The role of GPNMB ectodomain in breast cancer development

概要

Purpose (目的)
　To elucidate the role of GPNMB ectodomain, especially the Kringle-like domain (KLD) and the C-mannosylation motif, in breast cancer development

Material and Method (対象と方法)
1. Polymerase chain reaction
　To construct the mammalian expressing vector of mGPNMBΔKLD, mGPNMBW69H, mGPNMBW168H, and hGPNMBΔC mutants.

2. FACS analysis
　To analyze the surface expression of GPNMB-WT and GPNMBΔKLD protein.

3. Immunofluorescence assay
　To analyse the subcellular localization of GPNMB-WT and GPNMBΔKLD protein.

4. Western blotting
　To detect the protein expression in cell lysate or immunoprecipitated protein.

5. Transwell migration assay
　To evaluate the migratory ability of the cell lines.

6. 2D proliferation assay
　To evaluate the monolayer culture growth of the cell lines.

7. Sphere formation assay
　To evaluate the sphere forming ability as an in vitro tumorigenic assay of the cell lines.

8. In vivo tumor formation assay
　To evaluate the in vivo tumorigenic ability of the cell lines by injecting the cells subcutaneously to nude mice.

9. Immunohistochemical staining
　To analyse the histological features of the tumors.

10. Electron microscopy
　To analyse the cell junctions formed in the tumors.

11. Mass spectrometry
　To analyse the C-mannosylation status of GPNMB-WT and GPp-Fc proteins.

12. ELISA
　To analyse the antibodies produced by the mouse injected by GPp-Fc antigen.

13. Statistical analysis
　To show the significance of the results.

Result (結果)
1. GPNMB has conserved Kringle-like domain across species
　KLD of GPNMB is well conserved in across species, including the cysteines which are important in disulfide bond formation.

2. Deletion of KLD does not affect GPNMB physiological properties
　The deletion of KLD does not affect the surface expression, subcellular location, Src- induced tyrosine phosphorylation, and homodimerization of GPNMB.

3. KLD is important in GPNMB-induced tumor formation
　ΔKLD mutant expressing cells showed significantly lower sphere forming activity and had significantly smaller tumors with less incidence compared to those of WT.

4. ΔKLD mutants have less disruption of cell polarity than WT
　ΔKLD mutants could form epithelial tubular structure in tumors, meaning that they can partially maintain cellular polarity.

5. GPNMB-WT and GPNMB-ΔKLD mutants do not form tight junction
　The formation of tight junction was observed in mock, whereas none could be observed from WT and ΔKLD mutants; however, ΔKLD mutants were observed to have more tight- like junction compared to those of WT.

6. Deletion of KLD suppresses E-cadherin expression but impairs cellular migration ability
　Both WT and ΔKLD mutants had EMT phenotype as shown by the suppression of E- cadherin, however through immunofluorescence staining we observed that WT formed more stress fibers while ΔKLD mutants maintained cortical actin fibers. Moreover, ΔKLD7 mutant expressing cells had significantly lower migration ability compared to those of WT expressing cells

7. Conservation profile of the C-mannosylation motif of GPNMB
　hGPNMB has a WXXXW motif from amino acid 69 to 73, which is conserved among species and a WXXW motif, which is conserved in mouse and monkey.

8. GPNMB is C-mannosylated
　All tryptophan residues in both C-mannosylation motifs were shown to be mannosylated through GC-MS/MS analysis.

9. Tryptophan residue in the C-mannosylation consensus motif is essential in tumorigenesis
　Point mutation of the tryptophan residue in any C-mannosylation motifs could impair GPNMB-impaired tumorigenesis.

Discussion (考察)
Here, we provide the first experimental evidence that KLD and C-mannosylation motif of GPNMB are important in breast cancer development. The deletion of KLD, while not affecting GPNMB physiological properties, can impair GPNMB-induced tumorigenesis. Although the deletion of KLD suppresses E-cadherin expression, it somehow impairs the GPNMB-induced disruption of cellular polarity, hence reducing its tumorigenic activity. On another note, the mutation of the tryptophan residue at the C-mannosylation motif, which was shown to be uniquely mannosylated on both tryptophan residues, causes significant loss of tumorigenic activity induced by GPNMB. Taken together, these results suggest that both KLD and C-mannosylation motif of GPNMB, which are located at the ectodomain, could be potential targets for future breast cancer therapy in patients overexpressing GPNMB.

Conclusion (結論)
KLD and C-mannosylation motif of GPNMB are essential in breast cancer development and can be potential targets for future therapy.