BIOLOGICAL ROLE OF OSTEOACTIVIN, A PROTEIN EXPRESSED BY TUMOR-CONDITIONED MACROPHAGES, IN THE PROCESSES OF TUMOR GROWTH AND TISSUE REPAIR

Tumor-Associated Macrophages (TAM) are key orchestrators of the tumor microenvironment, directly affecting neoplastic cell growth, neo-angiogenesis, extracellular matrix remodeling and immunosuppression. In a gene profiling analysis on tumor-conditioned macrophages cultured in vitro with tumor cell supernatants, we identified a number of up-regulated genes. One of the most expressed gene was Gpnmb, coding for a protein called Human Glycoprotein non-metastatic melanoma protein B (GPNMB), also named Osteoactivin (OA). Osteoactivin is a trans-membrane and shed molecule with diverse biological functions, spanning from cell adhesion and migration, to immune-suppression and tissue repair. This study investigates the modulation of this protein and its functional role in monocytes/macrophages and TAM, in the tumor context. In human monocytes, expression of OA is up-regulated by anti-inflammatory stimuli, in particular IL-10, and corticosteroids. Immunostimulatory cytokines (IFNγ, IL-1β) or LPS are not stimulating its production. Accordingly, in vitro M2-polarized macrophages express more OA than M1-macrophages. A spontaneous mutation of the Gpnmb gene occurred in the DBA/2J mouse strain. The mutation causes a premature stop codon and generation of a truncated non-functional protein. This strain, and the reconstituted DBA/2J-Gpnmb+ mice with functional OA, are commercially available. OA-defective mice do not have obvious major problems, with the exception of the known rapid onset of glaucoma. To clarify the role of this protein in the tumor microenvironment, we generated methylcolantrene-induced fibrosarcoma in these mice. Both mouse strains produced tumors with a similar incidence. We established and characterized 2 cell lines from DBA/2JGpnmb+ mice and 2 from DBA/2J mice. Tumors from DBA/2J mice grew earlier in DBA/2JGpnmb+ mice, indicating that the protein Osteoactivin produced by stromal cells, including TAM, enhanced tumor growth. To better understand the function of this protein, we generated isogenic cell lines expressing or not the functional OA protein (G2 OA and G2 MOCK cells). Osteoactivin -expressing cells grew faster in vitro and under serum-free conditions were able to survive and to form spheroids which go on proliferating in an anchorage-independent manner. OA-expressing cells present typical cancer stem cell markers on their membranes such as Sca1, CD117 and SOX-2 and they are able to self-renew. The in vivo experiments demonstrated that Osteoactivin expression is associated with a significantly more aggressive phenotype, both in terms of tumor-take and tumor growth compared to OA-defective cell lines. We further demonstrated that OA-expressing tumors have higher mRNA levels of specific stem markers and in particular Nanog, SOX-2 and Brachyury. From these data we can speculate that the production of Osteoactivin and its secretion by macrophages in the tumor microenvironment might be involved in the maintenance of cancer cell stemness and their proliferative potential.