Yoshinori Imura

Combined targeting of mTOR and c-MET signaling pathways for effective management of epithelioid sarcoma.

BackgroundEpithelioid sarcoma (EpS) is a high-grade malignant soft-tissue sarcoma characterized by local recurrences and distant metastases. Effective treatments for EpS have not been established and thus novel therapeutic approaches against EpS are urgently required. mTOR inhibitors exert antitumor effects on several malignancies but AKT reactivation by mTOR inhibition attenuates the antitumor effects of mTOR inhibitors. This reactivation is receptor tyrosine kinase (RTK)-dependent due to a release of negative feedback inhibition. We found that c-MET was the most highly activated RTK in two human EpS cell lines, Asra-EPS and VAESBJ. Here we investigated the functional and therapeutic relevance of mTOR and/or c-MET signaling pathways in EpS both in vitro and in vivo.MethodsWe first examined the effects of an mTOR inhibitor, RAD001 (everolimus), on cell proliferation, cell cycle, AKT/mTOR signaling, and xenograft tumor growth in EpS cell lines. Next, we determined whether RAD001-induced AKT reactivation was blocked by silencing of c-MET or treatment with a selective c-MET inhibitor, INC280. Finally, we evaluated the antitumor effects of RAD001 combined with INC280 on EpS cell lines compared with either single agent or control in vitro and in vivo.ResultsConstitutive AKT phosphorylation was observed in Asra-EPS and VAESBJ cells. RAD001 suppressed EpS cell growth by inducing cell cycle arrest but enhanced AKT phosphorylation, which resulted in intrinsic resistance to mTOR inhibitors. In both EpS cell lines, RAD001-induced AKT phosphorylation was dependent on c-MET signaling. INC280 inhibited phosphorylation of c-MET and its downstream molecules, and decreased RAD001-induced phosphorylation of both AKT and ERK in EpS. Compared with a single agent or control, the combination of RAD001 and INC280 exerted superior antitumor effects on the growth of EpS cell lines in vitro and in vivo.ConclusionsTargeting of mTOR and c-MET signaling pathways significantly abrogates the growth of EpS in preclinical models and may be a promising therapeutic approach for patients with EpS.

Tailored therapeutic strategies for synovial sarcoma : Receptor tyrosine kinase pathway analyses predict sensitivity to the mTOR inhibitor RAD001

We examined efficacy of the mTOR inhibitor RAD001 to seek novel therapies for synovial sarcoma (SS). Although RAD001 had significant anti-tumor effects, its sensitivity differed among cell lines. Phospho-receptor tyrosine kinase (RTK) array analyses revealed c-MET phosphorylation in highly mTOR inhibitor-sensitive cells and PDGFRα (which induces intrinsic resistance to mTOR inhibitor) activation in less sensitive cells. Combined treatment with RAD001 and the PDGFR inhibitor pazopanib showed anti-tumor effects in xenograft models with less sensitive cells. Thus, evaluating activated RTKs in clinical samples may predict sensitivity to mTOR inhibitors, raising the possibility of a tailored therapy for SS.

Establishment of a novel clear cell sarcoma cell line (Hewga-CCS), and investigation of the antitumor effects of pazopanib on Hewga-CCS

BackgroundClear cell sarcoma (CCS) is a therapeutically unresolved, aggressive, soft tissue sarcoma (STS) that predominantly affects young adults. This sarcoma is defined by t(12;22)(q13;q12) translocation, which leads to the fusion of Ewing sarcoma gene (EWS) to activating transcription factor 1 (ATF1) gene, producing a chimeric EWS-ATF1 fusion gene. We established a novel CCS cell line called Hewga-CCS and developed an orthotopic tumor xenograft model to enable comprehensive bench-side investigation for intensive basic and preclinical research in CCS with a paucity of experimental cell lines.MethodsHewga-CCS was derived from skin metastatic lesions of a CCS developed in a 34-year-old female. The karyotype and chimeric transcript were analyzed. Xenografts were established and characterized by morphology and immunohistochemical reactivity. Subsequently, the antitumor effects of pazopanib, a recently approved, novel, multitargeted, tyrosine kinase inhibitor (TKI) used for the treatment of advanced soft tissue sarcoma, on Hewga-CCS were assessed in vitro and in vivo.ResultsHewga-CCS harbored the type 2 EWS-ATF1 transcript. Xenografts morphologically mimicked the primary tumor and expressed S-100 protein and antigens associated with melanin synthesis (Melan-A, HMB45). Pazopanib suppressed the growth of Hewga-CCS both in vivo and in vitro. A phospho-receptor tyrosine kinase array revealed phosphorylation of c-MET, but not of VEGFR, in Hewga-CCS. Subsequent experiments showed that pazopanib exerted antitumor effects through the inhibition of HGF/c-MET signaling.ConclusionsCCS is a rare, devastating disease, and our established CCS cell line and xenograft model may be a useful tool for further in-depth investigation and understanding of the drug-sensitivity mechanism.

Radiofrequency ablation of osteoid osteoma using a three-dimensional navigation system

OBJECTIVE This study aimed to report the clinical outcomes for patients with osteoid osteoma (OO) treated by radiofrequency ablation (RFA) using a three-dimensional (3D) navigation system. METHODS We performed RFA using a 3D navigation system on 32 patients with clinically and radiologically diagnosed OO. This study included 25 males and 7 females with a median age of 20 years (range, 10-39 years). The median duration of follow-up was 18 months (range, 1-65 months). We investigated technical specifications, tumor localization, technical success, clinical success, biopsy success, complications, incomplete treatment, and recurrences. RESULTS Eighteen tumors were located in the femur, seven in the tibia, two in the humerus, and one each in the fibula, scapula, patella, lumbar vertebra, and acetabula. All procedures were technically successful, and pain relief was achieved in all patients. However, local recurrence developed in one patient, needing additional RFA. The clinical success rate was 96.8%. Biopsy showed OO in 12 patients (37%). Complications occurred in three patients (9%), two cases of fractures and one of osteomyelitis. CONCLUSIONS A 3D navigation provides real-time imaging and enables us to set the RFA needle in the correct position, particularly in case of OO-aroused complex anatomical structures. Our initial results indicated that radiofrequency ablation using a 3D navigation system is feasible and safe for patients with OO.

Atypical femoral fracture associated with bone-modifying agent for bone metastasis of breast cancer: A report of two cases:

Atypical femoral fractures (AFFs) are recently observed as a complication of long-term bone-modifying agent (BMA; bisphosphonate or denosumab) therapy for bone metastases. We describe the cases of two women diagnosed with breast cancer who developed incomplete AFF associated with BMAs prescribed for bone metastases. Radiographs of their femurs revealed thickening of the lateral subtrochanteric cortex, and tomosynthesis revealed a visible fracture line in the thickened cortex. They were initially treated with conservative management; however, the incomplete fracture resulted in a complete fracture. These cases highlight two major implications. First, symptomatic incomplete AFF associated with BMAs prescribed for bone metastases should be treated with surgical prophylaxis, given the fact that fracture healing is expected to require a longer duration and an incomplete fracture might potentially progress to a complete fracture during long-term conservative management. Second, tomosynthesis is useful in identifying radiolucent fracture lines that are reliable predictors of fracture propagation.

Functional and therapeutic relevance of hepatocyte growth factor/c-MET signaling in synovial sarcoma.

Synovial sarcoma (SS) is an aggressive soft tissue sarcoma with a poor prognosis and, thus, novel therapeutic strategies for SS are urgently required. In the present study, we investigated the functional and therapeutic relevance of hepatocyte growth factor (HGF)/c‐MET signaling in SS. Both HGF and c‐MET were highly expressed in Yamato‐SS cells, resulting in activation of c‐MET and its downstream AKT and extracellular signal‐regulated kinase signaling pathways, whereas c‐MET was expressed but not activated in SYO‐1 or HS‐SY‐II cells. c‐MET‐activated Yamato‐SS cells showed higher anchorage‐independent growth ability and less sensitivity to chemotherapeutic agents than did c‐MET‐inactivated SYO‐1 or HS‐SY‐II cells. INC280, a selective c‐MET inhibitor, inhibited growth of Yamato‐SS cells both in vitro and in vivo but not that of SYO‐1 or HS‐SY‐II cells. INC280 induced cell cycle arrest and apoptosis, and blocked phosphorylation of c‐MET and its downstream effectors in Yamato‐SS cells. Co‐expression of HGF and c‐MET in SS clinical samples correlated with a poor prognosis in patients with SS. Taken together, activation of HGF/c‐MET signaling in an autocrine fashion leads to an aggressive phenotype in SS and targeting of this signaling exerts superior antitumor effects on c‐MET‐activated SS. HGF/c‐MET expression status is a potential biomarker for identification of SS patients with a worse prognosis who can benefit from c‐MET inhibitors.

A novel angiomatoid epithelioid sarcoma cell line, Asra-EPS, forming tumors with large cysts containing hemorrhagic fluid in vivo

BackgroundWhereas we can use several human epithelioid sarcoma (ES) cell lines for basic and preclinical research, an angiomatoid ES cell line has not been reported to date. We have treated a case of an angiomatoid ES developing in the right upper extremity of a 67-year-old man.MethodsAn angiomatoid ES cell line, Asra-EPS was newly established and characterized for its morphology, growth rate and chromosomal analysis. Tumorigenicity of Asra-EPS cells was also analyzed in athymic nude mice.ResultsAsra-EPS cells were round, polygonal or spindle-shaped with an abundant cytoplasm and have been maintained continuously in vitro for over 150 passages during more than 15 months. These cells secreted cancer antigen 125 (CA 125), interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) into the culture medium. Asra-EPS cells were tumorigenic when implanted in nude mice with tumors reaching a volume of 1000 mm3 at around 50 days. Histological features of tumors formed in mice were essentially the same as those of the original tumor, exhibiting a multinodular proliferation of eosinophilic epithelioid and spindle-shaped cells with prominent areas of hemorrhage and blood-filled cystic spaces strikingly corresponding to the potential of hemorrhagic cyst formation in the original tumor. They showed immunopositive staining for cytokeratins (AE1/AE3 and CAM5.2), epithelial membrane antigen (EMA), vimentin, CD31, CD34 and CA 125, but negative for integrase interactor 1 (INI-1) and factor VIII-related antigen.ConclusionsThe established cell line represents a biologically relevant new tool to investigate the molecular pathology of human angiomatoid ES and to evaluate the efficacy of novel therapeutics both in vitro and in vivo.

Trabectedin is a promising antitumour agent for synovial sarcoma

Synovial sarcoma (SS) is an aggressive soft tissue tumour with poor prognosis. Using five human SS cell lines, we examined the cytotoxic effects of trabectedin (ET-743; Yondelis®), a novel marine natural product, which was approved in Europe for the treatment of soft tissue sarcomas (STS). The significant growth inhibitory effects were observed in all SS cell lines below nanomolar concentration of trabectedin. Furthermore, trabectedin significantly suppressed the tumour growth in xenograft models. Flow cytometer analysis in vitro and immunohistochemical analysis in vivo revealed its effect of cell cycle inhibition and apoptosis induction. We also examined the expression of ERCC1, 5 and BRCA1 in SS cell lines and clinical samples, and majority of them showed highly trabectedin-sensitive pattern as previously reported in other cancers. Our preclinical data indicated that trabectedin could be a promising therapeutic option for patients with SS.

Trabectedin is a promising antitumor agent potentially inducing melanocytic differentiation for clear cell sarcoma

Clear cell sarcoma is an aggressive soft tissue sarcoma and highly resistant to conventional chemotherapy and radiation therapy. This devastating disease is defined by EWSR1‐ATF1 fusion gene resulting from chromosomal translocation t(12;22)(q13;q12) and characterized by melanocytic differentiation. A marine‐derived antineoplastic agent, trabectedin, inhibits the growth of myxoid liposarcoma and Ewing sarcoma by causing adipogenic differentiation and neural differentiation, respectively. In this study, we examined the antitumor effects and mechanism of action of trabectedin on human clear cell sarcoma cell lines. We showed that trabectedin decreased the cell proliferation of five clear cell sarcoma cell lines in a dose‐dependent manner in vitro and reduced tumor growth of two mouse xenograft models. Flow cytometry and immunoblot analyses in vitro and immunohistochemical analysis in vivo revealed that trabectedin‐induced G2/M cell cycle arrest and apoptosis. Furthermore, trabectedin increased the expression of melanocytic differentiation markers along with downregulation of ERK activity in vitro and the rate of melanin‐positive cells in vivo. These results suggest that trabectedin has potent antitumor activity against clear cell sarcoma cells by inducing cell cycle arrest, apoptosis, and, in part, by promoting melanocytic differentiation through inactivation of ERK signaling. Our present study indicates that trabectedin is a promising differentiation‐inducing agent for clear cell sarcoma.

Therapeutic potential of TAS-115 via c-MET and PDGFRα signal inhibition for synovial sarcoma

BackgroundThe prognosis of synovial sarcoma (SS), an aggressive soft tissue sarcoma, remains poor. We previously reported that c-MET or platelet-derived growth factor receptor α (PDGFRα) signalling pathway is related to SS progression based upon the findings of phospho-receptor tyrosine kinase (RTK) arrays. TAS-115 is a novel c-MET/ vascular endothelial growth factor receptor-targeting tyrosine kinase inhibitor that has been shown to inhibit multiple RTKs. Here we aimed to investigate the therapeutic potential of TAS-115 against SS.MethodsWe first evaluated which signalling pathway was relevant to the viability of three human SS cell lines: Yamato-SS, SYO-1 and HS-SY-II. Next, we assessed the anticancer activity and mechanism of action of TAS-115 in these SS cell lines. Finally, we compared the ability of TAS-115 to inhibit c-MET and PDGFRα phosphorylation with that of pazopanib.ResultsWe classified the SS cell lines as c-MET-dependent or PDGFRα-dependent based upon the differences in the signalling pathway relevant for growth and/or survival. We also found that c-MET and PDGFRα were the primary activators of both phosphatidylinositol 3-kinase/AKT and mitogen-activated protein kinase pathways in c-MET-dependent and PDGFRα-dependent SS cells, respectively. TAS-115 treatment blocked the phosphorylation of PDGFRα as well as that of c-MET and their downstream effectors, leading to marked growth inhibition in both types of SS cell lines in in vitro and in vivo studies. Furthermore, PDGFRα phosphorylation, on at least four representative autophosphorylation sites, was impeded by TAS-115 equivalently to pazopanib.ConclusionsThese experimental results have demonstrated the significance of c-MET and PDGFRα signalling for growth and/or survival of SS tumours. TAS-115 monotherapy may benefit SS patients whose tumours are dependent upon either c-MET or PDGFRα signalling by functioning as a multiple tyrosine kinase inhibitor to suppress c-MET as well as PDGFRα pathways.