Keiichiro Iida

Basic fibroblast growth factor in the bone microenvironment enhances cell motility and invasion of Ewing's sarcoma family of tumours by activating the FGFR1-PI3K-Rac1 pathway

Background:Ewings sarcoma family of tumours (ESFT) is a malignant small round-cell tumour of the bone and soft tissues. It is characterised by a strong tendency to invade and form metastases. The microenvironment of the bone marrow is a large repository for many growth factors, including the basic fibroblast growth factor (bFGF). However, the role of bFGF in the invasive and metastatic phenotype of ESFT has not been investigated.Methods:The motility and invasion of ESFT cells were assessed by a wound-healing assay, chemotaxis assay, and invasion assay. The expression and activation of FGF receptors (FGFRs) in ESFT cell lines and clinical samples were detected by RT–PCR, western blotting, and immunohistochemistry. The morphology of ESFT cells was investigated by phase-contrast microscopy and fluorescence staining for actin. Activation of Rac1 was analysed by a pull-down assay.Results:bFGF strongly induced the motility and invasion of ESFT cells. Furthermore, FGFR1 was found to be expressed and activated in clinical samples of ESFT. Basic FGF-induced cell motility was mediated through the FGFR1–phosphatidylinositol 3-kinase (PI3K)–Rac1 pathway. Conditioned medium from bone marrow stromal cells induced the motility of ESFT cells by activating bFGF/FGFR1 signalling.Conclusion:The bFGF–FGFR1–PI3K–Rac1 pathway in the bone microenvironment may have a significant role in the invasion and metastasis of ESFT.

miR-125b develops chemoresistance in Ewing sarcoma/primitive neuroectodermal tumor

BackgroundDiverse functions of microRNAs (miRNAs), including effects on tumorigenesis, proliferation, and differentiation, have been reported, and several miRNAs have also been demonstrated to play an important role in apoptosis. In this study, we investigated the possible role that miRNAs may play in the development of chemoresistance in Ewing sarcoma/primitive neuroectodermal tumor (EWS).MethodsWe screened doxorubicin (Dox)-resistant EWS cells to identify any distinct miRNA sequences that may regulate the chemoresistance of EWS cells. The effects of miRNAs were evaluated using a chemosensitivity assay. The possible target genes of the miRNAs were predicted using a web-based prediction program.ResultsWe found miR-125b to be upregulated in two different Dox-resistant EWS cell lines. The upregulation of miR-125b was also confirmed in the EWS tumors having survived chemotherapy regimen which includes doxorubicin. When miR-125b was knocked down in EWS cells, both the Dox-resistant and parental cells showed an enhanced sensitivity to doxorubicin, which was associated with the upregulation of the pro-apoptotic molecules, p53 and Bak. Inversely, the overexpression of miR-125b in parental EWS cells resulted in enhanced drug resistance, not only to doxorubicin, but also to etoposide and vincristine.ConclusionsOur findings suggest that miR-125b may play a role in the development of chemoresistance in EWS by suppressing the expression of the apoptotic mediators, such as p53 and Bak.

Y-box binding protein-1 regulates cell proliferation and is associated with clinical outcomes of osteosarcoma

Background:Prognosis of osteosarcoma (OS) with distant metastasis and local recurrence is still poor. Y-box binding protein-1 (YB-1) is a multifunctional protein that can act as a regulator of transcription and translation and its high expression of YB-1 protein was observed in OS, however, the role of YB-1 in OS remains unclear.Methods:Y-box binding protein-1 expression in OS cells was inhibited by specific small interfering RNAs to YB-1 (si-YB-1). The effects of si-YB-1 in cell proliferation and cell cycle transition in OS cells were analysed in vitro and in vivo. The association of nuclear expression of YB-1 and clinical prognosis was also investigated by immunohistochemistry.Results:Proliferation of OS cell was suppressed by si-YB-1 in vivo and in vitro. The expression of cyclin D1 and cyclin A were also decreased by si-YB-1. In addition, si-YB-1 induced G1/S arrest with decreased cyclin D1 and cyclin A in OS cell lines. Direct binding of YB-1 in OS cell lines was also observed. Finally, the nuclear expression of YB-1 was significantly related to the poorer overall survival in OS patients.Conclusion:Y-box binding protein-1 would regulate cell cycle progression at G1/S and tumour growth in human OS cells in vitro and in vivo. Nuclear expression of YB-1 was closely associated with the prognosis of OS, thus, YB-1 simultaneously could be a potent molecular target and prognostic biomarker for OS.

Tumour-associated macrophages correlate with poor prognosis in myxoid liposarcoma and promote cell motility and invasion via the HB-EGF-EGFR-PI3K/Akt pathways

Background:Myxoid liposarcoma (MLS) is the second most common subtype of liposarcoma, and metastasis occurs in up to one-third of cases. However, the mechanisms of invasion and metastasis remain unclear. Tumour-associated macrophages (TAMs) have important roles in tumour invasion, metastasis, and/or poor prognosis. The aim of this study was to investigate the relationship between TAMs and MLS.Methods:Using 78 primary MLS samples, the association between clinical prognosis and macrophage infiltration was evaluated by immunochemistry. The effects of macrophages on cell growth, cell motility, and invasion of MLS cell lines were investigated in vitro. In addition, clinicopathological factors were analysed to assess their prognostic implications in MLS.Results:Higher levels of CD68-positive macrophages were associated with poorer overall survival in MLS samples. Macrophage-conditioned medium enhanced MLS cell motility and invasion by activating epidermal growth factor receptor (EGFR), with the key ligand suggested to be heparin-binding EGF-like growth factor (HB-EGF). The phosphoinositide 3-kinase/Akt pathway was mostly involved in HB-EGF-induced cell motility and invasion of MLS. The expression of phosphorylated EGFR in MLS clinical samples was associated with macrophage infiltration. In addition, more significant macrophage infiltration was associated with poor prognosis even in multivariate analysis.Conclusions:Macrophage infiltration in MLS predicts poor prognosis, and the relationship between TAMs and MLS may be a new candidate for therapeutic targets of MLS.

Role of the VEGF-Flt-1-FAK pathway in the pathogenesis of osteoclastic bone destruction of giant cell tumors of bone

BackgroundGiant cell tumors (GCTs) of bone are primary benign bone tumors that are characterized by a high number of osteoclast-like multinuclear giant cells (MNCs). Recent studies suggest that the spindle-shaped stromal cells in GCTs are tumor cells, while monocyte-like cells and MNCs are reactive osteoclast precursor cells (OPCs) and osteoclasts (OCs), respectively. In this study, we investigated the pathogenesis of osteoclastic bone destruction in GCTs by focusing on the role of the vascular endothelial growth factor (VEGF)-Flt-1 (type-1 VEGF receptor)-focal adhesion kinase (FAK) pathway.MethodsThe motility of OPCs cells was assessed by a chemotaxis assay and the growth of OPCs was examined using a cell proliferation assay. The expression of VEGF and activation of Flt-1 and FAK in clinical GCT samples and in OPCs were detected by immunohistochemistry and immunoblotting. The correlation between the expression levels of activated Flt-1 and FAK and clinical stages of GCTs was investigated by immunohistochemistry.ResultsIn GCT samples, CD68, a marker of OPCs and OCs, co-localized with Flt-1. Conditioned media from GCT tissue (GCT-CM) enhanced the chemotaxis and proliferation of OPCs. GCT-CM also stimulated FAK activation in OPCs in vitro. Moreover, there was a correlation between the clinical stage of GCTs and the expression of tyrosine-phosphorylated Flt-1 and FAK.ConclusionsOur results suggest that the VEGF-Flt-1-FAK pathway is involved in the pathogenesis of bone destruction of GCTs.

Hypoxia-inducible factor 1 alpha is a poor prognostic factor and potential therapeutic target in malignant peripheral nerve sheath tumor

Background Malignant peripheral nerve sheath tumor (MPNST) is a rare soft tissue sarcoma with poor prognosis. Hypoxia-inducible factor 1 (HIF-1) plays a crucial role in the cellular response to hypoxia and regulates the expression of multiple genes involved in tumor progression in various cancers. However, the importance of the expression of HIF-1α in MPNSTs is unclear. Methods The expression of HIF-1α was examined immunohistochemically in 82 MPNST specimens. Cell culture assays of human MPNST cells under normoxic and hypoxic conditions were used to evaluate the impact of anti-HIF-1α–specific siRNA inhibition on cell survival. A screening kit was employed to identify small molecules that inhibited HIF-1α. Results The nuclear expression of HIF-1α was positive in 75.6% of MPNST samples (62/82 cases). Positivity for HIF-1α was a significant poor prognostic factor both in univariate (P = 0.048) and multivariate (P ≤ 0.0001) analyses. HIF-1α knockdown abrogated MPNST cell growth, inducing apoptosis. Finally, chetomin, an inhibitor of HIF-1α, effectively inhibited the growth of MPNST cells and induced their apoptosis. Conclusion Inhibition of HIF-1α signaling is a potential treatment option for MPNSTs.

Clinical Outcome and Prognostic Factors of Malignant Spinal Dumbbell Tumors

Introduction To investigate the clinical outcome and prognostic factors of malignant spinal dumbbell tumors (m-SDTs). Methods We retrospectively reviewed the clinical outcome of 22 consecutive cases of m-SDTs and analyzed the prognostic factors associated with worse outcome. Results Nineteen of the 22 cases were managed with surgery (86%), and gross total resection (GTR) was achieved in four cases (21%). The duration of overall survival (OS) ranged from 3 to 140 months, with a median survival time of 15.3 months. The 5 year OS rate was 55.6%. In multivariate analysis, histological subtype (high-grade malignant peripheral nerve sheath tumor) (hazard ratio [HR] 14.9, p = 0.0191), GTR (HR 0.07, p = 0.0343), and presence of local recurrences (HR 11.2, p = 0.0479) were significant and independent predictors of OS. Conclusions On the basis of clinical data, we propose that GTR and prevention of local recurrence may improve the clinical outcome of m-SDTs.

Effect of cartilaginous endplates on extruded disc resorption in lumbar disc herniation

Objective The aim of this study was to investigate the clinicopathologic features of lumbar disc herniation (LDH) with endplate degeneration and the association between cartilaginous fragments and inflammatory response to the herniated disc. Summary of background data LDH often involves hyaline cartilage fragments pulled from the vertebral endplates. Modic changes are closely associated with LDH that contains hyaline cartilage, and cartilaginous endplates seem to affect resorption of the herniated disc. Methods A total of 78 patients who underwent microscopic discectomy between 9 and 16 weeks after an occurrence of LDH were reviewed. Modic changes, disc degeneration, high-intensity zone, and vertebral corner defect were evaluated using magnetic resonance imaging (MRI). Histopathological observations of cartilaginous endplates and inflamed granulation tissue in the herniated disc were made. In cases with inflamed granulation tissue, neovascularization and macrophage infiltration were also evaluated using immunohistochemical analysis. Results Modic changes were observed in approximately one-third of the patients (26 cases: type 1, 7; type 2, 17; and type 3, 2). Cartilaginous endplates were observed in 32 cases (41%) and in the majority of cases with Modic changes compared with cases without Modic changes (65%, p = 0.001). Although inflamed granulation tissue was observed in 60 cases (77%), no significant differences were detected in patient age and the composition of the herniated material. Immunohistochemical analysis showed that fewer CD34-positive capillaries and CD68-positive cells were found in cases with cartilaginous fragments compared with those without cartilaginous fragments (p < 0.001). In addition, a higher immunoreactivity to CD34 and CD68 was found in herniated discs <25% of whose area was occupied by cartilaginous endplates compared with discs whose area was occupied at 25% or more (p < 0.001). Conclusion There is an association between LDH with endplate degeneration and cartilaginous herniation, with Modic type 2 predominating. Furthermore, neovascularization and macrophage infiltration, especially if the amount of cartilage is high, are likely to be less frequent in cartilaginous herniation, leading to failure in the spontaneous remission of clinical symptoms.

Class III β-Tubulin Overexpression Induces Chemoresistance to Eribulin in a Leiomyosarcoma Cell Line

Eribulin is a new drug to treat soft tissue sarcoma (STS) that exerts antitumor activity by binding to microtubules. The prognosis of STS is poor, and eribulin is expected to improve the treatment outcome. We observed several cases that exhibited resistance to eribulin and developed an eribulin-resistant leiomyosarcoma cell line to investigate the mechanism of resistance. The IC50 of eribulin was 125 times higher in the resistant cell line than in the parental cell line, and eribulin did not induce G2/M arrest in resistant cells. The resistant cell line showed increased expression of MDR1 transcript, but protein levels and functional analysis results were similar to the parental cell line. We found that class III β-tubulin (TUBB3) was overexpressed in the resistant cell line, and siRNA knockdown of TUBB3 partially recovered sensitivity to eribulin. TUBB3 expression in clinical samples varied, suggesting that TUBB3 has the potential to be a biomarker for selection of anticancer drugs and may be a target for overcoming resistance to eribulin.

Metabolic Tumor Volume by 18F-FDG PET/CT Can Predict the Clinical Outcome of Primary Malignant Spine/Spinal Tumors

Background and Purpose Primary malignant spine/spinal tumors (PMSTs) are rare and life-threatening diseases. In this study, we demonstrated the advantage of volume-based 18F-FDG PET/CT metabolic parameter, metabolic tumor volume (MTV), for assessing the aggressiveness of PMSTs. Materials and Methods We retrospectively reviewed 27 patients with PMSTs and calculated SUVmax, MTV, and total lesion glycolysis (TLG) to compare their accuracy in predicting progression-free survival (PFS) and overall survival (OS) by receiver operating characteristic (ROC) curve analysis. Univariate and multivariate analyses were used to compare the reliability of the metabolic parameters and various clinical factors. Results MTV exhibited greater accuracy than SUVmax or TLG. The cut-off values for PFS and OS derived from the AUC data were MTV 45 ml and 83 ml and TLG 250 SUV⁎ml and 257 SUV⁎ml, respectively. MTV above cut-off value, but not TLG, was identified as significant prognostic factor for PFS by log-lank test (p = 0.04). In addition, MTV was the only significant predictive factors for PFS and OS in the multivariate analysis. Conclusions MTV was a more accurate predictor of PFS and OS in PMSTs compared to TLG or SUVmax and helped decision-making for guiding rational treatment options.