk factor for heart failure. Regression of LVH has emerged as a major goal in the treatment of hypertensive patients. Here, we tested our hypothesis that the valosin‐containing protein (VCP), an ATPase associate protein, is a novel repressor of cardiomyocyte hypertrophy under the pressure overload stress. Left ventricular hypertrophy (LVH) was determined by echocardiography in 4‐month male spontaneously hypertensive rats (SHRs) vs. age‐matched normotensive Wistar Kyoto (WKY) rats. VCP expression was found to be significantly downregulated in the left ventricle (LV) tissues from SHRs vs. WKY rats. Pressure overload was induced by transverse aortic constriction (TAC) in wild‐type (WT) mice. At the end of 2 weeks, mice with TAC developed significant LVH whereas the cardiac function remained unchanged. A significant reduction of VCP at both the mRNA and protein levels in hypertrophic LV tissue was found in TAC WT mice compared to sham controls. Valosin‐containing protein VCP expression was also observed to be time‐ and dose‐dependently reduced in vitro in isolated neonatal rat cardiomyocytes upon the treatment of angiotensin II. Conversely, transgenic (TG) mice with cardiac‐specific overexpression of VCP showed a significant repression in TAC‐induced LVH vs. litter‐matched WT controls upon 2‐week TAC. TAC‐induced activation of the mechanistic target of rapamycin complex 1 (mTORC1) signaling observed in WT mice LVs was also significantly blunted in VCP TG mice. In conclusion, VCP acts as a novel repressor that is able to prevent cardiomyocyte hypertrophy from pressure overload by modulating the mTORC1 signaling pathway.