Abstract B62: ADAM8 promotes tumorigenesis, angiogenesis, and spreading of circulating tumor cells in breast cancer

Abstract

Abstract ADAM (a disintegrin and metalloprotease) proteins mediate cell adhesion and shedding of receptors and proteins of the extracellular matrix via their disintegrin and metalloproteinase domains, respectively. Recently, using microarray analysis we identified ADAM8 as a target of a pathway mediated by NF-κB RelB/Ras to B lymphocyte-induced maturation protein (Blimp-1) that promotes a more aggressive breast cancer cell phenotype. ADAM8 is a membrane-anchored protein that is synthesized as a pro-form and needs to multimerize to autocatalytically clip off its prodomain leaving an active processed form. Using the Oncomine and bc-GenExMiner databases, ADAM8 mRNA levels were found overexpressed in invasive breast tumors compared to normal breast tissue, and high ADAM8 levels were correlated with metastatic relapse in breast cancer patients. As ADAM8 is not essential under physiological conditions as evidenced by the lack of phenotype in ADAM8 deficient mice, here we tested the hypothesis that ADAM8 represents a novel target for the treatment of aggressive breast cancer. Immunohistochemical analysis revealed that ADAM8 is abundantly expressed in 34.0% (17/50) of triple-negative breast tumors and in 48.2% (27/56) of breast cancer metastases. In particular, ADAM8 was highly expressed in brain metastases (63.0%). In contrast, ADAM8 was not observed in normal mammary tissue and was only detected in 13.5% of Ductal Carcinoma In Situ tumors. Consistently, we found that ADAM8 promotes aggressive breast cancer cell behavior in culture. ADAM8 siRNA knockdown reduced the migratory and invasive properties of triple-negative breast cancer lines MDA-MB-231 and Hs578T in Boyden chamber and 3D Matrigel assays. MDA-MB-231 clones stably expressing an ADAM8 shRNA or Control shRNA showed equal rates of growth and survival in vitro. However, when these clones were used in a mammary fat pad mouse model (n=7/group), tumors derived from ADAM8 shRNA MDA-MB-231 clone uniformly failed to grow significantly beyond a palpable size over the 33 day-time course in contrast to the Control shRNA clone (0.08 g versus 2.0 g). Angiogenesis was also significantly decreased in ADAM8 shRNA cell-derived tumors as measured by CD31 staining. As the clones were stably expressing Green Fluorescent Protein, spreading of circulating tumor cells (CTCs) into the blood was analyzed by flow cytometry. The CTC numbers in mice bearing ADAM8 shRNA tumors did not exceed background level. In contrast, the Control shRNA group showed a high number of CTCs. To address the mechanism causing tumorigenesis and tumor cell spreading, we investigated the effects of hypoxia, which is an early event that promotes tumor angiogenesis. In vitro, hypoxia (1% oxygen) induced levels of ADAM8 pro-form and processed protein by ~3-fold in the Control shRNA clone, but not in the ADAM8 shRNA clone. As an early step in migration of cancers cells requires β1-integrin activation, we tested the effects of ADAM8 knockdown. Immunofluorescence demonstrated an essential role of ADAM8 in activation of β1-integrin in the MDA-MB-231 clones. Consistently, expression and activity of the matrix metalloprotease MMP-9, which is induced downstream of β1-integrin pathway, were reduced by ADAM8 knockdown, suggesting this is an essential pathway whereby ADAM8 promotes matrix remodeling and angiogenesis. Despite recent advances in diagnosis and treatment of breast cancer, in many patients the disease still progresses to invasive carcinoma with fatal metastatic spread of primary tumors to distant organs. Our findings suggest that ADAM8 constitutes a promising new target for the treatment of these aggressive breast cancers. Citation Format: Mathilde Romagnoli, Nora D. Mineva, Delphine Loussouarn, Sophie Barillé-Nion, Mike Polmear, Irene Georgakoudi, Catharina Conrad, Uwe Schlomann, Joerg W. Bartsch, Maddy Parsons, Gail E. Sonenshein. ADAM8 promotes tumorigenesis, angiogenesis, and spreading of circulating tumor cells in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B62.