Recombinant expression and tryptophan-assisted analysis of human sweet taste receptor T1R3's extracellular domain in sweetener interaction studies.

The human palate can discern multiple tastes; however, it predominantly perceives five fundamental flavors: sweetness, saltiness, sourness, bitterness, and umami. Sweetness is primarily mediated through the sweet taste receptor, a membrane-bound heterodimeric structure comprising T1R2-T1R3. However, unraveling the structural and mechanistic intricacies of the sweet taste receptor has proven challenging. This study aimed to address this knowledge gap by expressing an extracellular N-terminal domain encompassing the cysteine-rich domain of human hT1R3 (hT1R3-TMD) in Escherichia coli. The expressed protein was obtained as inclusion bodies, purified by metal affinity chromatography, and refolded using the dilution-refolding method. Through rigorous analysis, we confirmed the successful refolding of hT1R3-TMD and elucidated its structural characteristics using circular dichroism spectroscopy. Notably, the refolded protein was found to exist as either a monomer or a dimer, depending on its concentration. A tryptophan fluorescence quenching assay revealed that the dissociation constants for sucrose, sucralose, and brazzein were >9500 µM, 2380 µM and 14.3 µM, respectively. Our findings highlight the utility of this E. coli expression system for producing functional hT1R3-TMD for investigations and demonstrate the efficacy of the tryptophan fluorescence quenching assay in revealing complex interactions between sweet taste receptors and various sweeteners.

Neurotoxic effects of polystyrene nanoplastics on memory and microglial activation: Insights from in vivo and in vitro studies.

Nanoplastics, arising from the fragmentation of plastics into environmental pollutants and specialized commercial applications, such as cosmetics, have elicited concerns due to their potential toxicity. Evidence suggests that the oral ingestion of nanoplastics smaller than 100 nm may penetrate the brain and induce neurotoxicity. However, comprehensive research in this area has been hampered by technical challenges associated with the detection and synthesis of nanoplastics. This study aimed to bridge this research gap by successfully synthesizing fluorescent polystyrene nanoplastics (PSNPs, 30-50 nm) through the incorporation of IR-813 and validating them using various analytical techniques. We administered PSNPs orally (10 and 20 mg/kg/day) to mice and observed that they reached brain tissues and induced cognitive dysfunction, as measured by spatial and fear memory tests, while locomotor and social behaviors remained unaffected. In vitro studies (200 µg/mL) demonstrated a predominant uptake of PSNPs by microglia over astrocytes or neurons, leading to microglial activation, as evidenced by immunostaining of cellular markers and morphological analysis. Transcriptomic analysis indicated that PSNPs altered gene expression in microglia, highlighting neuroinflammatory responses that may contribute to cognitive deficits. To further explore the neurotoxic effects of PSNPs mediated by microglial activation, we measured endogenous neuronal activity using a multi-electrode array in cultured hippocampal neurons. The application of conditioned media from microglia exposed to PSNPs suppressed neuronal activity, which was reversed by inhibitors of microglial activation. Our findings offer detailed insights into the mechanisms by which nanoplastics damage the brain, particularly emphasizing the potential environmental risk factors that contribute to cognitive impairment in neurodegenerative diseases.

Association of overexpressed carboxyl-terminal amyloid precursor protein in brains with altered glucose metabolism and liver toxicity.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disease. The deposition of amyloid plaques mainly composed of amyloid beta (Aß) is observed in brain regions in AD patients. AD presents with similar pathophysiology to that of metabolic syndrome, including glucose and insulin resistance. In addition, epidemiological studies indicate diabetes, impaired glucose metabolism, and obesity increase the prevalence of AD. The liver is considered a key organ in the reciprocal relationship between AD and metabolic syndrome and is the major organ for the clearance of Aß in the periphery. Furthermore, liver dysfunction aggravates Aß-induced pathophysiology. Aß is produced in the brain and peripheral tissues and penetrates the blood-brain barrier. However, in vivo evidence showing the effect of Aß on the crosstalk between the brain and liver has not been reported yet. In the present study, we investigated the toxicity of brain-derived Aß on glucose metabolism and the liver using transgenic mice overexpressing the carboxyl-terminal of amyloid precursor protein in the brain. The transgenic mice were overweight, which was associated with impaired glucose metabolism and insulin resistance, but not due to increased food intake. In addition, transgenic mice had enlarged livers and reduced gene expressions associated with glucose and lipid metabolism. Thus, overexpressed amyloid precursor protein in the brain may promote being overweight and glucose resistance, possibly through liver toxicity.

Polypropylene microplastics promote metastatic features in human breast cancer.

Microplastics (MPs) are now a global issue due to increased plastic production and use. Recently, various studies have been performed in response to the human health risk assessment. However, these studies have focused on spherical MPs, which have smooth edges and a spherical shape and account for less than 1% of MPs in nature. Unfortunately, studies on fragment-type MPs are very limited and remain in the initial stages. In this study, we studied the effect that 16.4 µm fragment type polypropylene (PP) MPs, which have an irregular shape and sharp edges and form naturally in the environment, had on breast cancer. The detrimental effects of PPMPs on breast cancer metastasis were examined. Here, 1.6 mg/ml of PPMP, which does not induce cytotoxicity in MDA-MB-231, was used, and at this concentration, PPMP did not induce morphological changes or cellular migrating in the MDA-MB-231 and MCF-7 cells. However, PPMP incubation for 24 hours in the MDA-MB-231 cells significantly altered the level of cell cycle-related transcripts in an RNA-seq analysis. When confirmed by qRT-PCR, the gene expression of TMBIM6, AP2M1, and PTP4A2 was increased, while the transcript level of FTH1 was decreased. Further, secretion of the pro-inflammatory cytokine IL-6 from cancer cells was elevated with the incubation of PPMP for 12 hours. These results suggest that PPMP enhances metastasis-related gene expression and cytokines in breast cancer cells, exacerbating breast cancer metastasis.

Anti-inflammatory effect of Ailanthus altissima (Mill.) Swingle leaves in lipopolysaccharide-stimulated astrocytes.

ETHNOPHARMACOLOGICAL RELEVANCE: Activated astrocytes are involved in the progression of neurodegenerative diseases. Traditionally, Ailanthus altissima (Mill.) Swingle, widely distributed in East Asia, has been used as a medicine for the treatment of fever, gastric diseases, and inflammation. Although A. altissima has been reported to play an anti-inflammatory role in peripheral tissues or cells, its role in the central nervous system (CNS) remains unclear. AIM OF THE STUDY: In the present study, we investigated the anti-inflammatory effects and mechanism of action of A. altissima in primary astrocytes stimulated by lipopolysaccharide (LPS). MATERIALS AND METHODS: A nitrite assay was used to measure nitric oxide (NO) production, and the tetrazolium salt 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was performed to determine cytotoxicity. The expression levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and mitogen-activated protein kinase (MAPK) were determined with western blotting. Reverse-transcription PCR was used to assess the expression of inflammatory cytokines. The levels of reactive oxygen species were measured using 2,7-dichlorodihydrofluorescein diacetate. Luciferase assay and immunocytochemistry were used for assessing nuclear factor-kappa B (NF-κB) transcription and p65 localization, respectively. Memory and social interaction were analyzed using the Y-maze and three-chamber tests, respectively. RESULTS: The ethanol extract of A. altissima leaves (AAE) inhibited iNOS and COX-2 expression in LPS-stimulated astrocytes. Moreover, AAE reduced the transcription of various proinflammatory mediators, hindered NF-κB activation, and suppressed extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) activation without p38 activation. Ultra-high performance liquid chromatography with mass spectrometry analysis revealed that AAE comprised ethyl gallate, quercetin, and kaempferol, along with luteolin, which has anti-inflammatory properties, and repressed LPS-induced nitrite levels and the nuclear translocation of p65. Finally, oral administration of AAE attenuated LPS-induced memory and social impairment in mice and repressed LPS-induced ERK and JNK activation in the cortices of mice. CONCLUSION: AAE could have therapeutic uses in the treatment of neuroinflammatory diseases via suppression of astrocyte activation.

Analysis of microRNA signatures in ischemic stroke thrombus.

BACKGROUND: We investigated the microRNA expression pattern from thrombus retrieved by mechanical thrombectomy in acute stroke patients to understand the stroke mechanism. METHODS: This study included acute ischemic stroke patients who had undergone intra-arterial thrombectomy at Chung-Ang University Hospital in Seoul, Korea between February 2016 and March 2019. The thrombus was retrieved and stored at -70℃ after obtaining informed consent. MicroRNA microarray analysis was performed for the patients with identified stroke mechanisms including (1) large artery atherosclerosis, (2) cardioembolism with atrial fibrillation, and (3) cardioembolism with valvular heart disease. The microRNAs derived from microarray analysis were validated by quantitative real-time polymerase chain reaction (qRT-PCR) from different patient populations. The correlation analysis was performed between microRNA levels and laboratory data to understand the functional relevance of the altered microRNA. RESULTS: In total, 55 thrombi were obtained from 74 patients, and the microRNAs were analyzed in 45 samples. Microarray analysis of 2578 microRNAs revealed that 50 microRNAs were significantly altered among the three groups. Validation using qRT-PCR showed that miR-378f and miR-450b-5p were significantly elevated among the cardioembolic thrombi; both microRNAs were inversely correlated with the ejection fraction from echocardiography. Thrombi from patients with early neurological deterioration exhibited higher levels of miR-93-5p and lower levels of miR-629-5p than those from neurologically stable patients. CONCLUSIONS: The microRNA expression pattern can provide information regarding the mechanism of stroke by reflecting the underlying pathological status of the organ from which the thrombus was derived.

Selective and ATP-competitive kinesin KIF18A inhibitor suppresses the replication of influenza A virus.

The influenza virus is one of the major public health threats. However, the development of efficient vaccines and therapeutic drugs to combat this virus is greatly limited by its frequent genetic mutations. Because of this, targeting the host factors required for influenza virus replication may be a more effective strategy for inhibiting a broader spectrum of variants. Here, we demonstrated that inhibition of a motor protein kinesin family member 18A (KIF18A) suppresses the replication of the influenza A virus (IAV). The expression of KIF18A in host cells was increased following IAV infection. Intriguingly, treatment with the selective and ATP-competitive mitotic kinesin KIF18A inhibitor BTB-1 substantially decreased the expression of viral RNAs and proteins, and the production of infectious viral particles, while overexpression of KIF18A enhanced the replication of IAV. Importantly, BTB-1 treatment attenuated the activation of AKT, p38 MAPK, SAPK and Ran-binding protein 3 (RanBP3), which led to the prevention of the nuclear export of viral ribonucleoprotein complexes. Notably, administration of BTB-1 greatly improved the viability of IAV-infected mice. Collectively, our results unveiled a beneficial role of KIF18A in IAV replication, and thus, KIF18A could be a potential therapeutic target for the control of IAV infection.

Histidine residues at the copper-binding site in human tyrosinase are essential for its catalytic activities.

Tyrosinase is a copper-binding enzyme involved in melanin biosynthesis. However, the detailed structure of human tyrosinase has not yet been solved, along with the identification of the key sites responsible for its catalytic activity. We used site-directed mutagenesis to identify the residues critical for the copper binding of human tyrosinase. Seven histidine mutants in the two copper-binding sites were generated, and catalytic activities were characterised. The tyrosine hydroxylase activities of the CuA site mutants were approximately 50% lower than those of the wild-type tyrosinase, while the dopa oxidation activities of the mutants were not significantly different from that of wild-type tyrosinase. By contrast, mutations at CuB significantly decreased both tyrosine hydroxylation and dopa oxidation activities, confirming that the catalytic sites for these two activities are at least partially distinct. These findings provide a useful resource for further structural determination and development of tyrosinase inhibitors in the cosmetic and pharmaceutical industries.

3M-Brazzein as a Natural Sugar Substitute Attenuates Obesity, Metabolic Disorder, and Inflammation.

Obesity is a global chronic disease linked to various diseases. Increased consumption of added sugars, especially in beverages, is a key contributor to the obesity epidemic. It is essential to reduce or replace sugar intake with low-calorie sweeteners. Here, a natural sweet protein, 3M-brazzein, was investigated as a possible sugar substitute. Mice were exposed to 3M-brazzein or 10% sucrose of equivalent sweetness, in drinking water to mimic human obesity development over 15 weeks. Consumption of 3M-brazzein in liquid form did not cause adiposity hypertrophy, resulting in 33.1 ± 0.4 g body weight and 0.90 ± 0.2 mm fat accumulation, which were 35.9 ± 0.7 g (p = 0.0094) and 1.53 ± 0.067 mm (p = 0.0031), respectively, for sucrose supplement. Additionally, 3M-brazzein did not disrupt glucose homeostasis or affect insulin resistance and inflammation. Due to its naturally low-calorie content, 3M-brazzein could also be a potential sugar substitute that reduces adiposity.

Frequency spectrum of chemical fluctuation: A probe of reaction mechanism and dynamics.

Even in the steady-state, the number of biomolecules in living cells fluctuates dynamically, and the frequency spectrum of this chemical fluctuation carries valuable information about the dynamics of the reactions creating these biomolecules. Recent advances in single-cell techniques enable direct monitoring of the time-traces of the protein number in each cell; however, it is not yet clear how the stochastic dynamics of these time-traces is related to the reaction mechanism and dynamics. Here, we derive a rigorous relation between the frequency-spectrum of the product number fluctuation and the reaction mechanism and dynamics, starting from a generalized master equation. This relation enables us to analyze the time-traces of the protein number and extract information about dynamics of mRNA number and transcriptional regulation, which cannot be directly observed by current experimental techniques. We demonstrate our frequency spectrum analysis of protein number fluctuation, using the gene network model of luciferase expression under the control of the Bmal 1a promoter in mouse fibroblast cells. We also discuss how the dynamic heterogeneity of transcription and translation rates affects the frequency-spectra of the mRNA and protein number.

Author Correction: Loss of function of NCOR1 and NCOR2 impairs memory through a novel GABAergic hypothalamus-CA3 projection.

In the version of this article initially published, the Acknowledgements erroneously included a grant number that did not directly support the work in the article. The last sentence of the Acknowledgments should have read, "The authors' laboratories were supported by National Natural Science Foundation of China grants 31671222 and 31571556 (G.D.), a Taishan Scholarship (X.H.), the American Diabetes Association (ADA1-17-PDF-138) (Y.H.), the US Department of Agriculture (USDA) Cris6250-51000-059-04S (Y.X.), National Institutes of Health grants R01DK101379, R01DK117281, P01DK113954, R01DK115761 (Y.X.), the American Heart Association grant AHA30970064 (Z.S.), and grants R21CA215591 and R01ES027544 (Z.S.)." The error has been corrected in the HTML and PDF versions of the article.

Loss of function of NCOR1 and NCOR2 impairs memory through a novel GABAergic hypothalamus-CA3 projection.

Nuclear receptor corepressor 1 (NCOR1) and NCOR2 (also known as SMRT) regulate gene expression by activating histone deacetylase 3 through their deacetylase activation domain (DAD). We show that mice with DAD knock-in mutations have memory deficits, reduced anxiety levels, and reduced social interactions. Mice with NCOR1 and NORC2 depletion specifically in GABAergic neurons (NS-V mice) recapitulated the memory deficits and had reduced GABAA receptor subunit α2 (GABRA2) expression in lateral hypothalamus GABAergic (LHGABA) neurons. This was associated with LHGABA neuron hyperexcitability and impaired hippocampal long-term potentiation, through a monosynaptic LHGABA to CA3GABA projection. Optogenetic activation of this projection caused memory deficits, whereas targeted manipulation of LHGABA or CA3GABA neuron activity reversed memory deficits in NS-V mice. We describe de novo variants in NCOR1, NCOR2 or HDAC3 in patients with intellectual disability or neurodevelopmental disorders. These findings identify a hypothalamus-hippocampus projection that may link endocrine signals with synaptic plasticity through NCOR-mediated regulation of GABA signaling.

The HDAC3 enzymatic activity regulates skeletal muscle fuel metabolism.

Histone deacetylase 3 (HDAC3) is a major HDAC, whose enzymatic activity is targeted by small molecule inhibitors for treating a variety of conditions. However, its enzymatic activity is largely dispensable for its function in embryonic development and hepatic lipid metabolism. HDAC3 plays a pivotal role in regulating muscle fuel metabolism and contractile function. Here, we address whether these muscular functions of HDAC3 require its enzymatic activity. By mutating the NCoR/SMRT corepressors in a knock-in mouse model named NS-DADm, we ablated the enzymatic activity of HDAC3 without affecting its protein levels. Compared to the control mice, skeletal muscles from NS-DADm mice showed lower force generation, enhanced fatigue resistance, enhanced fatty acid oxidation, reduced glucose uptake during exercise, upregulated expression of metabolic genes involved in branched-chain amino acids catabolism, and reduced muscle mass during aging, without changes in the muscle fiber-type composition or mitochondrial protein content. These muscular phenotypes are similar to those observed in the HDAC3-depleted skeletal muscles, which demonstrates that, unlike that in the liver or embryonic development, the metabolic function of HDAC3 in skeletal muscles requires its enzymatic activity. These results suggest that drugs specifically targeting HDAC3 enzyme activity could be developed and tested to modulate muscle energy metabolism and exercise performance.

Cell surface vimentin-targeted monoclonal antibody 86C increases sensitivity to temozolomide in glioma stem cells.

Glioblastoma multiforme (GBM) is the most prevalent and aggressive brain tumor. The current standard therapy, which includes radiation and chemotherapy, is frequently ineffective partially because of drug resistance and poor penetration of the blood-brain barrier. Reducing resistance and increasing sensitivity to chemotherapy may improve outcomes. Glioma stem cells (GSCs) are a source of relapse and chemoresistance in GBM; sensitization of GSCs to temozoliomide (TMZ), the primary chemotherapeutic agent used to treat GBM, is therefore integral for therapeutic efficacy. We previously discovered a unique tumor-specific target, cell surface vimentin (CSV), on patient-derived GSCs. In this study, we found that the anti-CSV monoclonal antibody 86C efficiently increased GSC sensitivity to TMZ. The combination TMZ+86C induced significantly greater antitumor effects than TMZ alone in eight of 12 GSC lines. TMZ+86C-sensitive GSCs had higher CSV expression overall and faster CSV resurfacing among CSV- GSCs compared with TMZ+86C-resistant GSCs. Finally, TMZ+86C increased apoptosis of tumor cells and prolonged survival compared with either drug alone in GBM mouse models. The combination of TMZ+86C represents a promising strategy to reverse GSC chemoresistance.

Integrated omics approaches to characterize a nuclear receptor corepressor-associated histone deacetylase in mouse skeletal muscle.

Nuclear receptors regulate gene expression by differentially binding to coactivators or corepressors in a ligand-dependent manner, which further recruits a set of epigenome-modifying enzymes that remodel chromatin conformation. Histone acetylation is a major epigenomic change controlled by histone acetyltransferases (HATs) and histone deacetylases (HDACs). HDAC3 is the only HDAC that confers the enzymatic activity to the complexes nucleated by nuclear receptor corepressors NCoR and SMRT. To address the metabolic function of HDAC3, we have deleted it specifically in mouse skeletal muscles. We have performed the following omics profiling in skeletal muscles of these mice: (1) RNA-seq profiling of total RNA; (2) Global nuclear run-on (GRO-seq) analysis of nascent RNAs; (3) Chromatin immuno-precipitation (ChIP-seq) of HDAC3 at both early evening and early morning; (4) proteomics profiling with mass spectrometry; (5) snap-shot metabolomics profiling of water-soluble metabolites at the basal condition; (6) snap-shot metabolomics profiling of lipid species at the basal condition; (7) kinetic fluxomics analysis of glucose utilization using 13C6-glucose In vivo during treadmill running exercise. These approaches have provided several novel insights into how nuclear receptors regulate circadian rhythm of skeletal muscle fuel metabolism, which has been published elsewhere. Here we present the original datasets and technical details during the execution, analysis, and interpretation of these omics studies.

Dissociation of muscle insulin sensitivity from exercise endurance in mice by HDAC3 depletion.

Type 2 diabetes and insulin resistance are associated with reduced glucose utilization in the muscle and poor exercise performance. Here we find that depletion of the epigenome modifier histone deacetylase 3 (HDAC3) specifically in skeletal muscle causes severe systemic insulin resistance in mice but markedly enhances endurance and resistance to muscle fatigue, despite reducing muscle force. This seemingly paradoxical phenotype is due to lower glucose utilization and greater lipid oxidation in HDAC3-depleted muscles, a fuel switch caused by the activation of anaplerotic reactions driven by AMP deaminase 3 (Ampd3) and catabolism of branched-chain amino acids. These findings highlight the pivotal role of amino acid catabolism in muscle fatigue and type 2 diabetes pathogenesis. Further, as genome occupancy of HDAC3 in skeletal muscle is controlled by the circadian clock, these results delineate an epigenomic regulatory mechanism through which the circadian clock governs skeletal muscle bioenergetics. These findings suggest that physical exercise at certain times of the day or pharmacological targeting of HDAC3 could potentially be harnessed to alter systemic fuel metabolism and exercise performance.

Discovery of cell surface vimentin targeting mAb for direct disruption of GBM tumor initiating cells.

Intracellular vimentin overexpression has been associated with epithelial-mesenchymal transition, metastasis, invasion, and proliferation, but cell surface vimentin (CSV) is less understood. Furthermore, it remains unknown whether CSV can serve as a therapeutic target in CSV-expressing tumor cells. We found that CSV was present on glioblastoma multiforme (GBM) cancer stem cells and that CSV expression was associated with spheroid formation in those cells. A newly developed monoclonal antibody against CSV, 86C, specifically and significantly induced apoptosis and inhibited spheroid formation in GBM cells in vitro. The addition of 86C to GBM cells in vitro also led to rapid internalization of vimentin and decreased GBM cell viability. These findings were associated with an increase in caspase-3 activity, indicating activation of apoptosis. Finally, treatment with 86C inhibited GBM progression in vivo. In conclusion, CSV-expressing GBM cells have properties of tumor initiating cells, and targeting CSV with the monoclonal antibody 86C is a promising approach in the treatment of GBM.

SHOX2 is a direct miR-375 target and a novel epithelial-to-mesenchymal transition inducer in breast cancer cells.

MicroRNAs have added a new dimension to our understanding of tumorigenesis and associated processes like epithelial-to-mesenchymal transition (EMT). Here, we show that miR-375 is elevated in epithelial-like breast cancer cells, and ectopic miR-375 expression suppresses EMT in mesenchymal-like breast cancer cells. We identified short stature homeobox 2 (SHOX2) as a miR-375 target, and miR-375-mediated suppression in EMT was reversed by forced SHOX2 expression. Ectopic SHOX2 expression can induce EMT in epithelial-like breast cancer cells, whereas SHOX2 knockdown diminishes EMT traits in mesenchymal-like breast cancer cells, demonstrating SHOX2 as an EMT inducer. We show that SHOX2 acts as a transcription factor to upregulate transforming growth factor ß receptor I (TßR-I) expression, and TßR-I inhibitor LY364947 abolishes EMT elicited by ectopic SHOX2 expression, suggesting that transforming growth factor ß signaling is essential for SHOX2-induced EMT. Manipulating SHOX2 abundance in breast cancer cells impact in vitro invasion and in vivo dissemination. Analysis of breast tumor microarray database revealed that high SHOX2 expression significantly correlates with poor patient survival. Our study supports a critical role of SHOX2 in breast tumorigenicity.

COP1 and GSK3ß cooperate to promote c-Jun degradation and inhibit breast cancer cell tumorigenesis.

High abundance of c-Jun is detected in invasive breast cancer cells and aggressive breast tumor malignancies. Here, we demonstrate that a major cause of high c-Jun abundance in invasive breast cancer cells is prolonged c-Jun protein stability owing to poor poly-ubiquitination of c-Jun. Among the known c-Jun-targeting E3 ligases, we identified constitutive photomorphogenesis protein 1 (COP1) as an E3 ligase responsible for c-Jun degradation in less invasive breast cancer cells because depletion of COP1 reduced c-Jun poly-ubiquitination leading to the stabilization of c-Jun protein. In a panel of breast cancer cell lines, we observed an inverse association between the levels of COP1 and c-Jun. However, overexpressing COP1 alone was unable to decrease c-Jun level in invasive breast cancer cells, indicating that efficient c-Jun protein degradation necessitates an additional event. Indeed, we found that glycogen synthase kinase 3 (GSK3) inhibitors elevated c-Jun abundance in less invasive breast cancer cells and that GSK3ß nonphosphorylable c-Jun-T239A mutant displayed greater protein stability and poorer poly-ubiquitination compared to the wild-type c-Jun. The ability of simultaneously enforced expression of COP1 and constitutively active GSK3ß to decrease c-Jun abundance in invasive breast cancer cells allowed us to conclude that c-Jun is negatively regulated through the coordinated action of COP1 and GSK3ß. Importantly, co-expressing COP1 and active GSK3ß blocked in vitro cell growth/migration and in vivo metastasis of invasive breast cancer cells. Gene expression profiling of breast tumor specimens further revealed that higher COP1 expression correlated with better recurrence-free survival. Our study supports the notion that COP1 is a suppressor of breast cancer progression.

Role of urokinase receptor in tumor progression and development.

Elevated level of urokinase receptor (uPAR) is detected in various aggressive cancer types and is closely associated with poor prognosis of cancers. Binding of uPA to uPAR triggers the conversion of plasminogen to plasmin and the subsequent activation of metalloproteinases. These events confer tumor cells with the capability to degrade the components of the surrounding extracellular matrix, thus contributing to tumor cell invasion and metastasis. uPA-uPAR interaction also elicits signals that stimulate cell proliferation/survival and the expression of tumor-promoting genes, thus assisting tumor development. In addition to its interaction with uPA, uPAR also interacts with vitronectin and this interaction promotes cancer metastasis by activating Rac and stimulating cell migration. Although underlying mechanisms are yet to be fully elucidated, uPAR has been shown to facilitate epithelial-mesenchymal transition (EMT) and induce cancer stem cell-like properties in breast cancer cells. The fact that uPAR lacks intracellular domain suggests that its signaling must be mediated through its co-receptors. Indeed, uPAR interacts with diverse transmembrane proteins including integrins, ENDO180, G protein-coupled receptors and growth factor receptors in cancer cells and these interactions are proven to be critical for the role of uPAR in tumorigenesis. Inhibitory peptide that prevents uPA-uPAR interaction has shown the promise to prolong patients' survival in the early stage of clinical trial. The importance of uPAR's co-receptor in uPAR's tumor-promoting effects implicate that anti-cancer therapeutic agents may also be developed by disrupting the interactions between uPAR and its functional partners.