Ameloblastin Induces Tumor Suppression and Chemosensitivity in Osteosarcoma

Objectives: Ameloblastin (AMBN), the most abundant non-amelogenin enamel matrix protein, has a role in enamel matrix mineralization and ameloblast differentiation. Recently, it was reported that AMBN was expressed in bone tissue and we clarified that AMBN induced osteogenic differentiation through AMBN-CD63-Integrinβ1-Src axis by using osteosarcoma (OS) cell lines. OS affects jaw or long bones and doxorubicin is commonly used for the treatment. However, it still shows a poor prognosis with pulmonary metastasis. Here, we made a hypothesis that AMBN possess tumor suppressive role and enhances chemosensitivity to doxorubicin treatment in OS.
Methods: Tissue samples of 37 OS cases retrieved from Hiroshima University Hospital were used for correlating immunoexpression of AMBN and prognosis of patients. In vivo experiments, 143B-Luc cells were injected into five-week-old male BALB/cAnNCrlCrlj mice, and tumor growth and metastasis were analyzed by bioluminescent imaging. In vitro experiments, western blot, wound healing assay, soft-agar colony formation assay, and FACS analysis were used.
Results: (1) In immunohistochemical analysis of 37 clinical OS cases, AMBN expression showed lower frequency of pulmonary metastasis (chi-square test, P＜0.05) and better prognosis. (2) In vivo, AMBN stable 143B-Luc cells showed the inhibition of tumor growth and pulmonary metastasis by using mouse xenograft model (t-test, P＜0.05). (3) In vitro, AMBN stable 143B-Luc cells showed apoptosis with the cleavage of caspase-3, chemosensitivity to doxorubicin and suppressed colony formation ability, migration via the inactivation of Src-Stat3 pathway. Moreover, these phenotypes were rescued by the induction of constitutive active Src (t-test, P＜0.05).
Conclusions: We demonstrate that AMBN has a novel tumor suppressive role and induce chemosensitivity to doxorubicin via Src-Stat3 inactivation in OS. Our findings indicate that AMBN has a potential to be used as a therapeutic target of OS. (This study was supported by JSPS.)