Eiji Kozawa

Inhibition of hyaluronan synthesis in breast cancer cells by 4‐methylumbelliferone suppresses tumorigenicity in vitro and metastatic lesions of bone in vivo

Hyaluronan (HA) has been shown to play crucial roles in the tumorigenicity of malignant tumors. Previous studies demonstrated that inhibition of HA suppressed the tumorigenicity of various malignant tumors including breast cancer. 4‐methylumbelliferone (MU) has been reported to inhibit HA synthesis in several cell types. However, few studies have focused on the effects of HA inhibition in breast cancer cells by MU, nor the effects on bone metastasis. We hypothesized that MU would suppress the progression of bone metastasis via inhibition of HA synthesis. Here, we investigated the effects of MU on HA expression in MDA‐MB‐231 breast cancer cell line in addition to their tumorigenicity in vitro and in vivo. HAS2 mRNA expression was downregulated after 6 and 24 hr treatment with MU. Quantitative analysis of HA revealed that MU significantly inhibited the intracellular and cell surface HA. MU significantly inhibited cell growth and induced apoptosis as determined by cell proliferation and TUNEL assays, respectively. Phosphorylation of Akt was suppressed after 12 and 24 hr treatment with MU. MU treatment also inhibited cell motility as well as cell invasiveness. MU also inhibited cell growth and motility in murine fibroblast cell line NIH3T3. In vivo, administration of MU inhibited the expansion of osteolytic lesions on soft X‐rays in mouse breast cancer xenograft models. HA accumulation in bone metastatic lesions was perturbed peripherally. These data suggest that MU might be a therapeutic candidate for bone metastasis of breast cancer via suppression of HA synthesis and accumulation.

Inhibition of hyaluronan retention by 4-methylumbelliferone suppresses osteosarcoma cells in vitro and lung metastasis in vivo.

Background:Hyaluronan (HA) plays crucial roles in the tumourigenicity of many types of malignant tumours. 4-Methylumbelliferone (MU) is an inhibitor of HA synthesis. Several studies have shown its inhibitory effects on malignant tumours; however, none have focused on its effects on osteosarcoma.Methods:We investigated the effects of MU on HA accumulation and tumourigenicity of highly metastatic murine osteosarcoma cells (LM8) that have HA-rich cell-associated matrix, and human osteosarcoma cell lines (MG-63 and HOS).Results:In vitro, MU inhibited HA retention, thereby reducing the formation of functional cell-associated matrices, and also inhibited cell proliferation, migration, and invasion. Akt phosphorylation was suppressed by MU (1.0 mM). In vivo, although MU showed only a mild inhibitory effect on the growth of the primary tumour, it markedly inhibited (75% reduction) the development of lung metastasis. Hyaluronan retention in the periphery of the primary tumour was markedly suppressed by MU.Conclusion:These findings suggested that MU suppressed HA retention and cell-associated matrix formation in osteosarcoma cells, resulting in a reduction of tumourigenicity, including lung metastasis. 4-Methylumbelliferone is a promising therapeutic agent targeting both primary tumours and distant metastasis of osteosarcoma, possibly via suppression of HA retention.

Osteoarthritic change is delayed in a Ctsk‐knockout mouse model of osteoarthritis

OBJECTIVE Several studies have shown that cathepsin K (CTK) is overexpressed in osteoarthritic (OA) cartilage and subchondral bone. However, it has not been well established whether CTK expression is harmful or beneficial. We undertook this study to investigate the direct involvement of CTK in OA development using Ctsk-knockout (Ctsk(-/-)) mice in a joint instability-induced model of OA. METHODS We analyzed the natural course of the phenotype of 25-week-old Ctsk(-/-) mice. OA development was evaluated with a modified Mankin histologic score up to 8 weeks after surgery was performed to destabilize the knee in Ctsk(-/-) and Ctsk(+/+) mice. Histologic analysis was used to evaluate expression of CTK, matrix metalloproteinase 13 (MMP-13), ADAMTS-5, and tartrate-resistant acid phosphatase (TRAP) proteins in chondrocytes, synovial cells, and osteoclasts. Bone architecture was analyzed by histomorphometry. RESULTS Bone mineral content and bone volume were higher in Ctsk(-/-) mice at 25 weeks, whereas OA did not develop spontaneously in either Ctsk(-/-) or Ctsk(+/+) mice. In a model of destabilization-induced OA, OA progression was significantly delayed in Ctsk(-/-) mice. CTK was overexpressed in chondrocytes and synovial cells of knee joints developing OA in Ctsk(+/+) mice. MMP-13 and ADAMTS-5 were less strongly expressed in chondrocytes of Ctsk(-/-) mice, and MMP-13 was less strongly expressed in synovial cells. TRAP-positive osteoclasts were overexpressed in Ctsk(-/-) mice. CONCLUSION These results indicate that CTK plays crucial direct roles in the early to intermediate stage of OA development. CTK-positive chondrocytes and synovial cells may be a possible target to prevent disease progression in OA.

Cathepsin K Activity Controls Injury-Related Vascular Repair in Mice

Cathepsin K (CatK) is one of the most potent mammalian collagenases. We showed previously the increased expression of CatK in human and animal atherosclerotic lesions. Here, we hypothesized that ablation of CatK mitigates injury-induced neointimal hyperplasia. Male wild-type (CatK+/+) and CatK-deficient (CatK−/−) mice underwent ligation or a combination of ligation and polyethylene cuff-replacement injuries to the right common carotid artery just proximal to its bifurcation, and they were then processed for morphological and biochemical studies at specific time points. On operative day 28, CatK−/− significantly reduced neointimal formation and neovessel formation in both single- and combination-injured arteries compared with the Cat K+/+ mice. At early time points, CatK−/− reduced the lesion macrophage contents and medial smooth muscle cell proliferation, the mRNA levels of monocyte chemoattractant protein-1, toll-like receptor-2, toll-like receptor-4, chemokine ligand-12, and the gelatinolytic activity related to matrix metalloproteinase-2/-9. An aorta-explant assay revealed that smooth muscle cell movement was impaired in the CatK−/− mice compared with the CatK+/+ mice. In addition, the smooth muscle cells and macrophages from CatK−/− mice had less invasive ability through a reconstituted basement membrane barrier. This vasculoprotective effect was mimicked by Cat inhibition with trans-epoxysuccinyl-L-leucylamido-{4-guanidino} butane (E64d). These results demonstrate an essential role of CatK in neointimal lesion formation in response to injury, possibly via the reduction of toll-like receptor-2/-4–mediated inflammation and smooth muscle cell proliferation, suggesting a novel therapeutic strategy for the control of endovascular treatment–related restenosis by regulating CatK activity.

Therapeutic potential of hyaluronan oligosaccharides for bone metastasis of breast cancer

Hyaluronan (HA) oligosaccharides were reported to have suppressive effects on various malignant tumors via disruption of receptor HA interactions. However, no studies have focused on the effects of HA oligosaccharides on bone metastasis of breast cancer. In this study, we clarified the effective size of HA oligosaccharides required to inhibit cell growth in the highly invasive breast cancer cell line, MDA‐MB‐231 cells. Based on the results of cell growth assay, we subsequently analyzed the effects of HA tetrasaccharides, HA decasaccharides, and high molecular weight HA on the other breast cancer cell behaviors in vitro and breast cancer bone metastasis in vivo. HA decasaccharides significantly inhibited cell growth, motility, and invasion, whereas tetrasaccharides did not. HAS2 mRNA expression was altered after the treatment with both tetrasaccharides and decasaccharides. Phosphorylation of Akt was suppressed after 1 h treatment with HA decasaccharides, and the effect was partially reversed by anti‐CD44 monoclonal antibody. In vivo, local application of HA decasaccharides inhibited the expansion of osteolytic lesions in tibia on soft X‐rays using mouse bone metastasis model of breast cancer. Histological analysis revealed HA accumulation in bone metastatic lesions was perturbed by decasaccharides. These results suggest that HA oligosaccharides suppressed progression of bone metastasis in breast cancer via interruption of endogenous HA–CD44 interaction, and as such, can be a novel therapeutic candidate to limit bone metastasis of breast cancer.

CTNNB1 S45F mutation predicts poor efficacy of meloxicam treatment for desmoid tumors: a pilot study.

We hypothesized that patterns of CTNNB1 (β-catenin) mutations would affect the outcome of conservative therapy in patients with desmoid tumors. This study aimed to determine the significance of CTNNB1 (β-catenin) mutations in predicting the treatment outcome in patients with desmoid tumors treated with meloxicam, a cyclooxygenase-2 (COX-2) selective inhibitor. Between 2003 and 2012, consecutive thirty-three patients with extra-peritoneal sporadic desmoid tumors were prospectively treated with meloxicam as the initial systemic medical therapy. The efficacy of meloxicam was evaluated according to Response Evaluation Criteria in Solid Tumors (RECIST). DNA was isolated from frozen tissue or formalin-fixed materials. CTNNB1 mutation analysis was performed by direct sequencing. Positivity of nuclear β-catenin staining by immunohistochemistry was compared with the status of CTNNB1 mutations. The correlation between the efficacy of meloxicam treatment and status of CTNNB1 mutations was analyzed. Of the 33 patients with meloxicam treatment, one showed complete remission (CR), 7 partial remission (PR), 12 stable disease (SD), and 13 progressive disease (PD). The following 3 point mutations were identified in 21 of the 33 cases (64%): T41A (16 cases), S45F (4 cases) and S45P (one case). The nuclear expression of β-catenin correlated significantly with CTNNB1 mutation status (p = 0.035); all four cases with S45F mutation exhibited strong nuclear expression of β-catenin. S45F mutation was significantly associated with a poor response (all cases; PD) (p = 0.017), whereas the other mutations had no impact on efficacy. The CTNNB1 mutation status was of significant prognostic value for meloxicam treatment in patients with sporadic desmoid tumors.

Development and external validation of nomograms predicting distant metastases and overall survival after neoadjuvant chemotherapy and surgery for patients with nonmetastatic osteosarcoma: A multi‐institutional study

In this era of individualized cancer treatment, data that could be applied to predicting the survival of patients with osteosarcoma are still limited because of the rarity of the disease and the difficulty in accumulating a sufficient number of patients. Therefore, a multi‐institutional collaboration was implemented to develop and externally validate nomograms that would predict metastasis‐free survival (MFS) and overall survival (OAS) for patients with nonmetastatic osteosarcoma.

EMMPRIN co-expressed with matrix metalloproteinases predicts poor prognosis in patients with osteosarcoma.

Several studies have focused on the relationships between the expression of extracellular matrix metalloproteinase inducer (EMMPRIN) and the prognosis of patients with malignant tumors. However, few of these have investigated the expression of EMMPRIN in osteosarcoma. We examined expression levels of EMMPRIN immunohistochemically in 53 cases of high-grade osteosarcoma of the extremities and analyzed the correlation of its expression with patient prognosis. The correlation between matrix metalloproteinases (MMPs) and EMMPRIN expression and the prognostic value of co-expression were also analyzed. Staining positivity for EMMPRIN was negative in 7 cases, low in 17, moderate in 19, and strong in 10. The overall and disease-free survivals (OS and DFS) in patients with higher EMMPRIN expression (strong-moderate) were significantly lower than those in the lower (weak-negative) group (0.037 and 0.024, respectively). In multivariate analysis, age (P = 0.004), location (P = 0.046), and EMMPRIN expression (P = 0.038) were significant prognostic factors for overall survival. EMMPRIN expression (P = 0.024) was also a significant prognostic factor for disease-free survival. Co-expression analyses of EMMPRIN and MMPs revealed that strong co-expression of EMMPRIN and membrane-type 1 (MT1)-MMP had a poor prognostic value (P = 0.056 for DFS, P = 0.006 for OS). EMMPRIN expression and co-expression with MMPs well predict the prognosis of patients with extremity osteosarcoma, making EMMPRIN a possible therapeutic target in these patients.

Nuclear expression of β-catenin predicts the efficacy of meloxicam treatment for patients with sporadic desmoid tumors.

This study aimed to determine the prevalence of β-catenin nuclear positivity as a prognostic factor in patients with desmoid tumors (DTs) treated with meloxicam, a cyclooxygenase-2 (COX-2) selective inhibitor. Between 2003 and 2012, consecutive 31 patients with extraabdominal, sporadic DTs were prospectively treated with meloxicam as a systemic medical therapy. Immunohistochemistry was performed on formalin-fixed material to quantify the nuclear expression of β-catenin and Ki-67, and cytoplasmic expression of COX-2. All clinicopathological characteristics including the intensity of immunohistochemical staining were analyzed with respect to their prognostic value for meloxicam treatment. Of the 31 patients with meloxicam treatment, there was 1 with complete remission (CR), 7 with partial remission (PR), 12 with stable disease (SD), and 11 with progressive disease (PD). Higher nuclear expression of β-catenin was significantly associated with a poor response (PD/SD) (p = 0.017). The positivity of COX-2 and Ki-67 and none of the other clinical variables were associated with prognosis. The nuclear expression of β-catenin can predict the efficacy of meloxicam treatment for patients with sporadic DTs.

Versican V1 isoform regulates cell-associated matrix formation and cell behavior differentially from aggrecan in Swarm rat chondrosarcoma cells

Versican, a large chondroitin sulfate proteoglycan that binds hyaluronan and is composed of large extracellular matrix aggregates, has been shown to correlate with tumor progression. No studies have examined the roles of versican in chondrosarcoma nor compared them to those of aggrecan. In clinical specimens of human chondromatous tumors, versican expression was significantly increased in malignant tumors, moreover, as the tumor grade increased. To clarify the roles of versican in chondrosarcoma, versican splicing variant 1, variant 3 or only GFP was stably transfected to Swarm rat chondrosarcoma cells with Trap‐In System. Forced expression of versican V1 isoform in Swarm rat chondrosarcoma cells induced a marked increase of cell‐associated matrix compared to V3‐, GFP‐ transfected or RCS cells. Versican was immunolocalized in a fashion similar to that of hyaluronan and more diffusively than aggrecan. Anchor‐dependent and ‐independent growth was not affected by versican isoform expression, whereas cell motility and migration were significantly enhanced by V1 isoform transfection. Tumors formed in vivo with V1‐transfected cells exhibited more myxomatous area and included more spindle shaped cells. These results support the concept that versican has the capacity to form more extensive cell‐associated matrix than aggrecan, and the prominent matrix formation alters the cell behavior of chondrosarcoma more aggressively. These observations suggest that versican expression may serve as a marker of tumor grade determination in chondrosarcoma and possibly help to decide on therapeutic targets in higher grades of chondrosarcoma.