| Glycoprotein non-metastatic melanoma protein B (GPNMB)/Osteoactivin (OA) is a transmembrane protein that is commonly expressed in basal/triple-negative breast cancer. Our group discovered that GPNMB/OA is sufficient to enhance the migration and invasion of weakly metastatic murine breast cancer cells in vitro, and promotes the formation of bone and lung metastases in vivo. In this study, we sought to fully characterize the pro-invasive role of GPNMB/OA in human breast cancer cells by defining those motifs/domains within GPNMB/OA that are required to promote breast cancer cell invasion. In addition, we have sought the identity of putative GPNMB/OA interacting proteins that may be involved in promoting GPNMB/OA-mediated effects on breast cancer cell invasion and metastasis. To accomplish this, a panel of GPNMB/OA mutants were generated and expressed in the GPNMB/OA null BT-549 and MDA-MB-453 human breast cancer cells and subjected to in vitro invasion assays characterization. A candidate approach based on previous literature reports was used to facilitate the identification of GPNMB/OA protein binding partners in addition to attempting mass spectrometry analysis. Finally, a transgenic mouse model was created that expresses human GPNMB/OA under the control of the Mouse Mammary Tumor Virus (MMTV) promoter to further explore the role of GPNMB/OA on mammary gland development and tumorigenesis in vivo. Our results indicate that human GPNMB/OA is sufficient to induce enhanced invasion of BT549 breast cancer cells in vitro, which requires both the cytoplasmic tail and RGD recognition motif. Characterization of the MMTV-GPNMB/ OA transgenic mice revealed that GPNMB/OA does not negatively affect normal mammary duct development in virgin females and no mammary tumors have developed to date. We conclude that GPNMB/OA is indeed capable of inducing invasion of breast cancer cells in vitro, which may require the participation of integrins and/or the residues/motifs within the cytoplasmic tail responsible for recruiting signalling molecules to this region of GPNMB/OA. |
