High expression of COPZ2 is associated with poor prognosis and cancer progression in glioma.

Background: Coatomer protein complex zeta 2 (COPZ2) is a member of heptameric coatomer protein complex I and has been reported to be involved in various tumors. However, COPZ2's potential involvement in glioma remains to be explored. Methods: The COPZ2 expression and related clinical data were obtained from The Cancer Genome Atlas (TCGA). TIMER2.0 and the Ualcan database were utilized to assess the COPZ2 expression in various tumors. Univariable, multivariate Cox regression, Kaplan-Meier methods, nomogram analysis, and ROC curve analysis were carried out to assess the relationship of COPZ2 and other prognostic factors with glioma. The LinkedOmics database was used to predict the potential biological mechanism of COPZ2 in glioma. We also conducted in vitro experiments to evaluate the functional role and mechanism of COPZ2 in glioma cell lines. Results: We found that COPZ2 was highly expressed in glioma and it was associated with age and WHO grades. Kaplan-Meier survival curves, Cox analysis, nomogram analysis, and ROC curve showed that COPZ2 was a disadvantageous factor in poor glioma prognosis. The functions of COPZ2 and co-expression genes were significantly associated with neutrophil-mediated immunity, granulocyte activation, and response to interferon-gamma. In addition, COPZ2 knockdown significantly inhibited the proliferation, migration, and invasion of glioblastoma cells. Mechanistically, COPZ2 suppressed tumor development by participating in the regulation of the PI3K-AKT signaling pathway. Conclusion: Our results demonstrated that the elevation of COPZ2 was associated with the prognosis and progression of glioma, and it might be a potential diagnostic and prognostic biomarker for glioma.

A Comprehensive Pan-Cancer Analysis of the Regulation and Prognostic Effect of Coat Complex Subunit Zeta 1.

The Coatomer protein complex Zeta 1 (COPZ1) has been reported to play an essential role in maintaining the survival of some types of tumors. In this study, we sought to explore the molecular characteristics of COPZ1 and its clinical prognostic value through a pan-cancers bioinformatic analysis. We found that COPZ1 was extremely prevalent in a variety of cancer types, and high expression of COPZ1 was linked to poor overall survival in many cancers, while low expression in LAML and PADC was correlated with tumorigenesis. Besides, the CRISPR Achilles' knockout analysis revealed that COPZ1 was vital for many tumor cells' survival. We further demonstrated that the high expression level of COPZ1 in tumors was regulated in multi-aspects, including abnormal CNV, DNA-methylation, transcription factor and microRNAs. As for the functional exploration of COPZ1, we found a positive relationship between COPZ1's expression and stemness and hypoxia signature, especially the contribution of COPZ1 on EMT ability in SARC. GSEA analysis revealed that COPZ1 was associated with many immune response pathways. Further investigation demonstrated that COPZ expression was negatively correlated with immune score and stromal score, and low expression of COPZ1 has been associated to more antitumor immune cell infiltration and pro-inflammatory cytokines. The further analysis of COPZ1 expression and anti-inflammatory M2 cells showed a consistent result. Finally, we verified the expression of COPZ1 in HCC cells, and proved its ability of sustaining tumor growth and invasion with biological experiments. Our study provides a multi-dimensional pan-cancer analysis of COPZ and demonstrates that COPZ1 can serve as both a prospective target for the treatment of cancer and a prognostic marker for a variety of cancer types.

Copper Efflux System Required in Murine Lung Infection by Haemophilus influenzae Composed of a Canonical ATPase Gene and Tandem Chaperone Gene Copies.

Copper is an essential micronutrient but is toxic at high concentrations. In Haemophilus influenzae mechanisms of copper resistance and its role in pathogenesis are unknown; however, our previous genetic screen by transposon insertion-site sequencing implicated a putative cation transporting ATPase (copA) in survival in a mouse lung infection model. Here, we demonstrate that H. influenzae copA (HI0290) is responsible for copper homeostasis involving the merR-type regulator, cueR, as well as six tandem copies of the metallochaperone gene, copZ. Deletion of the ATPase and metallochaperone genes resulted in increased sensitivity to copper but not to cobalt, zinc, or manganese. Nontypeable H. influenzae (NTHi) clinical isolate NT127 has the same locus organization but with three copies of copZ. We showed that expression of the NTHi copZA operon is activated by copper under the regulatory control of CueR. NTHi single copA and copZ mutants and, especially, the double deletion copZA mutant exhibited decreased copper tolerance, and the ΔcopZA mutant accumulated 97% more copper than the wild type when grown in the presence of 0.5 mM copper sulfate. Mutants of NT127 deleted of the ATPase (copA) alone and deleted of both the ATPase and chaperones (copZ1-3) were 4-fold and 20-fold underrepresented compared to the parent strain during mixed-infection lung challenge, respectively. Complementation of cop locus deletion mutations restored copper resistance and virulence properties. NTHi likely encounters copper as a host defense mechanism during lung infection, and our results indicate that the cop system encodes an important countermeasure to alleviate copper toxicity.

BMI1 promotes the proliferation and inhibits autophagy of breast cancer cells by activating COPZ1

This study was designed to explore the role of COPZ1 in breast cancer as well as discuss its specific reaction mechanism.MethodsWith the help of RT-qPCR and western blot, the expression of BMI1 and COPZ1 were measured. Then, the proliferation, colony formation and apoptosis were evaluated by CCK-8, colony formation and TUNEL assays, separately. Luciferase reporter assay and ChIP were applied to assess the relative activity of COPZ1 promoter as well as its binding with BMI1. Moreover, western blot was utilized to measure the expression of proliferation-, apoptosis- and autophagy-related proteins.ResultsAccording to GEPIA2 database, COPZ1 was upregulated in breast cancer tissues and was associated with the poor prognosis (P = 0.03). Results obtained from RT-qPCR and western blot verified that COPZ1 expression was greatly increased at both mRNA and protein levels in breast cancer cells as compared to control cells (P < 0.05 or P < 0.001). COPZ1 knockdown inhibited the proliferation, induced the autophagy and promoted the apoptosis of breast cancer cells. HumanTFDB predicted the binding sites of BMI1 and COPZ1. The increased relative luciferase activity of COPZ1 promoter following BMI1 overexpression (P < 0.001) and the binding of BMI1 with COPZ1 promoter indicated that BMI1 could activate COPZ1. Further experiments suggested that the effects of COPZ1 knockdown on the proliferation, apoptosis and autophagy of breast cancer cells were reversed by BMI1 overexpression, implying that BMI1 promoted the proliferation and repressed the autophagy of breast cancer cells via activating COPZ1.ConclusionsTo sum up, BMI1 exhibited promotive effects on the malignant progression of breast cancer through the activation of COPZ1. These findings might offer a preliminary theoretical basis for COPZ1 participation in autophagy in breast cancer cells. (AU)

BMI1 promotes the proliferation and inhibits autophagy of breast cancer cells by activating COPZ1.

PURPOSE: This study was designed to explore the role of COPZ1 in breast cancer as well as discuss its specific reaction mechanism. METHODS: With the help of RT-qPCR and western blot, the expression of BMI1 and COPZ1 were measured. Then, the proliferation, colony formation and apoptosis were evaluated by CCK-8, colony formation and TUNEL assays, separately. Luciferase reporter assay and ChIP were applied to assess the relative activity of COPZ1 promoter as well as its binding with BMI1. Moreover, western blot was utilized to measure the expression of proliferation-, apoptosis- and autophagy-related proteins. RESULTS: According to GEPIA2 database, COPZ1 was upregulated in breast cancer tissues and was associated with the poor prognosis (P = 0.03). Results obtained from RT-qPCR and western blot verified that COPZ1 expression was greatly increased at both mRNA and protein levels in breast cancer cells as compared to control cells (P < 0.05 or P < 0.001). COPZ1 knockdown inhibited the proliferation, induced the autophagy and promoted the apoptosis of breast cancer cells. HumanTFDB predicted the binding sites of BMI1 and COPZ1. The increased relative luciferase activity of COPZ1 promoter following BMI1 overexpression (P < 0.001) and the binding of BMI1 with COPZ1 promoter indicated that BMI1 could activate COPZ1. Further experiments suggested that the effects of COPZ1 knockdown on the proliferation, apoptosis and autophagy of breast cancer cells were reversed by BMI1 overexpression, implying that BMI1 promoted the proliferation and repressed the autophagy of breast cancer cells via activating COPZ1. CONCLUSIONS: To sum up, BMI1 exhibited promotive effects on the malignant progression of breast cancer through the activation of COPZ1. These findings might offer a preliminary theoretical basis for COPZ1 participation in autophagy in breast cancer cells.

The CopA2-Type P1B-Type ATPase CcoI Serves as Central Hub for cbb 3-Type Cytochrome Oxidase Biogenesis.

Copper (Cu)-transporting P1B-type ATPases are ubiquitous metal transporters and crucial for maintaining Cu homeostasis in all domains of life. In bacteria, the P1B-type ATPase CopA is required for Cu-detoxification and exports excess Cu(I) in an ATP-dependent reaction from the cytosol into the periplasm. CopA is a member of the CopA1-type ATPase family and has been biochemically and structurally characterized in detail. In contrast, less is known about members of the CopA2-type ATPase family, which are predicted to transport Cu(I) into the periplasm for cuproprotein maturation. One example is CcoI, which is required for the maturation of cbb 3-type cytochrome oxidase (cbb 3-Cox) in different species. Here, we reconstituted purified CcoI of Rhodobacter capsulatus into liposomes and determined Cu transport using solid-supported membrane electrophysiology. The data demonstrate ATP-dependent Cu(I) translocation by CcoI, while no transport is observed in the presence of a non-hydrolysable ATP analog. CcoI contains two cytosolically exposed N-terminal metal binding sites (N-MBSs), which are both important, but not essential for Cu delivery to cbb 3-Cox. CcoI and cbb 3-Cox activity assays in the presence of different Cu concentrations suggest that the glutaredoxin-like N-MBS1 is primarily involved in regulating the ATPase activity of CcoI, while the CopZ-like N-MBS2 is involved in Cu(I) acquisition. The interaction of CcoI with periplasmic Cu chaperones was analyzed by genetically fusing CcoI to the chaperone SenC. The CcoI-SenC fusion protein was fully functional in vivo and sufficient to provide Cu for cbb 3-Cox maturation. In summary, our data demonstrate that CcoI provides the link between the cytosolic and periplasmic Cu chaperone networks during cbb 3-Cox assembly.

COPZ1 depletion in thyroid tumor cells triggers type I IFN response and immunogenic cell death.

The coatomer protein complex zeta 1 (COPZ1) represents a non-oncogene addiction for thyroid cancer (TC); its depletion impairs the viability of thyroid tumor cells, leads to abortive autophagy, ER stress, UPR and apoptosis, and reduces tumor growth of TC xenograft models. In this study we investigated the molecular pathways activated by COPZ1 depletion and the paracrine effects on cellular microenvironment and immune response. By comprehensive and target approaches we demonstrated that COPZ1 depletion in TPC-1 and 8505C thyroid tumor cell lines activates type I IFN pathway and viral mimicry responses. The secretome from COPZ1-depleted cells was enriched for several inflammatory molecules and damage-associated molecular patterns (DAMPs). Moreover, we found that dendritic cells, exposed to these secretomes, expressed high levels of differentiation and maturation markers, and stimulated the proliferation of naïve T cells. Interestingly, T cells stimulated with COPZ1-depleted cells showed increased cytotoxic activity against parental tumor cells. Collectively, our findings support the notion that targeting COPZ1 may represent a promising therapeutic approach for TC, considering its specificity for cancer cells, the lack of effect on normal cells, and the capacity to prompt an anti-tumor immune response.

Targeting COPZ1 non-oncogene addiction counteracts the viability of thyroid tumor cells.

Thyroid carcinoma is generally associated with good prognosis, but no effective treatments are currently available for aggressive forms not cured by standard therapy. To find novel therapeutic targets for this tumor type, we had previously performed a siRNA-based functional screening to identify genes essential for sustaining the oncogenic phenotype of thyroid tumor cells, but not required to the same extent for the viability of normal cells (non-oncogene addiction paradigm). Among those, we found the coatomer protein complex ζ1 (COPZ1) gene, which is involved in intracellular traffic, autophagy and lipid homeostasis. In this paper, we investigated the mechanisms through which COPZ1 depletion leads to thyroid tumor cell death. We showed that siRNA-mediated COPZ1 depletion causes abortive autophagy, endoplasmic reticulum stress, unfolded protein response and apoptosis. Interestingly, we observed that mouse tumor xenografts, locally treated with siRNA targeting COPZ1, showed a significant reduction of tumor growth. On the whole, we demonstrated for the first time the crucial role of COPZ1 in the viability of thyroid tumor cells, suggesting that it may be considered an attractive target for novel therapeutic approaches for thyroid cancer.

Identification of thyroid tumor cell vulnerabilities through a siRNA-based functional screening.

The incidence of thyroid carcinoma is rapidly increasing. Although generally associated with good prognosis, a fraction of thyroid tumors are not cured by standard therapy and progress to aggressive forms for which no effective treatments are currently available. In order to identify novel therapeutic targets for thyroid carcinoma, we focused on the discovery of genes essential for sustaining the oncogenic phenotype of thyroid tumor cells, but not required to the same degree for the viability of normal cells (non-oncogene addiction paradigm). We screened a siRNA oligonucleotide library targeting the human druggable genome in thyroid cancer BCPAP cell line in comparison with immortalized normal human thyrocytes (Nthy-ori 3-1). We identified a panel of hit genes whose silencing interferes with the growth of tumor cells, while sparing that of normal ones. Further analysis of three selected hit genes, namely Cyclin D1, MASTL and COPZ1, showed that they represent common vulnerabilities for thyroid tumor cells, as their inhibition reduced the viability of several thyroid tumor cell lines, regardless the histotype or oncogenic lesion. This work identified non-oncogenes essential for sustaining the phenotype of thyroid tumor cells, but not of normal cells, thus suggesting that they might represent promising targets for new therapeutic strategies.

Clinical relevance of miR-mediated HLA-G regulation and the associated immune cell infiltration in renal cell carcinoma.

In human tumors of distinct origin including renal cell carcinoma (RCC), the non-classical human leukocyte antigen G (HLA-G) is frequently expressed, thereby inhibiting the cytotoxic activity of T and natural killer (NK) cells. Recent studies demonstrated a strong post-transcriptional gene regulation of the HLA-G by miR-152, -148A, -148B and -133A. Standard methods were applied to characterize the expression and function of HLA-G, HLA-G-regulatory microRNAs (miRs) and the immune cell infiltration in 453 RCC lesions using a tissue microarray and five RCC cell lines linking these results to clinical parameters. Direct interactions with HLA-G regulatory miRs and the HLA-G 3' untranslated region (UTR) were detected and the affinities of these different miRs to the HLA-G 3'-UTR compared. qPCR analyses and immunohistochemical staining revealed an inverse expression of miR-148A and -133A with the HLA-G protein in situ and in vitro. Stable miR overexpression caused a downregulation of HLA-G protein enhancing the NK and LAK cell-mediated cytotoxicity in in vitro CD107a activation assays revealing a HLA-G-dependent cytotoxic activity of immune effector cells. A significant higher frequency of CD3+/CD8+ T cell lymphocytes, but no differences in the activation markers CD69, CD25 or in the presence of CD56+, FoxP3+ and CD4+ immune cells were detected in HLA-G+ compared to HLA-G- RCC lesions. This could be associated with higher WHO grade, but not with a disease-specific survival. These data suggest a miR-mediated control of HLA-G expression in RCC, which is associated with a distinct pattern of immune cell infiltration.

Metal ion homeostasis in Listeria monocytogenes and importance in host-pathogen interactions.

Listeria monocytogenes is responsible for one of the most life-threatening food-borne infections and the leading cause of food-poisoning associated deaths in the UK. Infection may be of the unborn/newly born infant where disease may manifest as listeric abortion, stillbirth or late-onset neonatal listeriosis, while in adults, infection usually affects the central nervous system causing meningitis. Crucial to the survival of L. monocytogenes, both inside and outside the host, is its ability to acquire metals which act as cofactors for a broad range of its cellular proteins. However, L. monocytogenes must also protect itself against the innate toxicity of metals. The importance of metals in host-pathogen interactions is illustrated by the restriction of metals (including zinc and iron) in vertebrates in response to infection and the use of high levels of metals (copper and zinc) as part of the antimicrobial defences within host phagocytes. As such, L. monocytogenes is equipped with various mechanisms to tightly control its cellular metal pools and avoid metal poisoning. These include multiple DNA-binding metal-responsive transcription factors, metal-acquisition, metal-detoxification and metal-storage systems, some of which represent key L. monocytogenes virulence determinants. This review discusses current knowledge of the role of metals in L. monocytogenes infections, with a focus on the mechanisms that contribute to zinc and copper homeostasis in this organism. The requirement to precisely control cellular metal levels may impose a vulnerability to L. monocytogenes which can be exploited in antimicrobials and therapeutics.