Glycoprotein NMB functions with growth factor signaling to induce tumorigenesis in breast cancer cells

概要

Purpose
Breast cancer is the most common cancer among the adult women worldwide. Glycoprotein NMB (GPNMB) is a type I transmembrane protein that is highly expressed in many cancers including breast cancer and one of the prognostic factors. We previously reported that GPNMB overexpression confer the tumorigenic abilities to non-tumorigenic mammary epithelial cells, NMuMG, through the induction of epithelial-to-mesenchymal transition (EMT). Therefore, this study focused on serine (S) 530 in the intracellular domain of human GPNMB isoform b which has been predicted by ELM database to clarify its involvement in GPNMB-induced tumorigenic abilities.

Materials and methods
In this study, mass-spectrometry was performed to identify phosphorylation of Serine 530 of human GPNMB. DNA construction and transfection was used to establish stable cell line. Immunoblot analysis was used to check the expression levels of proteins. Immunofluorescence was used to examine the expression pattern of GPNMB proteins. Quantitative real-time PCR was used to check mRNA expression levels. Sphere formation assay was used to identify stem cells. Migration assay was used to test the migratory ability of cells. Immunohistochemical staining was used to examine NMuMG-GPNMB tumor histology features. Animal experiment was performed to evaluate the malignant potential of cells.

Results
In this study, I focused on the serine (S) residue in the intracellular domain of GPNMB, S530 in human and S546 in mouse GPNMB, which are phosphorylated. Then, we made a point mutant which is replaced by an alanine residue (A), GPNMB(SA). Established GPNMB(SA) overexpressing cells showed the significant reduction of sphere formation in vitro and tumor growth in vivo, and we found that GPNMB(SA) expressing cells lose stemness-related genes expression compared to GPNMB(WT) expressing cells. Additionally, the GPNMB(SA) mutation impaired GPNMB-mediated cellular migration. Furthermore, we also found that tyrosine kinase receptor signaling triggered by epidermal growth factor (EGF) or fibroblast growth factor (FGF)-2 induce the phosphorylation of S530 through activation of downstream oncoproteins, RAS and RAF.

Discussion
In this study, we show that serine 530 in the intracellular domain of GPNMB is critical. We have previously shown that tyrosine residue of GPNMB are important for its tumorigenic activity. This study is the first to report that phosphorylation of S530 in human GPNMB isoform b affects its tumorigenicity.
In addition, we showed that S530 is one of the important switches of GPNMB-induced EMT, and it also affects the expression of stemness-related genes. EMT is one of the important events for tumor cell invasion and metastasis. The loss of phosphorylation of serine 530 significantly reduced the tumorigenic activity of GPNMB. However, we previously found that tyrosine phosphorylation in hemITAM motif is also required for the tumorigenic activity of GPNMB. Although S530 is not the only phosphorylation site that affects the EMT-inducing and tumorigenic functions of GPNMB, this finding is still important for the study of GPNMB function in cancer.
Furthermore, we found that this serine phosphorylation is stimulated by activation of RTK signaling upon EGF and FGF-2 treatment. Phosphorylation of proteins is one of the most common post-translational modifications. The expression level of pGPNMB(S530) is regulated by the RAS/RAF/MEK/ERK axis, one of the downstream signaling pathways of RTK. Therefore, we believe that blocking GPNMB(S530) downstream of RTK signal is a potential therapeutic target.

Conclusion
Serine530 is an important phosphorylation site located in the intracellular domain of human GPNMB. Meanwhile, the expression of this phosphorylated GPNMB can be regulated by growth factors EGF and FGF-2 and their downstream oncoproteins RAS and RAF. GPNMB(SA) inhibits EMT induced by GPNMB and reduces sphere in vitro formation and tumor growth in vivo. Moreover, GPNMB(SA) showed less stemness-related genes expression induction compared to GPNMB(WT) and cellular migration. These findings suggest that Serine530 plays an important role in the tumorigenic function of GPNMB, and it would be novel therapeutic target.