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Up-regulation of Imp3 confers in vivo tumorigenicity on murine osteosarcoma cells.

ABSTRACT: Osteosarcoma is a high-grade malignant bone tumor that manifests ingravescent clinical behavior. The intrinsic events that confer malignant properties on osteosarcoma cells have remained unclear, however. We previously established two lines of mouse osteosarcoma cells: AX cells, which are able to form tumors in syngeneic mice, and AXT cells, which were derived from such tumors and acquired an increased tumorigenic capacity during tumor development. We have now identified Igf2 mRNA-binding protein3 (Imp3) as a key molecule responsible for this increased tumorigenicity of AXT cells in vivo. Imp3 is consistently up-regulated in tumors formed by AX cells, and its expression in these cells was found to confer malignant properties such as anchorage-independent growth, loss of contact inhibition, and escape from anoikis in vitro. The expression level of Imp3 also appeared directly related to tumorigenic ability in vivo which is the critical determination for tumor-initiating cells. The effect of Imp3 on tumorigenicity of osteosarcoma cells did not appear to be mediated through Igf2-dependent mechanism. Our results implicate Imp3 as a key regulator of stem-like tumorigenic characteristics in osteosarcoma cells and as a potential therapeutic target for this malignancy.

SUBMITTER: Ueki A

PROVIDER: S-EPMC3511546 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Up-regulation of Imp3 confers in vivo tumorigenicity on murine osteosarcoma cells.

Ueki Arisa A Shimizu Takatsune T Masuda Kenta K Yamaguchi Sayaka I SI Ishikawa Tomoki T Sugihara Eiji E Onishi Nobuyuki N Kuninaka Shinji S Miyoshi Keita K Muto Akihiro A Toyama Yoshiaki Y Banno Kouji K Aoki Daisuke D Saya Hideyuki H

PloS one 20121130 11

Osteosarcoma is a high-grade malignant bone tumor that manifests ingravescent clinical behavior. The intrinsic events that confer malignant properties on osteosarcoma cells have remained unclear, however. We previously established two lines of mouse osteosarcoma cells: AX cells, which are able to form tumors in syngeneic mice, and AXT cells, which were derived from such tumors and acquired an increased tumorigenic capacity during tumor development. We have now identified Igf2 mRNA-binding protei ...[more]

PMID: 23226335

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Project description:BackgroundOsteosarcoma (OS) is the most common primary bone malignancy in children and adolescents, and hyperproliferation of cells is a major problem of OS. FBXO2 belongs to the family of F-box proteins, and is a substrate recognition component of the Skp1-Cul1-F-box protein (SCF) E3 ubiquitin ligase complex with specificity for high-mannose glycoproteins. The aim of the present study was to investigate the critical role of FBXO2 in OS cells.MethodsThe protein and mRNA expression levels of FBXO2 in clinic OS patients were measured by quantitative real time-polymerase chain reaction (qRT-PCR), Western blot and Immunohistochemical (IHC) staining assays, respectively. The FBXO2 overexpression model was constructed by retro-virus transfection in OS cells. FBXO2 knockout (KO) cells were generated by Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 9 (Cas9) assay. Cell counting and colony formation assays were used to analyze the effect of FBXO2 on the biological function of OS cells. FBXO2 KO cells were injected into nude mice to observe tumor growth in vivo. The interaction between FBXO2 and IL-6 was detected by immunoprecipitation. Luciferase assay was used to determine the transcriptional activity of STAT3.ResultsHere, we show that FBXO2 is significantly up-regulated in clinical OS samples compared to adjacent normal tissues. Ectopic expression of FBXO2 leads to increased OS cell proliferation and colony-forming ability, while FBXO2 knockout by CRISPR-Cas9-based gene editing has the opposite effect. In addition, the glycoprotein recognition activity of FBXO2 is required for its biological function in OS. In vivo experiments showed that FBXO2 knockout greatly impaired the tumorigenicity of OS cells in nude mice. At the molecular level, we found that knocking out FBXO2 can significantly inhibit STAT3 phosphorylation and downstream target gene expression through IL-6R stabilization.ConclusionTogether, these results indicate that FBXO2 promotes OS development by activating the STAT3 signaling pathway, suggesting that FBXO2 may be a new target for OS treatment.

Project description:BackgroundPhytoestrogens such as genistein, the most prominent isoflavone from soy, show concentration-dependent anti-estrogenic or estrogenic effects. High genistein concentrations (>10 μM) also promote proliferation of bone cancer cells in vitro. On the other hand, the most active component of the vitamin D family, calcitriol, has been shown to be tumor protective in vitro and in vivo. The purpose of this study was to examine a putative synergism of genistein and calcitriol in two osteosarcoma cell lines MG-63 (early osteoblast), Saos-2 (mature osteoblast) and primary osteoblasts.MethodsThus, an initial screening based on cell cycle phase alterations, estrogen (ER) and vitamin D receptor (VDR) expression, live cell metabolic monitoring, and metabolomics were performed.ResultsExposure to the combination of 100 μM genistein and 10 nM calcitriol reduced the number of proliferative cells to control levels, increased ERß and VDR expression, and reduced extracellular acidification (40%) as well as respiratory activity (70%), primarily in MG-63 cells. In order to identify the underlying cellular mechanisms in the MG-63 cell line, metabolic profiling via GC/MS technology was conducted. Combined treatment significantly influenced lipids and amino acids preferably, whereas metabolites of the energy metabolism were not altered. The comparative analysis of the log2-ratios revealed that after combined treatment only the metabolite ethanolamine was highly up-regulated. This is the result: a strong overexpression (350%) of the enzyme sphingosine-1-phosphate lyase (SGPL1), which irreversibly degrades sphingosine-1-phosphate (S1P), thereby, generating ethanolamine. S1P production and secretion is associated with an increased capability of migration and invasion of cancer cells.ConclusionFrom these results can be concluded that the tumor promoting effect of high concentrations of genistein in immature osteosarcoma cells is reduced by the co-administration of calcitriol, primarily by the breakdown of S1P. It should be tested whether this anti-metastatic pathway can be stimulated by combined treatment also in metastatic xenograft mice models.

Project description:BackgroundApart from the platelet/endothelial cell adhesion molecule 1 (PECAM-1, CD31), endoglin (CD105) and a positive factor VIII-related antigen staining, human primary and immortalized macro- and microvascular endothelial cells (ECs) differ in their cell surface expression of activating and inhibitory ligands for natural killer (NK) cells. Here we comparatively study the effects of irradiation on the phenotype of ECs and their interaction with resting and activated NK cells.Methodology/principal findingsPrimary macrovascular human umbilical vein endothelial cells (HUVECs) only express UL16 binding protein 2 (ULBP2) and the major histocompatibility complex (MHC) class I chain-related protein MIC-A (MIC-A) as activating signals for NK cells, whereas the corresponding immortalized EA.hy926 EC cell line additionally present ULBP3, membrane heat shock protein 70 (Hsp70), intercellular adhesion molecule ICAM-1 (CD54) and HLA-E. Apart from MIC-B, the immortalized human microvascular endothelial cell line HMEC, resembles the phenotype of EA.hy926. Surprisingly, primary HUVECs are more sensitive to Hsp70 peptide (TKD) plus IL-2 (TKD/IL-2)-activated NK cells than their immortalized EC counterpatrs. This finding is most likely due to the absence of the inhibitory ligand HLA-E, since the activating ligands are shared among the ECs. The co-culture of HUVECs with activated NK cells induces ICAM-1 (CD54) and HLA-E expression on the former which drops to the initial low levels (below 5%) when NK cells are removed. Sublethal irradiation of HUVECs induces similar but less pronounced effects on HUVECs. Along with these findings, irradiation also induces HLA-E expression on macrovascular ECs and this correlates with an increased resistance to killing by activated NK cells. Irradiation had no effect on HLA-E expression on microvascular ECs and the sensitivity of these cells to NK cells remained unaffected.Conclusion/significanceThese data emphasize that an irradiation-induced, transient up-regulation of HLA-E on macrovascular ECs might confer protection against NK cell-mediated vascular injury.

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Up-regulation of Imp3 confers in vivo tumorigenicity on murine osteosarcoma cells.

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Up-regulation of Imp3 confers in vivo tumorigenicity on murine osteosarcoma cells.

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