ONZIN Upregulation by Mutant p53 Contributes to Osteosarcoma Metastasis Through the CXCL5-MAPK Signaling Pathway.

BACKGROUND/AIMS: Gain-of-function of mutant p53 is associated with a high rate of lung metastasis in osteosarcoma. To investigate the mechanism of mutant p53-induced osteosarcoma metastasis, expression array analysis was performed, comparing non-metastatic osteosarcomas from p53+/- mice with metastatic osteosarcomas from p53R172H/+ mice. Onzin (Plac8) was identified as one of the genes upregulated in p53R172H/+ mouse metastatic osteosarcomas. Accordingly, we investigated the role of ONZIN in human osteosarcoma metastasis. METHODS: ONZIN function and its downstream targets were examined in osteosarcoma cell lines. Assays related to tumorigenesis and metastasis, including cell migration, invasion, clonogenic survival, and soft agar colony formation, were performed in osteosarcoma cells. Additionally, mouse xenograft models were used to examine the role of ONZIN overpression in tumorigenesis in vivo. Lastly, 87 osteosarcoma patients were recruited to investigate the clinical relevance of ONZIN overexpression in metastasis and prognosis. RESULTS: ONZIN overexpression enhanced osteosarcoma cell proliferation, clonogenic survival, migration, and invasion independent of p53 status. Furthermore, ONZIN overexpression induced CXCL5 upregulation and resulted in increased ERK phosphorylation, which contributed to more aggressive osteosarcoma metastatic phenotypes. More importantly, overexpression of ONZIN in human osteosarcoma patients was closely associated with lung metastasis, poor prognoses, and survival. CONCLUSIONS: Overexpression of ONZIN promotes osteosarcoma progression and metastasis, and can serve as a clinical biomarker for osteosarcoma metastasis and prognosis.

High expression of PLA2G16 is associated with a better prognosis in HER2-positive breast cancer.

BACKGROUND: PLA2G16 functions as a phosphatase in metabolism and its abnormal expression is closely associated with tumor progression. The aim of this study is to investigate the prognosis value of PLA2G16 in breast cancer. METHODS: A tissue microarray including 200 invasive ductal carcinoma specimens was constructed. Immunohistochemistry was performed to determine the PLA2G16 expression status. The Kaplan-Meier analysis and log-rank test were used to evaluate the prognostic value of PLA2G16. The Multivariate Cox regression analysis was performed to identify whether PLA2G16 was an independent prognostic factor. RESULTS: In our retrospective study, Kaplan-Meier analysis showed that elevated PLA2G16 expression was correlated with improved DFS (P=0.032) in the whole breast cancer patients. In further subgroup analysis, PLA2G16 overexpression was found to be associated with prolonged DFS (P=0.018) in HER2-positive breast cancer patients. More importantly, Multivariate analysis suggested that PLA2G16 was a significant independent prognostic factor in HER2-enriched patients [hazard ratio (HR) =0.151; 95% confidence interval (CI) =0.034-0.672; P=0.013]. CONCLUSIONS: Our study evaluated the prognostic significance of PLA2G16 in patients with HER2-positive breast cancer and confirmed the relevance of this metabolism-related gene in patient outcome.

PLA2G16 promotes osteosarcoma metastasis and drug resistance via the MAPK pathway.

The prognosis of metastatic osteosarcoma is dismal and a better understanding of the mechanisms underlying disease progression is essential to improve treatment options and patient outcomes. We previously demonstrated Pla2g16 overexpression in mouse osteosarcoma contributes to metastasis phenotypes and increased expression of PLA2G16 is associated with metastasis and poor prognosis in human tumors. To further examine the mechanisms through which PLA2G16 contributes to human osteosarcoma metastasis and explore the potential of PLA2G16 as a therapeutic target in osteosarcoma, we generated a panel of human osteosarcoma cell lines expressing different levels of PLA2G16. The functional analyses of these cell lines demonstrated high levels of PLA2G16 expression increased osteosarcoma cell migration, invasion, clonogenic survival, and anchorage-independent colony formation. Importantly, this activity was dependent on the phospholipase activity of PLA2G16. Additionally, PLA2G16 overexpression decreased the sensitivity of cells to a panel of chemotherapeutic agents. Analysis of downstream pathways revealed the pro-metastasis functions of PLA2G16 were mediated through the MAPK pathway, as knockdown of PLA2G16 decreased ERK1/2 phosphorylation and pharmacological inhibition of MEK significantly repressed PLA2G16 mediated cell migration and clonogenic survival. Furthermore, PLA2G16 overexpression promoted xenograft tumor growth in vivo, and these tumors exhibit increased ERK1/2 phosphorylation. Lastly, the expression of PLA2G16 is strongly correlated with the increased ERK1/2 phosphorylation in human osteosarcoma samples, and the combined lesions are associated with reduced overall and metastasis-free survival. Collectively, these results demonstrate increased PLA2G16 expression activates the MAPK pathway to enhance osteosarcoma metastasis and may be a novel therapeutic target for these cancers.

Coexpression of CXCR4 and MMP9 predicts lung metastasis and poor prognosis in resected osteosarcoma.

Osteosarcoma is a highly aggressive bone disease with a tendency to metastasize to the lung. The 5-year survival of patients with metastatic osteosarcoma is only 20 %. Many studies have demonstrated SDF-1/CXCR4 and MMP9 play important roles in the metastasis of malignant tumors, including osteosarcoma. The aim of this study was to investigate the association of CXCR4 and MMP9 expression with clinicopathological features and pulmonary metastasis in osteosarcoma. Using tumor tissue microarrays, we analyzed the expression of CXCR4 and MMP9 among 34 primary osteosarcomas with pulmonary metastasis and 62 primary osteosarcomas without metastasis. A median time of 57.5 months (range: 6 to 171 months) follow-up was performed to evaluate tumor metastasis and the patient survival. The prognostic values were determined by univariate Kaplan-Meier survival analysis and multivariate Cox proportional hazard model analysis. The accuracy of oncologic outcome prediction was evaluated by receiver-operating characteristics (ROC) curves (AUC). The expression of CXCR4 and MMP9 was significantly correlated in tumor tissues (P = 0.026). Both CXCR4 and MMP9 were independent predictors for overall survival and metastasis-free survival by Cox multivariate analysis, and high expression for both CXCR4 and MMP9 were even more significant and better biomarkers for osteosarcoma metastasis and survival. The combination of CXCR4 and MMP9 high expression is very likely to be a valuable independent predictor of lung metastasis and survival in osteosarcoma patients.

PLA2G16 Expression in Human Osteosarcoma Is Associated with Pulmonary Metastasis and Poor Prognosis.

BACKGROUND: Osteosarcoma is the most frequent type of malignant bone tumor in children and adolescents and is associated with a high propensity for lung metastasis. Recent experiments have indicated that PLA2G16 contributes to osteosarcoma progression and metastasis in both mouse and human osteosarcoma cell lines. The aim of this study was to compare the expression of PLA2G16 in non-metastatic and metastatic osteosarcomas to determine whether PLA2G16 expression can serve as a biomarker of osteosarcoma prognosis and metastasis. METHODS: Quantitative real-time PCR was used to examine PLA2G16 mRNA in primary osteosarcoma patients (18 patients without metastases and 17 patients with metastases), and immunohistochemistry (IHC) staining of PLA2G16 was performed on tissue microarrays from 119 osteosarcoma patients. Tumor metastatic behavior and survival of the patients were followed up for a minimum of 36 months and a maximum of 171 months. The prognostic value of PLA2G16 expression was evaluated by the Kaplan-Meier method and a log-rank test. Multivariate Cox regression analysis was used to identify significant independent prognostic factors. RESULTS: Osteosarcoma patients with metastasis showed a higher expression of PLA2G16 at both the mRNA and protein levels (both at P values< 0.05) than did patients without metastasis. Osteosarcoma patients with positive IHC staining of PLA2G16 expression at primary sites had shorter overall survival and metastasis-free survival (both at P values <0.02). Moreover, multivariate Cox analysis identified PLA2G16 expression as an independent prognostic factor to predict poor overall survival and metastasis-free survival (both P values < 0.03). CONCLUSIONS: This study indicated that PLA2G16 expression is a significant prognostic factor in primary osteosarcoma patients for predicting the development of metastases and poor survival.

Mitochondria-mediated apoptosis in mammals.

The mitochondria-mediated caspase activation pathway is a major apoptotic pathway characterized by mitochondrial outer membrane permeabilization (MOMP) and subsequent release of cytochrome c into the cytoplasm to activate caspases. MOMP is regulated by the Bcl-2 family of proteins. This pathway plays important roles not only in normal development, maintenance of tissue homeostasis and the regulation of immune system, but also in human diseases such as immune disorders, neurodegeneration and cancer. In the past decades the molecular basis of this pathway and the regulatory mechanism have been comprehensively studied, yet a great deal of new evidence indicates that cytochrome c release from mitochondria does not always lead to irreversible cell death, and that caspase activation can also have non-death functions. Thus, many unsolved questions and new challenges are still remaining. Furthermore, the dysfunction of this pathway involved in cancer development is obvious, and targeting the pathway as a therapeutic strategy has been extensively explored, but the efficacy of the targeted therapies is still under development. In this review we will discuss the mitochondria-mediated apoptosis pathway and its physiological roles and therapeutic implications.

Efficient soluble expression of secreted matrix metalloproteinase 26 in Brevibacillus choshinensis.

Matrix metalloproteinase 26 (MMP-26) is a novel member of the matrix metalloproteinase family with minimal domain constitution and unknown physiological function. The three-dimensional (3D) structure of the enzyme also remains to be deciphered. Previous studies show that MMP-26 may be expressed in Escherichia coli (E. coli) as inclusion bodies and re-natured with catalytic activity. However, the low re-naturation rate of this method limits its usage in structural studies. In this paper, we tried to clone, express and purify the pro form and catalytic form of MMP-26 (ProMMP-26 and CatMMP-26) in several widely used expression vectors and express the recombinant MMP-26 proteins in E. coli cells. These constructs resulted in insoluble expressions or soluble expressions of MMP-26 with little catalytic activity. We then used Brevibacillus choshinensis (B. choshinensis) as the host system for the soluble and active expression of MMP-26. The enzyme was secreted in soluble form in the supernatant of cell culture medium and purified via a two-step purification process that included Ni(2+) affinity chromatography followed by gel filtration. The yields of purified ProMMP-26 and CatMMP-26 were 12 and 18mg/L, respectively, with high purity and homogeneity. Both ProMMP-26 and CatMMP-26 showed gelatin zymography activity and the purified CatMMP-26 had high enzymatic activity against DQ-gelatin substrate. The large-scale soluble and active protein production for future structural studies of MMP-26 is thus feasible using the B. choshinensis host system.

Aluminum ammonium sulfate dodecahydrate purified from traditional Chinese medicinal herb Korean monkshood root is a potent matrix metalloproteinase inhibitor.

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases and key regulators for many physiological and pathological functions. The MMP inhibitors have been shown to modulate diseases such as cancer, inflammation, and cardiovascular diseases. In this paper we tracked the MMP inhibitory activities of the traditional Chinese medicinal herb Korean Monkshood Root. The purified active ingredient was identified by the elemental analysis, infrared spectrum (IR) and X-ray diffraction as aluminum ammonium sulfate dodecahydrate. This inorganic compound showed inhibitory activities toward a number of MMP family members. In particular, it has a strong inhibitory effect toward MMP-2 and MMP-9, with IC50 values of 0.54 and 0.50 µM, respectively. Further analysis suggested that the MMP inhibitory activity is mainly due to Al(3+). Cell viability assays using human fibrosarcoma HT1080 cells showed aluminum ammonium sulfate had minimal cyto-toxicity with a concentration up to 500 µM. However, within 50 µM, it exhibited significant inhibition of cell invasion. To our knowledge, there has been no previous report of inorganic form of the MMP inhibitor with strong inhibitory activity. Our results for the first time showed that aluminum ammonium sulfate is an inorganic form of MMP inhibitor with high potency, and can be used to interfere with MMP related cellular processes.

Ubiquitin-intein and SUMO2-intein fusion systems for enhanced protein production and purification.

Although most commonly used for protein production, expression of soluble and functional recombinant protein in Escherichia coli is still a major challenge. The development and application of fusion tags that can facilitate protein expression and solubility partly solve this problem, however, under most circumstance, the fusion tags have to be removed by proteases in order to use the proteins. Because the tag removal using proteases increases cost and introduces extra purification steps, it remains a significant problem that must be resolved before being widely used in industry production. Ubiquitin and SUMO have been successfully used to enhance protein expression and solubility. In the last decades, intein has also been widely used in protein production for its self-cleavage property, which could help to remove the fusion tag without any protease. Here, we take the advantages of ubiquitin, SUMO2 and intein in protein expression. We constructed tandem ubiquitin-intein and SUMO2-intein fusion tags, and chose human MMP13 (amino acid 104-274) and eGFP as the passenger proteins that fused to the C-terminus of the tags. These constructs were expressed in E. coli and both MMP13 and eGFP expression and solubility were evaluated. Both tags showed the ability to enhance the solubility of MMP13 and eGFP and improve the expression of eGFP, and the SUMO2-intein having a more significant effect. Both ubiquitin-intein-eGFP and SUMO2-intein-eGFP were purified using Ni-NTA column chromatography and self-cleavaged by changing pH. The recombinant un-tagged eGFP were released and eluted with high homogeneity. In summary, ubiquitin-intein and SUMO2-intein are convenient and useful fusion tags that can enhance the expression, solubility and improve the purification process of the model heterologous protein and these tags may have a good prospect in protein production.

Human SUMO fusion systems enhance protein expression and solubility.

A major challenge associated with recombinant protein production in Escherichia coli is generation of large quantities of soluble, functional protein. Yeast SUMO (small ubiquitin-related modifier), has been shown to enhance heterologous protein expression and solubility as fusion tag, however, the effects of human SUMOs on protein expression have not been investigated. Here we describe the use of human SUMO1 and SUMO2 as a useful gene fusion technology. Human SUMO1 and SUMO2 fusion expression vectors were constructed and tested in His-tag and ubiquitin fusion expression systems. Two difficult-to-express model proteins, matrix metalloprotease-13 (MMP13) and enhanced green fluorescence protein (eGFP) were fused to the C-terminus of the human SUMO1 and SUMO2 expression vectors. These constructs were expressed in E. coli and evaluation of MMP13 and eGFP expression and solubility was conducted. We found that both SUMO1 and SUMO2 had the ability to enhance the solubility of MMP13 and eGFP, with the SUMO2 tag having a more significant effect. Since fusion tags produce varying quantities of soluble proteins, we assessed the effect of SUMO2 coupled with ubiquitin (Ub). SUMO2-ubiquitin and ubiquitin-SUMO2 fusion expression plasmids were constructed with eGFP as a passenger protein. Following expression in E. coli, both plasmids could improve eGFP expression and solubility similar to the SUMO2 fusion and better than the ubiquitin fusion. The sequential order of SUMO2 and ubiquitin had little effect on expression and solubility of eGFP. Purification of eGFP from the gene fusion product, SUMO2-ubiquitin-eGFP, involved cleavage by a deubiquitinase (Usp2-cc) and Ni-Sepharose column chromatography. The eGFP protein was purified to high homogeneity. In summary, human SUMO1 and SUMO2 are useful gene fusion technologies enhancing the expression, solubility and purification of model heterologous proteins.