Role of CXC chemokine ligand 13 in oral squamous cell carcinoma associated osteolysis in athymic mice

Abstract

Oral squamous cell carcinomas (OSCC) are malignant tumors with a potent activity of local bone invasion; however, the molecular mechanisms of tumor osteolysis are unclear. In this study, we identified high level expression of chemokine ligand, CXCL13 and RANK ligand (RANKL) in OSCC cells (SCC1, SCC12 and SCC14a). OSCC cell-conditioned media (20%) induced osteoclast differentiation which was inhibited by OPG in peripheral blood monocyte cultures indicating that OSCC cells produce soluble RANKL. Recombinant hCXCL13 (10 ng/ml) significantly enhanced RANKL-stimulated osteoclast differentiation in these cultures. Trans-well migration assay identified that CXCL13 induces chemotaxis of peripheral blood monocytes in vitro which was inhibited by addition of anti-CXCR5 receptor antibody. Zymogram analysis of conditioned media from OSCC cells revealed matrix metalloproteinase-9 (MMP-9) activity. Interestingly, CXCL13 treatment to OSCC cells induced CXCR5 and MMP-9 expression suggesting an autocrine regulatory function in OSCC cells. To examine the OSCC tumor cell bone invasion/ osteolysis, we established an in vivo model for OSCC by subcutaneous injection of OSCC cells onto the surface of calvaria in NCr-nu/nu athymic mice, which developed tumors in 4-5 weeks. lCT analysis revealed numerous osteolytic lesions in calvaria from OSCC tumor-bearing mice. Histochemical staining of calvarial sections from these mice revealed a significant increase in the numbers of TRAP-positive osteoclasts at the tumor-bone interface. Immunohistochemical analysis confirmed CXCL13 and MMP-9 expression in tumor cells. Thus, our data implicate a functional role for CXCL13 in bone invasion and may be a potential therapeutic target to prevent osteolysis associated with OSCC tumors in vivo.Head and neck squamous cell carcinoma (HNSCC) are the most common malignant neoplasms, with a prevalence estimated to be greater than 40,000 cases annually in the US. Oral squamous cell carcinoma (OSCC), which contributes to 40% of all HNSCC is associated with mucosal surfaces of the oral cavity and oropharynx. 1 The etiology of OSCC involves both a genetic predisposition and exposure to environmental carcinogens such as tobacco, alcohol, chronic inflammation and viral infection. 2 Curcumin has been shown to suppress HNSCC growth both in vitro and in vivo, 3 and Fas-mediated apoptosis in HNSCC is a proven and efficient therapeutic approach in a xenograft animal model. 4 Furthermore, tamoxifen inhibition of OSCC cell growth in vitro 5 and a role for human longevity assurance gene 1 (LASS1) and C 18 -ceramide in chemotherapy induced cell death in HNSCC have been reported. 6 Malignant HNSCC tumors are known to have a potent activity of local bone invasion; however, the molecular mechanisms of tumor-associated osteolysis are unclear.The osteoclast is hematopoietic in origin and is the boneresorbing cell derived from monocyte/macrophage lineage. Tumor necrosis factor (TNF) family member, RANK ligand (RANKL), which is expressed on marrow stromal/osteoblast cells in respo...

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