ZNF692 promotes osteosarcoma cell proliferation, migration, and invasion through TNK2-mediated activation of the MEK/ERK pathway.

BACKGROUND: Osteosarcoma is a diverse and aggressive bone tumor. Driver genes regulating osteosarcoma initiation and progression remains incompletely defined. Zinc finger protein 692 (ZNF692), a kind of Krüppel C2H2 zinc finger transcription factor, exhibited abnormal expression in different types of malignancies and showed a correlation with the clinical prognosis of patients as well as the aggressive characteristics of cancer cells. Nevertheless, its specific role in osteosarcoma is still not well understood. METHODS: We investigated the dysregulation and clinical significance of ZNF692 in osteosarcoma through bioinformatic method and experimental validation. A range of in vitro assays, including CCK-8, colony formation, EdU incorporation, wound healing, and transwell invasion tests, were conducted to assess the impact of ZNF692 on cell proliferation, migration, and invasion in osteosarcoma. A xenograft mouse model was established to evaluate the effect of ZNF692 on tumor growth in vivo. Western blot assay was used to measure the protein levels of MEK1/2, P-MEK1/2, ERK1/2, and P-ERK1/2 in cells that had been genetically modified to either reduce or increase the expression of ZNF692. The relationship between ZNF692 and tyrosine kinase non-receptor 2 (TNK2) were validated by qRT-PCR, chromatin immunoprecipitation and luciferase reporter assays. RESULTS: Expression of ZNF692 was increased in both human osteosarcoma tissues and cell lines. Furthermore, the expression of ZNF692 served as an independent predictive biomarker in osteosarcoma. The results of the survival analysis indicated that increased expression of ZNF692 was associated with worse outcome. Downregulation of ZNF692 inhibits the proliferation, migration, and invasion of osteosarcoma cells, whereas upregulation of ZNF692 has the opposite impact. Western blot assay indicates that reducing ZNF692 decreases phosphorylation of MEK1/2 and ERK1/2, whereas increasing ZNF692 expression enhances their phosphorylation. U0126, a potent inhibitor specifically targeting the MEK/ERK signaling pathway, partially counteracts the impact of ZNF692 overexpression on the proliferation, migration, and invasion of osteosarcoma cells. In addition, ZNF692 specifically interacts with the promoter region of TNK2 and stimulates the transcription of TNK2 in osteosarcoma cells. Forcing the expression of TNK2 weakens the inhibitory impact of ZNF692 knockdown on P-MEK1/2 and P-ERK1/2. Similarly, partly inhibiting TNK2 counteracts the enhancing impact of ZNF692 overexpression on the phosphorylation of MEK1/2 and ERK1/2. Functional tests demonstrate that the suppressive effects of ZNF692 knockdown on cell proliferation, migration, and invasion are greatly reduced when TNK2 is overexpressed. In contrast, the reduction of TNK2 hinders the ability of ZNF692 overexpression to enhance cell proliferation, migration, and invasion. CONCLUSION: ZNF692 promotes the proliferation, migration, and invasion of osteosarcoma cells via the TNK2-dependent stimulation of the MEK/ERK signaling pathway. The ZNF692-TNK2 axis might potentially function as a possible predictive biomarker and a promising target for novel therapeutics in osteosarcoma.

Identification of allograft inflammatory factor-1 suppressing the progression and indicating good prognosis of osteosarcoma.

BACKGROUND: Osteosarcoma is one of the most common cancers worldwide. Intense efforts have been made to elucidate the pathogeny, but the mechanisms of osteosarcoma are still not well understood. We aimed to investigate the potential biomarker, allograft inflammatory factor-1 (AIF1), affecting the progression and prognosis of osteosarcoma. METHODS: Three microarray datasets were downloaded from GEO datasets and one was obtained from the TCGA dataset. The differentially expressed genes (DEGs) were identified. GO and KEGG functional enrichment analyses of overlapped DEGs were performed. The PPI network of overlapped DEGs was constructed by STRING and visualized with Cytoscape. Overall survival (OS) and Metastasis free survival (MFS) were analyzed from GSE21257. Finally, the effect of the most relevant core gene affecting the progression of osteosarcoma was examined in vitro. RESULTS: One hundred twenty six DEGs were identified, consisting of 65 upregulated and 61 downregulated genes. Only AIF1 was significantly associated with OS and MFS. It was found that AIF1 could be enriched into the NF-κB signaling pathway. GSEA and ssGSEA analyses showed that AIF1 was associated with the immune invasion of tumors. Cell experiments showed that AIF1 was underexpressed in osteosarcoma cell lines, while the malignant propriety was attenuated after overexpressing the expression of AIF1. Moreover, AIF1 also affects the expression of the NF-κB pathway. CONCLUSION: In conclusion, DEGs and hub genes identified in the present study help us understand the molecular mechanisms underlying the carcinogenesis and progression of osteosarcoma, and provide candidate targets for diagnosis and treatment of osteosarcoma.

Construction of a prognostic risk score model based on the ARHGAP family to predict the survival of osteosarcoma.

BACKGROUND: Osteosarcoma (OS) is the most common primary malignancy of bone tumors. More and more ARHGAP family genes have been confirmed are to the occurrence, development, and invasion of tumors. However, its significance in osteosarcoma remains unclear. In this study, we aimed to identify the relationship between ARHGAP family genes and prognosis in patients with OS. METHODS: OS samples were retrieved from the TCGA and GEO databases. We then performed LASSO regression analysis and multivariate COX regression analysis to select ARHGAP family genes to construct a risk prognosis model. We then validated this prognostic model. We utilized ESTIMATE and CIBERSORT algorithms to calculate the stroma and immune scores of samples, as well as the proportions of tumor infiltrating immune cells (TICs). Finally, we conducted in vivo and in vitro experiments to investigate the effect of ARHGAP28 on osteosarcoma. RESULTS: We selected five genes to construct a risk prognosis model. Patients were divided into high- and low-risk groups and the survival time of the high-risk group was lower than that of the low-risk group. The high-risk group in the prognosis model constructed had relatively poor immune function. GSEA and ssGSEA showed that the low-risk group had abundant immune pathway infiltration. The overexpression of ARHGAP28 can inhibit the proliferation, migration, and invasion of osteosarcoma cells and tumor growth in mice, and IHC showed that overexpression of ARHGAP28 could inhibit the proliferation of tumor cells. CONCLUSION: We constructed a risk prognostic model based on five ARHGAP family genes, which can predict the overall survival of patients with osteosarcoma, to better assist us in clinical decision-making and individualized treatment.

TRIM26 inhibited osteosarcoma progression through destabilizing RACK1 and thus inactivation of MEK/ERK signaling.

Osteosarcoma is a highly aggressive malignant tumor that is common in the pediatric population and has a high rate of disability and mortality. Recent studies have suggested that the tripartite motif-containing family genes (TRIMs) play critical roles in oncogenesis in several cancers. TRIM26, one of the TRIMs family genes, was more frequently reported to exert a tumor-suppressive role, while its detailed functional roles in the osteosarcoma progression were still unknown and require further investigation. Herein, we found that TRIM26 was markedly downregulated in osteosarcoma tissues and cells. Survival analysis revealed that higher expression of TRIM26 was associated with better prognosis and its expression was an independent protective factor in osteosarcoma. Functional analysis demonstrated that overexpression of TRIM26 inhibited osteosarcoma cell proliferation and invasion via inhibiting the EMT process and MEK/ERK signaling. In contrast, the silence of TRIM26 caused the opposite effect. RACK1, a member of the Trp-Asp repeat protein family, was identified as a novel target of TRIM26. TRIM26 could interact with RACK1 and accelerate the degradation of RACK1, thus inactivation of MEK/ERK signaling. Overexpression of RACK1 could attenuate the inhibitory effect of TRIM26 overexpression on p-MEK1/2 and p-ERK1/2, and silence of RACK1 could partly impair the effect of TRIM26 knockdown-induced upregulation of p-MEK1/2 and p-ERK1/2. Further, a series of gain- and loss-of-function experiments showed that decreased malignant behaviors including cell proliferation and invasion in TRIM26-upregulated cells were reversed when RACK1 was overexpressed, whereas RACK1 knockdown diminished the increased malignant phenotypes in TRIM26-silenced osteosarcoma cells. In conclusion, our study indicated that TRIM26 inhibited osteosarcoma progression via promoting proteasomal degradation of RACK1, thereby resulting in inactivation of MEK/ERK signaling, and impeding the EMT process.

A novel TSC1 frameshift mutation c.1550_1551del causes tuberous sclerosis complex by aberrant splicing and nonsense-mediated mRNA degradation (NMD) simultaneously in a Chinese family.

BACKGROUND: Tuberous sclerosis complex (TSC), belongs to autosomal dominant genetic disorder, which affects multiple organ systems in the body, including the skin, brain, lungs, kidneys, liver, and eyes. Mutations in TSC1 or TSC2 was proved to be associated with these conditions. METHODS: Gene-panel Sequence of NGS was used to detect the mutation in a Chinese family. The research further investigates whether aberrant splicing and nonsense-mediated mRNA degradation (NMD) could serve as a mechanism cause by TSC1 mutation. MINI-Gene assay apply by pcMINI-TSC1wt/mut plasmids delivered in HeLa and 293T cell lines. Recombinant plasmids expressing wild-type and mutant-type EGFP-TSC1 were constructed and transiently transfected into human embryonic kidney cells 293T by lipofectamine. Real-time PCR and Western Blot were performed to analyze the expression of mRNAs and proteins of EGFP-TSC1 and NMD factor UPF1. RESULTS: The gene test verified a novel heterozygous TSC1 frameshift mutation (TSC1 c.1550_1551del) in the proband and her mother. From MINI-Gene assay, the agarose gel showed that both the mutant and wild-type mRNA possess two main bands, indicating two splicing modes, named band A and B, respectively. The mutation c.1550_1551del has not produced new splicing site, but there is a selective splicing in varying degree significantly after mutation. On the contrary, function validation assay showed that cells transfected with the mutant TSC1 plasmids expressed significantly lower TSC1 in mRNAs and proteins levels, compared with the wild-type TSC1 plasmid transfection. A translation inhibitor cycloheximide and small interfering RNA of UPF1 (siRNA-UPF1) increased mRNA or protein expression of TSC1 significantly in cells transfected with the mutant plasmids. CONCLUSION: Our study demonstrated that the novel TSC1 frameshift mutation (TSC1 c.1550_1551del) trigger aberrant splicing and NMD simultaneously, causing decrease of hamartin, then, leading to tuberous sclerosis complex formation.

SPAG9 is overexpressed in human astrocytoma and promotes cell proliferation and invasion.

Sperm-associated antigen 9 (SPAG9) is a recently characterized oncoprotein involved in the progression of several human malignancies. The present study aims to investigate the expression pattern and biological roles of SPAG9 protein in human astrocytoma. SPAG9 expression was analyzed in 105 astrocytoma specimens by immunohistochemistry. We observed negative staining in normal astrocytes and positive staining of SPAG9 in 63 out of 105 (60 %) astrocytoma samples. Overexpression of SPAG9 correlated with tumor grade (p < 0.001). Small interfering RNA knockdown was performed in U251 and U87 cell lines with relatively high SPAG9 expression. Using methylthiazolyldiphenyl-tetrazolium bromide assay and Matrigel invasion assay, we were able to show that SPAG9 depletion in astrocytoma cell lines inhibited cell proliferation and invasion in both cell lines. In addition, mRNA and protein levels of matrix metallopeptidase 9 (MMP9) were downregulated, while the levels of tissue inhibitor of metalloproteinase 1 (TIMP1) and TIMP2 were not changed, indicating that SPAG9 might regulate invasion through MMP9. In conclusion, SPAG9 serves as an important oncoprotein in human astrocytoma by regulating cell proliferation and invasion.

The top cited articles on glioma stem cells in Web of Science.

BACKGROUND: Glioma is the most common intracranial tumor and has a poor patient prognosis. The presence of brain tumor stem cells was gradually being understood and recognized, which might be beneficial for the treatment of glioma. OBJECTIVE: To use bibliometric indexes to track study focuses on glioma stem cell, and to investigate the relationships among geographic origin, impact factors, and highly cited articles indexed in Web of Science. METHODS: A list of citation classics for glioma stem cells was generated by searching the database of Web of Science-Expanded using the terms "glioma stem cell" or "glioma, stem cell" or "brain tumor stem cell". The top 63 cited research articles which were cited more than 100 times were retrieved by reading the abstract or full text if needed. Each eligible article was reviewed for basic information on subject categories, country of origin, journals, authors, and source of journals. Inclusive criteria: (1) articles in the field of glioma stem cells which was cited more than 100 times; (2) fundamental research on humans or animals, clinical trials and case reports; (3) research article; (4) year of publication: 1899-2012; and (5) citation database: Science Citation Index-Expanded. Exclusive criteria: (1) articles needing to be manually searched or accessed only by telephone; (2) unpublished articles; and (3) reviews, conference proceedings, as well as corrected papers. RESULTS: Of 2 040 articles published, the 63 top-cited articles were published between 1992 and 2010. The number of citations ranged from 100 to 1 754, with a mean of 280 citations per article. These citation classics came from nineteen countries, of which 46 articles came from the United States. Duke University and University of California, San Francisco led the list of classics with seven papers each. The 63 top-cited articles were published in 28 journals, predominantly Cancer Research and Cancer Cell, followed by Cell Stem Cell and Nature. CONCLUSION: Our bibliometric analysis provides a historical perspective on the progress of glioma stem cell research. Articles originating from outstanding institutions of the United States and published in high-impact journals are most likely to be cited.

[Apoptosis inducing effect of ponicidin in leukemia K562 cells and its mechanisms of action].

OBJECTIVE: To investigate the apoptosis inducing effects of ponicidin (PON) on leukemic K562 cells and its mechanisms of action. METHOD: K562 cells in culture medium in vitro were given different concentrations of PON (10-50 micromol x L(-1)) for 24, 48 and 72 h. The inhibitory rate of the cells was measured by MTT assay, cell apoptotic rates were detected by flow cytometry (FCM) using Annexin V staining after K562 cells were treated with different concentrations of PON for 72 hours, and cell morphology was observed by Wright-Giemsa staining. Western blot was used to detect caspase-3 and poly(ADP-ribose) polymerase (PARP) expression, and the protein levels in mitogen-activated protein kinase signaling pathways (MAPKs, p-P38, p-ERK and p-JNK) as well as p-AKT and p-P85 in PI3K/AKT signaling pathways were also detected. RESULT: PON (over 30 micromol x L(-1)) could inhibit the growth of K562 cells in both time- and dose-dependent manner. FCM analysis revealed that apoptotic cells were gradually increased in a dose-dependent manner after treatment for 72 hours, and that marked morphological changes of cell apoptosis such as condensation of chromatin was clearly observed by Wright-Giemsa staining after treatment by 50 micromol x L(-1) PON. Western blot showed cleavage of the caspase-3 zymogen protein (32 kD), with the appearance of its 17 kD subunit, and a cleaved 89 kD fragment of 116 kD PARP was also found. Furthermore, Western blotting also showed that expression of p-AKT and p-P85 in PI3K/AKT signaling pathways was downregulated dramatically whereas the expression of p-P38 as well as p-ERK and p-JNK remained unchanged after the cells were treated by PON for 48 h. CONCLUSION: The results demonstrate that PON exhibits in vitro anti-leukemia effect by induction of apoptosis in K562 cells, and that PON induced apoptosis in K562 cells mainly related to activation of caspase-3 as well as inactivation of PI3K/AKT signaling pathway via down regulation of the expression of p-AKT and p-P85 protein levels. These results provide strong laboratory evidence for further anti-leukemia trials of PON.

Inactivation of PI3k/Akt signaling pathway and activation of caspase-3 are involved in tanshinone I-induced apoptosis in myeloid leukemia cells in vitro.

Tanshinone I (Tan I), a diterpene quinone extracted from herbal medicine Salvia miltiorrhiza Bunge, has recently been reported to have antitumor effects. As the mechanism of its proapoptotic effects on human myeloid leukemia cells has not been extensively studied, we performed an in-depth evaluation of the effects of Tan I on apoptosis in human K562 and HL-60 cells. The results revealed that Tan I could inhibit the growth of leukemia cells and cause apoptosis in a time- and dose-dependent manner. Apoptosis was observed clearly by flow cytometry and Hoechst 33258 staining, as well as DNA fragmentation analysis. After treatment by Tan I for 48 h, the percentage of disruption of mitochondrial membrane potential (Δψm) was increased in a dose-dependent manner. Western blotting analysis demonstrated the cleavage of caspase-3 zymogen protein and a dose-dependent cleavage of poly-(ADP-ribose) polymerase. Tan I-induced apoptosis was accompanied by a significant decrease in survivin and an increase in Bax. Moreover, Tan I treatment remarkably downregulated the phosphorylation of both P85/PI3K and Akt in a time-dependent manner, and the PI3K/AKT-specific inhibitor (LY294002) mimicked the apoptosis-inducing effects of Tan I. We therefore conclude that the induction of apoptosis by Tan I in these leukemia cells is mainly related to the disruption of Δψm, the upregulation of Bax expression, and the activation of caspase-3. This process is highly correlated with the inactivation of PI3K/Akt/survivin signaling pathways. The results indicate that Tan I may serve as an effective adjunctive reagent in the treatment of leukemia.