Objective. Notch signaling is implicated in the repression of mesenchymal stem cell (MSC) chondrogenic differentiation. The purpose of this study was to examine the mechanism of this repression and how it is modulated to permit chondrogenesis.Methods. Notch intracellular domain (NICD) protein levels were monitored via Western blotting throughout chondrogenic differentiation of human MSCs in pellet cultures. Overexpression of Notch signaling components and their effect on chondrogenesis was achieved by transfecting plasmids coding for NICD, HES-1, and HERP-2/HEY-1. COL2A1 and AGGRECAN expression was monitored via quantitative polymerase chain reaction analysis. Chromatin immunoprecipitation (ChIP) was used to test whether HES-1 and HEY-1 bind putative N-box domains in intron 1 of COL2A1.Results. High levels of NICD proteins were reduced during chondrogenesis of human MSCs, and this was mediated by transforming growth factor ␤3 (TGF␤3). COL2A1 gene expression was repressed following overexpression of NICD (2-fold) and HES-1 (3-fold) and was markedly repressed by overexpression of HEY-1 (80-fold). HEY-1 repressed AGGRECAN expression 10-fold, while NICD and HES-1 had no effect. We identified 2 putative N-box domains adjacent to, and part of, the SOX9 enhancer binding site located in intron 1 of COL2A1. ChIP studies showed that endogenous HES-1 and HEY-1 bound to these sites.Transducin-like enhancer, the HES-1 corepressor protein, was displaced during chondrogenic differentiation and following TGF␤3 treatment.Conclusion. These results reveal novel mechanisms by which Notch signaling represses gene expression. Notch signaling proteins act on the SOX9 binding site in the COL2A1 enhancer and prevent SOX9-mediated transcriptional activation of COL2A1 and, thus, chondrogenic differentiation.

Attention has recently focused on the critical role of inflammatory responses in the tumor stroma that provide favorable conditions for cancer-cell growth and invasion/metastasis. In particular, macrophages recruited into the tumor stroma and activated, known as tumorassociated macrophages, are suggested to promote tumorigenesis. In this study, we examined the effect of a decrease in the number of monocytes/macrophages in peripheral blood and the tumor stroma on the development of bone and muscle metastases by lung cancer cells. M etastases of several malignant cancers including those of the breast, lung, prostate, and kidney have high affinity for bone. Bone metastasis is often accompanied by serious complications such as pathological fractures, bone pain, spinal cord compression, and hypercalcemia. Organ metastasis, including that affecting bone, is a multistep process mediated through mutual interaction between cancer cells and the host microenvironment. In bone metastasis, cancer cells reach the bone via hematogenous spread, followed by osteoclastic bone resorption, and finally proliferate in the bone matrix.(1,2)Moreover, osteoclast-stimulating cytokines such as PTHrP have been shown to promote bone metastasis. Inflammatory responses in the tumor stroma play an important role by providing favorable conditions for cancer cell growth, invasion/metastasis, and angiogenesis as well as malignant progression.(4-6) In particular, monocytes/macrophages are recruited into the tumor stroma, and activated macrophages known as TAMs produce potent angiogenic factors, as well as inflammatory cytokines, growth factors, and proteases, resulting in a promotion of angiogenesis and invasion/metastasis.(7-9) Infiltrating TAMs are often closely associated with poor prognosis and tumor angiogenesis in patients with various tumor types.(9-11) A preparation of Cl 2 MDP-LIP has been reported to markedly inhibit angiogenesis in corneas in response to inflammatory cytokines through depletion of macrophages.(12) A recent study has demonstrated that administration of clodronate-liposomes depleted TAMs in mouse models resulting in significant inhibition of tumor growth and tumor angiogenesis, whereas free clodronate alone did not.(13) Clodronate-liposomes were also found to inhibit both tumor growth and tumor angiogenesis by lung cancer cells in a xenograft model when stimulated by inflammatory stimuli. (14) Angiogenesis in a tumor microenvironment in bone marrow also played a critical role in the induction of an angiogenic response and invasion/metastasis by cancer cells.(15) Furthermore, monocyte/macrophage precursor cells entered the osteoclastic lineage and expressed the osteoclastic marker TRAP under the influence of the RANK/RNAKL signaling pathway.(16) Tumor burden at bone metastatic sites was markedly decreased in preclinical models on treatment with inhibitors of the RANK/RANKL pathway and neutralizing antibodies against PTHrP as well as bisphosphonate, suggesting a central role for osteoclasts in bone metastasis. (17)(18)(19) ...

BackgroundGiant cell tumor of bone (GCTB) is an intermediate tumor known to be locally aggressive, but rarely metastasizing. To plan a prospective study of GCTB, we performed a questionnaire survey for institutions participating in the Bone and Soft Tissue Tumor Study Group (BSTTSG) in the Japan Clinical Oncology Group (JCOG) in 2015.MethodsWe reviewed 158 consecutive patients with primary GCTB treated with curettage without perioperative denosumab from 2008 to 2010 in Japan. We investigated local and distant recurrence rates after definitive curettage. We also investigated the recurrence rate after treatment with preoperative and/or postoperative denosumab with curettage in recent years. There were 40 patients treated with perioperative denosumab, and the factors affecting recurrence in them were investigated.ResultsAnswers were available from 24 of 30 institutions (80.0%) participating in JCOG BSTTSG. Thirty (19.0%) and 4 (2.5%) of 158 patients developed local and distant recurrence after curettage without perioperative denosumab from 2008 to 2010, respectively. Campanacci grade and embolization before surgery were significantly associated with increasing incidence of local recurrence after curettage (p = 0.034 and p = 0.022, respectively). In patients treated with perioperative desnosumab, 120 mg denosumab was administered subcutaneously for a median 6 (2–41) and 6 (1–14) times in preoperative and postoperative settings, respectively. The recurrence rates were 6 of 21 (28.6%), 2 of 9 (22.2%), and 0 of 10 (0.0%) in the preoperative, postoperative, and both pre- and postoperative denosumab treatment groups, respectively. With all of the preoperative treatments, administration exceeding five times was significantly associated with a decreased incidence of local recurrence after curettage (p < 0.001).ConclusionThe recurrence rate of GCTB was still high after curettage, especially in Campanacci grade III, and improvements in the therapeutic strategy are needed in this cohort. There is a possibility that a sufficient dose of preoperative denosumab can reduce recurrence after curettage. Recently, we have started a clinical trial, JCOG1610, to investigate the efficacy of preoperative denosumab in patients who can be treated with curettage in GCTB.

Purpose: The role of chemotherapy (CT) and radiotherapy (RT) for management of extraskeletal osteosarcoma (ESOS) remains controversial. We examined disease outcomes for ESOS patients and investigated the association between CT/RT with recurrence and survival. Patients and methods: Retrospective review at 25 international sarcoma centers identified patients ≥18 years old treated for ESOS from 1971 to 2016. Patient/tumour characteristics, treatment, local/systemic recurrence, and survival data were collected. Kaplan-Meier survival and Cox proportional-hazards regression and cumulative incidence competing risks analysis were performed. Results: 370 patients with localized ESOS treated definitively with surgery presented with mainly deep tumours (n = 294, 80%). 122 patients underwent surgical resection alone, 96 (26%) also received CT, 70 (19%) RT and 82 (22%) both adjuvants. Five-year survival for patients with localized ESOS was 56% (95% CI 51%-62%). Almost half of patients (n = 173, 47%) developed recurrence: local 9% (35/370), distant 28% (102/370) or both 10% (36/370). Considering death as a competing event, there was no significant difference in cumulative incidence of local or systemic recurrence between patients who received CT, RT, both or neither (local p = 0.50, systemic p = 0.69). Multiple regression Cox analysis showed a significant association between RT and decreased local recurrence (HR 0.46 [95% CI 0.26-0.80], p = 0.01). Conclusion: Although the use of RT significantly decreased local recurrences, CT did not decrease the risk of systemic recurrence, and neither CT, nor RT nor both were associated with improved survival in patients with localized ESOS. Our results do not support the use of CT; however, adjuvant RT demonstrates benefit in patients with locally resectable ESOS.

Refractory bone and soft tissue sarcomas are challenging diseases to treat because of their robustness to chemotherapy. Although cancer vaccines have the potential to become an attractive treatment modality, their progress has been hampered by the presence of many subtypes of sarcomas and different human leukocyte antigen (HLA)-types. We investigated whether personalized peptide vaccination (PPV) would be feasible for the vast majority of sarcoma patients. Twenty refractory bone and soft tissue sarcoma patients with nine different subtypes and 11 different HLA-class IA phenotypes were enrolled in this study. A maximum of four HLAmatched peptides showing higher peptide-specific IgG responses in pre-vaccination plasma were selected from 31 pooled peptide candidates applicable for the HLA-A2, -A3, -A11, -A24, -A26, -A31, and -A33 types, and were subcutaneously administered weekly for 6 weeks and bi-weekly thereafter. Measurement of peptide-specific CTL and IgG responses along with other laboratory analyses were conducted before and after vaccination. No patients were excluded by either sarcoma subtypes or different HLA-types. No severe adverse events associated with PPV were observed in any patients. Peptide-specific immunological boosting was observed in the post-vaccination samples from the majority of patients. Tumor reduction of the lung metastasis and a long stable disease was observed in each case, and the median overall survival time of the 20 cases was 9.6 months. Taken together, PPV could be feasible for the vast majority of refractory sarcoma patients because of the safety and higher rates of immunological responses regardless of the presence of different sarcoma subtypes and various HLA-types. (Cancer Sci 2013; 104: 1285-1294