No abstract

Background: CD44 plays a role in many physiological and pathological processes, including cell-cell and cell-extracellular matrix interactions, cell migration, and metastatic spread. A6 (KPSSPPEE) is a synthetic peptide that inhibits migration of human cancer cell lines and has anti-tumor activity in the absence of significant adverse events in tumor models and in a Phase 2 clinical trial in ovarian cancer. The current study was directed at identifying the mechanism of action of A6 through investigation of its effects on cell migration and metastasis. Methods: The effect of A6 on migration was investigated using human ovarian and breast carcinoma cell lines. Effects on cell migration in response to NIH3T3 conditioned media supplemented with VEGF were assessed using the Boyden chamber technique. To demonstrate the potential of A6 to inhibit cancer metastasis in vivo, the number of tumor nodules generated in the lungs of C57BL/6 mice intravenously inoculated with B16-F10 mouse melanoma cells was quantified. To characterize A6 binding and CD44 expression, standard FACS, cross-linking, and immunoprecipitation/blotting techniques were used. Results: A6 inhibited the migration of a subset of ovarian and breast cancer cell lines (e.g. SKOV3, OVCAR3, MDA-MB 361 and MDA-MB 468), exhibiting IC50 values in an in vitro chemotaxis assay in the pM to nM range. The ability of A6 to inhibit cancer cell migration in vitro correlated with CD44 expression: CD44 positive lines (e.g., SKOV3, OVCAR3, murine B16-F10 melanoma) were inhibited by A6 whereas cell lines expressing little or no CD44 (e.g., OVCAR4, OVCAR5, late passage A2780) were refractory to A6. Immunopreciptation studies demonstrate that CD44 binds biotin-tagged A6 peptide cross-linked to the plasma membrane. Although A6 perturbed the binding of a monoclonal anti-CD44 antibody (DF-1485) to SKOV3 cells it did not inhibit hyaluronic acid (HA)-induced adhesion of cancer cells through CD44 suggesting that A6 peptide is not a competitive antagonist of CD44/HA binding. In fact A6 potentiated the HA-induced adhesion of cancer cells. The ability of A6 to agonize the CD44 receptor and desensitize cells to migration signals represents a novel strategy for inhibiting CD44 function. In vivo, A6 (100 mg/kg delivered subcutaneously twice a day for 11 days) reduced the number of lung foci generated from the IV inoculation of 106 B16-F10 melanoma cells by 50% (pvalue= 0.003 in an unpaired t test), in a lung metastasis model dependent on the migration of B16-F10 melanoma cells from the circulation to the lungs. Conclusions: A6 potently blocks of the migration of CD44-positive cells in vitro through a novel mechanism of action. It binds to CD44 on the cell surface and in vivo it blocks the development of metastatic tumors at secondary sites. GOG study 170N is testing the activity of A6 in patients with recurrent ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5120.